Multiplexed protein detection using antibody-conjugated microbead arrays in a microfabricated electrophoretic device.
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.
Barbee
Kristopher D
KD
Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093-0412, USA.
Hsiao
Alexander P
AP
Roller
Eric E
EE
Huang
Xiaohua
X
D000818:Animals; D000941:Antigens / Q000032:analysis / Q000378:metabolism; D004574:Electromagnetic Fields; D046209:Electrophoresis, Microchip / Q000295:instrumentation* / Q000379:methods; D020342:Finite Element Analysis; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D006046:Gold / Q000737:chemistry; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008863:Microspheres; D011817:Rabbits; D012822:Silicon Dioxide / Q000737:chemistry
D000941:Antigens; D007074:Immunoglobulin G; D006046:Gold; D012822:Silicon Dioxide; D016650:Fluorescein-5-isothiocyanate
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