A LOW-COST PATHOGEN DETECTION SYSTEM IS PROPOSED COMPRISING AN INCUBATOR-READER UNIT AS WELL AS A LOW-COST CONSUMABLE PAPER-BASED INDICATOR TEST STRIP.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1010258
• Grant No.: 2016-33610-25679
• Project No.: COLW-2016-03885
• Proposal No.: 2016-03885
• Program Code: 8.5
• Project Start Date: Sep 1, 2016
• Project End Date: Aug 31, 2019
• Grant Year: 2016

Project Director
Reilly, T. H.

Recipient Organization
ACCESS SENSOR TECHNOLOGIES LLC
430 N COLLEGE AVE STE 410
FORT COLLINS,CO 80524

Performing Department
(N/A)

Non Technical Summary
Food safety is a global problem, which costs the world billions of dollars and over a million lives annually. This project seeks to develop a complete system for easy pathogenic bacteria detection for the food supply chain. The proposed kit will deliver exceptional ease-of-use as well as performance metrics (time, cost, labor). By delivering a complete pathogen detection solution, the kit will enable more testing locations and a higher frequency of testing. In addition, the kit is designed to be low-cost from the beginning so entire new segments of food processors may adopt the technology where currently it is not economically feasible to do so, such as rural fruit and vegetable producers that operate only seasonally. A safer food supply chain protects those who suffer the most from foodborne illness outbreaks such as the elderly and small children. Commercial applications of the technology will find a global customer base as current laboratory microbiology tests are cumbersome, expensive and time consuming. Additionally, the products will feature technology integration capability to support Laboratory Information Management Systems thus enabling food safety data to be widely disseminated and part of a modern tracking and traceability system.The technology proposed by AST will support several of the USDA strategic goals. Low-cost pathogen detection enables American agricultural success by reducing food safety risk to small co-operatives and independent rural operators. The ultimate goal of a low-cost product that can ensure greater food safety supports USDA strategic goals one, three, and for as outlined in the USDA Strategic Plan.

Animal Health Component

0%

Research Effort Categories

Basic

0%

Applied

20%

Developmental

80%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5010	1100	40%
712	5010	2000	30%
712	5010	2020	30%

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

Subject Of Investigation
5010 - Food;

Field Of Science
2020 - Engineering; 1100 - Bacteriology; 2000 - Chemistry;

Keywords

environmental screening

food safety

pathogen screening

post-harvest

pre-harvest

Goals / Objectives
A key desired outcome for this technology is to achieve AOAC certification of our instrument and methods under the AOAC Performance Tested Method Program. This milestone creates value for the product by enabling customer recognition and acceptance that our product carries an independent measure of quality assurance. To achieve this goal we will address four key development objectives during phase II that will prepare the product for testing after phase II. Phase I activities allowed us to de-risk the largest technical aspects of our product proposal. Phase II will further advance and customize the product for entry into specific target markets. To achieve these end goals for the phase II project we set out the following key objectives:Objective 1: Adapt single step enrichment growth medium for specific niche market applications.Our technology will use a broth to selectively grow harmful pathogens to a detectable level with our low cost device and consumables. Broths contain a number of reagents and we will determine the broth formulation that best matches our food and environmental applications.Objective 2: Define Low-cost mPAD Design, Manufacturing and Packaging Specifications.Our paper-based test strips give the indication of a positive or negative response to the enrichment broth. We will determine the most efficient manufacturing conditions to generate reproducible test strips. The consumable product will also be optimized for cost and the packaging requirements. Initial shelf life and storage conditions will also be determined.Objective 3: Design and prototype an integrated platform for sample incubation and imaging.This objective encompasses our instrument development effort. Here we will focus on arriving at a prototype design that can be tested by early adopters.Objective 4: Perform in-house Method Validation.In preparation for applying for third party testing of our technology we will perform in-house validation study that sets the stage for handing off our technology for AOAC evaluation. This activity will create an internal validation report with sub-reports including: inclusivity, exclusivity, specificity, sensitivity analysis.

