NANOTECHNOLOGY AND BIOSENSORS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010419
• Project No.: WIS01953
• Multistate No.: NC-_old1194
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2018

Project Director
Gunasekaran, S.

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Biological Systems Engineering

Non Technical Summary
Enteric bacteria such as Escherichia coli (E. coli) and salmonella cause unpredictable outbreaks of foodborne and waterborne diseases that can have dire consequences for our health and economy. Biosensor technology is the fastest growing area of pathogen detection. Though many sensors and detection techniques are available to meet the stringent detection sensitivity requirement, they fall short in terms of simplicity and rapidity. Many of these sensors require expensive and lab-use-only devices. Therefore, biosensors that provide a visually recognizable or colorimetric signal are the most appealing because they are comparatively simple to use and generate data that are easy to interpret. We aim to develop an easy-to-use biosensor will be developed to test food suspensions in out-of-lab settings for the presence or absence of target bacteria. The system will be optimized to detect various amounts of target organisms yielding a result that is visible to the naked eye.

Animal Health Component

0%

Research Effort Categories

Basic

50%

Applied

30%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	5010	2020	100%

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;

Keywords

biosensor

gold nanoparticles

pathogenic bacteria

visual signal

immunosensing

Goals / Objectives
Develop new technologies for characterizing fundamental nanoscale processes Develop devices and systems incorporating microfabrication and nanotechnology

Project Methods
We will use an immunogenic biosensing method.Part 1: Biosensor Development:We will employ a bifunctional linker that has the binding affinity to the target organism as well as to aggregate gold nanoparticles. The aggregation of gold nanoparticles, in the absence of target organism, will give raise to change in the system color -- from red to purple. Once optimized, the red-to-purple color change can be a visual indication of the absence of organism above a pre-set threshold level.Part 2: Evaluate and Optimize Biosensor Performance-We will evaluate assay performance as a function of various critical parameters including: the size and shape of AuNPs, the concentration of AuNPs, amount of streptavidin coating necessary on AuNPs, and the quantity of biotinylated antibody added to the system. The effects of variations in reaction time and temperature will also be examined.Part 3: Determine Biosensor Effectiveness in Real Matrices-We will consider a number of real matrices including fruit juices, milk and suspensions of solid foods. For establishing the detection effectiveness of the assay, known quantities of target organisms will be tested in a variety of water matrix interferents expected to be found at fresh produce production facilities.

Progress 10/01/16 to 09/30/18

Outputs
Target Audience:The target audiences are academic and research community and food and bioprocessing industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student was trained on this project. How have the results been disseminated to communities of interest?Results were disseminated through publications and professional society meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? On-site detection and rapid identification of foodborne pathogens on fresh products with minimal use of specialized instrumentation is crucial to the food industries. A switchable linker (SL)-based immunoassay was designed for the on-site detection of Salmonella in tomato samples. The assay is based on the large-scale aggregation of gold nanoparticles (AuNPs), which is induced by a quantitative relationship between the biotinylated Salmonella polyclonal antibody (b-Ab) used as the SL, the functionalized AuNPs(f-AuNPs), and Salmonella. Important factors, such as the concentration of SLs, time required for large-scale aggregation, and selectivity of b-Ab, were optimized to minimize the detection time (within 45 min with gentle agitation) and achieve the lowest limit of detection (LOD, 103CFU/200 g in tomato samples) possible. This SL-based immunoassay with its relatively low LOD and short detection time may answer the need for a rapid, simple on-site analysis for pathogens in various fresh produce.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang YC, Mohan CO, Guan JH, Ravishankar CN, Gunasekaran S. 2018. Chitosan and gold nanoparticles-based thermal history indicators and frozen indicators for perishable and temperature-sensitive products. Food Control 85:186-93.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wang YC, Lu L, Gunasekaran S. 2017. Biopolymer/gold nanoparticles composite plasmonic thermal history indicator to monitor quality and safety of perishable bioproducts. Biosens Bioelectron 92:109-16.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hahn J, Kim E, You YS, Gunasekaran S, Lim S, Choi YJ. 2017. A switchable linker-based immunoassay for ultrasensitive visible detection of salmonella in tomatoes. J Food Sci 82:2321-8.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Academic community and food industry personnel Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One PhD student was trained in nanotechnologyand food safety. How have the results been disseminated to communities of interest?The results were disseminated in two peer-review journal publications. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we will test and validate detectingpathogens in large sample volume without sample pre-treatment.

Impacts
What was accomplished under these goals? The results of our work for the past year show that we will be able to monitor degradation of food quality as affected by storage time and temperature non-invasively by visual observation of color change of out nonreactor affixed on the product or package. In our immunobiosensor we use biontinylated anti-bacteria antibodies as bifunctional linkers (BLs) to mediate aggregation of streptavidin-functionalized gold nanoparticles (st-AuNPs) to produce visually recognizable color change, due to surface plasmon resonance (SPR), rapidly within three hours of total assay time in quiescent conditions and in about 15 min when the sample is agitated. However, the aggregation of st-AuNPs, which produces the indication signal, is achieved very differently than in visual detection methods reported previously and allows for ultrahigh sensitivity. While BLs can both bind to the target and crosslink st-AuNPs, their latter function is essentially disabled when they bind to the target bacteria. Aqueous AuNPs, 13 nm in diameter, were synthesized according to the Turkevich method and colloidal gold was synthesized by reduction of a HAuCl4·3H2O aqueous solution by sodium citrate at ebullition. In an Erlenmeyer flask equipped with a hot plate, 10 mL of 1 mM HAuCl4·3H2O was brought to a rolling boil. 1 mL of 38.8 mM citrate sodium was added to 10 mL boiling solution of 1 mM HAuCl4·3H2O. The solution turned dark brown within 10 s; the final color change to burgundy occurred 50 s later. The solution was cooled down to room temperature and a red solution of AuNPs was obtained. To prepare st-AuNPs, 600 µL of colloidal AuNPs was added to 200 µL of streptavidin (0.2 mg mL-1 in borate buffer). The mixture was incubated at room temperature and centrifuged to remove any excess streptavidin present and were restored in 1 % (w v-1) of BSA dissolved in PBS. By varying the amount of st-AuNPs used, we were able to tailor the assay effectiveness in terms of improved limit of detection (LOD) down to 10 CFU mL-1 of E. coli and Salmonella. Test results obtained with real matrices such as tap and lake water and milk show that the assay performance is unaffected by the matrix effects. Therefore, our BL-based nanobiosensor is suitable for highly sensitive, rapid, and on-site detection of bacterial targets in real matrices.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: You Y, Lim S, Hahn J, Choi YJ, Gunasekaran S. 2018. Bifunctional linker-based immunosensing for rapid and visible detection of bacteria in real matrices. Biosensors and Bioelectronics, 100:389-395.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Hahn J, Kim E, You YS, Gunasekaran S, Lim S, Choi YJ. 2017. A Switchable Linker-Based Immunoassay for Ultrasensitive Visible Detection of Salmonella in Tomatoes. J. of Food Sci. (10):23212328. DOI: 10.1111/1750-3841.13861