PARTNERSHIP: DEVELOPMENT AND EVALUATION OF LOW-COST, EASILY DEPLOYABLE MOLECULARLY IMPRINTED POLYMER NANOPARTICLES FOR AGRICULTURAL VIRUSES AND TOXINS OF CONCERN

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: NEW
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1028014
• Grant No.: 2022-67021-36408
• Project No.: MASW-2021-08570
• Proposal No.: 2021-08570
• Program Code: A1511
• Project Start Date: Feb 1, 2022
• Project End Date: Jan 31, 2025
• Grant Year: 2022

Project Director
Moore, M. D.

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Dept: Food Sciences

Non Technical Summary
Foodborne illness causes considerable damage to public health and causes considerable economic loss in the United States and globally. The leading cause of foodborne infections in the US and globally is human norovirus, which causes millions of illnesses, results in hundreds of thousands of deaths, and losses in billions of dollars annually. These viruses can survive on surfaces for weeks, are resistant to most commonly used disinfectants, and require just a handful of particles to make someone sick.Mycotoxins are toxins that certain fungi can produce that cause illness in humans and agricultural animals. Although illness from mycotoxins does not immediately result in direct symptoms, these toxins can cause severe damage to peoples' liver and kidneys over time if consumed, making them even harder to prevent and control. Mycotoxins occur when certain fungi grow on foods that are eventually consumed, and the foods most commonly associated with these toxins include grains, legumes, and other plant-based products. Further, the conditions that climate change is causing have potential to further promote growth of these fungi and subsequent occurrence of mycotoxins in foods. This potential for higher presence of these toxins in foods due to climate change, as well as an increasing trend (and need for) of consumption of plant-based foods, makes mycotoxins and emerging existential public health threat. Despite their importance, routine and comprehensive testing for noroviruses and mycotoxins in foods is still fairly limited as a practice due to the lack of rapid, portable, and inexpensive testing methods and protocols; thus making controlling their consumption more difficult.A number of challenges exist in testing for noroviruses and mycotoxins in foods. One of which is that both are very diverse in the different types/variants of these contaminants that exist. One must be able to detect the broad class, while also being able to determine which type is detected. Further, many testing techniques either take a long time (limiting application), are not suitable for use in fields (require central lab testing), can be easily inhibited by other compounds found in foods, and are too costly to be realistically utilized for noroviruses and mycotoxins.Molecularly imprinted polymer nanoparticles (nanoMIPs), are an emerging technology that offers the potential for the development of inexpensive testing technologies that are also able to withstand harsh conditions (in-field). The application of nanoMIPs for norovirus and mycotoxin testing has not been investigated, and this project plans to develop this promising technology for the development of tests that are portable, inexpensive, and have potential to be utilized to target specific types of noroviruses and mycotoxins, thus enabling the ability to better detect noroviruses and mycotoxins in foods before they are served to people. The project will also evaluate the ability of the nanoMIP tests to withstand and perform in foods without the need to complicated manipulation that will add time to result and limit their application at the point of production or service. Overall, the development of such tests for noroviruses and mycotoxins has potential to reduce the considerable public health and economic loss that noroviruses and mycotoxins provides, as well as lead to knowledge that will aid development of nanoMIP sensors for other viruses and toxins of concern.This project also involves the establishment of an international partnership between two globally-leading research institutions, University of Massachusetts Amherst and Newcastle University. The Moore and Gibbons labs in the Department of Food Science at the University of Massachusetts are respective experts in foodborne viruses and fungi, respectively. The Peeters lab in the School of Engineering at Newcastle University has expertise in nanoMIPs and the development of rapid tests. Further, the Department of Food Science is ranked among the top departments in food science research in the US and globally, while Newcastle University is recognized as among the global leaders in the development of sensing technologies. This partnership is not only ideal for development of these novel testing methods for noroviruses and mycotoxins, but will also result in forming the foundation of a partnership that is anticipated to result in future scientific research. The partnership will also involve exchanges and training of the labs involved, and will also include additional activities to solidify collaboration between these two world-leading institutions.

Animal Health Component

100%

Research Effort Categories

Basic

30%

Applied

60%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4030	1101	50%
712	4020	1150	50%

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

Subject Of Investigation
4020 - Fungi; 4030 - Viruses;

Field Of Science
1101 - Virology; 1150 - Toxicology;