Project Methods
This project encompasses microbiology, chemistry, mechanical engineering, electrical engineering and firmware/software development. We will employ scientific methods from all these disciplines throughout the project to determine the best product development pathways. The microbiology efforts will rely on a deep library of previously characterized microorganisms which include pathogenic species and non-pathogenic species. These organisms will be used to systematically test broth enrichment improvements and used for inoculation of food and surface materials for recovery. Techniques used in conjunction with the micro-organisms are plating techniques where organisms are cultured in petri dishes containing specialty media. The plating techniques are useful for inclusivity/exclusivity analysis to see if broth modifications have been successful in enriching only the pathogens of interest.The colorimetric test strip development will rely on many traditional lab techniques common to analytical chemistry. Reagent combinations will be evaluated through single variable and multi-variable studies. Quantitative analysis of the results will be performed through digital imaging of the colorimetric strips. Color responses serve as a means to determine performance improvements achieved by altering the reagent mixtures present on the paper analytical devices. Image analysis also serves as the means to evaluate packaging, storage and shelf-life test results.The incubation and automated PAD analysis instrument will be evaluated for performance using control samples and fluids. In addition, imaging standards will be used to calibrate the image collection system. Split samples will be incubated using standard laboratory incubators and compared to our custom designed solution. Firmware and software development will be compared against defined instrument performance specifications.

Progress 09/01/16 to 08/31/19

Outputs
Target Audience:Target audiences include food safety practitioners, food producers, microbiologists and quality engineers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided several opportunities to advance the careers of early career scientists. The project supported a graduate student, several undergraduate summer interns and early career scientists at the Ph.D. and B.S. levels. How have the results been disseminated to communities of interest?The results have been disseminated through attendance of relevant conferences and presenting posters. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Several collection, enrichment and detection methods were developed under this project. This is important to advance the field of low-cost diagnostics for foodborne pathogen detection as well as other microbiology needs. Objective 2: Paper-based microfluidics is an active field of low-cost diagnostics. New designs were developed to support the easy interpretation and identification of pathogens. Objective 3: In support of the the easy determination and quantification of foodborne pathogen on paper-based substrates several instrument prototypes were designed and demonstrated.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Broten, Codi Jo, et al. "Colorimetric Detection of Listeria monocytogenes on Food Contact and Non-food Contact Surfaces Using Paper-based Microfluidics." IAFP 2020 (2020).
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Broten, Codi Jo, et al. "Colorimetric Detection of Clostridium perfringens in a Model Meat System Using Paper-based Microfluidics." IAFP 2019 Annual Meeting. IAFP, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Broten, Codi Jo, et al. "Colorimetric Detection of< em> Cronobacter< g class=." IAFP 2018 Annual Meeting. IAFP, 2018.

Progress 09/01/17 to 08/31/18

Outputs
Target Audience:During Year 2 we reached food safety professionals, local food and animal equipment vendors, large animal veterinarians (vets supporting farms), large food processing equipment vendors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project supported one graduate student at the University of Wyoming. The student benefitted from the guidance of her advisor (ProfessorBisha at U Wy) and the engagement with Access Sensor Technologies. We also employed early career scientists and engineers at the company. These young employees benefitted from the research and development experience as part of the product development process supporting this award. How have the results been disseminated to communities of interest?Results related to Chronobacter research was disseminated at the International Association for Food Protectionin the form of a poster presented by Codi Jo Broten. Individual contacts were made through meetings and discussions. What do you plan to do during the next reporting period to accomplish the goals?Objective 3 and 4 will be the main focus of the final (no-cost extension) yearof activity for this project. Our additional interviews and inputs from members of the food supply chain have affirmed the needs for very easy to use tools on the farm and in the manufacturing setting. In addition we will begin some the in-house method validation activities to demonastrateour new methods show comparable performance to those methods already in the marketplace. In addition we aim to deploy prototypes in the hands of potential customers and model users to confirm our value proposition. This activity is vital to developing future partners and investors to scale and launch our technology.

