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Bead-based immunoassays with microelectrode detection

Authors
  • Farrell, Svetlana1
  • Ronkainen-Matsuno, Niina J.1
  • Halsall, H. Brian1
  • Heineman, William R.1
  • 1 University of Cincinnati, Department of Chemistry, Cincinnati, OH, 45221-0172, USA , Cincinnati
Type
Published Article
Journal
Analytical and Bioanalytical Chemistry
Publisher
Springer-Verlag
Publication Date
Apr 30, 2004
Volume
379
Issue
3
Pages
358–367
Identifiers
DOI: 10.1007/s00216-004-2632-z
Source
Springer Nature
Keywords
License
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Abstract

The suitability of a microelectrode as the detector for a small-volume, bead-based enzyme-labeled immunoassay for later use in a microfluidic device was investigated. The microelectrode helps to overcome consumption of the electroactive species by the electrode (depletion) that is encountered with macroelectrodes such as the rotating disk electrode (RDE) and allows the volume of the detection cell to be reduced. Microelectrodes also allow the chemical reactions to be monitored in real time due to the electrodes’ close proximity to the assay site. A bead-based sandwich immunoassay for mouse IgG was developed with alkaline phosphatase (AP) as the enzyme label, p-aminophenyl phosphate (PAPP) as the enzyme substrate, and microelectrode detection. The diffusion coefficient of the product of enzymatic hydrolysis, p-aminophenol (PAP), was determined to be 7.2±0.9×10−6 cm2 s−1. The detection limits were determined for free (0.52 ng mL−1) and bead-bound AP (10 ng mL−1). The number of binding sites for AP per bead was calculated to be 9.6×104 molecules/bead, and under saturation conditions the minimum detectable number of beads was 2500. Lower detection limits could be achieved with the microelectrode than the RDE while maintaining similar reproducibility. The microelectrode also made it possible to work with lower sample volumes (down to 10 μL) than with the RDE (minimum volume of 40 μL). Depletion of PAP was not observed with the microelectrode. The results obtained here with a microelectrode showed great promise for later use of microelectrodes in microfluidic devices with limited sample volumes. RDE detection cannot be used in a microfluidic system due to its complex set-up that includes a motor for rotation.

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