NANOSCALE TOOLS FOR FOOD SAFETY, HEALTH AND ENVIRONMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007564
• Project Start Date: Oct 1, 2015
• Project End Date: Jun 30, 2017
• Program Code: [(N/A)]- (N/A)

Project Director
Irudayaraj, JO, .

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Ag & Biological Engineering

Non Technical Summary
Given the advances in materials and instrumentation there has been a significant improvement in the development of detection technologies. Sensors that can detect and quantify transcripts in live cells have also been developed (Lee et al., 2014). Nevertheless, given the complexity of the samples and the LOD expected and the need to perform this step at a rapid rate in the field, the area of sensors development is continually challenged. Sensors that can detect pathogens and toxins as low as a single cell in complex matrices at within a few minutes is the ultimate goal for the scientists and engineers involved in this technology development space.Devices for Food Pathogen Detection: Despite the fact that America's food supply is one of the safest in the world, 76 million cases of food borne illnesses, 325,000 cases of hospitalization and 5000 deaths have been estimated every year according to the Center for Disease Control (CDC). More recently the level of alertness has increased to counteract agroterrorism across the entire food chain, from farm to the table. The cost for the treatment and control is estimated to be between $1- 10 billion every year (Scallan et al., 2011). Part of the challenge facing the food industry, agencies, and institutions charged with protecting public health, is to develop appropriate strategies to identify contaminated products rapidly to ensure quality and safety at every step in the farm-to-consumer or farm-to-processing sequence. There is a universal need among regulators, food producers and/or processors, and researchers for rapid, precise, and accurate detection methods for foodborne pathogens (1 cell/25-325 g sample) and other foodborne hazards (Bhunia, 2014).While the development of sensor technologies is on the rise, a better understanding of the sensor materials in the context of biological interaction will help to develop sensors that are more specific and sensitive (Cho et al., 2014). Such accuracy is critical because of the zero-tolerance mandate. Sensitivity using conventional biosensors is in the range between 103-104 colony forming units (CFU)/ml. Labeled PCR products of various genes from one food borne pathogen could be probed on one DNA micro array to create a Multi-Locus micro array subtyping scheme for various food borne pathogens. To detect at 1 CFU/ml sensitivity, to be able to answer questions at the molecular level, micro and nano-based technologies should be examined in conjunction with the existing methods. In addition, bringing new and emerging sensor technologies and paradigms to the forefront will play a significant role in elucidating the complex structures and mechanisms involved in food and biological systems. The overall objective of this study is to utilize and integrate the concepts of biosensor and nanotechnology based methods to develop simple technologies that can be deployed onsite for rapid detection (< 30 minutes) of food pathogens at the lowest possible limit of detection (< 10 cfu/ml).Devices for Intracellular monitoring: The vascular system is one of the first organ structures to develop in an embryo. Vasculogenesis refers to generation of blood vessels de novo from mesodermal precursor cells, while angiogenesis refers to formation of new blood vessels from pre-existing blood vessels (Stainier et al., 1996; Dzubow et al., 2010). Vasculogenesis happens extensively during embryonic development, and studies over the last decade have convincingly demonstrated adult vasculogenesis (Drake, 2003). Although generation of blood vessels in adults is of rare occurrence, except during corpus luteum and wound healing, and widespread during tumorigenesis, it is possible that complex regulatory mechanisms involving 'switching on' and/or 'switching off' of critical angiogenesis/vasculogenesis pathways that are responsible for this transcriptional dynamism could dictate cell fate. The susceptibility of vascular cells to be imprinted has been studied in pregnancy-related disorders such as intra-uterine growth restriction, gestational diabetes and pre-eclampsia (Krause et al., 2009), and thus we hypothesize that vasculogenesis is heavily influenced by signaling and epigenetic mechanisms. While CVOCs such as trichloroethylene (TCE) among others and their metabolites have been implicated in regulating congenital cardiovascular defects (Dawson et al., 1993; Johnson et al., 2003), precisely how they induce changes in gene regulation, signaling pathways, and cellular functions during fetal heart development is unknown. Technologies that monitor kinase activity in live cells are rare. A critical first step in enhancing our understanding of signaling pathways is through methods that allow us to monitor signaling in live cells and in a quantitative manner.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	2020	40%
712	4030	2020	10%
712	5010	2020	10%
712	7010	2020	40%

