Electrochemical ELISA of testosterone using nitrocellulose membrane as a support for antibodies

Kumi Inoue, Pascal Ferrante, Yu Hirano, Tomoyuki Yasukawa, Hitoshi Shiku, Tomokazu Matsue

Research output: Contribution to journalArticlepeer-review


A quantitative electrochemical detection of testosterone was performed using a nitrocellulose membrane as a support for antibodies. Testosterone competitively reacted with horseradish peroxidase (HRP)-labeled testosterone, and was captured with an anti-testosterone antibody immobilized on the membrane. The activity of the labeled HRP was electrochemically estimated with a gold-disk electrode (diameter, 300 μm) by detecting the oxidized form of ferrocenemethanol (FcOH), which was produced by the HRP-catalyzed oxidation of FcOH with H2O2. The electrochemical detection chamber was fabricated in order to maintain a constant distance between the electrode and the membrane surface. We optimized some factors, especially nitrocellulose membranes and the concentration of bovine serum albumin (BSA) and HRP-labeled testosterone for the electrochemical analysis of testosterone with a high reliability. Testosterone was detected by a competitive amperometric measurement under the optimized conditions. As a result, the current response decreased with increasing testosterone concentration, and a linear current response was observed in the range of 0.5∼20 ng/mL on a semi-log graph. This method provides high sensitivity by one order of magnitude, compared with the conventional well plate assay.

Original languageEnglish
Pages (from-to)471-478
Number of pages8
Issue number6
Publication statusPublished - 2007


  • Amperometry
  • Immunoassay
  • Nitrocellulose membrane
  • Testosterone

ASJC Scopus subject areas

  • Analytical Chemistry


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