Skip to main content
Log in

Bead-based immunoassays with microelectrode detection

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript


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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others







Alkaline phosphatase


Nonspecific adsorption




p-aminophenyl phosphate


Rotating disk electrode


Nonspecific adsorption


Phosphate-buffered saline


  1. Matysik F-M, Nyholm L, Markides KE (1999) Comparison of μm and mm sized disk electrodes for end-column electrochemical detection in capillary electrophoresis. Fresenius J Anal Chem 363:231–235

    Article  CAS  Google Scholar 

  2. Ueno K, Kitamura N (2003) A spectroelectrochemical study on perylene cation radical in polymer microchannel–microelectrode chips. Analyst 128:1401–1405

    Article  CAS  PubMed  Google Scholar 

  3. Reichle C, Schnelle T, Muller T, Leya T, Fuhr G (2000) A new microsystem for automated electrorotation measurements using laser tweezers. Biochim Biophys Acta 1459:218–229

    Article  CAS  PubMed  Google Scholar 

  4. Vandaveer WR, Pasas SA, Martin SR, Lunte SM (2002) Recent developments in amperometric detection for microchip capillary electrophoresis. Electrophoresis 2002:3667–3677

    Article  Google Scholar 

  5. Rossier JS, Girault HH (2001) Enzyme-linked immunosorbent assay on a microchip with electrochemical detection. Lab Chip 1:153–157

    Article  CAS  PubMed  Google Scholar 

  6. Wightman RM, Amatore C, Engstrom RC, Hale PD, Kristensen EW, Kuhr WG, May LJ (1988) Real-time characterization of dopamine overflow and uptake in the rat striatum. Neuroscience 25(2):513–523

    Article  CAS  PubMed  Google Scholar 

  7. Satoh H, Okabe S, Norimatsu N, Watanabe Y (2000) Significance of substrate C/N ratio on structure and activity of nitrifying biofilms determined by in situ hybridization and the use of microelectrodes. Water Sci Technol 41(4–5):317–321

    Google Scholar 

  8. Ouvry A, Cachon R, Divies C (2001) Application of microelectrode technique to measure pH and oxidoreduction potential gradients in gelled systems as model food. Biotech Lett 23:1373–1377

    Article  CAS  Google Scholar 

  9. Abe T, Lau Y, Ewing A (1992) Characterization of glucose microsensors for intracellular measurements. Anal Chem 64:2160–2163

    CAS  PubMed  Google Scholar 

  10. Kennedy RT, Huang L, Atkinson MA, Dush P (1993) Amperometric monitoring of chemical secretions from individual pancreatic β-cells. Anal Chem 65:1882–1887

    CAS  PubMed  Google Scholar 

  11. Ewing AG, Dayton MA, Wightman RM (1981) Pulse voltammetry with microvoltammetric electrodes. Anal Chem 53:1842–1847

    CAS  Google Scholar 

  12. Kissinger PT, Heineman WR (1996) Laboratory Techniques in Electroanalytical Chemistry, 2nd edn. Marcel Dekker, New York

  13. Clark RA, Zerby SE, Ewing AG (1998) In: Bard AJ, Rubinstein I (eds) Electroanalytical Chemistry, vol 20; Marcel Dekker, New York, pp 227–294

  14. Hu Z, Heineman WR (2000) Oxidation-state speciation of [ReII (DMPE)3 ]2+ by voltammetry with a chemically modified microelectrode. Anal Chem 72:2395–2400

    Article  CAS  PubMed  Google Scholar 

  15. Masson M, Liu Z, Haruyama T, Kobatake E, Ikariyama Y, Aizawa M (1995) Immunosensing with amperometric detection, using galactosidase as label and p-aminophenyl-β-D-galactopyranoside as substrate. Anal Chim Acta 304:353–359

    Article  CAS  Google Scholar 

  16. Yao H, Halsall HB, Heineman WR, Jenkins SH (1995) Electrochemical dehydrogenase-based homogeneous assays in whole blood. Clin Chem 41(4):591–598

    CAS  PubMed  Google Scholar 

  17. Wijayawardhana CA, Purushothama S, Cousino MA, Halsall HB, Heineman WR (1999) Rotating disk electrode amperometric detection for a bead-based immunoassay. J Electroanal Chem 468:2–8

    Article  CAS  Google Scholar 

  18. Wijayawardhana CA, Halsall HB, Heineman WR (1999) Micro volume rotating disk electrode (RDE) amperometric detection for a bead-based immunoassay. Anal Chim Acta 399:3–11

