Source: UNIV OF MASSACHUSETTS submitted to
RAPID DETECTION OF MULTIPLE FOOD PATHOGENS IN A MICROFLUIDIC ASSAY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0223234
Grant No.
(N/A)
Project No.
MAS00996
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2010
Project End Date
Sep 30, 2015
Grant Year
(N/A)
Project Director
Nugen, SA, R.
Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003
Performing Department
Food Science
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.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71140101100100%
Goals / Objectives
The overall goal of the project is to develop a sensor which can rapidly detect pathogens in food resulting in increased food safety. 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. 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. 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.
Project Methods
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.

Progress 10/01/10 to 09/30/15

Outputs
Target Audience:We have reachedseveral target audiences during the course of the project. This includes undergraduate students, graduate students and visiting scientists. Undergraduates were trained by graduate studets which enabled increased technical learnings for the undergraduate as well and scientific instruction for the graduate student (with a future in academics). These technical learnings included chemistry, molecular genetics, microbiology and engineering as it pertains to food safety. Both the graduate student and undergraduate students also gained valuable experiences in technical writing in the form of manuscript preparation and laboratory notebook maintanance. The students have advanced technologies in the use of bacteriophages for the rapid detection of pathogens. These learnings have been presented to the food industry in scientific conferences and have been well-received. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The funding has provided significant training to the supported students. During the course of the project, two Ph.D. students were able to obtain faculty positions (without a postdoc), three obtained postdoc positions in relatred fields and one Ph.D. obtained a research position in industry. This was largely due to funding enabling research and travel. Therefore, the funding provided by this project will have a significant and long-lasting impact on the field. How have the results been disseminated to communities of interest?The progress of the research has been disseminated in several formats. We have had several popular press articles written up about the development. This has allowed direct dissemination to the general public. Additionally, we have had numerous presentations to the scientific community as well as publications in peer-reviewed articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have prototyped methods for continuous manufacturing of microfluidic chips for bacteria detection. This was performed to allow a more pragmatic and therfore manufacturable design which can be sooner realized for production. We have also designed bacteriophage-based detection which proved to be superior to the initial concept. This has been used for the ultra sensitive deteciton of E. coli in both a microfluidic format as well as a dipstick assay. We are approaching field testing for the dipstick assay as this has shown great promise.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nugen, S.R. Engineered bacteriophages for rapid bacteria separation and determination in agricultural samples Sensors in Food & Agriculture 2015 Cambridge, UK
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nugen, S.R. Engineering Bacteriophages for Rapid Food and Environmental Bacteria Sensing International Workshop on Biosensors and Bioanalytical Microtechniques for Environmental, Food and Clinical Analyses (BBMEC) 2015 Regensburg, Germany
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nugen, S.R. A Bacteriophage-based Nanosensor for Food & Agriculture International Association for Food Protection Annual Meeting 2015 Portland, OR
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Nugen, S.R. Bacterial Separation and Portable Detection: Preanalytical Consideration and Technology Sample Prep Technologies: Pre-Analytical Processing in Molecular Diagnostics and Biodetection. 2015 Bethesda, MD
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chen, J., Nugen, S.R. Development of Phage-Conjugated Magnetic Probes for Bacterial Separation The American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Wang, Z., Nugen, S.R. Development of a Novel Biomagnetic Separation Method for Rapid Detection of Escherichia coli by phage display technique The American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Wang, D., Nugen, S.R. Rapid Detection of Salmonella Using a Redox Cycling-Based Electrochemical Method American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Hinkley, T., Nugen, S.R. Engineering Bacteriophage for the Ultrasensitive Detection of Foodborne Pathogens American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Koo, C.K.W., Senecal, A., Senecal, K., and Nugen, S.R. Water soluble nanofibers for dehydration and storage of bacteriophage for decontamination of agricultural water The Institute of Food Technologists Annual Meeting and Food Expo, 2015, Chicago, IL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Koo, C.K.W., Nugen, S.R. Inexpensive, portable paper-fluidic devices for use in food safety and agricultural applications The 249th American Chemical Society Annual Meeting, 2015, Denver, CO
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chen, J., Nugen, S.R. Detection of Escherichia coli in Drinking Water Using T7 Bacteriophage-Conjugated Magnetic Probes American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Koo, C., Nugen, S.R. Electrospun Water-Soluble Nanofibers for Dehydration and Storage of Bacteriophage for Decontamination of Agricultural Water American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chen, J., Nugen, S.R. UV-Nanoimprint Lithography as a Tool to Develop Flexible Microfluidics for Electrochemical Detection American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Alcaine, S.D., Nugen, S.R. Engineering Bacteriophages to Develop Electrochemical Biosensors for Bacterial Pathogens American Chemical Society (ACS) 2015 Fall Meeting, Boston, MA


