A cerium-based metal-organic framework loaded gold nanocluster composite material, a preparation method and application thereof

Abstract

The present application relates to the technical field of nanobiological sensing, and in particular to a cerium metal organic framework loaded gold nanocluster composite material, a preparation method and application thereof. A fluorescent and colorimetric dual-channel sensing platform is formed by the combination of AuNCs and Ce-MOF for the detection of alpha-glucosidase and the screening of alpha-glucosidase inhibitors. The cerium metal organic framework loaded gold nanocluster composite material catalyzes the oxidation of the colorless substrate ABTS to generate the colored oxABTS, which has a characteristic ultraviolet absorption at 410 nm and can efficiently quench the fluorescence of the cerium metal organic framework loaded gold nanocluster composite material through the inner filter effect. Alpha-glucosidase catalyzes the hydrolysis of the substrate AAG to generate AA, which can inhibit the ABTS oxidation regulated by the cerium metal organic framework loaded gold nanocluster composite material, thereby causing the ultraviolet absorption of oxABTS to decrease and the fluorescence of ACM to recover. Therefore, the dual-channel detection of alpha-GLU is realized through the dual-signal output of colorimetric and fluorescent signals.

Description

Technical Field

[0001] This invention relates to the field of nanobiosensing technology, specifically to a cerium-based metal-organic framework-supported gold nanocluster composite material, its preparation method, and its application. Background Technology

[0002] Alpha-glucosidase (α-GLU) is an important carbohydrate hydrolase that converts polysaccharides into monosaccharides by hydrolyzing glycosidic bonds, and is particularly abundant in intestinal mucosal epithelial cells. Diabetes mellitus is a chronic metabolic disease characterized by long-term hyperglycemia. Based on different etiologies and pathogenesis, it can be subdivided into various types, among which type 2 diabetes (T2D) is closely related to abnormally elevated α-glucosidase activity. α-glucosidase inhibitors, as an important strategy for T2D treatment, can effectively delay carbohydrate hydrolysis and glucose absorption by inhibiting the activity of this enzyme, thereby effectively regulating postprandial blood glucose and reducing blood glucose fluctuations, demonstrating significant clinical application value. However, existing drugs can cause some degree of damage to the intestines or liver. Therefore, natural drugs have become an important resource for screening novel, safe, and effective α-glucosidase inhibitors, opening up new pathways for the innovative development of diabetes treatment drugs. Therefore, the development of highly sensitive and accurate α-glucosidase detection methods and the effective screening of inhibitors derived from natural products are of profound significance and important practical value for the accurate diagnosis and treatment of α-glucosidase-related diseases and the development of new diabetes drugs.

[0003] To date, commonly used methods for α-glucosidase analysis include fluorescence methods, colorimetric methods, and electrochemical methods. Among these, colorimetric and fluorescence methods have attracted considerable attention from researchers due to their excellent analytical performance, lack of complex instrumentation and operational requirements, visual observation capabilities of colorimetry, and rapid response and high sensitivity of fluorescence methods. Unfortunately, most existing α-glucosidase detection methods are based on a single colorimetric or fluorescence signal output. Due to the potential interference from various complex factors in real biological samples, these methods have limitations in achieving high-precision α-glucosidase detection. Therefore, in recent years, many researchers have combined colorimetry and fluorescence methods to construct dual-signal detection systems. This not only fully utilizes the advantages of both methods but also enables self-calibration using dual output signals. However, current dual-signal sensing methods using fluorescence and colorimetry employ the separate addition of nanozymes and fluorescent probes to form the dual-signal sensing system. However, the separate addition of nanozymes and fluorescent probes can introduce significant errors. Therefore, designing a composite material with both fluorescence and nanozyme properties is crucial for obtaining more reliable and accurate analytical results. Summary of the Invention

[0004] To overcome the above problems, this invention provides a cerium-based metal-organic framework-supported gold nanocluster composite material, its preparation method, and its application.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a cerium-based metal-organic framework-supported gold nanoclusters composite material, wherein a cerium-based metal-organic framework (Ce-MOF) is used as a carrier and gold nanoclusters (AuNCs) are loaded thereon.

[0007] Cerium-based metal-organic frameworks (Ce-MOFs) are composed of Ce 3+ Ce 4+ Metal-organic frameworks formed by coordination with ligand terephthalic acid.

[0008] In one or more embodiments, the mass ratio of gold nanoclusters to cerium-based metal-organic frameworks is 1:(1~2), preferably 1:1.5.

[0009] AuNCs and Ce-MOFs have different electrical properties, and ACM composites can be prepared through simple electrostatic recombination. Cerium-based metal-organic frameworks (Ce-MOFs) have excellent oxidase-like activity, while AuNCs have excellent fluorescence properties. The combination of the two not only enhances the fluorescence of AuNCs but also retains their oxidase-like activity.

[0010] A second aspect of the present invention provides a method for preparing the cerium-based metal-organic framework-supported gold nanocluster composite material described in the first aspect, comprising the following steps:

[0011] (1) The template glutathione and the gold source were placed in water, and after the reaction, they were dialyzed and dried to obtain gold nanoclusters;

[0012] (2) Cerium ammonium nitrate was reacted with ligand terephthalic acid to obtain cerium-based metal-organic frameworks;

[0013] (3) Place both gold nanoclusters and cerium-based metal-organic frameworks in water and react to obtain a cerium-based metal-organic framework-supported gold nanocluster composite material.

[0014] In one or more embodiments, in step (1), the gold source is selected from chloroauric acid (HAuCl4).

[0015] Preferably, the molar ratio of glutathione to chloroauric acid is (9~12):1, more preferably 10:1;

[0016] Preferably, the concentration of glutathione is 9-12 mol / L, and more preferably 10 mol / L.

[0017] In one or more embodiments, in step (1), the reaction temperature is 65~80 ℃, preferably 70 ℃; the reaction time is 20~30 h, preferably 24 h.

[0018] In one or more embodiments, in step (1), the molecular weight cut-off of the dialysis bag used during dialysis is 900~1200 Da, preferably 1000 Da.

[0019] In one or more embodiments, in step (2), the molar ratio of cerium ammonium nitrate to ligand terephthalic acid is 1:(0.8~1.2).

