Fanawmm. {3n . nun...“ 2min“ .9 x! .11.. c. fir .Yuwiw: . Ex, . 133.... .z .. . I y :3 $.13. .I,.£. .t \\v{ LIBRARY Michigan State University This is to certify that the dissertation entitled DEVELOPMENT OF A VERSATILE SILICON—BASED BIOSENSOR PLATFORM FOR PATHOGEN DETECTION presented by FINNY P. MATHEW has been accepted towards fulfillment of the requirements for the Ph.D. degree in Biosystems Engineering Major Professor’s Signature ”we; 5/ 2009 Date MSU is an Affinnative Action/Equal Opportunity Institution - -.-.-.- -- PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/C|RC/DateDue.indd-p.1 DEVELC DEVELOPMENT OF A VERSATILE SILICON-BASED BIOSENSOR PLATFORM FOR PATHOGEN DETECTION By Finny P. Mathew A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biosystems and Agricultural Engineering 2006 DEVELOP Bioscnscr that may Will“ of human and tell as Win detection of b. Emsducers it; in an electri conditions of functiona‘tizet antibodies an Ihebioscnsor CR' within ImmunnsenSt ABSTRACT DEVELOPMENT OF A VERSATILE SILICON-BASED BIOSENSOR PLATFORM FOR PATHOGEN DETECTION By Finny P. Mathew Biosensors are portable diagnostic tools used for the rapid detection of pathogens that may require up to 2-7 days to detect otherwise. Biosensors are important in the fields of human and animal diagnostics, bioterrorism preparedness, food and water safety, as well as environmental safety. So a versatile silicon-based biosensor platform for the detection of bacteria that could be used with different biological detecting elements and transducers was fabricated and demonstrated. Silicon (0.01 ohm-cm, p-type) was etched in an electrochemical cell containing hydrofluoric acid solution using anodizing conditions of 5 mA/cm2 for 1 h to fabricate nano-tubular Si (NTS). NTS chips were functionalized into biosensors using biological sensing elements such as enzymes, antibodies and nucleic acids. A chemiluminescence-based enzyme assay was adapted to the biosensor system for the detection of E. coli resulting in a lower detection limit of 102 CFU within 30 min. The NTS-based platform was also functionalized into an optical immunosensor to successfully detect a pure culture of Salmonella Typhimurium within 10 min. Finally, a DNA-based NTS biosensor was developed using an electrochemical transducer for the detection of Salmonella Enteritidis. The NTS DNA biosensor was able to detect S. Enteritidis DNA with a lower limit of detection of 1 pg/ul for PCR-amplified DNA and 10 pg/ul for pure culture-extracted DNA. The electrochemical detection process was completed within 60 min. The versatility of the NTS platform for pathogen detection “as lemme. anli" Dnelopmcnt bioseturit)‘ m. assurance in ti food produc : detection was successfully demonstrated using three different biological sensing elements (enzyme, antibody, and DNA) and two detection methods (optical and electrochemical). Development of commercial biosensing devices using the NTS platform will strengthen biosecurity measures, enable improvement in human and animal health as well as quality assurance in food industry, while also reducing costs associated with loss of productivity, food product recalls and lawsuits. Dedicat: Dedicated to my dear wife and my family in India, Chicago, and East Lansing iv First of L this degree. a: thank ti‘tQ'OI‘. to my famil) Slipped The and Chicago . times. Also. c Hill thC bcCl‘. dining 316 con I Would a “3.“ than one lhis endtal'or IWould It ACKNOWLEDGMENTS First of all, I thank my Lord and Savior, Jesus Christ, for the opportunity to pursue this degree, and for the strength and wisdom given to complete it. I would also like to thank everyone who helped me through this process. I want to extend my heartfelt thanks to my family - my parents, brother and sister as well as their families for their love and support. The same measure of thanks goes to my spiritual family here in East Lansing and Chicago without whose help it would have been tough getting through those low times. Also, celebration of all the joyous moments during the tenure of my degree would not have been possible without you all. I want to thank all of you who prayed for me during the course of my PhD program, couldn’t have done it without you. I would also like to thank Dr. Evangelyn C. Alocilja for being a mentor in more ways than one. Without your personal life example, constant encouragement and advice, this endeavor would definitely not have succeeded. I would like to thank my labmates - Lisa, Maria, Stephen, and Zarini who helped me in the lab, and also were friends I could talk to while spending the many hours in the lab. I also should thank Lori Kindschy for her help in providing journal articles in the electronic format; you helped me save a lot of time. Lastly, I have to acknowledge the love of my life - Sojo, my wife. You came into my life during the final stage of my PhD program and enriched my days with your phone calls, smile and jokes. You have also spurred me on to complete the corrections of this document for final submission. Thank you very much, everyone!! List olTahlc~ List of Figurt List of Abbrc Chapter 1: l.‘ Chapter 2: LlI 2.1 .lllt‘l' ' J 'J ' J (I) L’J (J) 'JJ ' J H F k 0-. 'i rs.“ f‘ .5. .1.) #9 ~ J—o 3 'J) I If.) J fi. 2.4.4 v .w L’. / Q. '5 .1... LI) (J p—a .’ ’K) I.) '2) (I: I'll I'll fl! 0“,; 0‘ ('7 *‘O) ' l- ! II I In {J [J r.) "J _L3‘ IO‘ '0‘ S. (,J (J ._‘ " ' {’7‘ TABLE OF CONTENTS List of Tables -- -- -- -- -- ............. - - x List of Figures -- - - xii List of Abbreviations -- - -- - -- - - - - .................. xx Chapter 1: INTRODUCTION _ _ -- 1 Chapter 2: LITERATURE REVIEW _ . ...................... - 3 2.1 Microbiological Detection Methods .................................................................... 3 2. 1 .1 Introduction ............................................................................................. 3 2.1.2 Rapid Detection Methods ....................................................................... 4 2.1.3 Electronic Nose ....................................................................................... 6 2. l .4 Biosensors ............................................................................................... 8 2.1 .4] Biological sensing elements ............................................................. 8 2.1.4.2 Transducers ..................................................................................... 17 2.2 Immobilization Techniques ................................................................................ 29 2.2.1 Enzyme Immobilization ........................................................................ 29 2.2.2 Antibody Immobilization ...................................................................... 31 2.2.3 DNA Immobilization ............................................................................ 34 2.3 Chemiluminescence and Biosensors .................................................................. 36 2.3. 1 Chemiluminescence .............................................................................. 36 2.3.2 Chemiluminescent Immunosensors ...................................................... 38 2.3.3 Chemiluminescent Enzyme-Based Sensors .......................................... 41 2.4 Electrochemical Biosensors ............................................................................... 46 2.4.1 Conductometric Biosensors .................................................................. 46 2.4.2 Potentiometric Biosensors .................................................................... 47 2.4.3 Amperometric Biosensors ..................................................................... 49 2.4.4 Cyclic Voltammetry and DNA Biosensors ........................................... 52 2.5 Nano-Tubular Silicon (NTS) .............................................................................. 58 2.5.1 Silicon (Si) ............................................................................................ 58 2.5.2 Silicon Etching ...................................................................................... 58 2.5.3 Nano-Tubular Si Porosity and Thickness Measurement ...................... 62 2.5.4 Nano-Tubular Silicon and Biosensors .................................................. 62 2.5.4.1 Enzyme-based NTS biosensors ...................................................... 63 2.5.4.2 Antibody-based NTS biosensors .................................................... 64 2.5.4.3 DNA-based NTS biosensors ........................................................... 65 2.6 Coliforms/Escherichia coli ................................................................................ 67 2.6.1 Introduction ........................................................................................... 67 2.6.2 Traditional Detection Methods ............................................................. 70 2.6.3 Rapid Detection Methods ..................................................................... 72 2.6.3.1 Antibody-based methods ................................................................ 72 vi Chapter 3: R 3.1 Inn. 3.: PM 3.3 Et'pt 3.4 H)“ 35 Spig't I Chapter-I:R 4.1 011:1 4.1.1 4.1.: 4.1.? 4.1.; 4.1.5 4.1.t? 41.. 41h 4.2 011,L 4.2.2 4.3.: 4‘3 0.5;. 4.3.1 2.6.3.2 Molecular methods ......................................................................... 74 2.6.3.3 Enzyme-based assays ...................................................................... 75 2. 7 Salmonella .......................................................................................................... 79 2.7.1 Introduction ........................................................................................... 79 2.7.2 Traditional Detection Methods ............................................................. 81 2.7.3 Rapid Identification of Salmonella: ...................................................... 82 Chapter 3: RESEARCH HIGHLIGHTS 87 3.1 Innovation and Novelty ...................................................................................... 87 3.2 Potential Difi‘iculties .......................................................................................... 88 3.3 Expected Significance ........................................................................................ 88 3.4 Hypothesis .......................................................................................................... 89 3.5 Specific Research Objectives ............................................................................. 90 Chapter 4: RESEARCH MATERIALS AND METHODS - _ 91 4.] Objective 1 ......................................................................................................... 91 4.1.1 Instrumentation ..................................................................................... 91 4.1.2 Culture Preparation ............................................................................... 92 4.1.3 Single Tube Chemiluminescence Assay Procedure .............................. 92 4.1.4 Determining Best Growth Media .......................................................... 93 4.1.5 Determining Assay Applications .......................................................... 93 4.1.6 Determining Amount of Sample to Use in the Assay ........................... 94 4.1.7 Assay Validation in Food Analysis ....................................................... 94 4.1.8 Statistical Analysis ................................................................................ 95 4.2 Objective 2 ......................................................................................................... 95 4.2.1 Nano-Tubular Silicon Fabrication ........................................................ 96 4.2.2 Characterization of Nano-Tubular Silicon ............................................ 96 4.3 Objective 3 ......................................................................................................... 99 4.3.1 Adaptation of CL Assay to the Biosensor Detection System - Procedure A ............................................................................................................ 99 4.3.1.1 Functionalization of nano-tubular silicon with Lumi-Gal® 530 ..... 99 4.3.1.2 Detection of E. coli on biosensor chip using CL detection .......... 100 4.3.2 Adaptation of CL Assay to the Biosensor Detection System - Procedure B .......................................................................................................... 100 4.3.2.1 Single-tube CL assay modification ............................................... 100 4.3.2.2 Functionalization of nano-tubular Si ............................................ 101 4.3.2.3 Biosensor testing ........................................................................... 101 4.3.2.4 Determining the biosensor sensitivity .......................................... 102 4.3.3 F unctionalization of the Nam-Tubular Silicon Platform with Salmonella Antibodies into an Immunosensor ................................... 103 4.3.3.1 Silanization of nano-tubular silicon platform ............................... 103 4.3.3.2 Immobilization of primary antibody ............................................. 104 4.3.3.3 Optical measurement method ....................................................... 104 vii . |_') l 4.4.: 4.4.: 4.. 4.4.: 4.4.: 4. 4.. 4.5 015.. 4.5.: 4.5.: 4.5.3 Chapter 5: R1 5.1.1 5.1.3 5.1.3 5.1.4 5.1.5 5.2 05/1. 5.2.1 5.3 0111,}; 5.3.1 5.3.2 ‘ 5.3.? 4.3.4 Functionalization of the Nano-Tubular Silicon Platform with Salmonella DNA Probes into a Nucleic Acid-Based Biosensor ......... 105 4.3.4.1 Bacterial strains ............................................................................ 105 4.3.4.2 DNA primers and probes .............................................................. 105 4.3.4.3 Polymerase chain reaction (PCR) ................................................. 105 4.3.4.4 Silanization of nano-tubular Si platform ...................................... 106 4.3.4.5 DNA probe immobilization .......................................................... 108 4.3.4.6 Characterization of the DNA ........................................................ 108 4.3.4.7 Target DNA hybridization ............................................................ 108 4.3.4.8 Electrochemical detection - cyclic voltammetry .......................... 109 4.3.4.9 Confirmation of results ................................................................. 109 4.3.4.10 Statistical analysis ......................................................................... 111 4.4 Objective 4 ....................................................................................................... 112 4.4.1 Sensitivity of the Biosensor Using PCR Amplified DNA .................. 112 4.4.2 Sensitivity of the Biosensor Using DNA from Pure Culture .............. 112 4.4.2.1 Isolation of genomic DNA from bacterial cultures ...................... 113 4.4.3 Confirmation of Results ...................................................................... 114 4.4.4 Statistical Analysis .............................................................................. 114 4.4.4.1 Analysis of curves (Modeling) ..................................................... 115 4.4.4.2 Analysis of Delta Q values ........................................................... 115 4.5 Objective 5 ....................................................................................................... 116 4.5.1 Specificity Testing .............................................................................. 116 4.5.2 Confirmation of Results ...................................................................... 116 4.5.3 Statistical Analysis .............................................................................. 117 Chapter 5: RESULTS AND DISCUSSION - - - - -- -- 118 5.1 Objective 1 ....................................................................................................... 118 5.1.1 Development of Chemiluminescence-Based Assay ........................... 118 5.1.2 Comparison Between Nutrient Broth and Lactose Broth ................... 120 5.1.3 Determining Assay Applications ........................................................ 123 5.1.4 Determining Amount of Sample Used in the Assay ........................... 126 5.1.5 Assay Validation in Food Analysis ..................................................... 130 5.2 Objective 2 ....................................................................................................... 134 5.2.1 Nano-Tubular Si Characterization ...................................................... 134 5.3 Objective 3 ....................................................................................................... 144 5.3.1 Adaptation of CL Assay to the Biosensor Detection System - Procedure A .......................................................................................................... 144 5.3.2 Adaptation of CL Assay to the Biosensor Detection System - Procedure B .......................................................................................................... 146 5.3.3 F unctionalization of the Nano-Tubular Si Platform with Salmonella . Antibodies into an Immune-Biosensor ............................................... 150 5.3.4 Functionalization of the Nano-Tubular Si Platform with Salmonella DNA Probes into a Nucleic Acid-Based Biosensor ............................ 156 5.3.4.1 Polymerase chain reaction ............................................................ 156 5.3.4.2 AF M characterization of nano-tubular Si DNA biosensor ........... 157 viii bioset: Chapter 6: ( 1 Chapter 7: F 1 Appendix A: :11 Fret; .13 5-4-. :13 69]}; Aphenom a; 1 B 1 Rt‘ug. 8: Ring. APltendit c; 1 C1 RUM 1 (.2 5.15.3 5.3.4.3 Electrochemical detection of DNA using nano-tubular Si biosensor. . . . .. ..................................................................................................... 158 5.3.4.4 Statistical analysis of CV data ...................................................... 165 5.4 Objective 4 ....................................................................................................... 166 5.4.1 Sensitivity of the Biosensor using PCR amplified DNA .................... 166 5.4.1.1 Cyclic voltammograms ................................................................. 166 5.4.1.2 Data analysis ................................................................................. 170 5.4.2 Sensitivity of the Biosensor Using DNA from Pure Culture .............. 177 5.4.2.1 Cyclic voltammograms ................................................................. 178 5.4.2.2 Analysis of Delta Q values ........................................................... 181 5.4.2.3 Interactive effects of variables ...................................................... 183 5.5 Objective 5 ....................................................................................................... 185 5.5.1 Specificity Testing .............................................................................. 185 5.5.2 Cyclic Voltammograms ...................................................................... 185 5.5.2.1 Analysis of Delta Q values ........................................................... 188 Chapter 6: CONCLUSION -- -- 192 Chapter 7: FUTURE RESEARCH 193 Appendix A: PROCEDURES -- - 195 A. 1 Fresh Produce Testing ..................................................................................... 195 A2 S-4 7001] Scanning Electron Microscope Operation ........................................ 197 A3 Gel Electrophoresis ......................................................................................... 198 Appendix B: REAGENTS/SOLUTIONS -- _ - - - 201 B. 1 Reagents for Enzyme-Based and Antibody-Based Testing .............................. 201 3.2 Reagents for DNA-based testing ...................................................................... 202 Appendix C: RAW DATA AND STATISTICAL ANALYSIS..- 204 C. 1 Raw Data: Cyclic voltammetry ........................................................................ 204 C2 SAS Statistical Data Analysis Output .............................................................. 226 REFERENCES __ -- __ - - ............... 255 ix Table 5-1. ”5 cc. Table 5-2. 11“.: Table 5-3. 1n:-1 D\ V0. 1ah1e5-4. Int. D.\ \ 0. 131‘165-5. IE1; D.\ \0; Table 56. [me D.\' Volt lab-165-] 1111c- 1C‘X‘ 0ft Table 5.8. Inn (ex Of( Table 5-1. Table 5-2. Table 5-3. Table 5-4. Table 5—5. Table 5-6. Table 5—7. Table 5-8. Table 5-9. LIST OF TABLES Light intensity for different amounts of overnight LB E. coli culture at various cell concentrations. ........................................................................................ 127 Thickness and porosity of nano-tubular Si chips fabricated from pH Si. ..... 141 Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with NTS biosensor at various concentrations using cycle 1 of cyclic voltammogram ............................................................................................... 175 Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with NTS biosensor at various concentrations using cycle 2 of cyclic voltammogram ............................................................................................... 175 Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with planar Si biosensor at various concentrations using cycle 1 of cyclic voltammogram ............................................................................................... 177 Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with planar Si biosensor at various concentrations using cycle 2 of cyclic voltammogram ............................................................................................... 177 Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with NTS biosensor at various concentrations using cycle 1 of cyclic voltammogram. ............................................................................... 182 Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with NTS biosensor at various concentrations using cycle 2 of cyclic voltammogram. ............................................................................... 182 Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with planar Si biosensor at various concentrations using cycle 1 of cyclic voltammogram. .................................................................. 184 Table 5-10. Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with planar Si biosensor at various concentrations using cycle 2 of cyclic voltammogram. ................................................................ 184 Table 5-1 1. 1r. (‘1 Vi Table 5-12. 1 Cl \ k Table 5-13. It d. Table 5-14. 11". di Table 5-11. Table 5-12. Table 5-13. Table 5-14. Integral charge (AQ, milli coulombs) values for E. coli, and S. Enteritidis-E. coli mixture (extracted) DNA with NTS biosensor using cycle 1 of cyclic voltammogram. ............................................................................................ 190 Integral charge (AQ, milli coulombs) values for E. coli, and S. Enteritidis-E. coli mixture (extracted) DNA with NTS biosensor using cycle 2 of cyclic voltammogram. ............................................................................................ 190 Integral charge (AQ, milli coulombs) values for S. Enteritidis DNA from different sources with NTS biosensor (Cycle 1). ........................................ 191 Integral charge (AQ, milli coulombs) values for S. Enteritidis DNA from different sources with NTS biosensor (Cycle 2). ........................................ 191 xi Figure 3 Figure 2- Figure 3-2 Figure 2-4 Figure 2-5. Figure 3-6. Figure 41. S Figure 4.1 8‘ bit I. ‘lE‘nre 43 Q1. Rm 4’6. SC ft. Figure 2-1. Figure 2-2. Figure 2-3. Figure 2-4. Figure 2-5. Figure 2-6. Figure 2-7. Figure 2-8. Figure 4-1. Figure 4-2. Figure 4-3. Figure 4-4. Figure 4-5. Figure 4-6. LIST OF FIGURES Simplified reaction mechanism of luminol (Dodeigne et al., 2000). ............. 39 The two modes of decomposition of 1,2-dioxetanes: (I) the diradical mechanism and (II) the chemically initiated electron exchange chemiluminescence (CIEEL) (Dodeigne et al., 2000). .................................. 42 Reaction mechanism of Lumi-Gal® 530 in the chemiluminescent assay. ..... 45 Excitation waveform for a cyclic voltammetry experiment (Adapted from (Robinson, 1987)). ......................................................................................... 54 Typical current response for a reversible couple obtained with cyclic voltammetry (Adapted from (Robinson, 1987)). ........................................... 54 Schematic of a three-electrode potentiostat for cyclic voltammetry (Adapted from (Wang, 2000)). ...................................................................................... 56 Typical J-V curve of p+-Si in dilute aqueous HF solution (Adapted from (Allongue, 1997)) ........................................................................................... 60 Reaction model for dissolution of nano-tubular silicon in aqueous HF solutions. ........................................................................................................ 61 Schematic of the F emtomaster FB 12 Luminometer .................................... 92 Biosensor schematic showing envisioned long-term and short-term research biosensors ....................................................................................................... 97 Single-tank cell set up used for tubular silicon fabrication. ........................... 98 Functionalization of planar Si and nano-tubular Si platforms into antibody- based biosensors ........................................................................................... 103 Functionalization of planar Si and nano-tubular Si platform into DNA-based biosensor. ..................................................................................................... 107 Schematic of target DNA hybridization to immobilized DNA probe on fimctionalized biosensor surface. ................................................................. 1 10 xii Figure 316. Si; F1EureS-7. SE CO FlEure 5-8. 81; CH Figure 4-7. Figure 5-1. Figure 5-2. Figure 5-3. Figure 5-4. Figure 5-5. Figure 5-6. Figure 5-7. Figure 5-8. Figure 5-9. Three-electrode electrochemical cell used for cyclic voltammetry measurements ............................................................................................... 1 11 Kinetics of light emitted by an overnight NB E. coli culture ....................... 119 Comparative kinetics of light emitted by 24 h and 48 h old E. coli cultures in NB and LB. .................................................................................................. 121 Kinetics of light emitted by an overnight generic E. coli and E. coli 0157:H7 (untreated and heat-killed) culture. .............................................................. 124 Normalized light emission and E. coli counts of food and water samples. Key: W - water, M - milk, SS - alfalfa sprout seeds. Normalized = (Sample - Blank) in RLU/s. ........................................................................................... 131 Normalized light emission of surface water samples direct assay and 12 h enrichment in LB prior to assay. Sample coliform counts are prior to enrichment. Normalized = (Sample — Blank) in RL U/s. .............................. 132 SEM image of bare p++ Si surface ................................................................ 135 SEM image of nano-tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 2 mA/cm2 for 1 h. ...................................... 135 SEM image of nano-tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 5 mA/cm2 for 20 min. ................................ 137 SEM image of nano-tubular Si produced from p” type Si. Anodizing conditions used were 15% HP, 5 mA/cm2 for 1 h. ...................................... 137 Figure 5-10. SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HF, 15 mA/cm2 for 1 min. .............................. 138 Figure 5-11. SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HF, 30 mA/cm2 for 1 min. .............................. 138 Figure 5-12. SEM image of nano-tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 50 mA/cm2 for 1 min. .............................. 139 Figure 5-13. SEM image of nano-tubular Si produced from p++ type Si . Anodizing conditions used were 15% HF, 100 mA/cm2 for 30 s ................................ 139 xiii Figure 5-15.. Figure 5-16. Figure 5-17. Figure 5-18, Fig-1365-19 Flg'ire 5-30 Figure 5-31. F Fight 5.1 1331"“ 5-73 , J. Figure 5-14. Figure 5-15. Figure 5-16. Figure 5-17. Figure 5-18. Figure 5-19. Figure 5-20. Figure 5-21. Figure 5-22. Figure 5-23. Figure 5-24. Cross-sectional SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HP, 5 mA/cm2 for l h. NTS chip cross-section was placed at 45° to capture the image. ............................... 142 Comparison of light emission of E. coli pure culture in nano-tubular Si and planar silicon biosensors against nano-tubular Si control. Different superscript over the bars indicates significantly different light emission (p<0.01). .................................................................................................... 145 Kinetics of light emitted by an overnight LB E. coli culture using the standard (non-mixed) and modified (pre-mixed with 15 min incubation) single-tube chemiluminescence assays. Different superscripts over the bars indicate significant differences (p<0.01). .................................................. 147 Comparison of light emission from serially diluted E. coli cultures on nano- tubular Si biosensor against nano-tubular Si control (blank). Different superscript over the bars indicates significantly different light emission (p<0.05). .................................................................................................... 149 Comparison of light emission for Salmonella pure culture with nano-tubular and planar silanized Si biosensor against nano-tubular silanized Si control. Different superscript over bars indicates significantly different light emission (p<0.05). ..................................................................................... 152 Comparison of light emission for Salmonella pure culture with silanized and non-silanized nano-tubular Si biosensor against silanized control. Different superscript over the bars indicates significantly different light emission (p<0.05). .................................................................................................... 154 Gel electrophoresis of PCR-amplified Salmonella: S1 and S2 - 5 ul culture used for PCR. Key: M — Marker, C — negative control, S — Salmonella 157 AF M image of NTS-based DNA biosensor chip prior to DNA probe immobilization. .......................................................................................... 1 59 AF M image of NTS-based DNA biosensor chip after DNA probe immobilization. .......................................................................................... 1 60 AF M image of NTS-based DNA biosensor chip after target DNA hybridization. ............................................................................................. 161 Cyclic voltammogram for a pure culture of Salmonella Enteritidis obtained with NTS-based DNA biosensor chip. ...................................................... 163 xiv . - m Figure :-... a IJI 1‘75. Figure Figure 5-27 Figure 5-28. Figure 5-29. Figure 5- 1 \ m-fir—vfl Hf“ Figure 5-25. Figure 5-26. Figure 5-27. Figure 5-28. Figure 5-29. Figure 5-30. Figure 5-31. Figure 5-32. Figure 5-33. Cyclic voltammograms for serially diluted PCR-amplified Salmonella Enteritidis DNA obtained with NTS-based DNA biosensor (sensitivity testing). ...................................................................................................... 167 Cyclic voltammograms for serially diluted PCR-amplified Salmonella Enteritidis DNA obtained with planar Si-based DNA biosensor chip (sensitivity testing) ..................................................................................... 169 Spline curve for PCR-amplified Salmonella Enteritidis DNA on nano- tubular Si biosensor at lug/pl concentration level. Key: 1 — No DNA, 2 — After DNA Hybridization, and 3 — Before DNA Hybridization. ................ 172 Spline curve for PCR-amplified Salmonella Enteritidis DNA with nano- tubular Si biosensor at 0.1 ng/ul concentration level. Key: 1 — No DNA, 2 — After DNA Hybridization, and 3 — Before DNA Hybridization. ................ 173 Cyclic voltammograms for serially diluted (extracted) pure culture Salmonella Enteritidis DNA obtained with NTS-based DNA biosensor chip (sensitivity testing) ..................................................................................... 179 Cyclic voltammograms for serially diluted (extracted) pure culture Salmonella Enteritidis DNA obtained with planar Si-based DNA biosensor chip (sensitivity testing). ............................................................................ 180 Gel electrophoresis of PCR-amplified Salmonella and extracted E. coli DNA. S1 to SS — 1 to 5 pl Salmonella-E. coli (1:1) culture mix used for PCR. Key: M — Marker, C — negative control, S — Salmonella-E. coli, EC — E. coli ......................................................................................................... 186 Comparison of cyclic voltammograms for Salmonella Enteritidis (target) DNA and E. coli (non-target) DNA obtained with NTS-based DNA biosensor chip (specificity testing). ........................................................... 187 Cyclic voltammogram for a mixture of Salmonella Enteritidis and E. coli DNA obtained with NTS-based DNA biosensor chip (specificity testing). ................................................................................................................... 189 Figure A-l. Experimental design for processing of produce samples ............................ 195 Figure 01. Mean cyclic voltarmnograms (Cycle 1) obtained with NTS biosensor chip for 1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3). ..................... 204 XV Figure C-Z. .\ 1 Figure C-4. .\ Figure C-S. .\ Figure C 6. .\ Figure C-7. .\ Figure C-8. _\. 1Figure C -9. ’/ Figure C‘IO . Figge C-I lg n 1 Figure c-11 ‘ Figme C-13.: Figure C-2. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 ng of PCR-amplified Salmonella Enteritidis DNA (n = 3). ..................... 204 Figure C-3. Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 0.1 ng of PCR-amplified Salmonella Enteritidis DNA (n = 3). .................. 205 Figure C-4. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 0.1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3). .................. 205 Figure C-S. Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 10 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3). ................... 206 Figure C-6. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 10 pg of PCR-amplified Salmonella Enteritidis DNA (11 = 3). ................... 206 Figure C-7. Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3). ..................... 207 Figure C-8. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 10 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3). ................... 207 Figure C-9. Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 0.1 pg of PCR-amplified Salmonella Enteritidis DNA (11 = 3). .................. 208 Figure C-10. Figure C-1 1. Figure C-12. Figure C-13. Figure C-l4. Figure C-15. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 0.1 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3). .......... 208 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 10 fg of PCR-amplified Salmonella Enteritidis DNA (n = 3). ............ 209 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 10 fg of PCR-amplified Salmonella Enteritidis DNA (n = 3). ............ 209 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 fg of PCR-amplified Salmonella Enteritidis DNA (n = 3). .............. 210 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 fg of PCR-amplified Salmonella Enteritidis DNA (n = 3). .............. 210 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor chip for 1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3) ....... 211 xvi Figure C -16 Figure C317. Figure C-18. EQMCJQ Figure C CO. Figure C -2 1 . Figure Cg: I Figure C-23. . Figure C534 Figure C.‘) q Figure (“-36 . Figure C-16. Figure C-17. Figure C-18. Figure C-19. Figure C-20. Figure C-21. Figure C-22. Figure C-23. Figure C-24. Figure C-25. Figure C-26. Figure C-27. Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor chip for 1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3) ....... 211 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor chip for 0.1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3).... 212 Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor chip for 0.1 ng of PCR-amplified Salmonella Enteritidis DNA (11 = 3).... 212 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor chip for 10 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3) ..... 213 Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor chip for 10 pg of PCR-amplified Salmonella Enteritidis DNA (11 = 3) ..... 213 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor chip for 1 pg of PCR-amplified Salmonella Enteritidis DNA (11 = 3) ....... 214 Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor chip for 1 pg of PCR-amplified Salmonella Enteritidis DNA (n = 3) ....... 214 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 ng of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 215 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 ng of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 215 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 0.1 ng of DNA extracted from pure culture of Salmonella Enteritidis (n = 3). ........................................................................................................... 216 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 0.1 ng of DNA extracted from pure culture of Salmonella Enteritidis (n = 3). ........................................................................... 216 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 10 pg of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 217 xvii Figure C {I} Figure C -3‘ Figure C ~31] igure 031 Figure C ~32. Figure 034. Figure C -35. Figure C'36 F1gure C9, 8 F 151-316 039 . it... M C40. . u Figure (2.28. Figure C-29. Figure C-30. Figure C-31. Figure C-32. Figure C-33. Figure (3-34. Figure C-35. Figure C-36. Figure C-37. Figure C-38. Figure C-39. Figure C-40. Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 10 pg of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 217 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 pg of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 218 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 pg of DNA extracted from pure culture of Salmonella Enteritidis (n = 3) ................................................................................................................ 218 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor for 1 ng mixture of S. Enteritidis and E. coli pure culture DNA (n = 3). .......... 219 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor for 1 ng mixture of S. Enteritidis and E. coli pure culture DNA (n = 3). .......... 219 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 ng of DNA extracted from pure culture of E. coli (11 = 3). ............... 220 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 ng of DNA extracted from pure culture of E. coli (n = 3). ............... 220 Mean cyclic voltammograms (Cycle 1) obtained with NTS biosensor chip for 1 pg of DNA extracted from pure culture of E. coli (n = 3). ............... 221 Mean cyclic voltammograms (Cycle 2) obtained with NTS biosensor chip for 1 pg of DNA extracted from pure culture of E. coli (11 = 3). ............... 221 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor chip for 1 ng of DNA extracted from pure culture of S. Enteritidis (n = 3). ................................................................................................................... 222 Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor chip for 1 ng of DNA extracted from pure culture of S. Enteritidis (n = 3). ................................................................................................................... 222 Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor for 0.1 ng of DNA extracted from pure culture of S. Enteritidis (n = 3)... 223 Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor for 0.1 ng of DNA extracted from pure culture of S. Enteritidis (n = 3)... 223 xviii HngJL Figure C 42. Figure C -43. Figure C -44. Figure C-41. Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor for 10 pg of DNA extracted from pure culture of S. Enteritidis (n = 3).... 224 Figure C—42. Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor for 10 pg of DNA extracted from pure culture of S. Enteritidis (n = 3).... 224 Figure C-43. Mean cyclic voltammograms (Cycle 1) obtained with Planar Si biosensor for 1 pg of DNA extracted from pure culture of S. Enteritidis (n = 3). ..... 225 Figure C-44. Mean cyclic voltammograms (Cycle 2) obtained with Planar Si biosensor for 1 pg of DNA extracted from pure culture of S. Enteritidis (n = 3) ...... 225 xix LIST OF ABBREVIATIONS CFU — colony forming units h — hour/hours min — minute/ minutes NTS — nano-tubular silicon s — second/seconds Si — Silicon XX The cau: metals “1111 neurological. Disease C on approximatel L'nited States Cnited States blood. which due 10 costs detection Of TradillOf‘. b10fhemical . 141656 method like 3100111 BiOSCTlSi ugS. h0m1<~ impolaling I CHAPTER 1: INTRODUCTION The causes of foodborne illness include viruses, bacteria, parasites, fungi, toxins, and metals with the symptoms ranging from mild gastroenteritis to life-threatening neurological, hepatic, and renal problems. According to a report from the Centers for Disease Control and Prevention (CDC), it is estimated that foodbome diseases cause approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the United States each year. Researchers at the Economic Research Service (ERS) of the United States Department of Agriculture (USDA) estimate that the total annual medical cost associated with foodborne illness caused by pathogens (known and unknown) is $65-$94 billion. The diagnosis of a foodbome disease is usually confirmed by cultures of stool or blood, which takes 2-3 days. Foodbome disease outbreaks lead to huge economic losses due to costs associated with lost work hours, lawsuits, and product recalls. Thus, detection of any foodborne pathogen is necessary before the contaminated product reaches the market. Traditional methods are laborious, require technical expertise, and often several biochemical and serological tests have to be performed to identify pathogens, making these methods very time consuming (2-7 days). Rapid methods being developed usually take about 18-24 h (including the time required for sample processing and pre- enrichment). Biosensors are portable diagnostic tools used for the rapid detection of metabolites, drugs, hormones, antibodies, and antigens. Biosensors are defined as analytical devices incorporating a biological material intimately associated with or integrated within a physicoch applicatio' pathogens Vario silicon die recently bi recent dex- with metal This procc penicillin. £ biosensor p This Stud} demonstrati: lhl’ee differg fabricated its characteristic fabrication c detectors. an mains USlng physicochemical transducer or transducing microsystem. While many of the biosensor applications are in the medical field, recent developments have been in the detection of pathogens especially for bioterrorism prepraredness. Various platforms have been employed to fabricate biosensors, such as silicon (Si), silicon dioxide, fiber optics, filter paper, quartz, and glass. Nano-tubular Si (NTS) has recently been investigated as a potential platform for biosensor applications. Most of the recent developments in porous silicon sensors carry out a deposition step to fill the pores with metallic, dielectric or semiconducting oxide, enzyme, or molecular receptor films. This process has resulted in porous silicon sensors capable of detecting or measuring penicillin, alkali metal ions, humidity, and hydrocarbons. However, the use of NTS as a biosensor platform for detecting pathogens has not been reported. This study was focused on developing a NTS-based biosensor platform and demonstrating its versatility by using three different biological sensing elements aimed at three different target pathogens and two transduction principles. The biosensor was fabricated using microfabrication and electrochemistry techniques and had the following characteristics: high sensitivity and specificity with an assay time of 60 min or less. The fabrication of NTS, its functionalization into biosensors using different biological detectors, and biosensor testing with Escherichia coli and two Salmonella enterica strains using optical and electrochemical transducers are described here. 2.1 315 2.1.1 L 310T caused b; er 01‘ N {Whom hosl‘imliz annual CC bactella: ‘8 and 005” billion ann' Tradliii preem'TChmc conlllmallor bizfore an." E a certain 1"" interpret and clinical 18501" sampling prt Therefore, Ll, problematic . response. 51: CHAPTER 2: LITERATURE REVIEW 2.1 MICROBIOLOGICAL DETECTION METHODS 2.1.1 Introduction More than 200 diseases are known to be transmitted through food, and these are caused by 40 different foodbome pathogens, including fungi, viruses, and bacteria (Mead et al., 1999). The Centers for Disease Control and Prevention (CDC) estimates that foodbome diseases cause approximately 76 million illnesses, including 325,000 hospitalizations and 5000 deaths in the US each year (Mead et al., 1999). The estimated annual cost of human illness caused by a group of seven pathogens (including six bacteria: Salmonella, Listeria, Campylobacter, E. coli 0157:H7, Staphylococcus aureus, and Clostridium perfringens) commonly associated with foodbome outbreaks is $5.6-9.4 billion annually (Buzby et al., 1999). Traditional methods for the detection of bacteria involve the following basic steps: preenrichment, selective isolation, and biochemical screening, as well as serological confirmation for certain pathogens. Hence, a complex series of tests is often required before any identification can be confirmed. These methods are laborious and may require a certain level of expertise to perform. The results of such tests are often difficult to interpret and not available on the time scale desired in the food quality assurance or clinical laboratory. Food sample testing also presents problems with the inadequacy of sampling protocols, and the presence of organic matter, as well as non-target bacteria. Therefore, the detection of target foodbome pathogens (for example, Salmonella) may be problematic due to low numbers present and may result in a false culture negative response. Moreover, four to seven days are generally required to confirm the presence of [he pathop implicatir pathogen: 2.1.2 & New length}: F used to an The PC R 1 requires pt er al. 19‘. Almeida. 2 In res} det'eIOpmer food and c1 Wing l'arior SFFCUOSCQP} for bacteria 1 Spficrroxop} reactions‘ 0; COmpOLmds . bitchem 1C3] “Valuer. 195 Shape. 1% the pathogens (Food and Drug Administration, 2000), which has safety, cost, and quality implications for the food, medical, and biodefense sectors. Rapid detection methods for pathogens have hence become a necessity. 2.1.2 Rapid Detection Methods New rapid detection technologies can be very sensitive, but analysis time is usually lengthy. For example, the polymerase chain reaction (PCR), 3 molecular method, can be used to amplify small quantities of genetic material to determine the presence of bacteria. The PCR method is extremely sensitive and can be used for various target pathogens, but requires pure samples and hours of processing and expertise in molecular biology (Meng et al., 1996; Sperveslage et al., 1996; Tsukamoto and Kanki, 1999; Almeida and Almeida, 2000; Narimatsu et al., 2001; Hong et al., 2003; Tims and Lim, 2003). In response to this problem, considerable effort is now directed towards the development of methods that can rapidly detect low concentrations of pathogens in water, food and clinical samples. For this purpose, several instruments have been developed using various principles of detection, e.g., chromatography, infrared or fluorescence spectroscopy, bioluminescence, flow cytometry, impedimetry, and many others (Nelson, 1985; Wyatt, 1995; Hobson et al., 1996; Perez et al., 1998). Initial instruments developed for bacterial identification counted the cells by microscope or by flow cytometry; infrared spectroswpy; measuring physical parameters by piezocrystals, impedimetry, redox reactions, optical methods, calorimetry, ultrasound techniques and detecting cellular compounds such as ATP (by bioluminescence), DNA, protein and lipid derivatives (by biochemical methods), radioactive isotopes (by radiometry) (Nelson, 1985; Rossi and Warner, 1985; Ramsay and Turner, 1988; Rodrigues and Kroll, 1990; Ding et al., 1993; Sharpe, 1994; Swaminathan and Feng, 1994; Hobson et al., 1996; Wang et al., 1997; Fratamit chemica naturallj ldentific produce (Suamir The used imr amounts related bi substrates process ca iHUIIurrodr'. aUTOmated, STULITCS “l ConVenllOng 80% agrees ImufflClem ELISA, rail Real-ti: diai‘i‘lOSlic [l Fratamico et al., 1998; Gunasekera et al., 2000). Among these, the primary physico- chemical methods of bacterial identification are those that involve the detection of some naturally occurring component of the bacterium. For example, the Microbial Identification System (MIS; Microbial ID, Inc., Newark, DE) uses gas chromatography to produce a fatty acid profile for detection and identification of microorganisms (Swaminathan and Feng, 1994; Crist Jr. et al. , 1996). The enzyme-linked immunosorbent assay (ELISA) is probably the most commonly used immunological assay because of its versatility, sensitivity (ability to detect small amounts of antigen or antibody), specificity (ability to discriminate between closely related but antigenically different molecules), and ease of automation. Chemicals and substrates used in ELISA are safer than radioisotopes used in radioimmunoassay. The process can be easily automated for performance of large numbers of tests. The VITEK immunodiagnostic assay system (V IDAS®, bioMe'rieux, Inc., Durham, NC) is an automated, qualitative, enzyme-linked fluorescent immunoassay method. Comparative studies with a variety of food types have found favorable correlation between conventional test methods and the VIDAS system (Blackburn et al., 1994; Curiale et al., 1997; De Medici et al., 1998; Walker et al., 2001). Wyatt et al. (1996) observed a 70- 80% agreement between the ELISA and the culturing method. The ELISA had a false- positive rate of 17% and a false-negative rate of 26%, which appeared to be due to insufficient growth of Salmonella spp. in the preenrichment broth employed in the ELISA, rather than lack of recognition by the antibodies. Real-time PCR approaches have recently emerged as a preferred DNA-based diagnostic tool in both medical and agricultural fields (Marin et al., 2004; Skof et al., 3004; “- Penders is increa 2003). 1 quantitat material detection 2003). T1 storage. v losses. Mt e«tilt-parent 2.13 & 'Electr application B'mcdetti 6. Electronic FlchOnics, 5iatistica] ar lo 130111 0d COHEQS [he Tliege dale I 1 2004; Wada et al., 2004; Whiley et al., 2004; Connor et al., 2005; Farrell et al., 2005; Penders et al., 2005; Rudi et al., 2005; Uchida et al., 2005). Real-time quantitative PCR is increasingly being used for pathogen detection and quantification in foods (Norton, 2002). There are still two major challenges with the widespread use of PCR for quantitative diagnostics. The detection limit is mainly determined by the amount of material that can be amplified in a single reaction. The other major limitation is the detection of DNA from dead cells (Herman, 1997; McKillip et al., 1999; Nogva et al., 2003). This is a particular problem for processed foods or foods subjected to long-time storage, wherein ‘false’ positives from dead cells could lead to unnecessary economic losses. Moreover, all the above methods require expensive, multiple, specialized, or large equipment. 2.1.3 Electronic Nose ‘Electronic nose’ systems have advanced rapidly during recent years, with most applications being within the food and beverage industries (Schaller et al., 1998; Benedetti et al., 2004; Garcia-Gonzalez et al., 2004; Lui et al., 2005; Marti et al., 2005). Electronic nose systems are comprised of sophisticated hardware, with sensors, electronics, pumps, flow controllers, and software, such as data pre-processing, and statistical analysis (Schaller et al., 1998). The sensor array of an electronic nose responds to both odorous and odourless volatile compounds. In the electronic nose, a computer collects the signal pattern from a sensor array, comprised usually of individual sensing elements with limited specificity, wherein the first pre-treatment of the data is carried out. These data are then further processed by suitable sofiware based on artificial neural networks approach for training and learning (Benedetti et al., 2004; Dutta et al., 2004). At present, different detection principles (heat generation, conductivity, electrochemical, optical. di electronic following compound short reco 1998). An; quality (M. al.. 2004 1. ; 2005). Gib resistive g3 More recen- nelu'ork “a 01572H7 151 and the my). (Younts e, a Afllfici; SIOrage qUa: ral‘id detec: changes in g for each ta: 15013163 0le optical, dielectric, and magnetic properties) are used in the basic sensing elements of the electronic nose. The ideal sensors to be integrated in an electronic nose should fulfill the following criteria: high sensitivity (down to 10"2 g/ml), must respond to different compounds present in the headspace of the sample, high stability and reproducibility, short recovery time, easy calibration, and must be robust and portable (Schaller et al., 1998). Artificial electronic noses have been used to assess cheese, and alcoholic beverage quality (Marti et al., 2005; Trihaas and Nielsen, 2005), beef spoilage (Balasubrarnanian et al., 2004), freeze damage in oranges (Tan et al., 2005), and fruit ripeness (Brezmes et al. , 2005). Gibson et al. (1997) reported on the use of an array of 16 conducting-polymer resistive gas sensors to detect 12 different bacteria from cultures grown on agar plates. More recently, an electronic nose based on gas sensors coupled with an artificial neural network was developed for E. coli 0157:H7 detection and its differentiation from non- OlS7:H7 isolates. The differences between the ‘gas signatures’ of the E. coli 0157:H7 and the non-015zH7 isolates were successfully detected using neural network algorithms (Younts et al. , 2002; Younts et al. , 2003). Artificial/electronic noses offer advantages such as simplicity of design, long shelf life of sensors, capability for long-term continuous monitoring (for example, in produce storage quality studies), and non-destructive testing. However, they cannot be used for rapid detection of pathogens as they take a minimum of 12-24 h to detect significant changes in gas patterns emitted from a sample. Also, identifying a unique gas signature for each target pathogen may not be possible, and variations in gas emission within isolates of the pathogen increase the probability of false positive/negative outputs. 2.1.4 310‘. The dis. electronic n' biosensors) emerged as to convert 1 biosensor c Transducers electrochem element user individual It Transducers dlSCuSSed b6 2.1.4 Biosensors The disadvantages with culture techniques, molecular and ELISA assays, as well as electronic noses have led to the development of several alternative methods (including biosensors) for the rapid detection of the presence of pathogens. Biosensors have emerged as highly promising for rapid diagnosis. The general function of a biosensor is to convert biological events into a quantifiable electrical response (Cahn, 1993). A biosensor consists of a platform, a biological sensing element, and a transducer. Transducers used in biosensors to report these biological events are electrical, electrochemical, optical, piezoelectric crystal, or acoustic wave in nature. The sensing element used in the construction of biosensors can vary from entire sections of tissue to individual molecules such as cells, enzymes, antibodies, and DNA oligonucleotides. Transducers used in biosensors with different kinds of biological sensing elements are discussed below. 2.1.4.1 Biological sensing elements 2.1.4.1.] Whole cells and tissues The use of tissue and whole (living) cells can impart certain advantages to a sensor, especially when subjected to suboptimal conditions. Biosensors based on whole cells as biorecognition elements are used as genotoxicity and toxicity sensors, targeting environmental pollutant analysis but have much far applications for food contaminant detection. Bacteria or yeast cells, genetically engineered to bear the lux or Iuc gene operon, express luminescence proteins such as the green fluorescence protein (GF P), and are used for detection of environmental pollutants, monitoring water quality, and stress response (Kohler et al., 2000; Gu and Choi, 2001; Weitz et al., 2001; Bechor et al., 2002; Horsburgh et al., 2002; Kostrzynska et al., 2002). Other signal reporter systems such as bacterial berm em; biosensor luminesce whole org range fror SU '- rgested 0 other emi drawback response ti 1 3.1. The lirr Of e“Dimes. PeSllCIdeS‘ t“ based 561150 agencies for 10 Con‘enli( example. at carbamateS 2. and {00d S‘ SChulZe 6’! u 200‘1). 11} fl tomato Past. bacterial luciferase, insect luciferase, B-galactosidase, and alkaline phosphatase have also been employed (Baeumner, 2003). Biran et al. (2000) developed an electrochemical biosensor for cadmium using genetically engineered microorganisms for non- luminescence signal generation with the lacZ gene. Plants and tissues as well as higher whole organisms have been used in some biosensor applications in recent years. These range from algae to soybeans, and nematodes. For example, Shvetsova et al. (2002) suggested the use of soybeans and their electrophysiological response to acid rain and other environmental stress conditions as a potential biosensor system. The biggest drawback of cell and tissue based biosensors is the problem of selectivity and slow response times, thus restricting their applicability. 2.1.4. 1.2 Enzymes The limitations of whole cell and tissue-based biosensors can be overcome by the use of enzymes, which represent the most commonly used sensing agent for chemical targets. Pesticides, especially insecticides, have traditionally been an application area for enzyme- based sensor systems. Many of them reach detection limits set forth by governmental agencies for environmental samples, infant food, etc., making them attractive alternatives to conventional analysis systems (Karube and Nomura, 2000; Rekha et al., 2000). For example, acetylcholine esterases were used for the detection of organophosphates and carbamates in single-use devices such as screen-printed electrodes in both, environmental and food samples (Albareda-Sirvent et al., 2001b, 2001a; Avrarnescu et al., 2002; Schulze et al., 2002a; Schulze et al., 2002b; Marques et al., 2004; Timur and Telefoncu, 2004). In food analysis, the freshness-test applied to the analysis of fish, sauerkraut, tomato paste, and instant food (Frebort et al., 2000; Niculescu et al., 2000; Kriz et al., 3002; La: lGustacSr MeB-ZZei. 2 Liter. biOSCIlSOr Sdfmang‘lj beef. and detected Chemilumj finx-jmnmc poo r Specn of such leg] 3.1. Biolog; in this Sec“ the 3113]}188 to hem}, m; Pesticide de descn‘be d [3 Murat er: 2002; Lange and Wittrnann, 2002; Mitsubayashi et al., 2004), antibiotic detection in milk (Gustavsson et al., 2002), and detection of phytotoxins found in seafood (Botre and Mazzei, 2000) are some of the other enzyme-based biosensor applications reported. Literature describing the detection of pathogenic bacteria using enzyme-based biosensors is limited. Sensors are found for the detection of E. coli 0157:H7 and Salmonella Typhimurium with a bienzyme electrode chicken carcass wash water, ground beef, and fresh-cut broccoli (Yang et al., 2001; Ruan et al., 2002a). Escherichia coli was detected in environmental samples based on culture-enhanced amperometric and chemiluminescence principles with detection limits of about 10,000 cells per 100 m1 of environmental sample within one working day (Nistor et al., 2002). Low sensitivity and poor specificity of enzyme-based biosensors for pathogen detection limit the application of such techniques. 2. 1.4. 1.3 Antibodies and receptors Biological sensing elements based on antibody and receptor molecules are grouped in this section, since their application area and functionality are very similar. Recently, the analytes targeted ranged from antibiotics in milk to general environmental monitoring to heavy metals to pathogens. The traditional application area of immunosensors is for pesticide detection in environmental samples and food matrices. Strachan et al. (2001) described the use of antibodies for the detection of atrazine, mecoprop, diuron, and paraquat emphasizing their superiority over other bioanalytical systems due to their functionality in aqueous and also organic solvents. Other environmental pollutants including PCBs were detected using screen-printed electrodes (Laschi and Mascini, 2002). In food contaminant analysis, sulfonamides (Bjurling et al., 2000) and toxins, such as aflatoxin, fumonisin B1, cholera toxin, and staphylococcal enterotoxin B, were 10 targeted in and Durst. been used penicillin. Haasnoot a Finall} some of th quartz crys‘ Salmonella resonance ( different b3 01 (J! 73117. u 5“ and tr. , 1m111$. In a 0157;H7 1r eIlrichmem An I'm:— On the sum Electrode c1: (3003) de:. membrane \, This 311910;; targeted in a variety of foods (Ho and Durst, 2000; Rasooly, 2001; Maragos, 2002; Ho and Durst, 2003; Medina, 2003; Rucker et al., 2005). Antibody-based biosensors have been used for the detection of antibiotic and other drug residues in food samples, e.g., penicillin, streptomycin, and gentamicin in milk samples (Delwiche et al., 2000; Haasnoot and Verheijen, 2001; Haasnoot et al., 2002). Finally, irnmunosensors have also been developed for pathogen analysis, but only some of these exhibit excellent detection limits with relatively short analysis times. A quartz crystal microbalance (QCM)-based biosensor was used to detect Salmonella sp. in milk samples with detection limits around 106 CFU/ml (Park et al., 2000). Listeria and Salmonella enterica were detected with a similar detection limit by a surface plasmon resonance (SPR) biosensor (Koubova et al., 2001b). QCM and SPR biosensors for different bacterial targets, such as Pseudomonas aeruginosa, Bacillus cereus, and E. coli 0157:H7, were later developed by researchers (Vaughan et al., 2003; Kim et al., 2004; Su and Li, 2004; Su and Li, 2005), and showed no significant improvement in detection limits. In a dipstick-type assay, Park and Durst (2000) were able to detect E. coli 0157:H7 in food matrices at a low detection limit of about 103 CFU/ml without any enrichment required. An impedance biosensor chip for detection of E. coli 0157:H7 was developed based on the surface immobilization of affinity—purified antibodies onto indium tin oxide (ITO) electrode chips, with a detection limit of 6 x 103 cells/ml (Ruan et al., 2002b). Shah et al. (2003) developed an amperometric immunosensor with a graphite-coated nylon membrane serving as a support for antibody immobilization and as a working electrode. This approach was used for detection of E. coli with a low detection limit of 40 CFU/ml. 11 Alocilja al- culture inf Alocilja 2C 3005b). An ant cells ollou generate a CPU ml for obtained at" mama-mg: “1111 10 to ] 3004), In at lmFILIA) ~. laggEd with generate If successful]; 3004). 1 An antibody-based conductimetric biosensor developed by Muhammad-Tahir and Alocilja also showed a similar detection limit of 50 CFU/ml for bacteria and 103 cell culture infective dose (CCID)/ml for virus within 10 min (Muhammad-Tahir and Alocilja, 2003a; Muhammad-Tahir and Alocilja, 2003b; Muhammad-Tahir et al., 2005a, 2005b) An antibody-based fiber-optic biosensor, to detect low levels of L. monocytogenes cells following an enrichment step, was developed using a cyanine 5-labeled antibody to generate a specific fluorescent signal. The sensitivity threshold was about 4.3x103 CFU/ml for a pure culture of L. monocytogenes grown at 37°C. Results could only be obtained after 2.5 h of sample processing. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 103 CFU/g after enrichment in buffered Listeria enrichment broth (Geng et al. , 2004). In another study, a microcapillary flow injection liposome immunoanalysis system (mFILIA) was developed for the detection of heat-killed E. coli 0157:H7. Liposomes tagged with anti-E. coli 0157:H7 and an encapsulating fluorescent dye were used to generate fluorescence signals measured by a fluorometer. The mFILIA system successfully detected as few as 360 cells/ml with a total assay time of 45 min (Ho et al., 2004) The ability to detect small amounts of materials, especially bacterial organisms, was demonstrated using micro electromechanical systems (MEMS) for the qualitative detection of specific Salmonella enterica strains with a functionalized silicon nitride microcantilever. Detection was achieved due to a change in the surface stress on the cantilever surface in situ upon binding of a small number of bacteria with less than 25 12 adsorbed b density mi: using 3 ch; 100 Hzo10 Alocilja If. The pc and selectir depending l in all type binding do. fluorescent are used to event. 1m,T antibodies 1 POSitive 5i! 1105110 Cauc 2.1, . A nucl; an M II Stable hid: mlelacu‘ 011:, lnueleotidt adsorbed bacteria required for detection (Weeks et al., 2003). A MEMS fabricated high- density microelectrode array biosensor, developed for the detection of E. coli 0157:H7 using a change in impedance caused by the bacteria measured over a frequency range of 100 Hz-lO MHz, was able to detect and discriminate 104-107 CFU/m1 (Radke and Alocilja, 2005). The potential use of immunosensors is due to their general applicability, specificity and selectivity of the antigen-antibody reaction, and the high sensitivity of the method, depending on the detection method used. The antigen-antibody complex may be utilized in all types of sensors. The physichochemical change induced by antigen-antibody binding does not generate an electrochemically detectable signal. Therefore, enzymes, fluorescent compounds, electrochemically active substrates, or avidin-biotin complexes are used to label either the antigen or the antibody to detect the biological recognition event. Immunosensors cannot be employed to specifically detect viable cells. The antibodies used are selective to the epitope on the antigen. If the epitope is present on a living or dead microorganism, the antibody will capture the antigen and register a positive signal. In the case of bacterial foodbome pathogens that must be ingested by the host to cause disease, a positive result from non-viable cells may raise false alarm. 