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: We have the goal of reaching several target audiences this year. This includes undergraduate students, graduate students and visiting scientists. Undergraduates were trained by graduate studets which enabled increased technical learnings for the undergraduate as well and scientific instruction for the graduate student (with a future in academics). These technical learnings included chemistry, molecular genetics, microbiology and engineering as it pertains to food safety. Both the graduate student and undergraduate students also gained valuable experiences in technical writing in the form of manuscript preparation and laboratory notebook maintanance. The students have advanced technologies in the use of bacteriophages for the rapid detection of pathogens. These learnings have been presented to the food industry in scientific conferences and have been well-received. Changes/Problems: We have included the use of bacteriophages in the project to allow for 1) target recognition of E. coli, 2) signal amplification and 3) separation. The phages will allow a much lower limit of detection than previously developed. What opportunities for training and professional development has the project provided? One graduate student, an undergraduate student and a visiting scientist have been involved in this project. They have increased their ability for technical writing, research methods, collaboration and oral presentations. The graduate student has even been mentored through a grant submission process which resulted in a federal award. How have the results been disseminated to communities of interest? We have presented our results through a manuscript submission, and several oral conference presentations including Institute of Food Technologists (IFT), Pittcon and the American Chemical Society (ACS). What do you plan to do during the next reporting period to accomplish the goals? By the next reporting period we expect to have demonstrated the working biosensor on food samples. To do this we will have engineered a bacteriophage to allow targeted infection and signal amplification for pathogen detection.

Impacts
What was accomplished under these goals? In order to improve the limit of detection, we are investigating the use of bacteriophages for signal amplification. We are developing a phage amplification-based scheme combined with a later flow assay for the detection of generic E. coli. The use of bacteriophage as a detection element has several advantages due to their potential broad host range, short amplification time, and ability to differentiate between viable and non-viable cells. The simplicity, portability, and reliability of paper fluidic devices make them an ideal platform for testing in resource limited settings like those found on-farm. In this study we (i) developed a later flow assay for the detection of T7 bacteriophage; (ii) investigated the impact of low E. coli concentrations and initial T7 inoculum on phage amplification; and ( iii) the subsequent impact on our schemes ability to detect E. coli. Our data indicates that the phage amplification-based lateral flow assay can detect 100 CFU/mL of E. coli following an 8 hour incubation, and 1000 CFU/mL following a 5 hour incubation. The results of this study suggest that, with some modification, a phage amplification-based lateral flow assay for bacterial detection in a format amenable testing in resource-limited setting is feasible, and potentially expandable to other bacterial targets and sample matrices.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2015 Citation: Alcaine, S.A., Law, K., Ho, S., Kinchla, A.J., Nugen, S.R. A Phage Amplification-Based Paper Fluidic Device for the Detection of generic E. coli 2014 (submitted).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Alcaine, S.D., Chen, J., Nugen, S.R. Low Cost Phage-Based Assays for Agricultural Testing Annual Meeting of the Institute of Food Technologists 2014 New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Alcaine, S.D., He, F., Nugen, S.R. An On-Farm Device for the Detection of Generic E. coli from Agricultural Water Sources Pittcon 2014, Chicago, IL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: He, F., Alcaine, S.D., Nugen, S.R. Disposable electrochemical microchip for on-farm detection of E. coli from agricultural water 247th National Meeting of the American Chemical Society 2014 Dallas, TX


Progress 10/01/12 to 09/30/13

Outputs
Target Audience: We have reached out to target audiences in the form of scientific presentations at conferences. Additionally, Professor Nugen and Professor Kinchla have published an article in a trade journal describing the need for affordable on-farm testing devices. Changes/Problems: The main problem in the current approach is the ability to acheive a low enough limit of detection in a rapid format. We are currently investigating phage amplification in order to provide a signal amplification to our testing format. What opportunities for training and professional development has the project provided? Professional development came in the form of graduate student training. Graduate students were able to gain scientific skills as well as presentation and writing skills. How have the results been disseminated to communities of interest? Dissemination to the scientific community have been accomplished through juouirnal publications and scientific presentations at conferences. What do you plan to do during the next reporting period to accomplish the goals? During the next reporting period, we will work torard multiplex detection with an extremely low limit of detection. We will use wash water as a sample matrix in an effort to enable farmers to conduct rapid testing of agricultural water as will soon be mandated by FSMA.