Impacts
What was accomplished under these goals? Objective 1 accomplishements. Objective 1 was largely completed during year 1. Some additional accomplishments investigated enrichment and detection of chronobacter sakazakii from powdered infant formula. Chronobacter is an emerging threat with limited products available for food safety professionals. Using the same platform as developed for other foodborne pathogens, dried infant formula was inoculated with Cronobacter sakazakii. After a 24 hour enrichment the presence/absence of cronobacter was determined using simple image analysis of the chromogenic response from alpha-glucosidase with two different chromogens. These reults generated interest at the IAFP summer conference. Objective 2 accomplishments. Objective 2 was largely completed in year 1. Additional activity focused on new porous media that can accommodate bacterial flow within pores of the media. Previous work has focused on cellulose based paper microfluidics but these pores are usually 20 microns or lessand may obstruct theflow of bacteria to incubation and detection zones. Several new media were used as prototypes for devices. Porous glass fiber media, typically used for air sampling, was laser cut and mounted within petri dishes. This media was very conductive for fluid and model bacterium. We also evaluated several other large pore media. These new device designs allow for multi-step sample prep driven by capillary action. Objective 3. Objective tasks included evaulating a new colorimetric approach to detecting the phospholipase enzyme using dye-embedded lecithin. Phospolipase is a key enzyme to detect as part of Listeria monocytogenes assays. Unfortunately some of the best chromogenic substrates are still patent protected. We attempted to exploit the natural activity of phospholipase to break down lecithin. It was hypothesized that upon break down of the lecithin the dye trapped in the matrix would be released and a visible migration of dye could be used as an indicator for phospolipase activity. Initial results are inconclusive. Several oil soluble dyes were traped within lecithin but the dyes were not liberated upon exposure to high loadings of listeria. We are still considering additional dyes that may have higher water solubility that will provide more obvious colorimetric response to the action of phospolipase enzymes on the lecithin matrix. If successful this would provide a very cheap way of identifying the phosholipase enzymes. Objective 4. Objective four is set to begin during 2019 as part of the final part of the project.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: International Association for Food Protection 2018 Conference (Salt Lake City, UT) Poster Presentation Colorimetric Detection of Cronobacter sakazakii in Artificially Contaminated Powdered Infant Formula Using Microfluidic Paper-based Analytical Devices Codi Jo Broten1, John B. Wydallis2, Thomas Reilly, III2 and Bledar Bisha1, (1)University of Wyoming, Laramie, WY, (2)Access Sensor Technologies, LLC, Fort Collins, CO

Progress 09/01/16 to 08/31/17

Outputs
Target Audience:During Year 1 we reached food safety professionals, including microbiologists, through on-site visits. Changes/Problems:No major changes or problems were encountered in Year 1. The project did commence a few months after originally proposed due to common issues such as finalizing our sub-award agreement and attracting a student and allocating resources at the company to be devoted to theproject. What opportunities for training and professional development has the project provided?The project supported one graduate student at the University of Wyoming. This student benefitted from the guidance of her advisor as well as the three Ph.D. scientists at the company. How have the results been disseminated to communities of interest?Given the proprietary nature of the research we have not widely disseminated the results from year 1. Our collaborators at the University of Wyoming are planning on attending conferences in Year 2 to present some of the research results from year one. What do you plan to do during the next reporting period to accomplish the goals?We plan on following our original development plans in Year 2. We will advance our designs and proecures to carry out in-house method validation data sets as we prepare to raise funds and submit our technology for external validation after the completion of the phase II project.

Impacts
What was accomplished under these goals? The purpose of this project is to develop a simple to use low-cost pathogen detection system for food safety applications. In year 1 of the project we advanced microbiology, chemistry and manufacturing engineering knowledge of our proposed system. Through prospective customer interviews we also improved our understanding of the specific customer product requirements that will satisfy a large segment of end-users in the food production space. In year 1 we worked closely with our sub-awardees at the University of Wyoming to develop the microbiology conditions and precedures that will allow for detection of very low levels of fodo borned pathogens. We also developed several iterations of prototypes for our system. The results are guiding our product closer to commercialization at the end of year 2. If successful we aim to develop a validated easy to use lower-cost pathogen detection system that can be broadly deployed throughout the food supply chain. Objective 1: Adapt single step enrichment growth medium for specific niche market applications. Major activities completed under this objective include evaluation of a large set of enrichment broths for recovery and growth of bacterial pathogens. Evaluation of pathogen recovery from surfaces and spiked milk products. Objective 2: Define Low-cost mPAD Design, Manufacturing and Packaging Specifications. We prototyped many different variations of the low-cost consumable component of the system and also developed prototypes for complimentary products that may increase the appeal of the product platform. Objective 3: Design and prototype an integrated platform for sample incubation and imaging. We collected more customer requirements information and began designing a low-profile simplified incubation system that can serve as a stand-alon enrichment incubator. Objective 4: Perform in-house Method Validation. No work on Objective 4 was planned for year 1.

Publications