[0020] In one or more embodiments, in step (2), the reaction temperature is 90~120 ℃, preferably 100 ℃; the reaction time is 0.5~2 h, preferably 1 h.

[0021] In one or more embodiments, in step (3), the mass ratio of gold nanoclusters to cerium-based metal-organic frameworks is 1:(1~2), preferably 1:1.5.

[0022] A third aspect of the present invention provides a probe composition for detecting α-glucosidase activity, comprising: the cerium-based metal-organic framework-supported gold nanocluster composite material described in the first aspect, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG), and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

[0023] A fourth aspect of the present invention provides a probe composition for screening α-glucosidase inhibitors, comprising: the cerium-based metal-organic framework-supported gold nanocluster composite material described in the first aspect, 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and α-glucosidase.

[0024] A fifth aspect of the present invention provides a product for detecting α-glucosidase activity, comprising the probe composition described in the third aspect.

[0025] A sixth aspect of the present invention provides a product for screening α-glucosidase inhibitors, comprising the probe composition described in the fourth aspect.

[0026] A seventh aspect of the present invention provides a method for detecting α-glucosidase activity, comprising:

[0027] The α-glucosidase solution to be tested was mixed with 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG) and incubated for the first time to obtain the first incubation solution;

[0028] A second incubation solution was obtained by adding cerium-based metal-organic framework-supported gold nanoclusters and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the first incubation solution and then performing a second incubation.

[0029] The second incubation solution was subjected to fluorescence and colorimetric detection. The activity of the α-glucosidase to be tested was obtained based on the working curve of α-glucosidase activity, the fluorescence intensity obtained from fluorescence detection, the standard curve of absorbance change, and the absorbance difference of the second incubation solution.

[0030] In one or more embodiments, the temperature of the first incubation is 37~55 °C, preferably 37 °C; the incubation time is 60~80 min, preferably 60 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG) is 50~70 µM, preferably 50 µM.

[0031] In one or more embodiments, the second incubation is carried out at room temperature for 12 to 20 minutes, preferably 15 minutes, and the pH value is 2.5 to 4.5, preferably 3.0.

[0032] In one or more embodiments, the concentration of the cerium-based metal-organic framework-supported gold nanocluster composite material is 10-200 µg / mL, preferably 50 µg / mL; the concentration of 2,2'-adiazonobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is 50-300 µM, preferably 100 µM.

[0033] In one or more embodiments, fluorescence detection is performed at a wavelength of 400 nm.

[0034] An eighth aspect of the present invention provides a method for screening α-glucosidase inhibitors, comprising the following steps:

[0035] The α-glucosidase inhibitor, α-glucosidase, and 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG) were mixed and incubated for a third time to obtain the third incubation solution.

[0036] A fourth incubation solution was obtained by adding cerium-based metal-organic framework-supported gold nanoclusters and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the third incubation solution and then performing a fourth incubation.

[0037] The fourth incubation solution was subjected to fluorescence and colorimetric detection. The half-inhibition concentration of the α-glucosidase inhibitor to be tested was obtained according to the calibration curve of the α-glucosidase inhibitor to be tested, thereby achieving the screening of α-glucosidase inhibitors.

[0038] In one or more embodiments, the α-glucosidase inhibitor includes hesperidin or apigenin.

[0039] In one or more embodiments, the temperature of the third incubation is 37~55°C, preferably 37°C; the incubation time is 60~80 min, preferably 60 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG) is 50~70 µM, preferably 50 µM.

[0040] In one or more embodiments, the fourth incubation is carried out at room temperature for 12 to 20 minutes, preferably 15 minutes, and the pH value is 2.5 to 4.5, preferably 3.0.

[0041] In one or more embodiments, the concentration of the cerium-based metal-organic framework-supported gold nanocluster composite material is 10-200 µg / mL, preferably 50 µg / mL; the concentration of 2,2'-adiazonobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is 50-300 µM, preferably 100 µM.

[0042] In one or more embodiments, fluorescence detection is performed at a wavelength of 400 nm.

[0043] The beneficial effects of this invention are as follows:

[0044] (1) AuNCs and Ce-MOFs have different electrical properties and can be combined by simple electrostatic recombination to prepare ACM composites. Cerium-based metal-organic frameworks (Ce-MOFs) have excellent oxidase-like activity, and AuNCs have excellent fluorescence properties. The combination of the two not only enhances the fluorescence of AuNCs, but also retains the oxidase-like activity.

[0045] (2) A dual-channel sensing platform for fluorescence and colorimetry was formed by combining AuNCs and Ce-MOF for the detection of α-glucosidase and the screening of α-glucosidase inhibitors. The cerium-based metal-organic framework-supported gold nanoclusters catalyzes the oxidation of the colorless substrate 2,2'-adiazonobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to generate the colored oxidation product (oxABTS). oxABTS has a characteristic UV absorption at 410 nm and can efficiently quench the fluorescence of the cerium-based metal-organic framework-supported gold nanoclusters through the internal filter effect (IFE). α-glucosidase catalyzes the hydrolysis of the substrate 2-O-α-D-glucopyranosyl-L-ascorbic acid (AAG) to generate ascorbic acid (AA). AA can inhibit the oxidation of ABTS regulated by the cerium-based metal-organic framework-supported gold nanoclusters, thereby causing a decrease in the UV absorption of oxABTS and the recovery of ACM fluorescence. Therefore, dual-channel detection of α-GLU is achieved through the dual signal output of colorimetric and fluorescence signals. Meanwhile, α-glucosidase inhibitors restrict the formation of AA from AAG catalyzed by α-GLU, thereby promoting the effective oxidation of ABTS by ACM and the fluorescence quenching of ACM, thus enabling the screening of α-glucosidase inhibitors.

[0046] (3) A dual-channel sensing platform for α-glucosidase detection was formed by combining AuNCs and Ce-MOFs. The detection limits for α-glucosidase were as low as 0.0919 U / L and 0.705 U / L, respectively. These detection limits are lower than most previous reports, indicating that the sensing platform has high sensitivity for α-glucosidase analysis. At the same time, the dual-channel sensing platform for α-glucosidase has good anti-interference ability. Before and after adding interfering substances to the detection system, the fluorescence intensity and absorbance signal response of the sensing platform to α-glucosidase are basically the same.

