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      Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device.

      Lab on a Chip
      Animals, Antigens, analysis, metabolism, Electromagnetic Fields, Electrophoresis, Microchip, instrumentation, methods, Finite Element Analysis, Fluorescein-5-isothiocyanate, chemistry, Gold, Immunoassay, Immunoglobulin G, Mice, Microscopy, Fluorescence, Microspheres, Rabbits, Silicon Dioxide

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          Abstract

          We report the development of a microfabricated electrophoretic device for assembling high-density arrays of antibody-conjugated microbeads for chip-based protein detection. The device consists of a flow cell formed between a gold-coated silicon chip with an array of microwells etched in a silicon dioxide film and a glass coverslip with a series of thin gold counter electrode lines. We have demonstrated that 0.4 and 1 μm beads conjugated with antibodies can be rapidly assembled into the microwells by applying a pulsed electric field across the chamber. By assembling step-wise a mixture of fluorescently labeled antibody-conjugated microbeads, we incorporated both spatial and fluorescence encoding strategies to demonstrate significant multiplexing capabilities. We have shown that these antibody-conjugated microbead arrays can be used to perform on-chip sandwich immunoassays to detect test antigens at concentrations as low as 40 pM (6 ng/mL). A finite element model was also developed to examine the electric field distribution within the device for different counter electrode configurations over a range of line pitches and chamber heights. This device will be useful for assembling high-density, encoded antibody arrays for multiplexed detection of proteins and other types of protein-conjugated microbeads for applications such as the analysis of protein-protein interactions.

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