In this application we propose to develop an improved method for detection of proteins secreted from single cells. The proposed method offers several significant advantages over the existing technologies;a simple biochemical protocol, detection of multiple secreted proteins, and real-time monitoring of the secretion process. Our approach is to employ optical amplification of fluorescence signal from a probe bound to the surface in the vicinity of a protein-secreting cell. The optical amplification will be achieved using metallic nanostructures deposited on the surface of a solid support. The method is termed MEFspot (Metal-Enhanced Fluorescence spot). This will avoid the use of more complex enzymatic amplification or biochemical amplifications used in current technologies;enzyme-linked immunospot (ELISPOT) and FLUOROSPOT, respectively. MEFspot represents a significant advance in single cell assays with sensitivity comparable to ELISPOT but utilizes a substantially simpler procedure, capabilities for real-time monitoring, and potential use for quantitative analysis. Specifically, we will demonstrate the application of MEFspot to the detection of cytokines from cells because cytokines play an important role during inflammation and diseases and they are regarded as the best tool to measure the activation of immune cells. It was found that a large number of diseases are associated with quantitative patterns of cytokine production. Cytokines have assumed increasing importance in cancer biology with the demonstration that many can be produced by tumor cells and can influence the malignant process both positively and negatively. The projected work will be accomplished by (1) Developing a procedure for reproducible fabrication of substrates with metallic nanostructures that perform in culture media for a prolonged length of time and result in the desired sensitivity for cytokine detection. (2) Evaluating MEFspot with several cytokines using model cell lines. (3) Demonstrating the multiplexing capability for detection dual (or possibly triple) cytokine secretion from a single T cell. (4) Validating the MEFspot method using clinical cell samples with comparison to the ELISPOT method. Various formats of MEFspot including real-time monitoring of secretion and quantitative analysis of single cell efficiency will be demonstrated.
The development of methods for high-throughput analysis of cell functionalities is highly in demand in current life science research, proteomics and its clinical applications, as well as in drug discovery. We propose to develop a significantly improved method for detection of proteins secreted from single cells. The method includes the optical amplification of signal from bound fluorescent probes allowing for real-time monitoring, multiplexing and quantization. It is anticipated that proposed method will be of broad use in immunology research, and in conjunction with other functional assays for detection of intracellular and blood level of cytokines, provide a better understanding of fine mechanisms of immune response.
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