DEVELOPMENT OF A NANOPARTICLE DNA BASED BIOSENSOR FOR THE SHIGALIKE TOXIN 1 DETECTION USING CO-POLYERMIZATION HYBRIDIZATION
READOUT AMPLIFICATION

By
Michael Jeffrey Anderson

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Biosystems Engineering
2012

ABSTRACT
DEVELOPMENT OF A NANOPARTICLE DNA BASED BIOSENSOR FOR THE SHIGALIKE TOXIN 1 DETECTION USING CO-POLYERMIZATION HYBRIDIZATION
READOUT AMPLIFICATION
By
Michael Jeffrey Anderson

Approximately 3.4 million deaths occur each year due to lack of clean water. The
Shigatoxigenic group of Escherichia coli (STEC) bacteria causes approximately 2.2 million of
these deaths and nearly a billion cases of illness. Current approved testing methods for
Escherichia coli require a 12-24 hours growth of the sample in order to test for an indicator
species. This indicator species only suggests possible E. coli O157:H7 presence, but does not
confirm it. Direct culture testing cannot identify the pathogenic STEC bacteria either.
Confirmation of toxicity requires molecular methods of identification leading to specialized
equipment needs and higher testing costs. Typical molecular methods use polymerase chain
reaction (PCR) to amplify a very specific target DNA sequence, indicating the presence of a
specific microorganism. PCR requires a thermocycler and a clean laboratory environment.
Recent work has been successfully explored using nanomaterial based systems to identify the
same genes used in a PCR reaction in a simpler detection system. The nanomaterials are used for
both signal amplification and as the detection molecule. In this dissertation work a biosensor
utilizing nanoparticles has been developed for the detection of Shiga-like toxin 1 gene that is
present in Escherichia coli O157:H7. The system has been successfully constructed using a
novel carbohydrate coated gold nanoparticle for delivery of the self assembling copolymerization DNA oligonucleotide reporters. A set of self-assembled single-stranded DNA

(ssDNA) molecules has been developed to amplify a target sequence without the use of enzymes.
The full detection system incorporates the gold nanoparticle and a magnetic microparticle into a
DNA recognition step. The gold nanoparticle is used for both co-polymerization detection and
electrochemical detection. Input material (genomic DNA) is extracted using a modified
commercial extraction method to produce PCR quality DNA from whole bacterial cells. When
extraction and recognition elements are combined, a limit of detection for E. coli O157:H7 of
5
1
10 cells/mL using co-polymerization and 10 cells/mL with electrochemical detection has been

achieved. Sample preparation from spiked culture samples until final detection takes a total of 7
hours. Electrochemical detection provides only a presence or absence indicator, where tethered
co-polymerization is able to provide quantitative values of the input bacterial concentration. So a
combination of co-polymerization amplification and electrochemical detection can provide the
potential for more sensitive and quantitative measurement without the need for enzymes as in
PCR applications.

Copyright by
MICHAEL JEFFREY ANDERSON
2012

ACKNOWLEDGEMENTS

I sincerely thank everyone who has contributed and supported this research over the years. I
would like to specially thank Dr. Evangelyn Alocilja for believing in me and making everything
possible. As a mentor she has been encouraging, supportive, and very patient in my research and
growth as a scientist. Equally important, Dr. Alocilja has been both a friend and taken me into
her family. Her support has been crucial in graduate career and shaping me as a responsible
scientist. I would like to thank my SMART scholarship sponsor, Dr. Clint Smith and his team,
for their support of my educational process and the opportunity to use my research in an applied
setting to help people. I would also like to thank the members of my Ph.D guidance committee:
Dr. Gary Blanchard, Dr. Xuefei Huang, and Dr. Renfu Lu for their support.

I would also like to extend my sincerest gratitude to my friends and family for all their support,
patience, and belief in me during my Ph.D pursuit. Everything I have accomplished has been a
combination of the support from all those in my life.

Michael Anderson

v

TABLE OF CONTENTS
LIST OF TABLES....................................................................................................................... viii
LIST OF FIGURES .........................................................................................................................x
Chapter 1: Introduction ....................................................................................................................1
1.1 Hypothesis..............................................................................................................................2
1.2 Research objectives................................................................................................................3
1.3 Research significance and novelty.........................................................................................3
Chapter 2: Literature review ............................................................................................................6
2.1 Escherichia coli and coliform detection ................................................................................6
2.1.1 Traditional methods of detection ....................................................................................9
2.1.2 Antibody detection........................................................................................................12
2.1.3
Molecular DNA methods of detection...................................................................14
2.2 Enzymatic amplification ......................................................................................................16
2.2.1 Polymerase chain reaction (PCR) .................................................................................17
2.2.2 Rolling circle amplification (RCA) ..............................................................................19
2.2.3 Loop-mediated isothermal amplification (LAMP) .......................................................20
2.3 Non-enzymatic amplification methods ................................................................................23
2.3.1 Hybridization chain reaction.........................................................................................23
2.3.2 Isothermal chain elongation..........................................................................................25
2.3.3 Triplex signal enhancement ..........................................................................................27
2.3.4 Branched DNA amplification .......................................................................................27
2.4 Nanoparticle based biosensor platforms ..............................................................................28
2.4.1 Nanoparticle sensing.....................................................................................................30
2.4.2 Fluorescence detection..................................................................................................36
2.4.3 Targets...........................................................................................................................37
2.5 Gold nanoparticle synthesis .................................................................................................37
Chapter 3: Synthesis and characterization of dextrin gold nanoparticles ......................................39
3.1 Introduction..........................................................................................................................39
3.2 Materials and methods .........................................................................................................40
3.2.1 Optimization of gold nanoparticle synthesis.................................................................40
3.2.2 Gold nanoparticle characterization ...............................................................................41
3.2.3 Gold nanoparticle functionalization..............................................................................41
3.3 Results and discussion .........................................................................................................42
3.3.1 Gold nanoparticle characterization and synthesis.........................................................42
3.3.2 Effects of dextrin concentration, pH, and temperature .................................................44
3.3.3 Reaction mechanism .....................................................................................................50
3.3.4 Gold nanoparticle functionalization..............................................................................57
3.4 Conclusions..........................................................................................................................59

vi

Chapter 4: Spectral co-polymerization mass amplification detection of Bacillus anthracis
Stern using sandwich assay identification .....................................................................................61
4.1 Introduction..........................................................................................................................61
4.2 Materials and methods .........................................................................................................62
4.2.1 DNA target generation..................................................................................................62
4.2.2 Sandwich assay .............................................................................................................65
4.2.3 Co-polymerization sequence design .............................................................................69
4.2.4 Co-polymerization detection.........................................................................................71
4.2.5 Quantum dot attachment ...............................................................................................72
4.3 Results and discussion .........................................................................................................74
4.3.1 Co-polymerization sequences .......................................................................................74
4.3.2 Dye specificity ..............................................................................................................74
4.3.3 Hybridization banding ..................................................................................................79
4.3.4 Co-polymerization sensitivity.......................................................................................81
4.3.5 Quantum dot-dye attachment........................................................................................84
4.3.6 Electrochemical detection of quantum dot-dye ............................................................86
4.4 Conclusions..........................................................................................................................87
Chapter 5: Co-polymerization hybridization sandwich assay for detection of the Shigalike toxin 1 in Escherichia coli O157:H7 ......................................................................................89
5.1 Introduction..........................................................................................................................89
5.2 Materials and methods .........................................................................................................90
5.2.1 Primer and probe generation for Shiga-like toxin 1 (stx1) gene...................................90
5.2.2 Assay particle generation..............................................................................................92
5.2.3 DNA target extraction...................................................................................................94
5.2.4 Sandwich assay .............................................................................................................95
5.2.5 Detection .......................................................................................................................96
5.3 Results and discussion .........................................................................................................99
5.3.1 Primer and probe generation evaluation for Shiga-like toxin 1 (stx1) gene .................99
5.3.2 Gold nanoparticles functionalization and detection mechanism ................................102
5.3.3 DNA extraction optimization......................................................................................106
5.3.4 Detection .....................................................................................................................107
5.4 Conclusions........................................................................................................................116
Chapter 6: Conclusion and future work .......................................................................................117
APPENDIX A: DATA.................................................................................................................123
A.1 CHAPTER 3 DATA .........................................................................................................123
A.2 CHAPTER 4 DATA .........................................................................................................153
A.3 CHAPTER 5 DATA .........................................................................................................157
REFERENCES ............................................................................................................................176

vii

LIST OF TABLES

Table 1-1. Research contribution of this dissertation project to the literature.

5

Table 2-1. Estimated infection from water related agents are taken from Gleick
(2002).

7

Table 2-2. Commonly used gene targets for coliform and Escherichia coli
detection.

15

Table 4-1. Probe sequences generated for pagA gene in Bacillus anthracis
Sterne.

63

Table 4-2. Thiol functionalized species of DNA for particle decoration.

66

Table 4-3. Engineered co-polymerization ssDNA strands.

74

Table 5-1. PCR primer properties and design optimization for the stx1 gene.

90

Table 5-2. stx1 primers and amplified sequence (95 bp).

91

Table 5-3. Sandwich assay particle probe sequences.

91

Table 5-4. Co-polymerization sequences. Thiol sequences are for AuNP
attachment. FAM was used for direct fluorescence testing, ABThiol and BA were used for standard co-polymerization detection
and C-Linker, AB, and BA were used for tethered copolymerization detection.

92

Table 5-5. Primer sequences for stx1 and stx2 used to test primers generated for
sandwich assay.

99

Table 5-6. Lane description for Figure 5-3. Lanes 1,7 and 13 do not contain
PCR reactants, lanes 2-6, 8-12, and 14 underwent PCR reaction.

100

Table 5-7. Comparison of DNA extraction methods.

107

Table A-1. Absorbance data of gold nanoparticles for Figure 3-1.

123

Table A-2. Additional absorbance data of gold nanoparticles for Figure 3-1.

131

Table A-3. Particle diameter data for Table 3-3.

139

viii

Table A-4. Dextrin generation conditions for gold nanoparticle size data for
Figure 3-4.

140

Table A-5. Data for pH generation conditions for gold nanoparticle size data
Figure 3-5.

141

Table A-6. Data for temperature generation of gold nanoparticle size data for
Figure 3-6.

142

Table A-7. Data for UV absorbance vs. time for Figure 3-7.

143

Table A-8. Data for UV-Vis absorbance of dextrin species for Figure 3-9.

144

Table A-9. Data for DNA efficiency attachment for Figure 3-10.

152

Table A-10. Fluorescence data for Figure 4-8.

153

Table A-11. Additional fluorescence data for Figure 4-8.

154

Table A-12. Differential pulse voltammetry data for Figure 4-10.

155

Table A-13. Differential pulse voltammety data for Figure 5-7.

157

Table A-14. Additional differential pulse voltammety data for Figure 5-7.

160

Table A-15. Differential pulse voltammetry data for Figure 5-8.

163

Table A-16. Additional differential pulse voltammetry data for Figure 5-8.

165

Table A-17. Differential pulse voltammetry data for Figure 5-9.

167

Table A-18.Additional differential pulse voltammetry data for Figure 5-9.

170

Table A-19. Fluorescence data for Figure 5-10.

173

Table A-20. Fluorescence data for Figure 5-11.

174

ix

LIST OF FIGURES
Figure 2-1. Schematic representation of a polymerase chain reaction. A doublestrand target is heated to create two single-stranded species.
Primers hybridize to each strand creating double-stranded regions
for enzyme binding. The polymerase enzyme binds and builds on
the primer creating a copy of the initial strand. (For interpretation
of the references to color in this and all other figures, the reader is
referred to the electronic version of this dissertation.) ..........................................18
Figure 2-2. Common RCA modes of reaction. (a) Initial binding; (b) Reaction
product; (c) Binding of internal DNA sequence; (d) Double RCA,
a second primer is added to create a traditional double-stranded
product (adapted and modified from Demidov, 2002). .........................................20
Figure 2-3: Cartoon illustration of the LAMP method. (a) Primer design location.
Six primer sites are required and noted as F1-F3 for forward
primer and B1-B3 for reverse primer. Primers notes with ‘c’ are
complimentary to the associated sites; (b) The first primer to bind
(FIP) adds a region during the first amplification, step 1. Step 2
uses an external primer (F3) to free the newly created DNA strand.
Step 3 uses a new primer (BIP) to add a second region to the other
end of the new DNA strand. A second external primer (B3) is used
to free the strand. Now is step 5, the added DNA and then end of
the target self-hairpin creating a self primed sequence (adapted and
modified from Notomi et al., 2000).......................................................................22
Figure 2-4: Hybridization chain reaction mechanism. (a) The two hairpin system
at start of reaction; (b) Addition of the target (initiator) and initial
hairpin opening; (c) Exposed sites on first hairpin cause the second
hairpin to open and hybridize (adapted and modified from Dirks
and Pierce, 2004). ..................................................................................................24
Figure 2-5. Isothermal chain elongation. (a) Single round of hybridization; (b)
Target is linked to the tethering molecule via reporter B.
Additional reporter B hybridizes and allows for additional target
binding and subsequent reporter B binding. Addition of a quantum
dot-streptavidin complex binds to biotin on the reporter B
molecule (adapted and modified from Song, Lau and Lu, 2012). .........................26
Figure 2-6. End product of branched DNA amplification. Target nucleic acid is
captured and hybridized to a second ssDNA to allow for additional
labeled probe binding (adapted and modified from Tsongalis,
2006). .....................................................................................................................28

x

Figure 2-7. Schematic diagram of the components in a biosensor system. ...................................29
Figure 2-8: DNA detection using the sandwich assay. The magnetic microparticle
(MMP) and gold nanoparticle (Au) are mixed with a DNA target.
A sandwich is formed between the MMP-DNA-Au which is
magnetically separated. The separated product contains Au
particles only when DNA target is present. Measurement is then
performed based on the reporter used (adapted and modified from
Nam, Stoeva, and Mirkin, 2004)............................................................................32
Figure 2-9: Bio-barcode detection overview. (a) Capture and release of the biobarcode based on target hybridization; (b) Bio-barcode is
hybridized to a surface, providing a tethering molecule for a
second gold nanoparticle (adapted and modified from Thaxton et
al., 2005) ................................................................................................................33
Figure 2-10: Electochemical detection of gold nanoparticles. (a) Formation of the
sandwich structure; (b) Gold nanoparticle recovery and detection
(adapted and modified from Torres-Chavolla and Alocilja, 2011)........................35
Figure 3-1: UV-Vis spectra time monitoring of gold nanoparticles synthesis.
Generation conditions 10 g/L dextrin, pH 9.0, 50°C (adapted and
modified from Anderson et al., 2011). ..................................................................43
Figure 3-2: Visual time monitoring of gold nanoparticles synthesis. Generation
conditions 10 g/L dextrin, pH 9.0, 50°C (adapted and modified
from Anderson et al., 2011)...................................................................................44
Figure 3-3: Transmission electron micrographs of gold nanoparticle. AuNP
synthesis using various dextrin concentrations. (a and b) 20 g/L, (c
and d) 10 g/L, and (e and f) 2.5 g/L. Scale bars of a, c, and e are
equal to 100 nm (adapted and modified from Anderson et al.,
2011). .....................................................................................................................46
Figure 3-4: Average gold nanoparticles diameter as a function of generation
dextrin concentration (adapted and modified from Anderson et al.,
2011). .....................................................................................................................47
Figure 3-5: Average gold nanoparticle diameter as a function of generation pH,
ranging from pH 7.0 – 11.0 (adapted and modified from Anderson
et al., 2011). ...........................................................................................................49
Figure 3-6: Average gold nanoparticles size as a product of generation
temperature and dextrin concentration (adapted and modified from
Anderson et al., 2011)............................................................................................50

xi

Figure 3-7: UV-Vis monitoring of reaction completion. ...............................................................52
Figure 3-8: UV-Vis monitoring of proposed reaction mechanism. ...............................................53
Figure 3-9: FTIR of capping agent. (a) Stock dextrin; (b) Pre-synthesis reactant;
and (c) Freed capping dextrin (adapted and modified from
Anderson et al., 2011)............................................................................................56
Figure 3-10: Comparison of dextrin and citrate generated gold nanoparticles for
ligand exchange compatibility. D=dextrin generated; C=citrate
generated; 12.4, 13, and 10.4 represent AuNP diameter used
(adapted and modified from Anderson et al., 2011)..............................................58
Figure 4-1: PCR of Bacillus anthracis DNA for the pagA gene. Lane 1: 500 µg of
100 bp ladder. Lane 2: blank. Lane 3: PCR product using primer in
Table 4-1. ...............................................................................................................64
Figure 4-2: SMCC labeling of the MMP with DNA. The amine labeled
microparticle attaches to the cross-linker sulfo-SMCC, releasing
sulfo-NHS. The reactive microparticle then bonds to the sulfhydryl
group on the DNA..................................................................................................67
Figure 4-3: Cartoon representation of co-polymerization. (a) Initial dsDNA
region; (b) Rearrangement of lower strand to created second copolymerization partner; (c) possible initial hybridization and
subsequent hybridization events (adapted and modified from
Anderson, Zhang, and Alocilja, 2011)...................................................................70
Figure 4-4: Possible terminal end configuration based on the two part copolymerization (adapted and modified from Anderson, Zhang, and
Alocilja, 2011). ......................................................................................................71
Figure 4-5: Agarose gel showing DNA dye testing. Lane 1: 1 µg of 100 bp ladder
with SYBR Gold. Lanes 2-4 were run with 45 ng BA ssDNA.
Lanes 5-7 were run with 15 ng of co-polymerized dsDNA. SYBR
Gold, PicoGreen, and ethidium bromide were used to stain lanes 2
and 5, 3 and 6, and 4 and 7 respectfully per manufacture
instructions (adapted and modified from Anderson, Zhang, and
Alocilja, 2011). ......................................................................................................76
Figure 4-6: Gel electrophoresis of SYBR 101-QD dye specificity. Lane 1: 100 µg
of 100 bp ladder. Lanes 2-4 and 5-6 are 450 ng of AB:BA at 10:1,
1:1, and 1:10 ratio respectively. Lane 1 stained with SYBR Gold,
lanes 2-4 stained with PicoGreen and lanes 5-7 stained with
SYBR101-QD (adapted and modified from Anderson, Zhang, and
Alocilja, 2011). ......................................................................................................78
xii

Figure 4-7: Optimization of co-polymer ratio. Lane 1: 1 µg of 100 bp ladder.
Lanes 2-8 contain 150 ng of total DNA with 10:1, 5:1, 2:1, 1:1,
1:2, 1:5, and 1:10 ratio of AB:BA respectively stained with 1x of
SYBR Gold (adapted and modified from Anderson, Zhang, and
Alocilja, 2011). ......................................................................................................79
Figure 4-8: Probe sensitivity. Line A is the BA ssDNA probe only. Lines B-F are
5, 10, 25, 50 and 100 ng respectively of BA probe challenged
against AB reporter (adapted and modified from Anderson, Zhang,
and Alocilja, 2011).................................................................................................82
Figure 4-9: Fluorescence of QD conjugated SYBR 101 dye. Dilutions of 60 bp
dsDNA stained with SYBR 101-AET or SYBR 101-QD. Lanes 1
and 2: 1 µg of 100 bp DNA ladder, lane 1 with SYBR 101-AET
and lane 2 with SYBR 101-QD. Lanes 3, 5, and 7 were run with
75, 30 and 15 ng of SYBR 101-AET stained dsDNA. Lanes 4, 6,
and 8 were run with 75, 30 and 15 ng of SYBR 101-QD stained
dsDNA. Samples were run in a 2% w/v agarose gel at 30 V for 2
hours in 1x TBE (adapted and modified from Anderson, Zhang,
and Alocilja, 2011).................................................................................................85
Figure 4-10: Differential pulse voltammetry of metallic particles. Peaks between 0.87 V and -0.90 V are seen indicating cadmium presence in all
three metallic samples (adapted and modified from Anderson,
Zhang, and Alocilja, 2011). ...................................................................................86
Figure 5-1: Probe attachment to magnetic particle via EDC/NHS cross-linker
reaction. DNA probe (R1) with 5’ end phosphate, Magnetic
particle (R2) with surface primary amine. .............................................................93
Figure 5-2: Schematic of the screen printed carbon electrode. Carbon working
electrode with silver/silver chloride combination reference/counter
electrode.................................................................................................................97
Figure 5-3: Agarose gel electrophoresis with PCR product. Combinations of stx
primer sets shown. Lane contents are listed in Table 5-6. ...................................100
Figure 5-4: Direct fluorescence AuNP construction. (a) AuNP after generation;
(b) Schematic of gold nanoparticle detection of end-labeled DNA
after sandwich assay. DNA is liberated from the AuNP via DTT
exchange. .............................................................................................................103

xiii

Figure 5-5: Standard co-polymerization AuNP construction. (a) AuNP after
generation; (b) Schematic of co-polymerization detection after
sandwich assay. The co-polymerization DNA reporter was
liberated and then reacted. ...................................................................................104
Figure 5-6: Tethered co-polymerization AuNP construction. (a) AuNP after
generation; (b) Schematic of co-polymerization tethering detection.
Co-polymerized DNA hybridizes to the AuNP before DTT
liberation and detection........................................................................................105
Figure 5-7: DPV response of gold nanoparticles after sandwich assay. Gold
response is seen as a peak between +0.2 and +0.4 V. The peak in
curve A is a result of the hydrochloric acid and MMP interaction
with the system with AuNPs................................................................................109
Figure 5-8: DPV response of thiol liberated AuNPs after sandwich assay. ................................110
Figure 5-9: DVP response from sandwich assay. AuNPs were separated using
95°C without DTT to break sandwich structure. .................................................112
Figure 5-10: Fluorescence detection of 6-FAM end labeled DNA AuNPs. DNA
was liberated using DTT exchange. Samples measured using
fluorescein filters set. ...........................................................................................113
Figure 5-11: Fluorescence detection of co-polymerization amplification of
sandwich assay. Blank samples are reported at the 0 cell count log
value. Samples measured using fluorescein filters set.........................................115

xiv

Chapter 1: Introduction

One in ten illnesses worldwide is attributed to water-borne sources, resulting in 3.4 million
deaths a year. The main contributors being enterohemorrhagic Escherichia coli (EHEC),
Shigatoxigenic group of Escherichia coli (STEC), and Salmonella [1, 2]. Rapid testing of water
supplies would help eliminate microorganism-associated illness through better water treatments
and awareness programs. The Environmental Protection Agency’s (EPA) current standard testing
and identification methods for microorganisms are all growth in a selective media [3, 4]. Growth
based methods are the standard for identification but are non-rapid, often requiring 18 or more
hours, and have a limited selectivity between related species. Culture plating based methods
identify bacteria by metabolic pathways, not pathogenic capabilities [5-7]. Antibody detection
involves the recognition of antigenic proteins on the cell surface of microorganisms and used in
both enzyme-linked immunosorbent assay (ELISA) assays and bio-barcode detection. Antibodies
are more rapid than culture techniques, but are limited by cost, cross-reactivities and storage
requirements [8]. Much like culture techniques, antibody recognition does not identify
pathogenic traits, but rather by binding surface antigens that are commonly found together with
pathogenic traits. More recent techniques have used DNA gene sequences for microorganism
detection and identification. DNA techniques currently require an amplification step for
detection, and polymerase chain reaction (PCR), isothermal amplification, and the bio-barcode
method have been explored for DNA detection [9-11]. PCR is an extremely sensitive technique,
but is often limited for laboratory use due to equipment and storage needs. Isothermal
amplification is less expensive to operate because of simplified equipment needs, but is still
limited to temperature sensitive enzyme storage. The bio-barcode technique has been used for
1

enzyme free detection and amplification of DNA, but DNA input and detection times are less
rapid.

This dissertation describes the generation of a fluorescence based biosensor for Shiga-like Toxin
1 (stx1) detection using gold nanoparticles and self assembling co-polymerization hybridization
amplification. Chapter 2 presents a review of the literature pertaining to technologies relevant to
this research project including gold nanoparticle generation, construction of a co-polymerization
hybridization DNA set, gold nanoparticle based detection, and detection of the stx1 gene from
bacterial culture. Chapter 3 describes the generation and size control of a carbohydrate gold
nanoparticle under alkaline reduction conditions. Chapter 4 describes the generation of a DNA
hybridization system and its use as a detection technology from a DNA hybridization assay. The
combination of an improved DNA extraction protocol, gold nanoparticle functionalized assay
particles and hybridization co-polymerization readout into a working biosensor is presented in
Chapter 5. Chapter 6 contains recommendations for future work and research extending from this
dissertation. The following section of this introduction describes the research hypothesis,
objectives, and novelty of the work performed.
1.1 Hypothesis
The research presented in this dissertation is based on the following hypotheses:
Gold nanoparticles can be synthesized with ‘green’ methodologies using a single step reaction,
under alkaline conditions, and a carbohydrate reduction and capping agent.
A set of short oligonucleotides can be engineered to self-assemble into longer double-stranded
DNA sequences detectable using fluorescence detection and intercalating DNA dyes.

2

A rapid DNA extraction method can be developed to provide quality genomic DNA input for
hybridization detection.
A biosensor platform can be developed using co-polymerizing DNA for readout in a
hybridization sandwich assay targeting the stx1 gene with increased detection compared to end
labeled oligonucleotides DNAs.
1.2 Research objectives
The overall objective of this dissertation research is to develop a rapid fluorescent detection
biosensor to detect the Shiga-like toxin 1 (stx1) gene.
The detailed objectives of this project are:
•

To synthesize gold nanoparticles under alkaline conditions using a carbohydrate capping
agent as an alternative to acidic citrate particle methodologies.

•

To develop a single-strand DNA detection system using a self assembly method called
co-polymerization hybridization.

•

To develop stx1 specific DNA probes and functionalize the carbohydrate generated gold
nanoparticles with the DNA probes.

•

To develop a fluorescent and electrochemical biosensor with the carbohydrate
functionalized gold nanoparticles based on co-polymerization hybridization.

1.3 Research significance and novelty
The novelty of the presented research in this dissertation relies on the use of a self-assembling
DNA system in conjunction with carbohydrate capped gold nanoparticles for use with genomic
DNA target and rapid fluorescence detection. Gold nanoparticles have been used as a carrier for
signal amplification previously, and most recently used for electrochemical detection through

3

reduction/oxidation potentiometry. Traditional DNA targets are enzymatically amplified
sequences utilizing multiple hybridization events or DNA reporters labeled with fluorescent
molecules. To the best of our knowledge, this is the first report of successful detection of a DNA
target from genomic input using carbohydrate generated gold nanoparticles with fluorescent copolymerization.

Detection of specific DNA sequences commonly requires extraction, purification and PCR
amplification of the target sequence. These steps often require the use of frozen enzymes,
expensive thermocyclers, or larger centrifuges limiting the range of physical locations the
detection can be accomplished. This approach is also more costly. Detection of fluorescently
labeled DNA systems has the ability to have high sensitivity but also requires larger equipment
needs with higher associated equipment costs. To the best of our knowledge, the techniques
described in this dissertation are the first time fluorescence detection has been accomplished
using extracted genomic DNA for the stx1 gene target using self assembling co-polymerization.
The following techniques require no special storage, no temperature requirements, and all
equipment can be portable. A summary of the presented research is listed in Table 1-1 and
comparison with current literature illustrates the novelty and scientific contribution [11-15].

4

Table 1-1. Research contribution of this dissertation project to the literature.

Subject
DNA hybridization detection
Self-assembling DNA for
fluorescent ssDNA detection
One step alkaline generated
glyco-nanoparticles
Biogenic AuNP DNA
functionalization
Sandwich assay detection
Direct fluorescence genomic
AuNP detection
Fluorescent detection of
genomic DNA by selfassembling DNA
amplification

References
[16]

[17]

[10]

[11]

[18]

[10]

[15]

[13]

[13]
[12]
[12]

This work

5

Chapter 2: Literature review

2.1 Escherichia coli and coliform detection
It has been argued that clean water is the most important resource for modern civilization. Each
year an estimated 3.4 million people die from water-borne diseases. Of these deaths,
approximately 2.2 million are due to diarrheal disease [19]. The main victims of diarrheal
disease are children five years and younger and the elderly. The causative agents of this disease
include viruses, protozoa, and bacteria [20]. The most common viral species include Adenovirus,
Enterovirus, Hepatitus, Norovirus and Rotaviruses with Adenovirus and Rotaviruses causing
more severe disease symptoms. Common Protozoa responsible for water-borne disease include
Cryptosporidium, Cyclospora, and Giardia. These two classes of water-borne disease agents are
resistant to disinfectants, have less severe effects than the bacterial agents, and are more stable in
water.

Bacterial causative agents include but are not limited to Escherichia, Legionella, Salmonella,
Shigellla, Vibrio and Yersinia geneses. Bacterial infections cause more severe diarrhea,
dehydration, and are life-threatening if left untreated. Hydration is an important part of treatment,
but without clean water, hydrating often reinforces the infection. Within the bacterial agents two
classes are most studied, as they are the major contributors to disease. The classes are the
enterohemorrhagic Escherichia coli (EHEC) and the Shigatoxigenic group of Escherichia coli
(STEC). The STEC group contains the Shiga toxin and Shiga-like toxin producing organisms
that include Escherichia coli (E. coli) O157:H7, Shigella dysenteriae, and various other non-

6

O157:H7 E. coli. Table 2-1 lists selected water related disease data, with diarrheal related cases
being the largest agent of death [21].

Table 2-1. Estimated infection from water related agents are taken from Gleick (2002).

Disease
Diarrheal
diseases
Intestinal
helminths

Yearly Estimated Yearly Estimated
Morbidity
Mortality
1,000,000,000

2,200,000

1,500,000,000

100,000

Schistosomiasis

200,000,000

200,000

Other

150,000,000

130,000

Relationship to
water
Unsafe drinking
water
Unsafe drinking
water
Unsafe drinking
water
Unsanitary washing
conditions

Water quality is often determined by measuring indicator organisms, since monitoring every
possible microbe is impractical and nearly impossible. Historically, the detection of a low
number of organisms has not been accurate. Water assessment is based on the detection of
bacteria that are normally found in high numbers along with the infectious targets. These
indicator organisms become detectable when individuals are infected and contagious, indicating
contamination and presence of the infectious agent. The currently measured bacterial indicators
are called coliform, coli-aerogens, or fecal coliforms [2]. Coliforms are a broad class of
microorganisms found naturally in water, soil, and warm-blooded animals and include
Escherichia, Enterobacter, Klebsiella, Citrobacter, Shigella and Salmonella. Many coliforms are
harmless to humans, but the presence of fecal coliforms in water often indicates contamination

7

through water run-off, improper sanitation, or insufficient treatment methods. The most common
fecal coliform group is Escherichia [22].

Within the coliform class, EHEC, STEC, and Salmonella groups are good indicator species for
water contamination. Both E. coli groups (EHEC and STEC) contain a conserved gene encoding
a Shiga-like toxin, which is also found in Shigella. The Shiga toxin is responsible for severe
diarrhea, abdominal pain, vomiting, bloody urine, and can cause death in children, the elderly
and immuno-compromised individuals through dehydration, hemolytic uremia, or renal failure
[23]. Each year, approximately 73,000 cases of illness specific to E. coli O157:H7 are reported in
the United States, with the more publicized cases being the food recalls of hamburger [24],
spinach [25] and cookie dough [26]. Domestically about 15% or 11,000 cases are specifically
attributed directly to water-borne sources [27].

The Environmental Protection Agency (EPA) has set a limit of zero detection of total coliform,
both harmful and harmless, with mandatory reporting when more than 5% of samples test
positive [28]. Testing of treated, bottled, and tap water can easily conform to zero tolerance.
Irrigation water, produce wash water, ground, and surface waters tend to contain non-fecally
derived bacteria that are ubiquitous in the environment. These non-fecal bacteria often pose no
risk to human health but will cause false positives in detection systems. Confirmation of coliform
presence has been limited to metabolic testing for governmental health monitoring which
requires between 18-24 hours for presence/absence determination. Identification of cell surface
markers has reduced detection time; molecular recognition methods have attempted to
investigate the genes responsible for pathogenicity.

8

The following sections discuss the accepted, traditional, unconventional, and novel methods of
coliform detection used in bacterial monitoring efforts.

2.1.1 Traditional methods of detection
Historically, determination of bacterial identification is based on metabolic differences.
Determination is through gas generation or colorimetric change. The EPA approved methods that
fall under this category are: multiple tube fermentation, membrane filtration, and
presence/absence testing.

Multiple tube fermentation involves growth of a sample in liquid media with an inverted tube
placed in the solution. The media used can be either lactose, lauryl tryptose, or lactose bile broth.
The coliforms are grown at 35-45°C for 24-48 hours under anaerobic conditions such that gas is
produced. The exact conditions determine if total coliform or fecal coliform are the identified
target. As a result of the growth conditions, results are reported as a most probable number,
which is a statistical estimate of the starting concentration. For example, this corresponds to a
37% likelihood of a positive reading when a sample containing 1 cell/mL is tested. Positive
quantification of fecal coliform requires a second fermentation of 24 hours to determine total and
fecal coliform amounts. Multiple tube fermentation remains useful for sample matrices that are
non-transparent or colored such as milk or environmental samples.

Membrane filtration is a combined concentration and growth assay. A typical detection would
filter a liquid sample through a 0.45 µm filter to capture all bacteria as well as larger particulates
9

on the filter membrane. EPA methods grow the filter on selective media agar, m-Endo-type agar,
containing lactose which produces red colonies after 24 hours of growth at 35°C. Other agar
media have been used including MacKonkey, MacKonkey-sorbital, ChromAgar, Rainbow agar,
and antibiotic augmented agars. These other types of media can be used with membrane
filtration, but are not approved currently by the EPA. Membrane filtration allows for sample
concentration, but has considerable limits for its use. Concentration on the filter membrane does
not provide optimal distribution of bacteria. This can cause over-crowding, uncountable plate
growth, and capture of non-target microbes that can out-compete weakened coliforms on the
filter. This method is used regularly for drinking water systems as it allows for large volumes of
dilute samples to be concentrated for detection.

Metabolic enzyme activity has been recently utilized for differentiation of microbe populations.
Two of the most common enzymes targeted in coliform detection are beta-D-glucuronidase and
beta-D-galactosidase. Detection is accomplished by conjugating a reporter molecule to a
metabolizable sugar. Upon proper metabolism, the reporter is released causing a change to the
system that is measured. It was found that the enzyme beta-D-glucuronidase was specific to
Enterobacteriaceae and contained in 95% of E. coli that were tested [29]. The enzyme beta-Dgalactosidase is contained by mainly E. coli, and conjugation of a reporter can give the probable
presence of E. coli when metabolized. Even though these enzyme systems are specific to the
organisms they target, false signals are possible. Shigella, Salmonella, and Yersinia are all
pathogenic Enterobacteriaceae which do not contain either enzyme. This would give a negative
result while still being capable of infection. False positives for E. coli can be generated by
Bacillus spp. and Aerococcus viridans when present [30]. A major advantage to enzyme linked

10

assays is multiple detections in a single sample. The commercial product, Colilert by IDEXX,
uses a dual substrate system. Coliform detection is accomplished by a yellow color change upon
metabolism of ortho-Nitrophenyl-β-galactoside, and E. coli determination is accomplished by
having a yellow color and also by a fluorescence signal after metabolism of 4Methylumbelliferyl-beta-D-glucuronide. This is just an example of a metabolic enzyme test that
has been approved by the EPA and is commercially available.

Various combinations of detection methods have been tried and include: enzymatic substrates,
selective growth media with membrane filtration, culture plating, and multiple tube fermentation.
Each of the possible pairings combines the various advantages and disadvantages of the different
methods involved. The combination of methods with light absorbency or fluorescence
measurements allows for detection of total coliforms in as little as 12 hours.

