We used very successfully as enzyme: alkaline phosphatase and as substrate: p-amino-phenyl-phosphate

this results in

reversible reduction with low overvoltage and no polymerization side-reaction

Forgot the picture

I agree with Karl! however p-amino phenyl phosphate is very unstable and often we prefer alfanaphtylphosphate which is stable and commerially available. the naphtol was detected by Differential Pulse Voltammetry.

Your question was slightly broader about other electrochemical approaches, given the build up of preciptant from the reaction perhaps impedence measurement might work.....?

little review on approaches

Iles RK, Kallichurn H (2012). What will be the Future Development of Electrochemical Biosensors for the Detection and Quantification of Biomarkers? J Bioengineer & Biomedical Sci 2:e110. doi: 10.4172/2155-9538.1000e110

The simplest and direct strategy is to use conducting polymers for entrapment of biomolecules and use potentiometry to detect changes.

So you are getting a good luminescent signal using luminol substrate but not a visual (blue) signal using TMB? If so, there is something wrong with the TMB solution. If this is a commercial preparation, it may have deteriorated. If this was made by you, it may not contain the correct hydrogen peroxide concentration and the appropriate pH (~4.6). TMB substrate activity is easy to test by adding a few microliters of diluted Ab2 directly to the substrate solution. As Benoit Limoges indicated TMB+ (actually a charge-transfer complex) should undergo reduction at -100 mV at the working electrode.

@Robert Rubin. I do get the blue color. I have solved the issue. Thanks to all colleagues to pay attention to my query. Well, I have another question, when I add stopping solution, it turn the blue into yellow. What is this yellow species? secondly, would it affect the amount of TMB+ which I want to detect electrochemically?

Hi Mohtashim

So you get blue colored product now? Is this different then when you wrote “…I can see the TMB color change visually of similar intensity for control and assay” which I interpreted as no response to peroxidase.

Regarding your other question, the yellow substance is 3,5,3’5’-tetramethylbenzidiimine, the 2 electron stable oxidation product of 3,5,3’5’-tetramethylbenzidine (which is the diamine “TMB”). It has an absorbance max of 450 mu, which makes it yellow. The blue substance is a charge-transfer complex between the diimine and the diamine, formed after a 1 electron oxidation of TMB to produce the free radical TMB cation. The charge-transfer complex has an absorbance max at 652 mu, which makes it blue, and it is this material that undergoes electrochemical reduction at -100mv. So you must measure the C-TC intermediate as it is being produced de-novo. It is possible that the stable diimine could be measured electrochemically, but I don’t know its reduction potential.

Number of substrates could be applied for this purpose:

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