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Bead-based immunoassays with microelectrode detection

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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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Abbreviations

Ab:

Antibody

Ag:

Antigen

AP:

Alkaline phosphatase

NSA:

Nonspecific adsorption

PAP:

p-aminophenol

PAPP:

p-aminophenyl phosphate

RDE:

Rotating disk electrode

NSA:

Nonspecific adsorption

PBS:

Phosphate-buffered saline

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Acknowledgements

This work was supported by the Doctoral Investment Award, Ohio Board of Regents. S.F. acknowledges the Stecker Fellowship and the University Research Council Fellowship sponsored by the University of Cincinnati.

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Authors and Affiliations

  1. Department of Chemistry, University of Cincinnati, P.O. Box 210172, Cincinnati, OH, 45221-0172, USA

    Svetlana Farrell, Niina J. Ronkainen-Matsuno, H. Brian Halsall & William R. Heineman

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  1. Svetlana Farrell
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  2. Niina J. Ronkainen-Matsuno
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  3. H. Brian Halsall
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  4. William R. Heineman
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Correspondence to William R. Heineman.

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Farrell, S., Ronkainen-Matsuno, N.J., Halsall, H.B. et al. Bead-based immunoassays with microelectrode detection. Anal Bioanal Chem 379, 358–367 (2004). https://doi.org/10.1007/s00216-004-2632-z

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  • DOI: https://doi.org/10.1007/s00216-004-2632-z

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