Source: AUBURN UNIVERSITY submitted to
DEVELOPMENT OF A MULTIPLEX BIOSENSOR SYSTEM WITH NANOBIOMOLECULAR PROBES FOR RAPID DETECTION OF COMMON FOODBORNE BACTERIAL PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1008986
Grant No.
2016-67021-25005
Project No.
ALAW-2015-07778
Proposal No.
2015-07778
Multistate No.
(N/A)
Program Code
A1511
Project Start Date
Mar 1, 2016
Project End Date
Feb 29, 2020
Grant Year
2016
Project Director
Suh, S.
Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Biological Sciences
Non Technical Summary
As demonstrated by frequent announcements of foodborne pathogen outbreaks and recalls of contaminated foods, we need better pathogen detection systems for identifying contaminated foods before they can be distributed for consumption by the unsuspecting public. The current systems are inadequate to be used by nonprofessionals for real-time pathogen detection because they require long time (hours to days and weeks), expensive instruments, and extensive trainining of the personnel. The detection system we propose to develop in this study requires only a few minutes, inexpensive, and require very little training to be adapted and used by anyone involved in food industry. In this study, we propose to develop a system for simultaneous detection of several most common foodborne bacterial pathogens. However, as we improve our system in future studies, it can be expanded to include viruses and toxins to become a comprehensive detection system for any foodborne pathogens to improve public safety.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7125010202050%
7124010110050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria; 5010 - Food;

Field Of Science
2020 - Engineering; 1100 - Bacteriology;
Goals / Objectives
In order to prevent frequent outbreaks of foodborne pathogens, accurate pathogen monitoring throughout the food supply chain is essential. In this study, we will develop an accurate, cost effective, user-friendly, and, most importantly, rapid pathogen detection system for simultaneous identification of multiple foodborne bacterial pathogens. The proposed magnetoelastic particle (MEP) biosensors are composed of nanobiomolecular recognition elements (phage-displayed oligopeptide probes) and magnetostrictive particle platforms. We have previously demonstrated the efficacy of our nanobiomolecular elements coated MEP biosensors for accurate and rapid detection of individual pathogens. In this study, we will develop a multiplex system capable of detecting the presence of several common foodborne bacterial pathogens including Salmonella enterica, Escherichia coli O157:H7, Clostridium perfringens, and Campylobacter jejuni in less than 20 minutes, including the sample preparation. We will achieve our goal via three Specific Aims:1. Construct singleplex MEP biosensors for rapid detection of common foodborne bacterial pathogens.2. Further improve MEP biosensors for multiplexing3. Demonstrate the efficacy of the multiplex MEP biosensors on various food items including meats, vegetables, fruits, and liquids.Success of the proposed research will yield a prototype multiplex biosensor that is highly accurate and can be used by anyone involved in food industry from farmers to food processors to consumers.
Project Methods
1. Isolation of phage-displayed oligopeptide probes. Pathogen specific phage-displayed oligopeptide probes will be isolated by a modified biopanning strategy that employs extensive pre- and post- clearing against similar organisms. To isolate probes that are specific against the target pathogens, we will follow the procedure we developed to isolate S. Typhimurium probes. The phage library will be initially pre-panned against plastic and BSA to eliminate nonspecifically binding oligopeptides. The remaining phages constitute the starting library. The library will be then extensively pre-cleared with a mixture of closely related bacteria to minimize isolating probes with cross-reactivity. Following pre-clearing, we will use a mixture of several different strains of each pathogen to isolate probes that recognize the particular species rather than a specific strain. In order to enhance isolation of probes with higher affinity, we will progressively change the ratio between the bacterial cells and phages following the second round of biopanning to limit the number of available target for the probes. We will also enhance the stringency by progressively increasing Tween 20 concentration from 0.1% to 0.5% to minimize nonspecific binding. After the fifth round of biopanning, we will sequence the isolated phage probes to determine common motifs among the peptide probes. These efforts have resulted in isolation of S. Typhimurium probes that bind the target cells with high specificity and affinity. We used a similar strategy to isolate probes that recognize multiple serovars of pathogenic S. enterica without binding to E. coli or Shigella species. The selectivity, affinity, and potential cross reactivity of each probe will be assessed via newly developed liquid-phase ELISA, competitive ELISA, and as a biosensor when immobilized on MEP. If any probe set demonstrates cross reactivity to a closely related organism, post-clearing step will be conducted to deplete the cross-reacting peptides.2. Evaluation of phage-displayed probes. The top probes for each target will be selected for further evaluation and development. Each identified set of probes will be tested for pathogen detection on the MEP biosensor. Each phage probe will be affinity tagged at GpIX and attached to MEPs. The MEP biosensors will be added to samples containing various concentrations of the pathogen of interest and the ability of our sensors to capture the target will be determined for each probe. To facilitate the analysis, each target pathogen will be manipulated to express one of three fluorescent proteins (GFP, YFP, or RFP). Both monoculture and mixed culture experiments will be performed with fluorescently tagged target bacteria. Following incubation of the MEP biosensors with fluorescent bacteria, we will isolate the sensors and determine selectivity via fluorescent microscopy as well as fluorometry readings. The effectiveness of our sensors will be also be determined via culturing and determining the efficiency of plating (EOP).3. Construction of a multiplex biosensor. In order to simultaneously detect multiple infectious agents, each probe set for a target organism will be attached to a specific and different MEP of designed resonant frequency. MEPs ranging from 1 mm to 1000 mm in length may be used, depending upon target agent size and mass. The corresponding characteristic resonant frequency range is from 2 GHz to 2 MHz. For a MEP 50 mm long x 2 mm wide x 1 mm thick (which has a characteristic resonant frequency of about 44 MHz), the attachment of one bacterial cell (mass of 1 bacterium = 2 picogram) would result in a resonant frequency shift of 56 KHz, or 0.125 %. For a MEP of 12 mm x 2 mm x 1 mm, the