2.1.4.1.4 DNA andRNA A nucleic acid (gene) probe is a segment of nucleic acid that specifically recognizes, and binds to, a nucleic acid target. The recognition is dependent upon the formation of stable hydrogen bonds between the two nucleic acid strands. This contrasts with interactions of antibody-antigen complex formation where hydrophobic, ionic, and hydrogen bonds play a role. The bonding between nucleic acids takes place at regular (nucleotide) intervals along the length of the nucleic acid duplex, whereas antibody- 13 protein 1 reflected an antib detection Skuridin different nucleic a. ligands as then reger Most nuc complemg The dc on their n BlOScnsors micromolar Cllifirtz Crys lEVels USin “mums. bc'tCten'a] It. lfuouA Climaimng ‘ I protein bonds occur only at a few specific sites (epitopes). The frequency of bonding is reflected in the higher association constant for a nucleic acid duplex in comparison with an antibody-protein complex, and thus indicates that highly specific and sensitive detection systems can be developed using nucleic acid probes (McGown et al., 1995; Skuridin et al., 1996). The specificity of nucleic acid probes relies on the ability of different nucleotides to form bonds only with an appropriate counterpart. Since the nucleic acid recognition layers are very stable, an important advantage of nucleic acid ligands as immobilized sensors is that they can easily be denatured to reverse binding and then regenerated simply by controlling buffer-ion concentrations (Graham et al., 1992). Most nucleic acid biosensors are based on this highly specific hybridization of complementary strands of DNA and also RNA molecules. The detection of bacteria and other pathogens in food, drinking water, and air, based on their nucleic acid sequences, has been explored using various detection systems. Biosensors have been developed to detect DNA hybridization at sub-picomolar to micromolar levels using gravimetric detection systems (Sung Hoon et al., 2001). A quartz crystal nanobalance system could detect DNA hybridization at 0.3 nanogram levels using frequency shift nanogravimetric measurement (N icolini et al., 1997). Tombelli et a1. (2000) developed a DNA piezoelectric biosensor for the detection of bacterial toxicity based on the detection of PCR amplified aer gene of Aeromonas hydrophila. The biosensor was applied to vegetables, environmental water, and human specimens. The biosensor was able to successfully distinguish between samples containing the pathogen and those not contaminated. Zhao et a1. (2001) developed a QCM biosensor using 50 nm gold nanoparticles as the amplification probe for DNA 14 detectior reported larger pa DNA f0] water in of genorr “hen us: could det. 0.02-0.14 developed have been PCR ampl. DNA hybr; Optica C'Ompared l fluOrQSCem detect 16-” l (Abel e! u mQIeCUlar larger Dx. commenter.I perfOTItied detection in the order of 10 M of target, which was higher than what has been ever reported using the same method. The high sensitivity was explained by the weight of the larger particles, and the larger area occupied by the larger particles that needed less target DNA for their binding. Another QCM biosensor applied to the detection of E. coli in water in combination with PCR amplification (of the lac gene) was able to detect a 10 fg of genomic E. coli DNA (few viable E. coli cells in 100 ml of water) (Mo et al., 2002). When used for detection of Hepatitis B virus, Zhou et al. (2002) observed that the QCM could detect frequency shifts of DNA hybridization as a linear relationship in the range 0.02-0.14 ug/ml with a detection limit of 0.1 ug/ml, similar to the QCM biosensor developed by He and Liu (2004) for Pseudomonas aeruginosa. While QCM biosensors have been demonstrated to successfully detect bacterial and viral targets, they rely on PCR amplification of the target due to poor sensitivity, in addition to requiring 3-12 h for DNA hybridization and detection. Optical biosensor systems developed for DNA detection exhibit higher sensitivity compared to the QCM biosensors. An automated optical biosensor system based on fluorescence excitation and detection in the evanescent field of a quartz fiber was used to detect l6-mer oligonucleotides in DNA hybridization assays. The detection limit for the hybridization with a complementary fluorescein-labeled oligonucleotide was 2 x 10'13 M (Abel et al., 1996). Another optical fiber evanescent wave DNA biosensor used a molecular beacon (MB) DNA probe that became fluorescent upon hybridization with target DNA. The detection limit of the evanescent wave biosensor with synthesized complementary DNA was 1.1 nM. Testing with environmental samples was not performed (Liu and Tan, 1999). Liu et al. later developed MB-DNA biosensors with 15 microme was higl detect 0. submicro Optic naditiona (3003.) de We bios emphasizi Were obta imF‘Ortam assessment “'35 det'elc membrane ‘ Stud." Comb as 3 nuclei Spore in 1 OlignUCleoz SallSllll'e (: The blOSe; entrapping plObe lhar . delECllon 0 ji micrometer to submicrometer sizes for DNA/RNA analysis. The MB-DNA biosensor was highly selective with single base-pair mismatch identification capability, and could detect 0.3 nM and 10 nM of rat gamma-actin mRNA with a 105-um biosensor and a submicrometer (0.1 pm) biosensor, respectively (Liu et al., 2000). Optical biosensors targeting RNA as the analyte offer an added advantage over traditional DNA-based detection methods, i.e., viable cell detection. Baeumner et al. (2003) detected as few as 40 E. coli cells/ml in samples using a simple optical dipstick- type biosensor coupled to Nucleic Acid Sequence Based Amplification (NASBA), emphasizing the fact that only viable cells were detected, and no false positive signals were obtained from dead cells present in a sample. The detection of viable cells is important in respect to safety, and also food and environmental sample sterilization assessments. Similarly, a biosensor for the protozoan parasite Cryptosporidium parvum was developed (Esch et al., 2001). Hartley and Baeumner (2003) developed a simple membrane strip—based biosensor for the detection of viable Bacillus anthracis spores. The study combined the optical detection process with a spore germination procedure as well as a nucleic acid amplification reaction to identify as little as one viable B. anthracis spore in 12 h. A quantitative universal biosensor was developed on the basis of olignucleotide sandwich hybridization for the rapid (30 min total assay time) and highly sensitive (1 nM) detection of specific nucleic acid sequences (Baeumner et al., 2004). The biosensor consisted of a universal (polyethersulfone) membrane, a universal dye- entrapping liposomal nanovesicle, and two oligonucleotides — a reporter and a capture probe that could hybridize specifically with the target nucleic acid sequence. Limits of detection of 1 nM per assay and dynamic ranges between 1-750 nM were obtained. While 16 the RNA-l: inherent di high susce; and need ft Other amperome'. 3001). car? microcantif amperomet 2000; Lee Several elt funher (Ya 20041 Lee t 2.1.4.: 2.1. This ; PhOtons as pTESEHCe Phages tha'l “ide ran“. .5; PTOSSer e! ,. 80 l rim] 8! (4‘ the RNA-based biosensor can be an excellent tool for detection of viable bacterial cells, inherent disadvantages to the technique include the short life span of the mRNA target, high susceptibility to contaminants and inhibitors from environmental and food samples, and need for complex detection systems. Other biosensors targeting DNA that have been developed include MEMS-based amperometric (Gau et al., 2001) and high throughput PCR biosensors (Nagai et al., 2001), carbon nanotube-based field effect transistor biosensor (Maehashi et al., 2004), microcantilever—based cyclic voltammetry biosensor (Zhang and Li, 2005), pulsed amperometry- (Ramanaviciene and Ramanavicius, 2004), capacitance- (Bemey et al., 2000; Lee et al., 2002), and absorbance-based biosensors (Mir and Katakis, 2005). Several electrochemical DNA biosensors have been developed that will be discussed further (Yan et al., 2001; Cai et al., 2002; Meric et al., 2002; Ye et al., 2003; Kara et al., 2004; Lee et al., 2004; Del Pozo et al., 2005; Wong and Gooding, 2005). 2.1.4.2 Transducers 2. 1.4.2.1 Bioluminescence sensors This phenomenon of bioluminescence is the ability of certain enzymes to emit photons as a byproduct of their reactions. Bioluminescence may be used to detect the presence and physiological condition of cells. The potential applications of bioluminescence for bacterial detection involve the development of luciferase reporter phages that infect host bacteria, conferring a bioluminescent phenotype to previously non-bioluminescent bacteria. Bioluminescence systems have been used for detection of a wide range of microorganisms and environmental toxicity markers (Prosser, 1994; Prosser et al., 1996; Ramanathan et al., 1998; Weitz et al., 2001; Bechor et al., 2002; Bolton et al., 2002; Shao et al., 2002; Farre and Barcelo, 2003). Folley-Thomas et al. 17 (1995) U5 paratuberc detectable approach. Griffiths. 1. of Salmon Increased cell marke orders of biolumines and the in} disadvanta Ofbacten‘a 2.1. Opticzi bacteria. 7 thickness ti SeVera] o; minding. ' lain 9’ al. El (1].. 200i 1. cl * . J04). Elli: ' at: ('SChneider (1995) used the TM4 bacteriophage to detect Mycobacterium avium and M. paratuberculosis. However, a concentration of 104 cells was required to produce a detectable luciferase signal, and the response declined after 2 b. Using the same approach, Salmonella spp. and Listeria were also detected (Turpin et al., 1993; Chen and Griffiths, 1996). A sensitive and specific method was developed for the specific detection of Salmonella Newport and E. coli (Blasco et al., 1998) using ATP bioluminescence. Increased sensitivity was obtained by focusing on the bacteria’s adenylate kinase as the cell marker instead of ATP. Light emission was proportional to cell numbers over three orders of magnitude, and 103 cells were readily detectable in a 0.1 ml sample. The bioluminescence approach is an attractive approach due to its extremely high specificity and the inherent ability to distinguish viable from non-viable cells. However, the main disadvantages are the relatively long assay time and lack of sensitivity when low numbers of bacteria are to be detected. 2. 1.4.2.2 Optical biosensors Optical transducers are attractive for application to direct, label-free, detection of bacteria. These sensors are able to detect minute changes in the refractive index or thickness that occur when cells bind to receptors immobilized on the transducer surface. Several optical techniques have been reported for detection of bacterial pathogens including: waveguides (Lukosz et al., 1991; Zhou et al., 1991; Amato et al., 2000; Rowe- Taitt et al., 2000; Demarco and Lim, 2002; Lim, 2003), surface plasmon resonance (Kai et al., 2000; Haes and Van Duyne, 2002; Chen et al. , 2003; Medina, 2003; Leonard et al. , 2004), ellipsometry (Swenson, 1993; Bae et al., 2005), the resonant mirror (Watts et al., 1994; Watts et al., 1995; Hirmo et al., 1998; Lathrop et al., 2003), and the interferometer (Schneider et al. , 1997; Liu et al., 2002). 18 Sueni free instru | optical un | Metabolir affected 13 tluorescer. The r bacteria (t refractive material. ,l index layer an Clamen reflé‘CtiorL occurs, it . Swenson (1993) utilized an ellipsometric technique for the development of a label- free instrument for detection of bacteria. The main component of the biosensor was an optical unit that consisted of an excitation source and a photodiode detection system. Metabolizing bacteria would result in an increased C02 concentration, which in turn affected an emulsion of aqueous colorimetric pH indicator, thus modulating the fluorescence detected at the photodiode. The resonant mirror is another technique that can be used for direct detection of bacteria (Cush et al., 1993). It is based on the use of a thin layer (~100 nm) of a high refractive index dielectric material and a thicker layer (~1 mm) of low refractive index material. At certain angles of incidence, light may be coupled into the high refractive index layer where it undergoes multiple internal reflections at the top interface, allowing an element of light, the evanescent wave, to penetrate to the sample overlayer. On reflection, the light undergoes a phase change, and by monitoring the angle at which this occurs, it is possible to detect changes within the evanescent field. A resonant mirror immunosensor was used for detecting S. aureus at 107cells/ml within 5 min (Watts et al. , 1994). The use of the resonant mirror biosensor for direct and rapid detection of DNA- DNA hybridization was also demonstrated, with the lowest detectable concentration of target oligonucleotide (40-mer) being 9.2 nM (Watts et al. , 1995). Schneider et al. (1997) described an evanescent wave interferometer that used a single planar wave of polarized light. Light from a diode laser source was coupled into the waveguiding film as a single broad beam. The light then passed through multiple sensing regions on the surface of the chip. An array of integrated Optical elements was used to combine light passing through adjacent regions that were functionalized with 19 specific Salmom The mai compror Piez detection Liu. 2004 and cost surface 01 bacteria 2 [he antibo r850fiance PZ ir Salmongm A tCCl’lniq deteloped CEllSiml ll Wand six ‘0 Set .- bl Washl'n F del‘eloped Will-body (i ' “l' l specific or non-specific receptors. Using this technique it was possible to directly detect Salmonella Typhmurium in the range of 5x108 CFU/ml with a detection time of 5 min. The main advantage of the above techniques is their short detection time, however this is compromised by their severe lack of sensitivity. 2. 1. 4. 2. 3 Piezoelectric biosensors Piezoelectric (PZ) biosensor systems, in principle, may be used for direct label-free detection of bacteria (Babacan et al. , 2002; Zhou et al., 2002; Lin and Tsai, 2003; He and Liu, 2004; Kim et al., 2004). This technology offers a real-time output, simplicity of use, and cost effectiveness. The theoretical basis of piezoelectricity is based on coating the surface of the Pl sensor with a selectively binding substance, for example, antibodies to bacteria, and then placing it in a solution containing bacteria. The binding of bacteria to the antibodies will increase the mass of the crystal while proportionally decreasing the resonance frequency of oscillation. PZ immunosensors were developed for Vibrio cholerae (Carter et al., 1995), Salmonella Typhimurium (Babacan et al., 2002), L. monocytogenes (Jacobs et al., 1995). A technique using a QCM sensor coated with a thin culture medium film was also developed and applied to determine Staphylococcus epidermidis in the range of 102-107 cells/ml (Bao et al., 1996). However, the Pl membrane was not strong enough to withstand several autoclavings. Prusak-Sochaczewski et al. (1990) developed a QCM biosensor for the detection of S. Typhimurium wherein the coated crystal was stable for six to seven assays. For repeated use, the bound bacteria were removed from the crystal by washing with 8 M urea. A flow-injection system, based on a PZ biosensor was also developed for detection of S. Typhimurium by. immobilizing anti-Salmonella spp. antibody onto a gold-coated quartz crystal surface (Ye et al., 1997). The biosensor had 20 responses CFL'fml. Possi interferer. sensors a: drying stc crystals n‘ disposable 2.. Micri Capacitant CODdUClar; and are ali bell’t’i‘en l responses of 23-47 Hz in 25 min with a dynamic detection range of 5.3x105 to 1.2x109 CFU/ml. Possible limitations of PZ technology include the lack of specificity, sensitivity, and interferences from the liquid media where the analysis takes place. Disadvantages of P2 sensors are the relatively long incubation time of the bacteria, the numerous washing and drying steps, and the problem of regeneration of the crystal surface. Regeneration of crystals may not be an issue if small crystals can be manufactured at low cost so that disposable transducers are economically feasible. 2. 1.4.2.4 Electrical impedance biosensors Microbial metabolism usually results in an increase in both conductance and capacitance, causing a decrease in impedance. Therefore, the concepts of impedance, conductance, capacitance, and resistance are different ways of monitoring the test system, and are all inter-related (Silley and Forsythe, 1996; Milner et al., 1998). The relationship between impedance (Z), resistance (R), capacitance (C), and frequency (f) for a resistor and a capacitor in series is expressed as follows (Bataillard et al., . Impedance is usually measured by a bridge circuit, with a 1988):Z2 = R2 + 1 (2 )2 reference module that measures and excludes non-specific changes in the test module. The reference module serves as a control for temperature changes, evaporation, changes in amounts of dissolved gases, and degradation of culture medium during incubation. This method is well suited for detection of bacteria in clinical specimens, to monitor quality and detect specific food pathogens, also for industrial microbial process control, and for sanitation microbiology (Swaminathan and Feng, 1994; Silley and Forsythe, 1996). This technique has been used for detecting microbial metabolism (Dezenclos et 21 al.1994 state of l instrume: in the Cl cells ml c A bi state of c based on structures changed 1 membrane the electrri PTOVided i Similar dc density IT disadyanu 2." Mien ~I techniques. I and [hen L al., 1994; Palmqvist et al., 1994), and for detecting the concentration and physiological state of bacteria (Desilva et al., 1995; Dupont et al., 1996; Ehret et al., 1997). Current instruments usually detect active metabolizing bacteria when 106—107 cell/ml are present in the culture media. Applied to bacterial detection in urine, concentrations of 105 cells/ml can be detected using the impedance technique with a detection time of 2.5 h. A biosensor for real-time monitoring of concentration, growth, and physiological state of cells in culture media was proposed by Ehret et al. (1997). This biosensor was based on impedance measurement of adherently growing cells on interdigitated electrode structures. Cell density, growth, and long-term behavior of cells on the electrodes changed the impedance of the biosensor due to the insulating property of the cell membrane. The presence of intact cell membranes on the electrodes and their distance to the electrodes determined the current flow, and thus the sensor signal. The biosensor provided information about spreading, attachment, and morphology of cultured cells. A similar detection principle was used by Radke and Alocilja (2005) to develop a high- density microelectrode array biosensor for detection of E. coli 0157 :H7. One disadvantage of using the impedence method is most analyses takes 20—25 h to complete. 2. 1.4.2.5 Fluorescence labeled biosensors Microorganisms are immunogenic due to the presence of proteins and polysaccharides in their outer coats. This permits the development of immunoassay techniques for bacterial detection. In fluorescent immunoassays (FIA), fluorochrome molecules are used to label antibodies. The fluorochrome absorbs short-wavelength light and then emits light at a higher wavelength that can be detected using fluorescent microscopy. Fluorescein isothiocyanate and rhodamine isothiocyanate-bovin serum albtunin are the most common fluorochromes used to tag antibodies. Direct and indirect 22 detection Donnelly samples I bacterial by food p fluoresce detection methods are used to test bacteria-containing samples (Colwell et al., 1985; Donnelly and Baigent, 1986; Brayton et al., 1987; Kaspar and Tartera, 1990). Food samples tested by F IA are typically from enrichment cultures because of insufficient bacterial numbers in the original sample to be directly detected, and interference caused by food particulates that produces background fluorescence. Identification of bacteria by fluorescent immunoassays (FIA) takes advantage of the high degree of specificity inherent in the immunological reaction. An immunomagnetic assay system (IMAS) was developed for detection of virulent bacteria in biological samples (Yu and Bruno, 1996; Vemozy-Rozand et al., 1997). The IMAS consisted of a magnetic separator for capturing the antigen, and an electrochemiluminescent detector. The IMAS was coupled to a flow cytometer and a continuous fluorimeter. This approach, like other chemiluminescence techniques, offered high signal-to-background ratios, and was comparable in sensitivity to radioisotopic methods. It had an advantage over other chemiluminescence techniques of being initiated by a voltage potential, and thus providing better-controlled luminescence (Yu and Bruno, 1996). The total assay time was one hour. The sensitivity of the IMAS for Bacillus anthracis spores, E. coli 0157:H7, and S. Typhimurium detection was approximately 100 cells/ml in phosphate buffered saline (PBS) and 1000 cells/ml in biological samples. Decreased sensitivity of the IMAS detection in biological samples was due to sample interference. 2. 1.4.2. 6 F low immunosensors Most microbial assays are currently based on solidphase enzyme-linked immunoassays (ELISA) using microtitration plates. This is a powerful analysis tool used in biomedical research due to its high reproducibility and possibility to simultaneously 23 conduct heterogen microtitra Also. the and other bacteria al pathogeni Administr desirable growing a arialuica] Concentrat lDUVerlie called the ELISA f0. difoSiona filtering a “tremen- VOlllme ra SOIid‘Phas; linked iWI detection " l and the E conduct a large number of assays (Hock, 1996). However, disadvantages of heterogeneous ELISA methods include the small sample volume (200 pl) that the microtitration plate holds and the long incubation time required for each ELISA step. Also, the sensitivity of ELISA methods is insufficient for direct measurement of bacteria and other microorganisms in the original samples. Because low numbers of pathogenic bacteria are often present in a biological sample, an analytical standard often used for pathogenic bacteria is to detect cells in 25 g of food (Wyatt, 1995; Food and Drug Administration, 2000). Therefore, in order to increase assay sensitivity, it would be desirable to concentrate the bacteria into a smaller volume prior to the assay or by growing a single cell into a colony. Several possible formats for concentrating cells in analytical systems were described by Wyatt (1995). The most attractive technique for the concentration of bacteria is membrane filtration in conjunction with flow systems (Duverlie et al., 1992; Paffard et al., 1996; Abdel-Hamid et al., 1999a). This procedure, called the flow immunofiltration assay, can be an excellent alternative to traditional ELISA for detection of bacterial pathogens, because it not only overrides the effects of diffusional limitations, but also allows the concentration of bacteria on the membrane by filtering a large volume of the sample. Heterogeneous flow immunofiltration assays offer extremely accelerated binding kinetics (Morais et al., 1997) due to high surface area to volume ratio, and the flowing stream actively bringing the sample in contact with the solid-phase antibody. Paffard et a1. (1996) developed an apparatus for use in an enzyme linked immunofiltration assay (ELIFA) for the detection of E. coli. Quantitative bacterial detection was based on precipitated chromogen determined by scanning densitometry, and the ELIFA method was capable of detecting 103 bacterial cells within 40 min. 24 — —-r afiig Another artificial noncorrt a limo touldbc the cell's order of procedu An includj and Sin lb. Variety i Causing 1 l'SCheu e Another flow injection immunosensor, developed for the determination of E. coli in artificially contaminated food samples (Bouvrette and Luong, 1995), was based on direct noncompetitive heterogeneous immunoassay of E. coli cells with an antibody column and a fluorescence detector. The advantage of this method was that bacterial concentrations could be determined without using any labeled compounds through the direct detection of the cell’s B-D-glucuronidase activity. However, the detection limit for E. coli was on the order of 5x107 CFU/ml, which is less than the detection limit of the standard ELISA procedure for microbial cells (typically 106 CFU/ml). Another promising format of immunoassay is based on the use of flow injection systems and antibody coated magnetic particles. This technique can be easily automated, the analyses performed quickly and continuously, and the renewal of the sensing surface of immunosensor was easily accomplished. The system has been applied to detect E. coli 0157 and S. Typhimurium (Brewster et al., 1996; Gehring et al., 1996; Yu and Bruno, 1996; Vemozy-Rozand et al., 1997; Brewster and Mazenko, 1998; Perez et al., 1998). This approach, though expensive, offers a number of advantages over the ELISA, including greater antibody binding capacity, higher sensitivity, reduction of assay time, and simpler operation. 2. 1.4.2. 7 Genomic sensors The detection of specific DNA sequences provides the basis for detecting a wide variety of bacterial pathogens. Gene probes have been used in the detection of disease- causing microorganisms in water supplies, food, and in plant, animal or human tissues (Scheu et al., 1998; Simpkins et al., 2000; Eun et al., 2002; Wang and Yeh, 2002; Jackson et al., 2004; Kotlowski et al., 2004). The original DNA hybridization test for 25 bacteria of radiol. and dis; probes i Hybridi/ positive hybridiz' required sensititir lm'olr'iné detected teal 5am 516133. th: and Fun rEttlioacti )ltKilli; Till mplOVe bacteria in foods used a radioactively labeled probe (Feng, 1992). The main disadvatages of radiolabelled probes are the short-shelf life of P32-labelled probes, high cost, hazards, and disposal problems associated with radioactive waste. The limitation of nucleic acid probes is also a problem associated with cultivating bacteria to a detectable level. Hybridization requires the presence of at least 105—106 bacteria in the sample to obtain a positive signal. Therefore, without pre-enrichment of the target organism, direct DNA hybridization approach does not provide the required sensitivity to detect bacteria at required levels (Tietjen and Fung, 1995). However, PCR extremely enhances the sensitivity of DNA probes, at least three orders of magnitude. To date, only methods involving PCR have been employed to detect foodbome pathogens. Bacteria can be detected directly, without cultivation, by extraction and isolation of nucleic acids from real samples, followed by hybridization with specific probes. Without any enrichment steps, the PCR method could detect less than 40 CF U/g of a given food sample (Tietjen and Fung, 1995). Alternative ways to detect nucleic acid hybridization by using non- radioactive labeled probes have been developed (Bruno and Kiel, 1999; Liu et al., 2000; McKillip and Drake, 2000; Seo et al., 2004). Two aims of DNA-based biosensor development should be emphasized: a) an improvement over conventional nucleic acid assay performance, and b) the design of special gene probe techniques for special applications under special conditions. Based on the nature of the physical detection principle used in the transducer, genosensing systems can be classified as optical, gravirnetric, and electrochemical. The principles of these transducer systems have been described above. 26 Eta et al.. 1‘ assays. l demons: “3V6 dc lntemati. based on interactit‘ The 5551 retlectio “11h a d lmroclur and p0 2002). Se KBTm; deb: r, "t Evanescent wave methods of total internal reflection fluorescence (TIRF) (Graham et al., 1992) and LAPS (Lee et al., 2000) were described as labeling methods for DNA assays. Direct (label-less) gravimetric monitoring of hybridization reactions has been demonstrated with SPR (Kai et al., 2000; Wang et al., 2004b) and piezoelectric acoustic wave devices (Zhou et al., 2002; He and Liu, 2004). The Biacore system (Biacore International SA, Neuchatel, Switzerland) is a commercially available optical sensor based on evanescent wave technology that has been used for the detection of DNA-DNA interactions (Kai et al., 2000; Jongerius-Gortemaker et al., 2002; Wang et al., 2004b). The system uses SPR that arises in thin metal films under conditions of total internal reflection. In the sensing element of this instrument, a gold transducer surface is modified with a dextran matrix on which the biological probe is immobilized. Oligonucleotides are introduced within a fluid flow system. Hybridization is carried out at room temperature, and positive signals are obtained within several minutes (Jongerius-Gortemaker et al., 2002). Several electrochemical DNA biosensors have been developed (Cai et al., 2002; Kerman et al., 2002; Cai et al., 2003a; Li and Hu, 2004; Wong and Gooding, 2005). Electrochemical detection of DNA hybridization usually involves monitoring of a current response resulting from the Watson—Crick base-pair recognition event, under controlled potential conditions. The probe-coated electrode is commonly immersed into a solution of a target DNA whose nucleotide sequence is to be tested. When the target DNA hybridizes, a duplex DNA is formed at the electrode surface. DNA hybridization is then detected via the increased current signal of an electroactive indicator (DNA intercalator) (Kerman et al., 2002), the use of enzyme labels or redox labels (Napier et al., 1997; Ju et 27 al.. 201. (for era Tu hybridiz as poro hybridiz solid-pi. the need is exper WSOIme “hen tes Eacl The focu different chntial biologic. hirh bu PTObabij Wilmer] lmmobr al., 2003), or from other hybridization-induced changes in electrochemical parameters (for example, capacitance or conductivity) (Cai et al., 2003b; Wei et al. , 2003). Two types of hybridization are currently used: (i) pseudo-homogeneous hybridization, which can be achieved in systems with high surface-to-volume ratio, such as porous membranes and highly dispersed immobilization carriers; (ii) solid-phase hybridization, which is preceded by transfer to a membrane. The main disadvantage of solid-phase relative to pseudo-homogeneous hybridization is the longer time required and the need for several manipulations (Aubert et al., 1997). The DNA hybridization method is expensive, complicated (multistep assay), time-consuming, and requires trained personnel to perform. There is also a problem of false amplifications from dead cells when testing samples subjected to sterilization/sanitization treatments. Each biological sensing element and transducer has its advantages and limitations. The focus of the current research will be to develop a versatile biosensor platform using different biological sensing elements and transducer systems. This will demonstrate the potential for future development of a biosensor system which can employ multiple biological sensing elements and transducer systems to provide a self-validating device with built-in output redundancy. This would be helpful in applications where a low probability of false positives and negatives is a must, for example, for bioterrorism countermeasures. One of the key stages of the biosensor development will involve the immobilization of the biological sensing element. 28 cross-lir 6’! al. , 1 depend: Ph}‘sicc 2.2.1 Er Ph‘SSic metho Platfo the er 2.2 IMMOBILIZATION TECHNIQUES The immobilization of the desired biological sensing element such as enzyme, antibody, and DNA probe is a key step in biosensor development. When the biological material produces the physicochemical changes in close proximity to a transducer, it can be successfully detected. Immobilization not only helps in achieving the required close proximity of the biomaterial with the transducer but also in stabilizing it for possible reuse. The biological material can be immobilized directly on the transducer or, in some cases, in membranes, which is subsequently mounted on the transducer. Biological materials can be immobilized either through adsorption, entrapment, covalent binding, cross-linking, or a combination of all these techniques (Hock, 1996; Cosnier, 1998; Thust et al., 1999; Bemey et al., 2000; Cai et al., 2004). Selection of a technique and platform depends on the nature of biological material, nature of substrate that produces the physicochemical change, and configuration of the transducer used. 2.2.1 Enzyme Immobilization Enzymes have normally been immobilized either by covalent binding, through physical adsorption, or by cross-linking. Physical immobilization, a simple and easy method, is the adsorption of enzymes onto solid supports (for example, biosensor platform). The enzyme is dissolved in solution, and the solid material is in contact with the enzyme solution for a fixed period of time. The unadsorbed enzyme is then removed by washing with a buffer. The adsorption mechanisms are governed by electrostatic attraction, hydrophobic interaction, and van derWaal’s forces (Choi, 2004). This method is simple, mild, and causes little or no enzyme inactivation; hence, the enzyme activity is maintained, and the sensitivity of the biosensor will be higher. 29 polymer enzyme or syntl: is smalfh pass th: binding bound alteratio be used “Width \‘ia thei Plfl't‘ntir enz)‘rne. Olganellt technjqu matrices Afier h Utilmrk been (let 3004a; h 2094). G Sillel e! Various enzymes have been entrapped within cross-linked organic or inorganic polymers. The preparation is done by cross-linking of the polymer in the presence of the enzyme; thus, the enzyme can be physically entrapped in the micro-pores of the natural or synthetic polymer (Koronczi et al., 2001; Ivekovic et al., 2004). The size of the pores is small, and this can prevent larger enzyme from leaching but allows small analytes to pass through to react with the entrapped enzyme (Kuswandi etal., 2001). When covalent binding or cross-linking is used for enzyme immobilization, the enzyme needs to be bound without significantly affecting its conformational flexibility, since minor alterations in conformational changes on binding a ligand to a protein molecule can often be used as a criteria in analytical determinations (Bonnington et al., 1995). Glycoprotein enzymes such as glucose oxidase, peroxides, and invertase have been covalently bound via their carbohydrate moiety (Melo and Dsouza, 1992; Husain and Jafri, 1995) preventing the chemical modification of functional groups in the protein moiety of the enzyme. Entrapment and adsorption techniques are also useful when cells or cellular organelles are used (Doktycz et al., 2003; Li et al., 2004). Novel sol-gel synthetic techniques have been developed to immobilize enzymes in stable, optically transparent matrices (Lobnik and Cajlakovic, 2001). The enzyme is added to the sol-gel solution. After the sol-gel has been gelled, the enzyme will be entrapped within the polymeric network of the porous gel (Wolfbeis et al., 2000). Enzyme-based sol-gel biosensors have been developed for detection of glucose (Zaitoun, 2005), hydrogen peroxide (Wang et al. , 2004a; Xu et al. , 2004), cholesterol (Shi et al., 2005), and creatinine (Pandey and Mishra, 2004). Gelatin has also been used as a natural polymeric support (Deshpande et al. , 1986; Svitel et al., 1998). However, the method ofien followed with gelatin is cross-linking 30 llSlll enzyme biomolc the surf; The excl chloride actir'atit eflZyme ETOUps residue Pennar Shelf li case c Witt. 2.2.2 using glutaraldehyde (GA), which is known to inactivate some of the enzymes requiring amino groups for activity. Chemical immobilization of enzymes can be achieved by covalently bonding the enzymes to fimctionalized solid materials or intermolecular cross-linking of the biomolecules. The binding of the enzymes to the solid support is achieved by activating the surface of the support, followed by coupling of the enzyme to the activated surface. The excess and unreacted enzymes are removed by washing with a buffer. Cyanuryl chloride (2,4,6-trichlorotriazine) is a versatile reagent that has been used for the activation of surfaces containing hydroxy groups and subsequent immobilization of enzyme layers. Similarly, solid surfaces can be functionalized to carboxylic acid or amino groups with subsequent attachment with enzymes. The enzyme is coupled with the amino residues surface by glutaraldehyde (Choi, 2004). In general, this technique provides permanent and stable attachment of enzyme molecules to the support, leading to longer shelf life and stable biosensors; however, the enzymatic activity often is not as good as in case of immobilized enzymes prepared by physical immobilization as some of the enzymes are prone to be deactivated in the coupling reaction (Kuswandi et al., 2001). 2.2.2 Antibody Immobilization Antibody immobilization is a vital step in successful development of an immunosensor. The immobilization method must preserve the biological activity of the antibody and enable efficient binding. These methods can be grouped into three main categories: (1) adsorption; (2) immobilization via entrapment in acrylarnide membranes (Ye et al., 1997; Vikholm, 2005); and (3) immobilization via glutaraldehyde (GA) and other cross-linking agents (Bhatia et al., 1989; Narang et al., 1997; Nashat et al., 1998; Slavik et al., 2002; Radke and Alocilja, 2005). 31 An hou'er'e 1 oil the Also. tl wealth The study 01 piezoele immobil reproduc immobil (Babaca select th Tu. flow in Whethj lmmobi Prorein Protein l€gj0n c binding Olanrjh Attachment of antibodies on quartz or glass can be achieved by simple adsorption; however, the immobilized proteins suffer partial denaturation, and tend to leach or wash off the surface (Bhatia et al., 1989; Huang et al., 2004; Zhou and Muthuswamy, 2004). Also, this approach does not provide permanent attachment because the complex is weakly bound to the solid support by adsorption. The selection of an effective method for a particular biosensor requires a detailed study of different immobilization methods. For example, when selecting a method for piezoelectric biosensors, the approach should address: (1) the best biological material for immobilization; (2) the optimum immobilization parameters for the highest yield and reproducibility of the attached layer on the crystal; and (3) the effect of the immobilization layers on the frequency and surface characteristics of the quartz crystal (Babacan et al., 2000). The results of these individual studies can then be combined to select the best method for the particular application. Two methods have been reported to have the best potential for use in piezoelectric flow injection analysis (FIA) system, immobilization on a precoated crystal with polyethylenimine (PEI) (Ye et al., 1997; Lin and Tsai, 2003; Tsai and Lin, 2005), and immobilization through Protein A coupling (Boltovets et al., 2002; Su and Li, 2005). Protein A is a cell wall protein, produced by most strains of Staphylcoccus aureus. Protein A is a directed immobilization method due to its natural affinity towards the Fc region of IgG molecules. This does not block the active sites of the antibodies for analyte binding (Babacan et al., 2000). In the PEI—glutaraldehyde (GA) method, immobilization of antibodies is achieved via surface aldehyde groups of GA on a quartz crystal precoated 32 mth P I antibod Thl proridel \tith ar reprodu Co the Stro surface have m directl} 1998).