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

Subject Of Investigation
7010 - Biological Cell Systems; 4010 - Bacteria; 4030 - Viruses; 5010 - Food;

Field Of Science
2020 - Engineering;

Keywords

biosensors

nanotechnology

detection

pathogens

single cells

interactions

Goals / Objectives
1) Develop effective onsite sensor technologies for enhanced onsite detection of pathogenic agents and disease markers. We will develop signal enhancement strategies utilizing multifunctional nanoparticle to significantly improve the limit of detection (LOD) of lateral flow immunochromatography (LF-IC) assays. Various (E. coli, Salmonella sp., Shigella sp., Listeria, Staphylococcus, Yersinia) foodborne and disease causing pathogens will be examined to achieve a LOD of < 10 cfu/ml (conventional LOD in LF-IC systems is ~106 cfu/ml) for pathogen detection and to the pictogram/ml LOD of protein biomarkers for cancer screening.2) Develop engineering tools to monitor signaling in live cells at single molecule resolution to understand intracellular signaling dynamics and cross-talk. In this aim we will use general purpose peptide biosensors to target specific kinase activity in live cells to monitor dynamic signaling events. Thus perturbation of signaling due to an environmental contaminant or a drug or a disease condition can be assessed and quantified by monitoring the degree of phosphorylation. Here we will monitor the effect of chlorinated volatile organic compounds on cell signaling with a potential implication in birth defects. We will use the same biosensor approach to monitor specific signaling pathways at different stages of cancer development and/or to assess the effect of drugs on signaling.