change in resonant frequency would be 1 MHz, or 0.5%, due to attachment of one bacterium. However, we expect much greater frequency changes because more than one cell will be bound to the biosensor. Our limit of detection on a 500 mm long sensor is 50 bacterial cells. The dimensions of the MEPs will be adjusted based on the properties of the target agents (principally mass and size) and the associated frequency change when the MEP biosensor is bound to the target. The principles used to define MEP width and thickness are: Rule 1) the resonant frequency shift due to the attachment of one target agent shall be more than 0.01%; Rule 2) the frequency shift due to the attachment of one layer of target agent (that is, 100% surface coverage of target species) is smaller than 30% of the resonant frequency.4. Automation of MSP measurement. We have recently completed construction of a prototype portable MSP measurement device. In addition, software to automatically measure the resonant frequency shift is being developed. This software automatically measures and compares the frequency shifts of control and measurement sensors and calculates whether there is a significant statistical difference between the frequency shifts. If there is a statistically significant difference in the frequency shifts, then the program indicates that the sample is contaminated with the pathogen of interest.5. Validation of multiplex biosensor in mixed cultures. Each MEP biosensor will be assessed initially for capturing of the cognate pathogen in a monoculture. Then each MEP biosensor will be tested in a mixed culture to determine the specificity using fluorescently labeled bacteria. Thus, we believe the multiplex biosensor composed of different MEP biosensors that had already been tested and characterized will behave as expected. To verify, if necessary, each set of MEP biosensors bound to pathogens will be isolated and the identity of the bacteria bound to each biosensor will be determined via 16S rDNA sequence analysis to assess the selectivity. To achieve this, bacteria will be stripped from the biosensor, 16S rDNA amplified using bacterial universal primers and sequenced. Each probe:MEP combination will continue to be optimized to improve sensitivity and to eliminate cross-reactivity.A) Detection of pathogens in liquids. We have already demonstrated the efficacy of our MEP biosensors in various liquids including milk and apple juice for capturing pathogens. Thus, we do not expect great difficulty of transitioning from singleplex to multiplex biosensor in vegetable and fruit washes, or in liquid foods. Experiments will be conducted with various water matrices including distilled water, tap water, vegetable and fruit rinse water, and meat rinse water. Additionally, liquid foods including milk and fruit juices will also be investigated. The liquids will be spiked with one or more flurorescently labeled pathogens varying in concentration (100 - 105 pathogens). Initially, only the liquid spiked with a single pathogen will be used to evaluate the system. As the project progresses, multiple pathogens will be spiked into the liquid to demonstrate simultaneous detection of multiple pathogens.B) Detection of pathogens on solid foods. We have already successfully demonstrated detection of individual pathogens on the surface of solid foods including egg shells and fresh produce. To validate the MEP biosensor, the captured pathogens on food contact surfaces will be evaluated as a function of pathogen type, MEP size, contact time, humidity, and surface roughness. The pathogens evaluated will be the same as for the liquid system. Pathogen capture will be tested singly (one pathogen type) and with multiple pathogens simultaneously seeded upon the surface. The contact time of the particle with the seeded surface will be varied up to 30 mins to study its effect. The effects of humidity (room humidity - 95%RH) and surface roughness (Ra of 0.2 µm - 1 µm) of standard stainless steel food processing surfaces will also be investigated.

Progress 03/01/16 to 02/29/20

Outputs
Target Audience:Food safety scientists, engineers, and stake holders Changes/Problems:This is the final report for my accomplishments at Auburn University which Ileft on June 15, 2019 to startmy new position at Texas A&M University College of Dentistry. Therefore, I am transferring the remains of my award to TAMUCOD. What opportunities for training and professional development has the project provided?Training of graduate students to conduct scientific research and presentation of their results. How have the results been disseminated to communities of interest?Presentations at scientific meetings and publication. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Construct singleplex MEP biosensors for detection ofClostridium perfringens(vegetative cells and spores),Escherichia coliO157:H7,Salmonella entericaserovar Enteritidis,S.Typhimurium, multiple pathogenic serovars ofS. enterica, andCampylobacterjejuni. Construction of ofSalmonella entericaserovar Typhimuriumbiosensor We isolated phage-displayed oligopeptide probes that are highly selective forS.Typhimurium. As shown in Figure 1 and 2, these probes are highlyeffective for detection ofS.Typhimurium. We compared the effectiveness of the TA1 probe against E2 landscape phage probe that has been used often in our biosensor system. TA1 was more sensitive and effective than the E2 phage. Isolation ofSalmonella entericaprobes We developed"parallel biopanning" and isolated probes for detection ofS. enterica.Using 14 commonly isolated pathogenic serovars ofS. enterica,we isolated two phage-displayed oligopeptide probes that apprear to be effective for detection of multiple serovars ofS. enterica. Isolation ofE. coliO157:H7 probes We finally succeeded in enriching for oligopeptide pool that binds to the target bacterium to 27% of the total phage population. Isolation of probes forCampylobacterspecies We took two approaches to isolate probes for Campylobacterspecies: biopanning approach and directed approach. The first approach yielded a phage pool of which 20% of the phages bind toCampylobacterspecies. The secondapproach was conducted as a collaboration with a protein engineer, Dr. Robert Pantazes, to design specificC. jejunibinding proteins. Over a six month period, we synthesized probes against CmeC and MOMP, respectively and displayed them M13 bacteriophage.Unfortunately we discovered that proteins were designedagainst common hydrophobic motifs of membrane proteins. Therefore, all of our probes bound to all gram-negative bacteria tested. Isolation ofListeria monocytogenesprobes We isolatedL. monocytogenesspecific oligopeptides that demonstrate good binding toL. monocytogenesthat have been adapted to both 4°C and 37°C (Figure 7). ***Although we took precautions, the move from Auburn University to Texas A&M University had a devasting effect. The most serious effect was the loss of several boxes with phage-displayed oligopeptide probes.We moved all of our temperature sensitive items several weeks before the official move and placed them in a departmental walk-in cooler.Unfortunately, the walk-in cooler malfunctioned andall of the boxes were distributed to multiple laboratories with refrigerator space.Ultimately, we were unable to recover all of our boxes including several boxes that contained the phage pools forCampylobacterspecies, putativeE. coliO157:H7 phages, andListeria monocytogenesprobes.Our immediate goal is to characterize these probes to determine their efficacy compared to the lost probes and to start reisolation to replace the lost probes forE. coliO157:H7,L. monocytogenes, andCampylobacterspecies. 