These EPA-approved and other metabolic methods have drawbacks however; the most apparent
is the required time from sample to detection. Common methods take 24-48 hours, and rapid
methods taking as little as 12 hours. Another issue is that these growth systems are subject to
contamination with other microorganisms. A high number of non-target microorganisms can
slow or retard growth of the target thus giving false negatives. When metabolic pathways are
shared among a large family of closely-related species, selective media may lack the specificity
needed for identification and give a false positive. Lastly, selective media do not always provide
optimal growth conditions thereby limiting growth of the target organisms. This can lead to false
negatives when there is low or stressed target present.

11

2.1.2 Antibody detection
Antibodies are proteins that have the ability to specifically bind to other molecules with a high
degree of specificity. The specificity of the antibody to the target can vary based on the species
of host used for generation, individual host differences, and the type of antibody. Antibody
recognition of bacteria uses surface antigens (features) such as the terminal sugars (O) on the cell
surface lipopolysaccharide (LPS), the capsule component (K) and the flagella antigens (H) as
common targets. When monoclonal antibodies are produced, each antibody is a clone and targets
the same antigen. Polyclonal antibodies are purified from blood serum and contain a mixture of
antibodies that can recognize different portions of the target antigen. Many antibodies available
for research use are uncharacterized, meaning the manufacturer does not know the exact target
antigen. These uncharacterized antibodies can lead to cross-reactivity, such as Klebsiella
pneumoniae, Klebsiella. oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter
amalonaticus, Citrobacter koseri, and some species of the Citrobacter freundii being reported as
E. coli O157:H7. Antibodies common to O157 have been reported to cross-react with O7 and
O116 LPS components [31] resulting in false positive identification of non-harmful
microorganisms.

To avoid the cross-reactivity of a single antibody binding event, two different antibodies can be
used in conjunction in a sandwich structure to increase specificity. The enzyme-linked
immunosorbent assay (ELISA) uses an antibody anchored to a surface. The target is allowed to
bind, and then a second antibody is challenged in the system. The second antibody is then
detected through an attached enzyme or by recognition with a third antibody attached to a
reporter. In an ELISA, two different antibodies are used for recognition. The double recognition
12

reduces non-specific binding and increases the specificity of the assay. Attachment of fluorescent
reporters to the antibodies allows for multiplex detection. Horseradish peroxidase and luciferase
are commonly used chemiluminescent reporters.

One downside of antibody recognition (capture) is that the target must come in close proximity
to be captured. For dilute solutions or large sample volumes the time for sample capture can be
on the scale of hours, providing bound target the chance to disassociate. To address this issue,
larger amount of antibody can be used to increase the chance of binding the target. A technique
called immunomagnetic separation (IMS) has been developed, combining antibodies and
magnetic particles. The antibodies are attached to the magnetic particle, allowed to mix in a
sample, and a magnetic field is applied and collects the magnetic particles. The magnetic
separation allows for good mixing of the sample and antibodies followed by a concentration step
on the magnet. IMS separation only provides the initial antibody recognition and requires
culturing, second antibody bonding, or other form of reporter for detection [32-34].

Antibody detection is rapid and can be specific with appropriate product selection, but it does not
measure pathogenicity. The use of surface antigens for detection is successful in many cases for
antibody binding, but surface antigens are not perfect. As mentioned before, cross-reactivities
can lead to false positive and false negatives, even with a two antibody system. The quick assay
times with higher antibodies or target concentrations, large target selection, and compatibility
with multiple reporter system make antibodies extremely useful tools for rapid screening, but not
always for identification of the pathogenicity of an organism.

13

2.1.3

Molecular DNA methods of detection

Molecular techniques of detection utilize the hybridization properties of DNA and RNA. DNA is
a more commonly used molecular species than RNA owing to the increased stability and ease of
amplification. Recognition is accomplished using Watson-Crick base pairing to bind to a known
DNA sequence. The DNA sequences targeted genetically encode for genes in the host organism.
Unlike antibodies, DNA hybridization can be used to look for genes targeting internal cellular
components such as toxins, enzymes, and proteins.

Identification of specific DNA sequences provides a detection technique with ~95% specificity
when properly designed primers and probes are used. Table 2-2 lists some common gene targets
for identification of waterborne coliforms.

14

Table 2-2. Commonly used gene targets for coliform and Escherichia coli detection.
Gene / Target Target Type

Coliform Target

Source

stx1

Toxin

O157 - Shiga Toxin

[9, 35, 36]

stx2

Toxin

O157 - Shiga Toxin

[9, 35, 36]

Surface Feature

O157:H7, O55:H7

[37]

fliC

Surface Feature

E. coli - H antigen

[38]

rfb

Surface Feature

E. coli - O antigen

[37]

lamB

Surface Feature

STEC - binding protein

[39]

eae

Surface Feature

E. coli - attachment protein

[40]

espB

Surface Feature

E .coli - attachment protein

[40]

phoE

Surface Feature

STEC - membrane protein

[41]

O157 LPS
antigen

Total coliform – betalacI

Enzyme

[42]
galactosidase

uidA

Enzyme

STEC - beta-glucuronidase

[43, 44]

STEC - beta-glucuronidase
uidR

Enzyme

[45]
regulation
Total coliform - lactose

lacZ

Enzyme

[39, 46]
metabolism

The use of pathogenic gene targets is a common method with molecular techniques, especially in
water and food safety. This safety concern arises when other detection methods give false
15

negatives and positives based on antigen recognition. Identification of a pathogenic gene, such as
Shiga toxin production, can be determined independently of the organism using DNA
hybridization. This becomes important when an antibody based assay is looking for E. coli but
misses Shigella. A Shiga toxin molecular assay would show positive and prevent possible illness
for both E. coli O157:H7 and Shigella.

When the above gene targets are in sufficient concentration, detection can be accomplished in a
number of hybridization based formats which include: polymerase chain reaction (PCR) [37],
real-time PCR [36], microarray hybridization [47], fluorescent labeled-DNA probes [48],
enzyme labeled DNA probes [49], Förster resonance energy transfer (FRET) [50], fluorescence
in situ hybridization (FISH) [51], colorimetric change [52], and change of mass systems [53].
The common requirement for most of the hybridization detection methods is sufficiently high
number of target for capture and/or relatively clean DNA input. Additional steps are required to
meet both of these input requirements. Commercially ready non-laboratory DNA testing methods
are limited and usually include enzymatic amplification [54].

2.2 Enzymatic amplification
Successful detection of any target requires a signal with sufficient strength for measurement.
Detection of STEC, E. coli, or stx1 containing organisms is currently accomplished through an
amplification reaction. Many novel methods have been developed for gene target amplification,
and have future potential use in microorganism detection. This section describes the enzyme
based amplification methods.

16

2.2.1 Polymerase chain reaction (PCR)
PCR is by far the most common enzyme based amplification method for DNA in use today.
Since its discovery in 1983, the use of PCR is now ubiquitous as a biological detection or
identification technique. The PCR method is described briefly, and is the basis for most of the
other enzyme-based methods. Figure 2-1 is a schematic representation of the PCR process.

17

5'

3'

5'

3'

5'

heat

3'

annealing

5'

3'

3'

5'

5'

3'
5'
5' 3'

5'

3'
5'

3'
3'

5'
3'

3'

3'

primer

denaturation

5'

5'

5'

primer
extenstion

3'
3'
5'

3'
5'

5'
3'

3' 5'

2nd cycle
Product = 2^n
n = # of cycles
5'
3'

3' 5'

Figure 2-1. Schematic representation of a polymerase chain reaction. A double-strand target is
heated to create two single-stranded species. Primers hybridize to each strand creating doublestranded regions for enzyme binding. The polymerase enzyme binds and builds on the primer
creating a copy of the initial strand. (For interpretation of the references to color in this and all
other figures, the reader is referred to the electronic version of this dissertation.)

18

The target is first heated to denature the DNA in to single-strands. Primers bind to each strand,
and the enzyme polymerase binds to the primer-target double-strand DNA region formed. The
polymerase polymerizes the primer in a 5’ to 3’ fashion completing new double-stranded DNA,
which is a copy of the original target. The process repeats between 20-30 cycles creating more
target exponentially.

PCR reaction conditions vary based on the target DNA sequence being amplified, the DNA
sequence of the primers used, and matrix interference. This highly sensitive amplification system
requires expensive amplification equipment and refrigerated reagents preventing the portability
of the system outside the laboratory environment.

2.2.2 Rolling circle amplification (RCA)
More advanced methods of primer design have been developed to reduce the complexity of the
amplification process. Rolling circle amplification utilizes a circular primer to bind to the target
DNA section. The polymerase enzyme then begins creating a copy of the bound section. Upon
exhausting the target sequence-circular primer region, the enzyme continues using the
circularized primer as the template. Once the entire circular primer is duplicated, the enzyme
displaces the recently generated double-stranded portion and continues replication and strand
displacement [55-57]. Strand displacement polymerases maintain attachment to the guide strand,
while removing the bound DNA in front of the polymerization direction. The product generated
from this style of amplification is a single-long single-stranded DNA sequence with a continually
repeated internal structure. Three methods of executing RCA are shown in Figure 2-2 [58].

19

Figure 2-2. Common RCA modes of reaction. (a) Initial binding; (b) Reaction product; (c)
Binding of internal DNA sequence; (d) Double RCA, a second primer is added to create a
traditional double-stranded product (adapted and modified from Demidov, 2002).

RCA benefits from simplified reaction conditions and equipment needs. Once the primer is
initially bound, only a single temperature is required for continuous amplification. Detection of
RCA product is common via electrophoresis or fluorescence measurement. Addition of labeled
primers is not well suited to RCA as the primer is not actually consumed in the reaction. Further
hybridization detection is also not well suited as the number of target molecules is not amplified
[59].

2.2.3 Loop-mediated isothermal amplification (LAMP)
Loop-mediated isothermal amplification is a novel method of using structure switching primers
to create a single long double-strand DNA output comprised of the target DNA sequence [60].
LAMP amplification is accomplished using a set of six primers and a strand displacement
20

polymerase enzyme. A set of primers initially binds to each of the single-strand targets in the
region of interest. Amplification is allowed to occur as it would in PCR for one cycle, Figure 23,b-1. A second primer set is then introduced into the solution and allowed to separate the
previously generated DNA, Figure 2-3,b-2. At this step, the first primer set has increased the
target size by introducing a possible loop structure. A third set of primers is added that upon
another round of amplification introduces a second possible loop structure into the target strand,
also increasing the product length, Figure 2-3, b2-5. When the second primer set is then added to
the double loop structure, the primer changes the loop structure opening up free DNA for a
polymerase to polymerize a new DNA strands, but unlike normal PCR, when the enzyme reaches
the loop structure, it continues back towards the point of origin. The strand displacement
polymerase pulls apart the structure until it reaches the point of origin, where a loop structure
exists also. This polymerase will continue along this cyclic amplification until the reactants are
exhausted.

21

B1
B2
B3

B1
B2
B3
B1c
B2c
B3c

F3c
F2c
F1c
F3c
F2c
F1c
F3
F2
F1

Figure 2-3: Cartoon illustration of the LAMP method. (a) Primer design location. Six primer
sites are required and noted as F1-F3 for forward primer and B1-B3 for reverse primer. Primers
notes with ‘c’ are complimentary to the associated sites; (b) The first primer to bind (FIP) adds a
region during the first amplification, step 1. Step 2 uses an external primer (F3) to free the newly
created DNA strand. Step 3 uses a new primer (BIP) to add a second region to the other end of
the new DNA strand. A second external primer (B3) is used to free the strand. Now is step 5, the
added DNA and then end of the target self-hairpin creating a self primed sequence (adapted and
modified from Notomi et al., 2000).

The LAMP reaction creates a single long double-stranded output. Once the initial primer set
creates the hair pinning target structure, the enzyme amplification reaction proceeds isothermally
until the reactants are exhausted or the polymerase degrades. For example, the system produces a
highly detectable target with fluorescent staining, but suffers from design constraints. The six
primer set is not compatible with all gene sequences, requires a large initial target (>200 bp), and
entails multiple steps with manual addition of the primers in a precise sequence. LAMP
demonstrates what careful primer design can accomplish though enzymatic polymerization.
22

2.3 Non-enzymatic amplification methods
Novel methods have been developed to remove the need for enzymatic amplification. The move
to create more stable assays based on DNA hybridization has provided some basic tools for
potential field-based methods. This section describes hybridization-based amplification methods.

2.3.1 Hybridization chain reaction
Hybridization chain reaction utilizes a dual hairpin DNA system that upon target binding causes
a chain reaction of hybridization. The system is based on the stored energy in the hairpin-loop
DNA reactant [17]. A single-stranded target binds to a free portion of the first hairpin structure
(H1). The target causes H1 to open, and the target continues to hybridize stabilizing H1 into a
linear structure. The newly exposed section on H1 then binds in the same fashion to the second
hairpin (H2). H1 and H2 then hybridize exposing a binding area on H2 that will then bind to H1.
This series of alternating hairpin opening and hybridization continues until the reactants are
exhausted. Figure 2-4 shows the hybridization scheme.

23

Figure 2-4: Hybridization chain reaction mechanism. (a) The two hairpin system at start of
reaction; (b) Addition of the target (initiator) and initial hairpin opening; (c) Exposed sites on
first hairpin cause the second hairpin to open and hybridize (adapted and modified from Dirks
and Pierce, 2004).

This system is enzyme free and produces large double-stranded DNA sequences. The hairpin
structure of the reagents provides for an inexpensive system of oligonucleotides that can produce
a large increase in size of the target. Detection in this system currently requires electrophoresis
readout. DNA intercalating dyes in solution used with this system do not show change in total
24

signal strength, as both the starting and end products are double-stranded. Even a single hairpin
opening could cause false negative detection. This opening event would be a thermodynamic
possibility leading to the same response as if the target were present. Readout would be possible
with a molecular beacon approach for fluorescence, but this would not alleviate the spontaneous
hairpin openings.

2.3.2 Isothermal chain elongation
Isothermal chain elongation is based on the concept of capturing a target single-strand DNA
sequence on a plate surface. The surface is decorated with a capture strand that hybridizes to the
target partially. The un-hybridized portion of the target is used to hybridize with a second strand
that is labeled with biotin. This second hybridization is also a partial hybridization, leaving a
portion of the strand open for binding to another target molecule [61]. Repeated hybridizations of
target generate a long double-stranded DNA sequence tethered to the plate surface. Detection is
achieved by binding of a quantum dot-streptavadin conjugate to the biotin attached to the second
hybridization molecule. Upon washing off the unbound quantum dots, the solution is
fluorescently detected. Figure 2-5 schematically details this method.

25

Figure 2-5. Isothermal chain elongation. (a) Single round of hybridization; (b) Target is linked to
the tethering molecule via reporter B. Additional reporter B hybridizes and allows for additional
target binding and subsequent reporter B binding. Addition of a quantum dot-streptavidin
complex binds to biotin on the reporter B molecule (adapted and modified from Song, Lau and
Lu, 2012).

This method has the advantage of being an enzyme free method capable of being conducted
without heating or temperature sensitive reactants. The quantum dot reporter system allows for
multiple different emission wavelengths, with possible use multiplexing in a microarray format.
The final sensitivity of this system is no better than microarray hybridization using a quantum
dot labeled probe. Since the target is half of the double-stranded product, at a 1:1 ratio with the
reporter, no increase in signal is observed over standard microarrays.

26

2.3.3 Triplex signal enhancement
Triplex signal enhancement is the process of electrostatic binding of a cationic polymer to a
single-strand DNA reporter. The thiopene polymer is quenched upon binding to a single-stranded
DNA molecule. Hybridization of the complimentary DNA sequence forms a triplex complex
creating a resonance energy transfer system with fluorescent properties. When each DNAcationic polymer duplex is labeled with green emitting fluorophore, a process of ‘super lighting’
can occur [62]. Super lighting happens when a single triplex structure is formed and acts as the
donor to multiple acceptors in close proximity. This process yields great sensitivity, but requires
equally sensitive photodetection equipment. The cationic polymer is not sequence specific in
duplex formation. Close proximity of non-target single-strand DNA and off-target hybridization
pose the possibility of a false positive in this system.

2.3.4 Branched DNA amplification
Branched DNA amplification is the technique of hybridizing a single-stranded target to an array
surface and through subsequent hybridizations, bind additional DNA reporter molecules. In
Figure 2-6, the method is shown in an abbreviated format [63]. A target hybridizes to multiple
bound surface probes. The multi-probe attachment creates a strong target-surface structure. A
preamplifier is then hybridized to the free portion of the target and stabilized though accessory
DNA strands (label extenders). The preamplifier DNA is then loaded by hybridizing labeled
probes.

27

Figure 2-6. End product of branched DNA amplification. Target nucleic acid is captured and
hybridized to a second ssDNA to allow for additional labeled probe binding (adapted and
modified from Tsongalis, 2006).

This enzyme free system provides a good deal of amplification, but at the cost of increasing
system complexity and material costs. The target DNA or RNA sequence must be sufficiently
long to bind both to the surface and for loading of the preamplifier sequence. Additionally, the
input DNA or RNA must be relatively pure so as to avoid off target binding from the many
different stabilizing, amplifying, and extension DNA sequences required for this system.

2.4 Nanoparticle based biosensor platforms
Biosensors are defined as sensing devices which integrate a biological recognition element and a
transducer to quantify a biochemical interaction. The biological recognition element is
responsible for capturing the signal. Once captured, the transducer changes the signal into
another form of energy that can be detected. Common recognition elements can be grouped
broadly into immunosensors, DNA hybridization sensors, enzyme based sensors, and
combinations of each. Transduction of the signal can be into many forms, with the most common
28

being light, electrical, and chemical change. Figure 2-7 shows a representative schematic of the
biosensor.

Figure 2-7. Schematic diagram of the components in a biosensor system.

DNA biosensor systems typically rely on hybridization to either a surface or a particle for
recognition. The hybridization event is then transduced into another form such as light via
fluorescent reporters, electrical via metallic particles, change in mass vibration frequency and
other physical properties through enzyme mediated events. The signal is then detected and
related back to the original binding event. As described in the previous sections, the only
governmental approved biosensors are based on metabolic changes via growth of the target
organism. Scientific biosensors have focused on antibody immobilization and DNA
amplification via PCR. Culture based methods are simple but time consuming and actually
produce more of the target organism. PCR techniques are more rapid but have very involved
processing steps and require a laboratory environment. The development of a rapid and simple
method for DNA detection is needed for public safety and water quality monitoring.

29

The following methods described show the current technology for a bio-sensing system based on
DNA recognition. They employ hybridization techniques which when used with the gene targets
in Table 2-2 would make viable bio-sensing systems for E. coli O157:H7.

2.4.1 Nanoparticle sensing
The recent use of nanoscale materials for biosensing elements, transducers and reporters have led
to a whole group of nanomaterial based sensing systems. The traditional role of nanoparticles has
been as reporters, delivery vehicles, and attachments surfaces. Quantum dots are a class of semiconducting nanoparticles that exhibit fluorescent properties. As previously described in section
2.3.2, isothermal chain elongation uses a quantum dot reporter in lieu of standard organic
fluorescent molecules. When nanoparticles were incorporated into a full sensor, a new field of
nanodiagnostics was created.

The most widely used of the nanoparticles is the gold nanoparticle. It has been used both for its
colorimetric application and its ability to tether a large array of molecules to the surface. Gold
nanoparticles exhibit a deep red color when in solution. As gold nanoparticles aggregate, or are
linked together in close proximity, the surface plasmon resonance between adjacent particles
causes a color change to purple. This change in color is used to report binding events [64-66].
Binding is accomplished by labeling two species of gold particles with different bio-elements
that recognize the physical molecule. The successful demonstration of the technique was shown
by the Mirkin group with both DNA and antibody probes [67]. When colorimetric detection is
used with DNA hybridization, no amplification of signal is seen. There is still a one-to-one
relation between target and reporter.
30

The use of gold nanoparticles for signal amplification has been recently achieved using a
technique called the bio-barcode [16, 68]. The bio-barcode (BBC) was the first truly enzyme free
DNA amplification assay. Selection and amplification of a DNA signal was accomplished with a
two particle system comprised of a magnetic microparticle and a gold nanoparticle. Each particle
is labeled with a different single-stranded DNA probe. The probes used are based on the same
sequences targeted in PCR amplification. The largest difference in probe design between the
BBC assay and PCR is that both particles are engineered to bind the same ssDNA sequence.
When a DNA target hybridizes, a bridge is formed linking the gold nanoparticle to the magnetic
microparticle. This DNA-bridge allows for magnetic separation of the solution, and only when a
target is present, are gold nanoparticles recovered, Figure 2-8.

31

MMP

MMP

Au

Au

Target DNA

Au

Readout

MMP
`

MMP

Figure 2-8: DNA detection using the sandwich assay. The magnetic microparticle (MMP) and
gold nanoparticle (Au) are mixed with a DNA target. A sandwich is formed between the MMPDNA-Au which is magnetically separated. The separated product contains Au particles only
when DNA target is present. Measurement is then performed based on the reporter used (adapted
and modified from Nam, Stoeva, and Mirkin, 2004).

This hybridization method uses the gold nanoparticle as a platform for amplification.
Amplification is achieved by attachment of a second molecule to the gold nanoparticle, either
through fluorescence or electrochemistry.

32

2.4.1.1 Bio-barcode detection
The bio-barcode was the first assay to popularize the use of gold nanoparticles in a complete
sensing system using enzyme free DNA amplification. Target recognition and recovery of the
gold nanoparticle are accomplished as shown in Figure 2-8 [69]. The bio-barcode amplification
is achieved by attachment of small single-strand DNAs (bio-barcodes, BBCs) to the gold
nanoparticle at a ratio of 100:1 with the probe DNA strand. The reporter BBCs are then
recovered by ligand exchange with dithiothreitol (DTT), Figure 2-9.

Figure 2-9: Bio-barcode detection overview. (a) Capture and release of the bio-barcode based on
target hybridization; (b) Bio-barcode is hybridized to a surface, providing a tethering molecule
for a second gold nanoparticle (adapted and modified from Thaxton et al., 2005)

33

Once the BBCs are freed, they are hybridized to a surface (Figure 2-9b.). The immobilized BBCs
act as a hybridization point for a further round of hybridization with a second gold nanoparticle.
This second gold nanoparticle is used as a growth point for standard silver enhancement and
subsequent reflective scanning readout. This platform has been successfully used for detection of
the pagA gene in Bacillus anthracis and viral targets [16, 70, 71]. Despite this assay method
being highly successful, it requires a relatively pure DNA input and the assay time after sample
is extracted is approximately 8 hours.

2.4.1.2 Gold reduction / oxidation
Electrochemical detection using the sandwich assay structure from the bio-barcode method has
been recently accomplished using the gold nanoparticle itself [10]. The same DNA hybridization
sandwich structure is used for target recognition and gold nanoparticle recovery. Once the gold
nanoparticle is recovered, it then is transferred to an electrode, as in Figure 2-10.

34

Au

MMP with first
DNA probe

MMP

Target DNA

Au
MMP

Au
Tracer
dissolution

Au

AuNP with
second DNA
probe

MMP

+
+

Au

+

Electrochemical
detection

Magnetic
separation
Figure 2-10: Electochemical detection of gold nanoparticles. (a) Formation of the sandwich
structure; (b) Gold nanoparticle recovery and detection (adapted and modified from TorresChavolla and Alocilja, 2011).

The gold nanoparticles are oxidized under positive potential and acidic conditions to generate a
detectable ion species. The gold ions are reduced under differential pulse voltammetry producing
a detectable current characteristic to the gold in the electronic system. This form of detection was
successfully used to detect target from Mycobacterium tuberculosis from enzymatically
amplified target. This detection system was a proof of concept for bacterial detection, but shows
great promise as a guide for a future rapid hybridization assays.

35

2.4.1.3 Other metallic nanoparticle electrochemical detection
An improvement to electrochemical detection is accomplished through the same methodology as
the bio-barcode assay. Attachment of nanoscale heavy metal-particles to the BBC produces an
amplified metallic system. The recovered gold nanoparticle-heavy metal particle conjugates are
oxidized in a similar fashion as the gold nanoparticle. The reduction of heavy metals (cadmium
or lead) is then measured as a current peak. Each metal in the system (gold, cadmium, lead)
produces a peak at a different reducing voltage. This enhanced nanoparticle system was used
successfully to detect PCR amplified target from Bacillus anthracis and Salmonella enteritidis.
Though highly sensitive, pure target from enzymatic amplification is still required for detection
from this assay system.

2.4.2 Fluorescence detection
The use of metallic nanoparticles and secondary hybridization structures creates a sensitive
detection assay, but adds steps to the process making it more complicated. Fluorescent readout
has been used to simplify the process. One approach to fluorescent output is to replace the gold
nanoparticle with a polystyrene (PSM) microparticle [72]. This PSM is labeled with
approximately 40,000 fluorescent reporter molecules. Other replacements for the gold
nanoparticle include DNA dendrimer agglomerates [73]. The dendrimer agglomerates contain
multiple biotin binding sites for a reporter molecule. The PSM molecules provide a greater
amplification factor than the gold nanoparticles (40,000x v. 100x) but do not suspend well in
water and provide higher amounts of non-specific binding. This non-specific binding reduces the
specificity of the assay as a whole. DNA dendrimers remain highly water soluble, but require

36

further conjugation of a reporter molecule to the biotin. This additional step also increases
system complexity.

2.4.3 Targets
Nanoparticle based DNA assay systems have been successfully used with short DNA targets
often resulting from enzymatic amplification. The steps required to generate a target for the
assay are not included in the total assay time. The extraction and preparation of a DNA target
requires an additional 2-3 hours to the total assay detection time. Focus is shifting to use of raw
genomic inputs [15], but have not included rapid detection methods.

2.5 Gold nanoparticle synthesis
The unique properties of gold nanoparticles make them well-suited for a variety of applications.
As described previously, they are used in spectral detection, colorimetrics, electrochemical
detection, and as platforms for other reporters. The utility of gold nanoparticles are a result of
their size and chemical reactivity. Controlled generation of gold nanoparticles is key to the
production of a uniform product for assay reproducibility. Generation of gold nanoparticles is
most commonly via citrate reduction. This process uses high temperatures and acidic conditions
for particle generation. Particle size is controlled through the surface capping agent (citrate) to
gold chloride concentration [74]. Other methods such as non-polar synthesis and biological
synthesis have been successful at generating gold nanoparticles [75-79]. Biological methods are
becoming more widely used as they provide a gentler or ‘green’ means of particle production.
The use of carbohydrate and protein capping agents has the potential for better bio-compatibility
in medical diagnostic systems [80, 81].
37

This chapter is adapted from our recently published work in the Journal of Nanoparticle
Research:

Anderson, J. Michael; Torres-Chavolla, Edith; Castro, A. Brian and Alocilja, C. Evangelyn. One
step alkaline synthesis of biocompatible gold nanoparticles using dextrin as capping agent.
Journal of Nanoparticle Research. 2011. 13(7):2843-2851.
DOI: 10.1007/s11051-010-0172-3

38

Chapter 3: Synthesis and characterization of dextrin gold nanoparticles

3.1 Introduction
Gold nanoparticles have been widely studied in recent years for their wide range of applications.
Gold nanoparticles exhibit surface plasmon resonance and have been used in colorimetric assays
and surface enhanced plasmon resonance (SERS) for fluorescent applications [82-84].
Electrochemical detection with gold nanoparticles has been accomplished using
reduction/oxidation potentiometry or as conductive elements in electrical circuits [85-87].
Assays involving gold nanoparticles all require attachment of recognition molecules for
detection. Surface molecules, or ligands, include but are not limited to DNA, proteins, polymers,
peptides and antibodies [11, 18, 88-90]. The attached ligands both aid in target recognition and
stabilize the gold nanoparticle cores by providing appropriate hydrophobicities.

Traditional gold nanoparticles synthesis has been achieved by three methods: 1) Brust method in
a non-polar media [76], 2) Turkevich method in aqueous media [91], and 3) biological synthesis
using microorganisms [78, 92]. Current aqueous generation is under aggressive conditions
(pH=3, 95°C), commonly requiring post-processing before biological materials can be attached.
Low energy input and milder generation conditions have been a recent focus for synthesis of
gold nanoparticles as trends towards ‘green’ processes are becoming more important. A second
benefit for milder generation conditions is the possibility for protein and DNA interaction during
particle generation. Generation of gold nanoparticles at pH 7.0-9.0, using temperatures between
20-50°C and with biomolecules would allow for exploration of gold nanoparticle generation and
surface functionalization with a minimum of processing steps.
39

Recent work into mild generation conditions has involved glyconanoparticles, or carbohydrate
coated particles and amines [93-95]. These particles are being tested in biomedicine, biolabeling,
and biosensings applications [10]. Cyclo-dextrins, dextrans, and glucose have been used recently
to generate gold nanoparticles [81, 96, 97]. This paper describes a generation process under
alkaline conditions using a dextrin, a carbohydrate, as a capping molecule under modest
temperatures.

3.2 Materials and methods
3.2.1 Optimization of gold nanoparticle synthesis
Gold nanoparticles were synthesized from a gold chloride (HAuCl4) stock solution of 20 mM
[Aldrich #520918-5G]. The gold chloride was prepared fresh weekly using deionized water and
stored under refrigeration. A stock solution of dextrin at 25 g/L was prepared in deionized water
and autoclave sterilized prior to use [Fluka #31400]. All reaction volumes were 50 mL and
carried out in 250 mL Erlenmeyer flasks that were washed and acetone rinsed before synthesis.

A volume of 5 mL of gold chloride was mixed with the stock dextrin to achieve 45 mL of
solution to achieve a final dextrin concentration between 2.5 and 20.0 g/L. The solution was pH
adjusted to pH 9 using filter sterilized sodium carbonate (Na2CO3). The reaction volume was
adjusted to 50 mL using pH 9 adjusted water, yielding a final gold chloride concentration of 2
mM. Each reaction flask was wrapped in aluminum foil to protect from light and continuously
agitated at 50°C for 8 hours.

40

Particle formation was observed visually through the following stages: yellow, clear, purple tint,
red tint, and finally red (520 nm), the same sequence reported for standard citrate generation of
gold nanoparticles [98]. Reaction temperature (25°C and 50°C) and pH (3 to 11) were varied to
explore the effects on gold nanoparticles generation.

3.2.2 Gold nanoparticle characterization
The gold nanoparticles were monitored during and after synthesis. Reaction completion was
evaluated using absorbance reading with a UV/Vis spectrometer. Size distribution was evaluated
using dynamic light scattering and transmission electron microscopy (TEM) imaging. Reported
size information was obtained from TEM images using a JEOL 100CX Transmission Electron
Microscope and a Zetasizer Nano series (Malvern Instruments). Samples were diluted 1:4 in
distilled water, and a volume of 5 µL was placed on to a formvar/carbon coated copper grid of
300 mesh. The samples were vacuum dried on the grids prior to imaging. TEM samples for
specific time points were prepared immediately after sample collection for proper reaction
monitoring.

3.2.3 Gold nanoparticle functionalization
Functionalization of the synthesized particles was accomplished using sulfur chemistries and was
necessary to evaluate particle compatibility within biosensor systems. Functionalization was
performed with a modified DNA molecule containing a three prime (3’) reactive thiol group and
a five prime (5’) fluorescent 6-carboxyfluorescein group (6-FAM, excitation peak = 495 nm,
emission peak = 520 nm). Standard DNA notation was used for all sequence and includes:
adenine (A), cytosine (C), thymine (T), and guanine (G). The sequence used was 5’ – TTA TTC
41

GTA GCT AAA AAA AAA A - 3’ (Integrated DNA Technologies, Coralville, IA). The thiol
group was chemically activated in a 1 M solution of dithiothreitol (DTT) for 2 hours and purified
through an Illustra NAP-5 desalting column (GE Healthcare #17-0853-02) producing a solution
of reactive DNA. The activated DNA was ligand exchanged for the capping dextrin molecules
over a 72 hour period before, with salting additions every 8 hours to promote close packing DNA
on the gold particle surface. The closed packed DNA creates a stable AuNP for potential assay
use. The procedure is the same used for citrate generated gold nanoparticles [11]. Successful
functionalization of the particles yielded a product with the same color as the initial dextrin
coated gold nanoparticles. Functionalization efficiency was determined by washing the
functionalized gold nanoparticles three times in distilled water and resuspension in a 5 M DTT
solution at 65°C for 15 minutes followed by a 45°C incubation at 25°C to remove the DNA from
the gold nanoparticle. The DTT treated samples were centrifuged at 13,000 x g for 30 minutes
and the supernatant was fluorescently excited on a VICTOR3 1420 Multilabel plate counter
(Perkin-Elmer) using a 492 ± 4 nm excitation filter and 535 ± 12.5 nm emission filter set for
detection.

3.3 Results and discussion
3.3.1 Gold nanoparticle characterization and synthesis
Gold nanoparticles were successfully generated with dextrin as a capping agent under alkaline
conditions. The baseline reaction conditions were: 10 g/L dextrin concentration, 2 mM gold
chloride, pH 9.0, 50°C and 24 hours for synthesis. The particle generation was monitored
through absorbance measurements of the 520 nm peak characteristic of gold nanoparticles of the
size range 5 – 100 nm (Figure 3-1). Absorbance at 520 nm was recorded after the visual color of
42

the solution began changing from a light purple to the light red tint between 4 – 6 hours of
reaction (Figure 3-2) [12]. The reactions were monitored for a full 24 hours to assess the degree
of completion of the reaction.

3.500

Absorbance Units (AU)

3.000

24 hr
12 hr

0 hr
1 hr
1.5 hr
2 hr
3 hr
4 hr
5 hr
6 hr
12 hr
24 hr

6 hr
4 hr

2.500
2.000
1.500
1.000

2 hr
1.5 hr

0.500
0.000
400

0 & 1 hr
450

500

550
600
Wavelength (nm)

650

700

Figure 3-1: UV-Vis spectra time monitoring of gold nanoparticles synthesis. Generation
conditions 10 g/L dextrin, pH 9.0, 50°C (adapted and modified from Anderson et al., 2011).

43

24 hrs

12 hrs

6 hrs

4 hrs

1.5 hrs

1 hr

0.5 hr

Figure 3-2: Visual time monitoring of gold nanoparticles synthesis. Generation conditions 10 g/L
dextrin, pH 9.0, 50°C (adapted and modified from Anderson et al., 2011).

The generation of particles required the presence of dextrin, alkaline pH, sodium carbonate and
gold chloride. Reactions lacking any of the mentioned reactants did not form gold nanoparticles.
When gold chloride was present in the reactions that either lacked dextrin, alkaline pH or sodium
carbonate, the gold would deposit in the neutral metallic state on the flask walls after several
days.

3.3.2 Effects of dextrin concentration, pH, and temperature
Successful generation of gold nanoparticles was accomplished under a large set of synthesis
conditions. Controlling the ratio of capping agents and reaction rate has been used to manage the
final particle size in nanoparticle production. The concentration of the dextrin was varied from
2.5 g/L to 20 g/L final concentration to determine the effect of capping agent on final particle
size. Production of 8.6 nm ± 1.2 nm, 10.6 nm ± 1.6 nm, and 12.4 nm ± 1.5 nm was accomplished
44

for dextrin concentrations of 20.0, 10.0 and 2.5 g/L respectively. Particle size determinations
were made from TEM image measurements. Figure 3-3 shows the TEM images of generation
conditions. Figure 3-4 shows the resulting data versus generation concentration.

