Construction of ADH@Pullulanase-AuNPs biosensor with the Cascade catalytic properties — Application in detecting the amylose/amylopectin ratios of Rice and its products

Starch is an important raw material in the food industry, and the ratios of amylose to amylopectin within it is closely related to the quality of starch products. Therefore, the development of rapid detection methods for the amylose/amylopectin ratios in starch is a practical demand to meet industrial development. Consequently, the development of a rapid, sensitive, and portable detection method is of great significance. In this study, an electrochemical detection method for the amylose-to-amylopectin ratios was established by utilizing alcohol dehydrogenase (ADH) and pullulanase to fabricate an ADH@pullulanase-AuNPs biosensor. Specifically, protein-metal nanoflowers were synthesized by regulating the interactions of Cu 2+ and PO 4 3− with the enzymes to achieve the spatially localized immobilization of ADH and pullulanase; meanwhile, AuNPs were modified to enhance electrical conductivity. Additionally, the applicability of this method to rice products was investigated. The experimental results showed that the ADH@pullulanase cluster exhibited a porous flower-like structure, good enzymatic activity, and excellent electrochemical performance. Under optimal conditions, this method exhibited a wide linear range (1 × 10 −6 –1 × 10 −4  mol/L) for the amylose/amylopectin ratios, a low limit of detection (0.5 × 10 −6  mol/L), and a short detection time (< 10 min). When applied to the detection of rice products, the average relative deviation between it and the iodine colorimetric method was 6.9%. The successful development of this method provides a technical reference and theoretical basis for the on-site rapid and sensitive detection of the amylose/amylopectin ratios.