2)Further improve the MEP biosensors. Each nanobiomolecular probe will be paired with a specific size of MEP to form singleplex biosensors with unique signals that can be easily detected. In addition, we propose to automate the detection system. Stability of magnetoelastic biosensors in wash water containing Clorox and chlorine dioxide We determined the efficacy of our ME biosensors in produce wash water. Our ME biosensorswere functional with no significant loss of activity in water that had been supplemented with Clorox (100 ppm free chlorine content) or chlorine dioxide (4 ppm) demonstrating the efficacy of our biosensors in a realistic setting. Capture efficiency as a function ofSalmonellaexposure time The capture efficiency was measured.Theexposure time ranged from 1 to 180 minutes as shown in the figure below.The capture efficiency was found to increase with increasing exposure time. Effects of washing on capture efficiency Three different washing methods, pipette washing, magnetic bar washing and dilution washing, were investigated. The dilution washing was found to show a roughly 15% higher capture efficiency than the other two methods. Phage immobilization i. Genetic approach for oriented immobilization We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The complete process of affinity tagging a phage probe takes less than five days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance. ii. Physical approach for immobilization In addition to the traditionally used physical adsorption method, a covalent bonding method was investigated to improve the robustness of phage immobilization. The phage was first chemically modified to have a sulfhydryl group on its outer surface. The thiolated phage then forms a covalent gold-sulfur bond on a gold-coated ME biosensor platform. We found that the covalent phage immobilization yielded a roughly 65% higher capture efficiency). Based on our data, we chose to use genetic approach of phage immobilization. Effect of temperature on our biosensor function Our biosensorswere tested over a wide range of temperatures: 19 to 50°C. The ability of our biosensors remainednearly constant at temperatures tested. Pathogen capture in flowing solutions The ability to detectpathogenin flowing solutions was also investigated.Spiked solutions containing known concentrations ofS.Typhimuriumwere passed over the sensors and the ability of the sensors to bind the bacteria measured as a function of fluid velocity.Figure 12 shows that velocities below 6 mm/sec have little effect on the pathogen capture %. Development of a phage filter system We developed a phage-filter system which can be used to capture pathogens using the magnetoelastic biosensors coated with phage-displayed oligopeptide probes in a flow-through system. This filter system is capable of testing a large volume of water. i. Initial Phage filter testing system setup We tested the initial filter systemconsisting of supporting frames with the solenoid coilsin a large square pipe. We discovered that formation ofME filter in a pipe with large diameter reduced pathogen capture. We also tested the effect of multiple layers and found thatthe capture rate increased with an increase in the number of phage filter layers. We discovered that usingsmall diameter pipe significantly increased pathogen capture to 94% capture rate. 3)Demonstrate the efficacy of the multiplex MEP biosensors on various food items. Each singleplex biosensors will be sequentially combined to make a multiplex system. With each successful expansion of multiplexing (from duplex to triplex, to tetraplex, etc), we expect to generate a multiplex system capable of simultaneously detecting various foodborne bacterial pathogens including two commonS. entericaserovars, various other pathogenicS. entericaserovars,C. jejuni,E. coliO157:H7, andC. perfringens(both vegetative and spore forms). Finally, our multiplex MEP biosensor system will be tested on various food items including meats, vegetables, fruits, and liquids.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Shin Horikawa, Songtao Du, Yuzhe Liu, Howard Clyde Wikle, James M. Barbaree, and Bryan A. Chin, Thermal stability of phage peptide probes Vs. aptamer for Salmonella detection on magnetoelastic biosensors platform, ECS Transactions 75 (2016) 165-173.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shin Horikawa, Songtao Du, Yuzhe Liu, I-Hsuan Chen, Yating Chai, Howard Clyde Wikle and Bryan A. Chin, The Bathtub Method for Detecting Small Quantities of Specific Pathogens, ECS Transactions, 75(2016) 183-192.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mi-Kyung Park and Bryan A Chin, Novel approach of phage-based magnetoelastic biosensor for the detection of Salmonella Typhimurium in soil, Journal of Microbiology and Biotechnology, 26 (2016) 2051-2059.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, Yating Chai, Howard C. Wikle, James M. Barbaree and Bryan A. Chin, Direct Detection of Bacteria on Fresh Produce, The 9th International Conference on Biomedical Electronics and Devices  BIODEVICES (February 2016)
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: I-Hsuan Chen, Shin Horikawa, Kayla Bryant, Rebecca Riggs, Bryan A. Chin and James M. Barbaree, Bacterial assessment of phage magnetoelastic sensors for Salmonella enterica Typhimurium detection in chicken meat, Food Control, 71 (2017) 273-278.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jianping He, Donald Sirois, Suiqiong Li, MariAnne Sullivan, Clyde Wikle and Bryan A. Chin, Ballistic impact welding of copper to low carbon steel, Journal of Materials Processing Technology, 232 (2016) 165-174.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, I-Hsuan Chen, Songtao Du, Yuzhe Liu, Howard C. Wikle, Sang-Jin Suh, James M. Barbaree and Bryan A. Chin, Method for detection of a few pathogenic bacteria and determination of live versus dead cells, SPIE Proceeding Vol. 9864, 98630H, Sensing for Agriculture and Food Quality and Safety VIII, (May 2016).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Jiajia Hu, Fengen Wang, Shin Horikawa, James M. Barbaree and Bryan A. Chin, Alternative soaking media for the FDA procedure in the detection of salmonella from tomatoes and spinach leaf using phage magnetoelastic biosensors, SPIE Proceeding Vol. 9864, 986412, Sensing for Agriculture and Food Quality and Safety VIII, (May 2016).