High-Throughput Single Molecule Analysis Instrument

The analysis and control of single macromolecules is an important goal of the DOE¿s Genome-to-Life Program. However, single molecule assays that use fluorescent dyes are not photostable, have low quantum yield, and have broad emission spectra. Conventional, confocal, fluorescence correlation microscopes use only one pair of detectors, have low throughput, lack multiparameter analysis, are inefficient, and cannot address multiple excitation and detection of the sample zones. This project will employ an array of fast microavalanche photodiodes to simultaneously detect several zones of the probing volume and use fluorescence-coincident analysis for high throughput screening. Innovative fluorescence-enhancing, nanostructured metallic substrates will be employed to enhance the quantum yield from any type of fluorescent molecule. Phase I will develop thin metallic substrates with 50-400 nm rectangular hole arrays to enhance single molecule fluorescence. Quantum dots will be conjugated to DNA molecules and will be used as fluorescent probes. Coincident burst analysis will be employed to detect the hybridization of a DNA to its conjugate. Optimal speed and the detection limits will be determined using 2-4 pairs of microavalanche photodiodes. Statistical analysis for quantum dot assays and enhancement factors will be performed. Commercial Applications and Other Benefits as described by the applicant: A microavalanche photodiode array with coincident analysis and sample scanning should increase the throughput for bioassay screening of extremely dilute amounts of pathogens. The resulting instrument should be useful for drug discovery and bioassaying, and for the study of biomolecular interactions, molecular and subcellular dynamics, enzyme kinetics, and structural/conformational changes in proteins or RNAs