BIOSENSING TOTAL BACTERIAL LOAD IN LIQUID MATRICES TO IMPROVE FOOD SUPPLY CHAIN SAFETY USING CARBOHYDRATE-FUNCTIONALIZED MAGNETIC NANOPARTICLES FOR CELL CAPTURE AND GOLD NANOPARTICLES FOR SIGNALING

Summary

Economical rapid nano-biosensing methods with expedited electrochemical signaling, were developed using carbohydrate-functionalized magnetic nanoparticles (MNP) and gold-nanoparticles (AuNP) to detect pathogenic bacteria in liquid or homogenized food samples. MNP functionalized with glycan- and amino/glycan ligands were able to rapidly extract bacteria, while concentrated dextrin-coated AuNP labeling improved detection sensitivity. Carbohydrate ligands are more stable than antibodies,... Show moreEconomical rapid nano-biosensing methods with expedited electrochemical signaling, were developed using carbohydrate-functionalized magnetic nanoparticles (MNP) and gold-nanoparticles (AuNP) to detect pathogenic bacteria in liquid or homogenized food samples. MNP functionalized with glycan- and amino/glycan ligands were able to rapidly extract bacteria, while concentrated dextrin-coated AuNP labeling improved detection sensitivity. Carbohydrate ligands are more stable than antibodies, permitting long shelf life of MNP at room temperature and minimized AuNP aggregation during simple refrigeration. Transmission electron microscopy (TEM) imaged the electrostatic binding between MNP and Salmonella Enteritidis, E. coli O157:H7, Bacillus cereus, Listeria monocytogenes and E. coli C3000, which mimics electrostatic binding by antibodies, although with lower specificity. Capture index (CI) is defined as the parts-per-thousand (ppt) of bacteria extracted per initial bacterial presence. TEM mages showed that attached milk matrix components did not interfere with microbial. capture. Salmonella, E. coli, and Bacillus (3 to 5 log CFU/mL) capture in three milks was 2 ppt to 120 ppt CI. Capture in beef juice and apple cider was 0.002 ppt to 0.011 ppt for E. coli and Listeria, respectively, at 10 log cfu/mL due to accelerated microbial growth immediately following the spike. Viscous homogenized eggs, though, impeded MNP-Salmonella migration to the magnet during separation. This phenomenon was a motivating factor in creating “dip-sticks”: plastic strips coated in MNP (MNP-strip). Rapid nano-biosensing of MNP-cell complexes in under 30 min from either suspended or strip capture was possible using electrochemical technology of spectrometry or a simple handheld potentiostat. Capture concentrates bacteria as MNP-cell from large volumes allowing strong cyclic voltammetric (CV) signaling. Normalized peak current responses (NPCR) for microbial detection from simple matrices (PBS and beef juices) showed sample (S) NPCR lower than negative controls (N) (S/N < 1.0). Whereas in complex matrices (milk, apple cider, and homogenized eggs), S/N were significantly greater than 1.0. NPCR for negative controls were found to be linearly related to matrix components fats, proteins, and sodium (R2 = 0.92). Except for E. coli in beef juices, all S/N were significant (p < 0.05) for contamination levels ranging between 6.2 to 12.3 log CFU/mL.Enhanced signaling of low pathogen presence in food was achieved using electrically active AuNP labeling. Electrochemical detection of MNP-cell-AuNP complexes with spectrophotometry or differential pulse voltammetry (DPV) was significantly more sensitive, detecting 3 log CFU/mL and 5 log CFU/mL E. coli contamination in milk (p < 0.20), respectively. Food component attachment to the complexes altered, but did not interfere, with distinguishing samples from negative controls . MNP carbohydrate ligands exposed to refrigerated milk matrix components (fats, lipids, sugars, protein and sodium) for up to 9-days still extracted bacteria. This makes possible future biocompatible tag-on nano-biosensors inside individual food packaging. Pathogen presence could be monitored over the lifetime of the product, reducing consumption of contaminated foods. Reliable frequent testing along the food supply chain would facilitate reduced human disease, while reducing industry financial losses due to foodborne outbreaks. Flexible carbohydrate-based MNP-cell/(CV) and MNP-cell-AuNP/(DPV or spectrometry) nano-biosensing with electrochemical detection can provide a truly rapid, economical test. Show less