Multidimensional microfluidic separation systems for differential proteome analysis.

摘要

High-density arrays of microfabricated channels are created for high-throughput genetic analysis and adapted to create a unique platform for the two-dimensional electrophoretic analysis of complex mixtures of bacterial proteins.; I tasted success early with the development of an ultra-high-density 384-lane bioanalyzer for high-throughput electrophoresis of DNA. The bioanalyzer was demonstrated by genotyping 384 individuals simultaneously for the H63D mutation in the human HFE gene in only 325 s. To date this represents the highest throughput and density for any microfabricated electrophoresis device.; This early success in genetic analysis led me to what I saw as a more challenging and worthwhile endeavor: proteomics. Chapter 3 chronicles my three-year microfluidic adaptation of the two-dimensional electrophoresis (2DE) technologies of isoelectric focusing (IEF) and protein electrophoresis. The electrical and chemical isolation of IEF from the contents of the 2nd-dimension proved necessary, as did the exclusion of the detergent SDS. To address these challenges, channels of the two dimensions were connected via a novel microfluidic interface (MFI) that consisted of microchannels one-tenth the depth of the separation channels. The MFIs act as a fluidic barrier to prevent mixing between the two dimensions and to facilitate discontinuous loading of IEF samples and 2nd-dimension gels while still allowing electrical connection.; I employed the optimized 2DE microdevice comprised of twenty 2 nd-dimension channels and a 3.75-cm long IEF channel to monitor protein expression in bacterial cell lysates. Chapter 4 tells how the move to cell lysates precipitated a collapse in 2DE reproducibility that was reversed by lowering the electric field of the 2nd-dimension and by including urea in the separation matrix. Using these optimized conditions, the 2DE microdevice successfully monitored the first three hours of differential expression between, lactose-induced and control populations of fluorescently-labeled protein samples from engineered E. coli. In the end, each 2DE run required only one hour and a 220-nL protein sample, marking more than a ten-fold reduction in time and volume when compared to conventional 2DE.; In Chapter 5, I propose a next-generation static-stack injector (SSI), based on the 2DE microdevice, to replace the standard inefficient cross-injector for on-chip electrophoretic analysis. The SSI combines MFIs and offset orthogonal channels to precisely define and electrophorese sample plugs in a single step. In superseding the cross injector, the SSI eliminates injection bias, increases resolution by sample stacking, and simplifies the supporting electronics. The dense channel arrays and microfluidic interfaces I developed are a significant step in broadening the capabilities of high-throughput microfabricated analysis systems.