Dear Rayan,

There is a lot of confusion regarding DET of enzymes with electrodes and lot of publication claiming DET of enzymes when in reality they are measuring or observing something else.

The scientific community expert on the area agrees on two experimental approaches to stablish wheter DET is occurring between redox enzymes and electrodes:

1. Direct evidence from observation of independent electrochemical activity of the redox cofactor comprising the active site in the absence of substrate, the so called non-turn over conditions.

2. Indirect evidence based on observing a catalytic response current in the presence of the enzyme substrate (turn-over voltammetry).

For full proof of DET between a redox enzyme and an electrode, these two conditions should be fulfilled.

To achieve this experimentally you need to do:

1. CV of your (Nafion/HRP/TiO2 film/ITO substrate)-electrode in the absence of H2O2. If you observe reversible electrochemical signals for the Fe-cofactor of HRP then you can say you fullfil condition 1.

2. CV or amperometry measurements of your (Nafion/HRP/TiO2 film/ITO substrate)-electrode in the presence of H2O2. Only If you observe electrocatalytical signal starting from the same potential as observerd for the non-turnover conditions you can say that you fullfill condition 2.

You can not evaluate more DET of enzymes with electrodes. When the two conditions are fulfilled then you can calculate the kinetics (changing the scan rate), biosensor response, ect.

I hope this helps you, anyway I recommend you to read the literature and some reviews on the subject.

Rayan Engg, you can use the cyclic voltammetry (CV) to evaluate the direct electron transfer occur or not?. When the direct electron transfer appear, the peak of CV will not show at the position of H2O2 reduction. It should shift to lower potential (near zero) and higher in current.

thanks Tanin for your consedering my question. do you mean the peak of the CV will shift to be lower potential and higher in current after adding H2O2?

Yes, the direct electron transfer of H2O2 via HRP, the peak of H2O2 present should be at the potential of Fe in HRP.

Rayan Engg,  To measure the direct electron transfer between enzyme and electrode, You need to record CVs in the presence and absence of HRP; In this analysis, H2O2 is not needed.  Further you can evaluate the direct electron transfer between enzyme and electrode from the CVs at various scan rates and from the shift in Ep at different scan rates.

You please refer this  following publication and details  and relevant references regarding Fig.3:  

K.V. Gobi, F. Mizutani Efficient Mediatorless Superoxide Sensors using Cytochrome c-modified Electrodes: Surface Nano-organization for Selectivity and Controlled Peroxidase Activity Journal of Electroanalytical Chemistry 484, 172-181 (2000)

Thanks k. V. Gobi

Dear Rayan,

There is a lot of confusion regarding DET of enzymes with electrodes and lot of publication claiming DET of enzymes when in reality they are measuring or observing something else.

The scientific community expert on the area agrees on two experimental approaches to stablish wheter DET is occurring between redox enzymes and electrodes:

1. Direct evidence from observation of independent electrochemical activity of the redox cofactor comprising the active site in the absence of substrate, the so called non-turn over conditions.

2. Indirect evidence based on observing a catalytic response current in the presence of the enzyme substrate (turn-over voltammetry).

For full proof of DET between a redox enzyme and an electrode, these two conditions should be fulfilled.

To achieve this experimentally you need to do:

1. CV of your (Nafion/HRP/TiO2 film/ITO substrate)-electrode in the absence of H2O2. If you observe reversible electrochemical signals for the Fe-cofactor of HRP then you can say you fullfil condition 1.

2. CV or amperometry measurements of your (Nafion/HRP/TiO2 film/ITO substrate)-electrode in the presence of H2O2. Only If you observe electrocatalytical signal starting from the same potential as observerd for the non-turnover conditions you can say that you fullfill condition 2.

You can not evaluate more DET of enzymes with electrodes. When the two conditions are fulfilled then you can calculate the kinetics (changing the scan rate), biosensor response, ect.

I hope this helps you, anyway I recommend you to read the literature and some reviews on the subject.