A cascade sensing platform based on ultra-stable metal-organic framework-integrated α-glucosidase for rapid and...

Accurate determination of amylase activity is crucial for controlling starch conversion in food production. However, although well-established methods exist for determining α-amylase and total amylase hydrolytic activity, methods for measuring the activity of β-amylase (BAM) remain inadequate and are often limited by insufficient selectivity or sensitivity and cumbersome procedures. This study developed a cascade colorimetric platform for BAM activity-sensing based on an α-glucosidase-integrated metal-organic framework (α-Glu@ZIF-8) synthesized via a biomimetic polyvinylpyrrolidone and L-cysteine-assisted mineralization method. In the cascade sensing system, BAM first hydrolyzes the specific substrate p-nitrophenyl-α-D-maltotriose (pNPG3) to p-nitrophenyl-α-D-glucopyranoside (pNPG), which is subsequently converted by α-Glu@ZIF-8 into yellow-colored p-nitrophenol (pNP). Structural characterization and spectroscopic analysis confirmed the formation of α-Glu@ZIF-8, with high enzyme-loading efficiency. Compared with free enzyme, the encapsulated α-Glu possessed higher physical and chemical stability with remarkable reusability. Under optimized conditions, α-Glu@ZIF-8 can detect BAM within the activity range of 100-1000 U/L (R² = 0.990). Common co-existing substances caused only minor signal changes, and relative responses from other interfering enzymes were gentle, indicating that the platform exhibits good selectivity. Analysis of actual fermentation samples further confirmed the feasibility of this platform for determining BAM activity in complex fermentation matrices.