Project Methods
?Methods for Objective 1:Lateral Flow Immunochromatography (LF-IC) utilizes the driving force of the liquid to present targets at a desired spot for signal generation. Additive forces could be helpful in achieving better sensitivity to realize the detection of complex analytes with LF-IC strips. LF-IC could be integrated with other detection modalities and devices however sensitivity and ease of use must still be the key considerations (Liang et al., 2014). The world market for LF-IC based tests is expected to reach $4.7 billion by 2015 and the demand will continue to rise (Wild, 2013). While the technology exists, future efforts need to address sensitivity, reproducibility, quantification, multiplexing, and cross-platform detection capability. The focus of the proposed effort is to develop a rapid and highly sensitive field-deployable LF-IC pathogen detection platform integrated with smart phone data processing tools for routine screening of food products to ensure the safety of the food chain. Requirements of the technology include detecting 10-50 cfu/ml as a first step and to extend the LOD to < 10 cfu/ml in a detection time of <30 minutes with appropriate pre-concentration steps and validation.A reason for the poor LOD of LF-IC platforms is the low surface reaction rate and time allowed for interaction between the analyte and antibody modified on to the strip at the detection zone. To overcome this, a strategy was proposed recently based on isotachophoresis (ITP) which can slow down the movement of the probe-labeled analytes at the test zone in the strip and to further improve the LOD (Moghadam et al., 2014; Schwartz and Bercovici, 2014). However, the detection system is complex due to the experimental setup and instrument needed to channel the probe labeled analytes. In our proposed strategy, we will use a magnetic focus concept induced by a simple magnet (from Sigma) that has been tested in our laboratory for preconcentration purposes to control the rate of flow of the magnetic probe labeled analytes and to increase the reaction time to achieve a highly improved LOD. Thus after a preconcentration step using magnetic particles functionalized with capture ligands (antibodies or aptamers) (Cho et al., 2014; Gu et al., 2006; Fang et al., 2014), the sample is applied to the lateral flow system and the targets are retained at the signal generation site with a simple magnet to complete the interaction and for target enrichment at the signal generation site. Horseradishperoxidase (HRP) will be used for signal generation. We will optimize the probes using the biotin-streptavidin conjugation to bear a given quantity of HRP molecules for signal generation. By enriching the target at the capture site and by increasing the number of signal generating molecules we expect to enhance the signal from the LF-IC system. Compared to the ITP-based method, our strategy does not require any complex experimental steps or additional instrumentation, thus retaining the practicality and simplicity of the LFIA; while improving the LOD to 10 cfu/ml or below, which will be a significant step in technology improvement.Bacterial cultures: Our laboratory has a large collection of microbial cultures (E. coli, Salmonella sp., Shigella sp., Listeria, Staphylococcus, Yersinia). Methods for culturing, sample preparation and dilutions are routinely done in their laboratory. In addition we will work with the Center for Food Safety Engineering (Purdue-USDA collaboration) investigators in the Department of Food Science with expertise in microbiology (Drs. Bhunia and Applegate) on sample selection, culturing, and sampling procedures.Testing methods: Different levels of testing will be planned to assess the sensitivity and specificity. Level 1 testing will be conducted in buffer solution to assess the limit of detection and to develop a calibration plot. We will also assess the exclusivity/inclusivity. Level 2 testing will be conducted in different food matrices: ground beef and spinach wash for E. coli O157:H7, boneless chicken for S. Typhimurium, and 2% reduced fat milk for L. monocytogenes using uninoculated foods as controls and by artificially inoculating each bacteria into the corresponding sample medium using standard protocols. Level 3 - Testing and validation in natural food systems. Testing will be conducted from samples obtained from beef and poultry carcass rinse. Testing of additional foods or food related samples may include carcass wash, chiller water, pH adjusted fruit juices, bottled water, hot dog juice, food processing facility (slaughterhouse, retail butcher, deli, etc.) swab samples for hygiene testing, etc.The detection platform can be integrated with a smartphone devise for quantification and for easy access and interpretation of the results. In the long-term we also envision expanding our detection scheme to a multiplex format and include an option to detect live and dead pathogens.Methods for Objective 2:We hypothesize that CVOCs, including TCE and its metabolites, induce impaired activation of key signaling pathways (focal adhesion kinase (FAK), Src/MAPK/Akt, or VEGF)) and the associated epigenetic mechanisms, resulting in abnormal vasculogenesis, in part by repressing the growth and survival of endothelial colony-forming cells, or ECFCs, including vessel formation. In this objective we will test the hypothesis, using multiplex lifetime imaging that exposure to CVOCs will induce impaired activation of focal adhesion kinase (FAK) and/or associated components (Akt, Src, MAPK, VEGF), causing alterations in developmental vasculogenesis pathways.The two key tasks involved are the design of the peptide biosensor and development of the technology for monitoring live cell kinase activity via phosphorylation. Peptide sequences will be designed based on our and other reports (Kemp and Pearson, 1991) and databases (UniProt, RSCB Protein Data Base,and PDB). We have extensive experience in the design and synthesis of Abl, Syk and Src kinase peptides using solid phase Fmoc synthesis as demonstrated in our preliminary work (Damayanti et al., 2013b) and discussed in US Patent No. 61/700,878.The detection strategy is detailed in (Damayanti et al., 2013b) for 2D cultures upon exposure to a drug. Cell-culture systems (2D/3D) or model organisms will be incubated with peptide biosensors. We have optimized the delivery conditions for Abl, Src, and Syc kinase peptide biosensor. Our preliminary results show stable cytoplasmic, nuclear and cell junction localization of peptide kinase sensors by incubating cells with 5 μM peptide for 1 hour. We will map phosphorylation events for each kinase sensor pixel-by-pixel by multi-exponential fitting (Chen and Irudayaraj, 2010; Damayanti et al., 2013a; Damayanti et al., 2013b), and construct average lifetime histogram for quantitative analysis using MATLAB and Origin Pro 8.6. We will validate the SH2 domain binding to phosphorylated kinase by performing FLIM-based Forster Resonance Energy Transfer (FRET) study to show interaction similar to our recent work (Damayanti et al., 2013a). Experiments will be conducted under normal physiological conditions at the prescribed doses with 4-6 replicates.Our experiments have a high probability of success because we expect that at least two or perhaps more of the chosen pathways (FAK/Src/Akt/MAPK/VEGF) to be critically affected will disrupt vasculogenesis. In the unlikely event that we find these pathways do not have a significant effect, an alternative is to examine the Rho/Rac pathway, which is also downstream from FAK in Endothelial Cells (Schmidt et al., 2013). In these studies, FAK was shown to regulate the fine balance between the activities of RhoA and Rac1 GTPases. Importantly, selective inhibition and gene transfer studies indicate that Cdc42 and Rac1 can also drive vacuole formation while RhoA appears to stabilize capillary tube networks (Bayless and Davis, 2002).?