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bryan A. Chin, Shin Horikawa, Yuzhe Liu, Songtao Du, I-Hsuan Chen, Michael S. Crumpler, Steve R. Best, Howard C. Wikle and Zhongyang Cheng, Rapid detection of small quantities of specific bacteria using phage-based wireless biosensors, 10th International Conference on Sensing Technology (November 2016).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Jiajia Hu, Fengen Wang, Shin Horikawa, James M. Barbaree and Bryan A. Chin, Alternative soaking media for the FDA procedure in the detection of Salmonella from tomatoes and spinach leaf using phage magnetoelastic biosensors, 9864-38, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Songtao Du, Shin Horikawa, Yuzhe Liu, M. Crumpler, Yating Chai, Jiajia Hu, Fengen Wang and Bryan A. Chin, Rapid detection of pathogens by a 3D biomolecular filter and automated biosensor measurement system for liquid, 9864-16, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, I-Hsuan Chen, Songtao Du, Yuzhe Liu, Howard C. Wikle, Sang-Jin Suh, James M. Barbaree and Bryan A. Chin, Method for detection of a few pathogenic bacteria and determination of live versus dead cells, 9864-17, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Horikawa, Y. Liu, S. Du, I. H. Chen, H. C. Wikle and B. A. Chin, Rapid detection of live versus dead bacteria, 1982, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I. H. Chen, S. Horikawa, S. Du, Y. Liu, H. C. Wikle, J. M. Barbaree, and B. A. Chin, Thermal Stability of Phage Peptide Probes Vs. Aptamer for Salmonella Detection on Magnetoelastic Biosensors Platform, PRiME 2016 Honolulu, Hawaii.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Horikawa, S., Chen, I.-H., Du, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Highly sensitive surface-scanning detector for the direct bacterial detection using magnetoelastic (ME) biosensors. Proceedings for SPIE 10217, Sensing for Agriculture and Food Quality and Safety IX, 1021703.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Du, S., Horikawa, S., Chen, I.-H., Chai, Y., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Application of 2-dimensional coil detector for the sensitive direct Salmonella detection on plastic board. ECS Transactions 80 (10): 1799-1807.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Y. Liu, S. Du, S. Horikawa, H. C. Wikle, J. Hu, F. Wang and B. A. Chin, Double-layer planar coil detector for improved detection of salmonella on food contact surfaces, 1989, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Du, S. Horikawa, Y. Liu, Y. Chai, J. Hu and B. A. Chin, Rapid Detection of Pathogens By a 3D Biomolecular Filter and Automated Biosensor Measurement System for Liquid, 1977, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Horikawa, S. Du, Y. Liu, I. H. Chen, Y. Chai, H. C. Wikle and B. A. Chin, The Bathtub Method for Detecting Small Quantities of Specific Pathogens, PRiME 2016 Honolulu, Hawaii.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Xi, J., Horikawa, S., Chen, I.-H., Du, S., Liu, Y., Lu, X., Suh, S.-J., Huang, T.-S., and Chin, B. A. 2017. Enhancement in the capture efficiency of magnetoelastic biosensors for Salmonella using a dilution method. ECS Transactions 80 (10): 1549-1555.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chen, I.-H., Du, S., Liu, Y., Xi, J., Lu, X., Horikawa, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Detection of Salmonella enterica with magnetoelastic biosensors in wash water containing Clorox and chlorine dioxide. ECS Transactions 80 (10): 1557-1564.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: I.-H. Chen, J. Xi, Y. Liu, S. Du, S. Horikawa, T.-S. Huang, B. A. Chin, and S.-J. Suh, Isolation of highly selective phage-displayed oligopeptide probes for detection of listeria monocytogenes in ready-to-eat food, presented at the SPIE Commercial+ Scientific Sensing and Imaging, 2018, vol. 10665, p. 106650N.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. Horikawa, Y. Liu, S. Du, I.-H. Chen, X. Lu, and B. A. Chin, Direct, surface-scanning detection of pathogenic bacteria using a wireless biosensor, presented at the SPIE Commercial+ Scientific Sensing and Imaging, 2018, vol. 10665, p. 106650C.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. Horikawa, I.-H. Chen, X. Lu, S. Du, Y. Liu, T.-S. Huang, Z. Cheng, and B. A. Chin, Direct Detection of Bacterial Pathogens on Fresh Fruits and Vegetables, presented at the 2018 IEEE SENSORS, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. Du, I.-H. Chen, Y. Liu, J. Xi, X. Lu, S. Horikawa, T.-S. Huang, S.-J. Suh, B.A. Chin, Comparison of Pathogens Capture by Different Bio-Receptors Immobilized Biomolecular Filter in a Large Volume of Liquid, ECS Meeting Abstracts, 2445-2445
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Xu Lu, I-Hsuan Chen, Yuzhe Liu, Shin Horikawa, Tung-Shi Huang, Bryan A. Chin. Biomolecular Phage Filter for the Detection of a Small Number of Pathogens in Large Volumes of Processing Water. Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2019): 108-113
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Xu Lu, Yuzhe Liu, I-Hsuan Chen, Shin Horikawa, Tung-Shi Huang, Bryan A. Chin. Biomolecular Phage Filter for the Detection of a Small Number of Pathogens in Processing Water by Two Different Models. SPIE conference poster (2019)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Yuzhe Liu, I-Hsuan Chen, Alana Maclachlan, Tung-Shi Huang, Bryan A. Chin. Effect of the Number of ME Filter Elements on Capture Rate of Phage Filter for a Large Volume of Liquid with a Small Number of Pathogens. ECS Atlanta conference poster (2019)


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:Scientists and researchers in food safety Changes/Problems:The major event that affected the progress in 2019 was my change of universities from Auburn University to Texas A&M University College of Dentistry. I left Auburn University on June 15, 2019 and started my new position at TAMU COD on July 1, 2019. Unfortunately, TAMU COD was slow in helping me get started. I did not get my office and the lab space until middle of September. My funding from TAMU did not get activated until Sept. 1, 2019. In addition, TAMU did not approve my IBC until February,2020. Therefore, my lab has been shut down effectively for almost 8 months. To further complicate the matter, my USDA funds still have not been transferred to my new university and I have no access to the funds yet. These unforeseen and unexpected problems have signicantly affected the progress of the study in 2019. What opportunities for training and professional development has the project provided?Training of graduate students. How have the results been disseminated to communities of interest?Through scientific publications and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals?Continue isolation of molecular probes and test the multiplex system.

