A Combinatorial Approach for Microbial Sensing Using Multi-layered Nanoparticles Based on Surface Enhanced Raman Spectroscopy (sers)

<p>NON-TECHNICAL SUMMARY: <br/>Maintaining the expected quality and safety of food products is often difficult due to potential chemical and biological contamination. Of particular concern is the threat of food poisoning. Over 40 different foodborne microbial pathogens cause an estimated 30 million cases of human illness each year costing >$12 billion annually. Over the last 20 years, food-borne diseases caused by microbes have created increasingly significant concerns in the national political agenda and gained media attention. In addition to food-borne diseases, the use of pathogenic microorganisms as weapons of mass destruction remains a threat throughout the world. Agencies like, the United States Department of Agriculture (USDA) and the Department of Homeland Security must seek refined protocols for the rapid characterization and identification of pathogenic bacteria.
Although, classical microbiological tests are available for detection and identification of pathogenic bacteria in samples, they usually involve a number of analytical steps of long duration and must be conducted by highly qualified scientific personnel. Therefore, new and faster techniques based on molecular biology principles have emerged during the last 10 years to supplement traditional methodology. This project will assess the feasibility to develop multi-layered nanoparticles that can be integrated into a Surface Enhanced Raman Spectroscopy (SERS) system for simple, accurate and sensitive detection of foodborne pathogens. Successful completion of this project will provide a new avenue for rapid bacterial detection along with a viable product to be developed for food safety markets. The advantageous characteristics of nanomaterials have made them an important component in a number
emerging technologies. There are significant opportunities for the use of nano-material biosensors for food product and environmental monitoring, among many other offshoot applications. Therefore, development, of nanomaterial-based SERS biosensors has a high probability of usefulness. This Phase I grant is designed to build on preliminary research to prove the concept that SERS active nanoparticles can be designed and manufactured that can be directly used in a combinatorial approach to isolate and concentrate pathogenic microorganisms from food products as well as serve to enhance and develop a unique specific signal in a SERS optical biosensor. Both the particles and the optical method used to detect microorganisms have potential for commercialization.

<p>APPROACH: <br/>Three types of magnetic particles will be synthesized using a sol-gel method including silica coated magnetic iron oxide NPs, SIONPs with attached silver NPs and AgSIO - NPs with silica encapsulation. Non-magnetic Raman active Ag-NPs will also be synthesized including plain Ag-NPs, Ag-NPs linked with Raman active organic compounds and AgRNPs coated with silica. These NPs will be characterized using transmission electron microscopy, scanning electron microscopy, X-ray diffraction, magnetometry, zeta potential - particle size analysis, Fourier transform infrared spectroscopy, and Raman spectroscopy. The surface active nanoparticles (silica or silver) will then be functionalized with bacterial specific antibodies. The attachment to the surface of the silica particles will be accomplished using two methods. Method 1 will link the primary amine groups of the
antibody with the hydroxyl groups of the silica surface using cyanogen bromide. Method 2 will accomplished by adding active chemical groups to the surface of the silica through silane chemistry and then linking the antibody to the surface with a bifunctional linker such as glutaraldehyde. Nanoparticles with exposed silver surfaces will be functionalized using direct attachment of nucleophilic amine or thiol groups on the antibody to the silver or by adding chemical groups to the surface of the silver through self-assembled monolayers and linking to antibody using a bifunctional linker. Functionalized particles will be analyzed to determine the presence of the antibody on the surface and the ability of the antibody functionalized particle to recognize its specific antigen using standard Enzyme Linked Immunoassay approaches. Escherichia coli O157:H7 bovine strain provided by Dr.Gregory
Bohach will be used as the model target organism in this project. Total plate counts will be utilized to provide quantitative data for analysis of the capture capacity (cells captured/ milligram of particle), efficiency (time to reach maximum capture capacity), and specificity of multi-layered nanoparticles to isolate and concentrate bacteria from solution phase. To quantify the number of viable cells in solution and attached to the particles, colony forming units will be determined using standard plate count techniques through the use of serial dilutions in phosphate buffer saline. Aliquots will be plated on the agar plates and then incubated for 16 h at 37C. The plates will be counted by photography using a UVP multi-doc-it instrument. For detection of bacteria using multi-layered nanoparticles with SERS, three different magnetic nanoparticles will be used to capture bacteria in
combination with one of three Raman active particles or without a secondary particle, resulting in a total of 12 possible combinations. The samples (5-10 ?l) will be placed on glass slides and dried at RT for Raman spectroscopy. The Raman spectra will be recorded using a WITec alpha300 Raman Microscopy System equipped with 532 nm and 785 mm laser and detector. SERS spectral data will be collected and analyzed for specific chemical fingerprints related to the bacterial surface or Raman active molecules.