Keywords

portable detection

molecularly imprinted polymer nanoparticles

norovirus

aflatoxin

fumonisin

Goals / Objectives
This projectseeks to develop and evaluate the potential of molecularly imprinted polymer nanoparticles (NanoMIPs) to serve as recognition elements for food and agricultural contaminants. We have chosen two targets of serious relevance to foodborne contamination, norovirus and mycotoxins that also serve as model targets in that they both are of smaller size, have a diversity of structures, and are more predominantly found in different food matrices. Further, the project will integrate these nanoMIPs into inexpensive, portable electrochemical sensors. We additionally hope to evaluate the degree to which nanoMIPs and nanoMIP sensors generated against these targets are able to withstand crudely processed food samples containing target contaminants, as well as the degree to which these sensors are capable of discriminating different variants/strains of noroviruses and mycotoxins. Additionally, the ability of the nanoMIP sensors to stand up to harsher field conditions will be evaluated. The project aims to result in a number (at least 4) of publications as well as posters and scientific presentations as a result of this work.One of the other major goals of the project is to establish a partnership between the University of Massachusetts, Amherst and Newcastle University. The project will include an exchange where PI and selected students/postdoc will conduct an exchange that will involve guest seminars/networking for future collaboration by the PIs with each other's respective institutions, as well as an expertise exchange between labs from the two institutions. The Moore lab (UMass) has expertise in applied microbiology, virology, and detection of pathogens in food and agricultural samples. The Peeters lab (Newcastle) has expertise in nanoMIP development, biosensor fabrication, and electrochemical sensing evaluation. The Gibbons lab (UMass) has expertise in fungal pathogens and their toxins. Students/postdocs from these two labs will spend at least one week in each other's' labs learning the techniques of each other's' labs. The Department of Food Science at the University of Massachusetts, Amherst is a global leader in food science, recently ranked first in the U.S. and second in the world in food science by U.S. News & World Report. Newcastle University is a noted global leader in electrochemical sensing and sensor engineering. Built in seminar and faculty meetings that will coincide with the visit will further promote collaboration. An additional goal of the project is to utilize what is learned from the evaluation of the nanoMIP technology into intellectual property and potential future devices for the agricultural market, as the devices themselves are anticipated to be inexpensive enough to be realistically utilized by the food production industry.The specific objectives of this project are:Objective 1. Development and evaluation of molecularly imprinted polymer nanoparticles (nanoMIPs) for detection of noroviruses and integration into an inexpensive electrochemical sensor.Objective 2. Development and evaluation of molecularly imprinted polymer nanoparticles for the detection of mycotoxins and integration into an inexpensive electrochemical sensor.Objective 3. Evaluation of the ability of electrochemical nanoMIP sensors to withstand common inhibitory substances as well as performance in crudely processed, real food samples in in-field conditions.

Project Methods
The methods of the project will include the specific activities outlined in the revised narrative submitted as a part of the response to panel comments. Specifically, numerous conditions involved in nanoMIP development and different aspects of the different selected targets will be executed. The performance of nanoMIP candidates will be evaluated by determination of binding affinity of the nanoMIPs for both the specific target norovirus/mycotoxin target strain/variant as well as others of varying degrees of relatedness (specificity). The ability of the nanoMIPs to withstand harsher field conditions (higher temperature) will also be determined in part by affinity and sensitivity for target when integrated into sensors. The ability of the nanoMIPs when incorporated into sensors to detect norovirus and mycotoxin targets will be evaluated based on sensitivity (limit of detection) and specificity (reactivity with different strains/variants). The ability of the nanoMIP sensors to withstand inhibitory components found in foods (both purified known inhibitors and crudely processed food samples) will also include sensitivity.