( the rear rePOrted aminobr mercapi. linkers Stitcimn.‘ lOdOacer 1989; Si: surface r ._ with PEI. Glutaraldehyde is a homobifimctional cross-linker. In this method, the antibodies are randomly oriented and bound to the active surface (Babacan et al., 2000). The self-assembled monolayer (SAM) technique offers one of the simplest ways to provide a reproducible, ultra thin, and well-ordered layer suitable for further modification with antibodies, which has potentials in improving detection sensitivity, speed, and reproducibility (Chen et al., 2003; Su and Li, 2004; Vikholm, 2005). Covalent binding is the preferred method of attaching an antibody to a surface due to the strong, stable linkage that is formed. Hydroxyl groups on the biosensor platform surface provide sites for covalent attachment of organic molecules. Several investigators have modified surface hydroxy] groups to provide a functionality that would react directly with antibodies (Bhatia et al., 1989; Shriver-Lake et al., 1997; Nashat et al., 1998). Coating the biosensor surface with a silane film provides a method for modifying the reactive hydroxyl groups on the surface to attach cross-linking agents. Silanes reportedly used include 4-arninobutyldimethylmethoxysilane, 4- aminobutyltriethoxysilane, 3-mercaptopropyltrimethoxysilane, mercaptomethyldimethylethoxysilane, and 3-aminopropyltriethoxysilane with cross- linkers such as glutaraldehyde, N-y-maleimidobutyryloxy succinimide ester, N- succinimidyl-3-(2-pyridyldithio) propionate, N-succinimidyl-(4- iodoacetyl)aminobenzoate, and succinimidyl 4-(p-aleimidophenyl) butyrate (Bhatia et al. , 1989; Shriver-Lake et al., 1997). Silanes utilized for the modification of the biosensor surface (gold, silicon, glass) introduce amino groups on it, which in turn provide reaction sites for covalently bonding to glutaraldehyde. Antibodies are then immobilized through Schiff base to the cross-linker, glutaraldehyde. Glutaraldehyde has been known to form 33 large p comple- lOWCFlE‘. Sportsr. I silane 1 letter biosensr 2.23 l Ar. . based or large polymers. These reactive polymers may bind many residues and form multi-protein complexes (Bhatia et al., 1989). These effects are likely to interfere with protein function, lowering the antibody activity on the biosensor surface. To obviate the problem, Sportsman and Wilson (1980) coated glass with glycidoxypropylsilane and oxidized the silane to produce aldehyde groups reacting directly with antibody. This method needs fewer functionalization steps and less processing time, while providing a functionalized biosensor with a high activity of immobilized antibodies. 2.2.3 DNA Immobilization Another immobilization technique used with DNA probes (as well as antibodies) is based on the avidin-biotin chemistry (Wang et al. , 2004b). Avidin is a basic glycoprotein with an isoelectric point (pI) of approximately 10, originally isolated from chicken egg white. It is also found in the tissues of birds. Biotin is a naturally occurring vitamin found in every living cell. The tissues with the highest amounts of biotin are the liver, kidney, and pancreas. Yeast and milk are also high in biotin content. Cancerous tumors have more biotin than normal tissue (Savage et al. , 1994). The most commonly used method in SPR-based optical sensing is the immobilization of biotinylated DNA probes or antibodies onto a layer of streptavidin. Streptavidin is a biotin—binding protein isolated from culture broth of Streptomyces avidinii. Streptavidin binds four moles of biotin per mole of protein, and has 3 pl of approximately 5-6 (Savage et al., 1994). Streptavidin is covalently linked to carboxylated dextran fixed onto the gold surface of the SPR chip on a self-assembled monolayer of 1 l-mercaptoundecanol (Bianchi et al. , 1997; Jordan et al. , 1997; Silin and Plant, 1997; Tombelli et al., 2000; Feriotto et al., 2002; Gao et al., 2004; Ruan et al., 2004). This method is very efficient in terms of sensitivity, selectivity, and stability of the realized sensor chip for DNA hybridization detection (Silin and Plant, 34 1997; proced‘ electrot gold St. surface al.. 19‘? Wang r hxmb pert‘orri compar. insrrunz. X” de higher :I sensorr when“ The iItlmobi: Platform imliltlbii; [other] biOSCllSrj . the tarm- fix 1997; Kukanskis et al., 1999; Mariotti et al., 2002), but remains a time consuming procedure (5 days) if one starts from the gold surfaces. Many transduction principles use gold surfaces, for example, SPR, piezoelectric, and electrochemical sensing. Self-assembly of terminally thiol-labeled oligonucleotides onto gold surfaces offers a direct method for chemisorption of DNA probes onto transducer surfaces based on the formation of gold-thiolate bonds (Heme and Tarlov, 1997; Yang et al., 1998; Steel et al., 2000; Cho et al., 2001; Hianik et al., 2001; Satjapipat et al., 2001). Wang et al. (2004b) investigated the suitability of direct coupling of probes by self- assembly of terminally thiol-labeled oligonucleotides for SPR-based sensor. The performance (sensitivity, reproducibility, and specificity) of thiol chemistry was compared to the streptavidin-biotin binding chemistry using commercially available SPR instruments, BIACORE X”, and SPREETATM. The results obtained on the BIACORE XTM device with synthetic oligonucleotides, using the thiolated probe method, showed higher reproducibility and stability, good sensitivity, specificity, and lifetime of the sensor chip (more than 100 measurements were performed on the same modified chip), when compared to the streptavidin-biotin method. The research reported in this study will investigate the performance of different immobilization methods with enzyme, antibody, and DNA probes on the biosensor platform to be developed. These techniques include adsorption, cross-linker mediated immobilization using glutaraldehyde, and direct coupling of antibodies and DNA probe to the silanizing agent. Once the biological sensing element has been immobilized, the biosensor fimctionalization step is completed. The biosensor will then be used to detect the target using appropriate transduction methods. 35 due to and ti. release selecti‘a system protol} llldl’orp intensit_ release chernil. Cl: PTOduc: chernia blue lit. eleetmr‘ Ofim a. mulecnj Iloll'bor. of the Chemo 2.3 CHEMILUMINESCENCE AND BrosgNsons 2.3.1 Chemiluminescence Optical immunosensors are one of the most popular sensors for bioanalysis. This is due to the advantages of applying visible radiation, the nondestructive operation mode, and the rapid signal generation and reading (Luppa et al., 2001). Luminescence is the release of energy by matter in the form of light. Luminescence transducers combine high selectivity and sensitivity to monitor changes of various parameters in the analytical system, such as concentration of the analytes and products, concentration of quenchers, protolytic and complexation reactions, membrane potential, and hydrophobicity or hydrophilicity. Main approaches in luminescence sensing utilize either luminescence intensity or lifetime measurement (Lin and Ju, 2005). When energy in the form of light is released from matter because of a chemical reaction, the process is called chemiluminescence. Chemiluminescence is the phenomenon observed when the vibronically excited product of an exoergic reaction relaxes to its ground state with emission of photons. The chemical reaction produces energy in sufficient amounts (approximately 300 kJ/mol for blue light emission and 150 kJ/mol for red light emission) to induce the transition of an electron from its ground state to an excited electronic state. This electronic transition is often accompanied by vibrational and rotational changes in the molecule. In organic molecules, transitions from a it bonding to a 1t* anti-bonding orbital (T! -> rt“) or from a non-bonding to an anti-bonding orbital (n -) 11*) are most frequently encountered. Return of the electron to the ground state with emission of a photon is thus called chemiluminescence. The excited molecule can also lose energy by undergoing chemical 36 reactit proces chemi'. In with (j trausdu tlinicai relatire dlhamr differen “Std in in then hOISErEK (Thorpc Chtmjlu Chemilu. the lifts atl‘lmol' reactions, by collision, internal conversion or inter—system crossing. These radiationless processes are undesirable from an analytical point of view as they compete with chemiluminescence (Dodeigne et al., 2000). In general, the chemiluminescence reaction can be represented as: A + B Catalyst > C“ C* —>C+hu Chemiluminescence occurs as a result of the oxidation of certain substances, usually with 02 or H202, to produce light in the absence of any exciting illumination. The optical transducer is configured to pick up the emitted light and transmitted to an optical detector for recording (Lin and J u, 2005). Chemiluminescent methods have been applied in routine food analysis, as well as in clinical and biomedical research, due to such advantages as no radioactive wastes, the relatively simple instrumentation required, the very low detection limit, and wide dynamic range (Baeyens et al., 1998; Kricka, 2003). It is versatile and flexible to many different approaches. In general, two major chemiluminescent analytical techniques are used in clinical analysis: (1) luminol, dioxetanes, or acridinium ester as label to be used in chemiluminescent immunoassay; or (2) chemiluminescent detection techniques using horseradish peroxidase (HRP) or alkaline phosphatase as label for enzyme immunoassay (Thorpe et al., 1985). The first technique detects directly a light flash of the chemiluminescent reagent to determine the analytes. In the second method, the chemiluminescent substrates undergo a peroxidase- or phosphatase-catalyzed oxidation in the presence of a suitable oxidant, and the detection limit for enzyme label is typically in attomolar range by the chemiluminescent detection. Several chemiluminescent sensors 37 and d. 1" lllll'lltll release reactin- Conjun. and their application have been reported (Ramanathan et al., 1998; Ye et al., 2002; Liu et al., 2003; Jain et al., 2004; Lin et al., 2004a, 2004b). Most of these sensors are based on immobilized enzymes, in which chemiluminescent reagents react with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescent light signal. 2.3.2 Chemiluminescent Immunosensors When immunoreagents are immobilized, the chemiluminescent sensors are called chemiluminescent immunosensors (Marquette and Blum, 2000). In this case, the chemiluminescent reagents or enzymes (catalyze) are labeled to the antibody (or antigen) molecules. After the immunological reaction, the chemiluminescent intensity of the label is detected by chemiluminescent method for the quantitative analysis of antibody or antigen. Ye et al. (2002) developed a biosensor, consisting of a chemiluminescence reaction cell, a fiber optics light guide, and a computer—linked luminometer, used in conjunction with immunomagnetic separation for rapid detection of Escherichia coli 0157:H7. HRP in the sandwich antibody-antigen complexes catalyzed the reaction between luminol and H202 in the reaction cell. The chemiluminescence biosensor was selective to E. coli 0157:H7 with a detection limit of 1.8 x 102 CFU/ml, and the assay was completed within 1.5 b. Figure 2-1 shows the simplified reaction mechanism of luminol, the substrate typically used for chemiluminescent immunoassays. The key intermediate is an oc- hydroxyperoxide obtained by oxidation of the heterocyclic ring. The decomposition pattern of this intermediate, leading to the excited state and the light emission, is unique and depends only on the pH of the system. In contrast, the first step is strongly dependent of the composition of the medium (Merenyi et al., 1990). In protic solvents (water, water 38 V solvent mixtures or lower alcohols), various oxygen derivatives (molecular oxygen, peroxides, superoxide anion) can oxidize luminol derivatives but catalysis either by enzymes or by mineral catalysts is required. Catalysts employed for immunoassays include enzymes such as microperoxidase, myeloperoxidase, horseradish peroxidase, catalase, and xanthine oxidase (Kozlov et al., 1990; Radi et al., 1990; Rongen et al., 1994). The bacterial peroxidase from Arthromyces ramosus, characterized by a very high tum-over, was claimed to provide a hundred times increase in sensitivity (Akimoto et al., 1990; Kim et al., 1991). O o NH _. N NH ll, NH 2 O NH2 0' OOH Luminol a—hydroxyperoxyde .N2 OH 0 OH O. O NH2 0 O- + light NH2 0 Figure 2-1. Simplified reaction mechanism of luminol: the key intermediate is an a- hydroxyperoxide obtained by oxidation of the heterocyclic ring. Its decomposition leads to the aminophthalate ion with light emission (Dodeigne et al., 2000). 39 V my to. anal) . tando: used i immu: techni. Many chemiluminescent immunoassay test kits and automated immunoassay analyzers have been developed and commercialized (Kricka, 2003). The fully automated, random-access, walk-way chemiluminescent immunoassay analyzers have been widely used in food and clinical analysis due to efficient use of labor, minimal sample and reagent manipulation, ease of automation, and flexibility (Rymer et al., 1999; Hendriks et al., 2000; Nandakumar et al., 2000). Flow injection immunoassay (F IA) has been shown to be useful for improving cumbersome, time-consuming, and labor-intensive traditional immunoassay (Xu et al., 1989; Pollema et al., 1992; Gunaratna and Wilson, 1993). This technique has been applied to many fields such as food, pharmaceutical, environmental, and clinical assays (Bjamason et al., 1997; Abdel-Hamid et al., 1999b; Burestedt et al., 2000; Nandakumar et al., 2000) due to the small volume required, the reduced sampling handling, good reproducibility, and easy automation for high sample throughput. In FIA heterogeneous immunoassays, a solid support is usually used to immobilize either the antibody or antigen. A wide variety of matrices, including inorganic surfaces, organic polymers, and other commercially available solid supports, have been used for the design of packing, while materials for preparation of flow injection immunosensors can be bead supports (silica, agarose, sepharose, and polystyrene particles with a magnetic nucleus) or membranes (Aberl et al., 1992; Bouvrette and Luong, 1995; Abdel-Hamid et al., 1999a; Choi et al. , 2000). These immunosensors have been used for detection of different targets such as E. coli, E. coli 0157:H7, and Salmonella. The main disadvantages of this type of optical transducer are finite lifetime due to reagent consumption, and steady-state mass transfer and laminar flow characteristics required to get a constant signal. 40 bioserj as Sui: a con perfor. Contir expect 2.33 1n sensitir describl Pouclte betllee The current research will focus on development of a chemiluminescent immuno- biosensor on a versatile silicon-based platform for detection of pathogenic bacteria such as Salmonella. The light output, recorded automatically using a luminometer connected to a computer, will be measured in a static (flow) environment to improve the sensor performance characteristics over existing flow injection immunoassay systems. Continuous automatic recording of the light output data will provide rapid results, expected within 5-10 min. 2.3.3 Chemiluminescent Enzyme-Based Sensors In many instances, enzymatically generated chemiluminescence offers greater sensitivity and more rapid detection of viable cells than antibody-based immunoassays described above, as well as fluorometry, colorimetry, and radiolabeled probes (Van- Poucke and Nelis, 1995). The emission of light due to the chemiluminescence reaction between reagent and analyte has been employed in enzyme-based optical biosensors. The light emitted can be measured using charged couple device (CCD) cameras, photo- multiplier tube detectors, avalanche photodiodes, fiber optics, laser detectors, etc. After having compared different luminescent and non-luminescent detection methods, different authors have concluded that dioxetanes are among the most sensitive ones (Nelson and Kacian, 1990; Gillespie and Hudspeth, 1991; Kricka, 1991). Figure 2-2 shows the decomposition mechanisms for 1,2-dioxetane compounds leading to generation of chemiluminescence. The dioxetanes decompose thermally, Chemically or enzymatically into two carbonylic compounds, one of which is in the excited sate (Bronstein et al., 1989a). Two distinct modes are observed (Figure 2-2). The diradical mechanism mainly occurs during thermal decomposition. Very high yields of excited states are obtained, but unfortunately, the often triplet excited state, T1, is rapidly 41 quene E112} r exchu COHCU carbo: obtain et al.. l:Tgurt media (CIEE Whhe Fr dldgm anenn phOSP delete {BTOns spilt) aC quenched in aqueous solutions, and therefore, of poor utility in diagnostic applications. Enzymatic or chemical decomposition is achieved through a chemically initiated electron exchange chemiluminescence (CIEEL) mechanism: a rigorous two-bond breaking concomitant process leads to an electronic redistribution and the formation of the two carbonylic products. Large singlet excited state (Sl) versus T1 ratios are generally obtained, which makes this reaction much more efficient in aqueous solutions (Dodeigne et al. , 2000) 0. O. O o * 1) \|““' '"nu 0—0 “H!" 30 2) ____, (f +MS1 A/K _ l Figure 2-2. The two modes of decomposition of 1,2-dioxetanes: (I) the diradical mechanism and (II) the chemically initiated electron exchange chemiluminescence (CIEEL). The diradical mechanism most often generates triplet excited states (T 1) while CIEEL generally results in singlet states (81) (Dodeigne et al., 2000). For the reasons listed above, thermally unstable dioxetanes are not suitable for diagnostic applications. Chemically triggered (thermally stable) dioxetanes have attracted attention from end-users for enzyme-based assays. B-D-galactosidase and alkaline phosphatase enzymes, used for a long time as labels in immunoassays and DNA-based detection methods, were adapted to chemiluminescent detection using dioxetanes (Bronstein et al., 1989b; Thorpe et al., 1989). The most widely used dioxetanes, 3-(2’- spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy)phenyl-1,2-dioxetane disodium salt 42 ,LV Sengm‘. (AMPPD) and 3-(2'-spiroadamantane) - 4-methoxy - 4-(3”-B-D- galactopyranosyloxyphenyl) - 1,2-dioxetane (AMPGD), are the substrates for high turnover enzymes currently used in immunoassays, alkaline phosphatase and 6-D- galactosidase, respectively (Bronstein et al., 1989a). In recent years, attention has shifted from the use of dioxetanes in immunoassays to enzyme-based assays for direct, easy, and highly sensitive detection of [SD- galactosidase. The lacZ gene from Escherichia coli is frequently used as a reporter gene in determining promoter strength in both transiently and stably transfected cells (Alam and Cook, 1990). Chemiluminescence-based methods are preferable to colorimetric and fluorometric assays for B-galactosidase detection as they are much more sensitive, and do not require sophisticated, expensive equipment. Jain and Magrath (1991) developed an AMPGD-based chemiluminescent assay for B-galactosidase that could detect as little as 2 fg of B-galactosidase activity in a single cell, stably transfected with a lacZ-expressing vector. Dioxetanes have also been used for detection of E. coli by using the B-galactosidase enzyme (a metabolic product) as the marker. Van Poucke and Nellis (1995) developed a chemiluminometric assay of B-galactosidase in coliform bacteria, using AMPGD as the substrate. By this method, one coliform seeded in 100 ml of sterile water could be detected after nine hours of pre-enrichment followed by a 45-min enzyme assay in the presence of polymyxin B. Compared with fluorometry and colorimetry, chemiluminometry allowed the detection of a smaller number of cells, the increase in sensitivity being 4- and 1,000-fold, respectively (Van Poucke and Nelis, 1995). 43 l derix. mice Lumi, itself (him: I galact gener; I depre: I alkalir‘l min» I result) I flu0re.l | L; Spec“ 1993‘). based . “13¢ k6: Indirect chemiluminescence is also possible with dioxetanes. A very hydrophobic derivative of a fluorescer (SN-tetradecanoyl-aminofluoresceine) can be included in the micelles of the surfactant, hexadecyltrimethyl ammonium bromide (Lumi-PhosTM from Lumigen Inc., Southfield, MI), and used in the dioxetane formulation, or the fluorescer itself can be conjugated to the dioxetane ring (Beck and Koster, 1990). Lumi-Gal 530 (Lumigen, Inc.), a commercial formulation of 4-methoxy-4-(3-B-D- galactosidephenyl)spiro[l,2-dioxetane-3,2'-adamantane], reacts with B-galactosidase to generate indirect chemiluminescence (Figure 2-3). The stable dioxetane is enzymatically deprotected by B-galactosidase (in step 1) to produce an unstable form of dioxetane under alkaline conditions (in step 2). This unstable form decomposes in local hydrophobic environment producing the singlet-excited ester (in step 3). Indirect chemiluminescence results when the excited ester decays (step 4), and this energy is transferred to the fluorescer in the Lumi-Gal® 530 cocktail (Beale et al., 1992). Lumi-Gal 530 was shown to have a 20-fold greater sensitivity over other standard spectrophotometric assays when used with a pure B-galactosidase solution (Beale et al., 1992). No further research has been reported for the development of a Lumi-Gal® 530- based chemiluminescent biosensor for E. coli detection using B-galactosidase as the marker, which will be the one of the aims of the research reported herein. 44 O—O Oocn3 o OCH3 B— galactosidase>N N 0 Step 1 Stable O-B-D-galactoside Stable 0H Base Step 2 Y Energy transfer OOCH3 OOCH3 -, Scan 1 Scan 2 Scan 3 a» Time Figure 2-4. Excitation waveform for a cyclic voltammetry experiment (Adapted from (Robinson, 1987)). E . c |pc~~ Fe3+ + e‘ —> Fe2+ {r} E (U A o 3 r 5 o E Fla2+ —> F83+ + e‘ 4 l a~~ p Epa —+ .—:L u—lp i i i 1 Potential (V) vs. SCE Figure 2-5. Typical current response for a reversible couple obtained with cyclic voltammetry (Adapted from (Robinson, 1987)). 54 Tm elec redc cadl anio direc back The current is measured in a three-electrode electrochemical cell discussed further in Chapter 4: Section 4.3.4.8. The schematic representation of a three-electrode potentiostat is shown in Figure 2-6. In a potentiostat, the current is supplied through the counter electrode and is measured through the working electrode. The response of the working electrode depends on the concentration and composition of the analyte in question. The third electrode is a reference electrode that provides a constant reference potential against which the working electrode measures the output current. The reference electrode draws little or no current from the system and is unresponsive to the composition of the species under measurement (Robinson, 1987). Cyclic voltammetry measures the faradaic current resulting from electron transfer. Two factors can affect the faradaic current: the rate the redox species diffuses to the electrode, and the rate of electron transfer. The rate of electron transfer for the common 3-/4- redox couple Fe(CN)6 is reasonably fast. The reaction at the working electrode or cathode is Fe(CN)6‘ + e“ <—-) Fe(CN)2‘ , where one electron is added to the ferricyanide anion to reduce iron from the +3 to the +2 oxidation state. This reaction proceeds in both directions (cyclic process) so that species can be oxidized to Fe(CN)63' , then reduced back to Fe(CN)64‘. 55 Scan mplifier _/\ (m r..— , Counter Electrode ..... Working Electrode FeedbaN Amplifier E, 1 Reference Output Electrode Cwmm Amplifier Figure 2-6. Schematic of a three-electrode potentiostat for cyclic voltammetry (Adapted from (Wang, 2000)). During the scan, the applied potential becomes sufficiently positive at point a (Figure 2-5 above) to cause oxidation of Fe2+ to occur at the electrode surface. This oxidation is accompanied by anodic current, which increases rapidly until the surface concentration of Fe2+ approaches zero, as signaled by peaking of the current at point (IpaaEpa) in Figure 2-5. The cm'rent then decays as the solution surrounding the electrode is depleted of Fe2+ due to its oxidation to Fe“. The magnitude of the current is described by the following 6C equation: I, = "FADo (EYihzw where: I, = Current at time, t (Amps). 11 = Number of electrons, eq/mole. F = Faraday's constant; 96,485 e/eq. A = Electrode area, cmz. C = Concentration of oxidized species, mol/cm3. Do = Diffusion coefficient of oxidized species, cmZ/s. 56 hi af t = Time (s). X = Distance from the electrode (cm). The product Do (6Co/6X) at x = 0,t is the flux or the number of moles of oxidized species diffusing per unit time to unit area of the electrode in units of mol/cm2 s (Shippy and Burns, 2002). During the positive scan in which 1362+ is oxidized to Fe“, the depletion of F e2+ in the vicinity of the electrode is accompanied by an accumulation of Fe”. After the direction of the potential scan is switched to a negative scan (reverse sweep), oxidation continues, as seen in Figure 2-4 above, until the applied potential becomes sufficiently negative to cause reduction of the accumulated Fe3+ (point b). Reduction of F e3+ is signaled by the appearance of cathodic current. Once again, the current increases as the potential becomes increasingly negative until all of the Fe3+ near the electrode is reduced. When the concentration of Fe3+ is significantly depleted, the current peaks, and then decreases. Thus, in the forward scan Fe3+ is electrochemically generated, as indicated by the anodic current. In the reverse scan, the Fe3+ is reduced back to Fe2+, as indicated by the cathodic current. The important parameters of cyclic voltammetry are the magnitude of the peak currents, Ipa and Ipc, and the potentials at which peaks occur, Epa and Epc (Shippy and Burns, 2002). The current research will investigate the use of cyclic voltammetry to detect DNA hybridization under the hypothesis that the parameter, diffusion coefficient D0, will be affected by the DNA probe immobilization and hybridization processes taking place on the biosensor platform surface, in turn influencing the output current, It, at any given time. 57 2.5 NANO-TUBULAR SILICON (NTS) 2.5.1 SiliconlSi) Silicon (Si) was discovered by Jons Jacob Berzelius, a Swedish chemist, in 1824 by heating chips of potassium in a silica container and then carefully washing away the residual by-products. Silicon compounds are the most significant component of the Earth’s crust. Silicon is recovered from an abundant resource, sand. Most pure sand is quartz, silicon dioxide (SiOz). Since sand is plentiful, easy to mine, and relatively easy to process, it is the primary ore source of silicon. Some Si is also retrieved from two other silicate minerals, talc and mica. The metamorphic rock, quartzite, is another source (quartzite is metamorphosed sandstone). Two allotropes of Si exist at room temperature: amorphous and crystalline. The amorphous form appears as a brown powder, while crystalline silicon has a metallic luster and a grayish color. Single crystals of crystalline silicon can be grown with a process known as the Czochralski process. These crystals, when doped with elements such as boron, gallium, germanium, phosphorus or arsenic, are used to manufacture solid- state electronic devices, such as transistors, solar cells, rectifiers, and microchips (Gagnon, 2005). Silicon is a semi-conductor. When silicon is doped with dopants, such as boron and gallium that make it conductive by providing more holes than electrons, it is referred to as p-type Si. Conversely, silicon doped with dopants, such as phosphorus and arsenic that make it conductive by providing more electrons than holes, is termed n-type Si. 2.5.2 Silicon Etching The etching of silicon surfaces in fluoride solutions is of both technological and fimdamental significance. It is essential to semiconductor processing in integrated circuit 58 manufacture. It can also create materials with different properties. Based on the etching conditions, either perfectly flat H-terminated surfaces or H-terrninated NTS is produced. Flat, clean Si surfaces are required for better device performance. NTS is of technological interest not only for its optical properties (Naftel et al., 2000; Nguyen et al., 2000; Setzu et al., 2000), but also as a biomaterial (Wolf et al., 1998), sensor (Lin et al., 1997; Dancil et al., 1999; Fichera et al., 2003; Martin-Palma et al., 2004), and substrate for laser desorption/ionization mass spectrometry (Shen et al., 2001). Silicon is the only material that spontaneously gives rise to well defined tubules with nm dimensions. Other materials such as silicon carbide (SiC), gallium arsenide (GaAs), gallium phosphate (GaP), and indium phosphate (InP) can be made porous but with great difficulty. NTS is typically obtained by anodization in aqueous or ethanoic hydrofluoric acid solution (HF). The versatility of fluoride etching of Si has prompted research to elucidate the mechanisms behind the chemical, electrochemical, and laser assisted chemistry at the fluoride/Si interface (Cullis et al., 1997; Chazalviel et al., 2000; Jungblut et al., 2002; Safi et al., 2002) A typical J-V curve is shown in Figure 2-7 for a diluted aqueous HF solution. Tubule formation occurs in the initial rising part of the curve for 0 < V < Vep- Vep is the potential of the small sharp peak corresponding to the current Jep- This peak is called the electropolishing peak. Jep depends on the solution composition but not so much on the silicon substrate itself. When V > Veps electropolishing occurs since the surface becomes covered by an oxide layer, whose composition and dielectric properties depend on the applied potential. This provides a flat, clean Si surface useful for integrated circuits fabrication (Allongue, 1997). 59 Current J, (mAlcm’) Potential (V) Figure 2-7. Typical J-V curve of p+-Si in dilute aqueous HF solution. Nano-tubular Si is obtainable for J < Jcp. (Adapted from (Allongue, 1997)) The dissolution of p-type silicon and formation of NTS may be described by the reaction model in Figure 2-8 (Allongue, 1997). As long as the voltage is not raised above the electropolishing range (J < Jepa so as to keep the supply of holes within a certain limit), steps A-B remain slower than steps C-E, and the surface remains H-terminated throughout etching (Chazalviel et al., 2000; Safi et al., 2002). Etching is initiated by hole capture at the surface (Gerischer and Lubke, 1987, 1988; Gerischer et al. , 1993). For each silicon atom, dissolved, two holes are consumed in steps A and B. One molecule of hydrogen (step E) and two protons (steps B and C) are also generated on the surface. Once etching is initiated by hole capture at the surface, the ensuing reactions proceed rapidly to dissolve the activated Si atom. After the initial substitution steps Si-H -) Si-OH -) Si-F (steps A to C), undissociated HF and H20 molecules chemically attack the strongly polarized Sif-Si+8 back bonds, leaving the H 60 atom attached to the Si‘3 atom on the surface (steps D). The final step occurs in solution where the Si complex is fin'ther hydrolyzed with production of molecular H2 (step E). Under most conditions, the hole capture step is the rate-determining step (RDS). Rate changes in response to solution composition changes must, therefore, be caused by one of the subsequent reactive steps. As the rate of substitution Si-H 9 Si-OH increases, by increasing the rate of hole supply, the density of Si-OH bonds increases and becomes such that the neighboring groups start to condense into Si-O-Si bridges. This is the early stage of oxide formation that corresponds to electropolishing (Allongue, 1997). A B Sl\ S'\ / \H \SI/ ”H +h‘ \S'/ H H* +h +H20 \S'\ /OH + H‘ \Si/ \Si/\ __p \Si/ \S.\ "' _._) \Si/ Si\H / \S' H / \Si H / \Si / l\H / \H / \H C D \Si\ \s. \ +l='I \s‘s/ +o/FH ”"20 \Si\ H +HO s H I did \$‘/ \Si\H \Si/ '1! F / \Si / \Si/ HO / \H / \H E x x +H,o,m= \s/ ' - ' + H2 (X = OH, F) (In solution) x/ \X Figure 2-8. Reaction model for dissolution of nano-tubular silicon in aqueous HF solutions. 61 Theories that species, such as (HF)2 and (HF )2', apart from F"1 and HF have played an important role in the etch mechanism have not yet been proven. In particular, consensus is that (HF )2 is not formed in aqueous solutions of HF, and consequently, any mechanism relying upon this species is incorrect. Species such as H2O, OH“, W, and F' are involved in the faster reaction steps (Figure 2-8) (Kolasinski, 2003). 2.5.3 Nana-Tubular Si Porosity and Thickness Measurement The porosity and thickness of the porous silicon layers are the most important parameters used to characterize porous silicon. The porosity is defined as the void within the porous silicon layer, and can be determined by weight measurements. The virgin wafer is first weighed before anodization (m1), then just after anodization etching (m2), and finally after complete dissolution of the porous layer in a l M NaOH aqueous solution (mg). The porosity is given by the following equation: 0 _ (ml "m2) P(/O)—(m1‘m3) From the above measurements, the thickness of the porous silicon layer can be (ml _ m3) x determined as follows: W = ; where d is the bulk silicon density, and S is the area of the wafer exposed to HF during anodization (Allongue, 1997). 2.5.4 Nana-Tubular Silicon and Biosensors Nano-tubular Si (NTS) has recently been investigated as a potential platform for biosensor applications. NTS has advantages over planar platforms in biosensor development due to the increased surface area, which will allow for higher sensitivity while using a smaller device. NTS can be easily synthesized directly from the same single-crystal silicon wafers used in the microelectronics industry, making it ideal for a 62 TV ______ silicon-based optoelectronic technology (the combination of optical measurement and silicon microelectronics). An NTS-based device is compatible with established solid-state fabrication technologies. Much of the developmental research has been related to the changes in the electronic and optical properties of NTS produced by quantum confinement. For example, light emitting devices (Hirschman et al. , 1996), photodetectors (Zheng et al., 1992), and wave guides (Loni et al. , 1996) have been reported. Recently, novel sensor applications of NTS have also been developed, taking advantage of the high internal surface area (Lin et al., 1997). Most of the recent developments in NTS sensors carry out a deposition step to fill the pores with metallic, dielectric or semiconducting oxide, enzyme, or molecular receptor films. This process has resulted in NTS sensors capable of detecting penicillin, alkali metal ions, humidity, and hydrocarbons (Stewart and Buriak, 2000). 2. 5. 