45

Figure 3-3: Transmission electron micrographs of gold nanoparticle. AuNP synthesis using
various dextrin concentrations. (a and b) 20 g/L, (c and d) 10 g/L, and (e and f) 2.5 g/L. Scale
bars of a, c, and e are equal to 100 nm (adapted and modified from Anderson et al., 2011).

46

Particle Diameter (nm)

15
y = -0.1842x + 12.669
R2 = 0.9805
10

5

0
0

5

10

15

20

25

30

Dextrin Concentration (g/L)
Figure 3-4: Average gold nanoparticles diameter as a function of generation dextrin
concentration (adapted and modified from Anderson et al., 2011).

The linear relation between dextrin concentration and particle diameter implies a direct
relationship between reaction conditions and size. Observation of the time for the initial red color
to form suggests that the dextrin is also responsible for the reduction of the gold chloride. The
normal reaction progression is for the yellow gold chloride solution to change to a clear or grey
tinted solution prior to particle formation. This same transition occurs when the sodium
carbonate is used for pH adjustment. The time for red color to form decreased as the dextrin
concentration was increased. Particle formation was observed between 5 and 6 hours for 2.5 g/L
and between 1 and 2 hours for 20 g/L dextrin generation conditions.

47

The generation of ground state gold atoms for nanoparticles production requires a reducing
agent. Citrate generated particles use citric acid as the reducing agent at acidic pH values, and
recent reports have used a sodium hydroxide pH adjustment and polyvinylpyrrolidone for gold
ion reduction [90]. The use of dextrin synthesis conditions was explored between the pH range
of 3 and 11 at 10 g/L dextrin concentration, 50°C, and 2 mM gold chloride. Control reactions
using sodium hydroxide for pH adjustment, lack of dextrin or lack of gold chloride did not form
gold nanoparticles with the common red color. The use of sodium hydroxide for particle
formation yielded a dark purple precipitate that was visible to the human eye. These large purple
particles that formed were unstable and caused ground state metallic gold deposition on the
glassware at the air-liquid interface. Upon 24 hours of generation gold nanoparticles were
generated from 7.0 nm ± 1.2 nm to 16.8 nm ± 2.3 nm in diameter. In Figure 3-5 the effect on size
resulting from pH and dextrin concentration variation is shown.

48

20

Paticle Diameter (nm)

20 g/L
10 g/L
2.5 g/L

15

10

5
6

7

8

9

10

11

12

pH
Figure 3-5: Average gold nanoparticle diameter as a function of generation pH, ranging from pH
7.0 – 11.0 (adapted and modified from Anderson et al., 2011).

Successful generation of particle using dextrin was accomplished between the pH range of 7.0
and 11.0. Reaction times were reduced with increasing pH from 1 minute at pH 11 to 24 hours at
pH 7.0.

Traditional citrate generated particles are produced at near boiling or boiling temperatures. The
effects of generation temperature on the particle size were also explored. Lower generation
temperatures required reduced energy input making the particle synthesis less expensive and

49

more environmentally friendly. Figure 3-6 shows the effects of temperature on the mean particle
size generated as a function of dextrin concentration.

15.0

Particle Diameter (nm)

25°C
50°C

10.0

5.0
0

5

10

15

20

25

30

Dextrin Concentration (g/L)
Figure 3-6: Average gold nanoparticles size as a product of generation temperature and dextrin
concentration (adapted and modified from Anderson et al., 2011).

3.3.3 Reaction mechanism

Gold particles were successfully generated over a large range of dextrin concentration, pH, and
temperature. The effects of the various parameters on the generated particle size can be
explained using the basic generation model. The paradigm is a four step generation modeled on
50

citrate particles and appears to be valid in the dextrin system [98]. The gold chloride is first
reduced, then stabilized, a slow growth phase followed by a fast growth phase. The initial
reduction is achieved upon the sodium carbonate pH adjustment. This is observed visually as the
solution turns from yellow (Au(III)) to a dark tinted solution (smoke grey). This grey color
persists and slowly turns darker with a purple tint, were ground state gold atoms are being
stabilized into individual particles. The particles begin the slow growth and the solution beings
turning a red tint. The fast growth phase usually last approximately 30 - 120 minutes (50°C) and
the solution develops the characteristic deep red color of 5-50 nm gold nanoparticles. The UVVis absorbance of the reaction was monitored with time and the 520 nm absorbance peak was
plotted in Figure 3-7.

51

Absorbance at 520 nm (AU)

A 20 g/L
6

B 15 g/L
C 10 g/L
D 7.5 g/L

4

E 5 g/L
F 2.5 g/L
F

E

2
A

B
D

C
0
0

1

2

3

4

5

6

Time (hours)
Figure 3-7: UV-Vis monitoring of reaction completion.

From Figure 3-7, a rapid increase in absorbance is seen after an initial lag phase. The absorbance
at 520 nm is used to measure article concentration. From the absorbance data, an ideal curve is
pictured in Figure 3-8 labeled with the proposed phases of growth. These conditions are the same
as line C from Figure 3-7.

52

Absorbance at 520 nm (AU)

6

4

Reduction

Rapid
Growth

Stabilization /
Slow growth

2

0
0

1

2

3

4

5

6

Time (hours)
Figure 3-8: UV-Vis monitoring of proposed reaction mechanism.

The rapid growth phase shown is Figure 3-8 is the most apparent stage. During the stabilization
and slow growth, a color change is observed visually (Figure 3-2, 1hr), but not seen in the UVVis data. The time at which rapid growth begins varies with temperature, pH, and dextrin
concentration. It was observed that higher amount of dextrin, higher pH and higher temperatures
shortened the time from the beginning of the reaction to when the rapid change in color occurred.
These observations agree with conventional particle generation theory.

Particle size is a combination of available capping agent, or stabilization agent, and reaction rate.
Using a higher ratio capping agent to gold concentration provides a greater surface area of gold

53

that can be stabilized, which allows generation of smaller particles. The higher capping agent
concentration also helps maintain the stability of the particles, by providing free capping agent in
solution to replace any molecules that disassociate from the particle surface. Faster reaction rates
create smaller particles by promoting more nucleation points. As the reaction rapidly proceeds,
all the available reactant, gold chloride, is consumed and a larger number of small particles are
formed. Smaller particles are generated with increasing pH. It is thought the higher pH creates a
more reactive dextrin molecule by promoting the opening of the terminal glucose molecule into
an aldehyde in the presence of sodium carbonate. This aldehyde group is thought to be the
reducing agent for the gold chloride. When the reaction temperature is increased, the rate of
reaction increases, but the particle size increases. This is contrary to the rate of reaction theory
proposed above. The increased temperature allows for the capping agent to disassociate/associate
more rapidly on the forming particles. The rapid exchange of capping agent may occur rapidly
enough to prevent the fusing of two free gold particle surfaces. This could explain why
increasing temperature also increased particle diameter.

The system used for alkaline generation utilizes sodium carbonate, dextrin, and gold chloride.
Determination by reactant substitution was conducted to help determine what components were
participating in the reaction. When the pH was adjusted with either sodium carbonate or
hydrochloric acid (HCl), the same grey tint formed in the gold chloride solution. Without
dextrin, the sodium carbonate reaction maintained the tint for 24-48 hours upon metallic gold
deposition on the gas/liquid interface. Without dextrin present, the HCl adjusted reaction
proceeded to precipitate into purple aggregates visible with the human eye. Previous work
suggests that standard citrate reactions reduce their reaction rates as pH increases by formation of

54

more stable and less reactive gold-hydroxyl species [98]. As the pH increases, the initial gold
chloride ion exchanges chlorides ions for hydroxyl ions. As the pH increases the gold ion goes
through the following species: [AuCl4] , [AuCl3OH] , [AuCl2OH2] , [AuClOH3] , to [AuOH4]

. As the pH increases, equilibrium in the carbonate-bicarbonate-carbonic acid systems shifts to
favor the carbonate species. Carbonate has pKa values of 6.33 and 10.35 for the range of pH
being used. The balance of carbonate/bi-carbonate between pH 6.33 and 11 has similar trending
in the same fashion as the reaction rate data show. The dextrin/carbonate system does not
produce particles below pH 7.0 while producing AuNPs within minutes at pH 11.0. The
combination of carbonate and alkaline pH are both required for synthesis. The presence of
dextrin in the system is required for stable particle formation. When dextrin is removed from the
system, a purple precipitate forms after 24-48 hours of synthesis time.

To determine the capping agent, FTIR readings were conducted on successfully generated
particles at 10 g/L dextrin, pH 9.0, 50°C and 2mM gold chloride. A volume of 1 mL of generated
particles was washed 3 times in water to remove excess reactants and pH adjusted with 50 µL of
0.1 M sodium hydroxide. The change in pH resulted in precipitate formation after 2 hours.
Samples were centrifuged to remove the metallic AuNP cores. The supernatant was compared to
stock dextrin and autoclaved dextrin samples. Figure 3-8 shows the FTIR data.

55

0.7
A

0.5
0.4
B
0.3
C

3000

2500

2000

1500

1000

500

0.1

4000

A Stock
B Autoclaved
C AuNP Bound

0.2

3500

Absorbance (%)

0.6

Wavenumber (cm-1)
Figure 3-9: FTIR of capping agent. (a) Stock dextrin; (b) Pre-synthesis reactant; and (c) Freed
capping dextrin (adapted and modified from Anderson et al., 2011).

From Figure 3-9, the recovered dextrin from the generated particles had the same absorbance
pattern as both the starting samples. Dextrin that has undergone a ring opening would be
-1
-1
-1
expected to have a carboxylic acid signature at 1714 cm , 1414 cm , or 1294 cm

wavenumber. The lack of the carboxylic group suggests the dextrin that is protecting the AuNP
core is unreacted. A lack of reacted dextrin on the AuNP surface does not remove dextrin as a
reduction agent for the gold chloride. Dextrin is a linear polysaccharide of glucose molecules
connected via alpha(1-4) and alpha(1-6) bonding. The monosaccharide glucose, a reducing
sugar, was used to generate AuNPs at 10 g/L. The synthesis was more rapid than the dextrin
56

reaction resulting in large purple precipitate. During the generation, the desired red color was
seen briefly, but it was not possible to stabilize the reaction at that stage. When dextrin and
glucose were mixed together (10 g/L dextrin, 5 g/L glucose) the reaction proceeded at a rate
between pure dextrin and pure glucose. The resulting particles generated were stable for
approximately 14 days, with significant precipitation beginning after 24 hours. The combination
of the different sugars suggests that the dextrin is a reactive species in AuNP formation.

The exact mechanism of AuNP was not empirically determined. From the data, stable gold
nanoparticle generation requires pH > 7.0, sodium carbonate, gold chloride and dextrin. Gold
nanoparticles formation may be accomplished by aldose / ketose reduction of gold chloride. The
degree of sugar ring opening may be increased by increased pH and carbonate concentration.
The higher pH may also stabilize the gold chloride for a slow and even particle growth. This
system of gold nanoparticles generation consistently produces uniform particles with controllable
size based on the generation conditions.

3.3.4 Gold nanoparticle functionalization
Common methodologies for utilizing gold nanoparticles involve replacing the capping agent for
a functional group or other recognition molecule. The process of ligand exchange involves
disassociation of the capping agent, such as dextrin or citrate, and the more permanent
attachment of the molecule of interest [11]. Thiols are most commonly used for attachment
chemistry to gold surfaces. Gold nanoparticles generated at 2.5 g/L and 10.0 g/L (12.4 nm and
10.4 nm respectively) were functionalized with 6-FAM labeled, thiol linked DNA. Both sizes of
57

particles were successfully functionalized, Figure 3-9 shows exchange efficiency compared to
citrate generated particles.

20%
15%
10%

2

3

4

C^-13

1

D^-10.4

0%

C*-13

5%

D*-12.4

Efficiency (% Total)

25%

5

Figure 3-10: Comparison of dextrin and citrate generated gold nanoparticles for ligand exchange
compatibility. D=dextrin generated; C=citrate generated; 12.4, 13, and 10.4 represent AuNP
diameter used (adapted and modified from Anderson et al., 2011).

Efficiency is reported in Figure 3-10 as the ratio of the amount of DTT liberated FAM-DNA
verses the total DNA used in the functionalization reaction. From Figure 3-10, the left grouping
of data, D*-12.4 nm and C*-13 nm, have the same efficiency. The right grouping, D^-10.4 and
C^-13, show a lower efficiency for the dextrin particles. The smaller 10.4 nm diameter dextrin

58

AuNP have ~69% of the available surface area for DNA-capture compared to a 13 nm diameter
particle. With surface area taken into account, the 77% signal seen from the smaller particles
showed comparable DNA capture to the larger 13 nm diameter citrate particles. Dextrin
generated gold nanoparticles were equivalent to citrate generated particles for use in assays after
ligand exchange.

3.4 Conclusions
Dextrin coated gold nanoparticles were successfully generated with sizes ranging from 5.9 and
16.8 nm ± 1.6 nm. Particle size was controllable by varying generation pH, dextrin
concentration, and reaction temperature. The produced AuNP were stable for 6 months when
stored at room temperature in the generation media. The particles were successfully
functionalized with DNA capping agents and used for the sandwich assay in Chapter 5.

59

This chapter is adapted from our recently published work in IEEE Transactions on
Nanotechnology:

Anderson, J. Michael; Zhang, Deng; and Alocilja, C. Evangelyn. Spectral and Electrical
Nanoparticle-Based Molecular Detection of Bacillus Anthracis Using Co-polymer Mass
Amplification. Journal of Nanoparticle Research. 2011. 10(1):44-49.
DOI: 10.1109/TNANO.2010.2061235

60

Chapter 4: Spectral co-polymerization mass amplification detection of Bacillus anthracis Stern
using sandwich assay identification

4.1 Introduction
Fluorescent methods of DNA reporting commonly rely on hybridization events with a separation
step. Hybridization is not a rapid process as it is driven by the concentration of products and the
DNA sequence being targeted [99]. The bio-barcode assay uses DNA specificity to achieve
proper sequence recognition, and the use of magnetic particles provides a simple separation
scheme [18]. The use of a second hybridization event and gold nanoparticles provides a vehicle
for a 100 times amplification of target signal via small single-stranded DNA (ssDNA) reporter
molecules (bio-barcodes) [100]. The signal from the bio-barcodes comes from a fluorescent
label on the DNA strand, and for more sensitive detection two additional rounds of hybridization
are required, with silver enhancement and laboratory specific detection hardware [101].
Simplification of the detection signal from the assay has been accomplished by electrochemical
detection of the gold nanoparticles [102]. An alternative fluorescent method using selfassembling small ssDNA molecules has been developed [13]. The detection method utilizes a
released co-polymerization partner from the gold nanoparticles and dye that have 1000 times
increase in signal when bound to the double-strand DNA (dsDNA) formed from copolymerization between the released gold nanoparticles DNA and a second probe DNA. Other
enzyme free hybridization schemes have been explored, but amplification is limited, they possess
high time requirements, or have long assay times [58, 60, 62, 63]. The method presented is
enzyme free, provides amplification and detection requires under 25 minutes.

61

4.2 Materials and methods
The DNA dyes SYBR Gold (S11494), PicoGreen (P7581), and SYBR 101 (S21500) and a 100
bp DNA ladder (15628-019) were obtained and used without purification from Invitrogen Life
Technologies. Single-stranded DNA sequences (oligonucleotides) and 6-carboxyflurescein (6FAM or FAM) labeled ssDNA was purchased from Integrated DNA Technologies. Preparation
and recovery of thiol-linked DNA was accomplished with 1,4-dithio-DL-threitol (DTT, D5545)
obtained from Sigma-Aldrich. Attachment of amine labeled DNA to magnetic particles was
conducted using sulfosuccinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate (sulfoSMCC, 22122) from Pierce-Thermo Scientific. Gold nanoparticles were synthesized using
hydrogen tetrochloroaurate (III) trihydrate (203386) and sodium citrate dehydrate (W302600)
from Sigma-Aldrich. Quantum dots (QDs, 7.2 nm evidot 490) suspended in toluene were
obtained from Evident Technologies and functionalized with aminoethanethiol (AET, AC153770259) from Fisher Scientific.

4.2.1 DNA target generation
The pagA gene was the target of the sandwich assay. A culture of Bacillus anthracis Sterne
(Bacillus) was refreshed and cultured over night in trypticase soy broth. A volume of 1 mL of
culture was digested with proteinase K for 75 minutes at 50°C. The resulting lysate was purified
using ethanol precipitation. The samples were suspended to 75% isopropanol / 25% lysate and
centrifuged for 30 minutes at 4°C, then decanted and the process repeated with 100% ethanol and
then 70% ethanol. Final resuspension was in double distilled 18 MΩ water. The recovered
genomic DNA was then amplified by polymerase chain reaction (PCR) to create 119 base pair
(bp) sequence for use in the sandwich assay. Table 4-1 listed the reaction conditions.
62

Amplification reactions were purified using a DNA binding column (Qiagen MinElute PCR
Purification Kit, 28004).

Table 4-1. Probe sequences generated for pagA gene in Bacillus anthracis Sterne.

Sequence Name

Sequence

Bac-FWDPrimer

5´ AAA ATG GAA GAG TGA GGG
TG 3´
5´ CCG CCT TTC TAC CAG ATT
Bac-REV-Primer TA 3'

Amplicon
Size
119 bp

DNA concentration was determined using a UV absorbance spectrophotometer (SmartSpec
3000, BioRad Laboratories), and the length of the purified sample was confirmed in a 2% w/v
agarose gel by electrophoresis in Figure 4-1.

63

1

2

3

Figure 4-1: PCR of Bacillus anthracis DNA for the pagA gene. Lane 1: 500 µg of 100 bp ladder.
Lane 2: blank. Lane 3: PCR product using primer in Table 4-1.

Upon size confirmation and purification from Figure 4-1, the PCR product was serially diluted
and then heated to 95°C for 10 minutes to denature the amplified DNA to ssDNA. Next, the hot
DNA was rapidly cooled in an isopropanol bath (-20°C) to create single-stranded product as
input for the sandwich assay.

64

4.2.2 Sandwich assay
4.2.2.1 Magnetic microparticle functionalization
The amine functionalized magnetic microparticles (MMP) (BM546, Bangs Laboratories, Inc.)
were conjugated to the 5’ end of the MMP-Bac-Probe via sulfo-SMCC shown in Figure 4-2 and
listed in Table 4-2. SMCC is a thiol to amine bifunctional cross-linking agent that introduces a
0.83 nm spacer arm. The MMPs were washed 3x times in buffer (0.1 M phosphate, 0.3 M
sodium chloride, pH 7.2) and 1 mg of MMP were resuspended in 1 mL in the same buffer. SulfoSMCC (0.3 g) was added to the MMP and allowed to react for 2 hours. The MMP-SMCC
particles were washed to remove excess sulfo-SMCC and released N-hydroxysulfosuccinimide
(sulfo-NHS) and the MMP-Bac-Probe was added and allowed to react for 8 hours. The thiolated
DNA probes were activated by reduction in 0.1 M DTT under constant agitation at room
temperature for 2 hours. Excess and reacted DTT was removed from the DNA reduction reaction
using a Sephadex filtration column (GE Healthcare #Nap-5) per manufacturer instructions. Upon
reaction, the MMP-DNA solution was washed three times and resuspended in blocking buffer
(0.2 M Tris, pH 8.5) for 1 hour. The MMP-DNA particles were washed again and stored in
reduced strength buffer (0.01 M phosphate, 0.2 M sodium chloride, pH 7.4) prior to use.

65

Table 4-2. Thiol functionalized species of DNA for particle decoration.
Sequence Name
MMP Probe
AuNP Probe
AuNP-6FAM
AuNP-AB

Sequence
5´-SH-AAA AAA AAA AGGA AGA GTG
AGG GTG GAT ACA GGC T-3´
5´ AGA TTT AAA TCT GGT AGA AAG
GCG GAA AAA AAA AA - Thiol 3'
5' 6FAM - ATC AGT CAG TCA GTC AGT
CA - Thiol 3'
5' Thiol - TTA TTC GTA GCG TGA TGC
CAA G 3'

66

+
Na O
O
S
O

Sulfo-SMCC

O
O
N

N O

O
O

O

HO

Sulfo-NHS
NH2

O
N

O
MMP

O

NH

Na+

-

O S O
O

N
O

O

HS DNA

MMP

S

O
NH

DNA

N
O

O
Figure 4-2: SMCC labeling of the MMP with DNA. The amine labeled microparticle attaches to
the cross-linker sulfo-SMCC, releasing sulfo-NHS. The reactive microparticle then bonds to the
sulfhydryl group on the DNA.

67

4.2.2.2 Gold nanoparticles generation and functionaliztion
Citrate generated gold nanoparticles were synthesized using a modified protocol from standard
citrate generate AuNPs [11]. A 50 mL solution of hydrogen tetrochloroaurate (III) trihydrate (1
mM) was heated on a hotplate in a clean 125 mL Erlenmeyer flask until boiling. A volume of 5
mL of sodium citrate (38.8 mM) was added slowly in a dropwise fashion to the gold chloride
stirring constantly. Upon development of the characteristic wine red color, the solution was taken
off the hot plate and allowed to cool on the bench top. After cooling, the gold nanoparticles
(AuNP) were stored in a 50 mL polypropylene centrifuge tube, protected from light at room
temperature. Particles were previously characterized using this generation technique and
successfully used in a biosensor [103].

The gold nanoparticles were functionalized with 0.05 nmol of AuNP-Bas-Probe and 5 nmol of
co-polymerization probe (Table 4-2) via ligand exchange [11]. Briefly, a reactive thiol bonds to a
free location on the gold surface after a weakly bound citrate molecule disassociates. The thiol
bond is sufficiently stronger than the electrostatic bonding of the citrate molecule and forms a
semi-permanent surface bond. Thiolated DNA was activated in the same manner as described in
section 4.2.2.1. The reduced and reactive DNA was mixed with 1 mL of washed gold
nanoparticles at 4 nM (1nM: 1 absorbance unit (AU) at 520 nm) in 18 MΩ water. Salt additions
were added over a 48 hour period to facilitate close packing of the DNA oligonucleotides on the
gold nanoparticles surface [15]. Particles were stored in the reaction solution for a maximum of
30 days on the bench top until use.

68

4.2.2.3 Target capture
The DNA input for the assay was prepared as described in section 4.2.1. The desired mass of
DNA was volume adjusted to 40 µL and mixed with 0.08 mg of probe functionalized DNA
microparticles and adjusted to a final volume of 200 µL using assay buffer (150 mM sodium
chloride, 10 mM phosphate, 0.1% sodium dodecyl sulfate, and pH 7.4). The samples were
inversion mixed at 45°C for 60 minutes. Upon hybridization, magnetic separation was used to
wash the unbound DNA from the particles and resuspended to 200 µL of assay buffer containing
40 µL of the desired functionalized gold nanoparticles (1 nM). The MMPs-DNA and AuNPs
were allowed to incubate and hybridize for 2 hours at 45°C under inversion mixing. The
reactions were magnetically separated and washed twice to obtain the final MMP-target-AuNP
sandwiches. The recovered sandwich structures were resuspended in 200 µL of 0.1 M DTT and
heated to 95°C for 10 minutes, and centrifuged at 13,000 x g for 30 minutes to pellet the now
free MMP and AuNPs. The supernatant containing the DNA was then used for detection.

4.2.3 Co-polymerization sequence design
Co-polymerization is defined as the process of having 2 or more ssDNA oligonucleotides
partially hybridize with a second strand leaving a portion of sequence for additional
hybridization events. By engineering overlapping sections that were complimentary to each
other, two co-polymerization probes were generated that upon hybridization formed long dsDNA
species with repeating subunits. The two probe system was designed as shown in Figure 4-3
[13].

69

Figure 4-3: Cartoon representation of co-polymerization. (a) Initial dsDNA region; (b)
Rearrangement of lower strand to created second co-polymerization partner; (c) possible initial
hybridization and subsequent hybridization events (adapted and modified from Anderson, Zhang,
and Alocilja, 2011).

A perfectly aligned 22 base pair (bp) DNA sequence was divided into two 11 base regions. One
stand of the dsDNA molecule remained unchanged. The second strand has a translation of the
bases. Bases 1-11 are moved into the 12-22 base position maintaining 5’-3’ order. When one of
each strand hybridizes, two different molecules can form. When sufficient quantity of both

70

strands is present a total of 4 different end groups can form as a result of the 5’-3’ directionality
of DNA, shown in Figure 4-4.

Figure 4-4: Possible terminal end configuration based on the two part co-polymerization
(adapted and modified from Anderson, Zhang, and Alocilja, 2011).
The two strands of DNA generated were tested and designed to prevent self-hair pinning and
improper self hybridization. Sequences were tested with the OligoAnalyzer (Integrated DNA
Technologies, http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/) and MFOLD
(http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/).

4.2.4 Co-polymerization detection
Sensitivity of the co-polymerization readout was accomplished using fluorescent detection with
PicoGreen DNA dye. The gold nanoparticles linked with the DNA species AuNP-AB were
tested from 0.1 ng to 100 ng against the hybridization probe (BA, Table 4-3) of 5 ng – 100 ng per
100 µL in hybridization buffer (10x PicoGreen, 10 mM Tris, 100 mM sodium chloride, 1 mM
ethylenediaminetetraacetic acid, pH 8). Hybridization samples were mixes and heated to 95°C in
a thermal cycler block for 3 minutes and allowed to cool to room temperature in the block. After
cooling, the 100 µL volume was measured in a plate based spectrophotometer (Victor 1420,

71

Perkin Elmer) with an excitation filter of 492 nm ± 4 nm and emission filter of 535 nm ± 12.5
nm.

All samples that were visualized using electrophoretic separation for detection were run in 2%
w/v agarose gels. Samples were separated using tric-boric-ethylenediaminetetraacetic acid (TBE)
buffer at 1x and run at 30 V for 2 hours. Images were excited at 254 nm and emission was
detected using a 540 nm long pass cut off ethidium bromide filter (Fotodyne 60-2030).

4.2.5 Quantum dot attachment
Cadmium/zinc core/shell quantum dots were purchased from Evident Technologies. The
QDs were stabilized with trioctylphosphine oxide (TOPO) in toluene by the manufacturer.
Before being conjugated to the SYBR101 dye, the QDs were functionalized with AET to provide
a reactive amine group with water solubility. A quantity of 5 nanomoles of quantum dots was
suspended in 5 mL of chloroform. Aminoethanethiol (0.5 M) in absolute ethanol was added to
the QD/chloroform solution drop wise until flocculation began. The solution was flushed with
nitrogen to purge oxygen and vortexed for 10 minutes. Then 0.5 mL of 18 MΩ water was added
and the tube was reflushed with nitrogen. The sample was then vortexed again for 10 minutes to
promote a phase change from the chloroform to water as the QDs were functionalized with AET.
The solution was allowed to separate on the bench top for 30 minutes into an upper water layer
containing AET-capped QDs and a lower chloroform phase. Capping was confirmed visually by
observing yellow coloration of the water phase being that was confirmed by excitation with a
254 nm light source because under 254 nm excitation the QDs fluoresce. Before AET capping,

72

the chloroform phase fluoresces and post capping only the aqueous phase fluoresces thereby
confirming AET functionalization.

Quantum dots were attached to the amine reactive dye SYBR 101. SYBR 101 is a double-strand
intercalating dye with a succinimidyl ester group which reacts with primary amines. The SYBR
101 was resuspended in dimethyl sulfoxide (DMSO) to 10 mg/mL. A volume of 10 µL of the
SYBR 101 dye was added to the 0.5 mL of AET water solubilized QDs and 0.5 mL reaction
buffer (0.2 M phosphate, 0.3 M sodium chloride, pH 7.2) and mixed. After 2 hours of reaction,
the SYBR 101 dye attached to the amine group on the QD’s and was centrifuged to pellet the
QD-SYBR particles. The pellet was resuspended in 500 µL of 0.1 M phosphate buffer. A volume
of 10 µL of SYBR 101 (10 mg/mL, estimated molecular mass 550 g/mol, estimated 180 nmol /
10 µL) was reacted with 400 nmol of AET in a volume of 100 µL of the same buffer as the QDSYBR reaction. This blocked the reactive ester of SYBR101 for use in DNA binding.

Electrochemical detection of the QDs was accomplished using a screen printed carbon electrode
(SPCE). The SPCE was measured using a benchtop potentiostat (Princeton Applied Research,
Potentiostat/galvanostat 263A). The fluorescence detection of the QD ions was accomplished
using differential pulse voltammetry (DPV), with voltage sweeping from +1.5 to 0 V at 33.3
mV/s (step potential 10 mV and modulation amplitude of 50 mV). Samples were first dried onto
the carbon electrode surface and then incubated for 5 minutes in 80 µL of 0.1 M hydrochloric
acid. Within the SPCE/QD/hydrochloric acid electrochemical system, cadmium has a
reduction/oxidation peak centered about -0.88 V.

73

4.3 Results and discussion
4.3.1 Co-polymerization sequences
The generated sequences are listed in Table 4-3. Results from MFOLD show an energy input is
required for both sequences to form self base pairing structures. The sequence AB has a change
in Gibbs free energy of +0.46 kcal/mol and BA has +1.07 kcal/mol as their most stable
structures. This required input of free energy indicates an unstable or unfavorable confirmation.
The sequences were then compared against each other using OligoAnalyzer for mispriming
alone, and with the other strand present. Free energy was minimized for self priming of each
sequence with AB and BA each having -3.61 kcal/mol. The most stable mis-priming between
AB and BA has a free energy of -4.95 kcal/mol, where proper priming is approximately 5 times
more stable with energies of -18.55 and -20.00 kcal/mol.

Table 4-3. Engineered co-polymerization ssDNA strands.

Sequence Name

Sequence

AB

5' TTA TTC GTA GCG TGA TGC CAA G 3'

BA

5’ GCT ACG AAT AAC TTG GCA TCA C 3’

4.3.2 Dye specificity
Determination of dye binding was accomplished by challenging each dye against ssDNA and
dsDNA. Separation via electrophoresis removed excess or unbound dye leaving only the stained
DNA to be visually imaged. Electrophoresis functions by applying a driving voltage to push
74

negatively charge materials though a polymer matrix. The ratio of charge to mass in a sample
will determine how far it moves in a fixed amount of time at a fixed amount of driving voltage.
DNA has a relatively constant charge per base value, giving the charge to mass ratio a constant
value. This results in band movement in the agarose gel being a consistent measure of the size
(length) for DNA.

The effects of three different DNA specific dyes were tested and the results are shown in Figure
4-5.

75

Figure 4-5: Agarose gel showing DNA dye testing. Lane 1: 1 µg of 100 bp ladder with SYBR
Gold. Lanes 2-4 were run with 45 ng BA ssDNA. Lanes 5-7 were run with 15 ng of copolymerized dsDNA. SYBR Gold, PicoGreen, and ethidium bromide were used to stain lanes 2
and 5, 3 and 6, and 4 and 7 respectfully per manufacture instructions (adapted and modified from
Anderson, Zhang, and Alocilja, 2011).

All three dyes in Figure 4-5 [13] have excitation and emission spectra compatible with the UV
excitation (SYBR Gold: 480 nm and UV-C excitation, 537 nm emission; SYBR 101 and
PicoGreen: 480 nm and UV-C excitation, 520 nm emission; ethidium bromide: 302 nm
excitation, 595 nm emission). PicoGreen dye binds to both ssDNA and dsDNA, but fluoresces
much greater when bound to dsDNA. This makes PicoGreen a very good dsDNA dye reporter.
76

SYBR Gold also bonds to both ssDNA and dsDNA, but was designed to fluoresce strongly when
bound to both. Lane 2 is stained with SYBR Gold and clearly showed the location of the ssDNA
BA probe, lanes 3 and 4 do not show signal for the same DNA when using PicoGreen or
ethidium bromide. Lanes 5-7 were run at a 2:1 ratio of BA:AB to ensure complete binding of the
proposed AB reporter. SYBR Gold has the strongest signal in lane 5, as it will bind and cause
fluorescence in both the dsDNA portion and the unhybridized ssDNA portion. Lane 6 showed a
strong signal using PicoGreen and lane 7 showed no signal. The lack of signal in lane 7 resulted
from being below the detection limit for ethidium bromide in the agarose gel.

When comparing lanes 2 and 3 to lane 5 and 6, it was seen that a third of the total DNA mass in
lane 5 and 6 was significantly brighter than in lanes 2 and 3. The increased quantum yield (or
efficiency) of SYBR Gold and PicoGreen upon binding to dsDNA was responsible. The lack of
signal in lane 3, but strong signal in lane 6 confirmed that PicoGreen could be used as a dsDNA
reporter for the co-polymerization detection.

Compatibility of the SYBR 101 dye system was tested with the co-polymerization product. The
SYBR 101-QD and PicoGreen dyes were used to stain three AB:BA ratios, which are shown
below in Figure 4-6.

77

Figure 4-6: Gel electrophoresis of SYBR 101-QD dye specificity. Lane 1: 100 µg of 100 bp
ladder. Lanes 2-4 and 5-6 are 450 ng of AB:BA at 10:1, 1:1, and 1:10 ratio respectively. Lane 1
stained with SYBR Gold, lanes 2-4 stained with PicoGreen and lanes 5-7 stained with
SYBR101-QD (adapted and modified from Anderson, Zhang, and Alocilja, 2011).

In Figure 4-6 [13], lanes 2-4 show similar banding as in Figure 4-7 confirming successful
hybridization co-polymerization. Lanes 5-7 show a less defined product in the same size region
suggesting proper reaction and staining with SYBR 101-QD conjugated dye. The lack of signal
higher up in the gel (larger fragments) may be the result of low sensitivity. As described in
Figure 4-9 in section 4.3.5, the SYBR 101-QD particle has severely reduced fluorescence
properties. The presence of banding in both dye types confirms compatibility of both dyes for
dsDNA specific reactions and with the co-polymerization detection.
78

4.3.3 Hybridization banding
Characterization of the two part hybridization system was conducted by varying the ratio of AB
to BA during hybridization. Each sample was run in an agarose gel and displayed in Figure 4-7
[13].

Figure 4-7: Optimization of co-polymer ratio. Lane 1: 1 µg of 100 bp ladder. Lanes 2-8 contain
150 ng of total DNA with 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10 ratio of AB:BA respectively
stained with 1x of SYBR Gold (adapted and modified from Anderson, Zhang, and Alocilja,
2011).

SYBR Gold stain was used in order to determine where ssDNA and dsDNA were located. The
lower bands in Figure 4-7, lanes 2, 3, 7 and 8 are the free, unhybridized ssDNA. When one DNA
species is in excess, sufficient unbound DNA is seen as the lower band. As the ideal 1:1 ratio is
79

approached (lane 5) the amount of signal increases. SYBR Gold stains dsDNA more brightly
than ssDNA, which is the cause of the darker bands in lanes 4-6. The formation of longer
dsDNA fragments also were seen as a 1:1 ratio was approached. The longer fragments migrate
slower in a gel and appear near the top of the gel. The dark band at approximately 25% down the
gel is the 1500 bp indicator, which shows that at a 1:1 ratio and 75 ng of each ssDNA species the
dsDNA fragments are close to 1500 base pairs in size. This longest size of generated dsDNA is
approximately 70 units in series. The signal in lane 7 was stronger than in lane 3, which
indicated that BA in excess creates more dsDNA than when AB is in excess. This follows primer
guidelines, BA contains a G or C within the last two bases, and C and G are stronger binders
with three hydrogen bonding partners each. The use of BA as the probe in excess was
determined to be optimal for later sensitivity limit detection.

