A new colorimetric assay for amylase based on starch-supported Cu/Au nanocluster peroxidase-like activity

In this paper, we present a new colorimetric technique as a novel assay for the easy and direct detection of -amylase activity. This detection system utilizes the interaction of -amylase with starch that is supporting copper/gold (Cu/Au) nanoclusters. The Cu/Au nanoclusters are synthesized using starch as a stabilizing agent at room temperature. These nanoclusters show robust peroxidase-like activity and are able to catalyze the oxidation of TMB (3,3,5,5-tetramethylbenzidine) in the presence of hydrogen peroxide (H2O2), leading to the generation of a blue-colored solution. The -amylase detection mechanism is based on the digestion of the starch by -amylase, which results in nanocluster aggregation, leading to increased nanoparticle size and thus decreased peroxidase-like activity of the Cu/Au NCs. Experiments showed that the gradual addition of -amylase causes the peroxidase activity to decrease step by step in a linear fashion. Using this method, colorimetric sensing of -amylase was achieved with a detection limit (LOD) of 0.04U/mL and a linear range of 0.1-10U/mL. This method is significantly selective for -amylase and could be affordably and conveniently applied to the detection of -amylase in blood serum.