4.1 Enzyme-based N T S biosensors In the last five years, NTS has been used as a biosensor platform for enzyme-based biosensors. EIS (electrolyte-insulator-sendconductor) structures of nano-tubular n- Si/SiO2/Si3N4 were fabricated to develop a capacitive pH sensor (Schoning et al., 2000) using photolithography-based etching. The average pH sensitivity of the NTS pH microsensor was in the concentration range between pH 4 and pH 8, with the electrodes showing a potential stability of more than 1 week in the long term. Further research (Liith et al., 2000) showed that the enlargement of the active sensor area increased the measured capacitance values up to a factor of 30. When used for glucose monitoring, the use of NTS as an efficient surface enlarging carrier matrix for immobilized enzymes was demonstrated. An increase in glucose turn- over of approximately 220 times as compared to an enzyme activated polished surface 63 was recorded. Glucose monitoring response was linear around 15 mM glucose with a satisfactory storage and operational stability (Bengtsson et al., 2000b). Fichera et al. (2003) also demonstrated Si-based micro-biosensors for glucose detection in water solutions using NTS as the surface to bind the glucose oxidase enzyme. Micro channels localised below a suspended and auto-supporting NTS membrane, fabricated using surface micromachining processes, allowed the buffer solution to flow through the porous matrix. Photoluminescence, absorbance, and optical microscopy measurements were used to demonstrate biosensor fimctionality. In a similar study, DeLouise and Miller (2005) used NTS to gain a quantitative understanding of the effects of immobilization on enzyme activity. The activity of glutathione-S-transferase immobilized in electrochemically etched NTS films was compared with the enzyme in solution. The Specific activity of the free enzyme in solution was observed to be four times higher than the immobilized enzyme, indicating that 25% of the enzyme was bound with the catalytic site in an inactive conformation or in a hindered orientation. To date, no studies have been reported employing NTS as a biosensor platform for bacterial detection with enzyme-based transduction. 2. 5. 4.2 Antibody-based N T S biosensors Tinsley-Bown et al. (2000) used NTS as an optical interferometric biosensor with anti-rabbit IgG and the analyte rabbit IgG conjugated to horseradish peroxidase (HRP). Later, NTS multilayer stacks were developed for use as interference filters in the visible range (Martin-Palma et al., 2004). The possibility of using these structures as biosensors was explored, based on the significant changes in the reflectance spectra before and after eXposing the NTS multilayer to antibodies. Significant reduction of reflectance from NTS Structures was observed when the sensor was exposed to polyclonal mouse antibodies. 64 V The authors claim the possibility of developing biosensors based on the variation of the shape and/or position of the optical or photoluminescent spectrum from NTS. Further studies have not been reported to demonstrate the use of NTS as an optical interference filter-based biosensor. Other biosensors developed with antibody-based detection have been demonstrated on planar silicon surfaces only. These include an ELISA (HRP)-based detection of thiabendazole (Flounders et al., 1995), an evanescent field-based fluorescence detection of rabit IgG (Hofrnann et al., 2002), an ion-sensitive field effect transistor (ISFET)-based immunosensor to detect B-Bungarotoxin (Selvanayagam et al., 2002), and a multi-analyte detection system using a protein biochip sensor array based on a complementary metal oxide silicon (CMOS) integrated circuit (Moreno-Bondi et al. , 2003). 2. 5. 4.3 DNA-based N T S biosensors A DNA biosensor was developed based on induced wavelength shifts in the Fabry- Perot fringes in the visible-light reflection spectrum of appropriately derivatized thin fihns of NTS semiconductors. Binding of molecules induced changes in the refractive index of the NTS. The validity and sensitivity of the system was demonstrated for small organic molecules (biotin and digoxigenin), and 16-nucleotide DNA oligomers, at picomolar analyte concentrations (Lin et al., 1997). Another DNA-based optical biosensor device was fabricated from a microcavity resonator made of various NTS layers. NTS containing silicon nanocrystals that could luminesce efficiently in the visible range was inserted between two Bragg reflectors (also made of NTS), to observe multiple and very narrow luminescence peaks. The position of these peaks was extremely Sensitive to a small change in refractive index, such as that obtained when a biological 65 element was attached to the large internal surface of porous silicon. The authors successfully demonstrated a DNA hybridization detection sensor. Further progress was made with the DNA biosensor to detect viral DNA (Chan et al., 2000; Chan et al., 2001). Change in electrical properties (capacitance and conductance) of NTS layers upon exposure to organic solvents and water have also been investigated for application in biosensors (Archer et al., 2003). The use of NTS as a biosensor platform for detecting foodbome pathogens in conjunction with enzyme- and electrochemical transduction-based methods, such as cyclic voltammetry, has not been reported. In the development of a sensor, selectivity/specificity is an important issue. The sensor must be able to distinguish the element/target being detected from other non-target elements at the appropriate concentration for the target. This research will develop a fabrication process to maximize the surface area of the biosensor platform to provide more reaction sites for the biological detection event. The NTS platform will be funtionalized into enzyme-, antibody-, and DNA-based biosensors, and used for detection of different bacterial targets. If appropriate transduction techniques are employed, such a biosensor platform will help achieve greater sensitivity while also improving the possibility of reducing the size of the sensor into a MEMS or nano- electromechanical system (NEMS) device. Specificity will be achieved by using enzymes, antibodies, and DNA probes specific to the target of interest, whenever possible. The versatility of the NTS platform will be demonstrated by using three biological sensing elements targeting three different pathogens with two immobilization techniques and two transduction mechanisms. 66 2.6 COLIFORMS/ESCHERICHIA cou 2.6.1 Introduction Direct testing of pathogenic bacteria and their toxins in foods is not routinely performed for quality control purposes (except for Salmonella enterica and Staphylococcus aureus). Instead, microbiological criteria for food safety often use tests for indicator organisms that suggest the possibility of a microbial hazard. Fecal colifonns are used as indicator organisms in water, foods, and beverages, the presence of which could suggest the possibility of microbial hazard (spoilage/pathogenic organisms). Coliforms are Gram-negative, rod-shaped, facultatively anaerobic bacteria that produce gas from glucose (and other sugars), and ferment lactose to acid and gas within 48 h at 3 5°C. Escherichia coli, originally known as Bacterium coli commune, was identified in 1 885 by the German pediatrician, Theodor Escherich. Escherichia coli is widely distributed in the intestine of humans and warm-blooded animals, and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host (Neill et al. , 1994). Escherichia coli is a member of the farme Enterobacteriaceae (Ewing, 1986), which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans (Feng et al., 2002). In 1892, Shardinger proposed the use of E. coli as an indicator of fecal Contamination based on the premise that E. coli is abundant in human and animal feces, and not usually found in other niches. Furthermore, since E. coli could be easily detected 67 by its ability to ferment glucose (later changed to lactose), it was easier to isolate than known gastrointestinal pathogens. Hence, the presence of E. coli in food or water became accepted as indicative of recent fecal contamination and the possible presence of pathogens. Although the concept of using E. coli as an indirect indicator of health risk was sound, it was complicated in practice, due to the presence of other enteric bacteria like Citrobacter, Klebsiella, and Enterobacter that can also ferment lactose and are similar to E. coli in phenotypic characteristics, so that they are not easily distinguished. As a result, the term "coliform" was coined to describe this group of enteric bacteria. Coliforrn is not a taxonomic classification but rather a working definition used to describe a group of bacteria. In 1914, the US. Public Health Service adopted the enumeration of coliforrns as a more convenient standard of sanitary significance (Feng et al. , 2002). Although coliforrns were easy to detect, their association with fecal contamination was questionable because some coliforrns are found naturally in environmental samples (Caplenas and Kanarek, 1984). This led to the introduction of the fecal coliforrns as an indicator of contamination. Fecal coliform is a subset of total coliforrns that grows and ferments lactose at elevated incubation temperature, hence also referred to as thermotolerant coliforrns. Fecal coliform analyses are done at 45.5°C for food testing, except for water, shellfish, and shellfish harvest water analyses, which use 445°C (Neufeld, 1984; Clesceri et al., 1998). The fecal coliform group consists mostly of E. coli but some other enterics, such as Klebsiella, can also ferment lactose at these temperatures and therefore, be considered as fecal coliforrns. The inclusion of Klebsiella spp. in the working definition of fecal coliforrns diminished the correlation of this group with fecal 68 contamination. As a result, E. coli has reemerged as an indicator, partly facilitated by the introduction of newer methods that can rapidly identify E. coli. In recent years, strains of pathogenic E. coli have emerged as a serious food-bome pathogen, and have been associated with numerous outbreaks of disease (Uyttendaele et al., 1999; Scotter et al., 2000). Enterohemorrhagic E. coli (EHEC) are recognized as the primary cause of hemorrhagic colitis (HC) or bloody diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome. EHEC produce verotoxin or Shiga toxins (Stx). Only those serotypes of Stx-producing E. coli that have been clinically associated with HC are designated as EHEC. Of these, E. coli 0157:H7 is the most often implicated in illness worldwide (Nataro and Kaper, 1998). The infectious dose for E. coli 0157:H7 is estimated to be 10 - 100 cells; but no information is available for other EHEC serotypes. EHEC have been implicated in outbreaks resulting from the consumption of water (Swerdlow et al., 1992), undercooked ground beef (Davis et al., 1993), cold sandwiches (Karmali, 1989), unpasteurized apple juice (Centers for Disease Control and Prevention, 1996), and sprouts and vegetables (Como—Sabetti et al., 1997; Itoh et al., 1 998). Raw milk and dairy products, such as pasteurized milk and yoghurt contaminated With E. coli 0157:H7, have been the main cause of several outbreaks of milk-home disease since the 19803 (Daly et al., 2002). EHEC 0157:H7 exhibit slow or no fermentation of sorbitol and do not have glucuronidase activity, making them phenotypically distinct from other E. coli (Feng and Weagant, 2002). Enterotoxigenic E. coli (ETEC) is recognized as the causative agent of travelers' diarrhea and illness, characterized by watery diarrhea with little or no fever. Found commonly in under-developed countries, it has been implicated as the etiologic agent in 69 El 2.6 (”Pr coll Unge Slant to 11] 00n. outbreaks due to consumption of soft cheeses, Mexican-style foods, raw vegetables, and water. The infective dose of ETEC for adults has been estimated to be at least 108 cells; but the young, the elderly, and the immunocompromised may be susceptible to lower levels (Feng and Weagant, 2002). Enteroinvasive E. coli (EIEC) closely resemble Shigella, and cause an invasive, dysenteric form of diarrhea in humans. No known animal reservoirs, except for infected humans, exist for EIEC. Volunteer feeding studies showed that at least 106 EIEC organisms are required to cause illness in healthy adults. Unlike typical E. coli, EIEC are non-motile and do not ferment lactose. Enteropathogenic E. coli (EPEC) cause a profuse watery diarrhea] disease. It is a leading cause of infantile diarrhea in developing countries. EPEC outbreaks have been linked to the consumption of contaminated drinking water as well as some meat products. Through volunteer feeding studies, the infectious dose of EPEC in healthy adults has been estimated to be 106 organisms (Feng and Weagant, 2002). Because of their high infectious dose, analysis for ETEC, EIEC, and EPEC are usually not performed unless high levels of E. coli have been found in a food. 2.6.2 Traditional Detection Methods Almost all the methods used to detect E. coli, total coliforrns (TC) or fecal coliforrns (F C) are enumeration methods that are based on lactose fermentation. Detection of coliforrns is used as an indicator of unsanitary quality of water or as a general indicator of unsanitary conditions in the food-processing environment. Fecal coliforrns are the Standard indicator of choice for shellfish and shellfish harvest waters; and E. coli is used to indicate recent fecal contamination or unsanitary processing (Downes and Ito, 2001). Traditionally, the detection of coliforrns (fecal or total) and E. coli is accomplished using non-selective and selective enrichment of samples in broth and enumeration on solid agar 7O medium. The Most Probable Number (MPN) method is a statistical, multi-step assay consisting of presumptive, confirmed, and completed phases. The method consists of inoculating a series of tubes with appropriate decimal dilutions of the water sample. Production of gas, acid formation or abundant growth in the test tubes after 48 h of incubation at 35°C constitutes a positive presumptive reaction. All tubes with a positive presumptive reaction are subsequently subjected to a confirmation test. The formation of gas in a brilliant green lactose bile broth fermentation tube at any time within 48 h at 35°C constitutes a positive confirmation test. Typically, only the first 2 phases are performed in coliform and fecal coliform analysis, while an additional phase is performed for E. coli. The 3-tube MPN test is used for testing most foods (Food and Drug Administration, 2000). The 5-tube MPN is used for water, shellfish, and shellfish harvest water testing, and there is also a 10-tube MPN method that is used to test bottled water or samples that are not expected to be highly contaminated (Neufeld, 1984; Clesceri et al., 1998). The three phases of the MPN procedure take five days to complete. There is also a solid medium plating method for coliforrns that uses Violet Red Bile Agar, which contains neutral red pH indicator, so that lactose fermentation results in the formation of pink colonies. This procedure takes 2-3 days for confirmation. There are also membrane filtration (MF) tests for TC and PC that measure aldehyde formation due to fermentation of lactose (Feng et al., 2002). The MP technique is fully accepted and approved as a procedure for monitoring drinking water microbial quality in many countries. This method consists of filtering a water sample on a sterile filter with a OAS-um pore size that retains bacteria, incubating this filter on a selective medium, and enumerating typical colonies on the filter. Media and incubation conditions for the MF 71 method tested for optimal recovery of coliforrns include m-Endo-type media (incubation 24 h at 35°C for TC) in North America, and the Tergitol-TTC medium (incubation for 24 and 48 h at 37°C and 44°C for TC and FC, respectively) in Europe (Clesceri et al., 1998; Rompre et al., 2002). Other media, such as MacConkey agar and the Teepol medium, have also been used. A significant advantage of the MF technique over the MPN method is that with MP, the examination of larger volumes of water is feasible, which leads to greater sensitivity and reliability. MF also offers a quantitative enumeration comparatively to the semiquantitative information given by the MPN method. However, the MF method takes 48 h to complete. While some raw foods would be expected to have some coliforrns because of their association with the environment, the presence of E. coli in heat-processed foods means either process failure or post processing contamination from equipment, employees, or from contact with contaminated food and food products. Contamination of E. coli implies a risk that other enteric pathogens may be present in the food. Thus, a more rapid detection of TC, FC, and E. coli (compared to MPN and MP) is very important to the food industry for quality control purposes. 2.6.3 Rapid Detection Methods Methods for rapid detection and enumeration of microorganisms on membrane filters based on either ATP-bioluminescence (Millipore, Bedford, MA) or esterase activity (Chemunex, Maisons-Alfort, France) with complementary instrument platforms have been commercialized (Raynolds and Fricker, 1999; Upperman et al., 1999). These methods, however, do not provide identification of the bacterial species. 2. 6.3.1 Antibody-based methods Rapid immunological methods are based on the specific recognition between antibodies and antigens, and the high affinity that is characteristic of this recognition 72 reaction. Depending on the taxonomic level of the targeted antigens, immunological methods permit detection of antigens at family, genus, species, or serotype levels. Two types of antibodies can be produced: polyclonal and monoclonal antibodies, which are more specific to the target organism. The properties of the antigen—antibody complex can be used to perform an immunocapture of cells or antigens by enzyme-linked immunosorbent assay (IMS or ELISA), or to detect targeted cells by immunofluorescence assay (IFA) or immuno-enzyme assay (IEA). Obst et al. (1989) developed an ELISA using a monoclonal antibody against the enterobacterial common antigen (ECA), a lipopolysaccharide that is linked within the outer membrane of Enterobacteriaceae. This method was not sensitive enough, however, and so pre-enrichment of the sample in a selective broth for 24 h was necessary in order to reach the detection limit of the ELISA (106 cells/ml). Cross-reactivity with different strains of Aeromonas, which are important competitors to coliform bacteria growing on m-Endo media, as well as Psedumonas and Bacillus, was also observed (Hfibner et al., 1992). Rapid methods, using the ELISA and EIA techniques in conjunction with luminescence and arnperometry, have been developed specifically for E. coli 06 and E. coli 0157:H7 strains, respectively (Abdel- Hamid et al., 1999a; Schalch and Stolle, 2000; Trevanich et al., 2000b). The ELISA is a rapid, simple, and quite sensitive test, which allows the detection of less than 10'9 g of antigenic-protein (Stryer, 1988). However, assay limitations are often associated with the specificity of the antibody used, the concentration of both antibody and antigen, and the type of reaction solution used. In addition, the solid matrix used often leads to non- specific binding of the antigen or of the second antibody (Kfir and Genthe, 1993). ELISA application to the detection of specific cells from a naturally contaminated sample is 73 limited. High levels of non-targeted microflora and diverse materials associated with the sample may interfere on the level of the specificity of the ELISA method (Hanai et al., 1997) 2. 6.3.2 Molecular methods Molecular and biochemical technologies have been developed to address the issues of both rapid and simultaneous detection, identification, and enumeration of microorganisms following membrane filtration. The biochemical methods utilize activities of cellular enzymes to detect individual cells (Van Poucke and Nelis, 2000a) or microcolonies following a short grth step (Sarhan and Foster, 1991; Bauters et al., 1999; Van Poucke and Nelis, 2000b). DNA-based molecular methods target various gene fragments unique to the bacteria. Primers based on the lacZ gene have been used for the polymerase chain reaction (PCR)—based detection of coliforrns because conventional coliform monitoring methods are based on the expression product (B-galactosidase) of this gene (Bej et al., 1990; Bej et al., 1991a; Bej et al., 1991b; Fricker and Fricker, 1994). For the specific detection of E. coli, a region of the malB gene that codes for a maltose transport protein was the first target sequence proposed (Bej et al., 1990). However, due to cross reactivity with members of the Shigella and Salmonella genera, the uidA gene has been used more extensively for the PCR-based detection of E. coli (Bej et al. , 1991a; Bej et al., 1991b; Tsai et al., 1993; Fricker and Fricker, 1994; Juck et al., 1996; Iqbal et al., 1997; Rodriguez and Alocilja, 2005). However, many food types contain PCR inhibitors which co-purify with the target DNA (Gonzalez et al., 1999), thereby requiring extensive sample preparation to remove, dilute or inactivate inhibitors prior to PCR amplification (Fratamico et al., 2000). In many cases, the low level of E. coli in food 74 types requires a pre-enrichment step to overcome poor sensitivity, which increases the overall assay time by up to 24 h (Seo et al., 1998; Scotter et al., 2000). Direct fluorescence in situ hybridization (FISH) methods based on fluorescently labeled DNA probes (Muruyama and Sunamura, 2000; Tottorello and Reineke, 2000), and indirect FISH methods using biotinylated peptide nucleic acid (PNA) probes (Prescott and Fricker, 1999), have been described for real-time analysis. FISH uses oligonucleotide probes to detect complementary nucleic acid sequences. This method exploits the ability of nucleic acids to anneal to one another in a very specific complementary way to form hybrids. PNA probes are DNA mimics with improved hybridization characteristics (Egholm et al., 1993; Nielsen et al., 1994), which have been exploited in variety of different molecular techniques (Thisted et al., 1996; Just et al., 1998; Taneja et al., 2001) and used for identification of microorganisms (Stender et al., 1999; Stender et al., 2001a). The popularity of the FISH technique is due to its advantages over radioactive labeling, which include sensitivity, speed of visualization of single cells (by means of microscopy or cytometrical devices), stability of the hybridization products, safety, diminished detection time, multiple labels (multiple colors), and ease of use (Richardson et al., 1991; DeLong, 1993; Swinger and Tucker, 1996). However, the practical applicability of FISH has been limited to samples with a high level of contamination as the results are interpreted by manual epifluorescence microscopy allowing only a small part of a membrane filter to be viewed within a reasonable time (Stender et al., 2001b). 2. 6.3.3 Enzyme-based assays Luminescence-based enzymatic assays are finding increased use in the food industry (V idon et al., 1994; Giese, 1995; Trevanich et al., 2000a; Vidon et al., 2001) due to their 75 .151”, fi simplicity and application to a wide variety of foods. Based on the enzymatic properties of coliforms, a defined substrate method has been developed to overcome some limitations of the traditional MTF and MF techniques. Unlike these techniques, which eliminate the growth of non-coliform bacteria with inhibitory chemicals, the defined substrate technology is based on the principle that only the target microbes (TC and E. coli) are fed and no substrates are provided for other bacteria. A defined substrate is used as a vital nutrient source for the target microbe. During the process of substrate utilization, a chromogen or a fluorochrome is released from the defined substrate, indicating the presence of the target microorganisms. Recently, Lee and Denninger (Lee and Denninger, 1999) developed a technique utilizing Adenosine Tri Phosphate (ATP) luminescence. Although this technique is sensitive and shows correlation to culture techniques, the bacteria must be viable to be detected. Bacteria that have been starved or damaged may not react in this test. Another method based on the enzyme B-D-glucuronidase produced by 94-96% of E. coli, which hydrolyses 4-methylurnbelliferyl-B-D-glucuronide (MUG) to form a blue fluorescent product, has been developed (Venkateswaran et al., 1996; Manafi, 2000). Members of the Salmonella, Shigella, and some Yersinia spp. produce B-D-glucuronidase as well, while Staphylococcus spp. can hydrolyze MUG, thus making the method only useful as a preliminary test for detection of E. coli. Moreover, a mutation in the uidA gene in E. coli leads to false-negative identification when using B-D-glucuronidase as a method of detection, while certain strains of E. coli, such as 0157, fail to produce this enzyme (Yokoigawa et al., 1999). Thus, a more widely found enzyme for detecting E. coli is necessary. 76 B-galactosidase (beta-D-galactoside galactohydrolase EC. 3.2.1.23; lactase) is an enzyme produced by all strains of E. coli as a part of its regular metabolic process. The enzyme catalyzes the hydrolysis of lactose and many beta-D-galactopyranosides. The enzyme is encoded by the LacZ gene, which encodes a 116 kD polypeptide of 1023 amino acids in addition to the N-terminal f-met (Wallenfels and Weil, 1972). B- galactosidase catalyzes the following hydrolysis: B-D-galactoside + H2O —> galactose + alcohol (Adhya, 1996). B-galactosidase is widespread in microorganisms, animals, and plants. However, Tryland and Fiksdal (1998b) observed that the B-D-galactosidase-positive, noncoliform isolates exhibited a mean B-D-galactosidase activity of at least 2 log units less than for E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in a rapid assay for detection of coliform bacteria. In foods, E. coli is usually present in numbers higher than noncoliform organisms that produce B-galactosidase. Thus, the enzyme can be used as a marker to develop rapid methods for E. coli detection. Thermally stable dioxetanes can be enzymatically "triggered" to produce chemiluminescence using B—galactosidase (Schaap et al., 1991). The use of such technique has been reported (Van Poucke and Nelis, 1995, 1997). A useful chemiluminescent substrate for B-galactosidase is a phenyl galactose-substituted dioxetane (Schaap et al., 1991). Lumi-Gal 530 (Lumigen, Inc., Southfield, MI), a commercial formulation of 4-methoxy-4-(3-[3-D-galactosidephenyl)spiro[1,2-dioxetane- 3,2'-adamantane], reacts with B-galactosidase (a 4-step process) to generate chemiluminescence, which decays with a half-life of 10 min at 37°C (Beale et al., 1992). 77 Lumi-Gal 530 was shown to have a 20-fold greater sensitivity over other standard spectrophotometric assays when used with a pure B-galactosidase solution (Beale et al., 1992). However, this dioxetane substrate was not evaluated any further to develop a rapid chemiluminescence-based detection assay for E. coli. In this research, Lumi-Gal 530 will be used to develop a single tube chemiluminescence-based assay for detection E. coli, which will then be used as the basis for development of a NTSlicon—based biosensor for E. coli detection. 78 2.7 SALMONELLA 2.7.1 Introduction Salmonella is a rod-shaped, motile bacterium, except for S. gallinarum and S. pullorum, nonsporeforming and Gram-negative. Salmonella is ubiquitous in the natural environment, widespread in animals, especially in poultry and swine. Environmental sources of the organism include water, soil, insects, factory surfaces, kitchen surfaces, animal feces, raw meats, raw poultry, and raw seafoods (Center for Food Safety & Applied Nutrition, 1992). The genus Salmonella consists of resilient microorganisms that readily adapt to extreme environmental conditions. Some strains of Salmonella grow at temperatures as high as 54°C while also exhibiting ability to grow in refrigerated foods stored at 2 to 4°C. Salmonellae have the ability to proliferate at pH values ranging from 4.5 to 9.5, with a pH optimum for growth between 6.5 and 7.5. High salt concentration exerts a bacteriostatic effect by decreasing the water activity (aw) leading to bacterial plasmolysis commensurate with the hypertonicity of the suspending medium. Foods with water activity of 0.93 or less do not support the growth of salmonellae. However, bacterial salt tolerance has been shown to increase at temperatures between 10 to 30°C. The growth or survival of this pathogen under extreme pH, temperature, and salt concentration conditions raises concerns of safety in foods, such as fermented products (e.g., sausages, fermented raw milk products), modified-atmosphere-packaged and vacuum-packaged foods that contain high levels of salt (D'Aoust, 1997). Salmonella Typhimurium and S. Enteritidis are the most common Salmonella serotypes found in the United States. According to CDC, salmonellosis is the most common foodbome illness (CDC, 2002b). Over 40,000 actual cases are reported yearly in the US. (CDC, 2002a). Approximately 500 (Mead et al., 1999) to 1,000 (CDC, 2001) 79 persons die annually from Salmonella infections in the US. The estimated annual cost of human illness caused by Salmonella is $3 billion (Mead et al., 1999). Salmonella Enteritidis has frequently been observed as a contaminant in foods, such as fresh produce (De Roever, 1998), eggs and poultry products (Cohen et al., 1994). While various Salmonella species have been isolated from the outside of egg shells, presence of S. Enteritidis inside the egg, in the yolk, is of great concern as it suggests vertical transmission, i.e., deposition of the organism in the yolk by an infected layer hen prior to shell deposition (Center for Food Safety & Applied Nutrition, 1992). Human Salmonella infection can lead to enteric (typhoid) fever, enterocolitis, and systemic infections by non-typhoid microorganisms. Typhoid and paratyphoid strains are well adapted for invasion and survival within host tissues, causing enteric fever, a serious human disease. Non-typhoid Salmonella causes salmonellosis, which is manifested as gastroenteritis with diarrhea, fever, and abdominal cramps. Severe infection could lead to septicemia, urinary tract infection, and even death in at-risk populations (young, elderly, and immunocompromised individuals). Raw meats, poultry, eggs, milk and dairy products, fish, shrimp, frog legs, yeast, coconut, sauces and salad dressing, cake mixes, cream-filled desserts and toppings, dried gelatin, peanut butter, cocoa, and chocolate are some of the foods associated with Salmonella infection (D'Aoust, 1997). All known strains (about 2400) of Salmonella are pathogenic with a very low infectious dose observed in some of the foodbome outbreaks traced back to Salmonella contamination. Newborns, infants, the elderly, and immunocompromised individuals are more susceptible to Salmonella infections than healthy adults (D'Aoust, 1997). The developing immune system in newborns and infants, the frequently weak and/or delayed 80 immunological responses in the elderly and debilitated persons, and low gastric acid production in infants and seniors facilitate the intestinal proliferation and systemic infection of salmonellae in this susceptible population (Blaser and Newman, 1982). Moreover, in recent years, concerns have been raised because many strains of Salmonella have become resistant to several of the antibiotics traditionally used to treat it, in both animals and humans, making Salmonella infections an important health concern in both developed and undeveloped countries. The majority of the increased incidence of resistance can be attributed to Salmonella Typhimurium DT104. Treatment of this infectious disease is complicated by its ability to acquire resistance to multiple antibiotics (Carlson et al., 1999). Evidence suggesting that ingestion of only a few Salmonella cells can develop a variety of clinical conditions (including death) is a reminder for food producers, processors, and distributors that low levels of salmonellae in a finished food product can lead to serious public health consequences, and undermine the reputation and economic viability of the incriminated food manufacturer. Thus, early and rapid detection of Salmonella is very important to the food industry so that appropriate measures can be taken to eliminate infection through food. 2.7.2 Traditional Detection Methods The method currently in use for Salmonella identification is a culture of the bacteria from the food samples, a time—consuming and laborious process. The conventional procedure of pre-enrichment broth, selective enrichment broth, and differential agar requires 3-5 days to achieve a presumptive identification of Salmonella bacteria. The biochemical identification of foodbome and clinical Salmonella species is generally coupled to serological confirmation, a complex and labor-intensive technique involving the agglutination of bacterial surface antigens with Salmonella-specific 81 antibodies. These include O lipopolysaccharides (LPS) on the external surface of the bacterial outer membrane, H antigens associated with peritrichous flagella, and the capsular (Vi) antigen. The diagnostic value of biochemical traits is generally combined with serological characterization, resistance to antibiotics, plasmid profiling, and phage— typing assays. The whole process for confirmation of Salmonella serovars can take as long as 7-10 days (Food and Drug Administration, 2000) and is likely to be replaced by molecular techniques. 