Comparison between lane 5 of Figure 4-7 and lane 3 of Figure 4-6 reveals a stronger signal with
less dsDNA in Figure 4-7. The difference in hybridization is the method of staining. The stronger
signal was the result of staining during the co-polymerization reaction, where in Figure 4-6 the
staining was done post-hybridization. This may be explained in how PicoGreen binds to dsDNA.
PicoGreen in an intercalating dye and requires approximately 4 bp per PicoGreen molecule
[104]. A range of 3 to 5 PicoGreen dye molecules are able to bind each 22 bp region based on
how closely the dye molecules are packed. Staining during hybridization may provide a more
directed and efficient binding of PicoGreen to the developing dsDNA regions. When stained
post-hybridization, the dye may bind less efficiently. Based on the data, PicoGreen was included
during hybridization in the co-polymerization hybridization sensitivity trials.

80

4.3.4 Co-polymerization sensitivity
Co-polymerization probe sensitivity was determined by challenging a fixed amount of BA probe
with a known amount of AB reporter. From the previous work, the BA species was in excess and
PicoGreen dye was present during the co-polymerization hybridization. BA probe amounts
between 5 – 100 ng and AB reporter amounts between 0.1 – 100 ng were tested and the detection
values are shown in Figure 4-8 [13].

81

Relative Fluorescence Units (RFU)

A 100 ng Probe
B 50 ng Probe
C 25 ng Probe
D 10 ng Probe
E 5 ng Probe
F Probe Only

50000
40000
30000

A

B
20000

D
C

10000

E
F

0
0

20

40

60

80

100

ssDNA Target (ng)

Figure 4-8: Probe sensitivity. Line A is the BA ssDNA probe only. Lines B-F are 5, 10, 25, 50
and 100 ng respectively of BA probe challenged against AB reporter (adapted and modified from
Anderson, Zhang, and Alocilja, 2011).

From Figure 4-8 the line indicating probe only (A) has little to no signal. This was expected as
PicoGreen does not have a strong fluorescent signal when only bound to ssDNA. The lines B-F
represent an increasing amount of BA probe in the system. As each probe concentration was
challenged with AB reporter, the signal increased in a linear fashion for 5-25 ng of probe.

82

Signals peaked and maintained strength beyond 30 ng of AB reporter for concentrations of BA
probe 50 ng and less. Only when 100 ng of probe where used, did the signal continue to increase
with reporter amount. The higher concentrations of probe, lines E and F, show irregularities
when 0.1 ng to 2 ng of target AB reporter were used. For assay purposes, it was desired to have a
constant signal increase and the probe amounts of 50 and 100 ng were removed from the
analysis.

Optimal concentration of the co-polymerization probe were chosen to be 25 ng of BA reporter
with 1x concentration of PicoGreen dye in 1x tris-borate-EDTA (TBE). At 25 ng of probe, the
signal to noise ratio was 3:1 at 100 pg of AB reporter amount. This indicated that the copolymerization had a detection sensitivity of 100 pg output from the sandwich assay.

To show improved fluorescent output, the co-polymerization method was compared to standard
end-labeled DNA release. Co-testing was conducted with Dr. Zhang, using the lower limits of
detection that he had achieved for co-polymerization detection [103]. Using 6-FAM liberated
DNA as the final reporter, the limit of detection was found to be 1 ng at <1000 counts / 0.1
seconds. The lower limit for co-polymerization was found to be 100 pg of PCR DNA target
yielding 1460 counts / 0.1 second. The copolymerization not only had a lower detection limit,
but also a stronger signal. The added 20 minutes of assay time yield a more sensitive detection
system by an order of magnitude with a stronger signal.

The local maxima in the 100 and 50 ng probe values from Figure 4-8 are thought to be a result of
the binding modes of PicoGreen to the semi-dsDNA region. At high probe values the maximum

83

amount of ssDNA-dsDNA ratio is achieve. Essentially, every target AB molecule is bound to
two BA probes. The PicoGreen binding near the ssDNA ends may be stabilized sufficiently to
avoid reduced ssDNA efficiencies. Secondly, PicoGreen will also act as a minor groove binder at
higher PicoGreen to DNA amounts. With an excess in BA probe, the ratio of PicoGreen to DNA
is lower, forcing intercalation as the major binding mode. This binding mode is though to allow a
greater number of dye molecules per dsDNA fragment. At higher PicoGreen to DNA ratios
(lower total DNA), PicoGreen may be intercalating and also minor groove binding. With a
stronger association from both binding modes, the number of dye molecules may be reduced per
dsDNA segment. With sample target concentration being unknown, the higher BA probe
concentrations were avoided to maintain a semi-dose response.

4.3.5 Quantum dot-dye attachment
Quantum dots were successful water solublized with AET. This was confirmed prior to SYBR
101 attachment. The quantum yield of the QDs was deceased after thiol surface
functionalization. Previous work (Pong and Lee 2008) has reported up to a 50% decrease in
signal after sulfur bonding. A quantity of 1 nmol of stock QD’s were dissolved into a total
volume of 100 µL in chloroform and compared against 1 nmol of AET capped QD’s and
measured in a fluorescent plate reader. The AET capped QDs showed a decrease in signal of
68.5% and were stable for 6 months at 4°C.

The fluorescent properties of the SYBR 101 dye were compared pre- and post-QD conjugation.
A quantified mass of dsDNA was stained with both the SYBR 101 reacted with AET or with
QDs and then separated with electrophoresis. The results are shown in Figure 4-9.
84

Figure 4-9: Fluorescence of QD conjugated SYBR 101 dye. Dilutions of 60 bp dsDNA stained
with SYBR 101-AET or SYBR 101-QD. Lanes 1 and 2: 1 µg of 100 bp DNA ladder, lane 1 with
SYBR 101-AET and lane 2 with SYBR 101-QD. Lanes 3, 5, and 7 were run with 75, 30 and 15
ng of SYBR 101-AET stained dsDNA. Lanes 4, 6, and 8 were run with 75, 30 and 15 ng of
SYBR 101-QD stained dsDNA. Samples were run in a 2% w/v agarose gel at 30 V for 2 hours in
1x TBE (adapted and modified from Anderson, Zhang, and Alocilja, 2011).

Fluorescence was maintained after conjugation, but was reduced. Comparison of lanes 3 and 4, 5
and 6, and 7 and 8 show the QD conjugated dye has only 38% signal compared to SYBR 101AET dye. This reduction in signal is a direct result of the QD conjugation. The QDs possess their
own inherent fluorescence and it would be expected to have seen greater signal after QD
conjugation, with a second fluorescent reporter. The severe reduction in signal is a result of
quenching between SYBR 101 and the QD. The QD-SYBR conjugate had reduced signal while
still binding DNA thus confirming successful conjugation of the dye and the quantum dot.
85

4.3.6 Electrochemical detection of quantum dot-dye
Proof of concept detection was accomplished with SYBR 101-QD dye in differential pulse
voltametrtry (DVP). Samples of Zinc/cadmium, SYBR 101-QD and cadmium powder were
tested as described in the methods section. Figure 4-10 [13] shows the plotted data from DPV.

A Water
B Zinc (Zn)

Current (dI)

C Cadmium (Cd)
E

0.0001
0.00008

D Zn & Cd
E SYBR101 & QD

D

0.00006
0.00004

C

0.00002

A&B
0
-1.1

-0.9

-0.7

-0.5

Voltage (V)

-0.3

-0.1

Figure 4-10: Differential pulse voltammetry of metallic particles. Peaks between -0.87 V and 0.90 V are seen indicating cadmium presence in all three metallic samples (adapted and modified
from Anderson, Zhang, and Alocilja, 2011).

86

Sample Zn/Cad in Figure 4-10 DPV contained 20 µL of 1 mM cadmium chloride and 1 mM of
zinc chloride, with a reduction peak of -0.87 V. When cadmium chloride alone was tested (1
mM), the same peak was seen indicating the -0.87 V peak was from cadmium alone. SYBR 101QD samples (approximately 7.5 nmol) contained a similar strength signal with a -0.90 V
reduction peak confirming the dye can be used in electrochemical detection. Electrochemical
detection using QD-dye was not accomplished in the original paper as a method to separate the
free dye from the bound dye was not available.

4.4 Conclusions
The successful generation of a co-polymerization hybridization system was accomplished. A
system of two ssDNA species was generated and shown to partially hybridize in a continuous
fashion generating longer dsDNA fragments. This system was incorporated into a sandwich
assay for detection of Bacillus anthracis from cultured samples. Compared to standard endlabeled 6-FAM DNA systems, co-polymerization increased the detection sensitivity by 10 fold
with a stronger signal and better signal to noise ratio than previous work. Optimal assay
conditions were determined to be 25 ng of BA probe in a total volume of 100 µL (1x Picogreen,
1x tris-borate-EDTA buffer).

To fully investigate the unusual spike in the co-polymerization hybridization additional work
will need to be conducted. The amount of dye used should be explored to try and force the
increase in signal but at lower probe amounts. The co-polymerization system if forced into the
conditions of appropriate dye to probe to target amount may provide a highly sensitive
absence/presence test using fluorescent detection. A possible future set of experiments would
87

include varying the PicoGreen amounts from 1x through 0.01x with probe values from 10 ng
through 1 pg. It would be expected to find a set of conditions at lower dye and probe levels that
exhibit similar increase in fluorescent signal as was seen at the 50 ng and 100 ng probe amounts
from Figure 4-8.

The successful attachment of quantum dots to a SYBR 101 dye molecule was accomplished and
was shown to be compatible with the co-polymerization system. The SYBR 101-QD system was
also shown to be detectable using electrical differential pulse voltammetry. The usage of the
SYBR101-QD system for electrochemical detection was not realized in this work. Successful
detection with the SYBR101-QD conjugate required a way to separate the bound dye from the
unbound dye. The SYBR101-QD conjugate formed a colloidal suspension, preventing separation
based on centrifugation. The size scale of the dye and DNA target remove the ability to filter the
dye-target product from the free dye. Future work to include the SYBR101-QD into the detection
system would require a plate based assay. An ssDNA strand tethered to a plate surface could act
as a hybridization point for co-polymerization. The resulting immobilized dsDNA would retain
bound SYBR101-QD when washed with buffer and separate the free SYBR101-QD dye from
bound dye.

88

Chapter 5: Co-polymerization hybridization sandwich assay for detection of the Shiga-like toxin
1 in Escherichia coli O157:H7

5.1 Introduction
In the previous Chapters, 3 and 4, the generation of dextrin coated nanoparticles and selfassembling co-polymerization hybridization was presented. The dextrin coated gold
nanoparticles (AuNPs) were successfully functionalized with DNA probes. These probes were
then shown to be removable for basic fluorescent detection. The use of the designed copolymerization DNA oligonucleotides was successful in detecting a target when allowed to
hybridize, and fluoresced after dye intercalation binding. Chapter 5 describes the combination of
these different technologies into a single biosensor system for detection of the Shiga-like toxin 1
(stx1) in Escherichia coli O157:H7 Sakai (E. coli O157:H7) strain. This biosensing system is
comprised of two particles, gold nanoparticles and magnetic microparticles (MMP), both of
which are labeled with DNA probes. These probes target the stx1 gene, which is present in shiga
toxigenic group of Escherichia coli (STEC) and enterohemorrhagic E. coli (EHEC). The Sakai
strain of E. coli O157:H7 was used in this assay as it contains both of the Shiga-like toxin (stx1
and stx2) genes common to the STEC and EHEC families of microorganisms. Shiga toxin and
Shiga-like toxin are responsible for the illness associated with STEC and EHEC disease.

The two DNA coated particles were linked together when simultaneously hybridized to the stx1
DNA target. The resulting sandwich structure of MMP-DNA-AuNP was magnetically separated
and the desired form of detection was performed. The detection system was evaluated with
electrical reduction-oxidation, standard end-label fluorescence, and co-polymerization detection
89

against genomic DNA extracted from E. coli O157:H7. While electrochemical detection has
been miniaturized for possible field use, fluorescent detection has potential for field use with
standard digital camera imaging and light emitting diode (LED) illumination.

5.2 Materials and methods
5.2.1 Primer and probe generation for Shiga-like toxin 1 (stx1) gene

The primer and probe sequences were designed to target the stx1 gene sequence. A modified
sequence was generated to target a 95 base pair (bp) region for hybridization assay detection [9].
The stx1 gene is highly conserved in STEC and EHEC organisms and was chosen for the target
sequence. Primers were designed with the properties listed in Table 5-1. Sequences are listed in
Table 5-2.

Table 5-1. PCR primer properties and design optimization for the stx1 gene.

Generated
95 bp
71.6 °C
47%
27
27
48%
44%
60.2°C
58.3°C

Target Size
Target Tm
Target GC%
FWD Primer Size
REV Primer Size
FWD Primer GC%
REV Primer GC%
FWD Primer Tm
REV Primer Tm

90

Optimal
90-110 bp
65-75°C
40-60%
18-24 bp
18-24 bp
40-60%
40-60%
45-55°C
45-55°C

Table 5-2. stx1 primers and amplified sequence (95 bp).
FWD Primer

5’ CAT CTG CCG GAC ACA TAG AAG GAA ACT 3’

REV Primer

5’ GGG AAG CGT GGC ATT AAT ACT GAA TTG 3’
5'CATCTGCCGG ACACATAGAA GGAAACTCAT CAGATGCCAT
TCTGGCAACT CGCGATGCAT GATGATGACA ATTCAGTATT

Generated Target:
AATGCCACGC TTCCC 3' Position 2,924,863 - 2,925,257 of gene sequence
[105].

The chosen primer set was used for generation of probes for the hybridization assay. The primer
sequences were incorporated into the probe set. Successful generation of the primers would also
indicate the availability for probe binding. The probes generated are listed in Table 5-3.

Table 5-3. Sandwich assay particle probe sequences.
5' Phosphate - AAA AAA AAA AAA CAT CTG CCG
MMP Probe
GAC ACA TAG AAG 3'
5' GTA TTA ATG CCA CGC TTC CCA AAA AAA AAA
AuNP Probe
AA - Thiol 3'

The probes generated included a spacer arm of poly-adenosine to distance the binding sequence
from the particle surface. The magnetic particle contained only a single species of DNA probe,
where the gold nanoparticle was conjugated with two different DNA species. The probe attached

91

to the AuNP allowed for the sandwich assay to occur, where the second species of DNA was
used for fluorescent reporting and detection. The second strand of DNA attached was determined
by the method of fluorescent detection. The sequences that were generated and their uses are
listed in Table 5-4 below.

Table 5-4. Co-polymerization sequences. Thiol sequences are for AuNP attachment. FAM was
used for direct fluorescence testing, AB-Thiol and BA were used for standard co-polymerization
detection and C-Linker, AB, and BA were used for tethered co-polymerization detection.
FAM

5' FAM - ATC AGT CAG TCA GTC AGT CA - Thiol 3'

C-Linker

5' GCT ACG AAT AAA TAG AAC AGT C - Thiol 3'

AB-Thiol 5' Thiol - TTA TTC GTA GCG TGA TGC CAA G 3'
AB

5' TTA TTC GTA GCG TGA TGC CAA G 3'

BA

5' GCT ACG AAT AAC TTG GCA TCA C 3'

5.2.2 Assay particle generation
The magnetic microparticles used were Fe3O4 particles of 1.5 µm diameter decorated with a
primary amine on the surface (Bangs Labs #BM546). The gold nanoparticles used were
generated as described in Chapter 3 (10 g/L dextrin, 50°C, pH 9.0 and 2 mM gold chloride).

92

5.2.2.1 Magnetic particle functionalization
The amine coated magnetic microparticles used in this assay were functionalized with the 5’phosphoralated probe listed in Table 5-3 (MMP-Probe). The probe was attached using the zero
length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and NHydroxysuccinimide (NHS) in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH
6.0, Figure 5-1. The magnetic particles (5 mg) were added to EDC (5 mg), NHS (50 mg), and 10
nmol of phosphorylated DNA in a 15 mL polypropylene centrifuge tube. The 5’ end phospohate
of a DNA chain reacts with EDC in the same manner as a carboxyl and provided a means of
covalent attachment. The reaction proceeded for 48 hours at 25°C under gentle agitation while
protected from light. The particles were washed three time in the assay buffer (described in
section 5.2.4 Sandwich assay) and resuspended to a final concentration of 0.8 mg/mL.

O
O P OH
R1
O

O

H2N R2

EDC

NHS

OH
P
O
HN R2
R1

Figure 5-1: Probe attachment to magnetic particle via EDC/NHS cross-linker reaction. DNA
probe (R1) with 5’ end phosphate, Magnetic particle (R2) with surface primary amine.

5.2.2.2 Gold nanoparticles functionalization
The gold nanoparticles were functionalized with DNA through ligand exchange in the same
fashion as citrate generated gold nanoparticles. Ligand exchange starts with a reactive thiol
which bonds to a free gold surface after a dextrin molecule disassociates. The thiol bond is
93

sufficiently stronger than the electrostatic bonding of the dextrin, and forms a semi-permanent
surface decoration. Three different species of gold nanoparticles were generated for each of the
fluorescent readout methods. All gold nanoparticles were functionalized with 0.05 nmol of
AuNP Probe (Table 5-3) and 5 nmol of a reporter (Table 5-4). The thiolated DNA probes were
reduced in 0.1 M dithiothreitol (DTT) under constant agitation at room temperature. Excess and
reacted DTT was removed from the DNA reduction reaction using a Sephadex filtration column
(GE Healthcare #Nap-5) per manufacturer instructions. The reduced and reactive DNA was
mixed with 1 mL of washed gold nanoparticles at 4 nM (1nM: 1 absorbance unit (AU) at 520
nm) in 18 megaohm (MΩ) water. Salt additions were added over a 48 hour period to facilitate
close packing of the DNA oligonucleotides on the gold nanoparticles surface [11]. Particles were
stored in the reaction solution for a maximum of 30 days on the bench top until use.

5.2.3 DNA target extraction
Bacterial cultures of E. coli O157:H7 Sakai were refreshed and allowed to grow for 4 hours
8
resulting in a culture of live E. coli at concentration of about 10 colony forming units per

milliliter (CFU/mL). The live culture was serially diluted eight times, each at 1:10, creating a
dilution series. A volume of 1 mL of each of the dilutions was treated with the Trizol (Invitrogen
#15596-026) reagent or proteinase K digestion followed by standard ethanol precipitation. Trizol
extraction was also compared against phenol/chloroform extraction as the Trizol protocol also
uses bi-phase extraction. Using Trizol, in brief, each culture sample was pelleted and
resuspended in the Trizol reagent and vortexed. Chloroform was then added and vortexed again.
The sample was allowed to separate in to a three phase system, with DNA in intraphase
boundary and the lower phase. The upper phase is removed and then 100% ethanol was mixed
94

into the lower phase. The sample was centrifuged and washed two times with citrate buffer (10%
ethanol, 0.1 M sodium citrate) following with a 70% ethanol precipitation. Following a final
centrifugation, the DNA was resuspended in 300 µL of 8 mM sodium hydroxide. The total
recovery time was 2 hours. Standard recovery starts by digestion with 100 µg of proteinase K
added to the bacterial pellet that has been resuspended in lysis buffer (200 mM sodium chloride,
100 mM Tris, 5 mM ethylenediamminetetraacetate (EDTA), and 0.2% sodium dodecyl sulfate
(SDS)). Digestion with proteinase K was accomplished at 65°C for 75 minutes. Samples were
then ethanol precipitated using three rounds of centrifugation at 13,000 x g. The first
centrifugation was carried out in 100% isopropanol, then 100% ethanol, and finally 70% ethanol,
with the supernatant being removed each time. The final DNA sample was dried for 2 hours in a
bio-safety cabinet and resuspended in the same fashion as the Trizol method. Total recovery time
for ethanol precipitation was 4.75 hours. Phenol chloroform extraction samples were mixed in a
phenol/chloroform solution (Invitrogen #15593-031), vortex for 3 minutes and then ethanol
precipitated, with recovery requiring 3.5 hours. The recovered DNA was measured on a
NanoDrop 1000 (Thermo Scientific) spectrophotometer.

5.2.4 Sandwich assay
The DNA input for the assay was prepared by heating the desired target (40 µL) in a thin walled
300 µL tube at 95°C for 5 minutes in a water bath to denature the DNA into single-stranded
DNA. The tubes were then rapidly cooled in a -20°C isopropanol bath for 30 seconds to stabilize
the denatured single-stranded DNA target. The 40 µL of DNA was mixed with 0.08 mg of probe
functionalized DNA microparticles and adjusted to a final volume of 200 µL using assay buffer
(150 mM sodium chloride, 10 mM phosphate, 0.1% SDS, and pH 7.4). The samples were
95

inversion mixed at 45°C for 60 minutes. Upon hybridization, magnetic separation was used to
wash the unbound DNA from the particles and resuspended to 200 µL of assay buffer containing
40 µL of the desired functionalized gold nanoparticles (1 nM). The MMPs-DNA and AuNPs
were allowed to incubate and hybridize for 2 hours at 45°C under inversion mixing. The
reactions were magnetically separated and washed twice to obtain the final MMP-target-AuNP
sandwiches. The choice of electrochemical detection or fluorescence amplification detection
method determines the next steps in the process are described in section 5.2.5.

5.2.5 Detection
5.2.5.1 Reduction oxidation electrochemical detection
Electrochemical detection was accomplished by placing the magnetically separated sandwich
structures from the previous section on to the carbon working electrode of a screen printed
carbon electrode (SPCE) and allowed to dry for 1 hours in a biosafety cabinet (Figure 5-2) [32].
A volume of 50 µL of 0.1 M hydrochloric acid was placed on the SPCE as the electrolyte.

96

Figure 5-2: Schematic of the screen printed carbon electrode. Carbon working electrode with
silver/silver chloride combination reference/counter electrode.

A voltage of +1.5 V was applied to the carbon electrode-sample for 120 seconds to condition the
sample. The +1.5 V converted the ground state gold nanoparticles into gold(III). The SPCE was
measured using a benchtop potentiostat (Princeton Applied Research, Potentiostat/galvanostat
263A). The readout of the gold ions was accomplished using differential pulse voltammetry
(DPV), with voltage sweeping from +1.5 to 0 V at 33.3 mV/s (step potential 10 mV and
modulation amplitude of 50 mV). Within the SPCE/gold/hydrochloric acid electrical system,
gold reduction appears as a differential pulse voltammetry signal centered around +0.3 V.

5.2.5.2 Direct carboxyfluorescein (6-FAM) attachment
Detection of fluorescence was quenched in the presence of gold nanoparticles and detection
requires liberation of the 6-FAM-DNA from the AuNP. The MMP-DNA-AuNP samples were
resuspended in 5 M DTT and heated to 95°C for 15 minutes and then agitated for 45 minutes at
21°C. The samples were then centrifuged at 13,000 x g for 30 minutes and the supernatant was
transferred into a 96 well plate. Detection was accomplished by excitation at 492 nm ± 4 nm and
with an emission filter at 535 nm ± 12.5 nm.

5.2.5.3 Co-polymerization growth
The sandwiched MMP-DNA-AuNP from the sandwich assay was resuspended in 5 M DTT and
the DNA was recovered in the supernatant the same way as in section 5.2.5.2. The recovered
DNA (AuNP-Probe, Target, and AB-Thiol) sequences were added to 50 ng of BA probe, and
97

heated again to 95°C for 5 minutes and allowed to air cool to room temperature over a 10 minute
period. The samples were then mixed with PicoGreen (Invitrogen #P7581) at 1x concentration
and allowed to stain for 5 minutes. The stained DNA was then plated and fluorescence detection
was conducted the same way as in section 5.2.5.2.

5.2.5.4 Co-polymerization tethering
The concentrated MMP-DNA-AuNP used with the “C-Linker” labeled AuNP was resuspended
in a solution of assay buffer with 20 nM AB and 20 nM BA. The AB/BA co-polymerization
solution was mixed to a concentration of 1 µM of each species in assay buffer and heated to
95°C for 15 minutes prior to the end of the sandwich assay. The AB/BA solution was allowed to
air cool slowly to room temperature before addition to the sandwich structures. A volume of 25
µL of the AB/BA solution was resuspended with the MMP-DNA-AuNP sandwiched to 200 µL
in assay buffer. The AB/BA sequences were allowed to hybridize to the C-Linker for 15 minutes
at room temperature under constant inversion. The samples were magnetically separated,
washed, and the DNA was liberated with DTT as described previously. The resulting supernatant
contained the AuNP-Probe, target DNA, C-Linker, and AB/BA complexes. This was heated to
95°C for 5 minutes and later air cooled to promote hybridization. Finally, PicoGreen was added
to a final concentration of 1x and the samples were read in a plate reader in the same fashion as
described in 5.2.5.2.

98

5.3 Results and discussion
5.3.1 Primer and probe generation evaluation for Shiga-like toxin 1 (stx1) gene
Primer sequences were generated from the gene sequence reported in Fode-Vaughan (2003) to
produce a 95 bp target. Primer compatibility was tested by polymerase chain reaction (PCR) of
genomic DNA extract using the Trizol extraction method. A mass of 50 ng of extracted E. coli
O157:H7 DNA was loaded into a PCR master mix (Invitrogen Fast SYBR® Green Master Mix
#4385610) with 300 nM of primer in a 50 µL reaction volume. Genomic E. coli O157:H7 DNA
was also amplified with a primer set reported in Sharma (2003) in combination for both the stx1
and stx2 genes. Table 5-5 listed the primers used to test the stx1 gene presence and potential
binding.

Table 5-5. Primer sequences for stx1 and stx2 used to test primers generated for sandwich assay.

Sequence Name
stx1-ForwardAnderson (FWD)
stx1-ReverseAnderson (REV)
stx1-ForwardSharma (S1F)
stx1-ReverseSharma (S1R)
stx2-ForwardSharma (S2F)
stx2-ReverseSharma (S2R)

Sequence
5’ CAT CTG CCG GAC ACA TAG
AAG GAA ACT 3’
5’ GGG AAG CGT GGC ATT AAT
ACT GAA TTG 3’
5' GAC TGC AAA GAC GTA TGT
AGA TTC G 3'
5' ATC TAT CCC TCT GAC ATC
AAC TGC 3'
5' ATT AAC CAC ACC CCA CCG 3'
5' GTC ATG GAA ACC GTT GTC
AC 3'

Source

Amplicon
Size
95 bp

Sharma
2003

150 bp

Sharma
2003

206 bp

The end product of PCR amplification reactions (10 µL) were loaded into a 2% w/v agarose gel
and run at 30 V for 120 minutes in buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA).
99

The samples were prestained with a fluorescent dye, SYBR Green, from the master mix PCR kit.
The results are show in Figure 5-3, with lane contents listed in Table 5-6.

Figure 5-3: Agarose gel electrophoresis with PCR product. Combinations of stx primer sets
shown. Lane contents are listed in Table 5-6.

Table 5-6. Lane description for Figure 5-3. Lanes 1,7 and 13 do not contain PCR reactants, lanes
2-6, 8-12, and 14 underwent PCR reaction.

100

Lane
1
2
3
4
5
6
7
8
9
10
11
12
13
14

Description
Ladder
Primer Only
Primer Only
Primer Only
Primer Only
Primer Only
Ladder
Template
Template
Template
Template
Template
Template
Template

Contents
10 bp per band
S1R / S1F
S2R / S2F
REV / FWD
S1R / S1F / S2R / S3F
REV / FWD / S2R / S3F
100 bp per band
S1R / S1F
S2R / S2F
REV / FWD
S1R / S1F / S2R / S3F
REV / FWD / S2R / S3F
Extracted Genomic DNA
PCR with No Primers

No significant product is seen in lanes 2-6 when the primers are run alone in the PCR reaction.
This establishes that no self-priming (binding) is occurring and creating off-target amplicons.
When the Sharma primers are run individually, as seen in lanes 8 and 9, only a single amplicon is
seen with the expected product size of about 150 and 200 bps. The developed primer, lane 10,
also creates the correct size amplicon of about 100 bp. When both the stx1 and stx2 primers from
Sharma are mixed together, lanes 11 and 12, two products are observed. In lane 11, a larger band
is generated that extends from the 150-200 bp size range. Lane 12 shows two distinct bands, both
100 and 200 bp in size, stx2 from Sharma and the stx1 primer generated. That two products are
formed indicates that both genes are present in the target E. coli genome. Both stx1 primer sets
function as expected. The generated FWD and REV primer set created for the sandwich assay
correctly bind the stx1 gene and were used to generate probes for the magnetic microparticles
and gold nanoparticles.

101

Probes were generated using the primer sequences with an appropriate poly-adenosine sequence
added to create a stand off distance from the particle. This stand-off distance allowed for
hybridization of the target DNA from E. coli while also having the attached fluorescent reporter
on the gold nanoparticles.

5.3.2 Gold nanoparticles functionalization and detection mechanism
The gold nanoparticles were functionalized as previously described in the methods section.
Three types of gold nanoparticles were generated based on the DNA species attached. Figure 5-4
shows the detection principle for 6-FAM labeled gold nanoparticles.

102

a.

FAM
AuNP Probe

b.
heat

centrifuge

time

DTT
Figure 5-4: Direct fluorescence AuNP construction. (a) AuNP after generation; (b) Schematic of
gold nanoparticle detection of end-labeled DNA after sandwich assay. DNA is liberated from the
AuNP via DTT exchange.

The 6-FAM labeled DNA wad recovered from the gold nanoparticles after treatment with DTT
as in Figure 5-4b and fluorescently detected as described in the methods section. Detection with
co-polymerization hybridization as described in Chapter 3 is shown in Figure 5-5.

103

AB-Thiol

a.

AuNP Probe
BA
PicoGreen dye

b.

heat

centrifuge

time

1. heat
2. cool

DTT
Figure 5-5: Standard co-polymerization AuNP construction. (a) AuNP after generation; (b)
Schematic of co-polymerization detection after sandwich assay. The co-polymerization DNA
reporter was liberated and then reacted.

Upon liberation of the attached DNA from the gold nanoparticles, the DNA was mixed with the
co-polymerization hybridization partner and PicoGreen dye and heated. The DNA was then
slowly cooled, forming double-stranded DNA sequences that fluoresce when bound to the dye.
Detection was accomplished in the same manner as with the 6-FAM samples. The third
fluorescent detection method was accomplished in the manner shown in Figure 5-6.

104

C-Linker

a.

AuNP Probe
Co-polymerized DNA
PicoGreen dye
b.

magnetic

heat

wash

time

DTT
c.

centrifuge

1. heat
2. cool

Figure 5-6: Tethered co-polymerization AuNP construction. (a) AuNP after generation; (b)
Schematic of co-polymerization tethering detection. Co-polymerized DNA hybridizes to the
AuNP before DTT liberation and detection.

The attachment of the C-Linker DNA species to the gold nanoparticles provided a hybridization
attachment point for pre-co-polymerized DNA to attach. The co-polymerized DNA hybridized to
the gold nanoparticles and separated while still part of the magnetic microparticle sandwich.
After the second magnetic separation, the system was treated in the same fashion as the ABThiol system.

105

Successful generation of each of the three gold nanoparticles species was confirmed by a stable
solution after ligand exchange, indicated by maintaining the original color. Control reactions
without stabilizing DNA molecules turn purple and precipitate out of solution after the salting
steps described in the methods of Chapter 5.

5.3.3 DNA extraction optimization
Optimization of a DNA extraction method was explored using Trizol, proteinase K digestion
with ethanol precipitation, and phenol/chloroform extraction. The previous method used was
enzyme digestion of the cellular material with ethanol purification. This method has a high
recovery of DNA, but requires a large amount of equipment and 4 hours to complete. The Trizol
kit uses a proprietary solvent system based on chloroform/phenol extraction. The Trizol kit is a
two hour procedure requiring low equipment demands. Phenol/chloroform was explored as a
control to the Trizol method. Table 5-7 lists the results of DNA recovery from 1 mL of 4 hour
8
culture (~1x10 CFU).

106

Table 5-7. Comparison of DNA extraction methods.
DNA Recovery

Standard

(ng)

Deviation (ng)

Method
Ethanol

19408

3982

Trizol

29980

6060

Phenol

600

420

From the data, the Trizol method performs reasonably well compared to the ethanol precipitation
in half the time. Trizol extraction also has the advantage of no perishable enzymes, no heating
steps, and only requires a low speed centrifuge, leading to possible field based extraction. The
standard phenol/chloroform extraction does not perform well compared to either of the other
methods. It is believed that additional lysis agents are present in the Trizol reagent that increases
DNA recovery. Trizol was used to obtain the target DNA input from the dilution series in the
sandwich assay.

5.3.4 Detection
Detection was accomplished by either of two methods, electrochemical or fluorescence.
Electrochemical detection uses the entire MMP-DNA-AuNP sandwich for detection, where
fluorescence uses the DNA removed by DTT from the metallic particles in the assay.

107

5.3.4.1 Reduction oxidation detection optimization
Electrochemical detection was performed as described in the methods section of Chapter 5. The
gold nanoparticle species used for electrochemical detection was the AB-Thiol particles. The
final product from the assay was dried on the SPCE and then conditioned with 0.1 M
hydrochloric acid for 120 seconds at +1.5 V. Dilutions were spilt between samples for the assay
and bacterial culture plating for actual determination of cell count. A volume of 1 mL from each
dilution was used for DNA extraction, assay hybridization, and gold reduction/oxidation
measurement. The detection is displayed in Figure 5-7 below.

108

0.00008

Current (dI)

0.00006
H

F

A
C

0.00004
G

0.00002

E

A Blank
B 5x107 CFU
C 5x106 CFU
D 5x105 CFU
E 5x10 3 CFU
2
F 5x10 CFU
1
G 5x100 CFU
H 5x10 CFU

B

D

0
0

0.2

0.4

0.6

0.8

1

1.2

Potential (V)
Figure 5-7: DPV response of gold nanoparticles after sandwich assay. Gold response is seen as a
peak between +0.2 and +0.4 V. The peak in curve A is a result of the hydrochloric acid and
MMP interaction with the system with AuNPs.

In Figure 5-7 most concentrations of target showed a positive capture. The low signal of sample
5
5x10 CFU/mL still had the reduction peak at 0.3-0.4 V characteristic of the SPCE system. This

set of data was from centrifuge tubes that leaked during hybridization, losing sample. Previous
use of the sandwich assay with PCR generated targets showed a linear response with target

109

concentration [32]. The DNA recovery and assay procedure above did not show the same linear
response.

The non-dose response of the reduction/oxidation plot may be the result of DNA extraction
inefficiency, sandwich assay losses, magnetic separation loss, and SPCE detection sensitivity.
The reduction/oxidation measurement was used to confirm proper functioning of the assay
system as a whole, including the new probes, target extraction, and nanoparticles recovery.
Detection of the thiol treated gold nanoparticles was attempted to verify the fluorescence in
tandem and compare the sensitivities on the same samples. Figure 5-8 shows the electrical
response from that system.

0.00005

A AuNP Standard
B Blank
C 5x107 CFU
D 5x106 CFU
E 5x105 CFU
F 5x10 4 CFU
3
G 5x10 CFU
1
H 5x10 CFU
0
I 5x10 CFU

G

0.00004

Current (dI)

F
D

0.00003
C

E

0.00002
A

0.00001

B
H&I

0
0

0.25

0.5

0.75

1

Potential (V)
Figure 5-8: DPV response of thiol liberated AuNPs after sandwich assay.

110

1.25

1.5

From Figure 5-8 no gold reduction peak was seen in the 0.3 – 0.4 V range for the samples. The
full amount of gold into the system showed the gold peak at +0.35 V confirming that the
electrical system was functioning. The thiol addition and gold particle recovery were
incompatible with the reduction of this system. The 5 M DTT in the system prevented the
measurement either through gold surface stabilization or masking of the electrochemical signal.
This result eliminated the ability to use both electrochemical and fluorescent detection of the
sandwich structure because of the thiol. A third method was explored to recover the gold
nanoparticles from the sandwich structure using high temperature (95°C) to break the hybridized
DNA, Figure 5-9. This method of gold recovery would only be compatible with the C-Linker copolymerization method, as the attached co-polymerized DNA would be freed and thus
recoverable. Without the addition of DTT into the sandwich structure system, little or none of the
thiol linked DNA would be free. The lack of DNA disassociation removed the direct FAM
attachment and AB-thiol recovery and subsequent fluorescent detection.

111

0.000018

A 1x107
6
B 1x10
5
C 1x10
4
D 1x10
3
E 1x10
1
F 1x10 0
G 1x10

A

Current (dI)

0.000013

C
B

0.000008

E
F

0.000003

G
D

-0.000002 0

0.5

1

1.5

Potential (V)
Figure 5-9: DVP response from sandwich assay. AuNPs were separated using 95°C without DTT
to break sandwich structure.

The gold containing fraction after 15 minutes of incubation at 95°C did not exhibit any
reduction/oxidation peaks or show appreciable signal. The corresponding MMP pellet was tested
under the same gold detection method. The Figure 5-9 shows the presences of gold in five of the
seven samples (lines A-C, E and F). The gold remaining after magnetic separation of the iron
fraction indicated that the hybridized gold nanoparticles were not successfully freed by boiling
alone. Based on the previous trials it was determined that electrochemical reduction/oxidation
measurement can be performed on the whole sandwich structure alone, and a sample cannot be
both electrochemically detected and fluorescently detected using this system.

112

5.3.4.2 Fluorescent readout
Fluorescent detection comparison was accomplished by splitting a serial dilution series of E. coli
O157:H7 between each of the three fluorescence methods. DNA was extracted as before and the
sandwich assay was performed using the appropriately generated gold nanoparticles. Detection
of the labeled DNA was accomplished after a thiol liberation step. The gold and magnetic
particles were pelleted at 13,000 x g for 25 minutes and the supernatant was readout. Figure 5-10

Relative Fluorescence Unit (RFU)

shows the signal from the dilution series for 6-FAM labeled gold nanoparticles.

600
500
400
300
200
100
0
0

2

4

6

8

Cell Count per mL (log)
Figure 5-10: Fluorescence detection of 6-FAM end labeled DNA AuNPs. DNA was liberated
using DTT exchange. Samples measured using fluorescein filters set.
113

The directly labeled DNA did not show a strong response to the amount of target after the
sandwich assay. The result seen above in Figure 5-10 is a combination of multiple factors. The
high amount of thiol used for liberating the DNA has the potential to attack the FAM reducing
the signal. The low amount of reporter on the gold nanoparticles in addition to photo-bleaching
during the assay and during gold-nanoparticle conjugation may also lead to the lower than
expected signal. Previous efforts showing effective conjugation of 6-FAM-DNA to both citrate
and dextrin generated gold nanoparticles utilized approximately 100 times the gold particles
[12]. This work used lower DTT concentrations and the detection was in a smaller volume.

The AB-Thiol and C-Linker gold nanoparticles were tested per the methods section previous
described and are shows in Figure 5-11.

114

15000

C-Linker
AB-Thiol

Relative Fluorescence Unit (RFU)

13000
11000
9000
7000
5000
3000
1000
0

2

4

6

Cell Count per mL (log)

8

Figure 5-11: Fluorescence detection of co-polymerization amplification of sandwich assay.
Blank samples are reported at the 0 cell count log value. Samples measured using fluorescein
filters set.

Both of the co-polymerization methods showed a dose response. The AB-Thiol and C-Linker
2
methods had detection in the 10 CFU/mL range. The difference between the two methods was

seen in the signal strength and the higher range values. The AB-Thiol method has a weaker
signal but was a simpler and faster method compared to the C-Linker amplification. The C-linker
had a more consistent signal and larger signal, but required more time for detection. Both
methods showed successful detection of the stx1 target in E. coli O157:H7.

115

5.4 Conclusions
The described nanoparticle based biosensor in this chapter was successfully used to detect the
stx1 gene in Escherichia coli O157:H7 Sakai culture samples. To accomplish detection, a rapid

and efficient DNA extraction method was determined and used to obtain PCR quality DNA
target. The extraction method finalized was using the Trizol reagent, which only requires room
temperature reactants and a low speed centrifuge. The nanoparticle sandwich assay was tested
against serially diluted E. coli culture. Detection methods compared were reduction/oxidation
and fluorescent co-polymerization hybridization. Both methods were capable of detecting target
2
DNA from a 10 CFU/mL sample. Total assay time from sample to electrochemical detection or

sample to fluorescence detection requires 7 hours.

The system developed and optimized in this study has comparable detection sensitivity and time
requirements to other systems previously developed. The main strengths of the described system
are the DNA extraction, low equipment needs, low cost, multiple detection formats, and an
additional amplification step with co-polymerization hybridization.

116

Chapter 6: Conclusion and future work

The combined research presented in Chapters 3-5 described the engineering and development of
a DNA based biosensor utilizing self assembling DNA sequences and carbohydrate coated gold
nanoparticles to target the stx1 gene in Escherichia coli O157:H7 Sakai. Current US government
standards for water testing require 12-24 hours before detection. This detection is not specific to
the pathogenic targets, but only identifies the presence of an indicator species commonly found
along side pathogenic species of bacteria. The research in this dissertation presents a complete
assay technique for DNA based identification in 7 hours, making it more specific and faster than
currently approved methods.

Traditional DNA based biosensors require lengthy and involved DNA extraction processes to
produce a pure input material for enzymatic amplification. In the presented system the extraction
is accomplished with a commercial kit. The kit produces a DNA output compatible with the
engineered assay system and requires less than 2 hours from sample to testing. The assay system
uses a novel carbohydrate coated nanoparticle for amplification of the input signal. Carbohydrate
generated gold nanoparticles were successfully generated under mild alkaline conditions and
used in the sandwich assay for detection of the stx1 gene in Escherichia coli O157:H7. The
synthesis procedure was accomplished using dextrin, a carbohydrate, and sodium carbonate to
initiate and form gold nanoparticles that were successfully decorated with DNA probes. Gold
nanoparticle size was controllable based on generation conditions. The resulting particles were
stable for over 6 months when left in the reaction solution and completely compatible for
replacement of citrate generated particles for the uses in this dissertation.
117

A novel reporter system of self-assembling DNA molecules was designed for co-polymerization
detection. Co-polymerization detection is currently capable of fluorescence measurement
through the use of a dye with high quantum efficiency when bound to double-stranded DNA
target. Co-polymerization is compatible with reduction / oxidation measurement through use of
metallic tracers linked DNA intercalation dyes. To fully utilize electrochemical detection with
co-polymerization, a means of separating free co-polymerization probe and excess dye needs to
be developed. Normally the fluorescent dye will bind to the DNA probes on both particles, but
does not contribute a fluorescent signal since the probes are single-stranded. The nanoparticle
linked dye would bind the same single-strand probes, and would contribute a metallic reduction
signal even in the absence of a target.

Successful detection using co-polymerization was accomplished when combined with a nano /
microparticle sandwich assay and proper DNA extraction. DNA was successfully and rapidly
extracted from live Escherichia coli O157:H7 Sakai and used directly as target in the sandwich
assay. A DNA probe set was created and used to recognize the stx1 gene from E. coli O157:H7
by the magnetic microparticle and gold nanoparticle. Both electrochemical detection of the gold
nanoparticles and fluorescence detection with co-polymerization were successfully
demonstrated. The hybridization based reporter system was shown to be capable of detecting as
5
little as 10 colony forming units per milliliter. Electrochemical detection was capable of a
1
presence / absence detection at 10 colony forming units per milliliter. The co-polymerization

118

method provided a rapid and sensitive fluorescent based detection method with increased
sensitivity over end labeled reporter systems.

Future work is required to optimize both the electrochemical and fluorescent detection methods
of co-polymerization hybridization. Further reduction in the DNA extraction and recovery time
may be possible. Possible reduction in times could include reduced centrifuge times at higher
speeds, additional wash steps with shorter washing times, reduction of the desalting step times,
or simple removal of desalting steps. The removal of salt may not be necessary with the high salt
hybridization buffer used during the sandwich assay. The dextrin gold nanoparticle generation
method has potential for functionalizing the nanoparticles with biomaterials during the
generation process. The conjugation of thiolated DNA and other functional groups during
generation should be explored. Addition of the thiol group at different reaction times, thiol
concentrations, reaction pH, or reaction temperatures may provide a one pot synthesis technique
that produces functionalized gold nanoparticles. A primary amine functional group would be an
ideal attachedment chemistry as it would allow for conjugation to carboxyl, hydroxyl, and thiol
groups.

Exploration into exploiting the large signal increase at low dye to probe ratio in copolymerization hybridization has potential for increased detection sensitivity. Optimization of
PicoGreen and co-polymerization probe amounts may lead to a better signal to noise ratio.
Reduction of background noise would also lead to higher sensitivities. The described assay
system utilizes a polystyrene 96-well plate for readout. The aromatic nature of polystyrene binds
free ssDNA probe and stabilizes the PicoGreen interaction with non-specifically bound ssDNA.

119

The stabilized dye fluorescence created higher background signal than polypropylene
microcentrifuge tubes. Future use of polypropylene 96-well plates would help reduce
background noise. The combination of optimized dye and probe concentration in polypropylene
plate would help achieve maximum sensitivity for the fluorescent detection readout of the copolymerization assay.

The sandwich assay was the longest step of the entire detection process. Reduction in time
during hybridization would have the greatest impact on improving the assay speed. Optimization
of hybridization time and temperature conditions may lead to a reduction of the assay time.
Currently, the assay is performed at a single temperature, which is 10°C less than the melting
temperature of the probes. Heating the initial binding step to a temperature twenty of more
degrees higher than the melting temperature and allowing for a slower rate of cooling may
promote faster hybridization of the magnetic microparticles in the same fashion as the copolymerization hybridization self-assembly. The same optimization would then be possible with
the gold nanoparticle hybridization step, providing further reduction in assay time.

The addition of electrochemical readout to the co-polymerization hybridization method would
further add additional sensitivity to the assay. The addition of a metallic nanoparticle to the copolymerization probe would be one way to introduce electrochemistry. Attachment of a QD
could be accomplished through a modification to the probe’s phosphate backbone. An internal
thiol or phosphorothioate would provide a reactive sulfur atom for QD attachment. By
introducing a QD-DNA structure to the tethered co-polymerization method in Chapter 5, a
method of free QD-DNA removal would be possible. The QD attachment would also present the

120

opportunity for multiplexed detection through different metallic compositions, allowing for
detection of multiple metals with differential pulse voltammetry.

In summary, this research demonstrates the successful integration of rapid DNA extraction,
specific target detection with a sandwich assay, and both electrochemical and fluorescent
detection capabilities. The use of electrochemical detection provides a rapid and sensitivity
absence/presence test, and the fluorescence detection provides a promising tool for sample
quantification.

121

APPENDICES

122

APPENDIX A: DATA
A.1 CHAPTER 3 DATA
Table A-1. Absorbance data of gold nanoparticles for Figure 3-1.
Time:
Wavelength
(nm)
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430

0 hr

1 hr

1.5 hr

2 hr

3 hr

4 hr

5 hr

2.063
2.059
2.057
2.054
2.051
2.047
2.044
2.043
2.041
2.038
2.035
2.032
2.030
2.029
2.024
2.022
2.022
2.021
2.020
2.018
2.017
2.015
2.015
2.014
2.011
2.011
2.011
2.008
2.009
2.009
2.007

2.088
2.080
2.077
2.075
2.071
2.068
2.065
2.064
2.064
2.060
2.057
2.055
2.052
2.049
2.047
2.045
2.043
2.041
2.040
2.036
2.035
2.033
2.031
2.031
2.030
2.029
2.028
2.026
2.026
2.026
2.026

2.029
2.026
2.023
2.020
2.017
2.014
2.009
2.007
2.006
2.002
1.999
1.997
1.995
1.994
1.991
1.990
1.987
1.984
1.982
1.981
1.980
1.979
1.977
1.976
1.974
1.973
1.972
1.972
1.974
1.973
1.971

Absorbance (AU)
0.395
0.393
0.391
0.389
0.388
0.387
0.385
0.383
0.382
0.381
0.379
0.378
0.377
0.376
0.374
0.373
0.372
0.371
0.369
0.368
0.367
0.367
0.366
0.364
0.363
0.363
0.362
0.361
0.360
0.359
0.359

0.393
0.392
0.390
0.389
0.387
0.386
0.385
0.383
0.382
0.381
0.379
0.378
0.377
0.376
0.374
0.373
0.373
0.372
0.370
0.369
0.368
0.368
0.367
0.366
0.365
0.365
0.364
0.363
0.362
0.361
0.361

0.438
0.436
0.435
0.433
0.432
0.430
0.429
0.427
0.426
0.425
0.423
0.422
0.421
0.420
0.419
0.417
0.417
0.416
0.415
0.413
0.412
0.412
0.411
0.410
0.409
0.409
0.408
0.406
0.406
0.405
0.405
123

0.740
0.738
0.735
0.733
0.731
0.730
0.728
0.726
0.724
0.722
0.720
0.719
0.717
0.716
0.714
0.713
0.712
0.710
0.708
0.707
0.705
0.705
0.704
0.702
0.701
0.700
0.699
0.698
0.697
0.696
0.695

Table A-1. Continued
431
0.358
432
0.356
433
0.356
434
0.355
435
0.354
436
0.353
437
0.352
438
0.351
439
0.351
440
0.350
441
0.349
442
0.348
443
0.348
444
0.347
445
0.345
446
0.344
447
0.344
448
0.344
449
0.343
450
0.342
451
0.340
452
0.340
453
0.340
454
0.339
455
0.338
456
0.337
457
0.337
458
0.337
459
0.337
460
0.336
461
0.335
462
0.335
463
0.335
464
0.335
465
0.334
466
0.333
467
0.333
468
0.333
469
0.334
470
0.333
471
0.332

0.360
0.359
0.358
0.358
0.357
0.356
0.355
0.354
0.354
0.353
0.352
0.351
0.351
0.350
0.349
0.348
0.348
0.348
0.346
0.345
0.344
0.344
0.344
0.343
0.342
0.342
0.341
0.341
0.341
0.340
0.339
0.339
0.340
0.340
0.339
0.338
0.338
0.338
0.338
0.338
0.337

0.404
0.402
0.402
0.402
0.401
0.399
0.399
0.398
0.398
0.397
0.396
0.395
0.395
0.394
0.393
0.392
0.392
0.392
0.391
0.390
0.388
0.388
0.388
0.388
0.387
0.386
0.385
0.385
0.385
0.384
0.384
0.384
0.384
0.384
0.384
0.383
0.383
0.383
0.384
0.383
0.382
124

0.694
0.692
0.692
0.691
0.690
0.689
0.688
0.687
0.687
0.686
0.685
0.684
0.684
0.683
0.682
0.681
0.681
0.681
0.680
0.679
0.678
0.678
0.678
0.678
0.677
0.677
0.676
0.677
0.677
0.677
0.676
0.676
0.678
0.678
0.678
0.677
0.678
0.679
0.680
0.680
0.680

2.007
2.009
2.008
2.006
2.006
2.005
2.006
2.008
2.008
2.010
2.012
2.012
2.012
2.015
2.018
2.020
2.022
2.024
2.027
2.029
2.032
2.036
2.037
2.040
2.044
2.045
2.050
2.057
2.063
2.072
2.079
2.084
2.090
2.096
2.106
2.115
2.121
2.129
2.139
2.148
2.158

2.024
2.023
2.024
2.025
2.025
2.024
2.024
2.024
2.025
2.027
2.030
2.033
2.034
2.035
2.038
2.040
2.040
2.044
2.047
2.051
2.054
2.057
2.061
2.064
2.068
2.073
2.078
2.082
2.089
2.096
2.103
2.108
2.116
2.126
2.134
2.141
2.150
2.161
2.172
2.181
2.190

1.969
1.968
1.969
1.970
1.970
1.969
1.970
1.972
1.972
1.974
1.976
1.976
1.977
1.979
1.980
1.983
1.985
1.987
1.990
1.993
1.996
1.999
2.002
2.006
2.009
2.011
2.016
2.021
2.026
2.033
2.040
2.046
2.054
2.063
2.070
2.076
2.083
2.094
2.103
2.111
2.121

Table A-1. Continued
472
0.332
473
0.332
474
0.333
475
0.333
476
0.332
477
0.332
478
0.332
479
0.333
480
0.333
481
0.333
482
0.332
483
0.332
484
0.332
485
0.334
486
0.334
487
0.333
488
0.333
489
0.333
490
0.334
491
0.335
492
0.335
493
0.335
494
0.334
495
0.334
496
0.335
497
0.336
498
0.336
499
0.336
500
0.336
501
0.336
502
0.337
503
0.337
504
0.338
505
0.338
506
0.337
507
0.337
508
0.338
509
0.338
510
0.339
511
0.339
512
0.339

0.337
0.337
0.338
0.338
0.338
0.337
0.337
0.338
0.339
0.339
0.338
0.338
0.339
0.340
0.340
0.340
0.339
0.340
0.340
0.341
0.341
0.341
0.341
0.342
0.343
0.343
0.344
0.344
0.343
0.343
0.344
0.345
0.345
0.345
0.345
0.345
0.345
0.346
0.347
0.347
0.347

0.382
0.383
0.384
0.384
0.383
0.383
0.384
0.384
0.385
0.385
0.385
0.385
0.385
0.387
0.387
0.387
0.387
0.387
0.388
0.389
0.390
0.390
0.390
0.390
0.391
0.392
0.393
0.393
0.393
0.393
0.394
0.395
0.395
0.396
0.395
0.395
0.396
0.397
0.398
0.398
0.398
125

0.681
0.682
0.684
0.685
0.686
0.686
0.687
0.690
0.692
0.693
0.694
0.695
0.696
0.699
0.701
0.702
0.703
0.705
0.708
0.710
0.711
0.713
0.714
0.716
0.719
0.722
0.723
0.725
0.726
0.727
0.730
0.732
0.734
0.736
0.737
0.738
0.740
0.742
0.744
0.745
0.745

2.171
2.183
2.195
2.206
2.216
2.232
2.248
2.261
2.276
2.290
2.308
2.323
2.339
2.356
2.373
2.390
2.409
2.428
2.445
2.461
2.479
2.496
2.515
2.536
2.552
2.568
2.586
2.597
2.615
2.634
2.647
2.661
2.674
2.689
2.703
2.710
2.718
2.727
2.732
2.748
2.760

2.204
2.217
2.230
2.245
2.259
2.273
2.290
2.303
2.323
2.343
2.356
2.373
2.394
2.413
2.428
2.449
2.471
2.489
2.511
2.536
2.558
2.581
2.604
2.629
2.655
2.682
2.702
2.717
2.741
2.756
2.773
2.802
2.821
2.842
2.853
2.863
2.885
2.898
2.898
2.909
2.920

2.133
2.145
2.156
2.167
2.180
2.192
2.204
2.221
2.237
2.250
2.267
2.284
2.299
2.314
2.329
2.341
2.358
2.380
2.398
2.416
2.436
2.449
2.466
2.484
2.496
2.511
2.531
2.551
2.563
2.581
2.592
2.597
2.609
2.622
2.627
2.640
2.653
2.653
2.667
2.681
2.681

Table A-1. Continued
513
0.338
514
0.339
515
0.339
516
0.340
517
0.340
518
0.340
519
0.340
520
0.340
521
0.340
522
0.341
523
0.342
524
0.342
525
0.341
526
0.341
527
0.341
528
0.342
529
0.343
530
0.343
531
0.343
532
0.342
533
0.342
534
0.343
535
0.343
536
0.344
537
0.345
538
0.344
539
0.344
540
0.344
541
0.344
542
0.345
543
0.345
544
0.346
545
0.346
546
0.346
547
0.345
548
0.345
549
0.346
550
0.346
551
0.347
552
0.348
553
0.348

0.346
0.347
0.348
0.349
0.349
0.349
0.348
0.348
0.349
0.349
0.350
0.350
0.350
0.350
0.350
0.351
0.351
0.352
0.352
0.351
0.351
0.351
0.352
0.353
0.353
0.353
0.353
0.353
0.353
0.354
0.355
0.355
0.355
0.355
0.354
0.354
0.355
0.356
0.357
0.357
0.356

0.398
0.398
0.400
0.400
0.401
0.400
0.400
0.400
0.401
0.402
0.403
0.403
0.402
0.402
0.402
0.403
0.404
0.405
0.404
0.404
0.404
0.404
0.405
0.406
0.406
0.406
0.406
0.405
0.406
0.406
0.407
0.407
0.407
0.407
0.406
0.406
0.407
0.407
0.408
0.408
0.407
126

0.746
0.747
0.749
0.751
0.752
0.753
0.753
0.753
0.754
0.755
0.756
0.757
0.756
0.756
0.756
0.756
0.757
0.757
0.757
0.756
0.755
0.755
0.755
0.756
0.755
0.754
0.753
0.751
0.751
0.750
0.750
0.749
0.748
0.746
0.744
0.743
0.741
0.741
0.740
0.739
0.737

2.760
2.760
2.769
2.779
2.769
2.769
2.779
2.779
2.779
2.779
2.779
2.762
2.743
2.750
2.750
2.734
2.727
2.717
2.700
2.687
2.673
2.659
2.646
2.626
2.597
2.582
2.570
2.543
2.514
2.486
2.455
2.423
2.389
2.357
2.322
2.283
2.248
2.209
2.170
2.129
2.088

2.920
2.920
2.934
2.960
2.960
2.948
2.960
2.960
2.948
2.960
2.960
2.948
2.948
2.934
2.920
2.920
2.909
2.883
2.858
2.839
2.819
2.802
2.777
2.750
2.725
2.695
2.667
2.636
2.600
2.560
2.525
2.487
2.441
2.402
2.364
2.322
2.277
2.232
2.189
2.148
2.105

2.681
2.681
2.689
2.697
2.697
2.689
2.689
2.697
2.697
2.697
2.697
2.697
2.689
2.681
2.674
2.661
2.653
2.646
2.631
2.619
2.607
2.589
2.574
2.558
2.537
2.517
2.498
2.476
2.446
2.419
2.397
2.365
2.327
2.293
2.260
2.226
2.190
2.153
2.113
2.075
2.037

Table A-1. Continued
554
0.347
555
0.347
556
0.347
557
0.348
558
0.349
559
0.349
560
0.349
561
0.348
562
0.348
563
0.348
564
0.348
565
0.349
566
0.349
567
0.350
568
0.349
569
0.349
570
0.348
571
0.348
572
0.349
573
0.349
574
0.349
575
0.350
576
0.349
577
0.348
578
0.348
579
0.347
580
0.348
581
0.348
582
0.348
583
0.349
584
0.348
585
0.347
586
0.346
587
0.346
588
0.345
589
0.346
590
0.346
591
0.346
592
0.346
593
0.345
594
0.344

0.356
0.356
0.356
0.357
0.357
0.358
0.358
0.357
0.357
0.356
0.357
0.357
0.358
0.358
0.358
0.357
0.357
0.356
0.356
0.357
0.357
0.358
0.357
0.356
0.355
0.355
0.355
0.355
0.356
0.356
0.355
0.354
0.353
0.353
0.352
0.352
0.353
0.353
0.352
0.351
0.351

0.407
0.407
0.407
0.407
0.407
0.408
0.407
0.407
0.406
0.406
0.406
0.406
0.407
0.406
0.406
0.405
0.405
0.404
0.404
0.404
0.404
0.404
0.403
0.402
0.401
0.401
0.401
0.401
0.400
0.400
0.400
0.398
0.397
0.396
0.396
0.396
0.396
0.395
0.395
0.393
0.392
127

0.734
0.732
0.731
0.730
0.729
0.727
0.725
0.723
0.720
0.718
0.717
0.715
0.713
0.712
0.709
0.706
0.704
0.701
0.700
0.698
0.696
0.694
0.692
0.689
0.686
0.683
0.682
0.680
0.678
0.675
0.673
0.670
0.668
0.665
0.662
0.660
0.659
0.657
0.654
0.652
0.648

2.051
2.012
1.970
1.931
1.891
1.850
1.811
1.773
1.734
1.696
1.656
1.618
1.581
1.545
1.509
1.474
1.439
1.406
1.373
1.339
1.307
1.277
1.247
1.219
1.190
1.161
1.134
1.108
1.081
1.055
1.029
1.005
0.981
0.958
0.935
0.913
0.892
0.871
0.851
0.831
0.811

2.063
2.020
1.975
1.932
1.891
1.849
1.808
1.768
1.728
1.688
1.647
1.609
1.572
1.534
1.498
1.463
1.428
1.394
1.360
1.327
1.295
1.264
1.234
1.204
1.176
1.148
1.121
1.094
1.067
1.041
1.015
0.991
0.967
0.944
0.922
0.900
0.879
0.858
0.838
0.817
0.798

1.996
1.955
1.915
1.875
1.836
1.796
1.757
1.718
1.678
1.640
1.601
1.563
1.527
1.491
1.456
1.422
1.387
1.352
1.318
1.286
1.254
1.223
1.193
1.164
1.136
1.107
1.079
1.051
1.024
0.998
0.972
0.947
0.923
0.899
0.877
0.855
0.833
0.811
0.791
0.770
0.750

Table A-1. Continued
595
0.343
596
0.343
597
0.343
598
0.343
599
0.343
600
0.343
601
0.343
602
0.342
603
0.340
604
0.339
605
0.339
606
0.339
607
0.339
608
0.339
609
0.339
610
0.338
611
0.337
612
0.336
613
0.335
614
0.334
615
0.333
616
0.333
617
0.334
618
0.334
619
0.333
620
0.332
621
0.331
622
0.330
623
0.329
624
0.329
625
0.329
626
0.328
627
0.328
628
0.328
629
0.327
630
0.326
631
0.325
632
0.323
633
0.322
634
0.322

0.350
0.349
0.348
0.349
0.349
0.349
0.348
0.347
0.346
0.345
0.344
0.344
0.344
0.344
0.344
0.343
0.342
0.341
0.339
0.339
0.338
0.338
0.337
0.337
0.337
0.336
0.335
0.334
0.333
0.332
0.331
0.331
0.331
0.331
0.330
0.329
0.327
0.326
0.325
0.324

0.391
0.390
0.389
0.389
0.389
0.389
0.388
0.386
0.385
0.383
0.382
0.382
0.381
0.381
0.381
0.380
0.378
0.377
0.375
0.374
0.373
0.373
0.372
0.372
0.371
0.370
0.368
0.367
0.365
0.365
0.364
0.364
0.363
0.363
0.362
0.360
0.358
0.357
0.355
0.354
128

0.645
0.643
0.641
0.638
0.636
0.635
0.633
0.630
0.627
0.624
0.621
0.619
0.617
0.615
0.613
0.611
0.608
0.605
0.602
0.600
0.598
0.596
0.594
0.592
0.591
0.588
0.585
0.582
0.580
0.578
0.576
0.575
0.573
0.571
0.569
0.566
0.563
0.560
0.558
0.556

0.792
0.773
0.755
0.737
0.720
0.703
0.687
0.670
0.654
0.639
0.624
0.610
0.596
0.582
0.569
0.555
0.543
0.530
0.517
0.505
0.493
0.482
0.470
0.459
0.448
0.437
0.427
0.416
0.406
0.396
0.386
0.377
0.368
0.360
0.352
0.344
0.336
0.328
0.321
0.313

0.779
0.760
0.742
0.725
0.708
0.691
0.674
0.658
0.642
0.627
0.612
0.598
0.584
0.571
0.557
0.544
0.531
0.519
0.506
0.494
0.482
0.471
0.459
0.448
0.437
0.427
0.416
0.405
0.395
0.385
0.375
0.366
0.358
0.349
0.341
0.333
0.325
0.318
0.310
0.303

0.731
0.712
0.693
0.676
0.659
0.642
0.626
0.610
0.594
0.579
0.563
0.549
0.535
0.521
0.507
0.493
0.480
0.467
0.455
0.443
0.431
0.419
0.408
0.396
0.385
0.373
0.363
0.352
0.342
0.332
0.322
0.313
0.305
0.297
0.289
0.281
0.274
0.266
0.259
0.251

Table A-1. Continued
635
0.322
636
0.322
637
0.321
638
0.321
639
0.321
640
0.320
641
0.318
642
0.317
643
0.316
644
0.316
645
0.315
646
0.315
647
0.315
648
0.315
649
0.314
650
0.313
651
0.312
652
0.311
653
0.310
654
0.309
655
0.309
656
0.308
657
0.308
658
0.308
659
0.308
660
0.307
661
0.305
662
0.304
663
0.303
664
0.302
665
0.301
666
0.301
667
0.301
668
0.300
669
0.300
670
0.300
671
0.299
672
0.297
673
0.296
674
0.295
675
0.294

0.324
0.324
0.323
0.323
0.322
0.321
0.320
0.318
0.318
0.317
0.316
0.317
0.316
0.316
0.315
0.314
0.312
0.311
0.310
0.309
0.308
0.309
0.309
0.308
0.307
0.306
0.305
0.304
0.302
0.301
0.300
0.300
0.300
0.299
0.299
0.298
0.297
0.296
0.294
0.293
0.292

0.354
0.353
0.353
0.352
0.351
0.350
0.348
0.346
0.345
0.345
0.344
0.343
0.343
0.342
0.341
0.340
0.338
0.336
0.335
0.334
0.334
0.333
0.333
0.332
0.331
0.330
0.329
0.327
0.326
0.324
0.323
0.322
0.322
0.322
0.321
0.320
0.318
0.317
0.316
0.314
0.313
129

0.554
0.553
0.551
0.550
0.547
0.545
0.542
0.540
0.538
0.536
0.535
0.533
0.531
0.530
0.528
0.526
0.523
0.521
0.519
0.516
0.515
0.514
0.512
0.511
0.509
0.507
0.505
0.502
0.500
0.498
0.496
0.495
0.493
0.492
0.491
0.489
0.487
0.485
0.482
0.480
0.478

0.306
0.299
0.292
0.285
0.278
0.272
0.265
0.258
0.252
0.246
0.240
0.234
0.228
0.222
0.216
0.211
0.205
0.200
0.195
0.189
0.184
0.180
0.175
0.170
0.166
0.161
0.157
0.152
0.148
0.144
0.140
0.135
0.132
0.128
0.124
0.120
0.116
0.113
0.109
0.106
0.102

0.296
0.289
0.282
0.275
0.268
0.262
0.255
0.249
0.242
0.236
0.230
0.224
0.218
0.212
0.207
0.201
0.196
0.191
0.185
0.180
0.175
0.170
0.166
0.161
0.156
0.152
0.147
0.143
0.139
0.134
0.130
0.126
0.122
0.118
0.115
0.111
0.107
0.103
0.100
0.096
0.093

0.244
0.237
0.230
0.223
0.216
0.209
0.202
0.196
0.189
0.183
0.177
0.170
0.164
0.159
0.153
0.147
0.142
0.136
0.131
0.125
0.120
0.116
0.111
0.106
0.101
0.097
0.092
0.088
0.083
0.079
0.075
0.071
0.067
0.063
0.059
0.056
0.052
0.048
0.044
0.041
0.038

Table A-1. Continued
676
0.293
677
0.292
678
0.292
679
0.292
680
0.291
681
0.291
682
0.290
683
0.289
684
0.287
685
0.286
686
0.284
687
0.283
688
0.283
689
0.282
690
0.282
691
0.282
692
0.281
693
0.280
694
0.279
695
0.278
696
0.277
697
0.275
698
0.274
699
0.273
700
0.272

0.291
0.290
0.290
0.290
0.289
0.288
0.288
0.286
0.285
0.283
0.282
0.281
0.280
0.279
0.279
0.278
0.278
0.277
0.275
0.274
0.273
0.272
0.270
0.269
0.268

0.312
0.311
0.310
0.310
0.309
0.308
0.307
0.306
0.304
0.302
0.301
0.299
0.298
0.298
0.297
0.297
0.296
0.295
0.293
0.292
0.291
0.289
0.287
0.286
0.285

130

0.476
0.475
0.473
0.472
0.471
0.469
0.467
0.465
0.463
0.461
0.458
0.457
0.455
0.454
0.453
0.451
0.449
0.448
0.446
0.444
0.442
0.440
0.438
0.436
0.434

0.099
0.096
0.092
0.089
0.086
0.083
0.080
0.077
0.074
0.071
0.068
0.066
0.063
0.060
0.058
0.055
0.053
0.050
0.048
0.046
0.043
0.041
0.039
0.037
0.035

0.090
0.086
0.083
0.080
0.077
0.074
0.071
0.068
0.065
0.062
0.059
0.057
0.054
0.052
0.049
0.046
0.044
0.042
0.039
0.037
0.034
0.032
0.030
0.028
0.026

0.034
0.031
0.028
0.025
0.022
0.019
0.016
0.013
0.010
0.007
0.004
0.001
-0.001
-0.004
-0.007
-0.009
-0.011
-0.014
-0.016
-0.019
-0.021
-0.023
-0.025
-0.028
-0.030

Table A-2. Additional absorbance data of gold nanoparticles for Figure 3-1.
Time:
Wavelength
(nm)
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436

6 hr

12 hr

24 hr

Absorbance (AU)
2.282
2.279
2.276
2.272
2.271
2.267
2.264
2.258
2.254
2.253
2.248
2.244
2.240
2.236
2.235
2.233
2.229
2.226
2.223
2.222
2.221
2.219
2.214
2.211
2.212
2.212
2.211
2.207
2.204
2.204
2.203
2.200
2.200
2.199
2.197
2.196
2.197

2.301
2.297
2.293
2.288
2.284
2.286
2.282
2.275
2.274
2.269
2.266
2.265
2.261
2.259
2.255
2.250
2.248
2.247
2.244
2.243
2.243
2.237
2.235
2.236
2.235
2.232
2.228
2.227
2.226
2.223
2.221
2.219
2.219
2.221
2.219
2.218
2.220

2.373
2.369
2.364
2.360
2.358
2.355
2.351
2.347
2.340
2.336
2.335
2.333
2.329
2.322
2.322
2.322
2.315
2.311
2.309
2.307
2.308
2.306
2.304
2.300
2.298
2.299
2.297
2.295
2.296
2.293
2.290
2.290
2.289
2.288
2.287
2.287
2.287
131

Table A-2. Continued
437
2.198
438
2.199
439
2.199
440
2.199
441
2.200
442
2.201
443
2.203
444
2.204
445
2.204
446
2.207
447
2.211
448
2.210
449
2.211
450
2.215
451
2.218
452
2.220
453
2.223
454
2.227
455
2.232
456
2.236
457
2.240
458
2.246
459
2.249
460
2.255
461
2.261
462
2.269
463
2.274
464
2.279
465
2.288
466
2.296
467
2.304
468
2.312
469
2.318
470
2.328
471
2.342
472
2.354
473
2.362
474
2.373
475
2.386
476
2.398
477
2.409

2.222
2.223
2.223
2.221
2.222
2.226
2.227
2.227
2.229
2.231
2.233
2.235
2.236
2.240
2.244
2.248
2.251
2.252
2.256
2.263
2.267
2.273
2.278
2.285
2.292
2.299
2.308
2.314
2.321
2.332
2.340
2.348
2.354
2.361
2.373
2.385
2.397
2.410
2.422
2.436
2.449

2.288
2.288
2.287
2.288
2.291
2.294
2.294
2.295
2.299
2.303
2.305
2.307
2.308
2.310
2.315
2.318
2.321
2.327
2.331
2.337
2.344
2.351
2.355
2.359
2.366
2.375
2.382
2.388
2.397
2.410
2.421
2.428
2.435
2.443
2.457
2.471
2.483
2.497
2.512
2.529
2.543
132

