CeMOF-on-CuMOF nano-enzyme, preparation method thereof and application of CeMOF-on-CuMOF nano-enzyme in alpha-glucosidase detection

Abstract

The invention provides a CeMOF-on-CuMOF nano enzyme, a preparation method thereof and application of the CeMOF-on-CuMOF nano enzyme in alpha-glucosidase detection, and belongs to the technical field of alpha-glucosidase detection. The MOF-on-MOF nano-enzyme (CeMOF-on-CuMOF) with double-enzyme activity is successfully synthesized, oxidase-like activity and laccase-like activity of the MOF-on-MOF nano-enzyme are both synergistically improved, a fluorescence and colorimetric dual-mode sensing method is established based on the enhanced double-enzyme activity, high-sensitivity detection of alpha-glucosidase is realized, and the detection limits are respectively as low as 0.35 U / L and 0.16 U / L. The dual-mode detection has a built-in self-correction function, so that the dual-mode detection is more reliable than the single-mode detection; and the dual-mode sensing technology based on the multifunctional MOF has a wide development prospect by virtue of the synergistic effect, the convenient multifunctional MOF synthesis method and the flexible multi-mode sensing design.

Description

Technical Field

[0001] This invention belongs to the field of α-glucosidase detection technology, specifically involving CeMOF-on-CuMOF nanozymes, their preparation methods, and their application in α-glucosidase detection. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Alpha-glucosidase (α-Glu) is a key enzyme responsible for catalyzing the cleavage of α-1,4-glycosidic bonds in oligosaccharides and disaccharides, playing a crucial role in controlling postprandial blood glucose levels and making it a primary therapeutic target for type 2 diabetes mellitus (T2DM). Therefore, developing a highly sensitive, reliable, and convenient method for α-Glu activity assay is of great significance for early diagnosis and basic biological research. Traditional α-Glu detection methods, such as high-performance liquid chromatography (HPLC) and electrochemical methods, typically require complex instruments, cumbersome sample preparation, and long analysis times, limiting their application in point-of-care testing. Optical sensing strategies, particularly colorimetric and fluorescence analysis, offer advantages such as simplicity, rapid response, and cost-effectiveness: colorimetry allows for visual semi-quantification, making it ideal for on-site testing, while fluorescence analysis provides superior sensitivity for precise quantification. However, single-mode detection systems are inherently susceptible to environmental interferences, such as sample autofluorescence or instrument fluctuations, which can reduce accuracy and reliability.

[0004] Nanozymes are nanomaterials with intrinsic enzyme-mimicking activities. Due to their excellent stability, lower cost, and ease of modification, they have become ideal natural enzyme alternatives for signal amplification. Among various nanomaterials, metal-organic frameworks (MOFs), porous crystalline materials formed by the self-assembly of metal ions and organic linkers, have emerged as a superior class of enzyme mimics. Their highly ordered and tunable structures allow for the rational design of catalytic sites, enabling precise modulation of their enzyme-like activities. To date, various enzyme activities have been mimicked, including peroxidase-like, oxidase-like, catalase-like, and superoxide dismutase-like activities. However, integrating multiple enzyme activities into a single nanomaterial is challenging, as enzyme activities or functions are often incompatible. Maintaining individual activities without interference remains a problem to be solved.

[0005] The existing technology "Construction and Application of a Portable Electrochemical Detection Device" prepared a prismatic rod-shaped tetrametal Fe / Co / Ni / Cu MOF@PPy, which was used to fabricate an electrochemical sensor and connected to a smart terminal device for electrochemical detection. However, the detection process requires a long incubation period, which cannot achieve the effect of rapid and convenient detection.

[0006] In recent years, MOF-on-MOF composites have become a research hotspot in the field of nanomaterials, focusing on the assembly of two or more homogeneous or heterogeneous MOFs with different structures and morphologies. MOF-on-MOF materials, composed of two or more MOFs, exhibit a richer diversity of metal ion centers and ligands compared to single MOF materials, thus displaying higher specific surface area and more diverse pore structures. These unique properties make MOF-on-MOF materials promising for applications in adsorption / separation and heterogeneous catalysis.

[0007] However, no research has been published on the application of MOF-on-MOF composites in the field of α-glucosidase detection. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the purpose of this invention is to provide CeMOF-on-CuMOF nanozymes, their preparation methods, and their application in α-glucosidase detection.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing CeMOF-on-CuMOF nanozymes: firstly, CuMOF is synthesized as a core and linked with polyvinylpyrrolidone for subsequent in-situ synthesis of CeMOF; subsequently, CeMOF is added sequentially. 4+ By mixing with 2-methylimidazole and stirring, CeMOF was formed on the CuMOF surface through the bridging effect of polyvinylpyrrolidone.

[0010] In embodiments of the present invention, the following steps are included: Step 1: Add CuMOF to water and mix ultrasonically; add polyvinylpyrrolidone and stir to obtain PVP@CuMOF; Step 2, add 2-methylimidazole, then add Ce dropwise. 4+ Salt solution was prepared and stirred at room temperature to obtain CeMOF-on-CuMOF.

[0011] In an embodiment of the present invention, in step 1, the mass ratio of CuMOF to polyvinylpyrrolidone is 1:2 to 2:1.

