Microfluidic Single-Cell Melting Curve Analysis for Broad-Scale Detection of Microbial Organisms

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Despite considerable progress made in developing microfluidictechnologies for biological analysis, including droplet microfluidics to enable scalable partitioning of fluidinto tiny compartments for single cell analysis, transforming the technology into practical applications arestill hindered by 1) inadequate macro-to-micro interface to cope with large volume biological samplescontaining complex matrix; 2) complicated post-PCR processing for multiplexed, broad-scale genotypicanalysis; and 3) insufficient progress toward system integration.In this project, we plan to develop a fully integrated micro total analysis system (��TAS) for highthroughput broad-scale microbial detection and genotypic identification from a mixed population at singlecell resolution. Our novel platform will incorporate a microfluidic droplet generator to encapsulate singlebacterial cells into a large quantity of pL-sized droplets where the cells are lysed and enzymatic reactionstake place to facilitate high throughput single cell PCR amplification. For broad-scale bacterial detectionand species identification, we will incorporate a previously validated 16S rRNA PCR assay coupled withhigh resolution melt analysis (HRMA) for amplicon sequence analysis. This platform will not only providehighly accurate quantification of bacterial load, but the incorporation of HRMA at the single cell levelpermits both broad-scale species identification, as well as unprecedented resolution in measuring relativeabundance of bacterial species present in a mixed population.