[0047] (4) The dual-channel sensing platform based on AuNCs and Ce-MOF composite fluorescence and colorimetry provided by this invention is simple to operate and does not require complex instrumentation for α-glucosidase activity detection or α-glucosidase inhibitor screening. Furthermore, the reliability and accuracy of detection can be improved by cross-validating the results of the two output signals. This work provides a convenient, highly sensitive, and reliable analytical method for clinical diagnosis and drug screening related to α-glucosidase activity. Attached Figure Description

[0048] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0049] Figure 1The synthesis of the ACM fluorescent composite material was confirmed by TEM and STEM. In the image, A is the TEM image of AuNCs, C is the TEM image of Ce-MOF, and D and E are the TEM images of ACM. B is the HR-TEM image of AuNCs, F is the HR-TEM image of ACM, and G~I are the STEM images and elemental mappings (C, N, O, S, Au, and Ce) of ACM.

[0050] Figure 2 A histogram of the size distribution of AuNCs;

[0051] Figure 3 The EDX spectrum of ACM;

[0052] Figure 4 In the figure, A represents the Zeta potential results, B represents the FT-IR spectrum, C represents the XRD patterns of Ce-MOF and ACM, D represents the XPS spectrum of ACM, and E~I represent the high-resolution XPS spectra of C 1s, N 1s, O 1s, Au 4f and Ce 3d.

[0053] Figure 5 XPS spectrum of S 2p;

[0054] Figure 6 Figure A shows the fluorescence emission spectra of Ce-MOF, AuNCs, and ACM, with the inset being the corresponding photographs taken under ultraviolet light; Figure B shows the fluorescence emission spectra of ACM at different mass ratios of AuNCs to Ce-MOF; Figure C shows the fluorescence intensity of ACM at different mass ratios of AuNCs to Ce-MOF, with the inset in Figure C being the corresponding digital photograph of ACM under ultraviolet light irradiation; Figure D shows the fluorescence decay curves of AuNCs and ACM.

[0055] Figure 7 In the figure, A represents the fluorescence excitation spectra of AuNCs and ACM, and B represents the fluorescence emission spectra of ACM at different excitation wavelengths.

[0056] Figure 8 The fluorescence intensity of ACM under different NaCl concentrations (A) and pH (B);

[0057] Figure 9 A diagram illustrating the preparation of ACM and the detection mechanism of α-GLU and its inhibitors;

[0058] Figure 10 In the diagram, A represents the UV absorption spectrum of oxABTS and the fluorescence excitation spectrum of ACM; B represents the fluorescence decay curves of ACM and ACM / ABTS; and C and D represent the UV absorption spectra (C) and absorbance difference ∆A of the ACM / ABTS / AAG system at different α-GLU concentrations. 410(D), The insets in C are the corresponding digital photographs under visible light; E and F are the fluorescence spectra (E) and fluorescence intensity ratio F / F0 (F) of the ACM / ABTS / AAG system at different α-GLU concentrations, and the inset in E is the corresponding digital photograph under ultraviolet light irradiation;

[0059] Figure 11 Fluorescence (A) and UV absorption (B) spectra of the ACM / ABTS system in the presence of AAG, α-GLU and AAG / α-GLU.

[0060] Figure 12 The effects of incubation temperature (A), time (B), and AAG concentration (C) on the absorbance difference of the ACM / ABTS / AAG system were investigated.

[0061] Figure 13 The detection method provided by this invention determines the selectivity (A, B) and anti-interference ability (C, D) of α-GLU, where A and C are fluorescence results, and B and D are ultraviolet absorption results; the horizontal axis of each graph from left to right in A to D are: blank, α-glucosidase (α-GLU), and sodium ion (Na+). + ), potassium ions (K) + ), magnesium ions (Mg 2+ ), manganese ions (Mn) 2+ ), cobalt ions (Co) 2+ ), ammonium ions (NH4+) + ), chloride ions (Cl) - ), iodide ions (I - ), nitrate ions (NO3) - ), sulfate ions (SO4) 2- Protein kinase A (PKA), alkaline phosphatase (ALP), pepsin, exonuclease III (ExoIII), lysozyme, urease, glutathione transferase (GST), tyrosine (Tyr), lysine (Lys), tryptophan (Trp), methionine (Met), glutamine (Gln), urea (Urea), glucose (Glu), galactose (Gal), sucrose (SUC), cholesterol (CHOL), and collagen (Collagen);

[0062] Figure 14In Figure A, the effect of hesperidin or apigenin on the ACM / ABTS or ACM / ABTS / AAG / α-GLU system is shown. The UV absorption spectra (B, F) and inhibition efficiencies (C, G) of the ACM / ABTS / AAG / α-GLU system at different concentrations of hesperidin and apigenin are also shown. The fluorescence spectra (D, H) and inhibition efficiencies (E, I) of the ACM / ABTS / AAG / α-GLU system at different concentrations of hesperidin and apigenin are also shown.

[0063] Figure 15 A is a flowchart of the portable kit for detecting α-GLU; B is an image of a swab with different α-GLU concentrations; C shows a linear relationship between the G / (R+B) value and the α-GLU concentration.

[0064] Figure 16 A is a schematic diagram of the α-GLU catalytic conversion of pNPG to yellow pNP; B is the UV-Vis absorption spectrum of the pNPG / α-GLU system after incubation with different amounts of α-GLU, with the inset showing the corresponding solution colors; C is the absorbance difference diagram of the pNPs generated in the pNPG / α-GLU system at 405 nm (∆A). 405 The relationship between the absorbance intensity difference of the pNPG system at 405 nm with and without α-GLU and the concentration of α-GLU is expressed. Detailed Implementation

[0065] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0067] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0068] Example 1

[0069] Preparation of cerium-based metal-organic framework-supported gold nanoclusters composite materials:

[0070] 0.3 mL of glutathione (100 mM) and 2.0 mL of chloroauric acid (HAuCl4, 10 mM) were added to 7.7 mL of ultrapure water and stirred at 25 °C for 5 min. The mixture was then heated to 70 °C and maintained with stirring for 24 h. Finally, the resulting gold nanoclusters (AuNCs) solution was dialyzed through a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h, and then freeze-dried to obtain solid gold nanoclusters (AuNCs).