Table A-2. Continued
478
2.421
479
2.436
480
2.448
481
2.464
482
2.480
483
2.492
484
2.510
485
2.526
486
2.542
487
2.563
488
2.582
489
2.601
490
2.617
491
2.633
492
2.653
493
2.676
494
2.695
495
2.715
496
2.736
497
2.752
498
2.773
499
2.789
500
2.802
501
2.821
502
2.841
503
2.856
504
2.868
505
2.883
506
2.898
507
2.915
508
2.934
509
2.948
510
2.954
511
2.954
512
2.966
513
2.979
514
2.979
515
2.985
516
2.991
517
3.004
518
3.018

2.463
2.480
2.493
2.506
2.522
2.543
2.564
2.582
2.593
2.612
2.636
2.658
2.676
2.689
2.708
2.734
2.756
2.773
2.795
2.812
2.826
2.846
2.865
2.883
2.906
2.920
2.928
2.946
2.963
2.972
2.982
2.991
3.001
3.011
3.011
3.021
3.031
3.031
3.031
3.031
3.031

2.556
2.574
2.588
2.607
2.627
2.644
2.665
2.687
2.707
2.727
2.748
2.771
2.795
2.819
2.844
2.865
2.888
2.912
2.937
2.963
2.979
2.995
3.014
3.046
3.068
3.080
3.101
3.127
3.154
3.154
3.154
3.184
3.205
3.205
3.216
3.227
3.227
3.227
3.244
3.244
3.227
133

Table A-2. Continued
519
3.018
520
3.018
521
3.008
522
3.008
523
3.008
524
3.008
525
3.008
526
2.998
527
2.998
528
2.991
529
2.982
530
2.969
531
2.951
532
2.945
533
2.940
534
2.923
535
2.901
536
2.878
537
2.863
538
2.846
539
2.821
540
2.802
541
2.781
542
2.756
543
2.731
544
2.702
545
2.669
546
2.635
547
2.604
548
2.572
549
2.536
550
2.503
551
2.469
552
2.431
553
2.391
554
2.355
555
2.322
556
2.286
557
2.247
558
2.207
559
2.169

3.031
3.031
3.031
3.031
3.031
3.031
3.031
3.024
3.018
3.008
2.988
2.969
2.960
2.954
2.931
2.906
2.893
2.873
2.844
2.819
2.791
2.764
2.739
2.712
2.679
2.640
2.600
2.561
2.523
2.488
2.451
2.408
2.368
2.325
2.282
2.243
2.202
2.161
2.120
2.079
2.036

3.227
3.232
3.238
3.238
3.238
3.238
3.221
3.205
3.205
3.179
3.154
3.150
3.136
3.101
3.065
3.046
3.028
2.998
2.964
2.931
2.894
2.854
2.817
2.778
2.736
2.697
2.652
2.604
2.559
2.515
2.472
2.426
2.379
2.334
2.289
2.244
2.200
2.156
2.112
2.068
2.024
134

Table A-2. Continued
560
2.131
561
2.093
562
2.055
563
2.019
564
1.982
565
1.945
566
1.907
567
1.871
568
1.836
569
1.801
570
1.768
571
1.733
572
1.700
573
1.667
574
1.633
575
1.601
576
1.569
577
1.538
578
1.510
579
1.482
580
1.454
581
1.425
582
1.396
583
1.369
584
1.341
585
1.315
586
1.290
587
1.265
588
1.241
589
1.218
590
1.194
591
1.170
592
1.148
593
1.126
594
1.105
595
1.085
596
1.064
597
1.045
598
1.025
599
1.005
600
0.986

1.995
1.956
1.916
1.876
1.838
1.800
1.762
1.725
1.689
1.653
1.618
1.584
1.551
1.518
1.486
1.454
1.424
1.394
1.366
1.338
1.310
1.282
1.255
1.228
1.203
1.178
1.155
1.132
1.109
1.086
1.064
1.042
1.020
0.999
0.979
0.960
0.942
0.924
0.907
0.889
0.871

1.980
1.939
1.898
1.858
1.817
1.777
1.738
1.700
1.663
1.626
1.590
1.556
1.522
1.488
1.455
1.423
1.392
1.362
1.332
1.303
1.275
1.247
1.220
1.192
1.166
1.142
1.117
1.093
1.070
1.047
1.025
1.002
0.980
0.959
0.940
0.921
0.903
0.884
0.866
0.848
0.830
135

Table A-2. Continued
601
0.967
602
0.949
603
0.932
604
0.916
605
0.899
606
0.883
607
0.867
608
0.851
609
0.835
610
0.820
611
0.805
612
0.791
613
0.778
614
0.764
615
0.751
616
0.738
617
0.724
618
0.711
619
0.699
620
0.686
621
0.675
622
0.664
623
0.654
624
0.643
625
0.632
626
0.622
627
0.611
628
0.600
629
0.590
630
0.580
631
0.571
632
0.562
633
0.553
634
0.545
635
0.536
636
0.527
637
0.519
638
0.510
639
0.502
640
0.493
641
0.486

0.854
0.838
0.822
0.807
0.792
0.777
0.761
0.747
0.732
0.718
0.705
0.692
0.680
0.668
0.656
0.644
0.632
0.621
0.610
0.599
0.589
0.580
0.570
0.561
0.551
0.542
0.532
0.522
0.513
0.504
0.496
0.488
0.480
0.472
0.464
0.456
0.448
0.441
0.433
0.426
0.419

0.813
0.796
0.780
0.765
0.750
0.735
0.720
0.705
0.690
0.676
0.663
0.651
0.638
0.627
0.615
0.604
0.592
0.581
0.570
0.559
0.549
0.540
0.530
0.521
0.512
0.502
0.492
0.483
0.473
0.465
0.457
0.449
0.441
0.434
0.426
0.418
0.411
0.403
0.396
0.389
0.383
136

Table A-2. Continued
642
0.479
643
0.472
644
0.465
645
0.457
646
0.450
647
0.443
648
0.436
649
0.429
650
0.422
651
0.416
652
0.410
653
0.405
654
0.399
655
0.394
656
0.388
657
0.382
658
0.376
659
0.370
660
0.365
661
0.360
662
0.355
663
0.350
664
0.346
665
0.342
666
0.337
667
0.332
668
0.328
669
0.323
670
0.318
671
0.313
672
0.310
673
0.306
674
0.303
675
0.300
676
0.296
677
0.292
678
0.288
679
0.284
680
0.281
681
0.277
682
0.273

0.413
0.407
0.401
0.394
0.388
0.381
0.375
0.369
0.363
0.358
0.353
0.348
0.344
0.338
0.333
0.328
0.323
0.318
0.313
0.309
0.305
0.301
0.297
0.293
0.289
0.285
0.281
0.277
0.273
0.269
0.265
0.262
0.259
0.256
0.253
0.250
0.247
0.243
0.239
0.236
0.233

0.377
0.371
0.366
0.359
0.353
0.347
0.341
0.335
0.330
0.325
0.320
0.316
0.311
0.307
0.302
0.297
0.293
0.288
0.283
0.279
0.275
0.272
0.268
0.265
0.261
0.257
0.253
0.249
0.245
0.241
0.238
0.235
0.233
0.230
0.227
0.224
0.221
0.218
0.214
0.211
0.208
137

Table A-2. Continued
683
0.270
684
0.267
685
0.264
686
0.261
687
0.258
688
0.256
689
0.253
690
0.250
691
0.246
692
0.243
693
0.240
694
0.237
695
0.234
696
0.232
697
0.230
698
0.228
699
0.226
700
0.224

0.230
0.227
0.225
0.223
0.220
0.218
0.215
0.212
0.209
0.206
0.204
0.201
0.199
0.197
0.195
0.193
0.192
0.190

0.205
0.203
0.201
0.199
0.197
0.194
0.192
0.189
0.186
0.184
0.181
0.179
0.177
0.175
0.174
0.172
0.170
0.169

138

Table A-3. Particle diameter data for Table 3-3.
Figure 3-3b
Size
(nm)
0-1
1-2
2-3
3-4
4-5
5-6
6-7
7-8
8-9
9-10
10-11
11-12
12-13
13-14
14-15
15-16
16-17
17-18
18-19
19-20

Count
(n)
0
0
0
1
9
45
134
206
154
70
14
2
0
0
0
0
0
0
0
0
8.64

Fraction (%)
0.0%
0.0%
0.0%
0.2%
1.4%
7.1%
21.1%
32.4%
24.3%
11.0%
2.2%
0.3%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
0.0%
Average
Diameter (nm)

Standard
1.21 Deviation
(nm)
Total Count
635
(n)

Figure 3-3d
Count
(n)

Fraction (%)
0.0%
0.0%
0.0%
0.0%
0.0%
0.5%
0.9%
3.3%
6.9%
9.8%
11.8%
10.4%
5.0%
0.9%
0.0%
0.2%
0.0%
0.0%
0.0%
0.0%
Average
11.19 Diameter
(nm)
Standard
1.58 Deviation
(nm)
Total Count
316
(n)
0
0
0
0
0
3
6
21
44
62
75
66
32
6
0
1
0
0
0
0

139

Figure 3-3f
Count
(n)

Fraction (%)
0.0%
0.0%
0.0%
0.0%
0.0%
0.2%
0.0%
0.0%
1.1%
4.3%
9.9%
8.7%
7.1%
5.4%
2.4%
0.0%
0.2%
0.0%
0.0%
0.0%
Average
12.56 Diameter
(nm)
Standard
1.53 Deviation
(nm)
Total Count
248
(n)
0
0
0
0
0
1
0
0
7
27
63
55
45
34
15
0
1
0
0
0

Table A-4. Dextrin generation conditions for gold nanoparticle size data for Figure 3-4.
Dextrin
Concentration
(g/L)

Diameter
(nm)

Standard
Deviation
(nm)

25
20
15
10
7.5
2.5

8.20
8.80
10.12
10.59
11.13
12.44

1.07
1.26
1.48
1.79
2.65
1.49

140

Table A-5. Data for pH generation conditions for gold nanoparticle size data Figure 3-5.
Dextrin
Standard
Generation
Diameter
Concentration
Deviation
pH
(nm)
(g/L)
(nm)
7
8
9
10
11
7
8
9
10
11
9
10
11

20
20
20
20
20
10
10
10
10
10
2.5
2.5
2.5

13.70
10.15
9.17
6.99
5.89
14.58
13.33
10.58
9.47
9.23
16.83
13.62
12.22

2.44
1.24
1.45
1.25
0.94
2.55
4.22
1.82
1.63
1.36
2.26
1.23
1.02

141

Table A-6. Data for temperature generation of gold nanoparticle size data for Figure 3-6.
Generation
Temp (°C)
25
25
25
50
50
50
50
50
50

Dextrin
Diameter
Concentration
(nm)
(g/L)
20
7.07
10
8.28
2.5
10.05
25
8.20
20
8.80
15
10.12
10
10.59
7.5
11.13
2.5
12.44

Standard
Deviation
(nm)
0.99
1.11
1.45
1.07
1.26
1.48
1.79
2.65
1.49

142

Table A-7. Data for UV absorbance vs. time for Figure 3-7.
Time
(hrs)
0.5
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
12
24

Dextrin generation condition
20g/L

15g/L

10g/L

0.708
2.345
5.858
5.482
5.486
5.436
5.454
5.868
5.306
5.246
6.348
6.410
6.300

0.578
1.293
3.780
5.468
5.454
5.490
5.394
5.394
5.520
5.254
6.036
6.036
5.862

0.357
0.407
0.753
5.406
5.558
5.454
5.920
5.362
5.394
5.282
6.152
6.062
6.488

7.5g/L
0.284
0.336
0.628
5.306
5.558
5.896
5.896
5.454
5.454
5.306
6.144
6.106
6.656

143

5g/L
0.178
0.189
0.221
0.453
2.033
4.996
5.976
5.468
5.436
5.362
6.202
6.152
6.548

2.5g/L
0.087
0.104
0.108
0.129
0.372
1.247
2.199
3.385
4.964
5.362
6.290
5.090
6.348

Table A-8. Data for UV-Vis absorbance of dextrin species for Figure 3-9.
Wave
Number
(cm-1)
501
507
513
519
525
530
536
542
548
553
559
565
571
577
582
588
594
600
606
611
617
623
629
634
640
646
652
658
663
669
675
681
687
692
698
704

Absorbance (%)
Stock
41.5%
41.9%
42.1%
42.5%
42.7%
42.5%
42.0%
41.5%
41.6%
41.8%
42.2%
42.6%
43.0%
43.0%
42.5%
41.9%
41.9%
41.7%
41.5%
41.3%
41.0%
40.4%
40.0%
39.6%
39.4%
39.1%
39.0%
38.9%
38.8%
38.7%
38.5%
38.2%
37.9%
37.8%
37.9%
38.0%

Autoclaved
36.3%
36.9%
36.9%
37.6%
37.7%
37.8%
37.6%
37.1%
37.4%
37.3%
38.0%
38.1%
38.8%
38.9%
38.6%
38.3%
38.1%
38.2%
37.9%
38.0%
37.5%
37.3%
36.9%
36.6%
36.5%
36.2%
36.3%
36.0%
36.2%
35.9%
35.9%
35.6%
35.4%
35.5%
35.4%
35.8%

AuNP
Bound
20.8%
20.7%
21.0%
21.1%
21.2%
21.4%
21.1%
21.4%
21.3%
21.5%
21.7%
21.6%
21.6%
21.5%
21.6%
21.7%
21.8%
21.8%
21.6%
21.5%
21.5%
21.3%
21.4%
21.2%
21.3%
21.0%
20.9%
20.9%
20.7%
20.8%
20.5%
20.5%
20.2%
20.2%
20.2%
20.0%
144

Wave
Number
(cm-1)
2225
2231
2237
2243
2249
2254
2260
2266
2272
2278
2283
2289
2295
2301
2306
2312
2318
2324
2330
2335
2341
2347
2353
2359
2364
2370
2376
2382
2387
2393
2399
2405
2411
2416
2422
2428

Absorbance (%)
Stock
28.7%
28.6%
28.6%
28.6%
28.6%
28.5%
28.5%
28.5%
28.5%
28.5%
28.5%
28.5%
28.5%
28.4%
28.4%
28.4%
28.4%
28.5%
28.5%
28.5%
28.5%
28.5%
28.6%
28.6%
28.6%
28.7%
28.7%
28.7%
28.8%
28.8%
28.8%
28.9%
28.9%
29.0%
29.0%
29.2%

Autoclaved
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.1%
33.2%
33.2%
33.2%
33.2%
33.2%
33.2%
33.2%
33.3%
33.3%
33.4%
33.4%
33.4%
33.5%
33.5%
33.5%
33.5%
33.6%
33.6%
33.7%
33.7%
33.8%
33.8%
33.9%
34.0%

AuNP
Bound
23.5%
23.5%
23.5%
23.5%
23.5%
23.5%
23.5%
23.6%
23.6%
23.6%
23.6%
23.6%
23.7%
23.7%
23.7%
23.7%
23.7%
23.8%
23.8%
23.8%
23.8%
23.8%
23.9%
23.9%
23.9%
23.9%
24.0%
24.0%
24.0%
24.1%
24.1%
24.1%
24.2%
24.2%
24.3%
24.3%

Table A-8. Continued
710 38.0%
715 37.8%
721 37.0%
727 36.4%
733 35.9%
739 35.6%
744 35.5%
750 35.8%
756 36.4%
762 37.0%
768 37.0%
773 36.3%
779
785
791
796
802
808
814
820
825
831
837
843
849
854
860
866
872
877
883
889
895
901
906
912
918
924
930
935

35.6%
35.0%
34.5%
33.9%
33.3%
32.8%
32.5%
32.3%
32.4%
32.9%
33.5%
34.0%
34.3%
34.6%
34.7%
34.0%
33.2%
32.9%
33.2%
34.0%
34.8%
35.5%
36.2%
37.2%
38.1%
38.6%
38.6%
38.5%

35.6%
35.6%
34.9%
34.5%
34.1%
33.8%
33.9%
34.0%
34.7%
34.9%
35.1%
34.4%

20.1%
19.9%
19.8%
19.6%
19.5%
19.5%
19.3%
19.3%
19.3%
19.5%
19.8%
19.7%

2434
2440
2445
2451
2457
2463
2468
2474
2480
2486
2492
2497

29.2%
29.3%
29.4%
29.5%
29.5%
29.6%
29.7%
29.8%
29.9%
29.9%
30.0%
30.1%

34.0%
34.1%
34.1%
34.2%
34.3%
34.3%
34.4%
34.5%
34.5%
34.5%
34.6%
34.6%

24.3%
24.4%
24.4%
24.5%
24.5%
24.5%
24.6%
24.6%
24.6%
24.7%
24.7%
24.7%

34.0%
33.4%
33.0%
32.7%
32.1%
32.0%
31.6%
31.7%
31.6%
32.1%
32.4%
32.8%
33.1%
33.3%
33.5%
32.9%
32.6%
32.0%
32.2%
32.5%
33.0%
33.4%
33.9%
34.6%
35.2%
35.8%
35.8%
35.9%

19.7%
19.5%
19.3%
19.2%
19.0%
19.0%
18.8%
18.7%
18.6%
18.5%
18.7%
18.6%
18.7%
18.7%
18.6%
18.7%
18.7%
18.8%
18.6%
18.6%
18.7%
18.7%
18.7%
18.7%
18.8%
18.7%
18.6%
18.6%

2503
2509
2515
2521
2526
2532
2538
2544
2549
2555
2561
2567
2573
2578
2584
2590
2596
2602
2607
2613
2619
2625
2630
2636
2642
2648
2654
2659

30.1%
30.2%
30.2%
30.3%
30.4%
30.4%
30.5%
30.6%
30.7%
30.8%
30.9%
30.9%
31.0%
31.1%
31.2%
31.3%
31.4%
31.4%
31.5%
31.6%
31.7%
31.9%
32.1%
32.2%
32.3%
32.5%
32.7%
32.8%

34.7%
34.7%
34.8%
34.8%
34.9%
34.9%
35.0%
35.1%
35.1%
35.2%
35.2%
35.3%
35.4%
35.4%
35.5%
35.6%
35.6%
35.7%
35.8%
35.8%
35.9%
36.0%
36.1%
36.2%
36.3%
36.4%
36.6%
36.7%

24.8%
24.8%
24.8%
24.9%
24.9%
24.9%
25.0%
25.0%
25.0%
25.1%
25.1%
25.1%
25.2%
25.2%
25.3%
25.3%
25.4%
25.4%
25.5%
25.5%
25.6%
25.7%
25.7%
25.8%
25.8%
25.9%
25.9%
26.0%

145

Table A-8. Continued
941 38.3%
947 38.9%
953 40.6%
958 42.5%
964 44.3%
970 45.8%
976 47.0%
982 47.7%
987 47.8%
993 47.7%
999 47.5%
1005 47.4%
1011 47.5%
1016 47.5%
1022 47.3%
1028 47.1%
1034 46.8%
1039 46.6%
1045 46.5%
1051 46.3%
1057 46.0%
1063 45.4%
1068 44.9%
1074 45.5%
1080 46.3%
1086 46.6%
1092 46.5%
1097 46.3%
1103 46.2%
1109 46.1%
1115 45.9%
1120 45.6%
1126 45.3%
1132 45.0%
1138 45.0%
1144 45.6%
1149 46.6%
1155 47.3%
1161 47.1%
1167 46.7%
1173 46.0%

35.5%
35.5%
36.3%
37.6%
39.0%
40.6%
42.4%
44.1%
45.6%
46.5%
47.0%
47.2%
47.8%
48.2%
48.2%
47.8%
47.3%
47.1%
46.7%
46.5%
45.7%
44.8%
44.1%
45.0%
45.9%
45.9%
45.4%
44.8%
44.6%
44.4%
44.2%
43.8%
43.6%
43.2%
43.3%
44.1%
45.3%
46.0%
45.7%
44.8%
43.5%

18.7%
18.8%
19.0%
19.2%
19.6%
19.9%
20.5%
21.2%
21.7%
22.1%
22.5%
23.1%
23.7%
24.2%
24.7%
25.0%
25.1%
25.1%
25.0%
25.0%
24.8%
24.9%
25.2%
25.6%
25.7%
25.5%
25.5%
25.5%
25.7%
25.7%
25.5%
25.3%
25.0%
25.1%
25.0%
25.2%
25.5%
25.3%
24.8%
23.9%
23.5%
146

2665
2671
2677
2683
2688
2694
2700
2706
2711
2717
2723
2729
2735
2740
2746
2752
2758
2764
2769
2775
2781
2787
2792
2798
2804
2810
2816
2821
2827
2833
2839
2845
2850
2856
2862
2868
2873
2879
2885
2891
2897

33.0%
33.1%
33.3%
33.4%
33.5%
33.7%
33.8%
34.0%
34.1%
34.3%
34.4%
34.6%
34.8%
35.0%
35.2%
35.4%
35.5%
35.8%
36.0%
36.2%
36.3%
36.6%
36.7%
37.0%
37.2%
37.4%
37.7%
37.9%
38.2%
38.5%
38.9%
39.2%
39.6%
40.0%
40.4%
40.9%
41.4%
41.8%
42.2%
42.5%
42.7%

36.7%
36.8%
36.9%
37.0%
37.1%
37.2%
37.3%
37.4%
37.5%
37.6%
37.7%
37.9%
37.9%
38.0%
38.2%
38.3%
38.4%
38.6%
38.7%
38.9%
39.0%
39.1%
39.3%
39.4%
39.6%
39.7%
39.9%
40.1%
40.3%
40.5%
40.7%
40.9%
41.2%
41.5%
41.8%
42.1%
42.5%
42.9%
43.3%
43.5%
43.7%

26.0%
26.1%
26.1%
26.2%
26.2%
26.3%
26.3%
26.4%
26.4%
26.5%
26.6%
26.7%
26.7%
26.8%
26.9%
27.0%
27.1%
27.3%
27.4%
27.6%
27.7%
27.8%
27.9%
28.0%
28.0%
28.1%
28.3%
28.3%
28.4%
28.6%
28.7%
28.8%
29.0%
29.1%
29.3%
29.4%
29.6%
29.8%
30.0%
30.2%
30.4%

Table A-8. Continued
1178 45.2%
1184 44.2%
1190 43.1%
1196 42.3%
1201 41.7%
1207 41.2%
1213 40.5%
1219 39.7%
1225 39.2%
1230 38.9%
1236 38.8%
1242 38.8%
1248 38.7%
1254 38.5%
1259 38.4%
1265 38.2%
1271 38.0%
1277 38.0%
1282 38.1%
1288 38.2%
1294 38.3%
1300 38.5%
1306 38.7%
1311 38.9%
1317 39.3%
1323 39.6%
1329 40.0%
1335 40.5%
1340 40.9%
1346 41.4%
1352 42.0%
1358 42.8%
1363 43.9%
1369 45.4%
1375 47.6%
1381 51.1%
1387 55.6%
1392 60.3%
1398 65.0%
1404 64.6%

42.2%
40.9%
39.9%
39.3%
39.0%
38.6%
38.0%
37.5%
37.2%
37.2%
37.1%
37.2%
37.1%
37.0%
36.9%
36.9%
36.8%
36.8%
37.0%
37.1%
37.3%
37.5%
37.8%
38.0%
38.3%
38.5%
38.9%
39.4%
39.7%
40.0%
40.4%
41.0%
41.8%
42.8%
44.3%
46.8%
49.9%
52.9%
56.3%
56.0%

23.0%
22.8%
22.7%
22.6%
22.6%
22.3%
22.1%
22.1%
22.0%
22.1%
22.0%
22.1%
22.1%
22.2%
22.2%
22.3%
22.4%
22.5%
22.7%
22.9%
23.0%
23.1%
23.3%
23.5%
23.9%
24.0%
24.0%
24.2%
24.4%
24.8%
25.2%
25.9%
26.7%
27.9%
29.7%
32.7%
36.8%
41.3%
46.1%
45.8%
147

2902
2908
2914
2920
2926
2931
2937
2943
2949
2954
2960
2966
2972
2978
2983
2989
2995
3001
3006
3012
3018
3024
3030
3035
3041
3047
3053
3059
3064
3070
3076
3082
3087
3093
3099
3105
3111
3116
3122
3128

43.0%
43.3%
43.7%
44.2%
44.6%
44.7%
44.8%
44.9%
44.7%
44.7%
44.7%
44.8%
44.9%
45.2%
45.4%
45.8%
46.3%
46.7%
47.2%
47.6%
48.0%
48.4%
48.7%
49.0%
49.4%
50.0%
50.7%
51.4%
52.4%
53.5%
54.6%
55.9%
57.1%
58.5%
59.7%
60.8%
61.7%
62.4%
62.9%
63.1%

43.9%
44.1%
44.4%
44.7%
45.1%
45.3%
45.4%
45.3%
45.1%
44.9%
44.8%
44.8%
45.0%
45.0%
45.2%
45.5%
45.7%
46.1%
46.3%
46.6%
46.8%
47.0%
47.3%
47.4%
47.7%
48.1%
48.6%
49.1%
49.7%
50.5%
51.2%
52.0%
52.9%
53.7%
54.6%
55.3%
55.9%
56.4%
56.8%
57.1%

30.5%
30.7%
31.0%
31.3%
31.5%
31.7%
31.9%
32.1%
32.3%
32.5%
32.7%
33.0%
33.3%
33.7%
34.1%
34.5%
34.9%
35.5%
35.8%
36.2%
36.4%
36.5%
36.7%
36.8%
37.1%
37.4%
37.9%
38.5%
39.3%
40.1%
41.0%
42.0%
43.1%
44.2%
45.3%
46.3%
47.2%
47.8%
48.3%
48.5%

Table A-8. Continued
1410 59.7%
1416 54.9%
1421 51.6%
1427 49.3%
1433 47.8%
1439 46.8%
1444 46.0%
1450 45.6%
1456 45.4%
1462 45.3%
1468 45.0%
1473 44.5%
1479 43.7%
1485 42.9%
1491 42.1%
1496 41.3%
1502 40.7%
1508 40.0%
1514 39.4%
1520 38.9%
1525 38.4%
1531 38.0%
1537 37.7%
1543 37.5%
1549 37.3%
1554 37.2%
1560 37.2%
1566 37.1%
1572 37.0%
1577 36.9%
1583 37.1%
1589 37.4%
1595 37.9%
1601 38.6%
1606 39.4%
1612 40.1%
1618 40.9%
1624 41.6%
1630 42.0%
1635 42.1%

52.8%
49.5%
47.2%
45.5%
44.4%
43.7%
43.2%
42.8%
42.6%
42.4%
42.0%
41.4%
40.7%
40.0%
39.4%
38.8%
38.3%
37.9%
37.5%
37.2%
36.9%
36.6%
36.5%
36.3%
36.3%
36.3%
36.3%
36.3%
36.2%
36.3%
36.5%
36.8%
37.2%
37.8%
38.4%
39.0%
39.7%
40.2%
40.5%
40.6%

41.2%
36.7%
33.7%
31.6%
30.3%
29.4%
28.8%
28.4%
28.0%
27.6%
27.1%
26.8%
26.3%
26.0%
25.6%
25.2%
25.0%
24.7%
24.6%
24.4%
24.3%
24.2%
24.2%
24.3%
24.3%
24.6%
24.8%
25.0%
25.1%
25.4%
25.8%
26.3%
27.0%
27.7%
28.4%
29.0%
29.5%
29.8%
29.8%
29.8%
148

3134
3140
3145
3151
3157
3163
3168
3174
3180
3186
3192
3197
3203
3209
3215
3221
3226
3232
3238
3244
3249
3255
3261
3267
3273
3278
3284
3290
3296
3302
3307
3313
3319
3325
3330
3336
3342
3348
3354
3359

63.3%
63.4%
63.4%
63.5%
63.4%
63.4%
63.3%
63.2%
63.1%
62.9%
62.6%
62.5%
62.2%
62.0%
61.7%
61.4%
61.0%
60.7%
60.2%
59.8%
59.4%
59.0%
58.7%
58.2%
57.8%
57.5%
57.2%
57.0%
56.8%
56.6%
56.5%
56.4%
56.4%
56.4%
56.6%
56.6%
56.7%
56.7%
56.7%
56.8%

57.2%
57.3%
57.4%
57.4%
57.5%
57.6%
57.6%
57.6%
57.5%
57.5%
57.5%
57.4%
57.3%
57.3%
57.2%
57.1%
56.9%
56.6%
56.6%
56.4%
56.1%
56.0%
55.8%
55.7%
55.5%
55.4%
55.3%
55.2%
55.1%
55.1%
55.0%
55.0%
55.1%
55.2%
55.2%
55.4%
55.3%
55.4%
55.5%
55.5%

48.7%
48.8%
48.8%
48.8%
48.9%
48.9%
48.9%
48.9%
48.8%
48.7%
48.4%
48.4%
48.1%
48.0%
47.7%
47.4%
47.2%
46.8%
46.5%
46.2%
45.9%
45.6%
45.3%
45.0%
44.6%
44.4%
44.1%
44.0%
43.8%
43.7%
43.7%
43.7%
43.8%
43.9%
43.9%
44.0%
44.1%
44.2%
44.3%
44.4%

Table A-8. Continued
1641 42.2%
1647 42.2%
1653 42.2%
1658 42.0%
1664 41.7%
1670 41.0%
1676 40.0%
1682 38.9%
1687 37.9%
1693 36.9%
1699 36.0%
1705 35.1%
1711 34.5%
1716 34.0%
1722 33.5%
1728 33.0%
1734 32.7%
1739 32.3%
1745 31.9%
1751 31.6%
1757 31.3%
1763 31.1%
1768 31.0%
1774 30.8%
1780 30.6%
1786 30.5%
1792 30.4%
1797 30.2%
1803 30.1%
1809 30.0%
1815 29.9%
1820 29.8%
1826 29.7%
1832 29.6%
1838 29.6%
1844 29.5%
1849 29.5%
1855 29.4%
1861 29.4%
1867 29.3%
1873 29.3%

40.6%
40.6%
40.6%
40.4%
40.1%
39.5%
38.7%
37.9%
37.1%
36.4%
35.8%
35.3%
34.8%
34.5%
34.2%
33.9%
33.7%
33.5%
33.3%
33.2%
33.0%
32.9%
32.9%
32.8%
32.7%
32.7%
32.6%
32.6%
32.5%
32.5%
32.4%
32.4%
32.4%
32.4%
32.3%
32.3%
32.3%
32.3%
32.3%
32.3%
32.3%

29.5%
29.4%
29.3%
29.0%
28.8%
28.2%
27.3%
26.5%
25.8%
25.1%
24.7%
24.2%
23.9%
23.7%
23.5%
23.4%
23.4%
23.2%
23.0%
22.8%
22.7%
22.6%
22.6%
22.5%
22.5%
22.4%
22.4%
22.3%
22.3%
22.2%
22.2%
22.2%
22.3%
22.2%
22.2%
22.2%
22.2%
22.3%
22.2%
22.3%
22.3%
149

3365
3371
3377
3383
3388
3394
3400
3406
3411
3417
3423
3429
3435
3440
3446
3452
3458
3464
3469
3475
3481
3487
3492
3498
3504
3510
3516
3521
3527
3533
3539
3545
3550
3556
3562
3568
3573
3579
3585
3591
3597

56.8%
56.8%
57.0%
57.0%
57.0%
57.0%
57.0%
57.1%
57.1%
57.2%
57.2%
57.1%
57.0%
56.9%
56.7%
56.6%
56.3%
56.0%
55.8%
55.5%
55.1%
54.9%
54.6%
54.2%
53.9%
53.7%
53.2%
52.9%
52.6%
52.2%
52.0%
51.7%
51.5%
51.3%
50.9%
50.5%
50.3%
49.8%
49.5%
49.0%
48.7%

55.5%
55.6%
55.7%
55.7%
55.7%
55.8%
55.7%
55.7%
55.7%
55.7%
55.7%
55.6%
55.6%
55.5%
55.4%
55.3%
55.2%
54.9%
54.7%
54.6%
54.4%
54.2%
54.1%
53.7%
53.5%
53.3%
53.1%
52.8%
52.6%
52.3%
52.2%
51.9%
51.6%
51.3%
51.0%
50.7%
50.3%
50.0%
49.6%
49.1%
48.7%

44.4%
44.7%
44.8%
44.8%
44.8%
44.9%
44.9%
44.9%
44.9%
44.8%
44.7%
44.7%
44.6%
44.4%
44.2%
44.0%
43.7%
43.3%
43.0%
42.7%
42.2%
41.8%
41.4%
41.0%
40.6%
40.1%
39.7%
39.2%
38.8%
38.4%
38.1%
37.7%
37.3%
36.8%
36.5%
36.0%
35.6%
35.2%
34.7%
34.2%
33.9%

Table A-8. Continued
1878 29.2%
1884 29.2%
1890 29.2%
1896 29.1%
1901 29.1%
1907 29.0%
1913 29.0%
1919 29.0%
1925 29.0%
1930 29.0%
1936 28.9%
1942 28.9%
1948 28.9%
1954 28.9%
1959 28.8%
1965 28.8%
1971 28.8%
1977 28.8%
1982 28.8%
1988 28.7%
1990 28.7%
1992 28.7%
1994 28.7%
1996 28.7%
1998 28.7%
2004 28.7%
2009 28.7%
2015 28.8%
2021 28.8%
2027 28.9%
2033 28.9%
2038 29.0%
2044 29.0%
2050 29.1%
2056 29.1%
2062 29.1%
2067 29.1%
2073 29.1%
2079 29.1%
2085 29.1%

32.3%
32.3%
32.3%
32.3%
32.3%
32.3%
32.3%
32.3%
32.3%
32.4%
32.4%
32.4%
32.4%
32.4%
32.4%
32.4%
32.4%
32.4%
32.4%
32.5%
32.5%
32.5%
32.5%
32.5%
32.5%
32.5%
32.6%
32.6%
32.6%
32.7%
32.7%
32.8%
32.8%
32.9%
32.9%
32.9%
32.9%
32.9%
33.0%
33.0%

22.3%
22.3%
22.3%
22.3%
22.3%
22.4%
22.4%
22.4%
22.4%
22.4%
22.5%
22.5%
22.5%
22.5%
22.6%
22.6%
22.6%
22.6%
22.7%
22.7%
22.7%
22.7%
22.7%
22.7%
22.7%
22.8%
22.8%
22.8%
22.8%
22.9%
22.9%
22.9%
23.0%
23.0%
23.0%
23.0%
23.0%
23.0%
23.0%
23.1%
150

3602
3608
3614
3620
3626
3631
3637
3643
3649
3654
3660
3666
3672
3678
3683
3689
3695
3701
3707
3712
3714
3716
3718
3720
3722
3728
3734
3739
3745
3751
3757
3762
3768
3774
3780
3786
3791
3797
3803
3809

48.4%
48.0%
47.5%
47.1%
46.7%
46.2%
45.8%
45.4%
45.0%
44.5%
44.3%
43.8%
43.5%
43.3%
43.0%
42.8%
42.5%
42.3%
42.1%
41.9%
41.8%
41.7%
41.7%
41.6%
41.6%
41.5%
41.4%
41.2%
41.2%
41.0%
40.9%
40.8%
40.7%
40.6%
40.3%
40.3%
40.3%
40.1%
40.1%
40.1%

48.4%
48.0%
47.5%
47.1%
46.7%
46.3%
45.9%
45.5%
45.2%
44.9%
44.5%
44.3%
44.0%
43.8%
43.5%
43.4%
43.2%
43.0%
42.9%
42.7%
42.7%
42.7%
42.6%
42.6%
42.6%
42.5%
42.4%
42.3%
42.3%
42.2%
42.1%
42.0%
42.0%
41.9%
41.8%
41.7%
41.7%
41.7%
41.7%
41.6%

33.4%
33.0%
32.5%
32.1%
31.8%
31.4%
31.0%
30.7%
30.5%
30.2%
30.0%
29.9%
29.7%
29.6%
29.4%
29.4%
29.3%
29.2%
29.1%
29.2%
29.1%
29.1%
29.1%
29.1%
29.1%
29.0%
29.1%
29.0%
29.0%
29.0%
28.9%
28.8%
28.8%
28.8%
28.9%
28.9%
28.9%
28.9%
28.9%
28.9%

Table A-8. Continued
2090 29.1%
2096 29.1%
2102 29.1%
2108 29.1%
2114 29.1%
2119 29.1%
2125 29.1%
2131 29.1%
2137 29.1%
2143 29.1%
2148 29.1%
2154 29.2%
2160 29.2%
2166 29.3%
2171 29.7%
2177 29.1%
2183 29.0%
2189 28.9%
2195 28.9%
2200 28.8%
2206 28.8%
2212 28.8%
2218 28.7%
2224 28.7%

33.0%
33.0%
33.0%
33.0%
33.0%
33.0%
33.1%
33.1%
33.1%
33.1%
33.1%
33.2%
33.2%
33.3%
33.6%
33.2%
33.2%
33.1%
33.2%
33.1%
33.1%
33.1%
33.1%
33.1%

23.1%
23.1%
23.1%
23.2%
23.2%
23.2%
23.2%
23.2%
23.2%
23.2%
23.3%
23.3%
23.4%
23.5%
23.8%
23.4%
23.4%
23.4%
23.4%
23.4%
23.4%
23.4%
23.5%
23.5%

151

3815
3820
3826
3832
3838
3843
3849
3855
3861
3867
3872
3878
3884
3890
3896
3901
3907
3913
3919
3950
3967
3982
3994
4000

40.0%
39.9%
39.8%
39.8%
39.7%
39.7%
39.6%
39.6%
39.5%
39.4%
39.4%
39.3%
39.2%
39.2%
39.2%
39.1%
39.1%
39.1%
39.1%
38.9%
38.8%
38.7%
38.7%
38.8%

41.5%
41.4%
41.5%
41.4%
41.4%
41.4%
41.3%
41.4%
41.2%
41.3%
41.3%
41.2%
41.2%
41.3%
41.3%
41.3%
41.2%
41.2%
41.2%
41.3%
41.1%
41.1%
41.1%
41.1%

28.9%
28.8%
28.8%
28.8%
28.9%
28.8%
28.8%
28.9%
28.8%
28.8%
28.8%
28.8%
28.8%
28.7%
28.8%
28.8%
28.8%
28.7%
28.8%
28.8%
28.8%
28.8%
28.7%
28.8%

Table A-9. Data for DNA efficiency attachment for Figure 3-10.

