DEVELOPMENT AND VALIDATION OF NOVEL NANOPARTICLES FOR DETECTION OF CAMPYLOBACTER

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1000776
• Grant No.: 2013-38821-21381
• Cumulative Award Amt.: $299,999.00
• Proposal No.: 2013-03631
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2017
• Grant Year: 2013
• Program Code: [EQ]- Research Project

Recipient Organization
TENNESSEE STATE UNIVERSITY
3500 JOHN A. MERRITT BLVD
NASHVILLE,TN 37209

Performing Department
Family and Consumer Sciences

Non Technical Summary
Campylobacter is one of the most common causes of foodborne illness. Billions in human illness costs could be saved each year in the United States by reducing Campylobacter in foods. The proposed project will apply nanotechnology in the development of an innovative method for Campylobacter isolation and detection. Experiments will be conducted to optimize aerobic enrichment conditions and biosensor protocols to improve the efficiency and time required to achieve a minimum detectable level. The antibody functionalized magnetic gold nanoparticles will be designed using monoclonal antibodies specific to Campylobacter surface antigens. Studies will be performed to validate the developed biosensor. A series of seminars and workshops on the applications of biosensor and nanotechnology will be presented to faculty and graduate students. This project is expected to build a capacity in applied nanotechnology in food safety research. Results from this project will enable PDs to attract support funding from interested industrial partners and to continue research activities to commercialize the developed technology.

Animal Health Component

50%

Research Effort Categories

Basic

0%

Applied

50%

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	3260	1100	100%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3260 - Poultry meat;

Field Of Science
1100 - Bacteriology;

Keywords

biosensor

food safety

food science

foodborne pathogens

poultry

Goals / Objectives
This project will apply nanotechnology in the development of an innovative method for Campylobacter isolation and detection to facilitate implementation of preventive measurements and intervention strategies. Objective1: To design a process to incorporate functionalized magnetic gold nanoparticles into the aerobic enrichment regime for rapid concentration of Campylobacter Objective 2: To develop a sensitive real-time Surface Plasmon Resonance (SPR) biosensor based on the functionalized magnetic gold nanoparticles (mAu-NP) in conjunction with the enrichment protocol Objective 3: To establish multidisciplinary partnerships to enhance research capacity in food safety and applied nanotechnology at TSU

Project Methods
Optimization of Aerobic Enrichment Conditions: Various levels of pure cultures of Campylobacter jejuni and Campylobacter coli will be inoculated onto retail chicken samples and recovery will be quantified. Each sample will be enriched in Bolton's broth with antimicrobial supplements and lysed horse blood. The samples will be incubated without the addition of microaerobic gas mix. All samples will be incubated at 42°C. Rates of recovery and growth after 6, 12, 24 and 48 h of enrichment will be determined by plate count method. Minimum time required to achieve a preset detectable level (100 CFU/ml) will be determined experimentally. Synthesis of Gold-Coated Magnetic Nanoparticles: The magnetic core-shell Fe3O4-Au nanoparticles will be prepared. Aqueous solution containing FeCl3, FeCl2, and HCl will be added drop wise into NaOH solution under vigorous stirring. Black Fe3O4 nanoparticles formed will be separated by magnet and washed by deionized water. The Fe3O4 nanoparticles will be collected by centrifugation, and resuspended in deionized water. Then HAuCl4 will be gradually reduced by NH2OH in Fe3O4 suspension to form a thin gold shell on the magnetite nanoparticle surface. The Fe3O4 gold-coated nanoparticles will be isolated by centrifugation and dried in a vacuum oven.16-MHA will be self-assembled on surface of nanoparticles by sonication of Fe3O4 gold-coated nanoparticles in ethanol solution of 16-MHA. The carboxylated nanoparticles will be washed with ethanol and stored at 4 °C for further use. Development of Functionalized mAu-NP: The surfaces of the nanoparticles will be modified with 11-MUA to form self-assembled monolayer. For the activation of self-assembled monolayer, nanoparticles will be treated with the solution of EDC/NHS in MES buffer. At the end of the activation period, nanoparticles will be separated magnetically and washed with MES buffer. Nanoparticles will then be incubated in avidin solution. Nanoparticles will be rinsed with MES buffer to remove unbound avidin. The avidin-coated nanoparticles will be mixed with biotin labeled Campylobacter specific monoclonal antibody. The antibody-coated particles will be removed magnetically from the solution and washed twice with MES buffer. The parameters of antibody immobilization on mAu-NP will be experimentally determined. Incorporation with Enrichment Procedures: The antibody-coated nanoparticles will be mixed with the enriched cultures and incubated at room temperature for 30 min on a vortex mixer. After the incubation, the complex will be collected magnetically and washed with PBST. After washing steps, the capture efficiency of functionalized mAu-NP will be determined by plating the Campylobacter bound mAu-NP onto modified charcoal cefoperazone deoxycholate agar. The rest of the Campylobacter bound mAu-NP will be used for SPR biosensor analysis. SPR Optimization: Changes of refractive index (RI) of mAu-NP will be determined by using a SensiQ SPR device after capturing of Campylobacter from enrichment media. By monitoring the response of the photodiode array, the sensor is able to monitor the SPR dip as it changes in response to refractive index. Baseline of the SPR system will be established by continuous flow of PBS buffer. The SPR responses of the Campylobacter bound mAu-NP will be obtained with injection of sample solutions. Intra-Laboratory Studies: The selectivity and sensitivity of the developed SPR biosensor will be determined using the inoculated chicken samples. In addition to Campylobacter, coliform group bacteria will be used to determine the selectivity of the SPR biosensor. The RI changes obtained from coliform group bacteria will be compared with the RI changes obtained from Campylobacter to establish a baseline for the background bacteria. The sensitivity of the SPR biosensor will be determined by serial dilutions of the enriched cultures and the results will be compared with that obtained from plate counting method. A regression analysis will be performed on the results from the SPR biosensor and the plate counts. The detection limit of the SPR biosensor will also be determined. Inter-Laboratory Studies: Three laboratories will evaluate the SPR biosensor protocol using the same sets of retail chicken samples. Retail chicken packages will be purchased from local stores. From each package, 100 g of meat will be cut aseptically and mixed thoroughly and divided into four 25 g portions of subsamples in individual, sterile bags. Three subsamples will be separately tested in each laboratory using the same protocols for enrichment and SPR biosensor. The results from the three laboratories will be compared. The fourth subsample will be tested by PCR and PFGE after enrichment to confirm the presence and subtype of Campylobacter. Presumptive isolates will be stored at -80°C for further analysis. PCR Identification of Campylobacter Isolates: Isolates will be identified with a PCR assay. Campylobacter jejuni ATCC 700819 and Campylobacter coli ATCC 43473 will be used as control strains to set up the PCR conditions. Amplified products will be detected by standard gel electrophoresis. DNA bands in the gels will be stained with ethidium bromide and the image of DNA profiles will be captured. Typing of Campylobacter Isolates with PFGE: Campylobacter isolates will be typed using pulsed-filed gel electrophoresis (PFGE). Briefly, DNA plugs will be digested with SmaI and the digested DNA fragments will be electrophoresed using CHEF-DR III Variable Angle Pulsed Field Gel Electrophoresis Systems. Gels will be stained and visualized and the images will be loaded into BioNumerics for analysis. Pairwise-comparisons will be done with the Dice correlation coefficient, and cluster analyses will be performed. Student Training in Food Safety Research: This project will provide research opportunities to students enrolled in the graduate programs in Agricultural Sciences and Agricultural Biotechnology at TSU. Two graduate students will be recruited and supervised by PD and Co-PDs to work on various aspects of the project. Students will be trained on isolation of Campylobacter from food and environment samples, identification of Campylobacter by molecular methods, and biosensor detection of Campylobacter. Students will gain knowledge and experience in molecular techniques, data analysis, and scientific communication. Students will be required to present their research results at professional conferences. Seminars on Nanotechnology in Agricultural Research: To stimulate applications of nanotechnology in agricultural research, PD and Co-PDs will present a series of special seminars on the applications of nanotechnology in food science, animal science and plant science. It is expected at least one special seminar will be presented at TSU University-Wide Research Symposium every year. This Symposium is the largest research forum for students and faculty around the campus. It is expected more than 200 students and faculty will attend the seminar. A total of six seminars will be conducted during the three-year project period. Workshops on Biosensor and Molecular Detection Methods: PD and Co-PDs will conduct a hand-on workshop for faculty and students at TSU on biosensor and molecular detection methods for Campylobacter during the second and third year of this project. Two workshops will be conducted one on SPR biosensing and another one on the molecular detection methods of Campylobacter.

