Multiplexed screening is a tool that finds broad use in applications such as drug discovery, genotyping, medical diagnostics, and blood typing for transfusion safety, and will be of utmost importance in the up-and-coming field of """"""""personalized"""""""" medicine. The two commercially available screening technologies offer either a high """"""""density"""""""" of analytes measured (i.e. planar microarrays) or high sample throughput (ie. bead-based systems), but not both. This application proposes the fundamental development of a new screening technology, based on multi-functional encoded particles, which could provide the density of microarrays and throughput of bead-based systems. Preliminary results show that particles composed of a spongy hydrogel material, with a punch-code barcode written on one half and a stripe for target capture on the other, can be used to simultaneously quantify targets in a single biological sample, with coding capabilities of over one million. In building upon a proof-of-concept demonstration, it is hypothesized that (1) increasing the size of pores in the hydrogel structure will allow targets to bind throughout the particle, increasing the sensitivity of each assay while decreasing required incubation times;(2) that hybridization conditions can be tuned to achieve performance competitive with existing technologies;and that (3) a flow-through system based on microfluidics and photomultiplier technologies can be used to rapidly scan particles (i.e. read codes and quantify targets).
The specific aims of the project are: (1) Enhance particle synthesis by exploring chemical variations and processing conditions to generate particles that are sufficiently porous and mechanically robust. Particles will be examined via microscopy and probed with FITC-conjugated dextrans. (2) Optimize the physical and chemical conditions of DNA hybridization assays to maximize sensitivity, specificity, and reproducibility. (3) Develop a microfluidic flow-based system for rapid scanning that integrates a flow-focusing microfluidic device, photomultiplier-aided fluorescence detection, and software to decode the acquired signal. The end goal of this project is to have a system capable of quantifying 2,500 nucleic acid targets per sample, detecting targets with single base-pair discrimination at a better sensitivity than commercially available systems, and scanning 5,000 particles at a rate of 500 particles per minute. The relevance of this project to public health is the development of a transformative technology for genomic medicine, ranging from disease diagnosis to drug discovery.
This project will develop a new technology that can be used to simultaneously detect thousands of biomolecules in a solution. This new technology will find potential use in disease diagnosis/treatment, blood typing for increasing the safety of transfusions and drug development.
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|Chapin, Stephen C; Doyle, Patrick S (2011) Ultrasensitive multiplexed microRNA quantification on encoded gel microparticles using rolling circle amplification. Anal Chem 83:7179-85|
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|Pregibon, Daniel C; Doyle, Patrick S (2009) Optimization of encoded hydrogel particles for nucleic acid quantification. Anal Chem 81:4873-81|
|Chapin, Stephen C; Pregibon, Daniel C; Doyle, Patrick S (2009) High-throughput flow alignment of barcoded hydrogel microparticles. Lab Chip 9:3100-9|
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