Progress 10/01/15 to 06/30/17

Outputs
Target Audience:Food processors, Industry consultants, Graduate students and postdoctoral scholars, Scientists and Engineers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several training opportunities for graduate students and postdoctoral associates exist. We have also had scholars from China visit our lab for our expertise in biosensors. We have in total 4 international scholars trained. How have the results been disseminated to communities of interest?Several companies are interested in the lateral flow devices we have developed. We are working in colalboration with other departments (Food Science) to test our devices in their laboratories. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have partially met the first major goal of our objective, in teh Chem Comm (2016) publication we have shown that 25-50 cfu/ml can be detected utilizing teh lateral flow system. We are now expanding tsi work to detect mRNA with a longterm goal to develop lateral flow microarrays. We have also been successful in demonstrating that the efficacy of our peptide biosensors. In our publictaion in Analyst (2017) we show that adhesion kinase senosrs can be successfully monitored by our peptide biosensors and lifetime imaging. This concept was also extended to AKY-VEGF kinase sensors.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Damayanti, N., Buno, K., Vyotik-Harbin, S., Deng, M., and Irudayaraj, J. 2017. Monitoring focal adhesion kinase phosphorylation dynamics in live cells, Analyst.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Bhandari, P., Ouyang, L., and Irudayaraj, J. 2017. Hypoxia re-programming oxygen nanobubbles sensitize human glioblastoma cells to temozolomide via methylation alterations. J. Bionanoscience, 11:1-9
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ren, W. Liu, W., and Irudayaraj, J. 2017. A netfishing enrichment strategy for colorimetric detection of E. coli O157:H7. Sensors and Actuators B. 247:923-929. http://dx.doi.org/10.1016/j.snb.2017.03.090
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, R., Xu, Y., Liu, H., Irudayaraj, J., and Cui, F. 2017. An integrated microsystem with dielectrophoresis enrichment and impedance detection for detection of Escherichia coli. Biomedical Microdevices, 19 (2): 34.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Scattering in Single Cells and Tissues. ACS Nano. http://dx.doi.org/10.1021/acsnano.6b07478.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ren, W., Cho, I-L., and Irudayaraj, J. 2016. Ultrasensitive detection of microbial cells by magnetic enhanced lateral flow sensors. Chemical Communications, 52:4930-4933.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Choudhury, S. and Irudayaraj, J. 2017. ZnO nanoparticles induced reactive oxygen species promotes multimodal cyto- and epigenetic toxicity. Toxicological Sciences. DOI: 10.1093/toxsci/kfw252.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang, R., Cui, Y., Xu, Y., and Irudayaraj, J. 2017. Basic studies on epigenetic carcinogenesis of low-dose exposure to 1-trichloromethyl-1,2,3,4-tetrahydro--carboline (TaClo) in vitro. PLoS ONE, 12(2): e0172243. doi:10.1371/journal.pone.0172243

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audience are food producers and processors, students, and faculty. Changes/Problems:We are pushing thelimits of technology. We will need to focus on thebasics to further improve the sensitivity and treatment efficacy. What opportunities for training and professional development has the project provided?Cross-disciplinary training is emphasized. Researchers and staff are provided opportunities to cross train in the fields of biological instrumentation, biophysics, and basic molecular biology. How have the results been disseminated to communities of interest?PUblications, conference presentations. What do you plan to do during the next reporting period to accomplish the goals?1) We will expand the work on on-site pathogen sensors to detect less than 10 cells/ml by optimization of the protocol. 2) Assessment of live cell dynamics and kinetics of DNA methylation 3) testing of oxygen nanobubles in increasing the efficacy of radiation therapy in cancer

