Targeted Deposition of Antibodies on a Multiplex CMOS Microarray and Optimization of a Sensitive Immunoassay Using Electrochemical Detection
COMBIMATRIX CORP MUKILTEO WA
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The CombiMatrix ElectraSenseregistered microarray is a highly multiplex, complementary metal oxide semiconductor with 12,544 electrodes that are individually addressable. This platform is commercially available as a custom DNA microarray and, in this configuration, it has also been used to tether antibodies Abs specifically on electrodes using complementary DNA sequences conjugated to the Abs. An empirical method is described for developing and optimizing immunoassays on the CombiMatrix ElectraSenseregistered microarray based upon targeted deposition of polypyrrole Ppy and capture Ab. This process was automated using instrumentation that can selectively apply a potential or current to individual electrodes and also measure current generated at the electrodes by an enzyme-enhanced electrochemical ECD reaction. By designating groups of electrodes on the array for different Ppy deposition conditions, we determined that the sensitivity and specificity of a sandwich immunoassay for staphylococcal enterotoxin B SEB is influenced by the application of different voltages or currents and the application time. The sandwich immunoassay used a capture Ab adsorbed to the Ppy and a reporter Ab labeled for fluorescence detection or ECD, and results from these methods of detection were different. Using Ppy deposition conditions for optimum results, the lower limit of detection for SEB using the ECD assay was between 0.003 and 0.01 pgml, which represents an order of magnitude improvement over a conventional enzyme-linked immunosorbant assay. In the absence of understanding the variables and complexities that affect assay performance, this highly multiplexed electrode array provided a rapid, high throughput, and empirical approach for developing a sensitive immunoassay.
- Genetic Engineering and Molecular Biology
- Medicine and Medical Research
- Electrical and Electronic Equipment
- Test Facilities, Equipment and Methods