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Rapid Detection of Multiple Food Pathogens in a Microfluidic Assay


<p>The overall goal of the project is to develop a sensor which can rapidly detect pathogens in food resulting in increased food safety.</p>

<p>The proposed project aims to develop and test a biosensor which uses complex nanoparticles for electrochemical detection of a pathogen's mRNA. Initial testing will use a magnetic bead (1micron) with surface immobilized DNA capture probe. Once the project moves to testing in the microfluidic device, the capture probes will be surface immobilized directly on the electrode surface.</p>

<p>We have previously developed gold-SiO2 core shell nanoparticles in our lab. Similar nanoparticles will be synthesized for this project. The dimensions of the nanoparticle will be optimized for maximum metal enhanced fluorescence. The SiO2 nanoparticle surface will be functionalized and conjugated to Ru(bpy)32+ and DNA probes. DNA probe density on the nanoparticle surface will also be optimized. The adsorption spectra of the nanoparticle will be matched with the emission spectra of the Ru(bpy)32+ in order to achieve MEF. An additional reporter incorporating 60,000 MW polyethyleneimine (PEI) as the backbone will be evaluated. The reporter DNA will be conjugated to the PEI and all remaining primary amines on the PEI will be tagged with Ru(bpy)32+. The resulting reporter probe will have better molecular mobility than the nanoparticle and thus possibly a better hybridization efficiency. The PEI reporter will be compared to the nanoparticle based reporter to determine differences in the limit of detection for each. A portable ECL reader will be designed and fabricated to perform the detection assay. The microfluidic device will be constructed from a rigid polymer. The chip will be designed and fabricated to allow for an enclosed electrode with surface immobilized DNA capture probes. The ECL reader will be constructed using a photomultiplier tube for light emission detection. Fluid delivery will be accomplished with integrated syringe pumps. Initial testing will use synthetic DNA target while optimizing the probes.</p>

<p>Once the probes are developed, bacterial cell lysate will be tested in the device. E. coli O157:H7 will be grown and lysed in a solution inhibiting RNase activity. The lysing of the bacterial cell will be investigated to preserve the RNA until testing. The limit of detection and sensitivity for the bacterial lysate will be determined. Spiked produce such as spinach will be used as a sample matrix to test for E. coli O157:H7. The sample will be mixed in broth and then the bacterial cells isolated with immunomagnetic separation. The isolated cells will then be lysed and fed into the finished device for mRNA detection. From this data, a dose response curve will be generated which will determine the limit of detection and sensitivity.</p>

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<p>NON-TECHNICAL SUMMARY: A recent study has shown that the annual cost of food borne illness in the Unites States is approximately $152 billion. This is a result of the estimated 76 million food-related illnesses which occur annually including approximately 5,000 deaths and 325,000 hospitalizations. Coast-to-coast and international distribution by megaprocessing plants puts potential outbreaks on a national and international scale. Therefore, monitoring of pathogen counts on processing surfaces is critical in maintaining low or zero counts in food products. There is a growing need for rapid and sensitive methods for the detection of pathogens and toxins in our food supply. Currently, most producers are using traditional testing methods which take days for results to be obtained. During this time, tons of product may have been distributed and consumed. Advances in nanotechnology have allowed more rapid and sensitive testing methods to be developed in the form of biosensors. These methods can be used to help identify potential dangers in food products prior to distribution. They would not only enable detection of pathogens and toxins, but allergens, antibiotics, hormones and genetically modified organisms. Not only will biosensors help protect against unintentional food contamination, but they could also help identify bioterrorist attacks. Analysis of an intentional bioterror attack on fluid milk using botulinum toxin concluded that the ability for rapid detection would have a significant impact on the reduction of fatalities. The proposed biosensor will identify pathogens using their unique mRNA sequences. The structure of mRNA is very similar to DNA and is used as a blueprint to make proteins in the cell. The mRNA is rapidly degraded within the cell. Due to the stability of DNA and antibody binding sights on an organism, both DNA and immune testing could result in significant false positives by detecting non-viable pathogens inactivated by heat treatment or other means. The short half-life of mRNA suggests that if detected, the target organism was recently viable. This distinction is important for products such as fluid milk which have undergone heat treatment, yet still contain non-viable bacteria with intact DNA. The field of microfluidics has been rapidly growing. This is especially true in biosensor development. The use of microfluidic technology allows miniaturization of a test which adds to portability. Electrochemiluminescence (ECL) is a detection method which causes a specific molecule to glow by stimulating it with an electrical potential. The proposed project aims to develop a microfluidic detection device. The device will be designed for rapid and portable testing. Both a core-shell nanoparticle and a polymer based reporter probe will be investigated for optimal sensitivity. The finished device will be used to test spiked food samples. The final biosensor will have two detection zones for multianalyte detection. One detection zone will target E. coli mRNA and the other will target Salmonella mRNA. The organisms will serve as indicators of contamination and will be independently quantified in a single assay.</p>

<p>APPROACH: Both gold and silver nanoparticles will be evaluated. Both have unique optical and plasmonic properties. The nanoparticles will be made using the standard Turkevich method. Particle size distribution and zeta potential will be conducted with a particle size analyzer. Size and uniformity will also be observed with transmission electron microscopy (TEM). The SiO2 shell will be formed around the silver nanoparticles using an improved Stober method. The thickness of the shell will be optimized for maximum metal enhanced fluorescence. The nanoparticle surface will be silane modified for surface conjugation. Nucleic acids will then be conjugated to the terminal nanoparticles resulting in a nanoparticle with surface-conjugated nucleic acid probes. The nucleic acid density on the surface will be optimized for maximum target hybridization. The PEI reporter probe will be made by initially conjugating 5' phosphate-terminated DNA to the 60,000 MW PEI in a 1:1 ratio using N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and imidazole chemistry. Bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthen ium N-succinimidyl ester will then be added in excess to the DNA/PEI solution and allow to conjugate to the remaining primary amines on the PEI. The assay conditions for nucleic acid hybridization will be determined using a M384 ECL analyzer (Bioveris). This includes formamide and sodium saline citrate (SSC) concentration as well as time and temperature. A dose response for each reporter probe will be determined. Once the assay condition has been determined, microfluidic testing will begin. Our lab has experience with the design and fabrication of microfluidic devices in rigid polymers such as poly(methymethacrylate). Channels will be embossed onto the polymer sheet and electrodes will be printed printed using conductive silver ink. The electrode pattern will be characterized and optimized using cyclic voltammetry experiments. For the microfluidic experiments, thiol-modified capture probe will be immobilized onto the electrode surface via chemadsorption. Capture probes will be printed onto the respective electrodes with a specialized plotter. Reaction solutions will be introduced into the chip via syringe pumps. This will allow the target sample, either synthetic DNA or cell lysate, to hybridize to the capture probes immobilized on the working electrode. Following the initial hybridization, the reporter probe will be introduced into the channel and allowed to hybridize to the target RNA. A potential will then be applied across the working and counter electrodes in order to stimulate the electrochemiluminescence. A photomultiplier tube placed above the chip will quantify the ECL. The chips will be designed to be single use to prevent false positives from sample carryover. The two working electrodes will be turned on at separate times to allow individual quantification of the two analytes. The assays will be tested initially on synthetic DNA targets. Following successful optimization, testing will continue on isolated RNA from bacterial cultures. This will conclude with RNA isolated from the surfaces of spiked produce.</p>

Nugen, Sam
University of Massachusetts - Amherst
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