Progress 02/01/22 to 01/31/23

Outputs
Target Audience:The target audience reached by the work in this reporting period consists primarily of fellow academics and researchers in the fields of detection/analytical biochemistry, microbiology, materials and bioengineering, and food safety. Secondary audiences reached in the different presentations/communications/publications involves members of the food/food safety industry, government, and academia. In particular, our data related to the potential of this technology to rapidly detect viruses as well as more fundamental developments in MIP technology to detect antibiotics in foods would be of interest to the broader infectious disease, polymer science, and food safety communities as a whole--including those who are focused on pathogen/contaminant control from a clinical perspective. Changes/Problems:The only problems encountered so far were related to scheduling, as originally the exchange was planned for reporting period 1; however, based on availability and timing, as well as research progress, the exchange was pushed back to the spring of 2023 and is anticipated to take place in April and May 2023. What opportunities for training and professional development has the project provided?We have scheduled a delayed exchange for spring 2023 between the labs, that also will coincide with the partner PIs delivering research seminars and meeting with other faculty at each others' respective institutions (UMass Amherst, May 31-June 6; Newcastle University, April 27-May 2). During the exchange each lab will be trained in respective disciplines (Peeters and group will be trained in microbiology, virology, and food safety techniques at UMass, while Moore and group will be trained in molecularly imprinted polymer nanoparticle formation, engineering, and microfluidic techniques while at Newcastle University). In addition to the training exchange and seminars, the date of the UMass visit to Newcastle was chosen to coincide with the International Association for Food Protection European Symposium being held May 3-5 in Aberdeen, Scotland, which relatively close to Newcastle, UK (See Products). Additionally, a number of graduate students and postdocs will get/have had opportunities to present this work in conferences held in North America (IAFP Annual Meeting in Toronto), Australia (Technical Talk,), Scotland (IAFP European Symposium), and Latvia (World Society for Virology). Based on timing as well as the availability of additional discretionary funds of the PI for travel, we have been able to leverage attendance at additional conferences this year. For example, PI Moore is heavily involved in the World Society for Virology (Treasurer, Co-Director, and Organizer for meeting) and will be able to attend on separate travel funds to present the poster to a different audience (virologists) compared to those who would normally attend IAFP. Similarly, the Peeters group presented the project to a polymer science audience. Two graduate students from the Moore lab, as well as members of the Peeters lab, will get to attend the IAFP European Symposium in Aberdeen, Scotland, as a consequence of the location being close to Newcastle, UK, and timed with the UMass visit for the expertise exchange. Additionally, both PIs Moore and Peeters have aided and supported applications for different awards and fellowships for students and postdoc funded by the project. Including a Fellowship from the Royal Society of Chemistry for which the postdoc has made the final round (still awaiting decision), and a prestigious fellowship to one of the graduate students at UMass (UMass Food Science Manley Fellowship). How have the results been disseminated to communities of interest?As mentioned above, we either have abstracts submitted or have presented this work to numerous scientific and academic communities internationally. We also have a textbook chapter under review for the food safety community, and a peer-reviewed manuscript under minor revision related to the same nanoMIP development for a food contaminant (Products). What do you plan to do during the next reporting period to accomplish the goals?We plan to continue evaluating the potential of the P domain peptide sequences to serve as targets for generation of nanoMIPs, as the fact the generated nanoMIPs show affinity for assembled viral capsid (VLPs) is quite exciting. We plan to evaluate the potential for cocktails of peptides to enable generation of more broadly reactive nanoMIPs for sensing. Additionally, we will covalently functionalize the norovirus nanoMIPs to low-cost and highly reproducible SPEs using previously established methods. These functionalized SPEs can then be utilized for electrochemical detection of norovirus with the potential for integration into low-cost, portable, and easy-to-use devices for in-field testing. Further, we aim to repeat the nanoMIP synthesis process to target mycotoxins after continuing optimization with norovirus. Depending on progress and after training/exchange, the Moore lab will attempt to evaluate the nanoMIP sensors generated against viruses in more complex matrices (foods).

