8/29/24

Dear Yaasir,

You are attempting to immobilize GOX on the surface of GCE using

Pt nanoparticles/Mxene. I'm not familiar with the chemistry of this process, but is it possible that the GOX is being denatured during the immobilization process? That would account for your observations. If that is the case, can you modifly the binding conditions to make them milder to prevent GOX denaturation?

Another possibility is the the GOX is simply not being bound at all. One way to find out is to test for soluble protein (GOX) in the liquid fraction after the binding step is over. If the initial level protein is recovered in the liquid fraction, it indicates that binding did not occur. If the protein is absent, it indicates that binding did occur, but that the immobilized GOX was inactivated during the immobilization step.

I hope this information helps you.

William J. Colonna, Dept. Food Science & Human Nutrition, Iowa State University, Ames, Iowa, USA [email protected]

Dear W.J. Colonna , i can thank you enough for your response, because at a point i was so lost and confused. As regards the immobilization process, lyophilized powder 100-250u/mg was purchased and dissolved in 0.1M PBS and this was immobilized on the Pt/mxene/GCE and left to dry at room temperature and the remainder stored in the freezer @ less than 4 degrees celsius.

Do you have a practical paper you could point me to as regards testing the soluble protein to determine if the GOx is being bound?

Thank you for response.

Regards.

9/3/24

Dear Yaasir,

Below are websites that describe 2 spectrophotometric methods for measuring soluble protein. One is the BCA (Bicinchoninic Acid) Method. The other is the Bradford Method that uses Coomassie Brilliant Blue G-250. Both assays are quick and easy to perform, and very sensitive. They can detect microgram quantities of protein. Either procedure should work for you.

Both methods require use of a pure protein that is used as a standard. You need to prepare a calibration curve by measuring the absorbance of a series of increasing amounts (i.e., micrograms) of the standard protein, then using the curve to measure the residual glucose oxidase in your test solution after the immobilization procedure.

A good protein to use is bovine serume albumin (BSA). This is relatively inexpensive and available in very high purity (>98% pure), and is a popular choice as a protein standard. One thing to keep in mind is that different proteins show different reactivities with protein detecting reagents. In other words, if you have equal concentrations of 2 different proteins, you may get calibration curves that are perfectly linear, but they do not overlap. You can see this regardless of the assay procedure you use. This is a common occurrence and is explained in the description of the BCA assay. It will be OK if you use BSA for measuring GOX, as long as you reference that your GOX determination was done using BSA as the standard.

An alternative protein to use as the standard is the GOX powder that you purchased for the immobilization trials. It would be good if you could find out from the supplier how pure the GOX powder is, i.e., is it pure protein, or does it also contain some other not-protein solids, e.g,salts, etc. The supplier should be able to provide you w/ that information. If it is not very pure, then using the BSA will be OK.

I hope this information helps you. Good luck w/ your research.

Bill Colonna

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011430_Pierce_BCA_Protein_Asy_UG.pdf

https://www.thermofisher.com/order/catalog/product/23225

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0011181_Coomassie_Bradford_Protein_Asy_UG.pdf