Analytical and Bioanalytical Chemistry

, Volume 382, Issue 2, pp 297–302

Amperometric detection in TMB/HRP-based assays

  • Pablo Fanjul-Bolado
  • María Begoña González-García
  • Agustín Costa-García
Special Issue Paper


3,3′,5,5′-Tetramethylbenzidine (TMB) is the most commonly used chromogen for horseradish peroxidase (HRP) and so its performance as an electrochemical substrate was evaluated. Measurements of HRP activity in solution were carried out by using an amperometric detector coupled to a flow injection analysis (FIA) system. The enzymatic product was easily detected at a potential of +0.1 V (vs. Ag-pseudoreference electrode) at a bare screen-printed electrode placed in a homemade electrochemical flow cell. A high flow rate (4.3 mL min−1) of 0.5 M H2SO4 was used to obtain repeatable signals and a short analysis time. The detection limit achieved after 15 min of incubation was 2×10−14 M of HRP. The applicability of the amperometric detector to ELISAs was demonstrated by using a commercially available kit for the quantification of interleukin-6 (IL-6) without modifying the kit manufacturer’s protocol or the reagents for this test.


Horseradish peroxidase 3,3′,5,5′-Tetramethylbenzidine Screen-printed carbon electrode Flow injection analysis ELISA 


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Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Pablo Fanjul-Bolado
    • 1
  • María Begoña González-García
    • 1
  • Agustín Costa-García
    • 1
  1. 1.Departamento de Química Física y AnalíticaUniversidad de OviedoOviedo, AsturiasSpain

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