2.7.3 Rapid Identification of Sglmonella_: Several techniques for improving the detection of Salmonella serovars in food as well as feces, such as the use of a selective culture medium, enzyme-linked immunosorbent assay (ELISA), and DNA-based techniques, have been developed (Araj and Chugh, 1987; Aspinall et al., 1992; Chiu and Du, 1996; Amavisit et al., 2001). Valdivieso-Garcia et al. (2003) used a combination of the above enrichment in selective culture medium and ELISA to develop a bioluminescent enzyme immunoassay (BEIA). The study used streptavidin-biotinylated firefly luciferase complex as a reporter. Salmonella cultures were grown for 24 h. After enrichment, the total test time for the BEIA was 1.5 h. The results obtained in the study indicated a sensitivity of 105-106 CFU/ml. The VITEK immunodiagnostic assay system (VIDAS) is an automated, qualitative, enzyme-linked fluorescent immunoassay method. Comparative studies with a variety of food types have found favorable correlation between conventional test methods and the VIDAS system (Blackburn et al., 1994; Curiale et al., 1997; De Medici et al., 1998; Walker et al., 2001). For Salmonella detection, the specific VIDAS reagent strip relies on immunocapture with specific antibodies raised against the O and H antigens of 82 the organism. A relative fluorescence value is determined for samples based on the measured fluorescence relative to background. This value is used for interpretation of results. Use of such a system has a number of advantages over conventional culture methods, including time to obtain a negative or presumptive positive result and technical ease of performing the test. Problems remain with sample processing, sensitivity, and specificity that have limited the routine use of the above antibody-based procedures. Polymerase chain reaction (PCR) technology, which allows amplification of a specific fragment of nucleic acid, has been used to identify the presence of specific pathogens directly from clinical as well as food specimens. PCR is an extremely sensitive test, able to amplify picograrn quantities of DNA. Although the PCR assay is sensitive, it may be susceptible to false negative results when applied to food and clinical samples. This may be due to a variety of inhibitory substances such as chelating agents and food components (Widjojoatrnodjo et al., 1992). To overcome the problem of false negative reactions, inclusion of an internal positive control (IPC) (Chadwick et al., 1998), and performing PCR afier preincubation in an enrichment broth has been investigated for human (Chiu and Du, 1996; Luk et al., 1997; Marsiglia et al., 1997; Lin and Tsen, 1999), animal (Stone et al., 1994; Stone et al., 1995; Cohen et al., 1996), fecal (Widjojoatrnodjo et al., 1992), and food samples (Chen et al., 1997; Bennett et al., 1998; Rijpens et al., 1999). This has been found as a useful and a more rapid method because the enrichment step increases the number of viable Salmonella in the sample, and therefore, the sensitivity of the assay (Gouws et al., 1998). PCR methods and DNA extraction procedures for Salmonella have been described for human, animal, and food samples 83 (Soumet et al., 1994). However, these extraction methods using lysozyme, proteinase K, and phenol-chloroform-isoamyl alcohol are expensive and time consuming, whilst methods using silica beads or glass particles to bind with DNA are more rapid but expensive (Boom et al. , 1990; Mahon and Lax, 1993; Tuchili et al., 1996). Recent advancements in PCR technology combined capillary therrnocycling and real-time fluorescence detection, and/or quantification of the PCR product in different formats such as SYBR Green I dye binding (Rantakokko-Jalava and J alava, 2001; Szuhai et al., 2001; De-Medici et al., 2003; Jothikurnar et al., 2003), and hybridization probe (Wittwer et al., 1997; Bellin et al., 2001; Eyigor and Carli, 2003). Carli et al. (2001) detected salmonellae in chicken feces by a tetrathionate broth (TTB) enrichment, capillary PCR, and capillary gel electrophoresis, and obtained positive results with high sensitivity. However, this process required separate units for capillary PCR and gel electrophoresis runs, and it was not real time. Eyigor et al. (2002) applied the real-time PCR technology with the SYBR Green I Dye format to TTB enrichment culture of a standard Salmonella isolation method from poultry. Salmonella was detected in 25 min from up to 32 18-h primary enrichment sample cultures from subclinically infected chickens. This PCR was about 10 times faster than the previously reported conventional PCR detection methods (Aabo et al., 1995; Cohen et al., 1996) and yielded more rapid results. The Light Cycler Real time PCR system’s (Roche Diagnostics, Mannheim, Germany) hybridization probe design allows sequence-specific detection of the amplicon with the use of fluorescence resonance energy transfer between the two fluorophores used in the probes. Also, this system reduces the detection time and enables the process of up to 32 samples simultaneously. 84 However, the need for expensive equipment (that are usually not portable) and expertise required in performing PCR makes these DNA-based detection methods cost prohibitive and limits their application in the field. Biosensors can provide a viable solution to the need for cheap, portable, sensitive, and specific rapid detection methods for pathogens. Specific and selective detection of Salmonella Typhymurimn based on the use of a polyclonal antibody immobilized by the Langmuir—Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated in liquid samples (Olsen et al., 2003). These biosensors were selective to S. Typhymurium at levels above 104-106 CF U/ml in the presence of large concentrations of Escherichia coli 0157:H7. A time-resolved fluoroirnmunoassay was developed for the simultaneous detection of E. coli 0157:H7, S. Typhimurium and S. Enteritidis in foods. Immunomagnetic beads were used to capture and concentrate the target pathogens. The captured bacteria were allowed to form sandwiched complexes with europium-labeled anti-E. coli 0157 antibodies and/or samarium-labeled anti-Salmonella antibodies. The specific time-resolved fluorescence associated with highly fluorescent europium or samarium micellar complexes were measured to estimate the quantity of E. coli and Salmonella, respectively, with resulting sensitivity of 103-104 cfu/ml for pure cultures of the target pathogens (Shu et al. , 2002). A Threshold Immunoassay System, using a light-addressable potentiometric sensor, was used to detect S. Typhimurium to levels as low as 119 CFUs (10-20 ul sample volume). This biosensor utilized solution-based binding of the biotin and fluorescein labeled antibodies to Salmonella, followed by filtration-capture of the 85 immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate was bound to the immunocomplex. Detection of the bound immunocomplex was made possible via a silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converted the substrate to ammonia and CO2, resulting in a pH change at the silicon surfacethat was monitored with time, and reported in mV/s. Total assay time was about 2 h. Sample debris was found to impede the sample flow through the membrane and hence, the signal output (Dill et al., 1999a). Salmonella Enteritidis and Listeria monocytogenes were detected in real time using a surface plasmon resonance (SPR) biosensor. The respective antibodies against the target pathogens were immobilized on the gold sensor surface as a covalently crosslinked double-layer or covalently bound on a crosslinked albumin layer. The target-binding event was detected through prism excitation of surface plasmons and spectral interrogation. Salmonella and Listeria could only be detected by the sensor at concentrations of 106 CF U/ml (Koubova et al. , 2001a). A rapid and sensitive primary screening method with a proper sampling plan is required to detect Salmonella in the food industry. While several biosensors are being developed for detection of Salmonella, very few biosensors are commercially available. With the recent advances in the silicon-based IC industry, biosensors using a silicon-based platform will be more amenable to commercialization using existing microfabrication processes. The current research will focus on developing a silicon-based biosensor with antibody and DNA probes as the biological recognition elements for Salmonella enterica. 86 CHAPTER 3: RESEARCH HIGHLIGHTS 3.1 INNOVATION AND NOVELTY The innovation of this research was the use of nanotechnology (nanO-tubular Si) combined with immuno-chemistry and molecular biology in the fabrication of a field- deployable nano-structured biosensor for food safety and quality assurance, health care, and bio-defense purposes. The research combined the use of chemiluminescence and electrochemical transduction mechanisms in a versatile NTS platform to build biosensors for detection of foodbome pathogens that were simple, easy to use (with no need for light source and filters), sensitive, and specific to the target. Three biological sensing elements (enzymes, antibodies, and nucleic acids) were used to functionalize the biosensors. The output light from the chemiluminescence transducer, measured using a photomultiplier tube (luminometer), was correlated with the number (cells) of the target being detected, thus indicating the presence or absence of the target, and in some cases, the number of target cells in the sample. Cyclic voltammetry was used to detect target DNA hybridization on the biosensor chip using intrinsic changes in DNA signals, a property not yet investigated in the literature for foodbome pathogen detection. In the long-term, a portable handheld nucleic acid-based biosensor could be built in collaboration with commercial companies. The biosensor will have a chip with a removable cartridge and on-board circuitry for data acquisition. The cartridge will consist of different chambers for reagent storage, performing PCR (if that is found to be necessary step before detection), and the chemiluminescence pathogen detection system. The detection system will consist of multiple detection “spots” immobilized with probes specific to different targets of interest. Each of the spots will be individually addressable 87 electronically to measure the chemiluminescence and electrochemical transducer principles that will be employed, thus eliminating the need for bulky and expensive equipment like scanners (which are needed in systems like DNA micro-arrays). The light output will be measured using a photodiode or an active pixel array sensor. Electrical contacts (aluminum) patterned along the edges of the patterned NTS platform will be used for electrical measurements (cyclic voltammetry). Using appropriate data acquisition, a computer interface will be provided using either a serial or USB port communication. 3.2 POTENTIAL DIFFICULTIES Going into the research, some difficulties were anticipated. Consistency in fabrication of NTS with uniform size and thickness characteristics could be difficult when using the anodization etching technique. This would introduce a large variation in the measured output of the biosensor from repeated experiments. Direct binding (immobilization) of enzymes, antibodies, and DNA probe to the NTS surfaces could provide possible challenges; therefore, two procedures were investigated. Other silanizing agents (e.g., 3-aminopropyltriethoxy silane) and cross-linking (e.g., glutaraldehyde, avidin-biotin) could be used if 3-glycidoxypropyltrimethoxy silane and diffusion/physical adsorption proved to be ineffective for binding the biological sensing elements to the silicon surface. 3.3 EXPECTED SIGNIFICANCE The expected outputs were the construction and validation of a "proof-of-concept" prototype NTS biosensor for rapid detection of E. coli and Salmonella that combined the current techniques in nanotechnology and biotechnology. This research would ultimately provide a highly self-contained, nucleic acid-based, specific, and sensitive, handheld 88 nanoscale-biosensor that has multiple, target-specific, electronically addressable, spots for on-site diagnosis of various pathogens (e.g., bedside diagnosis and point-of-care by health professionals). The prototype biosensor would be an added tool to protect the public from foodbome illness caused by contaminated food and water supply, reduce the health risk of microbial contamination, strengthen biosecurity measures, and enable quality assurance in food industry. The biosensor performance criteria included: (1) high sensitivity; (2) high specificity; (3) field-ready testing capacity; (4) simplicity ; and (5) short time requirements. The biosensor may be used for epidemiology, i.e., quick and effective diagnosis of disease outbreaks caused by pathogens either intentionally (bioterrorism) or inadvertently (lack of sanitation) added to air, food, and water. The biosensor could be easily adapted to several pathogens of concern, such as Escherichia coli 0157:H7, Listeria, and B. anthracis by changing the specificity of the nucleic acid primers and probes used. The current research developed the NTS biosensor platform, a versatile platform that was used for detection of three different targets with a high level of sensitivity (for example, pg level for DNA-based detection) and specificity (no cross reaction with related and non-related species), as well as detection time of 60 min or less. Two different kinds of transduction mechanisms and three biological sensing elements were employed successfully to develop the biosensors. 3.4 HYPOTHESIS The research was based on the following hypothesis: 0 A versatile nano-tubular platform can be fabricated using silicon and functionalized into a biosensor to detect foodbome pathogens using different biological sensing elements, such as enzymes, antibodies, and nucleic acids. 89 o The NTS platform can be used with multiple transducing mechanisms to report the biological detection event. 0 The biological detection event on the NTS biosensor will trigger a response that is unique from that of a ‘no detection’ event, and which can be processed and converted into an appropriate electronic signal output. 0 The NTS platform will provide greater sensitivity or lower limit of detection as compared to any planar platform of the same dimension (length and width). 3.5 SPECIFIC RESEARCH OBJECTIVES Objective 1: Development of a chemiluminescence-based detection assay external to the biosensor system. Objective 2: Design, fabrication and characterization of the NTS platform. Objective’3: F unctionalization of the NTS platform into a biosensor chip. Objective 4: Determination of sensitivity of the NTS biosensor chip in pure culture. Objective 5: Determination of specificity of the NTS biosensor chip using non-specific and mixed bacterial cultures. 90 CHAPTER 4: RESEARCH MATERIALS AND METHODS 4.1 OBJECTIVE 1 Development of a chemiluminescence-based detection assay external to the biosensor system to detect E. coli using B-galactosidase as the biological sensing element. This objective was aimed at developing and optimizing a chemiluminescence-based assay protocol, external to the biosensor system, prior to developing the biosensor in Objective 2. Development of this assay protocol would then limit confounding variables during biosensor development to those related to the biosensor fabrication and fimctionalization. 4.1.1 Instrumentation The chemiluminescence assay works on the basis of photon emission resulting from a 4-step reaction between the B-galactosidase enzyme produced by E. coli and the dioxetane in the Lumi-Gal® formulation. Total light emitted from the reaction is measured in a commercial luminometer (F emtomaster FB 12 Luminometer by Zylux Corp., Maryville, TN) equipped with a single photon-counter having a special measuring geometry that enables very high sensitivity (Figure 4-1). The sample is placed in a pullout sample holder that slides over a photo-multiplier tube (PMT). The sample holder focuses the light down toward the PMT. The PMT operates in the digital pulse mode wherein it acts as a photon counter. The light signal is processed and the photon counts are integrated over a period of 10 s. The digital readout is registered on the instrument; it can also be stored in a computer by using appropriate data acquisition software/hardware provided by the luminometer supplier. 91 Optional position Removable sample Bridge for for Injector Holder/Reflector light-tight shown with sample tube closure of measurement chamber (4’3 \ PMT Sample drawer Figure 4-1. Schematic of the F emtomaster FB 12 Luminometer 4.1.2 Culture Preparation Generic Escherichia coli (strain K-12) and E. coli 0157:H7 (strain AD317) were obtained from the Department of Food Science and Human Nutrition, Michigan State University. The cultures were maintained at -70°C in trypticase soy broth (Becton Dickinson and Co., Cockeysville, MD) containing 10% (v/v) glycerol (J. T. Baker, Phillipsburg, NJ) and subjected to two consecutive overnight transfers (18-24 h/35°C) in 9 ml of nutrient broth (NB) (Difco, Becton Dickinson and Co., Sparks, MD) or lactose broth (LB) (Difco). 4.1.3 Single Tube Chemiluminescence Assay Procedure Polymixin B sulfate (50mg/ml) solution (Sigma Chemical Co., St. Louis, MO) was added (as the E. coli cell wall permeabilizer) at a rate of 5 til/ml culture to 9 ml of the 92 above E. coli culture or uninoculated NB (used as control) prior to incubation at 37°C for 15 min. From this mixture, 25 ul was then pipetted into a 10x75mm culture tube. After the addition of 500 at Lumi-Gal® 53o (Lumigen, Inc., Southfield, M1), the tube was vortexed. The emitted light intensity was measured in relative light units per second (RLU/s) using the luminometer at 10 min intervals up to 60 min, and again at 75 and 90 min. Also, the number of E. coli was determined by spread plating the appropriately diluted sample on MacConkey agar (Difco). Bacterial counts were enumerated after 48 h of incubation at 35°C. 4.1.4 Determining Best Growth Media Escherichia coli cultures were grown overnight in LB as well as NB and assay procedure repeated as above in both cases. A comparison of the results between NB and LB was used to determine which broth was better for the assay in terms of higher light output. A significantly higher light output from a sample containing low number of target cells over the control sample would improve biosensor sensitivity. Also, a comparison of light emitted after 24 h and 48 h of incubation of E. coli cultures was made to investigate the effect of incubation period on light intensity. 4.1.5 Determining Assay Applications The above assay was also performed with E. coli 0157:H7 cultures to determine possible differences in light emission when compared to the generic E. coli strain resulting from different levels of B-galactosidase enzyme production and/or activity. Such differences could be used as a discriminatory test between E. coli 0157:H7 and generic E. coli. Also, overnight cultures of E. coli 0157:H7 were heat treated prior to 93 testing with the chemiluminescence assay to determine if whether the assay could be employed for sterility testing with heat-processed foods, such as pasteurized milk. 4.1.6 Determining Amount of Sample to Use in the Assay Following standardization of the assay procedure, a Randomized Complete Block Design (RBD) analysis, with 5 blocks of size 21 comprising a two-way treatment structure with 7 dilution and 3 E. coli culture levels, was used to assess the sensitivity of the assay. The randomized trials were performed for 5 consecutive days [determined by Power analysis of some preliminary data, performed using Statistical Analytical System (SAS© Version 8.0, SAS Institute, Inc., Cary, NC)] to nullify the effect of minor day-to- day variation in bacterial counts. Escherichia coli culture was serially diluted 5 times using 9 ml of 0.1% peptone dilution blanks, followed by the addition of 45 pl of polymixin B sulfate. From each dilution, 25 pl, 50 pl, and 55 p1 were pipetted separately into 10x75mm culture tubes, and the assay procedure described above was followed thereafter. Peptone blank was used as the control in each case. 4.1.7 Assay Validation in Food Analysis The chemiluminescence assay, developed and optimized using E. coli cultures in broth, was employed to detect E. coli in artificially contaminated food samples (pasteurized milk, sprouts, and water) so as to simulate a potential real-life usage scenario. Nine ml of water and pasteurized milk samples inoculated with different E. coli concentrations using overnight LB cultures were tested without any enrichment using the assay procedure. One gram of alfalfa sprouts inoculated with E. coli using the procedure of Beuchat et al. (2001) was added to 9 ml of sterile 0.1% peptone dilution blank. The mixture was hand mixed for 1 min. Five ml of liquid was removed and used for the assay 94 procedure as described above. All samples were tested in triplicate. Results of light emission from the samples were compared to coliform counts obtained from the MacConkey Agar plates for all samples (as described in the assay procedure). For assay validation, blind sample testing was performed using surface water samples collected at seven sites along the Red Cedar River (East Lansing, MI). The water samples were contained in sterile 100 ml sampling bottles and transported to the lab facility. Nine ml was removed from the samples (without any enrichment) and analyzed using the assay procedure. One ml of the water sample was also transferred into each of a series of tubes containing 9 ml of LB and incubated at 35°C for a period from 1 to 6 h (hourly intervals) with an additional tube incubated for 12 h. The assay was repeated following each enrichment period for all samples. 4.1.8 Statistical Anaksis Analysis of Variance (ANOVA) was performed on the data using the SAS software. Arithmetic means were compared using the Duncan grouping test at 95% confidence level (a=0.05). Interactive effects in RBD were analyzed using the Autoregressive Mixed Model (Proc Mixed) with Tukey adjustment for comparison of means (a=0.05). 4.2 OBJECTIVEZ Design, fabrication and characterization of a nano-tubular silicon platform. The prototype biosensor (in this research) was designed keeping in mind the long- term goal of building a self-contained nucleic acid-based individually addressable portable biosensor. In the current research, the process of PCR for DNA amplification was performed externally. The amplified product was then introduced to the prototype biosensor shown (Figure 4-2). The biosensor was fabricated on a p++-type silicon 4” 95 wafer with final chip dimensions of 1.5 cm x 1.5 cm. The main fabrication steps are described below. 4.2.1 Nano-Tubular Silicon Fabrication NTS was fabricated using the anodization process in a conventional single-tank cell (Figure 4-3). The O-ring was placed on the bottom hole of the inverted Teflon cell. The silicon wafer was then placed over the O-ring. Before the copper plate was placed over the silicon, the back surface of the silicon was scratched with a diamond-tipped scribe followed by application of graphite coating using a graphite pen to make the silicon more conductive. The copper metal contact was then made to the backside of the wafer and sealed. A platinum grid or wire placed inside the Teflon cell served as the cathode, and the copper plate served as the anode. The platinum grid (or wire) and the copper plate were connected to the negative and positive terminal of the power supply, respectively. After placing the Teflon cell upright, 15% hydrofluoric acid (in ethanol) was poured into the cell. Silicon was then electrochemically etched in the 15% hydrofluoric acid (HF) solution using different current and time conditions as follows: a) 100 mA — 30 s, b) 50 mA—lmin,c)30mA—1min,d)lSmA—lmin,e)5mA—20min,f)5mA—lh,and g) 2 mA — 1 h. These current-time conditions were chosen by modifying those used for porous silicon fabrication (Halimaoui, 1997) so as to obtain the desired NTS platform. 4.2.2 Characterization of Nana-Tubular Silicon SEM images of all NTS chips were taken using a Hitachi S-4700 11 Field Emission Scanning Electron Microscope to evaluate the quality of nanO-porous silicon chips obtained (detailed procedure provided in Appendix A:Section A.2). hnages were 96 £8583 £9.38.— ELoELoam can Scorn-S— 35E25 3327. 9:255» Lemuomfim .N... 2:»:— ch Lomcmmoa o_nmtOn_ Eosvzm—n— I semomom __ ommcm Ehoutmcom P532? :oEmE—coa Sun \ camowom x e _ Ommcm e a E9220 ‘ :ouxolatofo: o E333.“— mhz .0 .w 3.30:0:3m 97 Platinum grid (cathode) | +——— Teflon Cell / . «ta—— sr Wafer O-Rlng +—— Copper Plate (Back Contact) Figure 4-3. Single-tank cell set up used for tubular silicon fabrication. used to observe the size and shape of the tubes, inter-tube space, surface texture, and thickness of the nano-tubular layer obtained under different anodization conditions described above. The porosity and thickness of the porous silicon layers were also determined. The porosity was determined by weight measurements. The virgin wafer chip was first weighed before anodization (m1), then just after anodization etching (m2) and finally after complete dissolution of the NTS layer in NaOH solution (m3). The final dissolution process was carried out in the Teflon cell used for anodization. The chip, after weighing (m2), was carefully placed under the cell (as described above in Section 4.2.1) and exposed to an aqueous solution of 2.5 M NaOH for a period ranging between 30-90 min 98 (Vézsonyi et al., 2003) until the shiny clear surface of planar silicon was visible again. The chip was then weighed again (m3). The porosity is given by the following equation: Mxloo FC,/o) = (ml - m3) From the above measurements, the thickness of the porous silicon layer was also (ml —m3) X determined as follows: W = ; where, d = Bulk silicon density, (2.33 g/cm3), and S = Area of the wafer exposed to HF during anodization (circular area of 1.1 cm diameter = 0.95 cmz). 4.3 OBJECTIVE3 Functionalization of the nano-tubular silicon platform into a biosensor chip. 4.3.1 Adaptation of CL Assay to the Biosensor Detection System - Procedure A 4.3.1.1 F unctionalization of harm-tubular silicon with Lumi-Gal® 530 After fabrication of NTS, the platform was cleaned by rinsing with isopropyl alcohol and then dry toluene, followed by drying under a steady stream of nitrogen for 30 5. To functionalize the NTS platform, 50 pl of Lumi-Gal® 530 was carefully added to the NTS surface, which was then placed overnight in the refrigerator (4°C) to allow binding of Lumi-Gal® 530 by diffusion and adsorption. The biosensor chip was then dried by placing it in a vacuum oven (Isotemp vacuum oven 280A, Fisher Scientific, Pittsburg, PA) at 37°C for 1-2 h (25 in Hg). The biosensor chip was then ready to be used for detection of E. coli using B-galactosidase enzyme as the marker. Planar Si chips were 99 also functionalized using the above process and used to compare and evaluate the effectiveness of the NTS biosensor chip as a biosensor platform. 4.3.1.2 Detection of E. coli on biosensor chip using CL detection Polymixin B sulfate (50 mg/ml) solution (Sigma Chemical Co., St. Louis, MO) was added (as the E. coli cell wall permeabilizer) at a rate of 5 pl/ml culture to 9 ml of the overnight E. coli culture (Section 4.1.2 above) or uninoculated LB (used as control) prior to incubation at 37°C for 15 min. From this mixture, 5 pl was then added carefully to the biosensor chip. The biosensor was inverted before placing in the luminometer because the photomultiplier tube detects the light from below the sample drawer. The emitted light intensity was measured in relative light units per second (RLU/s) using the luminometer at 10 min intervals up to 60 min. Light emission of the functionalized NTS biosensor chip was also compared with that of a functionalized planar Si chip to evaluate the performance of the non-planar biosensor platform being developed. The light emission data collected at the third 10-min interval, established as ideal based on prior results (Mathew and Alocilja, 2002), were used for the comparisons. The trials were performed in triplicate. 4.3.2 Adaptation of CL Assay to the Biosensor Detection System - Procedure B 4. 3. 2.1 Single-tube CL assay modification In order to adapt the single-tube assay external to the biosensor (Section 4.1.1-4.1.6) (Mathew and Alocilja, 2004) to the NTS biosensor, the CL assay steps were modified as follows: polymyxin B sulfate was pre-mixed with dioxetane by adding 5 pl of polymyxin B sulfate (50 mg/ml) solution per ml of the dioxetane substrate. From this polymyxin B- dioxetane mixture, 500 pl was pipetted into a 10x75 mm culture tube followed by 100 addition of 55 pl of the E. coli culture (Section 4.1.2); the tube was then vortexed. The errritted light intensity was measured using the luminometer at 10 min intervals up to 60 min and compared with that of the single-tube assay procedure described above. These modifications were aimed at minimizing the assay steps and the assay time required to obtain an output from the biosensor. 4. 3. 2.2 F unctionalization of none-tubular Si After fabrication of the NTS chip, it was cleaned by rinsing with isopropyl alcohol and then dry toluene, followed by drying under a steady stream of nitrogen. To functionalize the NTS platform, 50 pl of the polymyxin B-dioxetane mixture (as described above) was carefully added to the porous silicon surface and placed overnight in the refrigerator (4°C) to allow binding of the mixture on the surface through diffusion and adsorption. The biosensor chip was then partially dried by placing it in a vacuum oven (Isotemp vacuum oven 280A, Fisher Scientific, Pittsburg, PA) at 37°C for 1-2 h (25 in Hg). At this stage, the biosensor chip was ready for use. Planar silicon chips were also functionalized using the above process and used to compare and evaluate the effectiveness of the porous silicon biosensor chip as a biosensor platform. 4.3.2.3 Biosensor testing Five rnicroliters of the overnight E. coli culture or uninoculated LB (used as control) was added carefully to the functionalized biosensor chip. The chip was placed in the lmninometer, and the emitted light was measured in RLU/s at 10 min intervals up to 60 min. Light emission of the functionalized NTS biosensor chip was also compared with that of a functionalized planar silicon chip to evaluate the performance of the NTS 101 biosensor platform being developed. The light emission data collected at the third 10-min interval, established as ideal based on prior results (Mathew and Alocilja, 2004), were used for comparison. The trials were performed in triplicate. 4. 3. 2.4 Determining the biosensor sensitivity Following standardization of the assay procedure for the biosensor, a completely randomized design (CRD) was used with 5 levels of dilution (including blank/control) to assess the sensitivity of the assay. The randomized trials were performed in triplicate to nullify the effect of minor day-to-day variation in bacterial counts. Escherichia coli culture was serially diluted 5 times using 9 ml of 0.1% peptone dilution blanks. From each dilution (101-104 cells), 5 pl were pipetted separately on to functionalized biosensor chips, and the modified assay procedure described above was followed thereafter. Peptone blank was used as the control. Statistical Analysis F-test was used to test equality of variances. Two-sample t-test was used to test the mean differences between the light emission observed using the standard and modified chemiluminescence assay procedures. Single-factor Analysis of Variance (ANOVA) was performed on the light emission data for the sensitivity analysis. Arithmetic means were compared using Duncan grouping at 95% confidence level (or=0.05). The performance of the biosensor using Procedures A and B were compared to determine possible significant differences in sensitivity. All statistical analyses were carried out using SAS® software (SAS, Gary, NC). 102 4.3.3 Functionalization of the Nano-Tubular Silicon Platform with Salmonella Antibodies into an Immunosensor 4. 3.3.1 Silanization of nano-tubular silicon platform The NTS surface was functionalized by Silanization and antibody immobilization based on a procedure described previously by Bhatia et al. (1989) (Figure 4-4). NTS was first cleaned by soaking in a 50/50 solution of methanol and hydrochloric acid. After rinsing with deionized water several times, NTS was treated with concentrated sulfuric acid for 30 min to hydrate the silicon surface. The chips were rinsed again with deionized water and then air dried. Following cleaning, NTS was silanized with a 2% solution of 3- glycidoxypropyltrimethoxysilane (GOPS) (Aldrich Chemicals, Allentown, PA) in dry toluene for2 h. Silanization was done in a glove box under inert atmosphere. The NTS substrate was then rinsed in dry toluene and allowed to air dry. O CH20H _ HOH c éi—ECH}O CH HC CH fl 2 — 2 _’ 2‘ 2 CH O . / \ CHZOH I ' Sir-0H , Si- -O—Si‘-[CH2}O—CH2—HC CH2 Silanization with GOPS (LHZOH 3 u—d L...- . ““2 SI or NTS Platform I / + —‘ Capture Antibody cleQOH OH Sit—o—si—Eszjl—o—CHz—clm——CH2——NH I CHZOH 3 I / e Antrbody 7 \ Immobilization ...._l Functionalized Si or NTS Platform Figure 4-4. Functionalization of planar Si and nano-tubular Si platforms into antibody-based biosensors. 103 4.3.3.2 Immobilization of primary antibody Following Silanization, 0.5 ml of a 0.15-mg/rnl solution of anti-Salmonella antibody, targeting the common structural antigen of Salmonella (KPL, Gaithersburg, MD), was carefirlly pipetted on to the active NTS area and then allowed to attach to the silanized NTS for 1 h. NTS was rinsed with phosphate buffered saline (PBS, pH 7.4) and stored at 4°C until used. In order to study the influence of Silanization on the functionalization of the biosensor, half of the NTS chips were subjected to all the above steps except for the 2 h Silanization. 4. 3. 3.3 Optical measurement method A CL-based Optical method was used to detect the presence of Salmonella on the biosensor chip surface. A half milliliter of overnight Salmonella Typhimurium DT104 (from Michigan State University collection) culture grown in lactose broth was added to the nano-tubular- and planar-silanized chips as well as the non-silanized NTS biosensor chips. A half milliliter of sterile buffer was added to the control chips. After five minutes, each biosensor chip was rinsed with 1 ml of 0.1% peptone dilution blank. Half milliliter of blocking solution (2% albumin in PBS) was then added. After 10 minutes, the blocking solution was drained. A 1:20000 solution of the secondary anti-Salmonella antibody conjugated with horseradish peroxidase (HRP) (KPL) was then added to cover the active porous silicon area (approximately 0.5 ml). After 15 minutes, the biosensor was rinsed 3-4 times with 1 ml each of PBS containing 0.1 % Tween-20 solution to remove unattached HRP-conjugate. One ml of Luminol® (Pierce Biotechnology, Rockford, IL) was pipetted into a 35 mm petri dish and then the biosensor was inverted into the petri dish. Chemiluminescence from the reaction between Luminol® and HRP was measured in the luminometer at 1 min intervals for 10 min. Bacterial numbers were 104 obtained by plating appropriately diluted samples on trypticase soy agar (Difco, Becton Dickinson and Co., Sparks, MD) containing 0.6% yeast extract (Difco). The trials were performed in triplicate. 4.3.4 Functionalization of the Nano-Tubular Silicon Platform with Salmonella DNA Propes into a Nucleic Acid-Based Biosensor 4.3.4.1 Bacterial strains A clinical strain of Salmonella Enteritidis (strain S-64) was obtained fi'om the Michigan Department of Community Health (Lansing, MI) and stored at —70°C. The pathogen was grown on trypticase soy agar containing 0.6% yeast extract (TSAYE) and/or broth (TSBYE) at 37°C, as appropriate. In broth culture, cells were grown to exponential phase, and enumerated by spiral plating appropriately diluted cultures on Bismuth Sulfite Agar and Brilliant Green Agar. The cultures were serially diluted for DNA extraction so that the number of bacterial cells ranged from 100 to 108 CFU/ml. 4. 3. 4.2 DNA primers and probes Primers used for PCR were designed for the detection of Salmonella Enteritidis from the insertion element (Iel) gene (Wang and Yeh, 2002). The single stranded forward and reverse primers were IelL - 5'- CTAACAGGCGCATACGATCTGACA -3' (positions 542 to 565, 24 bases) and IelR — 5’- TACGCATAGCGATCTCCTTCGTTG -3’ (positions 1047 to 1024, 24 bases). Capture probe used was 5’- [Amino link] AATATGCTGCCTACTGCCCTACGCTT -3’ (positions 690 to 716 of target, 26 bases). 4.3.4.3 Polymerase chain reaction (PCR) Polymerase chain reaction was performed with Taq DNA polymerase in a DNA thermal cycler. The 50 pl PCR reaction mix consisted of 0.5 pM of the above primers set, 25 pl of Promega Master Mix (Promega Corporation, Madison, WI), containing the 105 proprietary reaction buffer (pH 8.5), 200 pM deoxynucleotide triphosphates (dNTP), 3 mM MgCl2, and 2.5 U of Taq DNA polymerase. A Salmonella colony , grown ovemight on TSAYE, was suspended in 0.5 ml of DNA grade water (Fisher Scientific, Pittsburg, PA) containing 5 mM of NaOH, and boiled for 10 min to rupture the cells. Altemately, 0.1 ml Of an ovemight Salmonella culture grown in TSBYE was mixed with 0.4 ml of distilled DNA grade water containing 5 mM of NaOH and boiled for 10 min. As a template, 1 pl was added to the PCR reaction mixture. The cycling reaction was performed as follows: heat denature at 94°C for 5 min with 30 additional cycles of heat denature at 94°C for 30 5, primer annealing at 59°C for 30 s, and extension at 72°C for 45 5. After the final cycle, the samples were maintained at 72°C for 10 min to complete DNA strand synthesis and then cooled to 4°C, unless used immediately. 