Impacts
What was accomplished under these goals? Goal 1. 1.Characterization ofE. coliO157:H7 probes In 2018, we believed resolved the issues related to isolatingE. coliO157:H7 specific probes. Our problems were mainly due to poor enrichment of the target bacterium specific probes which resulted in overtaking of the population by the wildtype phages which, in turn, masked the desired probes. Using modified biopanning procedure to resolve this issue, we have successfully enriched for oligopeptide pool with 27% of the phages binding toE. coliO157:H7. As reported last year, we identified 25 phage clones from this pool that bind to the target at least 6-fold over non-targetE. coli. This year, we determined the sequence of top 17 candidates that boundE. coliO157:H7. Predicted amino acid sequence analysis of these 17 phage-displayed oligopeptides failed to demonstrate any common motif or sequence conservation (Table 1). This is not surprising since these peptides are only twelve-amino acids in length. All 17 potential probes bound the target bacterium at least 10-fold better than the M13KE parent phage in ELISA assays. Table 1. Predicted amino acid sequences of 17 topE. coliO157:H7 binding probes 2. Isolation ofCampylobacterprobes Last year, we reported our collaborative effort with Dr. Robert Pantazes to take a predictable approach of protein engineering to design phage-displayed protein probes forCampylobacterspecies.Dr. Pantazes designed potential binding proteins for CmeC and MOMP ofC. jenjuni.Over a six month period, we synthesized 1 probe and 3 probes against CmeC and MOMP, respectively and displayed them M13 bacteriophage.We were highly excited to test our probes and were severely disappointed when we discovered that the binding proteins were unfortunately designed against common hydrophobic motifs of membrane proteins. Therefore, all four of our probes bound to all gram-negative bacteria tested includingCampylobacter, E. coli,Salmonella enterica,andShigellaspecies. We have since abandoned this approach and have decided to stay with the unpredicted approach of isolating potential probes from a phage-display library. As shown in Table 2, we have made some progress in 2019. Using a mixture of 5Campylobacterspecies (C. jejuni,C. coli,C. lari,C. fetus, andC. mucosalis), we were able to enrich for phages that bind to these bacteria to almost 20% of the population.However, we are perplexed as to why it took us ten rounds of biopanning to achieve this enrichment. As can be expected, these many rounds of biopanning has resulted in a pool that also contains an overwhelming numbers of the wildtype M13 phages.We are currently in the process of developing a way to separate the phages of interest from wildtype phages. Table 2. Isolation ofCampylobacterspecific probes. We had limited progress of Goal 1 in 2019 because of my move to Texas A&M University College of Dentistry.Due to the move, my lab has been inactive from June, 2019 to February, 2020.My lab has finally been approved by the TAMU IBC and we are ready to resume our studies. Goal 2. My colleague, Bryan Chin (Co-PI), has developed a phage-filter system which can be used to capture pathogens using the magnetoelastic biosensors coated with phage-displayed oligopeptide probes in a flow-through system. This filter system is capable of testing a large volume of water. 1: Initial Phage filter testing system setup This past year, we were able to test the initial filter system,consisting of supporting frames with the solenoid coils,in a large square pipe.ME filter elements are aligned on the supporting frames layer by layer by the magnetic field produced from the solenoid coils. As the heat is produced by the solenoid coils, the solenoid coils are put outside of the pipe. The temperature variation was tested in three different location in testing system by thermocouple. The result showed that the average temperature was from 22.2?to 23.4?. The temperature variation was only less than 1.5?, so that there is no effect on thephage probes and the target bacteria. TheSalmonellaTyphimurium capturing test was explored after the temperature variation test. The ME filter elements with the size of 10mm were attached on the supporting frames to captureSalmonellafrom 200ml testing solution. The single layer, double layers, and triple layers were tested. The results showed that the capture rate increased with an increase of the number of phage filter layers. However, the highest capture rate was only 1.3%. Therefore, other methods were explored. 2: Testing the possibility of foodborne pathogen captured and filtered in small volume. Rocking experiment is used as the traditional method to capture and detect pathogens by Magnetoelastic (ME) biosensor. ME elements immobilized with phage probe were incubated in PCR tube with 200ul ofS.Typhimurium103cfu/ml for 30mins to determine the capture rate. The result showed that the capture rate increased with increase in the number of ME elements present. We were able to achieve approximately 98% capture rate. 3.The prototype of phage filter Based on the rocking experiments, a new frameless phage filter prototype was explored.The prototype of phage filter consisted of a long square glass pipe with a small diameter, phage probe immobilized-ME filter elements with the size of 1mm, and a pair of magnetic plates. The probe immobilized-ME filter elements were aligned in the square pipe by the magnetic field. Because of the high surface tension of the small diameter of pipe, a pump was used to introduce the bacteria from the top inlet to the bottom outlet. Thebacterialsolution was passed through the prototype phage filter once at a controlled flow rate. The experimental result verified the rocking experiment that the capture rate increases with an increase of the number of ME filter elements. We achieved approximately 94% capture rate.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tong, Z., Silo-Suh, L. A., Kalalah, A., Dawson, P., Chin, B. A., and Suh, S.-J. 2020. Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms. Applied Microbiology and Biotechnology 104 (3): 1201-1209. doi:10.1007/s00253-019-10338-8
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Xu Lu, I-Hsuan Chen, Yuzhe Liu, Shin Horikawa, Tung-Shi Huang, Bryan A. Chin. Biomolecular Phage Filter for the Detection of a Small Number of Pathogens in Large Volumes of Processing Water. Proceedings of the 12th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2019): 108-113
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Xu Lu, Yuzhe Liu, I-Hsuan Chen, Shin Horikawa, Tung-Shi Huang, Bryan A. Chin. Biomolecular Phage Filter for the Detection of a Small Number of Pathogens in Processing Water by Two Different Models. SPIE conference poster (2019)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Songtao Du, Yuzhe Liu, I-Hsuan Chen, Alana Maclachlan, Tung-Shi Huang, Bryan A. Chin. Effect of the Number of ME Filter Elements on Capture Rate of Phage Filter for a Large Volume of Liquid with a Small Number of Pathogens. ECS Atlanta conference poster (2019)


Progress 03/01/18 to 02/28/19

Outputs
Target Audience:Food safety scientists, engineers, and stake holders Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This past year, my relatively inexperienced graduate students had a chance to learn more about working in lab and learning how to combat foodborne pathogens. They have started to make good progress. How have the results been disseminated to communities of interest?Scientific publications and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals?We are very close to being able to attack our Goal 3 of assembling multiplex pathogen detection system. We would like to accomplish that in the upcoming year.

Impacts
What was accomplished under these goals? Goal 1. 1. Isolation ofE. coliO157:H7 probes As explained in the previous report, we've experienced some difficulty isolating phage-displayed oligopeptide probes for detection ofE. coliO157:H7.However, we have resolved this issue and have enriched for oligopeptide pool that binds to the target bacterium to 27% of the total phage population. We have performed ELISA and identified 25 phage clones that bind the target bacterium at least 6-fold over non-targetE. coli. The best binding clones bind to the target bacterium over 10-fold. We are currently in the process of determining the sequence of these oligopeptides. Therefore, we are in the last stages of finally isolating and characterizingE. coliO157:H7 probes.The initial ELISA data demonstrating potential of these putative phage probes are shown in Figures 1 and 2. 2. Isolation ofCampylobacterprobes We took two separate approaches to isolate phage-displayed oligopeptide probes for realtime detection ofCampylobacterspecies.In the first approach, we used the same method of using the 12-amino acid oligopeptide displayed on M13 that we have been using to isolate probes against other pathogens.After seven rounds of biopanning against a mixture of severalCampylobacterspecies, includingC. jejuni, C. lari,andC. coli, we have enriched our phage pool to over 20% of the total phage population that recognize these bacteria. We are in the process of performing ELISA to identify the best probes. Once we identify the generalCampylobacterprobes, we plan to further purify those that specifically recognizeC. jejuni, the leading cause of campylobacteriosis. In the second approach, we targetedC. jejunispecifically by synthesizing proteins that recognize either the CmeC protein or MOMP protein of the bacterium.We are pursuing this in collaboration with Dr. Robert Pantazes of Auburn University, Department of Chemical Engineering who specializes in protein engineering. He has designed several potential synthetic protein sequences of approximately 100 amino acids based on fibronectin binding protein 3 scaffold that may recognize CmeC. We synthesized the "genes" for the four proteins (1 against CmeC and 3 against MOMP) via SOE PCR and cloned them into a phagemid. We recently succeeded in producing phage particles displaying these synthetic proteins on their capsids. We plan to perform ELISA in near future to assess the binding potential of these synthetic proteins forC. jejuni. Fig 1: Comparison of ELISA data for PBS, phages (O157:H7 phage clone and M13KE) Fig 2: Comparison of ELISA data for PBS, phages (O157:H7 phage clone and M13KE) Goal 2. The following work has been completed on magnetoelastic biosensors during the last year.Our biosensors, immobilized with E2 phage forSalmonella, were tested over a wide range of temperatures to determine if detection performance varied with test temperature.Detection tests were conducted with spikedSalmonellasolutions at temperatures from 19 to 50°C.As seen in Figure 3, the ability of our biosensors to bind the targetSalmonellabacteria remains nearly constant over range of temperatures examined.Hence our biosensors will perform properly over all anticipated temperatures that may be encountered in real life field sensing applications.Theeffect of disinfectants on our biosensors was also tested.Disinfectants that are commonly added to washing solutions including Cl-100ppm, ClO2-4ppm and peracetic acid-70ppm were tested on E2 (phage forSalmonella) immobilized phage sensors.For Cl and ClO2there was no measured effect on the performance of the biosensors in detectingSalmonella(Figure 4).Only in peracetic acid disinfectant solutions was a measurable decrease in binding ofSalmonellaencountered.The ability to detectSalmonellain flowing solutions was also investigated.Spiked solutions containing known concentrations ofSalmonellawere passed over the sensors and the ability of the sensors to bindSalmonellameasured as a function of fluid velocity.Figure 5 shows that velocities below 6 mm/sec have little effect onSalmonellacapture %.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: I.-H. Chen, J. Xi, Y. Liu, S. Du, S. Horikawa, T.-S. Huang, B. A. Chin, and S.-J. Suh, Isolation of highly selective phage-displayed oligopeptide probes for detection of listeria monocytogenes in ready-to-eat food, presented at the SPIE Commercial+ Scientific Sensing and Imaging, 2018, vol. 10665, p. 106650N.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. Horikawa, Y. Liu, S. Du, I.-H. Chen, X. Lu, and B. A. Chin, Direct, surface-scanning detection of pathogenic bacteria using a wireless biosensor, presented at the SPIE Commercial+ Scientific Sensing and Imaging, 2018, vol. 10665, p. 106650C.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: S. Horikawa, I.-H. Chen, X. Lu, S. Du, Y. Liu, T.-S. Huang, Z. Cheng, and B. A. Chin, Direct Detection of Bacterial Pathogens on Fresh Fruits and Vegetables, presented at the 2018 IEEE SENSORS, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: [1] S. Du, I.-H. Chen, Y. Liu, J. Xi, X. Lu, S. Horikawa, T.-S. Huang, S.-J. Suh, B.A. Chin, Comparison of Pathogens Capture by Different Bio-Receptors Immobilized Biomolecular Filter in a Large Volume of Liquid, ECS Meeting Abstracts, 2445-2445


Progress 03/01/17 to 02/28/18

Outputs
Target Audience:Food safety scientists and engineers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two new microbiology graduate students were trained to work with bacteriophages. How have the results been disseminated to communities of interest?Scientific publications and presentations at SPIE and ECS meetings. What do you plan to do during the next reporting period to accomplish the goals?Once again, the project fell behind schedule due to the departure of experienced graduate students from the Suh lab. Two new graduate students had to be trained from scratch and therefore the probe isolation has fallen behind.

Impacts
What was accomplished under these goals? Goal 1. 1. Isolation of E. coli O157:H7 probes We determined that the putative E. coli probes we had isolated in the previous year all exhibited too much cross-reactivity with Shigella species and Salmonella enterica serovars. Thus, we reinitiated isolation of improved E. coli O157:H7 probes. 2. Isolation of Listeria monocytogenes probes We isolated L. monocytogenes specific oligopeptides displayed on bacteriophages using modified biopanning procedures that included extensive negative depletion against other Listeria species. In order to account for morphological alteration of L. monocytogenes at 4°C versus 37°C, we used bacterial cells adapted to either temperature as the ligand in our biopanning. To date, we have identified several putative oligopeptide displayed phages that demonstrate good binding to L. monocytogenes that have been adapted to both 4°C and 37°C. The figure below shows two putative phage probes. 3. Detection of Salmonella enterica with magnetoelastic biosensors in wash water containing Clorox and chlorine dioxide In order to be useful, biosensors have to be functional in wash water. Therefore, to determine the efficacy of our ME biosensors in testing for food-borne pathogens in produce wash water, we assessed the stability of our biosensors in the presence of Clorox and chlorine dioxide. As demonstrated in the figure, our ME biosensors coated with Salmonella enterica serovar Typhimurium detecting phage probe were functional with no significant loss of activity in water that had been supplemented with Clorox (100 ppm free chlorine content) or chlorine dioxide (4 ppm) demonstrating the efficacy of our biosensors in a realistic setting. Goal 2. The biomolecular recognition probe immobilized on the ME biosensor is responsible for capturing a specific pathogen. E2 phage, for instance, has been affinity-selected and used to capture Salmonella Typhimurium. During this reporting year, the following major investigations were conducted to ensure and improve biosensor's detection capabilities: 1. Capture efficiency as a function of Salmonella exposure time The capture efficiency was measured by exposing E2 phage-coated ME biosensors to a suspension of Salmonella Typhimurium (330 ul at 5x104 cfu/ml) with various times, followed by washing, elution and plate counting processes. In this work, E2 phage was immobilized on the biosensors via physical adsorption. The Salmonella exposure time ranged from 1 to 180 minutes as shown in the figure below. As has expected, the capture efficiency was found to increase with increasing exposure time. The capture efficiency reached ~10% after 12 minutes of exposure and began to plateau after 24 minutes. 2. Effects of washing on capture efficiency Three different washing methods, pipette washing, magnetic bar washing and dilution washing, were investigated and compared to see their influences on capture efficiency. Washing plays an important role in removing unbound or loosely bound Salmonella cells from the biosensor. However, washing too vigorously is likely to cause a harmful effect. As shown in the figure, the dilution washing, which is the gentlest washing method, was found to show a roughly 15% higher capture efficiency than the other two methods. 3. Phage immobilization via covalent bonding In addition to the traditionally used physical adsorption method, a covalent bonding method was investigated to improve the robustness of phage immobilization. E2 phage was first chemically modified to have a sulfhydryl group on its outer surface. This thiolated E2 phage can then form a covalent gold-sulfur bond on a gold-coated ME biosensor platform. By comparing the capture efficiencies of biosensors prepared via the two methods, we found that the covalent phage immobilization yielded a roughly 65% higher capture efficiency (25% for the covalent method against 14% for the physical adsorption method after a 60-minute Salmonella exposure and magnetic bar washing). The result indicates that some portion of physically adsorbed E2 phage might have been loosely bound on the biosensor and detached during the Salmonella capture process. By robustly immobilizing E2 phage via covalent bonding, the detection capability of the ME biosensor has been improved.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Horikawa, S., Chen, I.-H., Du, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Highly sensitive surface-scanning detector for the direct bacterial detection using magnetoelastic (ME) biosensors. Proceedings for SPIE 10217, Sensing for Agriculture and Food Quality and Safety IX, 1021703.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Liu, Y., Du, S., Horikawa, S., Chen, I.-H., Chai, Y., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Application of 2-dimensional coil detector for the sensitive direct Salmonella detection on plastic board. ECS Transactions 80 (10): 1799-1807.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Xi, J., Horikawa, S., Chen, I.-H., Du, S., Liu, Y., Lu, X., Suh, S.-J., Huang, T.-S., and Chin, B. A. 2017. Enhancement in the capture efficiency of magnetoelastic biosensors for Salmonella using a dilution method. ECS Transactions 80 (10): 1549-1555.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Chen, I.-H., Du, S., Liu, Y., Xi, J., Lu, X., Horikawa, S., Wikle, H. C., Suh, S.-J., and Chin, B. A. 2017. Detection of Salmonella enterica with magnetoelastic biosensors in wash water containing Clorox and chlorine dioxide. ECS Transactions 80 (10): 1557-1564.