Sample
D* -12.4
C* -13
D^ -10.4
C^ -13

Efficiency
(%)
5.1%
5.2%
14.2%
18.2%

Standard
Deviation
(%)
1.6%
1.9%
1.6%
1.9%

152

A.2 CHAPTER 4 DATA
Table A-10. Fluorescence data for Figure 4-8.
100ng Probe
Target
Mass (ng)
100
75
50
30
25
20
15
10
7.5
5
3
2
1
0.75
0.5
0.1
0

Average
(counts)
44617
44550
40148
26997
24024
20272
15844
13623
11770
11203
9430
9883
12358
11781
10930
8777
226

50ng Probe

25ng Probe

Standard
Deviation
(counts)

Average
(counts)

Standard
Deviation
(counts)

Average
(counts)

Standard
Deviation
(counts)

740.05
659.12
336.02
244.12
307.75
209.43
408.35
167.06
216.34
165.73
218.43
162.61
171.76
174.69
157.41
176.56
16.89

24731
23384
22128
17199
16893
15616
12368
10717
8861
6516
5036
4277
10255
7273
6008
3500
226

251.53
152.11
268.13
216.05
378.80
157.16
192.62
173.35
112.66
86.06
112.48
87.82
534.89
96.58
86.18
103.70
16.89

15036
14152
12801
11541
10337
10212
9072
7527
5865
4419
3568
2760
1642
1663
1560
1461
226

285.08
304.13
221.00
243.50
218.17
203.40
122.34
373.82
151.11
142.72
614.30
232.81
76.13
146.06
35.70
32.41
16.89

153

Table A-11. Additional fluorescence data for Figure 4-8.
10ng Probe
Target
Mass (ng)
100
75
50
30
25
20
15
10
7.5
5
3
2
1
0.75
0.5
0.1
0

Average
(counts)
18653
13996
10629
5063
3416
2503
1851
1181
982
394
551
410
436
426
1002
586
226

5ng Probe

Probe only

Standard
Deviation
(counts)

Average
(counts)

Standard
Deviation
(counts)

Average
(counts)

Standard
Deviation
(counts)

286.05
844.08
272.59
217.26
177.93
176.75
58.72
80.74
470.91
28.90
189.43
26.21
90.15
34.15
22.07
19.85
16.89

7271
6129
5279
3711
3487
2787
2886
1932
1874
1471
1123
1108
450
476
398
314
226

507.36
519.05
386.11
222.22
255.16
175.06
180.76
82.27
139.23
119.45
168.76
147.69
41.06
59.24
29.11
24.16
16.89

5150
3128
2081
832
441
342
252
286
260
225
196
249
198
259
225
187
226

613.33
419.53
238.14
62.01
16.61
14.46
13.58
68.49
14.06
17.34
6.82
51.74
10.08
14.82
9.36
6.50
16.89

154

Table A-12. Differential pulse voltammetry data for Figure 4-10.

Voltage
-1.13
-1.12
-1.11
-1.10
-1.09
-1.08
-1.07
-1.06
-1.05
-1.04
-1.03
-1.02
-1.01
-1.00
-0.99
-0.98
-0.97
-0.96
-0.95
-0.94
-0.93
-0.92
-0.91
-0.90
-0.89
-0.88
-0.87
-0.86
-0.85
-0.84
-0.83
-0.82
-0.81
-0.80
-0.77

Water
0.0000580
0.0000530
0.0000500
0.0000440
0.0000430
0.0000390
0.0000380
0.0000350
0.0000330
0.0000320
0.0000310
0.0000290
0.0000290
0.0000280
0.0000290
0.0000300
0.0000260
0.0000300
0.0000300
0.0000270
0.0000280
0.0000280
0.0000260
0.0000270
0.0000270
0.0000270
0.0000260
0.0000260
0.0000250
0.0000260
0.0000270
0.0000270
0.0000270
0.0000270
0.0000270

Differential pulse current
Cadmium
Zinc (Zn)
Zn/Cd
(Cd)

SYBR101
& QD

0.0000760
0.0000690
0.0000620
0.0000580
0.0000510
0.0000460
0.0000420
0.0000390
0.0000370
0.0000350
0.0000330
0.0000320
0.0000300
0.0000290
0.0000280
0.0000280
0.0000270
0.0000270
0.0000260
0.0000270
0.0000270
0.0000260
0.0000250
0.0000250
0.0000250
0.0000250
0.0000240
0.0000250
0.0000250
0.0000240
0.0000230
0.0000240
0.0000220
0.0000240
0.0000240

0.0000530
0.0000510
0.0000480
0.0000460
0.0000440
0.0000420
0.0000400
0.0000390
0.0000380
0.0000380
0.0000370
0.0000390
0.0000400
0.0000430
0.0000440
0.0000440
0.0000450
0.0000480
0.0000530
0.0000560
0.0000620
0.0000680
0.0000740
0.0000740
0.0000730
0.0000700
0.0000630
0.0000520
0.0000420
0.0000380
0.0000330
0.0000280
0.0000280
0.0000270
0.0000270

0.0000730
0.0000690
0.0000660
0.0000630
0.0000590
0.0000550
0.0000520
0.0000480
0.0000450
0.0000420
0.0000390
0.0000360
0.0000340
0.0000320
0.0000310
0.0000300
0.0000290
0.0000290
0.0000280
0.0000270
0.0000270
0.0000260
0.0000280
0.0000330
0.0000400
0.0000460
0.0000540
0.0000510
0.0000480
0.0000440
0.0000400
0.0000360
0.0000320
0.0000310
0.0000270
155

0.0000690
0.0000660
0.0000640
0.0000610
0.0000600
0.0000570
0.0000550
0.0000550
0.0000510
0.0000490
0.0000470
0.0000450
0.0000430
0.0000400
0.0000400
0.0000380
0.0000370
0.0000350
0.0000340
0.0000330
0.0000330
0.0000340
0.0000390
0.0000440
0.0000510
0.0000570
0.0000570
0.0000550
0.0000490
0.0000420
0.0000360
0.0000300
0.0000260
0.0000250
0.0000190

Table A-12. Continued
-0.74 0.0000270
-0.71 0.0000260
-0.68 0.0000230
-0.65 0.0000210
-0.62 0.0000180
-0.59 0.0000150
-0.56 0.0000130
-0.53 0.0000110
-0.50 0.0000100
-0.47 0.0000040
-0.44 0.0000070
-0.41 0.0000050
-0.38 0.0000050
-0.35 0.0000060
-0.32 0.0000040
-0.29 0.0000030
-0.26 0.0000030
-0.23 0.0000030
-0.20 0.0000030
-0.17 0.0000030
-0.14 0.0000030
-0.11 0.0000040
-0.08 0.0000040
-0.05 0.0000030
-0.02 0.0000040
0.00 0.0000050

0.0000250
0.0000240
0.0000220
0.0000210
0.0000190
0.0000160
0.0000140
0.0000110
0.0000100
0.0000080
0.0000070
0.0000060
0.0000060
0.0000050
0.0000050
0.0000040
0.0000040
0.0000030
0.0000030
0.0000030
0.0000040
0.0000040
0.0000040
0.0000030
0.0000040
0.0000050

0.0000240
0.0000220
0.0000200
0.0000170
0.0000140
0.0000130
0.0000110
0.0000100
0.0000080
0.0000070
0.0000060
0.0000050
0.0000040
0.0000040
0.0000030
0.0000030
0.0000030
0.0000020
0.0000020
0.0000030
0.0000020
0.0000020
0.0000020
0.0000030
0.0000030
0.0000040

156

0.0000200
0.0000190
0.0000190
0.0000180
0.0000170
0.0000160
0.0000140
0.0000110
0.0000100
0.0000080
0.0000080
0.0000050
0.0000050
0.0000040
0.0000040
0.0000040
0.0000030
0.0000040
0.0000030
0.0000020
0.0000030
0.0000030
0.0000030
0.0000040
0.0000030
0.0000040

0.0000270
0.0000270
0.0000270
0.0000270
0.0000270
0.0000270
0.0000250
0.0000240
0.0000220
0.0000210
0.0000190
0.0000170
0.0000160
0.0000140
0.0000130
0.0000120
0.0000110
0.0000100
0.0000090
0.0000090
0.0000080
0.0000080
0.0000080
0.0000070
0.0000070
0.0000070

A.3 CHAPTER 5 DATA
Table A-13. Differential pulse voltammety data for Figure 5-7.
Differential pulse current (dI)
Voltage
(V)
1.49
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87

Blank
0.00004563
0.00004277
0.00003980
0.00003663
0.00003337
0.00003007
0.00002660
0.00002307
0.00001967
0.00001640
0.00001330
0.00001053
0.00000820
0.00000627
0.00000477
0.00000360
0.00000273
0.00000223
0.00000203
0.00000200
0.00000227
0.00000270
0.00000330
0.00000413
0.00000530
0.00000677
0.00000850
0.00001070
0.00001313
0.00001577
0.00001850
0.00002137

7

6

5

5x10
CFU/mL

5x10
CFU/mL

5x10
CFU/mL

0.00005740
0.00005393
0.00005037
0.00004693
0.00004367
0.00004083
0.00003823
0.00003617
0.00003437
0.00003287
0.00003167
0.00003080
0.00003017
0.00002967
0.00002933
0.00002900
0.00002877
0.00002867
0.00002857
0.00002850
0.00002857
0.00002863
0.00002873
0.00002890
0.00002910
0.00002920
0.00002957
0.00002997
0.00003030
0.00003070
0.00003123
0.00003160

0.00004233
0.00004167
0.00004100
0.00004033
0.00003933
0.00003800
0.00003733
0.00003667
0.00003567
0.00003533
0.00003467
0.00003467
0.00003433
0.00003333
0.00003333
0.00003300
0.00003300
0.00003333
0.00003333
0.00003333
0.00003367
0.00003400
0.00003400
0.00003433
0.00003533
0.00003533
0.00003600
0.00003533
0.00003633
0.00003700
0.00003700
0.00003767

0.00001347
0.00001343
0.00001337
0.00001327
0.00001320
0.00001317
0.00001310
0.00001303
0.00001307
0.00001300
0.00001293
0.00001290
0.00001283
0.00001280
0.00001273
0.00001273
0.00001270
0.00001267
0.00001263
0.00001260
0.00001257
0.00001260
0.00001267
0.00001260
0.00001263
0.00001260
0.00001260
0.00001263
0.00001270
0.00001270
0.00001273
0.00001273

157

Table A-13. Continued
0.85
0.00002423
0.83
0.00002707
0.81
0.00002993
0.79
0.00003263
0.77
0.00003513
0.75
0.00003743
0.73
0.00003953
0.71
0.00004140
0.69
0.00004313
0.67
0.00004457
0.64
0.00004557
0.62
0.00004623
0.60
0.00004670
0.58
0.00004670
0.56
0.00004623
0.54
0.00004557
0.52
0.00004457
0.50
0.00004313
0.48
0.00004140
0.46
0.00003953
0.45
0.00003850
0.44
0.00003743
0.43
0.00003630
0.42
0.00003513
0.41
0.00003387
0.40
0.00003263
0.39
0.00003130
0.38
0.00002993
0.37
0.00002850
0.36
0.00002707
0.35
0.00002567
0.34
0.00002423
0.33
0.00002290
0.32
0.00002133
0.31
0.00001970
0.30
0.00001803
0.29
0.00001663
0.28
0.00001540
0.27
0.00001427

0.00003207
0.00003257
0.00003300
0.00003340
0.00003363
0.00003393
0.00003400
0.00003397
0.00003383
0.00003343
0.00003297
0.00003243
0.00003187
0.00003137
0.00003110
0.00003103
0.00003113
0.00003160
0.00003243
0.00003363
0.00003450
0.00003540
0.00003633
0.00003720
0.00003797
0.00003850
0.00003890
0.00003907
0.00003900
0.00003863
0.00003803
0.00003737
0.00003663
0.00003603
0.00003547
0.00003500
0.00003457
0.00003417
0.00003390
158

0.00003833
0.00003867
0.00003933
0.00003967
0.00004000
0.00004033
0.00004067
0.00004100
0.00004133
0.00004133
0.00004167
0.00004167
0.00004133
0.00004200
0.00004233
0.00004200
0.00004233
0.00004200
0.00004200
0.00004167
0.00004133
0.00004133
0.00004133
0.00004100
0.00004067
0.00004033
0.00004033
0.00004000
0.00004000
0.00004000
0.00003967
0.00003967
0.00003967
0.00004000
0.00004033
0.00004067
0.00004167
0.00004233
0.00004300

0.00001273
0.00001280
0.00001283
0.00001277
0.00001277
0.00001277
0.00001277
0.00001277
0.00001277
0.00001277
0.00001277
0.00001273
0.00001270
0.00001270
0.00001270
0.00001273
0.00001277
0.00001273
0.00001277
0.00001280
0.00001283
0.00001287
0.00001290
0.00001297
0.00001307
0.00001317
0.00001327
0.00001337
0.00001350
0.00001363
0.00001373
0.00001383
0.00001383
0.00001390
0.00001397
0.00001410
0.00001417
0.00001420
0.00001417

Table A-13. Continued
0.26
0.00001320
0.25
0.00001230
0.23
0.00001073
0.21
0.00000937
0.19
0.00000817
0.17
0.00000710
0.15
0.00000633
0.13
0.00000567
0.11
0.00000510
0.09
0.00000460
0.07
0.00000420
0.05
0.00000397
0.03
0.00000363
0.01
0.00000333
-0.01
0.00000320
-0.03
0.00000297
-0.05
0.00000280
-0.07
0.00000273
-0.09
0.00000270
-0.11
0.00000270
-0.13
0.00000263
-0.15
0.00000263
-0.17
0.00000257
-0.19
0.00000260
-0.21
0.00000270
-0.23
0.00000290
-0.25
0.00000340
-0.27
0.00000530
-0.29
0.00001207
-0.31
0.00001650
-0.33
0.00001357
-0.35
0.00001140
-0.37
0.00000937
-0.39
0.00000763
-0.41
0.00000870
-0.43
0.00000770
-0.45
0.00000783
-0.47
0.00000793
-0.49
0.00000857

0.00003370
0.00003353
0.00003310
0.00003263
0.00003210
0.00003170
0.00003130
0.00003093
0.00003067
0.00003047
0.00003030
0.00003033
0.00003027
0.00003027
0.00003043
0.00003047
0.00003040
0.00003037
0.00003030
0.00003013
0.00002987
0.00002960
0.00002940
0.00002910
0.00002897
0.00002880
0.00002863
0.00002833
0.00002810
0.00002800
0.00002793
0.00002770
0.00002740
0.00002723
0.00002710
0.00002683
0.00002670
0.00002667
0.00002650
159

0.00004333
0.00004367
0.00004433
0.00004367
0.00004233
0.00004200
0.00004167
0.00004200
0.00004133
0.00004167
0.00004167
0.00004167
0.00004167
0.00004200
0.00004200
0.00004200
0.00004267
0.00004300
0.00004367
0.00004400
0.00004400
0.00004400
0.00004367
0.00004367
0.00004333
0.00004300
0.00004267
0.00004267
0.00004300
0.00004267
0.00004267
0.00004267
0.00004233
0.00004300
0.00004267
0.00004300
0.00004267
0.00004267
0.00004300

0.00001417
0.00001413
0.00001410
0.00001407
0.00001390
0.00001373
0.00001363
0.00001353
0.00001353
0.00001333
0.00001347
0.00001343
0.00001343
0.00001343
0.00001343
0.00001343
0.00001343
0.00001350
0.00001350
0.00001353
0.00001370
0.00001380
0.00001383
0.00001383
0.00001397
0.00001400
0.00001397
0.00001403
0.00001400
0.00001397
0.00001390
0.00001393
0.00001383
0.00001373
0.00001377
0.00001380
0.00001363
0.00001370
0.00001367

Table A-14. Additional differential pulse voltammety data for Figure 5-7.
Differential pulse current (dI)
Voltage
(V)
1.49
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81

3

2

1

0

5x10
CFU/mL

5x10
CFU/mL

5x10
CFU/mL

5x10
CFU/mL

0.00003900
0.00003867
0.00003800
0.00003733
0.00003633
0.00003467
0.00003333
0.00003233
0.00003200
0.00003100
0.00003100
0.00003033
0.00003000
0.00003000
0.00002900
0.00002900
0.00002933
0.00002933
0.00002900
0.00002900
0.00002933
0.00002967
0.00002933
0.00002967
0.00003000
0.00003033
0.00003067
0.00003100
0.00003167
0.00003200
0.00003267
0.00003267
0.00003333
0.00003367
0.00003367

0.00004733
0.00004600
0.00004500
0.00004400
0.00004233
0.00004133
0.00003933
0.00003800
0.00003667
0.00003567
0.00003433
0.00003400
0.00003333
0.00003233
0.00003233
0.00003233
0.00003267
0.00003167
0.00003167
0.00003200
0.00003133
0.00003167
0.00003167
0.00003200
0.00003233
0.00003267
0.00003233
0.00003267
0.00003300
0.00003333
0.00003433
0.00003500
0.00003567
0.00003600
0.00003667

0.00003833
0.00003700
0.00003700
0.00003667
0.00003567
0.00003533
0.00003433
0.00003333
0.00003300
0.00003167
0.00003167
0.00003100
0.00003067
0.00002967
0.00003000
0.00002967
0.00002933
0.00002967
0.00002933
0.00002967
0.00002967
0.00003033
0.00002967
0.00003000
0.00003033
0.00003067
0.00003033
0.00003133
0.00003167
0.00003100
0.00003167
0.00003333
0.00003300
0.00003300
0.00003300

0.00004233
0.00004100
0.00004000
0.00003900
0.00003767
0.00003667
0.00003567
0.00003433
0.00003367
0.00003333
0.00003300
0.00003233
0.00003200
0.00003200
0.00003200
0.00003133
0.00003167
0.00003200
0.00003167
0.00003167
0.00003167
0.00003200
0.00003200
0.00003233
0.00003300
0.00003300
0.00003300
0.00003333
0.00003333
0.00003433
0.00003433
0.00003433
0.00003533
0.00003567
0.00003567

160

Table A-14. Continued
0.79
0.00003400
0.77
0.00003433
0.75
0.00003500
0.73
0.00003533
0.71
0.00003567
0.69
0.00003533
0.67
0.00003500
0.64
0.00003567
0.62
0.00003600
0.60
0.00003567
0.58
0.00003600
0.56
0.00003567
0.54
0.00003533
0.52
0.00003533
0.50
0.00003567
0.48
0.00003500
0.46
0.00003467
0.45
0.00003467
0.44
0.00003467
0.43
0.00003467
0.42
0.00003467
0.41
0.00003467
0.40
0.00003533
0.39
0.00003600
0.38
0.00003667
0.37
0.00003733
0.36
0.00003800
0.35
0.00003900
0.34
0.00004000
0.33
0.00004067
0.32
0.00004100
0.31
0.00004133
0.30
0.00004167
0.29
0.00004200
0.28
0.00004167
0.27
0.00004133
0.26
0.00004067
0.25
0.00004000
0.23
0.00003933

0.00003700
0.00003767
0.00003833
0.00003900
0.00003933
0.00004000
0.00003967
0.00004000
0.00004000
0.00004000
0.00004000
0.00004000
0.00003967
0.00003867
0.00003833
0.00003900
0.00003933
0.00003933
0.00003933
0.00003967
0.00004033
0.00004067
0.00004167
0.00004267
0.00004367
0.00004467
0.00004533
0.00004633
0.00004667
0.00004733
0.00004767
0.00004767
0.00004733
0.00004667
0.00004600
0.00004533
0.00004467
0.00004400
0.00004267
161

0.00003367
0.00003400
0.00003433
0.00003467
0.00003500
0.00003500
0.00003500
0.00003500
0.00003500
0.00003500
0.00003533
0.00003500
0.00003467
0.00003500
0.00003467
0.00003467
0.00003367
0.00003367
0.00003367
0.00003367
0.00003333
0.00003300
0.00003300
0.00003333
0.00003400
0.00003467
0.00003500
0.00003533
0.00003567
0.00003667
0.00003733
0.00003800
0.00003833
0.00003833
0.00003867
0.00003867
0.00003900
0.00003833
0.00003733

0.00003667
0.00003700
0.00003733
0.00003700
0.00003767
0.00003800
0.00003767
0.00003767
0.00003800
0.00003767
0.00003767
0.00003733
0.00003767
0.00003700
0.00003633
0.00003600
0.00003600
0.00003567
0.00003567
0.00003567
0.00003633
0.00003700
0.00003800
0.00003900
0.00004000
0.00004067
0.00004133
0.00004167
0.00004233
0.00004267
0.00004300
0.00004300
0.00004267
0.00004233
0.00004167
0.00004133
0.00004067
0.00004000
0.00003867

Table A-14. Continued
0.21
0.00003867
0.19
0.00003767
0.17
0.00003733
0.15
0.00003667
0.13
0.00003600
0.11
0.00003567
0.09
0.00003567
0.07
0.00003600
0.05
0.00003600
0.03
0.00003600
0.01
0.00003633
-0.01
0.00003633
-0.03
0.00003667
-0.05
0.00003667
-0.07
0.00003733
-0.09
0.00003767
-0.11
0.00003700
-0.13
0.00003767
-0.15
0.00003767
-0.17
0.00003700
-0.19
0.00003667
-0.21
0.00003700
-0.23
0.00003667
-0.25
0.00003600
-0.27
0.00003667
-0.29
0.00003667
-0.31
0.00003667
-0.33
0.00003667
-0.35
0.00003667
-0.37
0.00003667
-0.39
0.00003700
-0.41
0.00003667
-0.43
0.00003667
-0.45
0.00003700
-0.47
0.00003733
-0.49
0.00003667

0.00004200
0.00004167
0.00004233
0.00004200
0.00004133
0.00004133
0.00004100
0.00004133
0.00004100
0.00004100
0.00004100
0.00004133
0.00004167
0.00004200
0.00004200
0.00004200
0.00004200
0.00004200
0.00004200
0.00004233
0.00004167
0.00004167
0.00004200
0.00004200
0.00004200
0.00004200
0.00004200
0.00004200
0.00004200
0.00004167
0.00004167
0.00004200
0.00004233
0.00004233
0.00004233
0.00004233

162

0.00003633
0.00003600
0.00003567
0.00003567
0.00003467
0.00003500
0.00003533
0.00003467
0.00003467
0.00003467
0.00003433
0.00003433
0.00003433
0.00003433
0.00003467
0.00003467
0.00003500
0.00003500
0.00003467
0.00003500
0.00003433
0.00003400
0.00003467
0.00003467
0.00003500
0.00003500
0.00003467
0.00003500
0.00003433
0.00003467
0.00003500
0.00003500
0.00003533
0.00003533
0.00003500
0.00003500

0.00003833
0.00003700
0.00003700
0.00003667
0.00003633
0.00003667
0.00003633
0.00003633
0.00003667
0.00003633
0.00003633
0.00003633
0.00003567
0.00003500
0.00003533
0.00003567
0.00003533
0.00003500
0.00003433
0.00003433
0.00003400
0.00003367
0.00003367
0.00003367
0.00003267
0.00003267
0.00003300
0.00003300
0.00003267
0.00003200
0.00003233
0.00003233
0.00003233
0.00003233
0.00003233
0.00003233

Table A-15. Differential pulse voltammetry data for Figure 5-8.
Differential pulse current (dI)
Voltage
(V)
1.49
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81

AuNP
Standard
0.00005067
0.00004900
0.00004600
0.00004367
0.00004033
0.00003700
0.00003333
0.00002967
0.00002567
0.00002200
0.00001833
0.00001567
0.00001233
0.00001033
0.00000867
0.00000700
0.00000633
0.00000567
0.00000533
0.00000533
0.00000600
0.00000667
0.00000733
0.00000833
0.00000933
0.00001033
0.00001167
0.00001300
0.00001467
0.00001633
0.00001867
0.00001967
0.00002067
0.00002200
0.00002400

7

6

5

5x10
CFU/mL

Blank
0.00001033
0.00001533
0.00001533
0.00001500
0.00001467
0.00001433
0.00001433
0.00001367
0.00001367
0.00001300
0.00001267
0.00001300
0.00001333
0.00001367
0.00001400
0.00001467
0.00001367
0.00000900
0.00000733
0.00000733
0.00000667
0.00000667
0.00000733
0.00000633
0.00000533
0.00000533
0.00000533
0.00000533
0.00000600
0.00000533
0.00000567
0.00000533
0.00000533
0.00000533
0.00000500
163

5x10
CFU/mL

5x10
CFU/mL

0.00003033
0.00003033
0.00003067
0.00003167
0.00003133
0.00002933
0.00003200
0.00003133
0.00003067
0.00002867
0.00003167
0.00002900
0.00003000
0.00003067
0.00003067
0.00002967
0.00003133
0.00003067
0.00002767
0.00002633
0.00002600
0.00002700
0.00002700
0.00002600
0.00002500
0.00002467
0.00002500
0.00002433
0.00002433
0.00002433
0.00002400
0.00002367
0.00002333
0.00002267
0.00002200

0.00004200
0.00004167
0.00004133
0.00004133
0.00004067
0.00004000
0.00004000
0.00003967
0.00003933
0.00003900
0.00003867
0.00003767
0.00003700
0.00003633
0.00003533
0.00003433
0.00003367
0.00003333
0.00003267
0.00003200
0.00003133
0.00003033
0.00003033
0.00003033
0.00003033
0.00002967
0.00002933
0.00002900
0.00002867
0.00002800
0.00002800
0.00002867
0.00002833
0.00002867
0.00002900

0.00003600
0.00003633
0.00003600
0.00003600
0.00003533
0.00003500
0.00003500
0.00003433
0.00003367
0.00003367
0.00003267
0.00003200
0.00003133
0.00003067
0.00003000
0.00002967
0.00002900
0.00002900
0.00002800
0.00002767
0.00002700
0.00002733
0.00002733
0.00002700
0.00002667
0.00002633
0.00002600
0.00002533
0.00002567
0.00002600
0.00002600
0.00002633
0.00002633
0.00002600
0.00002567

Table A-15. Continued
0.79
0.00002500
0.77
0.00002667
0.75
0.00002733
0.73
0.00002833
0.71
0.00002867
0.69
0.00002867
0.67
0.00002867
0.65
0.00002800
0.63
0.00002700
0.61
0.00002600
0.59
0.00002400
0.57
0.00002200
0.55
0.00001933
0.53
0.00001700
0.51
0.00001567
0.49
0.00001433
0.47
0.00001333
0.45
0.00001333
0.43
0.00001433
0.41
0.00001600
0.39
0.00001833
0.37
0.00002067
0.35
0.00002233
0.33
0.00002267
0.31
0.00002100
0.29
0.00001733
0.27
0.00001367
0.25
0.00001100
0.23
0.00000967
0.21
0.00000967
0.19
0.00000933
0.17
0.00000867
0.15
0.00000833
0.13
0.00000833
0.11
0.00000867
0.09
0.00000867
0.07
0.00000833
0.05
0.00000867
0.03
0.00000900

0.00000500
0.00000533
0.00000533
0.00000533
0.00000533
0.00000500
0.00000500
0.00000500
0.00000533
0.00000600
0.00000500
0.00000467
0.00000467
0.00000500
0.00000500
0.00000500
0.00000500
0.00000500
0.00000567
0.00000633
0.00000600
0.00000600
0.00000600
0.00000567
0.00000567
0.00000600
0.00000600
0.00000600
0.00000567
0.00000600
0.00000567
0.00000567
0.00000600
0.00000633
0.00000533
0.00000567
0.00000633
0.00000633
0.00000633
164

0.00002133
0.00002067
0.00002000
0.00002000
0.00001933
0.00001900
0.00001900
0.00001867
0.00001833
0.00001833
0.00001833
0.00001800
0.00001800
0.00001833
0.00001800
0.00001800
0.00001800
0.00001800
0.00001800
0.00001833
0.00001833
0.00001833
0.00001867
0.00001900
0.00001867
0.00001900
0.00001933
0.00001933
0.00002000
0.00002033
0.00002100
0.00002100
0.00002167
0.00002167
0.00002233
0.00002267
0.00002200
0.00002233
0.00002300

0.00002867
0.00002900
0.00002833
0.00002867
0.00002867
0.00002833
0.00002867
0.00002900
0.00002933
0.00002967
0.00003000
0.00003000
0.00003067
0.00003100
0.00003100
0.00003100
0.00003100
0.00003033
0.00003000
0.00003067
0.00003067
0.00003000
0.00003000
0.00002967
0.00003000
0.00003033
0.00003033
0.00003033
0.00003000
0.00003000
0.00003000
0.00003000
0.00003000
0.00003000
0.00003000
0.00003000
0.00003000
0.00003033
0.00003000

0.00002567
0.00002567
0.00002567
0.00002567
0.00002600
0.00002600
0.00002600
0.00002667
0.00002700
0.00002700
0.00002700
0.00002700
0.00002767
0.00002800
0.00002867
0.00002933
0.00003000
0.00003000
0.00002933
0.00002900
0.00002900
0.00002900
0.00002833
0.00002800
0.00002800
0.00002800
0.00002800
0.00002800
0.00002767
0.00002733
0.00002800
0.00002733
0.00002767
0.00002800
0.00002767
0.00002700
0.00002700
0.00002733
0.00002733

Table A-16. Additional differential pulse voltammetry data for Figure 5-8.
Differential pulse current (dI)
Voltage
(V)
1.49
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81

4

3

1

0

5x10
CFU/mL

5x10
CFU/mL

5x10
CFU/mL

5x10
CFU/mL

0.00004933
0.00004900
0.00004867
0.00004833
0.00004733
0.00004667
0.00004600
0.00004567
0.00004433
0.00004300
0.00004200
0.00004100
0.00003933
0.00003800
0.00003700
0.00003533
0.00003467
0.00003333
0.00003233
0.00003133
0.00003033
0.00003033
0.00002967
0.00002933
0.00002967
0.00002933
0.00002933
0.00002933
0.00002967
0.00003000
0.00003000
0.00003000
0.00003033
0.00003000
0.00003000

0.00007033
0.00006933
0.00006833
0.00006700
0.00006533
0.00006400
0.00006233
0.00006067
0.00005933
0.00005733
0.00005467
0.00005233
0.00005000
0.00004800
0.00004633
0.00004500
0.00004233
0.00004100
0.00003967
0.00003767
0.00003633
0.00003600
0.00003567
0.00003500
0.00003367
0.00003400
0.00003367
0.00003367
0.00003367
0.00003333
0.00003367
0.00003367
0.00003400
0.00003367
0.00003367

0.00000005
0.00000006
0.00000007
0.00000006
0.00000006
0.00000005
0.00000003
0.00000004
0.00000004
0.00000003
0.00000004
0.00000003
0.00000003
0.00000004
0.00000004
0.00000004
0.00000004
0.00000004
0.00000004
0.00000004
0.00000004
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000002
0.00000002
0.00000003
0.00000003
0.00000003
0.00000002
0.00000002
0.00000002
0.00000002

0.00000040
0.00000040
0.00000037
0.00000037
0.00000040
0.00000037
0.00000030
0.00000037
0.00000033
0.00000033
0.00000033
0.00000037
0.00000037
0.00000033
0.00000033
0.00000040
0.00000037
0.00000040
0.00000040
0.00000027
0.00000033
0.00000033
0.00000037
0.00000033
0.00000023
0.00000023
0.00000023
0.00000027
0.00000027
0.00000033
0.00000030
0.00000030
0.00000027
0.00000027
0.00000030

165

Table A-16. Continued
0.79
0.00003033
0.77
0.00003033
0.75
0.00003100
0.73
0.00003033
0.71
0.00003100
0.69
0.00003100
0.67
0.00003133
0.65
0.00003167
0.63
0.00003167
0.61
0.00003200
0.59
0.00003200
0.57
0.00003233
0.55
0.00003267
0.53
0.00003367
0.51
0.00003400
0.49
0.00003400
0.47
0.00003400
0.45
0.00003467
0.43
0.00003500
0.41
0.00003500
0.39
0.00003467
0.37
0.00003467
0.35
0.00003533
0.33
0.00003500
0.31
0.00003500
0.29
0.00003467
0.27
0.00003433
0.25
0.00003500
0.23
0.00003433
0.21
0.00003400
0.19
0.00003400
0.17
0.00003433
0.15
0.00003433
0.13
0.00003433
0.11
0.00003467
0.09
0.00003467
0.07
0.00003367
0.05
0.00003433
0.03
0.00003500

0.00003400
0.00003400
0.00003400
0.00003400
0.00003433
0.00003433
0.00003467
0.00003500
0.00003567
0.00003667
0.00003767
0.00003900
0.00004000
0.00004067
0.00004133
0.00004200
0.00004167
0.00004100
0.00004100
0.00004100
0.00004067
0.00004033
0.00004067
0.00004033
0.00004000
0.00004000
0.00003967
0.00003933
0.00003967
0.00003933
0.00003933
0.00003900
0.00003900
0.00003900
0.00003900
0.00003900
0.00003900
0.00003900
0.00003967
166