[0071] 0.53 g of terephthalic acid was dissolved in 25 mL of N,N-dimethylformamide solution and magnetically stirred. Then, 8 mL of aqueous solution containing 1.74 g of cerium ammonium nitrate was added. After stirring for 10 min, the mixture was transferred to a polytetrafluoroethylene-lined autoclave, sealed, and reacted at 100 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature, and the precipitate was collected by centrifugation (10,000 rpm, 10 min). The precipitate was washed repeatedly with water to remove unreacted substances. Finally, cerium-based metal-organic framework (Ce-MOF) powder was obtained by freeze-drying.

[0072] AuNCs (0.5 mL, 1 mg / mL) and Ce-MOF (0.75 mL, 1 mg / mL) were added to 8.75 mL of ultrapure water. The mixture was reacted at room temperature for 2 h. After centrifugation (10000 rpm, 15 min), the supernatant was removed. The precipitate was then diluted with water to 10 mL and dispersed by ultrasonication to obtain a cerium-based metal-organic framework-supported gold nanoclusters composite material (ACM).

[0073] Example 2

[0074] The successful synthesis of ACM fluorescent composite material was confirmed by TEM and STEM. The morphology and particle size of AuNCs obtained by TEM were also analyzed. Figure 1 As shown in Figure A, AuNCs are approximately spherical, well dispersed, and have an average particle size of approximately 2.06 nm. Figure 2 High-resolution transmission electron microscopy (HRTEM) images ( Figure 1 The lattice fringes in (B) are characterized by a spacing of 0.235 nm, which is attributed to the (111) lattice spacing of face-centered cubic (fcc) gold. From the TEM image of Ce-MOF ( Figure 1 As can be seen from Figure C, Ce-MOF exhibits an irregular shape and an aggregated state. After Ce-MOF is compounded with AuNCs, the shape of Ce-MOF remains unchanged, and the ACMs still exhibit an aggregated state. Figure 1 (Middle D). Magnified TEM image from ACM ( Figure 1In the HR-TEM image, it can be clearly observed that AuNCs are uniformly distributed on the Ce-MOF surface. Figure 1 The (111) lattice spacing (0.235 nm) of AuNCs can be clearly observed in the middle (F). Figure 1 The image in G shows that AuNCs (white dots) are uniformly distributed throughout the Ce-MOF matrix. Furthermore, through element-mapped images ( Figure 1 The figure (G~I) shows the C, N, O, S, Au and Ce elements in the ACM, which also indicates the uniform distribution of AuNCs on Ce-MOF. Figure 3 The EDX spectrum shown further indicates that ACM is composed of C, N, O, S, Au, and Ce elements. Furthermore, the contents of Ce and Au in ACM are 3.5% and 9.02% (weight percentage), respectively. These results confirm the successful preparation of ACM.

[0075] The successful preparation of ACM was further confirmed by zeta potential measurement, Fourier transform infrared (FT-IR), spectroscopic X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD). Figure 4 Zeta potential analysis in A showed that AuNCs were negatively charged with an average zeta potential of -30.0 mV, while Ce-MOF was positively charged with an average zeta potential of +16.4 mV. Therefore, AuNCs can be adsorbed and confined on Ce-MOF via electrostatic interactions, forming the ACM composite material. Furthermore, the zeta potential of ACM was -10.7 mV, falling between that of Ce-MOF and AuNCs, confirming their electrostatic recombination.

[0076] Fourier transform infrared spectroscopy (AuNCs, CeMOF, and ACM) Figure 4 As shown in Figure B, the characteristic peak in the FT-IR spectrum of AuNCs is at 1231 cm⁻¹. -1 (CO stretching vibration), 1650 cm -1 and 1734 cm -1 (C=O stretching vibration), 1535 cm -1 (NH tensile vibration) and 3430 cm -1 (OH stretching vibration). In the FT-IR spectrum of Ce-MOF, 1547 cm⁻¹ -1 (-COO- asymmetric stretching vibration), 1387 cm -1 (-COO- symmetric stretching vibration), 747 cm -1 (Ce-O stretching vibration) and 3400 cm -1(OH stretching vibration). After the synthesis of ACM, specific absorption peaks from AuNCs and CeMOF can be observed, indicating the successful preparation of the ACM composite probe.

[0077] The electronic structure and surface elemental composition of the ACM were analyzed using XPS. Figure 4 In D, there are obvious peaks at 285 eV (C1s), 401 eV (N1s), 531 eV (O1s), 905 / 885 eV (Ce3d), 88 eV / 84 eV (Au4f), and 163 eV (S2p). Figure 4 The middle E image shows high-resolution C1s spectra, revealing different structural compositions: CC (284.8 eV), CN / CO / CS (286.9 eV), and C=O (288.2 eV). N 1s spectra ( Figure 4 The presence of two nitrogen states, CN (399.7 eV) and NH (401.8 eV), was confirmed by the O1s spectrum. Figure 4 The γ-ray dihydrogen ion (γ-O) exhibits three peaks at 530.7, 531.5, and 532.7 eV, attributed to lattice oxygen (Ce-O), CO, and C=O, respectively. Notably, Au(… Figure 4 The middle H)) exhibits a unique signal: Au(I) Au 4f 7 / 2 (84.8 eV) and Au 4f 5 / 2 (88.5 eV), Au(0) Au 4f 7 / 2 (84.2 eV) and Au 4f 5 / 2 (87.9 eV). Figure 4 The middle I indicates that the Ce 3d spectrum can be divided into six small peaks; the binding energies of 904.9, 899.9, 886.8, and 881.7 eV correspond to Ce 4+ The peak values ​​at 903.4 and 884.8 eV belong to Ce 3+ S 2p spectrum ( Figure 5 The results showed two peaks: -SH (162.2 eV) and Au-S (163.4 eV).

[0078] The crystal structures of Ce-MOF and ACM were investigated using XRD. Figure 4In the B-wavelength XRD pattern, characteristic diffraction peaks of Ce-MOF were observed mainly at 7.07°(111) and 8.15°(200), consistent with previous studies. After ACM formation, the XRD results were largely consistent with the spectrum of pure Ce-MOF, with a slight decrease in intensity, indicating that AuNCs adsorption did not affect the structure of the crystalline Ce-MOF. This means that Ce-MOFs essentially maintained the excellent crystallinity and stability of its composite material, thus providing a uniform and stable surface area, effectively adsorbing AuNCs, and improving the fluorescence intensity of AuNCs.

[0079] Example 3

[0080] The fluorescence properties of ACM and the fluorescence enhancement mechanism of AuNCs were explored in depth. Figure 6 In A, after AuNCs were electrostatically confined on Ce-MOF, the fluorescence emission of AuNCs was significantly enhanced, and the fluorescence emission peak showed a blue shift, moving from 601 nm to 585 nm. For example... Figure 6 As shown in the inset A, the final ACM exhibits orange-red fluorescence. Figure 6 Figure B clearly demonstrates the relationship between the fluorescence enhancement effect of AuNCs and the weight ratio (w / w) of AuNCs and Ce-MOF. Figure 6 The results showed that with increasing Ce-MOF content, the fluorescence intensity of AuNCs initially increased and then decreased. The fluorescence intensity of ACM reached its maximum at an AuNCs:Ce-MOF ratio of 1:1.5 (w / w), which was 7 times that of the original AuNCs. Correspondingly, the quantum yield of ACM significantly increased to 18.60%, which was 13.4 times that of the control group AuNCs (QY=1.39%). Furthermore, in... Figure 7 The results in Figure A show that the optimal fluorescence excitation spectrum of AuNCs is located at 405 nm, while that of ACM is located at 400 nm. Furthermore, the fluorescence spectra of ACM at different excitation wavelengths (…) Figure 7 (B) also confirms that the fluorescence intensity reaches its maximum when the excitation wavelength is 400 nm. From... Figure 6 Table D and Table 1 summarize the fluorescence lifetime data of AuNCs and ACMs. The average fluorescence lifetimes of AuNCs and ACMs were 8.61 µs and 9.87 µs, respectively, indicating that energy transfer occurred in AuNCs after recombination with Ce-MOF. On the other hand, the fluorescence of AuNCs was enhanced after recombination with Ce-MOF. This may be because the confinement effect of Ce-MOF can effectively suppress intramolecular motion and reduce nonradiative transitions in AuNCs, thereby enhancing the fluorescence of AuNCs. These results indicate that Ce-MOF confinement significantly improves the fluorescence performance of AuNCs. In addition, the stability of ACMs was also investigated. Figure 8This indicates that the fluorescence intensity of ACM remains largely unaffected by different NaCl concentrations and pH levels. Therefore, the confinement of Ce-MOF endows AuNCs with excellent fluorescence properties and stability, enhancing the application prospects of ACM in the field of biosensing.

[0081] Table 1. Fitting parameters for fluorescence lifetime measurements of AuNCs, ACM, and ACM+oxABTS

[0082]

[0083] Example 4

[0084] A dual-channel signal sensing platform for detecting α-GLU and its inhibitors was constructed using ACM as a peroxidase mimic and a fluorescent signal indicator. Figure 9 As shown, this sensing system is mainly based on the fact that the product generated by α-GLU catalyzes its substrate can inhibit the blue-green reaction of Ce-MOF-catalyzed ABTS. In short, Ce-MOF catalyzes the colorless substrate ABTS to form the oxidation product oxABTS. oxABTS has strong absorption at 410 nm, thus the generated oxABTS effectively quenches the fluorescence of AuNCs through the internal filtration effect (IFE). However, in the presence of α-GLU, the α-GLU catalyzes the hydrolysis of the substrate AAG to AA. AA has reducing properties and can significantly inhibit the Ce-MOF-catalyzed oxidation of ABTS. Since the production of oxABTS is inhibited, the absorbance of the system decreases, and the fluorescence quenching of ACM induced by oxABTS is also suppressed, resulting in fluorescence recovery. Therefore, by utilizing the changes in both fluorescence and colorimetric signals of this system, the sensing of α-GLU and its inhibitors can be achieved.

[0085] The internal filtration effect, as an effective fluorescence quenching mechanism, arises from the absorption of the emitted light and / or excitation light of the fluorophore by the absorbing substance when the ultraviolet absorption spectrum of the absorbing substance in the analytical system overlaps with the fluorescence emission spectrum and / or excitation spectrum of the fluorophore. Figure 10 The A-value shows a significant overlap between the UV absorption of oxABTS and the fluorescence excitation spectrum of AuNCs, which is a prerequisite for the formation of an internal filtering effect between ACM and oxABTS. Figure 10 As shown in Figure B, the fluorescence lifetime of ACM remained almost unchanged after the addition of oxABTS, indicating that no electron or energy transfer occurred between ACM and oxABTS. Based on these results, it can be concluded that the fluorescence quenching mechanism of ACM caused by oxABTS is attributed to the internal filtering effect.

[0086] Example 5

[0087] Methods for detecting α-GLU:

[0088] To detect α-GLU, different concentrations of α-GLU and AAG solution (50 μL, 1 mM) were incubated at 37 °C for 60 min. Then, ACM (80 μL, 0.625 mg / mL), ABTS (100 μL, 1 mM), and acetate buffer (pH=3.0, 50 μL, 100 mM) were added. After 15 min, fluorescence and UV-Vis absorption spectra were recorded at an excitation wavelength of 400 nm.

[0089] This further validates the feasibility of this method for detecting α-GLU. Figure 11 Figures A and B show that α-GLU and AAG alone have no effect on the UV absorption and fluorescence spectra of the ABTS / ACM system, but the addition of coexisting α-GLU and AAG to the system significantly reduces the absorbance at 410 nm. Figure 11 (A) Conversely, Figure 11 The fluorescence intensity of the B-mode system increased significantly to four times the original value, clearly demonstrating the feasibility of this dual-signal sensing platform for detecting α-GLU. Under the optimal conditions for α-GLU-catalyzed AAG (… Figure 12 The detection capability of this dual-signal sensing platform for α-GLU was studied. Figure 10 The absorbance of the detection system at 410 nm gradually decreased with increasing α-GLU concentration, as shown by the C-ray discretization assay. Figure 10 The inset in Figure C shows that the obvious fading of the green color is easily discernible to the naked eye. Figure 10 The value of absorbance difference ΔA at 410 nm is shown in the figure. 410 It exhibits a good linear relationship with α-GLU concentration in the range of 1–100 U / L. The fitted linear equation is ∆A. 410 =0.0181+0.0036[α-GLU](U / L), R 2 =0.993. Figure 10 E observed that the fluorescence of the detection system increased with increasing α-GLU concentration, and the inset showed that the fluorescence intensity of the solution also showed an increasing trend. Figure 10 As can be seen from F, the fluorescence intensity ratio F / F0 is related to the α-GLU concentration in the range of 0.1~100 UL. -1 The relationship is linear within the range. The regression equation is F / F0 = 1.29502 + 0.032[α-GLU] (U / L), R 2=0.990. Based on the colorimetric and fluorescence dual-mode approach, the limits of detection (LOD) for α-GLU were 0.705 U / L and 0.0919 U / L, respectively. The LOD of this dual-channel strategy is lower than most previously reported methods, indicating that the system has high sensitivity for the detection of α-GLU. In particular, this dual-signal output detection mode can achieve self-calibration to eliminate systematic errors caused by fluctuations in environmental factors, providing a strong foundation for achieving high-accuracy determinations.

[0090] Optimization of α-GLU detection conditions:

[0091] To obtain good α-GLU detection performance, the following parameters were further optimized: (A) enzymatic culture temperature; (B) time; and (C) AAG concentration. Figure 12 Figure A shows the absorbance difference (∆A) of the system when the reaction catalyzed by α-GLU on AAG reaches the enzyme culture temperature of 37°C. 410 Reaching the platform. Furthermore, such as... Figure 12 As shown in Figure B, the system's ∆A 410 As time gradually increases, the enzyme reaction reaches a plateau at approximately 60 minutes. Figure 12 C indicates that as the AAG concentration increases, the system's ΔA... 410 The concentration was gradually increased and then stabilized after reaching 50 µM. Therefore, the following experimental conditions were selected to obtain better α-GLU analysis results: (A) Enzymatic culture temperature of 37℃; (B) Time of 60 minutes; (C) AAG concentration of 50 µM.

[0092] Selective recognition capability is a crucial indicator for evaluating the practicality of a sensing platform, especially for analytical methods involving complex biological samples. Therefore, to simulate the real-world application environment of α-GLU detection, common substances, including ions, amino acids, other enzymes, and biomolecules, were selected to systematically study the selectivity and anti-interference capabilities of this method. Figure 13 As shown in Figures A and B, the fluorescence intensity and absorbance of the system only change significantly in the presence of α-GLU. In the absence of α-GLU, there is no significant change in fluorescence intensity and absorbance, indicating that the sensing system has high selectivity for α-GLU. Furthermore, interference experiments were conducted by adding the aforementioned substances in the presence of α-GLU. Figure 13 As can be seen from C and D, the fluorescence intensity and absorbance signal response of the sensing platform to α-GLU are basically the same before and after the addition of interfering substances to the detection system, indicating that the α-GLU sensing platform has good anti-interference ability.

[0093] Example 5

[0094] α-GLU inhibitor detection methods:

[0095] To detect α-GLU inhibitors, different concentrations of α-GLU inhibitors (hesperidin or apigenin) were reacted with α-GLU (10 μL, 10 U / mL) and AAG solution (50 μL, 1 mM) at 37 °C for 60 min. Afterwards, ACM (80 μL, 0.625 mg / mL), ABTS (100 μL, 1 mM), and acetate buffer solution (pH=3.0, 50 μL, 100 mM) were added. After 15 min, fluorescence and UV-Vis absorption spectra were recorded at an excitation wavelength of 400 nm.

[0096] The inhibition efficiency (IE, %) can be calculated using the following equation.

[0097] IE (%) = (F' − F) / (F' –F0) × 100

[0098] IE (%) = (A' − A) / (A' – A0) × 100

[0099] In the formula, F0 (or A0) is the fluorescence intensity (or absorbance) of the ACM / ABTS / AAG system, F' (or A') is the fluorescence intensity (or absorbance) of the ACM / ABTS / α-GLU / AAG system, and F (or A) is the fluorescence intensity (or absorbance) of the ACM / ABTS / α-GLU / AAG system containing the inhibitor (hesperidin or apigenin).

[0100] Plant-derived natural products serve as a valuable source for lead compound exploration and are of great significance for the discovery and screening of natural enzyme inhibitors. To date, a variety of structurally diverse α-glucosidase inhibitors have been isolated from abundant plant medicinal resources. These inhibitors encompass polyphenols, alkaloids, flavonoids, and polysaccharides. To further explore the potential application of this dual-signal analysis method in the screening of α-GLU inhibitors, hesperidin and apigenin, two flavonoid inhibitors, were selected as typical models to study its feasibility. Figure 14 Hesperidin or apigenin have virtually no effect on the ACM / ABTS system. However, the fluorescence is quenched upon the addition of hesperidin or apigenin to the ACM / ABTS / α-GLU / AAG detection system. This is because hesperidin or apigenin restricts the α-GLU-catalyzed formation of AA from AAG, thereby promoting the effective oxidation of ABTS by ACM and the fluorescence quenching of ACM. Figure 14As shown in Figures B and D, with a fixed α-GLU concentration of 100 U / L, as the hesperidin concentration increased from 0 to 100 µM, the UV absorption of the ABTS / ACM / α-GLU / AAG system gradually increased, while the fluorescence intensity gradually decreased. The same trend was observed when the apigenin concentration increased from 0 to 80 µM. Figure 14 (F and H). IC50 of hesperidin 50 The value (the inhibitor concentration required to inhibit 50% of enzyme activity) was calculated using a dual-channel mode to be 93.14 µM (colorimetric method). Figure 14 (C) and 91.39µM (fluorescence method) Figure 14 (E), apigenin IC 50 The value was calculated to be 46.51 µM (colorimetric method). Figure 14 (medium G) and 47.52µM (fluorescence method) Figure 14 (I). The above results indicate that this method has potential applications in screening α-GLU inhibitors.

[0101] Example 6

[0102] Applications of portable reagent kits:

[0103] Based on the above research, this embodiment encapsulates ACM onto a cotton swab and designs and develops a detection kit. Figure 15 The process of assembling the test kit was demonstrated in section A. First, ACM mixed with gelatin was sealed onto a cotton swab. Then, AAG solution and α-GLU solution were added dropwise to the inside of a centrifuge tube and allowed to react at 37 °C for 1 h. After the reaction, ABTS and buffer solution were added, and the cotton swab was then inserted to allow the reacted solution to permeate the inside of the swab. After the reaction, the cotton swab was removed, and an optical image of the swab was obtained using a smartphone. This image was then combined with colorimetric software to plot a quantitative curve of RGB data versus α-GLU concentration, thus achieving quantitative detection of α-GLU. Figure 15 In Figure B, a clear gradient change in the color of the cotton swab was observed with increasing α-GLU concentration. Further image digitization was performed using colorimetric software to plot quantitative curves of RGB values ​​versus α-GLU concentration. Figure 15 As shown in Figure C, the α-GLU concentration exhibits a good linear relationship with the G / (R+B) value in the range of 5~200 U / L: G / (R+B) = 0.5895 - 3.1132 [α-GLU] (U / L) (R 2 = 0.998). This kit has the advantages of simple operation and low cost, providing a new method for the clinical monitoring of α-GLU.

[0104] Example 7

[0105] To investigate the feasibility of this sensing platform for detecting α-GLU in real samples, we applied the established method to detect α-GLU in serum samples from healthy adults. Furthermore, to evaluate the accuracy of this method, we compared the detection results with those of the traditional colorimetric detection method for α-GLU (pNPG method). Figure 16 A comparison was made with traditional colorimetric detection methods, which rely on α-GLU's ability to hydrolyze PNPG to PNP and monitor the absorbance of PNP at 410 nm to detect α-GLU activity. As shown in Table 2, the recoveries of α-GLU ranged from 95.1% to 104.2%, with relative standard deviations less than 5.26% (n = 3). These results demonstrate that this dual-signal detection method can quantitatively detect the α-GLU content in complex biological samples with high reliability and accuracy.

[0106] Table 2 Comparison of analytical results of α-GLU in human serum

[0107]

[0108] pNPG-based α-GLU activity analysis:

[0109] The activity of α-glucosidase (α-GLU) in human serum was determined using p-nitrophenyl-α-D-glucopyranoside (pNPG) as a reference method. This method relies on α-GLU catalysis to convert pNPG to yellow p-nitrophenol (pNP). Figure 16 (A). pNPG has a characteristic absorption peak at 405 nm. In short, different concentrations of α-GLU were mixed with pNPG (60 μL, 5 mM) and incubated at 37 °C for 60 min. Then, Tris-HCl buffer (pH 9.0, 50 μL, 50 mM) was added to the mixture, and the UV-Vis absorption spectra of the pNPG / α-GLU systems containing different amounts of α-GLU were measured. Figure 16 (B). A linear regression equation was obtained between the absorbance difference at 405 nm (∆A405, representing the difference in absorbance intensity of the pNPG system at 405 nm with and without α-GLU) and α-GLU activity. Figure 16 (C). α-GLU activity in serum samples was detected using a pNPG-based method by replacing pure α-GLU with spiked serum samples (Table 2). α-GLU activity in human serum was determined using p-nitrophenyl-α-D-glucopyranoside (pNPG) as a reference method.

[0110] in conclusion:

[0111] A high-performance ACM composite material was prepared by combining the fluorescence properties of AuNCs with the highly efficient oxidase-mimicking activity of Ce-MOF through electrostatic interaction. A dual-channel sensing platform using both fluorescence and colorimetric methods was constructed for the detection of α-GLU and its inhibitors. The limits of detection for α-GLU in the fluorescence and colorimetric modes were as low as 0.0919 U / L and 0.705 U / L, respectively, which are lower than most previously reported limits, indicating that the sensing platform has high sensitivity for α-GLU analysis. This dual-signal detection method using colorimetry and fluorescence was used to sensitively detect α-GLU and its inhibitors in human serum. The method is simple to operate, requires no complex instrumentation, and the reliability and accuracy of the detection can be improved by cross-validating the results of the two output signals. This work provides a convenient, highly sensitive, and reliable analytical method for the clinical diagnosis and drug screening of α-GLU-related diseases.

[0112] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A probe composition for detecting α-glucosidase activity, characterized in that, include: A cerium-based metal-organic framework-supported gold nanocluster composite material, 2-O-α-D-pyranose glucosyl-L-ascorbic acid and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). The cerium-based metal-organic framework-loaded gold nanocluster composite material uses a cerium-based metal-organic framework as a carrier and loads gold nanoclusters. The cerium-based metal-organic framework is composed of Ce 3+ Ce 4+ Metal-organic frameworks formed by coordination with ligand terephthalic acid.

2. The probe composition for detecting α-glucosidase activity as described in claim 1, characterized in that, The mass ratio of gold nanoclusters to cerium-based metal-organic frameworks is 1:(1~2).

3. The probe composition for detecting α-glucosidase activity as described in claim 2, characterized in that, The mass ratio of the gold nanoclusters to the cerium-based metal-organic framework is 1:1.

5.

4. The probe composition for detecting α-glucosidase activity as described in claim 1, characterized in that, The preparation method of the cerium-based metal-organic framework-supported gold nanocluster composite material includes the following steps: (1) The template glutathione and the gold source were placed in water, and after the reaction, they were dialyzed and dried to obtain gold nanoclusters; (2) Cerium ammonium nitrate was reacted with ligand terephthalic acid to obtain cerium-based metal-organic frameworks; (3) Place both gold nanoclusters and cerium-based metal-organic frameworks in water and react to obtain a cerium-based metal-organic framework-supported gold nanocluster composite material.

5. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (1), the gold source is selected from chloroauric acid.

6. The probe composition for detecting α-glucosidase activity as described in claim 5, characterized in that, In step (1), the molar ratio of glutathione to chloroauric acid is (9~12):

1.

