Rapid and ultrasensitive activity detection of α-amylase based on γ-cyclodextrin crosslinked aminated Fe-Ni bimetallic MOF nanozyme.

α-Amylase (α-Amy) is a clinically important biomarker for the diagnosis of acute pancreatitis and related disorders. However, currently available methods for α-Amy detection often require sophisticated instrumentation, labor-intensive procedures, and prolonged assay times, thereby limiting their applicability in rapid and point-of-care analysis. Herein, we developed a simple, sensitive, and rapid nanozyme-based colorimetric assay for the detection of α-Amy. This sensing strategy is based on a substrate-shielding and enzymatic deprotection mechanism, in which γ-cyclodextrin (γ-CD) serves as both a crosslinking agent and a catalytic site blocker for the Fe₃Ni-MOF-NH₂ (FNMN) nanozyme, forming the composite Fe₃Ni-MOF-NH₂@γ-CD (FNMNCD). The specific hydrolysis of γ-CD by α-Amy induces the disassembly of the aggregated FNMNCD structure and restores the peroxidase-like activity of the nanozyme, thereby modulating the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in a concentration-dependent manner. Under optimal conditions, the proposed assay exhibited a wide linear detection range from 2 to 800 U/L, with a low detection limit of 1.1 U/L and relative standard deviation below 4.21%. Furthermore, the method was successfully applied to the determination of α-Amy activity in urine samples from healthy individuals and patients with pancreatitis. Therefore, we believe that the γ-CD-crosslinked FNMN nanozyme system could be applicable for the sensitive and quantitative detection of α-Amy in complex biological samples.