Microfabricated two-dimensional electrophoresis device for differential protein expression profiling.

A microfluidic separation system is developed to perform two-dimensional differential gel electrophoretic (DIGE) separations of complex, cellular protein mixtures produced by induced protein expression in E. coli. The micro-DIGE analyzer is a two-layer borosilicate glass microdevice consisting of a single 3.75 cm long channel for isoelectric focusing, which is sampled in parallel by 20 channels effecting a second-dimension separation by native electrophoresis. The connection between the orthogonal separation systems is accomplished by smaller channels comprising a microfluidic interface (MFI) that prevents media leakage between the two dimensions and enables facile loading of discontinuous gel systems in each dimension. Proteins are covalently labeled with Cy2 and Cy3 DIGE and detected simultaneously with a rotary confocal fluorescence scanner. Reproducible two-dimensional separations of both purified proteins and complex protein mixtures are performed with minimal run-to-run variation by including 7 M urea in the second-dimension separation matrix. The capabilities of the micro-DIGE analyzer are demonstrated by following the induced expression of maltose binding protein in E. coli. Although the absence of sodium dodecyl sulfate (SDS) in the second-dimension sizing separation limits the orthogonality and peak capacity of the separation, this analyzer is a significant first step toward the reproducible two-dimensional analysis of complex protein samples in microfabricated devices. Furthermore, the microchannel interface structures developed here will facilitate other multidimensional separations in microdevices.

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice.

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.

High-throughput single-strand conformation polymorphism analysis on a microfabricated capillary array electrophoresis device.

A high-density 384-lane microfabricated capillary array electrophoresis device is evaluated for high-throughput single-strand conformation polymorphism (SSCP) analysis. A delayed back bias direct electrokinetic injection scheme is used to provide better than 10-bp resolution with an 8.0-cm effective separation length. Separation of a HaeIII digest of PhiX174 yielded theoretical plate numbers of 4.0 x 10(6). Using 5% PDMA containing 10% glycerol and 15% urea, 21 single-nucleotide polymorphisms (SNPs) from HFE, MYL2, MYL3, and MYH7 genes associated with hereditary hemochromatosis (HHC) and hereditary hypertrophic cardiomyopathy (HCM) are discriminated at two running temperatures (25 degrees C and 40 degrees C), providing 100% sensitivity. The data in this study demonstrate that the 384-lane microCAE device provides the resolution and detection sensitivity required for SSCP analysis, showing its potential for ultrahigh-throughput mutation detection.

Capillary electrophoresis chips with a sheath-flow supported electrochemical detection system.

Microfabricated capillary electrophoresis chips containing an integrated sheath-flow electrochemical detector are developed with the goal of minimizing the influence of separation voltages on end-column detection while maintaining optimum performance. The microdevice consists of an upper glass wafer carrying the etched separation, injection, and sheath-flow channels and a lower glass wafer on which gold- and silver-plated electrodes have been fabricated. The sheath-flow channels join the end of the separation channel from each side, and gravity-driven flow carries the analytes to the electrochemical detector placed at working distances of 100, 150, 200, and 250 microm from the separation channel exit. The performance of this detector is evaluated using catechol and a detection limit of 4.1 microM obtained at a working distance of 250 microm. Detection of DNA restriction fragments and PCR product sizing is demonstrated using the electroactive intercalating dye, iron phenanthroline. Additionally, an allele-specific, PCR-based single-nucleotide polymorphism typing assay for the C282Y substitution diagnostic for hereditary hemochromatosis is developed and evaluated using ferrocene-labeled primers. This study advances the feasibility of high-speed, high-throughput chemical and genetic analysis using microchip electrochemical detection.

Microfabricated 384-lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis.

A microfabricated 384-lane capillary array electrophoresis device is developed and utilized for massively parallel genetic analysis. The 384 capillary lanes, arrayed radially about the center of a 200-mm-diameter glass substrate sandwich, are constructed using scalable microfabrication techniques derived from the semiconductor industry. Samples are loaded into reservoirs on the perimeter of the wafer, separated on the 8-cm-long poly(dimethylacrylamide) gel-filled channels, and detected with a four-color rotary confocal fluorescence scanner. The performance and throughput of this bioanalyzer are demonstrated by simultaneous genotyping 384 individuals for the common hemochromatosis-linked H63D mutation in the human HFE gene in only 325 s. This lab-on-a-chip device thoroughly exploits the power of microfabrication to produce high-density capillary electrophoresis arrays and to use them for high-throughput bioanalysis.

High throughput DNA sequencing with a microfabricated 96-lane capillary array electrophoresis bioprocessor.

High throughput DNA sequencing has been performed by using a microfabricated 96-channel radial capillary array electrophoresis (microCAE) microchannel plate detected by a 4-color rotary confocal fluorescence scanner. The microchannel plate features a novel injector for uniform sieving matrix loading as well as high resolution, tapered turns that provide an effective separation length of 15.9 cm on a compact 150-mm diameter wafer. Expanded common buffer chambers for the cathode, anode, and waste reservoirs are used to simplify electrode addressing and to counteract buffering capacity depletion arising from the high electrophoretic current. DNA sequencing data from 95 successful lanes out of 96 lanes run in parallel were batch-processed with basefinder, producing an average read length of 430 bp (phred q > or = 20). Phred quality values were found to exceed 40 (0.01% probability of incorrectly calling a base) for over 80% of the read length. The microCAE system demonstrated here produces sequencing data at a rate of 1.7 kbp/min, a 5-fold increase over current commercial capillary array electrophoresis technology. Additionally, this system permits lower reagent volumes and lower sample concentrations, and it presents numerous possibilities for integrated sample preparation and handling. The unique capabilities of microCAE technology should make it the next generation, high performance DNA sequencing platform.