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We will concentrate our effort on achieving several specific objectives during the funding period. <OL> <LI> To develop off-chip protocols that clean and concentrate food-derived bacterial samples (product rinses) for analysis on chip. <LI> To develop microfluidic platform for capture and lysis of bacterial cells and for immunoassays. When high specificity is desired, we will incorporate one additional immunoassay targeting at a surface antigen of L. monocytogenes during the cell capture step on chip. <LI>To develop processes for fabricating nanoporous silicon sensor array for label-free antigen detection. The nanoporous silicon sensor array will be fabricated by combining microfabrication and electrochemical etching. We will also optimize the protocol for immobilizing antibodies on the inner surface of nanoporous silicon.<LI> To systematically study the relationship between nanoscale dimensions of nanoporous silicon and the sensitivity and specificity of the sensor. Listeria species will be detected based on detecting an intracellular antigen (alcohol acetaldehyde dehydrogenase (Aad), also known as Listeria adhesion protein (LAP)) from cell lysate using nanoporous silicon sensors. We will fully characterize and optimize the sensitivity and specificity of the sensor using Aad and its monoclonal antibody MAb-H7 together with model antigens and antibodies such as horseradish peroxidase (HRP) and anti-HRP, streptavidin and biotin. We will also demonstrate simultaneous detection of multiple antigens using the sensor array.
NON-TECHNICAL SUMMARY: Nanoporous silicon is an attractive material for label-free biosensing due to its high surface area, convenient surface chemistry, and optical signal transduction capability. The shift in the Fabry-Perot interference fringes due to change in the refractive index of a nanoporous silicon film has been demonstrated as a transduction mechanism for specific detection of antigen-antibody binding. In this proposal, our goal will be to develop a prototype integrated sensor system which has nanoporous silicon sensor array incorporated onto a microfluidic platform for simultaneous detection of multiple antigens. We will apply this system to bacteria detection relevant to food safety by demonstrating detection of Listeria monocytogenes based on recognition of an intracellular antigen. After off-chip treatment of biological samples, the microfluidic platform will capture and lyse the cells and the released antigen will bind to its antibody immobilized on the inner surface the nanopores. This specific binding will introduce significant change in the reflective interferometric spectra yielded by the nanoporous silicon sensors. We will fully characterize and optimize the sensitivity and specificity of the device. The relationship between the size of the nanopores and the performance of the biosensor will be systematically studied. We will demonstrate detecting multiple antigens by immobilizing different antibodies in the sensor array. We expect that our approach will yield a new generation of nanoscale biosensor systems for sensitive, rapid, and simultaneous detection of multiple pathogens. We believe that such portable nanosensor systems will be very valuable for point-of-care applications in food industry. This project addresses Priority 1.
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APPROACH: In the proposed study, our goal will be to develop a prototype chip-based sensor system which has nanoscale sensor array incorporated onto a microfluidic platform for bacteria detection. We will demonstrate simultaneous detection of L. monocytogenes and two other molecular species (horseradish peroxidase and streptavidin) using the device with a limit of detection of ~20 cells for the bacterium. The highly integrated chip will include two main components: the nanoporous silicon sensor array and the microfluidic platform for cell capture and lysis. Briefly, bacterial suspension after off-chip cleaning and concentration will flow into the chip and be filtered by a packed bed of microbeads and the bacterial cells will be retained in the bead array. The cells will then be lysed by applying electric pulses and intracellular materials (containing the target antigen) will be released to the downstream of the bead array where the nanoporous silicon sensor array is located. The reflective interferometric spectra will be taken at each spot of the array and the effective optical thickness (EOT) will be extracted by doing Fourier transform of the data. The results will indicate the presence of target antigen(s).