Alpha-amylase (α-1,4-glucan-4-glucan hydrolase; EC 3.2.1.1) is a classical calcium-containing enzyme that cleaves the α-1,4-glycosidic bonds within starch, glycogen, and other glucose polymers to produce several polysaccharides, such as maltose and glucose [[1], [2], [3]]. As one of the longest and broadest enzymes with the largest yield, α-amylase is widely present in animals, plants, and microorganisms, and it has a wide range of applications in starch processing, pasta manufacturing, ultra-high maltose syrup production, pharmaceutical industry, alcoholic beverage brewing, and other fields [[4], [5], [6], [7]]. In the field of brewing, α-amylase hydrolyzes the starch in raw materials into sugar, accelerates the decomposition of starch and increases the alcohol content, affecting the taste and flavor of liquors [8,9]. Therefore, the rapid determination of α-amylase activity is essential to improving the quality and yield of liquors.

So far, the commonly used methods to determine α-amylase include viscosity/turbidity detection based on starch decomposition, the iodine-starch method, and Bernfeld reducing sugar method [10]. However, these traditional methods have the following problems in practical application in the brewing process: (a) the multiple detection steps are time-consuming and labor-intensive, and relevant indicators cannot be comprehensively and accurately quantified; (b) these methods are non-stoichiometric with variable reaction conditions and are usually interfered with by endogenous substances, such as glucose, making it difficult to precisely evaluate enzyme activity; (c) large volumes of samples and reagents are required, resulting in low flux and inability to perform real-time monitoring. Recently, some new analytical methods were developed, such as chromatography [11], ultrasound [12], colorimetry [13], isothermal quantitative calorimetry [14], and electrochemical methods [15]. These strategies are sensitive and accurate for α-amylase detection but require relatively more expensive reagents, precise instruments, and complicated operational procedures. Even more unacceptable is the fact that the activity of the target enzyme is susceptible to loss during these complex processes, and the accuracy of the results cannot be ensured [16,17]. Therefore, the development of a highly sensitive and practical real-time analysis method for α-amylase activity is urgently needed.

Cascade reactions have higher reaction efficiency than conventional sequential reactions due to the closer active sites, higher local concentrations of intermediates, and faster mass transfer, which results in reduced diffusion losses [18,19]. In recent years, more and more cascade reactions have been used for various applications, including accelerated catalysis and enhanced biosensing/detection [20,21]. As a typical example, β-galactosidase, glucose oxidase, and horseradish peroxidase have been encapsulated in metal-organic frameworks (MOFs), enabling cascade enzyme reactions to occur with better performance than free enzymes [22]. However, natural enzymes have some intrinsic disadvantages, such as high cost, low stability, and easy inactivation [23]. To address these problems and broaden the practical applications in harsh environments, nanozyme has appeared as a new emerging field instead of natural enzymes, which possess enzyme-like activity with high stability, low cost, easy storage, simple preparation, and tunable catalytic performance [24,25]. Inspired by this, the researchers replaced some of the enzymes in cascades and constructed hybrid multi-enzyme cascade systems, which combined the high stability and controllable catalytic activity of nanozymes with the good selectivity of natural enzymes to mimic biocatalytic processes [26]. Such hybrid systems provide opportunities to study the co-operative effects between chemo-catalysts and natural enzymes and have been applied in bioanalysis and biomedicine [27].

In addition to being an active participant in cascade reactions, nanozymes (e.g., precious metal/metal oxides, MOFs, and graphene) also serve as carriers for immobilization and loading of single or multiple enzymes in multi-enzyme systems [28]. Among them, MOFs are a class of porous crystalline materials consisting of organic linkers and metal clusters. Due to their ultra-high-specific surface area, remarkable functional diversity, satisfactory biocompatibility, tunable pore sizes, and excellent structural stability, MOFs have already made significant breakthroughs in nanozyme-related catalysis, therapeutics, and biosensing [29]. MOFs have also been used to immobilize enzymes by adsorption, covalent binding embedding, and encapsulation [30,31]. Encapsulating the enzymes within MOF cavities paves the way to solving the inherent problem of traditional enzyme immobilization methods (e.g., unavoidable enzymatic conformation changes, activity loss, and unfavorable tolerance towards hostile environments) because: (a) the ultra-high porosity of MOFs provides enough space for hosting; (b) the tightly surrounding MOF layer can significantly stabilize the enzymes by conformational confinement; and (c) the tailorable porous network allows for selective mass transfer of the substrate, facilitating the catalytic process. In a word, these attractive advantages of MOF-encapsulated multi-enzyme systems endow them with great potential in the detection of cascade amplification.

In this work, two natural enzymes, α-glucosidase (Glu) and glucose oxidase (Gox), were simultaneously embedded in Cu-MOF-74 crystals using a mild aqueous-based in situ encapsulation method in 15 min at room temperature to construct a cascade biocatalytic platform Glu&Gox@Cu-MOF-74 (Fig. 1a). Cu-MOF-74 was not only used to encapsulate enzymes and protect them from harsh conditions, but also acted as nanozyme, participating in the cascade reaction and triggering the generation of fluorescent product due to its excellent peroxidase-like catalytic activity. Benefitting from the satisfactory activity and stability, the synthesized Glu&Gox@Cu-MOF-74 was used successfully for fluorescent detection of α-amylase: in the presence of α-amylase, Glu&Gox@Cu-MOF-74 converts starch into glucose, and the generated H2O2 directs oxidation reaction of o-phenylenediamine to produce fluorescence signal. Finally, real fermentation samples were assayed to demonstrate the practicability of the method.