Progress 03/01/16 to 02/28/17

Outputs
Target Audience: Food safety scientists and engineers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?New microbiology graduate students were trained to work with bacteriophages. New materials engineering graduate student was trained to fabricate and test magnetoelastic biosensors. How have the results been disseminated to communities of interest?Scientific publications and presentations at meetings. What do you plan to do during the next reporting period to accomplish the goals?The project has fallen behind in probe isolation. The major cause of this is due to the departureof experienced graduate students from the Suh lab and training of inexperiencedf first year students. As students gain more experience, further progress is expected.

Impacts
What was accomplished under these goals? Goal 1. 1. Isolation ofE. coliO157:H7 probes: We started by depleting the phage population that binds to plastic and BSA. This became our starting phage pool. We followed this by extensive negative biopanning against 2 species ofShigellaand 14 serovars ofSalmonella entericato eliminate non-specific phages. Group Input (pfu/ml) Output (pfu/ml) 1 4 x 1011 1.5 x 1011 2 1.5 x 1011 1.4 x 1011 3 1.4 x 1011 1 x 1011 4 1 x 1011 8.1 x 1010 5 8.1 x 1010 6.8 x 1010 6 6.8 x 1010 5.4 x 1010 7 5.4 x 1010 4.1 x 1010 8 4.1 x 1010 3 x 1010 Table 1: Phage titer post negative depletion against closely related bacteria. We performed six rounds of biopanning against a mixture of 3 isolates ofE. coliO157:H7. Round Input (pfu/ml) Output (pfu/ml) Binding Percentage 1 1 x 1011 1.15x106 0.00015% 2 1 x 1011 8.3x106 0.0083% 3 1 x 1011 1.7x108 0.17% 4 1 x 1011 1.2x109 12% 5 1 x 1011 3.4x1010 34 % 6 1 x 1011 4.7x1010 47% Table 2. Enrichment ofE. coliO157:H7 specific phages Following six rounds of biopanning, individual phages were tested via ELISA. Several phages that demonstrated high degree of binding toE. coliO157:H7 were selected and retested for specificity. Unfortunately, we discovered that somehow ourE. coliO157:H7 pool was contaminated withS. entericaand so all of our putative phages probes also boundS. entericawith high specificity. Thus, we have to restart the phage probe isolation. Figure 1. ELISA of putativeE. coliprobes 2. Isolation ofL. monocytogenesphage probes. As stated forE. coliO157:H7, we precleared the starting phage pool against fiveotherListeriaspecies. The precleared phage library was then used to biopan forL. monocytogenesprobes. As target, we used a mixture of four isolates ofL. monocytogenes. In addition, becauseL. monocytogenesgoes through morphological and physiological change based on temperature, we used the bacterium at 4 C as well as 37 C as target organism. After four rounds of biopanning, we acquired putative pool ofL. monocytogenesspecific phages. Figure 2.Comparison of phage concentrations in three rounds of biopanning against L. monocytogenes Unfortunately, we discovered that our phage pool was contaminated with high levels of wildtype phage. We are currently in the process of eliminating the wt phages from our pool. Goal 2. Excellent progress to allow on-site measurement of Multi-plexed Magnetoelastic (ME) Biosensors for the detection of multiple pathogens has been completed. The accomplishments for this year include major advancements in: 1). Fabrication of Different Size Magnetoelastic Biosensors for Multi-plex Detection 2). Development of a Flat Coil Detection for In-situ Measurement 3). Detection of Salmonella pathogen using Flat Coil 4). Demonstration of the Robustness and Repeatability of ME Resonator Measurements 5). Commercialization of ME Biosensor Technology I. Fabrication of Different Size Magnetoelastic Biosensors A new method of manufacturing different size ME resonators was engineered under this project. Five different sizes of ME resonators were manufactured varying from (4mm to 0.5 mm in length). A simple, effective and inexpensive mechanical dicing method was used to fabricate the ME resonators as shown in Figure 1. From the success of the experiments it has been demonstrated that different lengths and widths of resonators with accuracy of ± 20 microns can be produced repeatedly. Figure 3: Photograph of ME resonators fabricated ranging in length from 4 mm to 0.5 mm. II. Development of a Flat Coil Detector for On-site Measurement A new coil that allows on-site measurement of the above as fabricated ME biosensors has been developed. This new flat coil is fabricated by the electrochemical deposition of copper on a flexible substrate such as Kapton film. Figure 5shows several different detector coils microelectronically fabricated on a single sheet of Kapton film. The Kapton film is then cut with ordinary scissors to obtain a single coil. The new coil is impendence matched to cover a specific range of sensor size. For instance 1 mm and .75mm sensors can be measured using the same detection coil, but 4 mm and 1 mm sensors would require different sizes of detection coils for measurement. The advantage of flat (2-D) detection coils over older (3D) detection coil designs is that the ME biosensors can be placed directly on the flat detector coil for measurement or the flat coil placed horizontally above the ME biosensors for measurement. Previously the biosensor had to be placed into a glass tube which was then inserted into the middle of the 3D detection coil for measurement. An additional advantage of the 2D flat coil is that multiple ME biosensors can be measured simultaneously. Figure 4: Six flat Detector Coils Microelectronically Fabricated on a single sheet of Kapton. To demonstrate detection using the flat coil detector a control and ME Salmonella biosensor were placed on the Salmonella spiked surface of a tomato. A flat coil detector was then used to immediately monitor both sensor the resonance frequencies (control and Salmonella biosensor) as a function of time. Figure 5: Detection of Salmonella on surface of Spiked Tomato Using ME Biosensors (Control and Measurement Biosensors) Figure 6: Measured Resonant Frequencies of Control and Measurement Biosensor. Notice Control Sensor Resonant peak remains constant while Measurement Biosensor resonant peak shifts (decreases with time) indicating the binding of Salmonella to the sensor surface. Demonstration of the Robustness and Repeatability of ME Resonator Measurements Using the flat coil demonstrated above, 500 measurements of a single ME resonator was made. Each measurement involved: the movement of the same ME resonator to the measurement position on the flat coil measurement of the resonant frequency removal of the ME resonator from the measurement position repeat step 1 The flat coil enables on-site measurement with excellent robustness, repeatability and reliability. Figure 7: The measurements and histogram statistical evaluation. The histogram shows a standard deviation of 96 Hz out of 2.2 MHz. This is a variation of ± 0.0042%.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: I. H. Chen, S. Horikawa, S. Du, Y. Liu, H. C. Wikle, J. M. Barbaree, and B. A. Chin, Thermal Stability of Phage Peptide Probes Vs. Aptamer for Salmonella Detection on Magnetoelastic Biosensors Platform, PRiME 2016 Honolulu, Hawaii.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: I-Hsuan Chen, Shin Horikawa, Kayla Bryant, Rebecca Riggs, Bryan A. Chin and James M. Barbaree, Bacterial assessment of phage magnetoelastic sensors for Salmonella enterica Typhimurium detection in chicken meat, Food Control, 71 (2017) 273-278.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shin Horikawa, Songtao Du, Yuzhe Liu, I-Hsuan Chen, Yating Chai, Howard Clyde Wikle and Bryan A. Chin, The Bathtub Method for Detecting Small Quantities of Specific Pathogens, ECS Transactions, 75(2016) 183-192.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mi-Kyung Park and Bryan A Chin, Novel approach of phage-based magnetoelastic biosensor for the detection of Salmonella Typhimurium in soil, Journal of Microbiology and Biotechnology, 26 (2016) 2051-2059.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, Yating Chai, Howard C. Wikle, James M. Barbaree and Bryan A. Chin, Direct Detection of Bacteria on Fresh Produce, The 9th International Conference on Biomedical Electronics and Devices  BIODEVICES (February 2016)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, I-Hsuan Chen, Songtao Du, Yuzhe Liu, Howard C. Wikle, Sang-Jin Suh, James M. Barbaree and Bryan A. Chin, Method for detection of a few pathogenic bacteria and determination of live versus dead cells, SPIE Proceeding Vol. 9864, 98630H, Sensing for Agriculture and Food Quality and Safety VIII, (May 2016).
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jianping He, Donald Sirois, Suiqiong Li, MariAnne Sullivan, Clyde Wikle and Bryan A. Chin, Ballistic impact welding of copper to low carbon steel, Journal of Materials Processing Technology, 232 (2016) 165-174.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Shin Horikawa, Songtao Du, Yuzhe Liu, Howard Clyde Wikle, James M. Barbaree, and Bryan A. Chin, Thermal stability of phage peptide probes Vs. aptamer for Salmonella detection on magnetoelastic biosensors platform, ECS Transactions 75 (2016) 165-173.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Jiajia Hu, Fengen Wang, Shin Horikawa, James M. Barbaree and Bryan A. Chin, Alternative soaking media for the FDA procedure in the detection of salmonella from tomatoes and spinach leaf using phage magnetoelastic biosensors, SPIE Proceeding Vol. 9864, 986412, Sensing for Agriculture and Food Quality and Safety VIII, (May 2016).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Bryan A. Chin, Shin Horikawa, Yuzhe Liu, Songtao Du, I-Hsuan Chen, Michael S. Crumpler, Steve R. Best, Howard C. Wikle and Zhongyang Cheng, Rapid detection of small quantities of specific bacteria using phage-based wireless biosensors, 10th International Conference on Sensing Technology (November 2016).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I-Hsuan Chen, Jiajia Hu, Fengen Wang, Shin Horikawa, James M. Barbaree and Bryan A. Chin, Alternative soaking media for the FDA procedure in the detection of Salmonella from tomatoes and spinach leaf using phage magnetoelastic biosensors, 9864-38, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Songtao Du, Shin Horikawa, Yuzhe Liu, M. Crumpler, Yating Chai, Jiajia Hu, Fengen Wang and Bryan A. Chin, Rapid detection of pathogens by a 3D biomolecular filter and automated biosensor measurement system for liquid, 9864-16, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shin Horikawa, I-Hsuan Chen, Songtao Du, Yuzhe Liu, Howard C. Wikle, Sang-Jin Suh, James M. Barbaree and Bryan A. Chin, Method for detection of a few pathogenic bacteria and determination of live versus dead cells, 9864-17, SPIE April 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Horikawa, Y. Liu, S. Du, I. H. Chen, H. C. Wikle and B. A. Chin, Rapid detection of live versus dead bacteria, 1982, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Y. Liu, S. Du, S. Horikawa, H. C. Wikle, J. Hu, F. Wang and B. A. Chin, Double-layer planar coil detector for improved detection of salmonella on food contact surfaces, 1989, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Du, S. Horikawa, Y. Liu, Y. Chai, J. Hu and B. A. Chin, Rapid Detection of Pathogens By a 3D Biomolecular Filter and Automated Biosensor Measurement System for Liquid, 1977, 229th ECS June 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: S. Horikawa, S. Du, Y. Liu, I. H. Chen, Y. Chai, H. C. Wikle and B. A. Chin, The Bathtub Method for Detecting Small Quantities of Specific Pathogens, PRiME 2016 Honolulu, Hawaii.