0.00000002
0.00000002
0.00000002
0.00000002
0.00000002
0.00000002
0.00000002
0.00000003
0.00000003
0.00000003
0.00000004
0.00000004
0.00000005
0.00000006
0.00000005
0.00000005
0.00000005
0.00000005
0.00000005
0.00000005
0.00000004
0.00000004
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000003
0.00000002
0.00000002
0.00000002
0.00000002
0.00000002
0.00000003
0.00000002

0.00000030
0.00000027
0.00000023
0.00000027
0.00000023
0.00000023
0.00000017
0.00000013
0.00000000
0.00000000
0.00000000
-0.00000003
-0.00000007
0.00000000
0.00000000
-0.00000003
0.00000007
0.00000010
0.00000000
-0.00000007
0.00000007
0.00000000
0.00000003
0.00000003
0.00000003
-0.00000007
-0.00000010
-0.00000007
-0.00000003
0.00000000
0.00000003
-0.00000003
-0.00000003
-0.00000003
-0.00000003
-0.00000003
-0.00000003
0.00000000
0.00000000

Table A-17. Differential pulse voltammetry data for Figure 5-9.
Differential pulse current (dI)
Voltage
(V)
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81
0.79

7

6

5

0.00001629
0.00001614
0.00001557
0.00001486
0.00001400
0.00001314
0.00001257
0.00001214
0.00001186
0.00001143
0.00001114
0.00001100
0.00001029
0.00001000
0.00000971
0.00000986
0.00000971
0.00000957
0.00000943
0.00000943
0.00000986
0.00000986
0.00001000
0.00001014
0.00001014
0.00001043
0.00001029
0.00001057
0.00001086
0.00001114
0.00001157
0.00001200
0.00001229
0.00001257
0.00001257

0.00001143
0.00001129
0.00001086
0.00001043
0.00001000
0.00000943
0.00000900
0.00000871
0.00000829
0.00000814
0.00000786
0.00000786
0.00000786
0.00000786
0.00000786
0.00000771
0.00000786
0.00000786
0.00000757
0.00000743
0.00000743
0.00000757
0.00000786
0.00000800
0.00000800
0.00000786
0.00000786
0.00000786
0.00000786
0.00000814
0.00000843
0.00000871
0.00000900
0.00000900
0.00000929

0.00001143
0.00001129
0.00001100
0.00001086
0.00001057
0.00001029
0.00001000
0.00001000
0.00000986
0.00000971
0.00000943
0.00000929
0.00000971
0.00001000
0.00001014
0.00001043
0.00001029
0.00001014
0.00001000
0.00001000
0.00001014
0.00001014
0.00001014
0.00001014
0.00001000
0.00001014
0.00001029
0.00001043
0.00001043
0.00001043
0.00001029
0.00001043
0.00001014
0.00001043
0.00001014

4

5x10 CFU/mL 5x10 CFU/mL 5x10 CFU/mL 5x10 CFU/mL

167

-0.00000029
0.00000000
0.00000043
0.00000071
0.00000071
0.00000071
0.00000057
0.00000043
0.00000014
-0.00000029
0.00000000
-0.00000029
-0.00000029
-0.00000029
-0.00000043
-0.00000029
-0.00000014
0.00000000
-0.00000014
-0.00000029
0.00000000
0.00000014
0.00000014
0.00000000
-0.00000029
-0.00000014
-0.00000014
0.00000014
-0.00000014
0.00000000
-0.00000014
0.00000029
0.00000043
0.00000043
0.00000043

Table A-17. Continued
0.77
0.00001271
0.75
0.00001300
0.73
0.00001329
0.71
0.00001357
0.69
0.00001400
0.67
0.00001414
0.65
0.00001414
0.63
0.00001400
0.61
0.00001414
0.59
0.00001443
0.57
0.00001471
0.55
0.00001443
0.53
0.00001429
0.51
0.00001386
0.49
0.00001371
0.47
0.00001371
0.45
0.00001386
0.43
0.00001357
0.41
0.00001343
0.39
0.00001357
0.37
0.00001371
0.35
0.00001414
0.33
0.00001429
0.31
0.00001457
0.29
0.00001457
0.27
0.00001514
0.25
0.00001543
0.23
0.00001571
0.21
0.00001557
0.19
0.00001543
0.17
0.00001514
0.15
0.00001471
0.13
0.00001443
0.11
0.00001414
0.09
0.00001386
0.07
0.00001371
0.05
0.00001386
0.03
0.00001400
0.01
0.00001414

0.00000929
0.00000957
0.00000957
0.00000971
0.00001000
0.00001000
0.00001000
0.00001000
0.00000986
0.00001000
0.00001000
0.00001000
0.00001000
0.00001014
0.00001029
0.00001029
0.00001014
0.00000971
0.00000971
0.00000943
0.00000943
0.00000957
0.00001000
0.00001029
0.00001043
0.00001043
0.00001057
0.00001071
0.00001114
0.00001100
0.00001100
0.00001057
0.00001029
0.00001014
0.00001000
0.00001000
0.00001000
0.00000986
0.00000986
168

0.00001043
0.00001057
0.00001071
0.00001071
0.00001071
0.00001057
0.00001057
0.00001057
0.00001071
0.00001043
0.00001029
0.00001029
0.00001043
0.00001057
0.00001071
0.00001057
0.00001057
0.00001043
0.00001071
0.00001086
0.00001114
0.00001100
0.00001086
0.00001114
0.00001114
0.00001143
0.00001143
0.00001157
0.00001186
0.00001200
0.00001186
0.00001186
0.00001171
0.00001157
0.00001114
0.00001114
0.00001114
0.00001129
0.00001129

0.00000043
0.00000043
0.00000029
0.00000000
0.00000000
0.00000014
0.00000043
0.00000029
0.00000014
0.00000000
0.00000014
0.00000000
0.00000000
-0.00000029
-0.00000029
0.00000014
0.00000029
0.00000043
0.00000029
0.00000043
0.00000029
0.00000029
0.00000057
0.00000029
0.00000014
-0.00000029
-0.00000029
-0.00000014
0.00000000
-0.00000029
0.00000000
0.00000000
0.00000014
0.00000000
-0.00000014
0.00000014
0.00000000
-0.00000014
-0.00000043

Table A-17. Continued
-0.01
0.00001457
-0.03
0.00001486
-0.05
0.00001514
-0.07
0.00001529
-0.09
0.00001543
-0.11
0.00001571
-0.13
0.00001571
-0.15
0.00001586
-0.17
0.00001571
-0.19
0.00001557
-0.21
0.00001543
-0.23
0.00001529
-0.25
0.00001500
-0.27
0.00001514
-0.29
0.00001500
-0.31
0.00001514
-0.33
0.00001500
-0.35
0.00001486
-0.37
0.00001471
-0.39
0.00001457
-0.41
0.00001457
-0.43
0.00001457
-0.45
0.00001457
-0.47
0.00001457

0.00000971
0.00000986
0.00001029
0.00001043
0.00001086
0.00001057
0.00001086
0.00001071
0.00001086
0.00001086
0.00001057
0.00001057
0.00001043
0.00001043
0.00001029
0.00001029
0.00001043
0.00001043
0.00001029
0.00001029
0.00001014
0.00001014
0.00001014
0.00001043

169

0.00001100
0.00001100
0.00001129
0.00001129
0.00001143
0.00001129
0.00001143
0.00001157
0.00001186
0.00001171
0.00001157
0.00001157
0.00001143
0.00001157
0.00001157
0.00001157
0.00001143
0.00001129
0.00001114
0.00001129
0.00001129
0.00001129
0.00001157
0.00001171

-0.00000029
0.00000000
0.00000029
0.00000029
0.00000029
0.00000043
0.00000029
0.00000043
0.00000014
0.00000029
0.00000014
-0.00000029
-0.00000014
0.00000000
0.00000043
0.00000029
0.00000014
0.00000014
-0.00000014
0.00000014
0.00000000
-0.00000029
0.00000000
-0.00000043

Table A-18.Additional differential pulse voltammetry data for Figure 5-9.
Differential pulse current (dI)
Voltage
(V)
1.47
1.45
1.43
1.41
1.39
1.37
1.35
1.33
1.31
1.29
1.27
1.25
1.23
1.21
1.19
1.17
1.15
1.13
1.11
1.09
1.07
1.05
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
0.85
0.83
0.81
0.79

3

1

0

0.00000714
0.00000700
0.00000671
0.00000671
0.00000671
0.00000657
0.00000629
0.00000600
0.00000600
0.00000600
0.00000586
0.00000586
0.00000586
0.00000600
0.00000586
0.00000571
0.00000571
0.00000557
0.00000557
0.00000557
0.00000543
0.00000571
0.00000586
0.00000571
0.00000600
0.00000586
0.00000571
0.00000571
0.00000571
0.00000571
0.00000600
0.00000600
0.00000600
0.00000614
0.00000629

0.00000614
0.00000629
0.00000614
0.00000614
0.00000600
0.00000600
0.00000614
0.00000614
0.00000614
0.00000571
0.00000557
0.00000557
0.00000557
0.00000543
0.00000557
0.00000557
0.00000571
0.00000571
0.00000543
0.00000529
0.00000500
0.00000529
0.00000557
0.00000557
0.00000571
0.00000557
0.00000571
0.00000557
0.00000543
0.00000557
0.00000571
0.00000586
0.00000600
0.00000600
0.00000586

0.00000229
0.00000243
0.00000257
0.00000214
0.00000200
0.00000214
0.00000229
0.00000243
0.00000214
0.00000186
0.00000186
0.00000186
0.00000200
0.00000200
0.00000200
0.00000214
0.00000214
0.00000214
0.00000214
0.00000200
0.00000186
0.00000186
0.00000171
0.00000157
0.00000143
0.00000129
0.00000143
0.00000171
0.00000171
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200

5x10 CFU/mL 5x10 CFU/mL 5x10 CFU/mL

170

Table A-18. Continued
0.77
0.00000629
0.75
0.00000629
0.73
0.00000643
0.71
0.00000629
0.69
0.00000643
0.67
0.00000657
0.65
0.00000657
0.63
0.00000657
0.61
0.00000657
0.59
0.00000643
0.57
0.00000643
0.55
0.00000657
0.53
0.00000700
0.51
0.00000686
0.49
0.00000671
0.47
0.00000643
0.45
0.00000614
0.43
0.00000629
0.41
0.00000629
0.39
0.00000657
0.37
0.00000657
0.35
0.00000671
0.33
0.00000686
0.31
0.00000686
0.29
0.00000686
0.27
0.00000686
0.25
0.00000671
0.23
0.00000671
0.21
0.00000686
0.19
0.00000714
0.17
0.00000700
0.15
0.00000686
0.13
0.00000671
0.11
0.00000686
0.09
0.00000657
0.07
0.00000643
0.05
0.00000657
0.03
0.00000671
0.01
0.00000686

0.00000586
0.00000586
0.00000586
0.00000586
0.00000586
0.00000571
0.00000557
0.00000571
0.00000571
0.00000571
0.00000586
0.00000586
0.00000571
0.00000571
0.00000586
0.00000586
0.00000614
0.00000614
0.00000614
0.00000600
0.00000614
0.00000614
0.00000629
0.00000643
0.00000643
0.00000643
0.00000657
0.00000657
0.00000657
0.00000671
0.00000671
0.00000657
0.00000643
0.00000657
0.00000643
0.00000629
0.00000629
0.00000629
0.00000629
171

0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000200
0.00000214
0.00000243
0.00000243
0.00000243
0.00000200
0.00000186
0.00000200
0.00000214
0.00000214
0.00000214
0.00000200
0.00000171
0.00000186
0.00000200
0.00000214
0.00000243
0.00000243
0.00000257
0.00000243
0.00000229
0.00000214
0.00000186
0.00000186
0.00000186
0.00000200
0.00000214
0.00000214
0.00000214

Table A-18. Continued
-0.01
0.00000686
-0.03
0.00000686
-0.05
0.00000686
-0.07
0.00000686
-0.09
0.00000700
-0.11
0.00000729
-0.13
0.00000757
-0.15
0.00000786
-0.17
0.00000800
-0.19
0.00000786
-0.21
0.00000771
-0.23
0.00000757
-0.25
0.00000729
-0.27
0.00000757
-0.29
0.00000757
-0.31
0.00000743
-0.33
0.00000729
-0.35
0.00000700
-0.37
0.00000700
-0.39
0.00000714
-0.41
0.00000743
-0.43
0.00000743
-0.45
0.00000743
-0.47
0.00000714

0.00000629
0.00000614
0.00000614
0.00000643
0.00000643
0.00000657
0.00000671
0.00000643
0.00000657
0.00000629
0.00000629
0.00000643
0.00000657
0.00000671
0.00000643
0.00000643
0.00000629
0.00000629
0.00000629
0.00000629
0.00000629
0.00000629
0.00000629
0.00000600

172

0.00000214
0.00000214
0.00000229
0.00000243
0.00000257
0.00000229
0.00000214
0.00000229
0.00000229
0.00000243
0.00000243
0.00000243
0.00000271
0.00000271
0.00000271
0.00000271
0.00000257
0.00000257
0.00000243
0.00000243
0.00000243
0.00000229
0.00000243
0.00000257

Table A-19. Fluorescence data for Figure 5-10.
Sample
Control
8

5x10 CFU/mL
7

5x10 CFU/mL
6

5x10 CFU/mL
5

5x10 CFU/mL
4

5x10 CFU/mL
3

5x10 CFU/mL
2

5x10 CFU/mL
1

5x10 CFU/mL

Fluorescence
(RFU)

Standard
Deviation

424

109

188

55

204

20

185

15

183

23

255

98

310

192

173

7

169

1

173

Table A-20. Fluorescence data for Figure 5-11.

Concentration

Control

Method

Fluorescence Standard
(RFU)
Deviation

C-linker

9965

391

C-linker

11347

276

C-linker

11950

389

C-linker

11206

311

C-linker

11006

428

C-linker

10684

442

C-linker

10394

320

C-linker

10147

356

5x10 CFU/mL

C-linker

10003

1223

Control

AB-thiol

5031

195

AB-thiol

7616

4240

AB-thiol

5521

2239

AB-thiol

6590

1061

AB-thiol

6028

1456

AB-thiol

5711

384

AB-thiol

5867

642

AB-thiol

5735

788

AB-thiol

4809

482

8

5x10 CFU/mL
7

5x10 CFU/mL
6

5x10 CFU/mL
5

5x10 CFU/mL
4

5x10 CFU/mL
3

5x10 CFU/mL
2

5x10 CFU/mL
1

8

5x10 CFU/mL
7

5x10 CFU/mL
6

5x10 CFU/mL
5

5x10 CFU/mL
4

5x10 CFU/mL
3

5x10 CFU/mL
2

5x10 CFU/mL
1

5x10 CFU/mL

174

REFERENCES

175

REFERENCES
1.

Abu-Ali, G.S., et al., Genomic diversity of pathogenic Escherichia coli of the EHEC 2
clonal complex. BMC Genomics, 2009. 10: p. 296.

2.

Leclerc, H., et al., Advances in the bacteriology of the coliform group: their suitability as
markers of microbial water safety. Annual Review of Microbiology, 2001. 55: p. 201-34.

3.

Eaton, A.D., Standard methods for the examiniation of water and wastewater, W.E.
Federation, Editor. 1992, American Public Health Association.

4.

March, S.B. and S. Ratnam, Sorbitol-MacConkey medium for detection of Escherichia
coli O157:H7 associated with hemorrhagic colitis. Journal of Clinical Microbiology,
1986. 23(5): p. 869-72.

5.

Federal, R., Quality standards for foods with no identity standards; bottled water, F.a.D.
Administration, Editor. 1993. p. 52042-52050.

6.

Yakub, G.P., et al., Evaluation of Colilert and Enterolert defined substrate methodology
for wastewater applications. Water Environment Research, 2002. 74(2): p. 131-5.

7.

Federal, R., Guidelines establishing test procedures for the analysis of pollutants;
analytical methods for the biological pollutants in wastewater and sewage sludge; final
rule. 2007. 72(157): p. 14220-14233.

8.

Rompre, A., et al., Detection and enumeration of coliforms in drinking water: current
methods and emerging approaches. Journal of Micobiological Methods, 2002. 49: p. 3154.

9.

Fode-Vaughan, K.A., et al., Direct PCR detection of Escherichia coli O157:H7. Letters
in Applied Microbiology, 2003. 37(3): p. 239-43.

10.

Torres-Chavolla, E. and E.C. Alocilja, Nanoparticle based DNA biosensor for
tuberculosis detection using thermophilic helicase-dependent isothermal amplification.
Biosensors & Bioelectronics, 2011. 26(11): p. 4614-8.

11.

Hill, H.D. and C.A. Mirkin, The bio-barcode assay for the detection of protein and
nucleic acid targets using DTT-induced ligand exchange. Nature Protocols, 2006. 1(1): p.
324-36.

12.

Anderson, M.J., et al., One step alkaline synthesis of biocompatible gold nanoparticles
using dextrin as capping agent. Journal of Nanoparticle Research, 2011. 13(7): p. 28432851.

176

13.

Anderson, M.J., D. Zhang, and E.C. Alocilja, Spectral and electrical nanoparticle-based
molecular detection of Bacillus anthracis using copolymer mass amplification. Ieee
Transactions on Nanotechnology, 2011. 10(1): p. 44-49.

14.

Goluch, E.D., et al., A bio-barcode assay for on-chip attomolar-sensitivity protein
detection. Lab Chip, 2006. 6(10): p. 1293-9.

15.

Hill, H.D., R.A. Vega, and C.A. Mirkin, Nonenzymatic detection of bacterial genomic
DNA using the bio-barcode assay. Analytical Chemistry, 2007. 79(23): p. 9218-23.

16.

Nam, J.M., S.I. Stoeva, and C.A. Mirkin, Bio-bar-code-based DNA detection with PCRlike sensitivity. Journal of the American Chemical Society, 2004. 126(19): p. 5932-3.

17.

Dirks, R.M. and N.A. Pierce, Triggered amplification by hybridization chain reaction.
Proceedings of the National Academy of Sciences of the United States of America, 2004.
101(43): p. 15275-8.

18.

Goluch, E.D., et al., A bio-barcode assay for on-chip attomolar-sensitivity protein
detection. Lab on a Chip, 2006. 6(10): p. 1293-9.

19.

Sujakhu, K. and U.T. Shrestha, Micrbial (Coliform and Vibrio cholerae) analysis of
drinking water collected from outbreak region of Bhaktapur municipality. 2011.

20.

Woodall, C.J., Waterborne diseases - Whar are the primary killers? Desalination, 2008.
252: p. 199-204.

21.

Gleick, P.H., Dirty water: Estimated deaths from water-related diseases 2000-2020.
2002, Pacific Insitute for Studies in Development, Environment, and Security. p. 1-12.

22.

Envirometnal_Protection_Agency, Drinking Water, National Primary Drinking Water
Regualtion, Total Coliforms (Including Fecal Coliforms and E. Coli); Final Rule, E.P.
Agency, Editor. 1989. p. 26.

23.

Beutin, L., et al., Prevalence and some properties of verotoxin (Shiga-like toxin)producing Escherichia coli in seven different species of healthy domestic animals. J Clin
Microbiol, 1993. 31(9): p. 2483-8.

24.

McIntire, R.J. California Firm Recalls Ground Beef Products Due to Possible E. coli
O157:H7 Contamination. 2011 [cited 2011 Feb. 02 2011]; Available from:
http://www.fsis.usda.gov/News_&_Events/Recall_008-2011_Release/index.asp.

25.

Ready_Pac_Foods (2010) Ready Pac Foods, Inc. Announces Voluntary Product Recall
because of Possible Health Risk. Volume,

26.

FDA, FDA Warns Consumers Not to Eat Nestle Toll House Prepackaged, Refrigerated
Cookie Dough, B.o.C.E.H.F. Protection, Editor. 2009.
177

27.

EPA, Economic Analysis for the Proposed Revised Total Coliform Rule. 2010.

28.

Federal, R., Rules and regulations, E.P. Agency, Editor. 1989. p. 27544-27568.

29.

Kilian, M. and P. Bulow, Rapid diagnosis of Enterobacteriaceae. I. Detection of
bacterial glycosidases. Acta Pathologica et Microbiologica Scandinavica, 1976. 84B(5):
p. 245-51.

30.

Tryland, I. and L. Fiksdal, Enzyme characteristics of beta-D-galactosidase- and beta-Dglucuronidase-positive bacteria and their interference in rapid methods for detection of
waterborne coliforms and Escherichia coli. Applied and Environmental Microbiology,
1998. 64(3): p. 1018-23.

31.

Navarro, A., et al., Antibody responses to Escherichia coli O157 and other
lipopolysaccharides in healthy children and adults. Clin Diagn Lab Immunol, 2003.
10(5): p. 797-801.

32.

Zhang, D., et al., Nanoparticle-based Bio-barcoded DNA Sensor for the Rapid Detection
of pagA Gene of Bacillus Anthracis. Ieee Transactions on Nanotechnology, Accepted
2011.

33.

Pal, S. and E.C. Alocilja, Electrically active magnetic nanoparticles as novel
concentrator and electrochemical redox transducer in Bacillus anthracis DNA detection.
Biosensors & Bioelectronics, 2010. 26(4): p. 1624-30.

34.

Setterington, E.B. and E.C. Alocilja, Rapid electrochemical detection of polyanilinelabeled Escherichia coli O157:H7. Biosensors & Bioelectronics, 2011. 26(5): p. 2208-14.

35.

Olsvik, O., et al., Use of automated sequencing of polymerase chain reaction-generated
amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae O1 strains.
Journal of Clinical Microbiology, 1993. 31(1): p. 22-5.

36.

Bellin, T., et al., Rapid detection of enterohemorrhagic Escherichia coli by real-time
PCR with fluorescent hybridization probes. Journal of Clinical Microbiology
2001. 39(1): p. 370-4.

37.

Desmarchelier, P.M., et al., A PCR specific for Escherichia coli O157 based on the rfb
locus encoding O157 lipopolysaccharide. Journal of Clinical Microbiology, 1998. 36(6):
p. 1801-4.

38.

Prager, R., et al., Subtyping of pathogenic Escherichia coli strains using flagellar (H)antigens: serotyping versus fliC polymorphisms. International Journal of Medical
Microbiology, 2003. 292(7-8): p. 477-86.

178

39.

Bej, A.K., S.C. McCarty, and R.M. Atlas, Detection of coliform bacteria and Escherichia
coli by multiplex polymerase chain reaction: comparison with defined substrate and
plating methods for water quality monitoring. Applied and Environmental Microbiology,
1991. 57(8): p. 2429-32.

40.

Wieler, L.H., et al., Shiga toxin-producing Escherichia coli strains from bovines:
association of adhesion with carriage of eae and other genes. Journal of Clinical
Microbiology, 1996. 34(12): p. 2980-4.

41.

Spierings, G., et al., Polymerase chain reaction for the specific detection of Escherichia
coli/Shigella. Research in Microbiology, 1993. 144(7): p. 557-64.

42.

Wahlberg, J., et al., General colorimetric method for DNA diagnostics allowing direct
solid-phase genomic sequencing of the positive samples. Proceedings of the National
Academy of Sciences of the United States of America, 1990. 87(17): p. 6569-73.

43.

Juck, D., et al., Nested PCR protocol for the rapid detection of Escherichia coli in
potable water. Canadian Journal of Microbiology, 1996. 42(8): p. 862-6.

44.

Iqbal, S., et al., Efficiency of the polymerase chain reaction amplification of the uid gene
for detection of Escherichia coli in contaminated water. Letters in Applied Microbiology,
1997. 24(6): p. 498-502.

45.

Blanco, C., P. Ritzenthaler, and M. Mata-Gilsinger, Cloning and endonuclease restriction
analysis of uidA and uidR genes in Escherichia coli K-12: Determiniation of
transcription direction for the uidA gene. Journal of Bacteriology, 1981. 149(2): p. 587594.

46.

Fricker, E.J. and C.R. Fricker, Application of the polymerase chain reaction to the
identification of Escherichia coli and coliforms in water. Letters in Applied
Microbiology, 1994. 19(1): p. 44-6.

47.

Geiss, G.K., et al., Direct multiplexed measurement of gene expression with color-coded
probe pairs. Nature Biotechnology, 2008. 26(3): p. 317-25.

48.

Sassolas, A., B.D. Leca-Bouvier, and L.J. Blum, DNA biosensors and microarrays.
Chemical Reviews, 2008. 108(1): p. 109-39.

49.

Murakami, A., et al., Highly sensitive detection of DNA using enzyme-linked DNA-probe.
1. Colorimetric and fluorometric detection. Nucleic Acids Research, 1989. 17(14): p.
5587-95.

50.

Vaknin, A. and H.C. Berg, Single-cell FRET imaging of phosphatase activity in the
Escherichia coli chemotaxis system. Proceedings of the National Academy of Sciences of
the United States of America, 2004. 101(49): p. 17072-7.
179

51.

Tang, Y.Z., K.Y. Gin, and T.H. Lim, High-temperature fluorescent in situ hybridization
for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide
probes and flow cytometry. Applied and Environmental Microbiology, 2005. 71(12): p.
8157-64.

52.

Quinones, B., et al., Identification of Eschericia coli O157 by using a novel colorimetric
detection method with DNA microarrays. Foodborne Pathogens and Disease, 2011. 8(6):
p. 705-711.

53.

Fritz, J., et al., Translating biomolecular recognition into nanomechanics. Science, 2000.
288(5464): p. 316-8.

54.

Metzgar, D., et al., Evaluation and validation of a real-time PCR assay for detection and
quantitation of human adenovirus 14 from clinical samples. PLoS One, 2009. 4(9): p.
e7081.

55.

Walker, G.T., et al., Strand displacement amplification--an isothermal, in vitro DNA
amplification technique. Nucleic Acids Research, 1992. 20(7): p. 1691-6.

56.

Walker, G.T., et al., Isothermal in vitro amplification of DNA by a restriction
enzyme/DNA polymerase system. Proceedings of the National Academy of Sciences of
the United States of America, 1992. 89(1): p. 392-6.

57.

Bergmann, J.S. and G.L. Woods, Clinical evaluation of the BDProbeTec strand
displacement amplification assay for rapid diagnosis of tuberculosis. Journal of Clinical
Microbiology, 1998. 36(9): p. 2766-8.

58.

Demidov, V.V., Rolling-circle amplification in DNA diagnostics: the power of simplicity.
Expert Review of Molecular Diagnostics, 2002. 2(6): p. 542-8.

59.

Thomas, D.C., G.A. Nardone, and S.K. Randall, Amplification of padlock probes for
DNA diagnostics by cascade rolling circle amplification or the polymerase chain
reaction. Archives of Pathology & Laboratory Medicine, 1999. 123(12): p. 1170-6.

60.

Notomi, T., et al., Loop-mediated isothermal amplification of DNA. Nucleic Acids
Research, 2000. 28(12): p. E63.

61.

Song, W., C. Lau, and J. Lu, Quantum dot-based isothermal chain elongation for
fluorescence detection of specific DNA sequences via template-dependent surfacehybridization. The Analyst, 2012. 137(7): p. 1611-7.

62.

Dore, K., M. Leclerc, and D. Boudreau, Investigation of a fluorescence signal
amplification mechanism used for the direct molecular detection of nucleic acids. Journal
of Fluorescence, 2006. 16(2): p. 259-65.
180

63.

Tsongalis, G.J., Branched DNA technology in molecular diagnostics. American Journal
of Clinical Pathology, 2006. 126(3): p. 448-53.

64.

Sato, K., K. Hosokawa, and M. Maeda, Colorimetric biosensors based on DNAnanoparticle conjugates. Analytical Sciences, 2007. 23(1): p. 17-20.

65.

Zhao, W., M.A. Brook, and Y. Li, Design of gold nanoparticle-based colorimetric
biosensing assays. Chembiochem, 2008. 9(15): p. 2363-71.

66.

Nam, J.M., K.J. Jang, and J.T. Groves, Detection of proteins using a colorimetric biobarcode assay. Nature Protocols, 2007. 2(6): p. 1438-44.

67.

Elghanian, R., et al., Selective colorimetric detection of polynucleotides based on the
distance-dependent optical properties of gold nanoparticles. Science, 1997. 277(5329):
p. 1078-81.

68.

Nam, J.M., C.S. Thaxton, and C.A. Mirkin, Nanoparticle-based bio-bar codes for the
ultrasensitive detection of proteins. Science, 2003. 301(5641): p. 1884-6.

69.

Thaxton, C.S., et al., A bio-bar-code assay based upon dithiothreitol-induced
oligonucleotide release. Analytical Chemistry, 2005. 77(24): p. 8174-8.

70.

Stoeva, S.I., et al., Multiplexed detection of protein cancer markers with biobarcoded
nanoparticle probes. Journal of the American Chemical Society, 2006. 128(26): p. 83789.

71.

Stoeva, S.I., et al., Multiplexed DNA detection with biobarcoded nanoparticle probes.
Angewandte Chemie (International ed), 2006. 45(20): p. 3303-6.

72.

Oh, B.K., et al., A fluorophore-based bio-barcode amplification assay for proteins.
Small, 2006. 2(1): p. 103-8.

73.

Borucki, M.K., et al., Suspension microarray with dendrimer signal amplification allows
direct and high-throughput subtyping of Listeria monocytogenes from genomic DNA.
Journal of Clinical Microbiology, 2005. 43(7): p. 3255-9.

74.

Turkevich, J., P.C. Stevenson, and J. Hillier, A study if the nucleation and growth
processes in the synthesis of colloidal gold. Discussion of the Faraday Society, 1951. 11:
p. 55-75.

75.

Brust, M., et al., Synthesis and reactions of functionalized gold nanoparticles. Journal of
the Chemical Society - Chemical Communications, 1995: p. 1655-1656.

181

76.

Brust, M., et al., Synthesis of thiol-derivatized gold nanoparticles in a 2-phase liquidliquid system. Journal of the Chemical Society - Chemical Communications, 1994: p.
801-801.

77.

Sanyal, A. and M. Sastry, Gold nanosheets via reduction of aqueous chloroaurate ions by
anthracene anions bound to a liquid-liquid interface. Chemical Communications
(Cambridge, England), 2003(11): p. 1236-7.

78.

Ahmad, A., et al., Extracellular biosynthesis of monodisperse gold nanoparticles by a
novel extremophilic Actinomycete, Thermomonospora sp. Langmuir, 2003. 19: p. 35503553.

79.

Ma, Z. and H. Han, One-step synthesis of cystine-coated gold nanoparticles in aqueous
solution. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2008.
317(1-3): p. 229-233.

80.

de la Fuente, J.M. and S. Penades, Glyconanoparticles: types, synthesis and applications
in glycoscience, biomedicine and material science. Biochim Biophys Acta, 2006.
1760(4): p. 636-51.

81.

Huang, Y.J., D. Li, and J.H. Li, Beta-cyclodextrin controlled assembling nanostructures
from gold nanoparticles to gold nanowires. Chemical Physics Letters, 2004. 389(1-3): p.
14-18.

82.

Li, Z., et al., Multiple thiol-anchor capped DNA-gold nanoparticle conjugates. Nucleic
Acids Research, 2002. 30(7): p. 1558-1562.

83.

Rechberger, W., et al., Optical properties of two interacting gold nanoparticles. Optics
Communications, 2003. 220(1-3): p. 137-141.

84.

Kneipp, J., et al., In vivo molecular probing of cellular compartments with gold
nanoparticles and nanoaggregates. Nano Letters, 2006. 6(10): p. 2225-31.

85.

Dudak, F.C., Rapid and label-free bacteria detection by surface plasmon
resonance (SPR) biosensors. Biotechnology Journal, 2009(4): p. 1003-1011.

86.

Pal, S., E.C. Alocilja, and F.P. Downes, Nanowire labeled direct-charge transfer
biosensor for detecting Bacillus species. Biosensors & Bioelectronics, 2007. 22(9-10): p.
2329-36.

87.

Xiliang, L., et al., Application of nanoparticles in electrochemical sensors and
biosensors. Electroanalysis, 2006. 18(4): p. 319-326.

88.

Chah, S., M.R. Hammond, and R.N. Zare, Gold nanoparticles as a colorimetric sensor
for protein conformational changes. Chemistry & Biology
2005. 12(3): p. 323-8.
182

89.

Slocik, J.M., M.O. Stone, and R.R. Naik, Synthesis of gold nanoparticles using
multifunctional peptides. Small, 2005. 1(11): p. 1048-52.

90.

Zhou, M., et al., Minute synthesis of extremely stable gold nanoparticles.
Nanotechnology, 2009. 20(50): p. 505606.

91.

Turkevich, J., P.C. Stevenson, and J. Hillier, A study of the nucleation and growth
processes in the synthesis of colloidal gold. Discussions of the Faraday Society,
1951(11): p. 55-57.

92.

Das, S.K., A.R. Das, and A.K. Guha, Gold nanoparticles: Microbial synthesis and
application in water hygiene management. Langmuir, 2009. 25(14): p. 8192-8199.

93.

Aslan, K., J.R. Lakowicz, and C.D. Geddes, Nanogold plasmon resonance-based glucose
sensing. 2. Wavelength-ratiometric resonance light scattering. Analytical Chemistry,
2005. 77(7): p. 2007-14.

94.

de la Fuente, J.M. and S. Penades, Glyconanoparticles: types, synthesis and applications
in glycoscience, biomedicine and material science. Biochimica et Biophysica Acta, 2006.
1760(4): p. 636-51.

95.

Newman, J.D. and G.J. Blanchard, Formation of gold nanoparticles using amine
reducing agents. Langmuir, 2006. 22(13): p. 5882-7.

96.

Li, X., et al., Enhancement of cell recognition in vitro by dual-ligand cancer targeting
gold nanoparticles. Biomaterials, 2011. 32(10): p. 2540-5.

97.

Porta, F., et al., Gold-ligand interaction studies of water-soluble aminoalcohol capped
gold nanoparticles by NMR. Langmuir, 2008. 24(14): p. 7061-4.

98.

Polte, J., et al., Mechanism of gold nanoparticle formation in the classical citrate
synthesis method derived from coupled in situ XANES and SAXS evaluation. Journal of
the American Chemical Society, 2010. 132(4): p. 1296-301.

99.

Ishkanian, A., et al., A tiling resolution DNA microarray with complete coverage of the
human genome. Nature Genetics 2004. 36: p. 299-303.

100.

Taton, T.A., C.A. Mirkin, and R.L. Letsinger, Scanometric DNA array detection with
nanoparticle probes. Science, 2000. 289(8): p. 1757-1760.

101.

Thaxton, C.S., D.G. Georganopoulou, and C.A. Mirkin, Gold nanoparticle probes for the
detection of nucleic acid targets. Clinica Chimica Acta, 2006. 363(1): p. 120-126.

102.

Zhang, D., et al., Nanoparticle-based bio-barcoded DNA sensor for the rapid detection of
pagA gene of Bacillus anthracis. Ieee Transactions on Nanotechnology, 2011.
183

103.

Zhang, D., D.J. Carr, and E.C. Alocilja, Fluorescent bio-barcode DNA assay for the
detection of Salmonella enterica serovar Enteritidis. Biosensors & Bioelectronics, 2009.
24(5): p. 1377-1381.

104.

Dragan, A.I., et al., Characterization of PicoGreen interaction with dsDNA and the
origin of its fluorescence enhancement upon binding. Biophysical journal, 2010. 99(9): p.
3010-9.

105.

Hayashi, T., et al., Complete genome sequence of enterohemorrhagic Escherichia coli
O157:H7 and genomic comparison with a laboratory strain K-12. DNA Research, 2001.
8(1): p. 11-22.

184