    Article  CAS  Google Scholar 

  19. Purushothama S, Kradtap S, Wijayawardhana CA, Halsall HB, Heineman WR (2001) Small volume bead assay for ovalbumin with electrochemical detection. Analyst 126:337–341

    Article  CAS  PubMed  Google Scholar 

  20. Kradtap S, Wijayawardhana CA, Schlueter KT, Halsall HB, Heineman WR (2001) Bugbead: an artificial microorganism model used as a harmless simulant for pathogenic microorganisms. Anal Chim Acta 444:13–26

    Article  CAS  Google Scholar 

  21. Jenkins SH, Halsall HB, Heineman WR (1988) Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level. Anal Biochem 168:291–294

    Google Scholar 

  22. Halsall HB, Heineman WR, Jenkins SH (1988) Capillary immunoassay with electrochemical detection. Clin Chem 34:1701–1702

    Google Scholar 

  23. Thompson RQ, Barone III GC, Halsall HB, Heineman WR (1991) Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection. Anal Biochem 192:90–95

    CAS  PubMed  Google Scholar 

  24. Tang HT, Lunte GE, Halsall HB, Heineman WR (1988) p-Aminophenyl phosphate: an improved substrate for electrochemical enzyme immunoassay. Anal Chim Acta 214:187–195

    Article  CAS  Google Scholar 

  25. Rosen I, Rishpon J (1989) Alkaline phosphatase as a label for a heterogeneous immunoelectrochemical sensor. J Electroanal Chem 258:27–39

    Article  CAS  Google Scholar 

  26. Yu Z, Xu YI, Ip MPC (1994) An ultra-sensitive electrochemical enzyme immunoassay for thyroid stimulating hormone in human serum. J Pharm Biomed Anal 12(6):787–793

    Article  CAS  PubMed  Google Scholar 

  27. Kronkvist K, Lovgren Ulf, Edholm LE, Johansson G (1993) Determination of drugs in biosamples at picomolar concentrations using competitive ELISA with electrochemical detection: application to steroids. J Pharm Biomed Anal 11(6):459–467

    Article  CAS  PubMed  Google Scholar 

  28. Niwa O, Xu Y, Halsall HB, Heineman WR (1993) Small-volume voltammetric detection of 4-aminophenol with interdigitated array electrodes and its application to electrochemical enzyme immunoassay. Anal Chem 65(11):1559–1563

    CAS  PubMed  Google Scholar 

  29. Aguilar ZP, Vaveer WR, Fritsch I (2002) Self-contained microelectrochemical immunoassay for small volumes using mouse IgG as a model system. Anal Chem 74(14):3321–3329

    Article  CAS  PubMed  Google Scholar 

  30. Moore EJ, Pravda M, Kreuzer MP, Guilbault GG (2003) Comparative study of 4-aminophenyl phosphate and ascorbic acid 2-phosphate, as substrates for alkaline phosphatase based amperometric immunosensor. Anal Lett 36(2):303–315

    Article  CAS  Google Scholar 

  31. Stulik K, Amatore C, Holub K, Marecek V, Kutner W (2000) Microelectrodes. Definitions, characterization, and applications. Pure Appl Chem 72(8):1483–1492

    CAS  Google Scholar 

  32. Oldham KB, Myland JC (1994) Fundamentals of electrochemical science. Academic, San Diego

  33. Robinson D, Anderson JE, Lin J (1990) Measurement of diffusion coefficients of some indoles and ascorbic acid by flow injection analysis. J Phys Chem 94:1003–1005

    CAS  Google Scholar 

  34. Snead WK, Remick AE (1957) Studies on oxidation-reduction mechanism. II. The anodic oxidation of p-aminophenol. J Am Chem Soc 79:6121–6127

    CAS  Google Scholar 

  35. Cornish-Bowden A (1979) Fundamentals of enzyme kinetics, 2nd edn. Butterworth, London

  36. Aslam M, Dent A (1998) Bioconjugation. Macmillan Reference Ltd

Download references


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.

Author information

Authors and Affiliations


Corresponding author

Correspondence to William R. Heineman.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Farrell, S., Ronkainen-Matsuno, N.J., Halsall, H.B. et al. Bead-based immunoassays with microelectrode detection. Anal Bioanal Chem 379, 358–367 (2004).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: