Immobilisation of esterase purified from mung bean (Vigna radiata L. Wilczek) onto chitosan/glutaraldehyde/glycine and its application in bi-enzyme colorimetric assay for dichlorvos detection

Reliable methods for detecting organophosphorus pesticides (OPs) are urgently demanded to guarantee food safety. Herein, a purified alpha-naphthyl acetate esterase from mung bean (mANAE) was prepared and successfully immobilized on the functionalized chitosan (CTS)-glutaraldehyde(GA)-glycine(Gly). Superior to free enzymes, the immobilized enzyme exhibited more excellent surface morphology characteristics, pH and temperature stability, higher affinity to α-naphthol acetate (1-NA). Furthermore, a new colorimetric method was firstly developed by using the immobilized mANAE (I-mANAE) and hydrogen peroxide (H₂O₂)/horse radish peroxidase (HRP)/3,3′,5,5′-tetramethylbenzidine (TMB) color system. This method gives an associated response of inhibition ratio that is linearly proportional to the concentrations (10–100 ng/mL) of dichlorvos (DDVP), with a low limit of detection (1.32 ng/mL). Moreover, the performance of the method was challenged for DDVP detection in real agricultural products (cabbage, tomato and jujube) with excellent recoveries. The results indicated the advantages of the constructed method (strong stability, low cost, high sensitivity, ease of visual identification), which provide a promising alternative for the screening of OPs in foods.