Impacts
What was accomplished under these goals? We have investigated and generated a number of different nanoMIP formulations against a norovirus epitope on the exposed outer portion of a norovirus epidemic genotype (GII.4) capsid protein (YQEAAPAQSDV) as the target for NanoMIP synthesis. This allowed for low-cost and safe synthesis as only a tiny virus fragment was required to develop the synthetic receptors; if successful, this could serve as a much cheaper and more efficient means of generating norovirus-specific ligands, as well as generate broadly reactive ligands against a cocktail of different strains. NanoMIP synthesis begins by immobilizing the target (norovirus epitope) to functionalized glass beads, which act as a solid-phase support during synthesis. The monomers are then allowed to self-assemble around the target before crosslinkers/initiators are added to lock the polymer structure in place. A low-temperature elution is initially performed with room-temperature water to remove any unreacted monomers or low-affinity nanoMIPs from the solution. This is followed by a high-temperature elution (70 °C), which separates the high-affinity nanoMIPs from the target. The collected high-affinity nanoMIPs contain cavities within them that are the correct size, shape, and functionality to selectively rebind with the target upon exposure. Essentially, they mimic the lock-and-key mechanism observed in biology. To develop high-performance nanoMIPs, it is vital to optimize monomer selection. This can be performed experimentally or by using computational techniques. Wherein, we developed innovative nanoMIPs using electroactive monomers [N-Isopropylacrylamide (NIPAM), N-(Tert-Butyl)Acrylamide (TBAM), Ferrocenylmethyl methacrylate (FMMA), N-(3-Aminopropyl)methacrylamide hydrochloride (NAMPA), Acrylic acid (AAc), and Dopamine Methacrylamide (DPMA)], along with a cross-linker [N, N'-methylenebisacrylamide (BIS)]. This means that they can be utilized for electrochemical detection which is optimal for portable, low-cost, and rapid in-field testing. Three batches of nanoMIPs were developed (two different electroactive and one non-electroactive). As these nanoMIPs are novel, the protocol development and optimization accounted for a considerable time period. NIPAM (mg) TBAM (mg) FMMA (mg) DPMA (mg) NAMPA (mg) AAc (µL) BIS (mg) TEMED (µL) APS (mg) Batch 1 (Standard Batch) 20 17 - - 4 1.1 1.5 15 24 Batch 2 (Ferrocene Batch) 20 17 20 - 4 1.1 1.5 15 24 Batch 3 (Dopamine Batch) 20 17 - 20 4 1.1 1.5 15 24 As confirmed by SEM, the nanoMIPs showed spherical morphology. Furthermore, DLS showed relatively homogenous nanoMIPs with average sizes of 90, 100, and 110 nm for the standard, ferrocene, and dopamine nanoMIPs, respectively. This characterization confirms the nanoMIP synthesis protocol was effective and that the nanoMIPs possess the necessary morphology for favorable sensing performance. SPR was performed on the three types of prepared nanoMIPs. The results demonstrate that all nanoMIP types showed high binding affinities for the norovirus epitope, P-domain, and VLPs. Furthermore, measurements were performed using a similar epitope (non-target) and binding affinity was two orders of magnitude larger. Consequently, this demonstrates that the prepared nanoMIPs can selectively bind to a range of norovirus targets (different sizes) with high affinity. Sample Epitope KD (M) Selectivity KD (M) P-domain KD (M) VLPs KD (M) Standard Batch 3.28 × 10-7 - 6.65 × 10-7 5.12 × 10-7 Ferrocene Batch 7.50 × 10-7 2.67 × 10-5 5.75 × 10-7 7.95 × 10-7 Dopamine Batch 1.92 × 10-6 3.51 × 10-5 1.37 × 10-6 1.76 × 10-6 Thermal detection (heat transfer method) was used to confirm the sensing capabilities of the standard nanoMIPs (non-electroactive). The method shows receptor-target interactions through increases in thermal resistance (Rth) at the functionalized-electrode surface. More target binding = greater thermal resistance. The results above clearly show there is no statistically significant difference in Rth during multiple PBS additions. However, when a VLP-spiked PBS solution (100 ng/mL) is added, the thermal resistance increases significantly, whereby it is many times greater than the standard deviation of the baseline signal (3x and 6x baseline SD on graph). This demonstrates that nanoMIPs can detect the VLPs using thermal detection methods.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Singla, P.; Kaur, S.; Jamieson, O.; Dann, A.; Garg, S.; Mahon, C.; Crapnell, R.D.; Banks, C.E.; Kaur, I.; Peeters, M. Electrochemical�and�Thermal Detection of Allergenic Substance Lyzosyme with Molecularly Imprinted Nanoparticles. Analytical and Bioanalytical Chemistry, 2023, In revision  minor revisions.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2023 Citation: Dann, A.; Kaur, S.; Singla, P.; Stoufer, S.; Kim, M.; Kaur, I.; Moore, M.D.; Peeters, M.; McClements, J. Molecularly� Imprinted Polymers for Detection of Chemical and Microbial Contaminants in Foods. Encyclopedia of Food Safety, 2nd Edition, 2023, Under Editorial Review.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Jamieson, O.; McClements, J.; Kaiya, G.E.; Stoufer, S.; Moore, M.D.; Bell, J.; P�rez-Padilla, V.; Rurack, K.; Peeters, M. The Devolvement of Polymer-based Sensors for Detecting Antibiotics in Food. International Association for Food Protection Annual Meeting, 07/16/23-07/19/23, Toronto, Ontario, Canada. Oral abstract submitted.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Kaur, S.; McClements, J.; Singla, P.; Dann, A.; Minji, K.; Stoufer, S.; Moore, M.D.; Kaur, I.; Peeters, M. Development and Evaluation of Low-Cost, Easily Deployable Molecularly Imprinted Polymers for Norovirus Detection. International Association for Food Protection European Symposium on Food Safety, 05/03/23-05/05/23, Aberdeen, Scotland, United Kingdom. Oral abstract submitted.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kaur, S.; Singla, P.; McClements, J.; Minji, K.; Stoufer, S.; Moore, M.D.; Kaur, I.; Peeters, M. Development of Molecularly Imprinted Polymer (MIP) Technologies for Norovirus Detection. The 17th Pacific Polymer Conference, 11/12/22-14/12/22, Brisbane, Queensland, Australia. Oral presentation delivered.
• Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Kaur, S.; Singla, P.; McClements, J.; Minji, K.; Stoufer, S.; Moore, M.D.; Kaur, I.; Peeters, M. Application of Molecularly Imprinted Polymer Nanoparticles for Viral Pathogen Detection. World Society for Virology Meeting 2023, 15/6/2023-17/6/2023, Riga, Latvia. Poster, Abstract Under Review.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Moore MD. Developments in Detection and Control of Viral Pathogens. Invited Talk, Advanced Strategies to Control Microorganisms in Seafood Session. Korean Society of Food Science and Technology Annual Meeting 2022. Busan, South Korea. 7/6/2022.