Tuesday, May 12, 2009

May 15, 2009; Homewood, Clark 110
-- Chris Puleo

Title: "Accessible Single Molecule Detection Technologies: Microfluidic Interfaces
for Diagnostic and Single Cell Applications"

Abstract: The long term goal of this proposal is to develop microfluidic technologies that enable the widespread use of confocal fluorescence spectroscopy (CFS) for single molecule detection (SMD) in vital applications including, single molecule diagnostics and single cell analysis. Currently amplification techniques have been used to determine the presence of rare biomolecules in such applications; however, the cost, complexity, and time requirements associated with these tests limit clinical value and general utility. Direct molecule-by-molecule assessment using SMD remains an intriguing replacement for amplification technologies due to high sensitivity, assay simplicity, and low costs; however, technical challenges continue to limit the utility of these single molecule techniques. We use microfluidic interfaces for SMD platforms to expand applicability in the analysis of rare molecules from complex biological fluids. The overall goal of this work is apply microfluidics to SMD assays in order to obtain the lowest possible detection limits from the smallest possible sample volume. Direct implications are clear, high-throughput biomolecular analysis from the most precious and rare biological samples. In theory, single molecule sensitivity confers infinite detection limits in CFS platforms; however, in practice SMD platforms rely on continuous flow formats and bulk probe-target reactions. These design flaws yield significant fundamental pitfalls and result in practical limitations: 1) Analyte delivery to the microscale sensing elements is wasteful, leading to extremely low measurement efficiencies and the need for excessive sample volumes. 2) Passive probe-target interactions result in slow reaction kinetics and prohibitive assay run times. 3) Unoptimized and manual processing of target molecules results in low molecular throughput and incompatibility with arrayed formats. Experiments will be performed using two microfluidic platforms, multilayer soft lithography and water-in-oil droplets. Coupling these discrete volume control technologies with SMD provides direct control over probe hybridization, efficient transfer of target molecules to optical detection volumes, and automated processing of large numbers of sample in relatively short assay times. These improvements serve to open the door to practical use of single molecule assays in areas of vital need, such as, non-invasive diagnostic screening and investigation of cell-to-cell heterogeneity. Our lab stands in a unique position to take advantage of this potential due to practical experience in all three aspects of this challenge, including single molecule probe design, microfluidic device design and fabrication, and optical CFS platform engineering.