3,3′,5,5′-Tetramethylbenzidine as electrochemical substrate for horseradish peroxidase based enzyme immunoassays. A comparative study

Abstract

The use of 3,3A,5,5A-tetramethylbenzidine (TMB) as an electrochemical substrate for horseradish peroxidase (HRP) was investigated. HRP activity has been detected using flow injection analysis at a glassy carbon working electrode polarised at +100 mV versus Ag/AgCl in 0.1 mol l 21 citrate-phosphate buffer (pH 5.0). The optimum concentrations were 2 3 10 24 mol l 21 TMB and 10 23 mol l 21 H 2 O 2 . The detection limit obtained after 15 min of incubation was 8.5 3 10 214 mol l 21 HRP with the amperometric method. This limit was lower than that obtained using hydroquinone as HRP substrate and comparable to that with the p-aminophenyl phosphate-alkaline phosphatase system. Better performance was achieved with amperometric than spectrophotometric detection using TMB in a competitive ELISA for rabbit immunoglobulin G as a model analyte.

Inkjet-printed microtiter plates for portable electrochemical immunoassays

“…Briefly, PAPP is irreversibly oxidised at the electrode to para-iminoquinone (PIQ) at around 0.4 V, which is electrochemically reduced to PAP at − 0.15 V. In the second CV scan, PAP is electrochemically oxidised at around − 0.1 V. The addition of MBs to the PAPP solution did not present any influence on the CVs and the PAP detection can be performed at the potential equal to 0.05 V. The CVs for the TMB substrate solution (Fig. 4b) presented two oxidation peaks corresponding to the two-electron transfer reaction of TMB [58]. When using the IJP microchip in the absence of any MBs, the TMB oxidation peaks can be observed at potentials around 0.15 V and 0.35 V while the TMB DI reduction takes place at around 0.2 V and 0 V. By adding MBs into the system the measured redox potentials of TMB were shifted by approximately 0.1 V to higher values, probably due to poor polymeric coating of Fe 3 O 4 core in MBs (for more details see SI-II).…”

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

“…The CVs are consistent with those presented previously in literature. 25,32 The applied potential provides a sufficient driving force for the Fig. 4 Cyclic voltammograms of TMB (1) and oxidized TMB (2).…”

Imunossensor amperométrico

“…Peroxidase (HRP): catalisa a desidrogenação de diversos compostos aromáticos como fenóis, hidroquinonas e derivados da benzidina; a atividade da enzima também tem sido determinada por técnica amperométrica usando hidroquinona, 1,2-fenilendiamina ou pirocatecol 40 , tetrametilbenzidina 41 , ou através da transferência de elétrons por mediadores [42][43][44] .…”

“…Estudos com diferentes substratos da fosfatase alcalina [57][58][59] e da peroxidase 41 têm sido realizados no sentido de desenvolver imunossensor com bom desempenho.…”