Impacts
What was accomplished under these goals? 1) An onsite sensor to detect at 100 CFU/ml was developed based on magnetic lateral flow technology. Extensive specificity experiments were conducted. Initial evaluation of the sensor in juices was demonstrated. 2) Fluorescence microscopy techniques were applied to assess teh effect of TCE in epigenetic regulation. Such highly sensitive methods will be used to examine theeffect of contaminants in key epigenetic proteins. 3) We have developed a concept of trapping oxygen in cellulosic particles for delivery of oxygen to hypoxic tumor cells. Preliminary experiments in mice shows tremendous promise in teh utilization of these oxygen nanobubbles in treating hypoxia. 4) Optogenetic and CRISPR-Cas9 constructs were developed to demonstrate epigenetic regulation of DNA methylation at a specific gene.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Chowdhury, B., Arun, G., Thimmapuram, J., Lossie, A., and Irudayaraj, J. 2016. A study of alterations in DNA epigenetic modifications (5mC and 5hmC) and gene expression influenced by simulated microgravity in human lymphoblastoid cells. PLOS One, 11 (1), e0147514.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Wirbisky, S., Damayanti, N., Mahapatra, C., Sepulveda, M., Irudayaraj, J., and Freeman, J. 2016. Mitochondrial dysfunction, disruption of F-Actin polymerization, and transcriptomic alterations in zebrafish larvae exposed to trichloroethylene. Chemical Research in Toxicology. 29 (2): 169-179. DOI:10.1021/acs.chemrestox.5b00402.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mohammed, S., Ren, W., Rajwa, B., Chibwesha, C., Parham, G., and Irudayaraj, J. 2016. Point- of-care test for cervical cancer in LMICs. Oncotarget. DOI: 10.18632/oncotarget.7709.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cui, Y., Chowdhury, S., and Irudayaraj, J. 2016. Epigenetic toxicity of trichloroethylene  a single molecule perspective. Toxicology Research. 5: 641-655.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ren, W., Cho, I-L., and Irudayaraj, J. 2016. Ultrasensitive detection of microbial cells by magnetic enhanced lateral flow sensors. Chemical Communications, 52:4930-4933.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Choudhury, S., Cui, Y., Lubeca, K., Stefanska, B., and Irudayaraj, J. 2016. CRISPR-dCas9 mediated TET1 targeting for selective DNA demethylation at BRCA1 promoter. Oncotarget. 7(29): 46545-46556.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Liu, J. and Irudayaraj, J. 2016. Non-fluorescent quantification of single mRNA with transient absorption microscopy. Nanoscale. DOI:�10.1039/C6NR04433F.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lo., S., Resendiz, M., Chen, Y., Choudhury, S., Irudayaraj, J., Zhou, F. 2016. Epigenetic mechanism of alcohol on neural development demonstrated in a site-specific manner. Alcoholism-Clinical and Experimental Research. 40: 299A.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Choudhury, S., Cui, Y., Narayanan, A., Gilley, D., Huda, N., Lo, C., Zhou, F. 2016. Optogenetic regulation of site-specific subtelomeric DNA methylation. Oncotarget, 7(31):50380-50391.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hu, Y., Xuan, Y., Wang, X., Deng, B., Saei, M., Jin, S., Irudayaraj, J., and Cheng, G. 2016. Superplastic formulation of matal nanostructure arrays with ultrafine gaps. Advanced Materials. DOI: 10.1002/adma.201602497.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Ouyang, L., Hu, Y., Cheng, G., and Irudayaraj, J. 2016. A reusable laser wrapped graphene-Ag array based SERS sensor for trace detection of genomic DNA methylation. Biosensors and Bioelectronics. http://dx.doi.org/10.1016/j.bios.2016.09.072
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Cui, Y., Li, J., Weng, L., Wirbisky, S., Freeman, J., Liu, J., Liu, Q., Yuan, X., and Irudayaraj, J. 2016. Regulatory Landscape and Clinical Implication of MBD3 in Human Malignant Glioma. Oncotarget (In Press)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Mahapatra, C., Serafin, S., Guffey, S., Irudayaraj, J., and Sepulveda, M., 2016. Comparative in vitro toxicity assessment of perfluorinated carboxylic acids. Journal of Applied Toxicology. (In Press)