Amperometric detection in TMB/HRP-based assays

Abstract

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.

Use of Electrochemical DNA Biosensors for Rapid Molecular Identification of Uropathogens in Clinical Urine Specimens

“…Detector probes coupled to oxidoreductase reporter enzymes allow amperometric detection of redox signals by the sensor electrodes (28,34). When a fixed potential is applied between the working and reference electrodes, enzyme-catalyzed redox activity is detected as a measurable electrical current (11,16,27). The current amplitude is a direct reflection of the number of target-probe-reporter enzyme complexes anchored to the sensor surface.…”

Micro-flow-injection analysis (μFIA) immunoassay of herbicide residue 2,6-dichlorobenzamide – towards automated at-line monitoring using modular microfluidics

“…The concentration of the surface bound antibody (inversely proportional to the concentration of BAM in a sample) is then detected by the peroxidase reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate which is both optically and electrochemically active. 24,25 In microtitre plate format, the developed assay is suitable for extensive parallelisation. However, transfer into a serial FIA environment requires that the immunosorbent be able to withstand multiple regeneration cycles.…”

Sensitive electrochemical immunosensor based on three-dimensional nanostructure gold electrode

“…Notably, because of the site of HRP redox, which was shielded within insulating peptide backbones, the site of HRP redox did not directly exchange electrons with the electrode. 30,31 Therefore, small redox molecule TMB can easily diffuse in and out of the redox site of HRP as an electron shuttle, and coupling with the redox reaction of TMB, the catalytic reduction of H 2 O 2 occurs on the electrode. 30,31 Thus, the HRP modified on the surface of 3D-Au electrode could efficiently catalyze reduction reactions of H 2 O 2 with the redox reaction of TMB, leading to significant amplification of current.…”

“…30,31 Therefore, small redox molecule TMB can easily diffuse in and out of the redox site of HRP as an electron shuttle, and coupling with the redox reaction of TMB, the catalytic reduction of H 2 O 2 occurs on the electrode. 30,31 Thus, the HRP modified on the surface of 3D-Au electrode could efficiently catalyze reduction reactions of H 2 O 2 with the redox reaction of TMB, leading to significant amplification of current. 31,32 However, due to the absence of the detection antibody, this catalytic reduction current on the capture antibody-modified 3D-Au electrode was not obvious.…”

“…30,31 Thus, the HRP modified on the surface of 3D-Au electrode could efficiently catalyze reduction reactions of H 2 O 2 with the redox reaction of TMB, leading to significant amplification of current. 31,32 However, due to the absence of the detection antibody, this catalytic reduction current on the capture antibody-modified 3D-Au electrode was not obvious. The phenomenon at the flat gold electrode surface ( Figure 3, curves a and c) was also similar, but the current signals decreased significantly and the difference in current value between the two curves was only 0.63 μA.…”