Topical application question

[Wanderer0]

I'm currently reproducing an experiment for a case study, and it concerns a topical application of horseradish peroxidase (lyophilised, 200IU/mg, dissolved in water, activated by H2O2 15%, in a 15% H2SO4 buffer).

H2O2 was stored at 4°C, diluted with H2O stored at room temperature, the buffer was also at room temperature, and the process was conducted at 20°C.

Working with peroxidase for the first time, I'm having doubts regarding application and handling. I've diluted the HRP with water 1 ml/mg, activated and added the buffer in a ceramic dish, and applied the mixture at room temperature using a PVC syringe, and covered over the area with a cellulose gause to ensure that the surface would remain soaked for 30 minutes). Total fluid volume was 50 cubic centimetres.

I have had no surface effect, possibly due to storing HRP at 4°C for 10 weeks?

Also, could the transfer in a pvc syringe affect the HRP activity, or could the covering of the surface with gauze draw HRP activity from the surface.

Is there a more preferred way of applying the composition on the surface (mixing the diluted HRP, activator and buffer on the surface itself than leaving them on for 30 minutes? Or preparing them beforehand in a pvc spray bottle and applying them for 30 minutes without gauze cover? Or using glassware, avoiding PVC (which makes the application a bit trickier).

Thank you in advance for your replies.