Impacts
What was accomplished under these goals? The rapid, specific and sensitive detection of nucleic acids is of utmost importance for the identification of infectious agents, diagnosis and treatment of genetic diseases, and the detection of pathogens related to human health and safety. Here we report the development of a simple and sensitive nucleic acid sequence-based and Ru(bpy)(3)(2+)-doped silica nanoparticle-labeled lateral flow assay which achieves low limit of detection by using fluorescencent nanoparticles. The detection of the synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, was utilized to demonstrate this assay. The 30 nm spherical Ru(bpy)(3)(2+)-doped silica nanoparticles were prepared in aqueous medium by a novel method recently reported. The nanoparticles were modified by 3-glycidoxypropyl trimeth oxysilane in order to conjugate to amine-capped oligonucleotide reporter probes. The fluorescent intensities of the fluorescent assays were quantified on a mictrotiter plate reader using a custom holder. The experimental results showed that the lateral flow fluorescent assay developed was more sensitive compared with the traditional colloidal gold test strips. The limit of detection for the fluorescent lateral flow assay developed is approximately 0.066 fmols as compared to approximately 15 fmols for the colloidal gold. The limit of detection can further be reduced about one order of magnitude when "dipstick" format was used. Although the potential role of microfluidics in point of care diagnostics is widely acknowledged, the practical limitations to their use still limit deployment. Here, we developed a capillary flow microfluidic with on-chip reagent delivery which combines a lateral flow assay with microfluidic technology. The horseradish peroxidase tagged antibody was electrospun in a water-soluble polyvinylpyrrolidone nanofibers and stored in a microfluidic poly(methyl methacrylate) chip. During the assay, the sample containing Escherichia coli on immunomagnetic beads came in contact with the nanofibers causing them to dissolve and release the reagents for binding. Following hybridization, the solution moved by capillary flow toward a detection zone where the analyte was quantified using chemiluminescence. The limit of detection was found to be approximately 10(6) CFU/mL of E. coli O157. More importantly, the ability to store sensitive reagents within a microfluidic as nanofibers was demonstrated. The fibers showed almost instant hydration and dissemination within the sample solution.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Alcaine, S., He, F. & Nugen, S.R. 2013, "Design of a low-cost and user friendly nanosensor for on-farm pathogen detection", Abstracts of Papers of the American Chemical Society, vol. 245.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: He, F., Jin, S. & Nugen, S.R. 2012, "Low cost, portable lab-on-a-chip device for detection of food adulterants", Abstracts of Papers of the American Chemical Society, vol. 244.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Kinchla, A. & Nugen, S.R. 2013, "Challenges and Innovations for On-Farm Bacterial Testing", Food Safety Magazine, [Online], vol. Aug/Sept


Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: We have been fabricating a device which can be used for on-farm detection of pathogens from fresh produce. Over the past year, we have fabricated prototypes of these devices and we are about to run our validation tests. We have also investigated using phage amplification for the detection of the pathogens. Professor Nugen and four graduate students attended a workshop to learn new methods of developing diagnostic tools for low-resource settings such as farms. Professor Nugen attended an additional workshop in Seattle, WA on this topic. The results of experiments so far have been presented in the following annual meetings and conferences: He, F., Merian, T., Goddard, J.M. and Nugen, S.R. 2012 "Development of an Electrowetting Valve in Capillary-Driven Microfluidic Biosensor for Nucleic Acid Detection " Clinical and Translational Research Retreat, Shrewsbury, MA Nugen, S.R. 2012 "Nanotechnology for Improved Pathogen Isolation and Detection in Foods" International Association of Food Protection Annual Meeting, Providence, RI. Nugen, S.R. 2012 "Food Biosensors for Low Resource Settings" Point of Care Diagnostics for Global Health, Seattle, WA Wang, Y. and Nugen, S.R. 2012 "Development of Lateral Flow Fluorescence Assay for the Detection of Trypanosoma" F. Clinical and Translational Research Retreat, Shrewsbury, MA Dai, M. and Nugen, S.R. 2012 "Water-soluble electrospun reagent containing nanofibers for on-chip storage" Gordon Research Conference on Analytical Sensors, Newport, MA Gilbert, J.T., Goddard, J.M. and Nugen, S.R. 2012 "Design of a bicinchoninic acid sensor to determine milk protein concentration" ACS Spring Meeting, San Diego, CA He, F., Jin, S. and Nugen, S.R. 2012 "Low cost, portable lab-on-a-chip device for detection of food adulterants" ACS Fall Meeting, Philadelphia, PA He, F. and Nugen, S.R. 2012 "Development of a capillary-driven microfluidic biosensor for foodborne pathogen detection" ACS Fall Meeting, Philadelphia, PA Wang, Y., Koo, C., and Nugen, S.R. 2012 "Fluorescent lateral flow assays for food safety" ACS Fall Meeting, Philadelphia, PA PARTICIPANTS: The Investigators associated with the project are: Nugen, S. R. - Investigated the microfluidic device development. McLandsborough, L. - Investigated the microbiology efforts. Goddard, J. - Investigated antimicrobiol surfaces. Kinchla, A. - collected produce rinse water samples for testing. Students who have worked on the project include: F. He - Graduate Student who has developed the microfluidic device. Y. Chang - Post doc who performed microbiology experiments. Y. Wang - Post doc who developed fluorescent nanoparticles. TARGET AUDIENCES: The ultimate audience for the project will the produce and livestock farmers. At the completion of the project, we will have developed new methods to diagnose pathogens on-farm. Dissemination of the efforts will take place later in the project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We have developed new tools to detect pathogens in a low-resource setting. We have developed microfluidic valves which can be incorporated into a user-friendly disposible device. The valves allow more complex reactions to be performed without the need for additional user input. These electrowetting valves were also realized onto nitrocellulose paper to fabricate more sophisticated lateral flow assays. This will now allow an ultra low-cost sensor which can perform complex reactions with multiple reagents. We have developed a method to store reagents in a microfluidic device using water soluble nanofibers. The fibers dissolve rapidly once the aqueous sample comes in contact. Enzymes and antibodies stored in the fibers demonstrated good stability over time. Fluorescent nanoparticles doped with a fluorescent dye have been synthesized using a new method developed in the Nugen Lab. This method is a significant improvement over the current method to make such particles. No organic solvents are used in the synthesis and the particles can be made in much higher concentrations than before. These particles can then be produced at low-cost and therefore they are applicable towards routine tests of agricultural products.

Publications

  • Chang, Y., Gu, W. Fischer N. and McLandsborough L. 2012. "Identification of genes involved in Listeria monocytogenes biofilm formation by mariner-based transposon mutagenesis" Appl. Microbiol. Biotechnol. 93:2051-2062
  • Barish, JA and Goddard, JM. 2011. "Polyethylene Glycol Grafted Polyethylene: A Versatile Platform for Non-Migratory Active Packaging Applications" Journal of Food Science. 76 (9): E586-E591.
  • Talbert, JN and Goddard, JM. 2012. "Enzymes on Material Surfaces". Colloids and Surfaces B: Biointerfaces. 93:8-19
  • Mancuso, M., Goddard, JM, Erickson, D. 2012. "Nanoporous Polymer Ring Resonators for Biosensing" Optics Express. 20 (1): 245-255
  • Merian, T. and Goddard, JM. 2012. "Advances in non-fouling materials: perspectives for the food industry" Journal of Agricultural and Food Chemistry 60 (12) 2943-2957
  • Tian, F, Decker, E.A. and Goddard, JM. 2012. "Development of an iron chelating polyethylene film for active packaging applications". Journal of Agricultural and Food Chemistry. 60: 2046-2052
  • Goddard, JM. 2011. "Improving the Sanitation of Food Processing Surfaces". Food Technology. October 2011 Issue, p. 40-46.
  • Barish, JA and Goddard, JM. 2011. "Polyethylene Glycol Grafted Polyethylene: A Versatile Platform for Non-Migratory Active Packaging Applications." Journal of Food Science. 76 (9): E586-E591
  • Talbert, JN and Goddard, JM. 2012. "Enzymes on Material Surfaces". Colloids and Surfaces B: Biointerfaces. 93:8-19
  • Mancuso, M., Goddard, JM, Erickson, D. 2012. "Nanoporous Polymer Ring Resonators for Biosensing." Optics Express. 20 (1): 245-255.
  • Dai, M., Jin, S. and Nugen, S.R. 2012. "Water-Soluble Electrospun Nanofibers as a Method for On-Chip Reagent Storage" Biosensors 2(4), 388-395.
  • Wang, Y., Fill, C. and Nugen S.R. 2012 "Development of Chemiluminescent Lateral Flow Assay for the Detection of Nucleic Acids" Biosensors 2(1), 32-42.
  • Jin, S., Dai, M., He, F., Wang, Y., Nugen, S.R. 2012 "Development and characterization of a capillary-flow microfluidic device for nucleic acid detection" Microsystem Technologies 18(6) 731-737.
  • Merian, T., He, F., Yan H., Chu D., Talbert, J.N., Goddard, J.M., and Nugen, S.R. 2012 "Development and surface characterization of an electrowetting valve for capillary-driven microfluidics" Colloids and Surfaces A: Physicochemical and Engineering Aspects 414, 251-258.
  • Wang, Y.H., Fan, W. and Nugen, S.R. 2012 "A novel method for the preparation of Ru(bpy)32+-doped silica nanoparticles" Materials Letters 92(1) 17-20
  • Wang, Y.H., Fan, W. and Nugen, S.R. 2012 "Low-cost fluorescent nanoparticles for lateral flow detection of nucleic acids" Accepted Biomedical Microdevices
  • Jin, S., Dai, M., He, F., Wang, Y., Nugen, S.R. 2012 "Development of a capillary flow microfluidic E. coli biosensor with on-chip reagent delivery using water-soluble nanofibers" Submitted Microsystem Technologies
  • Chang, Y., McLandsborough L. 2012 "Low Concentration of Ethylenediaminetetraacetic Acid (EDTA) Affects Biofilm Formation of Listeria monocytogenes by Inhibiting its Initial Adherence" Food Microbiology 29:10-17


Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: The results of research by the Nugen Research Group have been disseminated to the public using both scientific literature as well as conferences. This year, the group submitted five publications and presented their research five times. Professor Nugen has also met with various food producers to discuss his research results. This allowed a direct demonstration of technology to industry. The conference presentations are as follows: 1. Fill, C., Wang, Y., Johnson, K. and Nugen, S.R. "Development of a novel lateral-flow test to detect yeast in yogurt" Institute of Food Technologists Annual Meeting, 2011, New Orleans, LA 2. He, F. and Nugen, S.R. "Development of a capillary-driven microfluidic nucleic acid biosensor" NanoTech Conference & Expo, 2011, Boston, MA 3. He, F. and Nugen, S.R. "A surface-driven microfluidic biosensor for resource-limited setteings" Gordon Research Conference on microfluidics, 2011, Waterville, NH 4. Wang, Y. and Nugen, S.R. "Development of nucleic acid sequence-based and horseradish peroxidase-labeled lateral flow assay for the detection of Trypanosoma". 2nd Annual Clinical and Translational Science Research Retreat, 2011, Shrewsbury, MA 5. Jin, S., Dai, M., He, F., Wang, Y. and Nugen, S.R. "Development of Pumpless Capillary Microfluidic Biosensor" Institute of Food Technologists Annual Meeting, 2011, New Orleans, LA PARTICIPANTS: Sam R. Nugen - Primary Investigator. Professor Nugen directed the projects and met weekly with his staff for updates and advising. Yuhong Wang - Postdoc. Dr. Wang designed the chemiluminescent lateral flow assay. Catherine Fill - Graduate Student. Catherine was involved in the design and execution of the projects. Shenquan Jin and Fei He are graduate students who designed and implemented microfluidic experiments. Julie M. Goddard - Collaborator. Professor Goddard was involved in electrowetting valve project. Dr. Goddard's postdoc performed the surface analysis of the silver electrode. TARGET AUDIENCES: The target audiences for these projects include food safety individuals and general diagnostics practitioners. These groups were addressed in the presentations as technical presentations during 2011. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The Nugen Research Group has developed rapid diagnostic methods to detect contamination in foods. The microfluidic biosensors was driven exclusively by capillary flow and did not require the use for external pumping which resulted in a low-cost and portable detection platform. The design of low-voltage electrowetting microfluidic valves is a capillary-driven sensor will also aid to rapid and portable diagnostics. In this project, the Nugen Research Group was able to design a valve which uses no moving parts to control fluid flow in a microfluidic device. The valve is low cost and will allow more complex reactions on a microfluidic device where timed reagent delivery is imperative. A lateral flow assay utilizing chemiluminescent signal transduction has also been developed. This assay allowed the detection of target nucleic acids an order of magnitude lower than traditional assays using gold nanoparticles. The technologies listed above will allow increased field testing of fruits and vegetables without the need for complicated and time consuming diagnostics. Increased testing on farm along with sufficient prevention strategies are vital to limiting food borne illness associated with fresh produce.

Publications

  • No publications reported this period