Robust oxidase mimicking activity of protamine-stabilized platinum nanoparticles units and applied for colorimetric sensor of trypsin and inhibitor

In this study, we developed a simple, sensitive and label-free colorimetric sensor for trypsin and its inhibitor using oxidase mimicking activity of protamine-stabilized platinum nanoparticles units (Pro-PtNPs units). Protamine is designed to stabilize PtNPs and served as the substrate of trypsin hydrolysis. The results show that protamine combines several PtNPs together to form active units, which have stronger oxidase mimicking activity than other PtNPs. In addition, the Michaelis constant of the Pro-PtNPs units is smaller than negatively charged citrate capped PtNPs, suggesting that the positively charged Pro-PtNPs units have better affinity with 3,3′,5,5′-tetramethylbenzidine (TMB), which is inconsistent with previous reports. The protamine stabilizer could be hydrolyzed in the presence of trypsin, and the Pro-PtNPs units would be decomposed and aggregated, resulting in a decrease of catalytic activity of Pro-PtNPs units. The color of the solution would be reduced according to the decrease of catalytic activity, so the catalytic activity is linearly related to UV absorbance. The linear range of trypsin detected by this new method is 0.06 μg/mL˜0.6 μg/mL, and the detection limit down to 0.03 μg/mL. Pro-PtNPs units have been successfully applied to the determination of serum trypsin and screening of inhibitors.