Amperometric detection in TMB/HRP-based assays

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 inje...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2005-05, Vol.382 (2), p.297-302
Main Authors: Fanjul-Bolado, Pablo, González-García, María Begoña, Costa-García, Agustín
Format: Article
Language:English
Subjects:
Benzidines - chemistry
Electrochemistry - methods
Enzyme-Linked Immunosorbent Assay
Horseradish Peroxidase - chemistry
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Summary: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 2x10(-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.
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-005-3084-9