Self-assembling heterostructured MOF@HOF nanozyme for ultrasensitive α-amylase activity detection

α-Amylase not only has a wide range of applications in the food industry (especially baking, alcoholic beverage manufacturing and sugar production), but also serves as a core enzyme in the human digestive system, serving as an important parameter in the diagnosis of pancreatic diseases. Nevertheless, current methods still face challenges of numerous reagents, complex operation process, high cost and unsatisfactory sensitivity, restricting their utilization in on-field detection. Thus, the development of facile, low-cost, and sensitive strategies to monitor α-amylase activity is desperately required. Herein, we report the fabrication of a hybrid nanozyme through the in situ assembly of a hydrogen-bonded organic framework (HOF) onto an amino-functionalized metal-organic framework (MOF). The enhanced substrate channels and introduced affinity groups significantly improved the biocatalytic performance of the hybrid nanozyme NMOF@DHOF, leading to a strengthened colorimetric signal. Leveraging its high activity and stability, an ultrasensitive colorimetric platform was developed for the rapid detection of α-amylase activity. The assay platform utilized the interaction between α-amylase and starch-cross-linked NMOF@DHOF (NMOF@DHOF@Starch), where α-amylase hydrolyzed starch and led to the aggregation of nanoclusters, resulting in a weakening of the peroxidase activity. The proposed colorimetric strategy produced a linear response over a relevant range of α-amylase levels (1–30 U·L −1 ) with a low detection limit (0.68 U·L −1 ) and significant specificity. The platform's sensitivity and applicability were validated for α-amylase detection in complex matrices, including fermentation samples and human serum. This work establishes a novel biosensing platform design framework with significant potential to advance rapid analysis of α-amylase in both fermentation process monitoring and clinical disease diagnostics. Through facile replacement of particular crosslinking agents, the proposed platform is able to be utilized to quantify the activity of other types of enzymes, offering a beneficial guideline for the establishment of other enzyme analysis systems.