<p>PROGRESS: 2011/09 TO 2012/04
<p>OUTPUTS: <br/>The overall goal of this project was to develop multi-layered nanoparticles for integration into a Surface Enhanced Raman Spectroscopy (SERS) system that could provide simple, accurate and sensitive detection of foodborne pathogens. This Phase I grant was designed to build on preliminary research to prove the concept that SERS active nanoparticles could be designed and manufactured for direct use in a combinatorial approach to isolate and concentrate pathogenic microorganisms from food products as well as serve to enhance and develop a unique specific signal in a SERS optical biosensor. A number of nanoparticles were designed to be synthesized with variations in physical composition, size and Raman activity. A total of 6 base particles were utilized in this project. Silver nanoparticles of sizes 20, 40 and 60 nanometers and
paramagnetic particles of 2.8 micron and 1 micron diameter were used to produce a total of 20 different particle types. Nine of the silver nanoparticles were developed to be Raman active through labeling with 5,5 dithiobis (succinimidyl-2-nitrobenzoate) (DSNB) synthesized in our laboratory. All of the particles produced were shown be reactive against Escherichia coli (E. coli) O157 antigens following modification with antigen specific antibodies. The activity of the antibody was measured in a standard ELISA using heat killed E. coli O157 as a positive control and heat killed E. coli O111 as a negative control. The selectivity of the antibodies was shown to be very good in this approach (1000:1). A surface-based immunoassay was developed to show that dual functionalized (DSNB and antibodies) could be utilized for detection of E. coli O157. Particles with or without DSNB were shown to give
a visual signal related to the presence of the antigen. Only the complexes with DSNB labeled particles resulted in a measurable SERS signal. Twenty nanometer particles resulted in the best signal to background ratio. All paramagnetic particles showed the capacity to capture the bacteria. Based on the use of a standard ELISA it was determined that the maximum signal increased as the quantity of particles was increased up to 30 micrograms of magnetic microparticles per 200 microliters of sample. A SERS procedure was then developed to detect E. coli O157 using immunomagnetic particles and DSNB activated silver nanoparticles. After capture of the E. coli, the specific immunomagnetic complexes were separated from solution, silver enhanced, dried and analyzed using SERS. No discernible signal was achieved with non-silver enhanced samples regardless of particle type or size. DSNB containing
silver particles in combination with the 2.8 micron non-decorated immunomagnetic particles, showed a significantly higher SERS signal at several wave numbers for those samples containing the bacteria. There appeared to be no difference in the response when using different sizes of the silver particles. Immunomagnetic particles pre-coated with silver particles showed high Raman signal enhancements but had higher backgrounds.
PARTICIPANTS: Josh Branen- Program director, assay design, development and integration of assays with SERS detection, lead on nanoparticle modification; Larry Branen: co- Program director, data analysis, technical support and assay design; Shiva Rastogi- co- Program director, chemical synthesis and chemical analysis. Collaborators and contacts: D. Eric Aston, Andrew Weakley.
TARGET AUDIENCES: Large and small scale food producers, biotechnology based companies, and
current diagnostic and food safety platform developers. PROJECT MODIFICATIONS: The original intention was to fabricate nanoparticles de novo on site. It was determined the time and cost to acieve a high quality product was better focused in other areas of assay development and design. As a result, base particles were purchased from commercial sources and further modifications were completed in our laboratory.