7. The probe composition for detecting α-glucosidase activity as described in claim 6, characterized in that, The molar ratio of glutathione to chloroauric acid is 10:

1.

8. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (1), the concentration of glutathione is 9~12 mol / L.

9. The probe composition for detecting α-glucosidase activity as described in claim 8, characterized in that, The concentration of glutathione is 10 mol / L.

10. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (1), the reaction temperature is 65~80 ℃; the reaction time is 20~30 h.

11. The probe composition for detecting α-glucosidase activity as described in claim 10, characterized in that, The reaction temperature is 70 °C; the reaction time is 24 h.

12. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (1), the molecular weight cut-off of the dialysis bag used during dialysis is 900~1200 Da.

13. The probe composition for detecting α-glucosidase activity as described in claim 12, characterized in that, The dialysis bag used during the dialysis process has a molecular weight cutoff of 1000 Da.

14. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (2), the molar ratio of cerium ammonium nitrate to terephthalic acid ligand is 1:(0.8~1.2).

15. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (2), the reaction temperature is 90~120 ℃; the reaction time is 0.5~2 h.

16. The probe composition for detecting α-glucosidase activity as described in claim 15, characterized in that, The reaction temperature is 100 °C; the reaction time is 1 h.

17. The probe composition for detecting α-glucosidase activity as described in claim 4, characterized in that, In step (3), the mass ratio of gold nanoclusters to cerium-based metal-organic frameworks is 1:(1~2).

18. The probe composition for detecting α-glucosidase activity as described in claim 17, characterized in that, The mass ratio of the gold nanoclusters to the cerium-based metal-organic framework is 1:1.

5.

19. A probe composition for screening α-glucosidase inhibitors, characterized in that, include: A cerium-based metal-organic framework-supported gold nanocluster composite material, 2-O-α-D-pyranoglutinyl-L-ascorbic acid, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and α-glucosidase; The cerium-based metal-organic framework-loaded gold nanocluster composite material uses a cerium-based metal-organic framework as a carrier and loads gold nanoclusters. The cerium-based metal-organic framework is composed of Ce 3+ Ce 4+ Metal-organic frameworks formed by coordination with ligand terephthalic acid.

20. A product characterized in that, This includes the probe composition for detecting α-glucosidase activity as described in claim 1 or the probe composition for screening α-glucosidase inhibitors as described in claim 19.

21. A method for detecting α-glucosidase activity, characterized in that, The use of the probe composition for detecting α-glucosidase activity according to claim 1 comprises the following steps: The α-glucosidase solution to be tested was mixed with 2-O-α-D-glucopyranosyl-L-ascorbic acid and then incubated for the first time to obtain the first incubation solution. A second incubation solution was obtained by adding cerium-based metal-organic framework-supported gold nanoclusters and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) to the first incubation solution and then performing a second incubation. The second incubation solution was subjected to fluorescence and colorimetric detection. The activity of the α-glucosidase to be tested was obtained based on the working curve of α-glucosidase activity, the fluorescence intensity obtained from fluorescence detection, the standard curve of absorbance change, and the absorbance difference of the second incubation solution.

22. The method as described in claim 21, characterized in that, The temperature for the first incubation was 37–55 °C; the incubation time was 60–80 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid was 50–70 µM.

23. The method as described in claim 22, characterized in that, The temperature of the first incubation was 37 °C; the incubation time was 60 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid was 50 µM.

24. The method as described in claim 21, characterized in that, The second incubation was carried out at room temperature for 12-20 minutes, and the pH value for the second incubation was 2.5-4.

5.

25. The method as described in claim 24, characterized in that, The second incubation period was 15 minutes, and the pH value of the second incubation was 3.

0.

26. The method as described in claim 21, characterized in that, The concentration of the cerium-based metal-organic framework-supported gold nanoclusters composite material was 10–200 µg / mL; the concentration of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) was 50–300 µM.

27. The method as described in claim 26, characterized in that, The concentration of the cerium-based metal-organic framework-supported gold nanocluster composite material is 50 µg / mL; the concentration of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) is 100 µM.

28. The method as described in claim 21, characterized in that, Fluorescence detection was performed at a wavelength of 400 nm.

29. A method for screening α-glucosidase inhibitors, characterized in that, Using the probe composition for screening α-glucosidase inhibitors according to claim 19 includes the following steps: The α-glucosidase inhibitor, α-glucosidase, and 2-O-α-D-glucopyranosyl-L-ascorbic acid were mixed and incubated for a third time to obtain the third incubation solution. A fourth incubation solution was obtained by adding cerium-based metal-organic framework-supported gold nanoclusters and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) to the third incubation solution and then performing a fourth incubation. The fourth incubation solution was subjected to fluorescence and colorimetric detection. The half-inhibition concentration of the α-glucosidase inhibitor to be tested was obtained according to the calibration curve of the α-glucosidase inhibitor to be tested, thereby achieving the screening of α-glucosidase inhibitors.

30. The method as described in claim 29, characterized in that, The α-glucosidase inhibitors include hesperidin or apigenin.

31. The method as described in claim 29, characterized in that, The temperature for the third incubation was 37–55 °C; the incubation time was 60–80 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid was 50–70 µM.

32. The method as described in claim 31, characterized in that, The temperature of the third incubation was 37 °C; the time of the third incubation was 60 min; and the concentration of 2-O-α-D-glucopyranosyl-L-ascorbic acid was 50 µM.

33. The method as described in claim 29, characterized in that, The fourth incubation was performed at room temperature for 12-20 minutes, and the pH value was 2.5-4.

5.

34. The method as described in claim 33, characterized in that, The fourth incubation lasted for 15 minutes, and the pH value of the fourth incubation was 3.

0.

35. The method as described in claim 29, characterized in that, The concentration of the cerium-based metal-organic framework-supported gold nanoclusters composite material was 10–200 µg / mL; the concentration of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) was 50–300 µM.

36. The method as described in claim 35, characterized in that, The concentration of the cerium-based metal-organic framework-supported gold nanocluster composite material is 50 µg / mL; the concentration of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) is 100 µM.

37. The method as described in claim 29, characterized in that, Fluorescence detection was performed at a wavelength of 400 nm.

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