Progress 09/01/13 to 08/31/17

Outputs
Target Audience:The developed biosensor technology has been presented to food safety professional, food industry and regulatory food testing laboratories at the International Association for Food Protection Annual Meeting. This project has trained two graduate students the knowledge and skills in biosensor and nanotechnology research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students were trained in using SPR biosensor for the detection of Campylobacter. Both students presented their research findings at the Annual University-Wide Research Symposium at Tennessee State University, April 19, 2017 and IFT Vol Section 2017 Spring Meeting, April 28, 2017, Nashville, TN with the support from this project. How have the results been disseminated to communities of interest?The SPR method developed from this project has been presented to the food safety professionals interested in advanced detection technology at International Association for Food Protection Annual Meeting, Tampa, FL, July 9-12, 2017. A seminar on new development in biosensor for detection of Campylobacter was presented to graduate students and faculties in Agricultural Biotechnology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Experiments were conducted to validate the developed biosensor protocol for detection of Campylobacter in inoculated retail chicken samples. Samples of raw chicken products (total 120 samples; 60 samples of whole chicken and 60 samples of chicken parts) were artificially contaminated with low level of mixed strains of Campylobacter between 10-100 cfu/g. Samples were tested in batches of ten samples and prepared fresh every day. Each sample was divided into six subsamples. From each subsample, a portion was used to verify the number of Campylobacter by culture methods and another portionwas used for the extraction procedures for SPR analysis. Samples were independently tested by using the cultural methods and SPR biosensor protocol. All testing samples were analyzed blindly. Samples then were independently analyzed and the results were compared with culture methods. The developed biosensor protocol showed comparable sensitive and specificity to the cultural methods. This project has allowed the development of rapid and reliable detection technologies, which will provide effective alternatives to the current cultural methods for the detection of Campylobacter in poultry products. The researchers have communicated with food industry interested in the advanced detection technologies to further optimize and commercialize the developed technologies.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chen, F.C., and Bridgman, R.C. Magnetic Nanoparticles-Enhanced Biosensor for the Detection of Campylobacter in Raw Poultry Products. International Association for Food Protection Annual Meeting, Tampa, FL, July 9-12, 2017.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The biosensor and related detection technologies is intended to be used by regulatory agencies, meat and poultry processors to facilitate the detection of Campylobacter in the processing facilities and food products. This project is also to train students at TSU the knowledge and skills in biosensor and nanotechnology research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained two graduate students in SPR biosensor for detection of Campylobacter, isolation of Campylobacter from food samples, and identification of Campylobacter by molecular methods. How have the results been disseminated to communities of interest?The findings from this project have been communicated with the food safety professionals interested in advanced detection technology at the Institute of Food Technologists Annual Meeting, Chicago, IL, July 16-19, 2016. The findings from this project have been published in a book chapter. What do you plan to do during the next reporting period to accomplish the goals?Further studies will be conducted to validate the developed biosensor protocol for detection of Campylobacter in inoculated and retail chicken samples.