4. 3. 4.4 Silanization of nano-tubular Si platform Figure 4-5 illustrates the functionalization of the NTS platform into a DNA biosensor, comprising two main steps — Silanization and DNA oligonucleotide probe immobilization. For Silanization, the NTS substrates were first cleaned in boiling acetone for 5 min, boiling methanol for 5 min, and then dried under nitrogen. The substrates were immersed in sulfochromic acid (strong oxidizing agent) for 15 min to hydrate and prepare the surface for Silanization. Following the acid treatment, the substrates were washed twice in deionized water and dried under nitrogen. The substrates were then placed in a clean oven for 1 h at 140 °C. Irrunediately after heating, the substrates were transferred to a glove box under a dry nitrogen atmosphere. A 10% 3-glycidoxypropyltrimethoxysilane (GOPS) solution (in dry toluene) was placed on the substrates. The substrates were left to react under nitrogen for 4 h, then rinsed with toluene and left to dry. A background 106 cnon '5 HOHZC—Si—ECHgl—O— CHz—HC/ OH2 “"' CHZOH ’ CHZOH giro” , Si —O—Si—[Cl-1230—CH2—HC/ CH2 Silanization with GOPS CHZOH ..... -4 + $1 or NTS Platform 5 end O E 3 g g a V ””2 5' and \N it .4 o—P—o {.— HNW I- NH2 01on 0 /N \N Si--O—Si{CH230 O—CHz—CH—CHZ 3 < l 2 ‘13"ch OH —T—°—CHZO N N —.l O- 9 Functionalized Si or NTS Platform 0H 3' end Figure 4-5. Functionalization of planar Si and nano-tubular Si platform into DNA- based biosensor. - 107 electrochemical cyclic voltammetry (CV) reading was then taken prior to DNA probe immobilization. 4. 3. 4.5 DNA probe immobilization Immobilization of the probe layer was performed by coating each substrate with 50 pl of 150 nM aqueous DNA probe solution (in 1 mM NaOH) specific to the pathogen of interest, and leaving them to react overnight at room temperature in the glove box. The substrates were frnally placed in boiling water for 2 min to remove excess unreacted DNA probe and dried under nitrogen. The unreacted aldehyde groups of GOPS were then saturated by dipping the chips in a 0.1 M glycine solution for 20 min. Chips were washed using a washing solution (1X SSC containing NaCl and Na-citrate — see Appendix B:Section B.2 for more details) and dried under nitrogen. Electrochemical cyclic voltammetry (CV) reading was taken prior to target DNA hybridization. The prototype biosensor was stored under nitrogen at 25 °C for maximum stability, until used. 4.3.4.6 Characterization of the DNA Following PCR, the size of the PCR amplified DNA product was determined using gel electrophoresis (procedure in Appendix A:Section A3). The DNA concentration was also determined using Bio-Rad SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA) readings at 260 and 280 nm. PCR amplified DNA was diluted to 10'9 pg/pl in hybridization buffer (5X SSC, 0.1% SDS, 0.5% BSA) and used for initial characterization of the NTS DNA-based biosensor. 4. 3. 4. 7 Target DNA hybridization DNA in the hybridization buffer was denatured in a 95°C water bath, and cooled to 59°C (just below melting temperature of the capture probe). The hybridization of complementary DNA strands (obtained by extracting DNA from the sample of interest) 108 with the immobilized oligonucleotide probe layer was performed by dipping the DNA modified chip in a solution of the target DNA at the appropriate concentration in hybridization buffer for 45-60 min at 59°C (Figure 4-6). The non-specifically adsorbed strands were then removed by extensive washing in Wash solution A (0.1X SSC, 0.1% SDS — see Appendix for more details) followed by Wash solution B (0.1X SSC — see Appendix for more details). The chips were then dried under nitrogen and CV readings measured. Optimum operating conditions for the biosensor were determined. 4. 3. 4.8 Electrochemical detection - cyclic voltammetry Before adding the target DNA, a baseline was obtained for each sensor (after DNA probe immobilization) using a blank containing an aqueous solution of 5 mM potassium ferrocyanide in 1 M potassium nitrate. A volume of 25 ml was added to a three-electrode electrochemical cell (Figure 4-7). Cyclic voltammetry was performed on the blank solution using a Versastat II Potentiostat/Galvanostat (Princeton Applied Research, Oak Ridge, TN). The working electrode was the treated biosensor platform, the reference electrode was the Ag/AgCl electrode, and the counter electrode was a graphite electrode rod. The potentiostat was run in the ramp, one vertex multi mode. The potential was cycled between —1.2 V to +1.5 V at a scan rate of 20 mV/s. The resulting current was measured and plotted against the potential. After measurement, the blank solution was discarded. The procedure was repeated with the biosensor following target DNA hybridization. 4.3.4.9 Confirmation of results Atomic Force Microscopy images were used to determine surface level changes occuning due to immobilization of the DNA probe and the target DNA hybridization. 109 693...; .8238:— eon=a=oro=5 .5 2.9:— 0.05) when grown in NB or LB. Differences in E. coli numbers were also not significant (8.37 :1: 0.10 Log CFU/ml and 8.23 :t 0.09 Log CFU/ml for NB after 24 and 48 h, respectively; versus 8.29 d: 0.08 Log CFU/ml and 8.20 i 0.10 Log CF U/ml for LB afier 24 and 48 h, respectively). However, the intensity of light emitted by E. coli cultures grown in NB and 120 oow .5 .5 mz 5 8.5.3 28 .m 2.. ._ a. is a z E 35.5 E»: a. 88:: 3.2255 .3 95“: 05¢ m4 le :wv m2 IXI cvw m2 III :3. m.— III EN m.— IOI 3.5 as: 8 8 E 8 on ow om on S o _ _ p p _ h h _ b m I '0. (’0 93 m._ 85:85:: \ ;. l ‘l ‘l l ~\ j “3 ¢ cwv Ucmcvm-mz 5 :00M I I ‘0. l0 .5 .0 (Sims 601) natsswla W5!'I I '0. (D c wv Dem: VN- ml. 5 :00 .m 121 LB were significantly different (p<0.05). Immediately after mixing the sample with the Lumi-Gal® 530 dioxetane substrate, the light emitted by the 24h and 48 h cultures of E. coli grown in LB was one log higher than that of the uninoculated LB as well as the 24 h and 48 h cultures of E. coli grown in' NB. Light intensity for E. coli cultures grown in LB increased with time to 6.3 Log RLU/s with no significant increase or decrease observed after 60 min (p>0.05). As a result, for all later experiments, the light intensity measurement period was limited to 60 min. At the end of the light measurement period (90 min), the light emitted by the LB culture was two orders of magnitude higher than that of the NB culture (p<0.01) (Figure 5-2). Tryland and Fiksdal (1998a) observed that various strains of E. coli required 1-4 days to reach stationary phase, thus, influencing the chemiluminescence. However, in the current study, increasing incubation time from 1 to 2 days had no significant effect on chemiluminescence. Escherichia coli cultures grew rapidly in both NB and LB to reach the stationary phase of grth within 24 h (as evidenced by no significant differences in E. coli numbers between 24 h and 48 h cultures), thus explaining the results obtained in the current study. A 100-1000 fold increase in the light emitted by the LB E. coli culture in comparison to the NB culture supported the hypothesis that lactose broth would be a better medium for the assay. Availability of lactose as a substrate for E. coli increases B-galactosidase production. The lac operon in E. coli consists of one regulatory gene (the I gene) and three structural genes (Z, Y, and A). The lac I gene codes for the repressor of the lac operon. The lac Z gene codes for B-galactosidase, which is primarily responsible for the hydrolysis of lactose into its monomeric units, galactose and glucose. The lac Y gene 122 "v.1: codes for permease, which increases permeability of the cell to B-galactosides. The lac A gene encodes a transacetylase that has a known, but not essential, enzymatic activity. During normal grth on a glucose-based medium, the lac repressor is bound to the operator region (lac 0) of the lac operon, preventing transcription. However, in the presence of an inducer (lactose) of the lac operon, the repressor protein binds to the inducer and is rendered incapable of interacting with the operator region of the operon. RNA polymerase is thus able to bind at the promoter region (lac P), and transcription of the operon ensues, resulting in increased production of the B-galactosidase enzyme (King, 2002). 5.1.3 Determining Assay Applications When the two E. coli cultures were tested with the chemiluminescence assay, no significant difference in light emission was observed between the light emissions for E. coli K-12 and E. coli 0157:H7 throughout the entire 60-min period of light measurement (Figure 5-3). B-galactosidase enzyme activity for the pathogenic strain of E. coli was higher in LB versus that in NB, as observed with the generic E. coli strain grown in LB. However, with the heat-killed E. coli 0157:H7 culture, the light emission was significantly lower than that of the untreated E. coli and E. coli 0157:H7 cultures (p<0.01). The light intensity for the heat-killed E. coli 0157:H7 culture was also significantly lower than that of the uninoculated broth (used as the control) (p<0.05) (Figure 5-3). Lack of significant differences in light emitted for an overnight E. coli 0157:H7 compared to E. coli K-12 indicates that the assay cannot be used as a discriminatory test for non-pathogenic versus pathogenic strains of E. coli. The use of other enzymes, such 123 .2518 €25-32— eaa $83.55: Swarm—O m3.» .N ES ~39 .M 3.69% Bum—:95 .3 .3 68:59 Eu: no 832.3— .m-m 95$...— Efimro 3:581]?! xcsm lxl EH35 __oo.m . .o. . :8 .mllul 3:5 as: on om om ov om cm or o _ a _ r _ _ m Hi A I? lImT |T in .. V M N T N1 T N I m I m (sin-Ia 601) uorssma tufin 124 as B-glucuronidase, to discriminate between non-pathogenic strains of E. coli and E. coli 0157:H7 needs to be investigated. Significant differences in light emission for heat-killed E. coli 0157:H7 cultures as compared to untreated E. coli 0157:H7 and the LB control indicate that the assay can be used for quality control of heat-processed foods. The assay might be used to assess possible cross-contamination with raw product or ineffective heat treatment of foods such as pasteurized milk or apple cider. Significantly lower light intensity of the heat-treated sample compared to the uninoculated control could be explained by presence of organic matter (from the dead cells) in the sample. Lumi-Gal® 530 (the dioxetane substrate) has been carefully formulated to optimize enzyme activity, chemiluminescence intensity and lifetime for high sensitivity, which also makes it sensitive to the sample matrix. The organic matter present in the sample quenches the light emitted, lowering the light emission output for the heat-treated sample compared to the background signal emitted for the uninoculated control devoid of any organic matter (Beale et al., 1992). This could potentially be exploited to determine presence of unusually high levels of organic matter in a food sample that also contains E. coli or other B-galactosidase producing organisms as a part of its normal microflora. For example, a mastitic milk sample from an infected cow normally has a high somatic cell count in addition to bacteria (Tsenkova et al., 2001). Testing pasteurized mastitic milk (pooled with normal milk) could yield a chemiluminescence signal significantly lower than that of normal pasteurized milk (an untested hypothesis), which in turn can be used as an indicator of potential problems with animals in a dairy farm. 125 5.1.4 Determining Amount of Sample Used in the Assay Preliminary trials conducted using 25 ul of E. coli culture for the assay showed a very low sensitivity of 105 CFU (4x106 CFU/ml for a liquid sample) after 60 min. Randomized block design (RBD) analysis comprising three levels of culture was used to optimize the assay performance in terms of sensitivity. The interactive effect of the amount of culture and the light emission was analyzed using an Autoregressive mixed model in SAS, the results of which are summarized in Table 5-1. The intensity of chemiluminescence was directly proportional to the E. coli cell concentration (B-galactosidase) in the sample. With decreasing cell numbers, the light intensity decreased exponentially. For example, with 50 ul of culture, light intensity for 108 CFU/m1, 107 CFU/ml and 106 CFU/ml was 5.99, 5.07, and 4.38 log RLU/s, respectively, after 10 min (Table 5-1). The intensity of light emitted increased with the amount of E. coli culture used in the assay; for example, light emission of the 106 CFU/ml E. coli culture using 25, 50 and 55 ul was 4.3, 4.38, and 4.41 log RLU/s, respectively, after 10 min (Table 5-1). The mean coliform count of the pure culture for the sensitivity trials was 8.19 :1: 0.09 Log CFU/ml. The intensity of light emission for all samples at all serial dilution levels increased with time (10-60 min). Readings of light emission were stable after 30 min, thus 30-60 min readings were used for data analysis. The lower limit of detection based on significantly different light emission values [highlighted in Table 5-1] for 25 ul sample was 104 CFU at 60 min; for 50 pl culture level was 103-10“ CFU between 30 to 60 min; and for 55 ul was 102-103 CFU (30-60 min). 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The sensitivity of the assay was observed to be low (105 CFU) using 25 pl of E. coli culture, resulting in the investigation of different levels of the culture to maximize the sensitivity. As recommended by Lumigen (the supplier), Lumi-Gal® 530 was not to be diluted by more than 10% of the total volume with the culture or enzyme. The three E. coli culture amounts — 25, 50 and 55 pl - were chosen accordingly. Maximum sensitivity of the assay required B-galactosidase and stabilized dioxetane to be mixed and incubated for sufficient time to allow the product (detected by luminometer) to approach steady state. About 30 min after mixing Lumi-Gal® 530 and the perrneabilized culture mix, the light emissions were stable; therefore, the readings from 30-60 min were considered in the interpretation of results. Beale et al. (1992) followed a similar procedure while testing Lumi-Gal® 530 with purified B-galactosidase. The enzyme was mixed with Lumi-Gal® 530, followed by sample incubation at 37°C for 30 min and then the chemiluminescence response was measured (Beale et al. , 1992). Sensitivity of the assay improved with the increasing amount of E. coli culture used for the assay. Higher cell numbers, and thus higher amount of B-galactosidase enzyme, in 55 pl of E. coli culture may explain maximum sensitivity of 103 CFU. This means that any sample analyzed (using 55 pl) had to contain a minimum of 103 CFU in the reaction mixture for successful detection. Thus, the assay would require filtration or similar processes to concentrate the cells for samples with low numbers (100-102 CFU). An enrichment process to elevate the E. coli or coliform numbers to detectable levels could also be used. Tryland and Fiksdal (1998b) used isopropyl-B-D-thiogalactopyranoside 129 (IPTG), a B-galactosidase inducer, and observed that the B-D-galactosidases of all non- target environmental isolates were less inducible than the enzymes of coliforrns and E. coli when grown in tryptic soy broth. So, the addition of B-galactosidase inducers, such as IPTG, to LB during an enrichment step could increase B-galactosidase activity of E. coli in samples contaminated at low levels, potentially reducing enrichment time required and improving the sensitivity of the assay. This is yet to be investigated. 5.1.5 Assay Validation in Food Analysis Based on the results of the sensitivity trials, a sample of sprouts (SS), three samples of milk (M1, M2, M3), and three samples of water (W1, W2, W3) inoculated with E. coli were tested using 55 pl sample and polymixin B sulfate mixture with 500 pl Lumi-Gal® 530. Escherichia coli levels below 103 CFU/ml could not be detected in the samples tested (Figure 5-4) as the difference in intensity of light emitted between the samples and the blank was not significantly different (p>0.05). At E. coli levels above 103 CFU/ml, the intensity of light emitted increased exponentially with the bacterial numbers. Moreover, the sample matrix did not have any significant effect on the assay. Figure 5-5 shows the light output measured 60 min after addition of Lumi-Gal® to the Red Cedar River water samples collected from seven sites. Based on analysis of the sensitivity study, a guideline was used to assess the presence or absence of coliforrns in a sample with unknown number of coliforrns. If the light emission from the sample was consistently greater than that of the blank/control by at least 1000 RLU/s (between 30-60 minutes), the sample was considered positive. Based on this guideline, when the water samples were analyzed directly (without pre-enrichment), a conclusive 130 (lenao 601) aunoo .1109 '3 (O V |\\\\\\\\\ Log(RLU/s) + Log(CFU/ml) l l OOOOOOO (Sin-Ia 601) WWW; tufin 65$ E 3:35 I mafia.» u vantage? 458:3..5 8 a3...— oaa 3.58 8.3.33 «Ea—am :32: 3 hem...— ma— 5 2.25—3.50 : N— can .32: «3.5. 3.953. .52; 3%...» no nova—no Eu: comm—«ESZ .m-m 95w:— oEEam n macaw m waEmw m macaw v 0.993 m macaw N maEmw _. oEEmw O x I O i F Flt 3 n l 6 m N .q. I. 3 M N -- l m m. u w. 9. o ) u m t." 6 m it II V o o B ._n. w I x n w h m m m -- 958 56:60 IT ufiotcm ; N? I 695 a -- n 132 interpretation of the data (presence/absence and/or quantification) could not be made for five of the seven samples. Only sample 1 and sample 2 could be interpreted as positive. Subsequently, the coliform counts for samples 1 and 2 (from MacConkey agar plates) were found to be ~1 log higher than the rest of the samples; counts ranged from 550 CFU/ml (for sample 5) to 13,500 CFU/ml (for sample 2). Light emission from samples 3, 5, and 6 was very close to that of the blank (Figure 5-5). When the assay was repeated following enrichment in LB for 1-6 h, no significant difference in light emission was observed between enriched samples and those without enrichment (data not shown). However, after a 12-h enrichment of all samples in LB, the light emission increased by 2.5 orders of magnitude for all samples compared to the direct assay. Interpretation of results from the 12-h enrichment assay was conclusive in detecting presence/absence of coliforrns in all the samples (compared with plate counts). When surface water samples, contaminated at low levels (101-103 CFU/ml), were analyzed using the assay without any pre-enrichment in LB, the assay indicated a presumptive positive/negative result. This was due to the stationary (or lag) phase condition of the E. coli cells in the water samples. Production of B-galactosidase by E. coli is affected by several factors, such as cell growth phase, availability of nutrients like glucose versus lactose in the growth medium, ATP levels in the E. coli cells, strain of E. coli, all of which influence the amount of enzyme that can be detected definitively by the assay procedure. When enriched in LB, at first the E. coli cells are quiescent; they do not metabolize the lactose, their other metabolic activities decline, and cell division ceases. During the quiescent interval, the cells begin to produce three enzymes (with transcription of the lac 133 operon gene as explained above) in large amounts that have not been previously produced. The culture then begins growing rapidly again with the lactose being rapidly consumed (Adhya, 1996; Kimball, 2003). This grth behavior during enrichment explains the inability to obtain significantly higher light emission with samples enriched for 1-6 h. In conclusion, the development of a chemiluminescence-based assay and its application in food samples for detection of coliforrns (E. coli), outside of the biosensor system has been demonstrated. The detection limit of the assay was 103 CFU. The assay was completed within 60 min. This assay will be adapted to the biosensor system in the third research objective. 5.2 OBJECTIVE 2 Design, fabrication and characterization of a nano-tubular silicon platform. 5.2.1 Nana-Tubular Si Characterization NTS was fabricated from a p”;+ type wafer using different anodization conditions. These conditions were chosen by modifying those used for porous silicon fabrication (Shimasaki et al., 1996; Thust et al., 1996; Halimaoui, 1997) reported previously. Scanning electron microscope (SEM) images (Figures 5-6 to 5-13) of the chips were used to determine the best NTS biosensor fabrication conditions based on nano-tube size, shape and uniformity, nano-tubular surface texture and inter-nano-tube space. No features were observed on the bare (non-nano-tubular) Si (Figure 5-6). NTS fabricated using anodization conditions of 2 mA/cm2 for 1 h (Figure 5-7) showed a uniform structure, slightly uneven surface texture with noticeably less inter-nano-tube spaces (5-20 nm), and nano-tube size ranging from 5—30 nm. Anodization conditions of 5 mA/cm2 for 20 min 134 I 15.0kV 11.7mm x500k SE(U) 7/24/2003 15:38 Figure 5-6. SEM image of bare pH Si surface. ,4 I .. . llllll’llllli l‘SQkV 10.8mm XSOOR SElU) 7/23/2003 16:08 9, 100nm Figure 5-7. SEM image of nano-tubular Si produced from pH- type Si. Anodizing conditions used were 15% HF, 2 mA/cm2 for 1 h. 135 (Figure 5-8) and 5 mA/cm2 for 1 h (Figure 5-9) provided the best NTS chip characteristics with uniform nano-tube structure and nano-tube sizes ranging from 10-30 nm, a smooth (even) surface, and an inter-nano-tube space of 10—20 nm. NTS fabricated using 15 mA/cm2 (Figure 5-10) and 30 mA/cm2 for 1 min (Figure 5-11) showed an uneven surface texture and non-uniform structures. Nano-tubes were elliptical or rod-shaped with large inter-nano-tube spaces and nano-tube sizes ranging from 5—20 nm in length. NTS fabricated using 50 mA/cm2 for 1 min (Figure 5-12) showed non-uniform structures, highly uneven texture, and round, elliptical or rod- shaped nano-tubes with less inter-nano-tube spaces compared to 15 mA/cm2 and 30 mA/cmz. Nano-tube sizes ranged from 5—30 nm in length. All the properties of a NTS layer are strongly dependent on anodizing conditions such as HF concentration, pH of the solution and its chemical composition, current density, wafer type and resistivity, temperature, anodization duration, and illumination (Halimaoui, 1997). NTS fabricated using 100 mA/cm2 for 30 s (Figure 5-13) had a poor and uneven surface structure. Uneven shaped nano-tubes with very large inter-nano-tube size spaces were observed. Visual inspection of the NTS chip showed discoloration as well as partial etching of the top NTS surface. This result contradicted previously reported literature where porous silicon was successfiilly fabricated using 100 mA/cm2 for 30 s to 1 min (Halimaoui, 1997; J anshoff et al., 1998). This discrepancy could be due to variations in the resistivity of the wafers used and the concentration of the HF solution. 136 I I I I I I I I I I I 15.01'V 'llflfimr‘r‘l XSClC'l’ SEtU} 75' [2003. 1.5 l4 liiil'lrm‘. Figure 5-8. SEM image of nano-tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 5 mA/cm2 for 20 min. I I I I I I I I I I I lSDLV 10.9mm XSOCIl: SEIU) 77232003 15:21 100nm Figure 5-9. SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HF, 5 mA/cm2 for 1 h. 137 15.0kV 11.7mm x350k $E(U) WES/2003 17:08 100nm Figure 5-10. SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HF, 15 mA/cm2 for 1 min. ~w3 .1? ' 15.0kV 11.8mm x350k SE(U) 7124/2003 16:14 100nm Figure 5-11. SEM image of nano-tubular Si produced from pH type Si. Anodizing conditions used were 15% HF, 30 mA/cm2 for 1 min. 138 15.0kV11.8I’hm x350l< SE(U) 7124/2003 16:53 "- I l ' I lObnlml. I Figure 5-12. SEM image of nano—tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 50 mA/cm2 for 1 min. 15.0kV 11.8mm x350k $E(U) 7/24/2003 17:24 100nm Figure 5-13. SEM image of nano—tubular Si produced from p++ type Si . Anodizing conditions used were 15% HF, 100 mA/cm2 for 30 s. 139 Based on the characterization of the NTS using SEM images, 5 mA/cm2 for 1 h was chosen for the fabrication of the NTS biosensor chip in further testing. Anodizing conditions of 5 mA/cm2 for 1 h was chosen over 5 mA/cm2 for 20 min because longer anodization with the same current density yields a thicker NTS layer (longer nano-tubes) (Halimaoui, 1997) and thus more surface area for the biological sensing element to bind and for the biochemical reaction during the detection process. The porosity and thickness of the NTS obtained using anodization conditions of 5 mA/cm2 for 1 h with 15% ethanoic solution of HF was determined gravimetrically (Table 5-2). The percent porosity of the NTS ranged from 70.00 % (Chip 10) to 87.23 % (Chip 4), and the thickness of the NTS layer varied from 13.09 pm (Chip 3) to 21.67 pm (Chip 1). The mean percent porosity of the NTS layer for the 14 chips was 80.21 % :t 4.29 % with a corresponding mean thickness of 17.54 pm :I: 2.82 pm. The percent porosity of the NTS was similar to that reported previously under similar anodizing conditions (Halimaoui, 1997; Janshoff et al., 1998; Amato et al., 2000). The variation in porosity and thickness observed was possibly due to batch-to-batch variations in composition of the ethanoic HF used or slight fluctuations in the current density applied to the silicon chip. A cross-sectional SEM image was taken by carefully dicing the NTS chip with a diamond saw and mounting the cross-section of the chip at a 45° angle. The scanning electron micrograph provided additional confirmation of the thickness obtained by gravimetric analysis (Figure 5-14). The micrograph shows the NTS structures to be about 15-20 pm thick/deep and interconnected to some degree. 140 Table 5-2. Thickness and porosity of nano-tubular Si chips fabricated from p++ Si. Thickness Chip m. (g) m. (g) M. (g) Area (on?) (um) Porosity (%) 1 0.2747 0.2708 0.2699 0.950714 21.67 81.25 2 0.3365 0.3329 0.3322 0.950714 19.41 83.72 3 0.3697 0.3674 0.3668 0.950714 13.09 79.31 4 0.32 0.3159 0.3153 0.950714 21.22 87.23 5 0.345 0.3413 0.3406 0.950714 19.86 84.09 6 0.2981 0.2946 0.2938 0.950714 19.41 81.40 7 0.3369 0.3336 0.3327 0.950714 18.96 78.57 8 0.3306 0.328 0.3272 0.950714 15.35 76.47 9 0.3087 0.3062 0.3055 0.950714 14.45 78.12 10 0.3171 0.315 0.3141 0.950714 13.54 70.00 11 0.3281 0.3249 0.3242 0.950714 17.61 82.05 12 0.3523 0.3491 0.3484 0.950714 17.61 82.05 13 0.3084 0.3059 0.3051 0.950714 14.90 75.76 14 0.2673 0.2639 0.2632 0.950714 18.51 82.93 Anodizing conditions used were 15% HF, 5 mA/cmz for 1 h. 141 Top porous surface 0 .',-.w{v,-"“ $1., . (‘ross section of NTS Sample mounting support Figure 5-14. Cross-sectional SEM image of nano—tubular Si produced from p++ type Si. Anodizing conditions used were 15% HF, 5 mA/cm2 for 1 h. NTS chip cross-section was placed at 45° to capture the image. When using doped p-type silicon to produce a porous silicon-based bioreactor for glucose detection, Bengtsson et al. (2000a) produced a porous surface with a maximum thickness of 10 pm, while DeLouise and Miller (2005) could only obtain a 6.5 pm thick porous layer for enzyme immobilization. Other investigators have also reported similar thickness when fabricating porous silicon layers fiom highly doped p-type wafers (Splinter et al., 2001; Martin-Palma et al., 2004). Halimaoui (1997) predicted the possibility of producing a porous silicon layer 13 pm thick using 25% HF and a fixed current density of 50 mA/cm2 for 4 min. However, a porous silicon layer with more than 10 pm thickness using highly doped p-type silicon has not been reported, especially with 142 lower current densities. The current research has shown a significant improvement (50- 100 %) in the thickness of the porous layer over currently reported values, thus justifying the name NTS. This improvement was made possible by maintaining the potential, V, less than Vep, the electropolishing peak potential. As long as the current density, J < Jepa the supply of holes was restricted to maintain the H-terminated surface throughout etching (Chazalviel et al. , 2000; Safi et al., 2002). Ethanoic HF solution was used as the electrochemical etching agent, as opposed to purely aqueous HF solution, because ethanol had been shown to act as a surfactant, improving the porous silicon layer uniformity by eliminating hydrogen bubbles (formed during the process of porous silicon layer formation), improving the penetrability of the electrolyte (etching) solution in the pores (Halimaoui, 1997). Also, the use of the ethanoic HF enabled fabrication of porous silicon at higher currents for longer times than that used typically with diluted aqueous HF solutions. Longer anodization time with the same current density had been shown to provide a thicker (deeper) porous silicon layer (Halimaoui, 1997) and thus more surface area for the biochemical reactions during the detection process. When anodization times greater than 60 min were employed to fabricate NTS, the process was not repeatable; a porous layer of silicon was obtained in some trials while electropolishing occurred in others (data not shown). As the anodization time was increased, current density, J, eventually peaked beyond Jcp, leading to electropolishing (complete etching of the silicon surface layers) instead of pore formation (Allongue, 1997). Hence, an anodization condition of 5 mA/cm2 for 60 min was used to fabricate the NTS platform for the biosensors, yielding porous silicon layers consistently with the maximum depth of the pores possible (before electropolishing occurred). 143 5.3 OBJECTIVE3 Functionalization of the nano—tubular silicon platform into a biosensor chip. 5.3.1 Adaptation of CL Assay to the Biosensor Detection System - Procedure A Figure 5-15 shows the light emission of the control NTS biosensor chip (blank with no E. coli culture) as well as that of NTS- and planar Si biosensor chips tested with an overnight E. coli pure culture. Light emission for the control functionalized NTS biosensor chip (with no E. coli) was significantly lower (p<0.01) than that for both the planar Si and the NTS biosensor chips, thus indicating that both chips were able to detect E. coli present in the sample using B-galactosidase produced as a marker (biological sensing element). However, light emission for the nano-tubular biosensor chip was significantly (about three times) higher than that of the planar biosensor chip. For example, light emission of the control biosensor chip, the planar Si chip and the NTS biosensor chip after 30 min were 5480 RLU/s, 173,666 RLU/s and 561,868 RLU/s, respectively. The higher light emission of the NTS biosensor chip as compared to the planar Si biosensor chip was due to the higher surface area available for the Ltuni-Gal® 530 to diffuse and bind to during the functionalization step. With greater amounts of Lumi-Gal® 530 available to react with B-galactosidase in the sample, the amount of light emitted from the NTS biosensor chip, when compared with that of the planar Si chip, was higher. The external CL assay (Objective 1, Table 5-1 on page 127) had higher light emission than that of the NTS biosensor chip for an E. coli pure culture by about one log RLU/s. This can be explained by the lower amounts (1/ mm) of sample and Lumi-Gal® 530 used in the biosensor system as compared to that of the single-tube external CL of assay. However, this also reduces the cost of the assay by 90%. Also, per unit volume 144 .Qodvmv aofimmao Em: Eobbfi .Azcmociwmm 85.8me was 05 .8>o Etofiomsm 580me 495:3 mm hat—E5553 3.3:»: 28585 :35... Luau—a can mm 53.55.55:— 5 95:3 9::— ..~50 .m a: nevi—:5 Eu: he scumbag—=60 .me «Sufi 5 Bear”. D 5 390a I £55 a 3:5 25... on (Sin-Ia 601) minimal :IIBI'I Fo 145 dioxetane substrate, light emitted from the NTS biosensor was slightly higher (4.05 Log RLU) than that from the single-tube assay (3.97 Log RLU). The single-tube external CL-based assay developed in Objective 1 (sections 5.1.1 to 5.1.5) was successfully adapted to the NTS biosensor chip system to detect E. coli using B-galactosidase as the biological sensing element. The NTS biosensor platform is practically an array of single-tube external assays in the nano scale. 5.3.2 Adaptation of CL Assay to the Biosensor Detection System - Procedure B Figure 5-16 shows the comparative kinetics of light emission for an overnight E. coli culture grown in LB using the standard chemiluminescence assay, developed previously, and the modified chemiluminescence assay where the Polymyxin B sulfate and dioxetane substrate were mixed prior to addition of the E. coli culture. Mean light emission for E. coli with the modified chemiluminescence assay was equivalent to that obtained with the standard chemiluminescence assay immediately after addition of the E. coli culture to the dioxetane-Polymyxin B sulfate mixture. However, an incubation period of 15 min after addition of culture to the Polymyxin-dioxetane mixture significantly improved the light emission over the standard chemiluminescence assay procedure. Light emission for the modified chemiluminescence assay procedure (afier incubation) was significantly greater than the standard assay throughout the 60 min period of measurement (p<0.01) (Figure 5-16). 146 (mm 601) minimal mm 147 Time (min) LI Pre-Mixed E Non-mixefl g the standard (non-mixed) and modified (pre- says. Different superscripts over the bars indicate significant ight LB E. coli culture usin Figure 5—16. Kinetics of light emitted by an overn cubation) single-tube chemiluminescence as mixed with 15 min in differences (p<0.01). Figure 5-17 shows the light emission from serially diluted E. coli cultures on NTS biosensor chips. With increasing cell numbers, the light intensity also increased. For example, light intensity of samples containing 101 CFU, 102 CFU, 103 CFU and 104 CFU of E. coli were 3.38, 3.52, 3.85 and 4.22 log RLU/s, respectively, at the 40 min reading (Figure 5-17). The amount of B-galactosidase in the sample extracted from the cells would be higher with increasing E. coli cell numbers, thus leading to a greater chemiluminescence intensity. Beale et al. (1992) also observed a linearly increasing relationship between B-galactosidase concentration and chemiluminescence using Lumi- Gal® 530. Following addition of the sample to the functionalized biosensor chips, light emission increased with time and had lower variability after 30 min. This trend was similar to that observed previously with the single-tube chemiluminescence assay external to the biosensor (Mathew and Alocilja, 2004) and the study by Beale et al. (1992). Hence, light emission values obtained after 30 min of sample addition in this study were used to determine the sensitivity of the biosensor to E. coli. The average bacterial count of the E. coli pure culture for the sensitivity trials was 5.66 x 107 CFU/ml. The lower limit of detection based on significantly different light emission values was 102 CPU at 30 and 50 min and lOl at 40 and 60 min. This was determined using the same equation as before: Sensitivity (CF U) = [Average coliform count (in CFU/ml corresponding to bold light emission value) x volume of sample (pl)]/1000 pl. The lower detection level of 101-102 CFU was a significant improvement over that of the single-tube chemiluminescence assay determined external to the biosensor (103 cells, (Mathew and Alocilja, 2004)). This improvement could be explained 148 u 22’7//////////////////////////////////2 0 .