The escalating global prevalence of obesity in the later part of the 20th century precipitated a substantial increase in type 2 diabetes mellitus [1]. This metabolic ailment is marked by hyperglycemia, posing a risk for various conditions detrimental to human health [2,3]. The α-glucosidase, present throughout the small intestine, serves as a crucial enzyme in organismal carbohydrate digestion. It contributes to an elevation in postprandial blood glucose levels by facilitating the breakdown of various intestinal carbohydrates, such as starch, sucrose, and maltose, into monosaccharides. These monosaccharides are absorbed by the epithelial cells in the upper small intestine, subsequently entering the bloodstream [4,5]. In recent years, α-glucosidase inhibitors have been reported to delay glucose absorption and improve postprandial hyperglycemia [6]. Acarbose, the most effective oral hypoglycemic agent on the market, is widely used in clinical practice. Although acarbose is effective in reducing postprandial blood glucose, it is also associated with side effects such as diarrhea, bloating, and liver dysfunction [7,8]. Therefore, the development of new α-glucosidase inhibitors is a promising approach for disease prevention.

At present, enzyme inhibitor screening methods widely used include UV–Vis spectroscopy [9], nuclear magnetic resonance (NMR) technology [10,11], fluorescence technology screening [12,13] and electrochemical methods [14,15]. Although the development is relatively mature, there are some inevitable shortcomings. For instance, UV–Vis spectroscopy determines α-glucosidase activity through the hydrolysis of p-nitrophenyl-α-D-glucopyranoside (pNPG), producing p-nitrophenol (PNP) measurable at 400 nm. However, substances with similar absorption spectra can impact detection accuracy. While the long time required for NMR techniques makes it difficult to achieve rapid screening of inhibitors. In addition, both fluorescence technology and electrochemical methods have serious interference issues [16,17]. Therefore, there is an urgent need for a rapid and accurate method for screening active compounds with inhibitory effects. Designing enzyme activity sensors based on enzyme reaction products is a common strategy. Sensor analysis has been widely used for enzyme activity assays and inhibitor screening because the procedure can quickly identify whether complex samples contain inhibitor components or not [18,19]. Chen et al. developed a simple strategy for sensitive detection of DNA MTase activity (using M.SssI as an example) by using a blood glucose meter as a signal sensor [20]. Nevertheless, the challenge of extracting individual compounds from complex samples cannot be ignored.

Affinity chromatography has been successfully introduced to isolate and identify the target substance from complex samples. It is a chromatographic method that separates and purifies substances based on specific recognition and interactions between bioaffinity molecules and target substances. In general, enzymes, antibodies, cell membranes, etc. are employed as bioaffinity molecules [21]. By fixing one of the ligands on the filler, the corresponding target substances can be adsorbed from the initial sample and then dissociated through appropriate elution to achieve identification and purification [22]. The screening method of bioactive compounds based on affinity chromatography has received increasing attention in the past few years [23,24]. Yue et al. established an enrichment method based on Ti-IMac IV material, which enriched complete O-GalNAc glycopeptides through hydrophilic and affinity interactions. This method can identify nearly 200 integral O-GalNAc glycopeptides from only 0.1 μL of human serum [25]. However, affinity chromatography screening also has drawbacks, such as low screening efficiency and false positives [26,27].

High-performance liquid chromatography (HPLC) is a multifunctional separation technique that can provide a large amount of data during the analysis process [28,29]. Simultaneously, HPLC can quickly determine trace components in complex matrices with high selectivity [30,31]. By combining HPLC detection, Chen et al. screened seven potential active ingredients targeting three enzymes from Polygoni Cuspidati Rhizoma et Radix [32]. To date, it has been widely used in traditional Chinese medicine and biological samples analysis [9,[33], [34], [35]].

In this study, an analytical model that integrates activity screening and affinity component capture and identification was reported based on the respective advantages of sensing screening and affinity chromatography. This model achieved quick identification of active ingredients in complex samples through sensing screening, improving the efficiency and accuracy of affinity chromatography screening. At the same time, enzyme activity measurement and affinity recognition were integrated through immobilization of α-glucosidase. Carboxyl-functionalized multi-walled carbon nanotubes (MWCNTs), with their unique combination of physical, chemical, electrical, and optical properties, usually serve as ideal scaffolds for immobilizing biomolecules on their surface [36,37]. During the dual sensing process, α-glucosidase decomposes maltose to produce glucose, which in turn leads to significant numerical and color changes in blood glucose and urine glucose test strips, enabling rapid identification of inhibitors in complex samples. Then α-glucosidase@MWCNTs were used as a chromatographic identification medium and placed at the tip of the pipette, achieving miniaturization of the affinity recognition screen and greatly reducing the screening cost. The working principle of this study was shown in Fig. 1. In summary, the dual sensing model ensured the rapid capture of active ingredients and achieved the separation and identification of target substances through affinity chromatography.