David Roach

Virginia Polytechnic Institute and State University, Блэксбург, Virginia, United States

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Publications (4)19.59 Total impact

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    ABSTRACT: We describe a whole-capillary, multicolor laser-induced fluorescence scanner for microfluidic protein analysis systems. Separation of proteins is achieved by isoelectric focusing in a short length of fused-silica capillary after which the resolved proteins are immobilized to the capillary wall using photochemistry. The capillary is then evacuated, and fluorescently labeled antibodies are flowed through the capillary to bind to the immobilized proteins. This technique provides high sensitivity, the ability to spatially resolve and quantify proteins, and provides the opportunity for complete automation. Results obtained by fluorescence detection are compared to those obtained by chemiluminescence while offering enhanced resolution and signal stability.
    Analytical Chemistry 01/2008; 79(24):9478-83. DOI:10.1021/ac071537z · 5.64 Impact Factor
  • SE HOOPER · David Roach · MR ANDERSON
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    ABSTRACT: Use of a square-wave potential program for time-dependent amperometric detection of analyte zones in capillary electrophoresis (CE) is described. Electrochemical detection for CE requires that the separation field be isolated from that of the electrochemical detection. This is generally done by physically separating the CE separation field from that of the detection. By applying a time variant potential program to the detection electrode, the detector current has a time dependence that can be used to help isolate the electrochemical detection current from that of the separation. When using a 20 μm inner-diameter capillary, we find that a square-wave potential program decreases the RMS baseline current from 4.5×10−10 A, found with a constant potential amperometric detection, to 1.1×10−10 A when using a square-wave potential program. With a 75 μm inner-diameter capillary, the improvement is even more dramatic, from 2.3×10−9 A with amperometric detection to 2.06×10−10 A when using a 1 Hz square-wave potential program. When not using the time-dependent detection with the 75 μm capillary, the analyte zones were beneath the S/N for the system and not detected. With the square-wave potential program and time-dependent detection, however, the analyte zones for an electrokinetic injection of 200 μM solution of 2,3-dihydroxybenzoic acid were observed with the 75 μm inner-diameter capillary. The improvement in the ability to discriminate the analytical signal from the background found experimentally is consistent with modeling studies.
    Electroanalysis 01/2008; 20(1):102-106. DOI:10.1002/elan.200704077 · 2.14 Impact Factor
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    ABSTRACT: A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.
    Proceedings of the National Academy of Sciences 11/2006; 103(44):16153-8. DOI:10.1073/pnas.0607973103 · 9.67 Impact Factor
  • David M. Roach · Stephanie E. Hooper · Mark R. Anderson
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    ABSTRACT: Electrochemical detection in capillary electrophoresis requires decoupling the voltage applied to the working electrode from the separation voltage applied across the capillary. End-capillary electrochemical detection achieves this by placing the electrode just outside the ground end of the separation capillary. Obtaining adequate signal-to-noise in this arrangement requires using small inner diameter capillaries. Decreasing the inner diameter of the separation capillary, however, increases the difficulty of aligning the microelectrode with the open end of the capillary. Using scanning electrochemical microscopy (SECM), the position of the capillary opening is determined while electroactive material is continuously emerging from the end of the capillary. The SECM instrument is then used to place the electrode at the position of maximum current for subsequent separations. Subsequent measurements found that the best signal-to-noise is obtained when the detection electrode is placed directly opposite the capillary opening and just outside of the capillary opening. When the electrode is further above the opening (but still opposite the capillary opening), the signal-to-noise does not dramatically decrease until the electrode is more than 30 μm above the 10 μm inner-diameter capillary.
    Electroanalysis 12/2005; 17(24):2254-2259. DOI:10.1002/elan.200503368 · 2.14 Impact Factor