\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\¥ 60 min .— *— D .. .5 “g“. .. \g m g :—m ‘“ 3 .. .s :3 .\g .. :_v2 or E V: .. /.s"3 .. g a. .3 _«> a .o E (9:) a s\x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ ; a =—~ .. .. a .5 E . g . . =—- _ (sln'lu 601) UOIssrwa ruBI‘I 149 Figure 5-17. Comparison of light emission from serially diluted E. coli cultures on nano-tubular Si biosensor against nano- tubular Si control (blank). Different superscript over the bars indicates significantly different light emission (p<0.05). by the lower background noise observed with the control sample (102-103 RLU/s, Figure 5-16) for the modified chemiluminescence assay on the biosensor chip as compared to the background light emission for the single-tube chemiluminescence assay (104 RLU/s). With lower background noise, the assay could detect smaller differences in light emission that resulted from samples contaminated with low numbers of E. coli, thus improving the sensitivity of the assay. In conclusion, functionalization of the NTS into a biosensor was achieved using difiusion and physical adsorption of the dioxetane substrate as confirmed by a chemiluminescence-based assay with B-galactosidase produced by E. coli as a marker. Light emission of the NTS biosensor chip was higher than that of the planar Si biosensor chip. Escherichia coli could be detected using the modified assay procedure within 30 min after addition of the sample to the functionalized NTS biosensor chip. The lower detection limits (sensitivity) of the biosensor for E. coli were 101 and 102 cells at 40 and 30 min, respectively, which was an improvement over the sensitivity of 103 cells for the single-tube chemiluminescence assay developed previously. Future research will involve adapting the biosensor to detect disease-causing agents, such as Salmonella, E. coli 0157:H7 and Listeria. 5.3.3 Functionalization of the Nano-Tubular Si Platform with Salmonella Antibodies into an Immuno-Biosensor The NTS surface was functionalized into an immuno-biosensor by physical adsorption of antibodies as well as a combination of Silanization and antibody immobilization processes. The biosensor chips were then used to successfully detect Salmonella. 150 Figure 5-18 shows the light emission of a control NTS biosensor chip (with no Salmonella) as well as that of nano-tubular- and planar silanized Si biosensor chips tested with an overnight culture of Salmonella. The average bacterial count determined by standard plating was 8.68 :t 0.23 Log CFU/ml of Salmonella. The intensity of light emitted for the NTS biosensor chip with Salmonella increased for the first 2 minutes and then decreased slowly while that of the control and planar Si chip decreased slowly over the entire 10 min period of light measurement. Light emission from the NTS chip was higher than that of the planar Si chip throughout the measurement period. However, the difference in light emission was not statistically significant (p>0.05) due to high variability in light emission for the NTS chip (coefficient of variance ranging from 2.04 to 8.92% for NTS chip as compared to 0.38 to 1.48% for planar silicon chip). Standardization of biosensor functionalization steps and improving the efficiency of blocking/washing steps during chemiluminescence assay can reduce this variability. However, there may not be a significant advantage when using the NTS biosensor chip as an immuno-biosensor because the size of the bacterial target (approximately 0.5 x 3 pm in diameter or length) exceeds the size of the nano-tubes greatly, thus reducing the chances of penetrability of the target into the nano-tubes. Overall, light emission of the nano-tubular and planar silicon chips was significantly greater than that of the control chip (p<0.05), indicating presence of Salmonella in the sample. However, the NTS could easily be adapted to detect viruses of significance to human health. Viral particles are much smaller than the bacterial agent used in the current research, and are reported to have a particle size ranging fi'om 20-250 nm or greater depending on the size of the single— or double-stranded RNA or DNA genome 151 ooooooooooooooooooooooooooooooooo ---------------------------------- 0 222222222’/////////////////////////// ‘ oooooooooooooooooooooooooooooooooooo " 32,227 ooooooooooooooooooooooooooooooooooooooo nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 52222222!7///////////////////////////// s '222227///////////////////////////////// . vvvvvvvvvvvvwvvvvvvv vvvvvvvvvvvvw uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu ...................................... ooooooooooooooooooooooooooooooooooooooo a 5227///////////////////////////////////////. l I T l 1 fl to co no In In v o :0 <- (sm'lu 601) UOIssuua win 152 10 Time (mln) [I Control fi Porous silicon El Planar silicofl against nano-tubular silanized Si control. Different superscript over bars indicates significantly different light emission (p<0.05). Figure 5-18. Comparison of light emission for Salmonella pure culture with nano-tubular and planar silanized Si biosensor (Balch et al., 2000; Yamaguchi et al., 2003; Brussaard et al., 2004). Macroporous (up to 1 pm diameter pores) NTS-based immuno-biosensor can be fabricated by using appropriate anodization conditions to detect the larger sized viral particles. Another possible medical application for the NTS immuno-biosensor would be detection of antibodies produced against viral and bacterial targets in blood samples (Yamaguchi et al., 2003). The NTS biosensor can then be used as a nano-biosensor device for detection of nano particles to provide increased speed and sensitivity in biological sensing applications, compared to their macro or micro counterparts (Ilic et al., 2004; Lee and Chang, 2005). Light emission of the control NTS biosensor chip (with no Salmonella) was also compared against that of silanized and non-silanized (physically adsorbed antibodies) NTS biosensor chips tested with an overnight Salmonella pure culture (Figure 5-19). Light emission of the control chip and the non-silanized chip tested with Salmonella was approximately one log lower than that of the silanized NTS chip with Salmonella. For example, light emitted from the control, non-silanized and silanized chips after 5 min were 4.97, 5.09, and 6.04 Log RLU/s, respectively. Light emission for the Salmonella pure culture on the silanized NTS chip was significantly greater (p<0.05) than the control as well as the non-silanized NTS chip with Salmonella for all time measurements, indicating successfiil Silanization and detection of Salmonella. HRP-Luminol® reaction kinetics showed an exponential decrease in light emission with the light remaining intense for about 20 minutes (data not shown). Based on reaction kinetics, the half-life of the HRP-Luminol® reaction was reported to be 60 min (Pierce Chemical Company, 2001). When the NTS biosensor chips were tested with Salmonella, 153 .Amodvfi nommmmEo Em: “cutaway bacon—mafia 3:365 was 05 .8>o firewood”; Bogota 46.5.3 105—3:... umfiawa 32.3.53 5 hiss—5-2::— eonmnazméea can 355:» .53 9:55 9:3 53.3530 .5. 53.389 Eu: no acmmnanEeU .a—-m ensur— umscgwn um~_cm__m-=oza .228- Eé 2:: m e n a P (Sin-Ia 601) UOISSIwa rufin 154 light was measured for only 10 min, because significant difference in light emission could already be observed afier the first minute in silanized chips with Salmonella when compared to the control. Light readings were measured to test the hypothesis that Silanization of NTS was an essential step in functionalization of the biosensor chip, accomplished by comparing the kinetics of Salmonella on non-silanized chips against that of silanized chips. Light emission from a non-silanized chip comparable to that of a silanized chip would indicate successful functionalization by physical adsorption of antibodies directly onto the NTS surface or through entrapment within the nano-tubes. However, no significant difference (p>0.05) in light emission of the non-silanized chip (with Salmonella) was observed when compared to the silanized and non-silanized control (no Salmonella), indicating that physical adsorption or entrapment of antibodies was not a reliable method of antibody immobilization. The physical adsorption process used for antibody binding explains the lower light emission of the non-silanized NTS biosensor chip when compared to that of the silanized chip. No (or significantly lower) binding of the antibody resulted from physical adsorption due to lack of a stable bond/link between the NTS surface and the antibody. Silanization of NTS enables formation of a stable covalent bond between the cross linker and the silicon surface, which in turn binds the antibody to the surface (Bhatia et al., 1989). When the NTS surface is not silanized, antibodies are not covalently bound to the surface. Physically entrapped antibodies in the non-silanized biosensor chip could easily get removed during any of the blocking and washing steps used in the optical detection method, thus explaining the lower light emission of the chip. As observed in the current study, other investigators have successfully employed Silanization for 155 immobilization of biological molecules on silica (Bhatia et al., 1989) and SiOz-TiOz optical wave-guide (Trummer et al., 2001) surfaces. Functionalization of the NTS into an immuno-biosensor was achieved using Silanization and immobilization of antibodies as confirmed by the chemiluminescence- based optical detection method. Comparison of light emission from silanized and non- silanized biosensor chips proved Silanization to be an essential step in the functionalization of the biosensor. Light emission of the NTS biosensor chip was not significantly higher than that of the planar Si chip, indicating limited advantage of NTS as an immuno—biosensor platform when used for bacterial detection. Application of the NTS biosensor to virus and antibody detection is yet to be investigated. 5.3.4 Functionalization of the Nano-Tubular Si Platform with Salmonella DNA Probes into a Nucleic Acid-Based Biosensor 5.3.4.1 Polymerase chain reaction A clinical strain of Salmonella Enteritidis (strain S-64, MDCH collection) was used as the target for. development of the nucleic acid-based biosensor. The chosen PCR primers and the DNA capture probe were from the insertion (iel) gene of Salmonella Enteritidis with a very high degree of specificity for Salmonella species (B value of 2.0 x 10'5 for Salmonella versus 5.1 for the closest non Salmonella target with the capture probe). PCR was successfully performed with overnight cultures of S. Enteritidis grown in TSBYE as confirmed by gel electrophoresis (Figure 5-20). The PCR amplified DNA product should be 505 bp long (positions 542 to 1047 of the iel gene of Salmonella Enteritidis). Figure 5-20 shows that the PCR product (Lanes 8] and S2) corresponds to the 500 bp DNA band of the marker (Lane M), confirming the 156 1.2 Kbp” an. 1.0 Kbp “ are. 700 bp “ 500 bp 1 m 300 bp 100 bp Figure 5-20. Gel electrophoresis of PCR-amplified Salmonella: 81 and 82 - 5 pl culture used for PCR. Key: M — Marker, C — negative control, S — Salmonella length of the PCR product. Following PCR and gel electrophoresis confirmation, the concentration and purity of DNA in the PCR amplified (pooled) sample were determined using spectrophotometer measurements. The DNA concentration in the PCR amplified (SI and S2 pooled together) sample was 132.61 pg/ml with an Azeo/zso ratio of 1.99, indicative of the high quality of the PCR DNA product. 5.3.4.2 AFM characterization of nano-tubular Si DNA biosensor The NTS platform was functionalized into a DNA-based biosensor by immobilizing the DNA capture probe specific to S. Enteritidis. The above PCR amplified DNA product was diluted appropriately to obtain a 1 ng/ pl solution of DNA and allowed to hybridize to the capture probe immobilized on the NTS DNA biosensor. The process of functionalization and target DNA hybridization was confirmed using atomic force microscopy (AFM) images. Figures 5-21 to 5-23 show the surface profiles of NTS DNA 157 biosensor chips prior to DNA probe immobilization, afier DNA probe immobilization, and following target DNA hybridization at the nanometer scale. The profile of the NTS surface prior to DNA probe immobilization (immediately after Silanization) confirmed the SEM image characterization of NTS with pores distributed evenly throughout the surface (Figure 5-21). After probe immobilization, a small change in the surface profile was observed, with some surface leveling and deposition of matter in the pores and inter- pore spaces occurring on the chip surface (Figure 5-22). Following target DNA hybridization on the NTS biosensor chip (and after washing steps and drying under nitrogen gas stream), AF M showed that the surface profile changed drastically with smoothening of the surface observed at the nanometer scale, indicating extensive deposition of matter in the pores as well as on the inter-pore spaces (Figure 5-23). This is hypothesized to be from the hybridization of DNA target to the biosensor chip surface, as care was taken to prevent non-specific binding of organic matter to the biosensor surface through use of surface blocking agents (bovine serum albtunin), washing steps, and a high degree of hybridization specificity by maintaining the hybridization temperature at 59°C (melting point of capture probe was 60°C) with a standard hybridization buffer. 5. 3. 4.3 Electrochemical detection of DNA using nano-tubular Si biosensor A cyclic voltammogram (CV) is a valuable and convenient tool to monitor the barrier of the modified electrode because electron transfer between the solution species and the electrode must occur by tunneling either through the barrier or through the defects in the barrier. When an electrode surface is modified by some materials, the electron-transfer kinetics of F e(CN)'45 is perturbed, in turn affecting the output current. Therefore, cyclic voltammetry was chosen as a method to investigate the changes 158 .gzaufinofia: 2.5.:— TU\E: ooo.Om N o o >mu\Ec ooo.OOH X . E: 159 29.... En: WM. wrocm 26 .5 D E: oo.Om wrmum mumo ucowo: mama wmmEH 0mm moansmm mo Lonszz NI omH.H cum; cmum E: 0.00m oNrm :mum onoumocmz mucosagumCH hmumomo 5:353:55 2.9:. <2: .8... 35 558:. <75 833-32 a cage 2%. .33 2:»...— >wU\Ec ooo.Om N o 325: 000 .03 x 03 0 EC . orocm usomr WMM 71: orocm 3om> _mu Ec oo.Om wFMUm muwo usmwwx mumo wmmEH 0mm mw—aEmm mo LwnE32 N: omH.H mum; :muw E: 0.00m onm zoom waoumocmz mucosagumCH quwuwo 160 £23552 <75 :35 .8: 35 .8582 <2: 3322.2 .8 one... 2.: .m~-m Ema >mu\5c ooo.Om N o >wu\e: ooo.OOH x 8H 0 EC wanes uEOWF me wrocm 3om> film E: oo.Om waUm mumo usurp: wumo wmmEH 0mm mwFQEmm mo LonEzz NI me.H mum: :wum E: 0.00m onm :mum oaoumocmz mucosagum:H quwmmo 161 in electrochemical behavior of the system after each step, from Silanization to DNA target hybridization. Figure 5-24 illustrates the CV obtained with the NTS biosensor chip using 5 mM potassium ferrocyanide as the redox couple marker in 1 M potassium nitrate after different processing steps. Unlike the typical CV (Section 2.4.4 above, Figure 2-5 on page 54), the CV obtained with the NTS biosensor did not have a characteristic cathodic (1pc) or anodic peak current (Ipa), thus showing signs of irreversibility of reactions. The current went to a steady-state maximum on the cathodic side and was not peak-shaped (Figure 5-24). When the rate of electron transfer is sufficiently slow so that the potential no longer reflects the equilibritun activity of the redox couple (Fe(CN)6' + e’ <—> Fe(CN)2') at the working electrode surface, the reaction is considered irreversible. In such a case, the potentials corresponding to the cathodic and anodic current peaks (Ep) would change as a function of the scan rate. This steady-state current could be explained by envisioning the working electrode (NTS biosensor) placed at the bottom of the 3-electrode electrochemical cell (Chapter 4, Figure 4-7 on page 111) as a "dot", with the diffusion boundary layer being hemispherical in shape extending up into the solution. The amount of ferrocene diffusing to the working electrode surface would be defined by the voltune enclosed by the hemisphere, which is much smaller than a plane projecting into the solution as in the case of a planar electrode immersed completely into the electrochemical cell (Wang, 2000). The atypical CV response could thus be explained by the limited area available for the diffusion layer at the bottom of the cell for electron transfer combined with the use of silicon as the working electrode (lower conductivity) instead of platinum 162 .35 non—.083 <2: 683m; .53 353:: $3.283— ~S~§E-i .3 9:53 9::— a he Hugues—88.5.» 9:950 .v~-m 95w:— E 35:90.. N 3 F no o no- 7 3- P _ _ E _ c ”Crux?! F Boa <20 - v N 8.26 9: mm - m - mama..- F296 9: mmé <20 02 -F ”1.. . «EN- 0 m m w w . mom..- ( n . 80.0 e molm: 163 that is used to generate typical CV curves. Irreversibility of the reaction could also be observed from the CV profiles for the two consecutive cycles (Lines 2 and 3 in Figure 5-24). There was a consistent reduction (decay) in the current output on the anodic side from cycle 1 to cycle 2 for each set of hybridization conditions at various DNA concentrations (data shown in Sections 4 and 5). Running more than two CV cycles reduced the current output even further (data not shown). Without any DNA deposited on the NTS surface, the resistance or barrier offered to the flow of electrons was the least, resulting in a CV with a high current output (Line 1 in Figure 5-24). Afier immobilization of the single-stranded DNA capture probe, the current drastically decreased on the anodic side (Line 4 in Figure 5-24). After hybridization of the DNA target (1 ng/ul), the obtained CV showed relatively higher current values on the anodic side (Lines 2 and 3 in Figure 5-24) when compared to the chip with the immobilized DNA probe only. Immobilization of the single-stranded DNA would form an insulating diffusion barrier on the working electrode surface, hindering electron transfer through the working electrode. This insulating phenomenon was similar to that observed in previous studies that employed physical parameters (besides the conductivity of the working electrode or reporting molecules employed) for direct label-free electrochemical detection of specific DNA sequences. For example, Souteyrand et al. (1997) used a probe-coated field-effect silicon device for in situ impedance measurements of DNA sequences. The device displayed well-defined shifts of the impedance curves, corresponding to changes in surface charge induced by base-pair recognition. Similarly, Berggren et al. (1999) demonstrated a positive change in the capacitance of a thiolated (insulating) 164 oligonucleotide modified gold electrode caused by hybridization of the complementary DNA strand (and the corresponding displacement of solvent molecules from the surface). An even greater insulating property of a single-stranded DNA monolayer on the gold electrode was observed in a previous study by the same authors (Berggren and Johansson, 1997) The intrinsic electroactivity of DNA (Palecek, 1996) restores the anodic current upon hybridization of the target DNA. Of the four nucleobases, guanine and adenine are readily oxidized. The decreased guanine and adenine response of the immobilized oligonucleotide probe restored upon hybridization of the complementary strand has been used for detecting RNA hybridization (Wang et al., 1995). Other studies have also demonstrated a similar increase in signal with hybridization of the target DNA strand with and without reporting molecules such as Hoechst 33258 or [Ru(bpy)3]3+ (Wang, 1999; Kobayashi et al. , 2004; Lee et al., 2004; Li and Hu, 2004). 5. 3. 4.4 Statistical analysis of CV data As a typical CV curve with anodic and cathodic peak currents could not be obtained with the NTS biosensor chip, the usual statistical method of peak analysis could not be employed to determine significant differences between mean current outputs before and after target DNA hybridization. The alternate procedures described in Chapter 4:Section 4.4.4 were employed to determine the performance of the NTS and planar Si biosensor chips in terms of sensitivity and specificity. These are described in greater detail in the results and discussion for Objectives 4 and 5. 165 5.4 OBJECTIVE 4 Determination of sensitivity of the nano-tubular silicon DNA biosensor in pure culture. 5.4.1 Sensitivig of the Biosensor using PCR amplified DNA PCR-amplified Salmonella DNA from Objective 3 (Section 5.3.4.1 above) was serially diluted so that the final concentration ranged from 10'9 ug/ul to 10'15 ug/ul. Following NTS and planar Si DNA biosensor functionalization and pre-hybridization cyclic voltammetry measurements, each of these DNA concentrations was tested with the two Si-based biosensors. The specific DNA concentration to be tested with the biosensor was diluted to a total volume of 5 ml using the DNA hybridization buffer and then heated to 59°C. The chips to be tested were then placed in the hybridization buffer solution containing the target DNA and allowed to hybridize for 45 min. Preliminary testing with a hybridization time of 60 min versus 45 min showed no added advantage in terms of signal output. Thus, a hybridization time of 45 min was chosen for all experiments giving a total assay time of 60 min (45 min for hybridization and 15 min for the cyclic voltammetry testing procedure). After hybridization, the NTS and planar Si chips were subjected to cyclic voltammetry for the final time. 5. 4. 1.] Cyclic voltammograms Figure 5-25 shows the CV curves for the NTS biosensor chips tested for different PCR-amplified Salmonella DNA concentrations with the mean current values plotted against the corresponding applied potential. For the sake of clarity of the cyclic voltammograms (CVs) in Figure 5-25, the standard errors of the curves are not shown. 166 .3938» 535303 hem—.365 <2: 583..th 5m? 52:an <75 fluttoaam Sfiaeeaum. cocanaJ—Um @833 5:33... .8.— nfiaaueafiaxg Ego .muum Baum...— E .3232. N 3 F was o no- 7 3- _ _ C p p _ mo.:m.# Boa o_ grab—59:8 3»: 2. a .8583 a 5.53-82 5:» <75 auntie snagstu 2535.QO he «:3 25% a3 2:5 E aucsom N F 0 Fl ml _ _ _ _ _ .mdl -wdl Im.OI xvdl (V) JUNJNO Im.O| IF.OI -od 173 the points in the curve as opposed to a single peak point (anodic and/or cathodic current). Moreover, when calculating the integral charge value, the time factor was also used along with the voltage, incorporating any effects of the time factor into the model automatically. Unlike the SPLINE curve smoothening procedure, calculation of the Delta Q did not affect the original curve data values, thus providing a measure that could easily be used for comparison under different experimental conditions. Table 5-3 and Table 5-4 provide AQ values for PCR-amplified S. Enteritidis DNA from cycle 1 and cycle 2, respectively, of the CVs obtained for various DNA concentrations using the NTS biosensor chip. Delta Q values for cycles 1 and 2 were analyzed separately because of the seemingly significant differences observed in the current output for the two cycles (due to irreversibility of the redox couple reaction). Results of ANOVA performed on the AQ values are shown in the tables. The standard errors provided in the Tables are common to each experimental group. One of the assumptions of AN OVA was homogeneity of variances, i.e., it assumed that the three hybridization treatments (blank, before and after) have the same variance and by doing so, it increased the power of the analysis. The overall variance was obtained from the residuals of all treatments, and consequently, had more degrees of freedom than any individual variance. In both cycles, the AQ values for CVs corresponding to those ‘After’ DNA target hybridization decreased in magnitude with a decrease in DNA concentration. For example, for cycle 1, the mean AQ values for CVs corresponding to 1 ng, 0.1 ng and 0.01 ng were -45.767j:3.72, -14.467:i:2.33, and -12.633d:1.79 mC, respectively. This trend was observed in Cycle 2 as well. Delta Q values corresponding to CVs ‘Before’ DNA hybridization remained fairly steady at -18 to -57 uC irrespective of the concentration of 174 Table 5-3. Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with NTS biosensor at various concentrations using cycle 1 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) After, AQ (mC) 1 ng -l73.333i3.72° -0.23li3.72“ -45.767:t3.72b 0.1 ng —60.767i2.33° -O.306i2.33a -14.467:t2.33b 0.01 ng -29.867:t1.79° -0.237i1.79“ 426332179“ 1 pg -77.167i19.ZSa -0.183:l:19.25" 777344925“ 0.1 pg -23.600:t2.73b 057691.73“ -5.933i2.73“ 0.01 pg 484005.40“ -0.571=t5.40“ 00765.40“ 1 fg -15.867i1 .22b 0461421 .22“ -0.030i1.22“ Different superscripts for means across the row indicate significantly diflerent charge values (p<0. 05). Table 5-4. Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with NTS biosensor at various concentrations using cycle 2 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) Afier, AQ (mC) 1 ng -148.000i:2.70° 02032.70“ -39.667i2.70b 0.1 ng -56.833:t1.24c -O.249:l:l .24“ -l3.733:i:1.24b 0.01 ng -25.000i2.49“ -0.174i2.49“ 979712.49“ 1 pg -51.100:t1.15° -0.153:t1.15“ -6.267i1.15b 0.1 pg -24.l33:t1.94b -O.210i1.94a -4.770i1.94“ 0.01 pg -49.000:t6.17b -O.349:t6.17a -0.066i6.17a 1 fg -l4.267:tl.55b -0.192i1.55“ -0.040=t1.55“ Dzflerent superscripts for means across the row indicate significantly different charge values (p< 0. 05). 175 target DNA hybridized later (Table 5-3). ‘Blank’ AQ values prior to DNA immobilization on the NTS biosensor chips varied the most from about -16 to -173 mC for various DNA concentrations. However, this variation did not affect the biosensor performance as the focus of the assay was the mean difference between AQ values for the ‘Before’ and the ‘After’ target DNA hybridization steps. The NTS biosensor chips could detect PCR- amplified S. Enteritidis DNA in the dynamic detection range of 1 ng to 10 pg with cycle 1, and 1 ng to 1 pg with cycle 2. Thus, the lower limit of detection (sensitivity) for NTS biosensor chips with PCR-amplified Salmonella DNA was between 1 pg and 10 pg. This difference in the dynamic range of detection and sensitivity could be due to the larger variation in AQ values observed during cycle 1 as compared to cycle 2 under all experimental conditions investigated. The sensitivity for NTS was similar to that established in previous studies using modified gold surfaces as the biosensor platform in combination with various reporting molecules (Wang et al. , 1996; Li and Hu, 2004; Fu et al,2005) Sensitivity analysis of the planar Si showed that the biosensor chip was unable to successfully detect the PCR-amplified DNA target even at 1 ng level ( Table 5-5). With both the cycles, there was no significant difference between the “Before” and “After” hybridization steps (Table 5-6) at any DNA concentration level. The low current CV outputs for planar Si biosensor chips (resulting from significantly lower surface area available for the electrochemical reaction) makes planar Si more susceptible to variations in the data from replicate to replicate (biological or technical). 176 Table 5-5. Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with planar Si biosensor at various concentrations using cycle 1 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) After, AQ (mC) 1 ng —0.197i0.l3a O.132:t:O.13a -O.014:l:0.13a 0.1 ng -O.l67:t0.11a -O.037i0.11a -0.027:t0.11a 0.01 ng 0.094zt0.108 0.09Oi0.10a -0.021:I:O.10a 1 pg 0.038:I:O.11a 0.022:I:O.11a 0.069i0.1la Different superscripts for means across the row indicate significantly different charge values (p<0. 05). Table 5-6. Integral charge (AQ, milli coulombs) values for PCR-amplified S. Enteritidis DNA with planar Si biosensor at various concentrations using cycle 2 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) After, AQ (mC) 1 ng -0.041i0.12“ 0.174i0.128 -0.018iO.IZa 0.1 ng -0.005=+:0.08“ 0.0562t0.08“ 001120.08“ 0.01 ng 0.012i005“ 0.051i0.05“ 000320.05“ 1 pg 0.034i:0.08‘ 0.079zt0.08‘ 0.004i0.08" Different superscripts for means across the row indicate significantly diflerent charge values (D< 0. 05 ). 5.4.2 Sensitivity of the Biosensor Using DNA from Pure Culture The DNA concentration in the overnight Salmonella pure culture was 113.85 ug/ml with an A260/230 ratio of 2.34, indicative of very high quality DNA. This stock solution was appropriately diluted for sensitivity testing with NTS and planar Si biosensors. 177 5.4.2.1 Cyclic voltammograms Figure 5-29 and Figure 530 show the CV curves for the NTS biosensor and planar Si chips, respectively, tested with different pure culture (extracted) Salmonella DNA concentrations obtained by plotting mean current values against the corresponding applied potential. As with CVs for PCR-amplified DNA, standard errors for the curves are not shown. The CVs obtained for pure culture DNA with NTS as well as with planar Si followed the same pattern as observed with CV output for PCR-amplified DNA. The anodic current for the CV before DNA probe immobilization (Line 1) was the highest of all the samples tested with the NTS biosensor platform. The output current cycled between 10 11A on the cathodic side to about -1 mA (slightly lower than that for PCR-amplified DNA) on the anodic side. The CV for the NTS biosensor chip after DNA probe immobilization (Line 9) was significantly lower (as determined statistically through delta Q analysis in Section 4.2.2) than the CV prior to probe immobilization with the current output cycling from 1 uA to —l uA. After the target DNA hybridization, anodic current increased again as observed previously, with the increase in magnitude of the anodic current being the highest for 0.01 ng/ul DNA. The other three concentrations also showed a change over the anodic current from the CV for the DNA probe. When planar Si biosensor chips were tested, the anodic current for the CV before DNA probe immobilization (Line 1 — No DNA) was again lower than that obtained with NTS biosensor chip (Figure 5-30). The output current cycled between 25 11A on the cathodic side to about ~20 uA on the anodic side. The CV for the planar Si biosensor chip after DNA probe immobilization (Line 9) and that after the target DNA hybridization were all in the same range. Based on Figure 5-29 and Figure 5-30, there appears to be a 178 .958. Eganpe .59 .8583 <75 3.2-th its 6053:: <75 mmEzuouum 53—353.». 0.5.15 9::— Goaoabxov 682:. .2328 .8.“ 3.9.3833?» 9:9»0 .aN-m 95w?— E .333. 3 F no 0 3. F- 3- _ _ _ _ . _ MOIW.NI . Boa 0.05). Again, the low current CV outputs for planar Si biosensor chips made planar Si more susceptible to variations in the data and unable to detect the DNA hybridization event at any of the levels tested. 5.4.2.3 Interactive effects of variables Investigation of Single or Main effects using factorial ANOVA analyses provided some insight into possible interactive effects of variables on the output of the NTS and planar Si platforms. Four-way interaction of DNA hybridization event, DNA source, DNA concentration and the platform was shown to be highly significant (p<0.01). Further investigation of the Single effect of platform showed NTS to have a highly Significant effect on DNA hybridization (p<0.0001), as did the DNA source (PCR or 183 pure) (p<0.05). No interactive effects between DNA source and platform were found to be significant (p=0.70). Complete SAS data analysis output is provided in the Appendix C: Section C.2. Table 5-9. Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with planar Si biosensor at various concentrations using cycle 1 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) After, AQ (mC) 1 ng 008510.098 -0.088i0.09°b -0.4l4:l:0.09b 0.1 ng -0.307:t0.16" 008010.16“ -0.330i0.16’ 0.01 ng -0.298i0.12a -0.081i0.12" -o.212io.12“ 1 pg -o.113io.11a -o.1 17i0.1 1a -O.502:l:0.l 1° Different superscripts for means across the row indicate significantly different charge values m<0. 05). Table 5-10. Integral charge (AQ, milli coulombs) values for pure culture S. Enteritidis (extracted) DNA with planar Si biosensor at various concentrations using cycle 2 of cyclic voltammogram. DNA Hybridization Condition Concentration Blank, AQ (mC) Before, AQ (mC) After, AQ (mC) 1 ng 0.073zt0.058 -0.083i0.05b’ -o.23610.05b 0.1 ng -0.131i0.08‘ -0.056i0.08a -O.377i:0.088 0.01 ng -0.057:eo.03a -o.o14¢o.03“ -0.095+.o.03“ 1 pg (1017:0041a -o.o34io.041m -o.170:1:0.04b Different superscripts for means across the row indicate significantly different charge values (p<0. 05). 184 5.5 OBJECTIVE 5 Determination of specificity of the nano-tubular silicon biosensor using mixed bacterial cultures. 5.5.1 Specificity Testing In order to determine the specificity of the biosensor, DNA from Salmonella cultures was mixed with that of related bacteria (Escherichia coli) and tested with the NTS Salmonella biosensor. Figure 5-31 shows the gel electrophoresis image of PCR amplified DNA from an overnight S. Enteritidis mixed 1:1 with generic E. coli culture, as well as non-specific bacterial cultures (only E. coli). In the lane EC, no DNA bands were observed. Hence, there was no PCR amplification of the E. coli genome, indicative of the specificity of the PCR primers used. The thickness of the PCR-amplified ‘Salmonella-E. coli mix’ DNA bands increased with the amount of culture inoculum used for PCR (1-5 ul). The DNA bands in lanes Sl-SS were observed parallel to the 500 bp band in marker lane (M). For specificity testing, extracted genomic E. coli DNA as well as the mixture of Salmonella and E. coli DNA was allowed to hybridize with the NTS biosensor. The signal generated was compared statistically against negative and positive control samples of Salmonella Enteritidis DNA to determine the specificity of the biosensor. 5.5.2 Cyclic Voltammograms Figure 5-32 shows the CV curves for the NTS biosensor chips tested with genomic E. coli DNA at 1 ng/ul and 1 pg/ul concentrations obtained by plotting mean current values against the corresponding applied potential. For comparison, the CVs for 1 ng/ul 185 MC SISZS3 S4 SSEC 1.2 Kbp 1.0 Kbp ' 500 bp 300 bp 100 bp Figure 5-31. Gel electrophoresis of PCR-amplified Salmonella and extracted E. coli DNA. S] to S5 — l to 5 ul Salmonella-E. coli (1:1) culture mix used for PCR. Key: M — Marker, C — negative control, S — Salmonella-E. coli, EC — E. coli and 1 pg/pl of Salmonella are included along with the CVs for the two E. coli DNA concentrations. The anodic current for the CV before DNA probe immobilization (Line 1) was the highest of all the samples tested with the NTS biosensor platform. The CV for the NTS biosensor chip after DNA probe immobilization (Line 6) was significantly lower (as with all previous experiments) than the CV prior to probe immobilization. Afier the E. coli non-target DNA hybridization at both levels were tested (Lines 4 and 5), anodic current did not increase, as it did with Salmonella DNA previously (Section 5.4.1.2 above and Figure 5-32), because there was no hybridization of a complementary target to the NTS biosensor chip. However, when a 1:1 mixture of lng/ul solution of Salmonella and E. coli DNA was tested, an increase in the magnitude of the anodic current from that of 186 Amaze. £95925 9% 5883 «z: 3...“..-th a? 353.... <75 cow—3-55 £8 .m can <75 Cow-:5 mmeumaouam— 53:35am. .5. mfiauweafiaxg 9:93 a: comm-.3590 .Nm-m 95$...— 2.8.5.... N m... P no o 3. F- .3- _ _ _ _ . . 8m? . Boa