Impacts
What was accomplished under these goals? Experiments were conducted to improve the SPR sensor protocol and to increase the sensitivity. A highly sensitive SPR sensor combining antibody- functionalized magnetic gold nanoparticles was developed for rapid detection of Campylobacter. The sensor was based on a sandwich format. The magnetic gold nanoparticles used in this protocol were functioned not only as probes to selectively bind and separate Campylobacter, but also as amplification regent to enhance SPR signal. Campylobacter can be detected at the concentration as low as 100 cfu/mL and the signal showed a linear range between 1.0 × 102 and 8.5 × 107 cfu/mL. The use of magnetic gold nanoparticles in the SPR protocol provided three orders of magnitude in the improvement of sensitivity toward Campylobacter compared to the regular SPR sensor with direct detection format. The specificity of the SPR sensor was examined with two non-target bacteria, Escherichia coli and Enterobacter cloacae. No significant signal was detected in the presence of these bacteria. The recovery of Campylobacter from raw chicken samples was 98.5%. The developed SPR sensor has potential to provide a simple, low-cost and sensitive method for detection of Campylobacter in poultry products.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Chen, F.C., S. Godwin, and M. Wakefield. 2016. Persistence of Campylobacter in Chicken Meat Juice on the Contaminated Kitchen Surfaces. Institute of Food Technologists Annual Meeting, Chicago, IL, July 16-19, 2016.
• Type: Theses/Dissertations Status: Submitted Year Published: 2016 Citation: Richard Yorke. 2016. Antibiotic Sensitivity of Campylobacter jejeni and Effect of Allicin on Succinate Dehydrogenase Activity. Tennessee State University, June 10, 2016.
• Type: Book Chapters Status: Published Year Published: 2016 Citation: Chen, F.C., Zhou, S., Nahashon, S., and Bridgman, R.C. 2016. Detection of Salmonella and Campylobacter in chicken rinse water using a surface plasmon resonance sensor. In Microbes in the spotlight: recent progress in the understanding of beneficial and harmful microorganisms (pp.186-190). A. Mendez-Vilas, Ed, BrownWalker Press, Barcelona, Spain.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:This project is intended to develop a laboratory method for rapid identification of dangerous foodborne pathogens in the processing facilities and final products. The PD has communicated the new development of the SPR sensor with food safety professionals and testing laboratories. Students and faculty participated in the project have learned knowledge and skills in biosensor and nanotechnology research. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained two graduate students in SPR biosensor for detection of Campylobacter, isolation of Campylobacter from food samples, and identification of Campylobacter by molecular methods. How have the results been disseminated to communities of interest?The results from this project have been communicated with the food safety professionals interested in advanced detection technology at the International Association for Food Protection Annual Meeting in Portland, OR, July 25-28, 2015. What do you plan to do during the next reporting period to accomplish the goals?Experiments will be conducted to improve the SPR sensor protocol and to increase the sensitivity. Further studies will be conducted to validate the developed biosensor protocol for detection of Campylobacter in inoculated and retail chicken samples.

Impacts
What was accomplished under these goals? Experiments were conducted to develop an extraction protocol to prepare the sample for the sensor analysis and to increase the sensitivity of SPR responses. The protocol as described below was tested. The Campylobacter captured by the functionalized magnetic nanoparticles from the enrichment media were extracted by glycine-hydrochloride solution and the cell debris was removed by centrifugation. The buffer conditions of the extracted flagellar antigens were exchanged to running buffer by gel filtration. The elution from the gel filtration was injected into the SPR sensor. Various concentrations of Campylobacter were tested. The sensor specifically detected Campylobacter and a response of 25 µRIU was recorded when Campylobacter at the concentration of 103 CFU/ml was tested. The sensor responses were positively related to the concentrations Campylobacter. A linear relationship between the µRIU and log CFU was observed within the range between 102 and 105 CFU/ml. The baseline noise was 2 µRIU peak-to-peak, therefore the lowest concentrations that could be detected by the sensor was estimated at 65 CFU/ml. The sensor was operated continuously with various samples and the regenerations were required between samples by injecting 0.5 ml of 20 mM hydrochloric acid followed by 0.5 ml of running buffer. The same sensor was regenerated for more than 50 times and the average of responses in the middle log-linear range (103 CFU/ml) remained above 90% of the initial level. The repeatability of sensor responses was demonstrated. The percent coefficient of variation (% CV) was 4.8% within the 50 runs. The SPR sensor provided reproducible quantitative results and was able to detect Campylobacter at concentrations of 102 CFU/ml from the enriched cultures and all procedures could be performed in less than 1 hr. The developed sensor analysis, which requires minimal instrument investment and less labour intensity than other molecular methods, has practical applications in poultry processing facility and industrial laboratories. In addition, a study was conducted to evaluation of poultry packages from grocery stores in Nashville, Tennessee. Packages of raw poultry were purchased from thirty-five grocery stores in metropolitan area of Nashville, Tennessee. Three Packages were purchased from each store including whole chicken, chicken breast, and ground turkey. Information on the brands and packaging types was recorded and package conditions were inspected to note any leaking of meat juices. Microbiological contaminations on the packages were evaluated. Of the 105 packages, 24 had aerobic plate count (APC) higher than 106 CFU on the packages and 18 had coliform count (CF) and 8 had E. coli count (EC) higher than 103 CFU on the packages. Campylobacter was found on 7 and pathogenic E. coli was found on 6 of the packages while no Salmonella was found. This study found Campylobacter and pathogenic E. coli are more often found on poultry packages than Salmonella and leaking packages clearly increase the risk of cross contamination.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Chen, F-C., Godwin, S., Green, A., Chowdhury, S., and Stone, R. 2015. Microbiological evaluation of poultry product packages from grocery stores in Nashville, Tennessee. Journal of Food Protection Supplement A. 78:181.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The method to be developed is intended to be used by regulatory agencies, meat and poultry processors to facilitate identification of dangerous foodborne pathogens in the processing facilities and final products. Students and faculty at TSU will learn knowledge and skills in biosensor and nanotechnology research. Interested industrial partners will acquire the opportunity of the new technology developments. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two graduate students were trained in using cultural and molecular methods for detection and identification of Campylobacter in foods. How have the results been disseminated to communities of interest? A seminar on new development in biosensor for detection of Campylobacter was presented to graduate students in Agricultural Biotechnology. What do you plan to do during the next reporting period to accomplish the goals? SPR biosensor will be optimized to detect the Campylobacter captured by the functionalized magnetic nanoparticles from the enrichment media. Intra- and inter-laboratory studies will be conducted to validate the developed biosensor protocol for detection of Campylobacter in inoculated and retail chicken samples. Students will be trained on isolation of Campylobacter from food and environment samples, identification of Campylobacter by molecular methods (PCR and PFGE), and biosensor detection of Campylobacter. One special seminar will be presented at TSU University-Wide Research Symposium.

Impacts
What was accomplished under these goals? The antibody functionalized magnetic nanoparticles were produced using monoclonal antibodies specific to Campylobacter surface antigens. The process to incorporate the functionalized nanoparticles into the enrichment procedures for fast separation of bacteria cells from the enriched media was investigated. Experiments were conducted to optimize aerobic enrichment conditions to improve the efficiency and time required to achieve a minimum detectable level. Two enrichment protocols were studied; with and without immunomagnetic separation before enrichment. Minimum time required to achieve a preset detectable level (100 CFU/ml) were determined experimentally. Inclusion of immunomagnetic separation before enrichment was able to reduce the time to reach the preset detectable level by 6 hours.

Publications