[0012] In an embodiment of the present invention, in step 1, CuMOF is mixed with polyvinylpyrrolidone and stirred for 10 to 14 hours.

[0013] In an embodiment of the present invention, in step 2, the Ce 4+ It is added in the form of Ce(NH4)2(NO3)6.

[0014] In an embodiment of the present invention, in step 2, the molar ratio of 2-methylimidazole to Ce(NH4)2(NO3)6 is 105:20 to 105:60.

[0015] In an embodiment of the present invention, in step 2, after adding 2-methylimidazole and Ce(NH4)2(NO3)6, the mixture is stirred at room temperature for 0.5 to 1.5 hours.

[0016] In an embodiment of the present invention, the mass ratio of Ce(NH4)2(NO3)6 added in step 2 to CuMOF in step 1 is 5:15~80.

[0017] In step 2, the obtained CeMOF-on-CuMOF was washed with deionized water and the solid was dried at 37°C.

[0018] Secondly, the present invention provides a CeMOF-on-CuMOF nanozyme, wherein CeMOF and CuMOF are linked by polyvinylpyrrolidone (PVP) and have strong laccase-like and oxidase-like activities.

[0019] In an embodiment of the present invention, the CeMOF-on-CuMOF nanozyme is prepared by the method for preparing CeMOF-on-CuMOF nanozyme described in the first aspect.

[0020] The prepared CeMOF-on-CuMOF uses CuMOF sheets as a substrate, with CeMOF nanoparticles loaded on its surface.

[0021] Thirdly, this invention provides the application of CeMOF-on-CuMOF nanozymes in the detection of α-glucosidase.

[0022] Fourthly, the present invention provides a method for detecting α-glucosidase, comprising colorimetric and fluorescence dual-mode detection of α-glucosidase, the operation of which is as follows: For colorimetric detection, α-glucosidase, Phenyl-α-Glc and Tris-HCl were mixed. After the reaction was completed, Tris-HCl, 4-aminoantipyrine (4-AAP) and CeMOF-on-CuMOF were added in sequence. After the reaction was completed, the absorbance at 510 nm was measured. For fluorescence detection, α-glucosidase, 2-O-α-D-glucopyranoside-L-ascorbic acid (AAG) were mixed with acetate buffer. After the reaction, acetate buffer, CeMOF-on-CuMOF and 10-acetyl-3,7-dihydroxyphenazine (AR) were added sequentially. The reaction was carried out in the dark, and the fluorescence at 585 nm was measured. The excitation light was 525 nm.

[0023] Its detection principle is based on the enzymatic hydrolysis of phenyl-α-D-glucopyranoside (Phenyl-α-Glc) and 2-O-α-D-glucopyranoside-L-ascorbic acid (AAG).

[0024] The method of this invention integrates a dual-mode sensing platform for colorimetric and fluorescence signals, providing a built-in self-calibration mechanism that significantly improves detection accuracy, anti-interference capability, and reliability.

[0025] In the solution provided by this invention, multi-enzyme activity promotes mass transfer, thereby enhancing cascade reaction performance and increasing sensitivity. In addition to multiple activities, the integration of additional functions is also convenient, facilitating the construction of multimodal sensing methods.

[0026] This patent develops a CeMOF-on-CuMOF nanozyme that combines the oxidase-like activity of CeMOF with the laccase-like activity of CuMOF, enhancing both the oxidase-like and laccase-like activities of individual MOFs. It is used for the dual-channel detection of the anti-diabetic enzyme α-glucosidase (α-Glu). Specifically, CeMOF catalyzes the oxidation of 10-acetyl-3,7-dihydroxyphenazine (AR) to a fluorescent product oxAR emitting fluorescence at 585 nm, while α-glucosidase hydrolyzes 2-O-α-D-glucopyranoside-L-ascorbic acid (AAG) to ascorbic acid (AA). AA inhibits this oxidation reaction, allowing detection via fluorescence at 585 nm. Simultaneously, the α-glucosidase activity hydrolyzes phenyl-α-D-glucopyranoside to release phenol. CuMOF catalyzes the oxidation of phenol and its coupling with 4-aminoantipyrine (4-AAP) to generate a red quinone imine with UV absorption at 510 nm, thereby enabling colorimetric detection of α-Glu. The detection method provided by this invention has detection limits as low as 0.35 U / L for fluorescence and 0.16 U / L for colorimetry. This sensing method has been further applied to the detection in human serum, demonstrating its potential application in real samples.

[0027] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention successfully synthesized a MOF-on-MOF nanozyme (CeMOF-on-CuMOF) with dual enzyme activity, in which both oxidase-like and laccase-like activities were synergistically enhanced, demonstrating the effectiveness of the MOF-on-MOF strategy in achieving spatial separation and synergistic effects.

[0028] 2. Based on enhanced dual-enzyme activity, this invention establishes a fluorescence and colorimetric dual-mode sensing method, achieving highly sensitive detection of α-glucosidase. Dual-mode detection, due to its built-in self-calibration function, is more reliable than single-mode detection; and dual-mode sensing technology based on multifunctional MOFs, with its synergistic effect, convenient multifunctional MOF synthesis method, and flexible multimode sensing design, has broad development prospects. Therefore, this research opens a new path for the vigorous development of MOF-on-MOF structures. Attached Figure Description

[0029] 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.

[0030] Figure 1 The synthesis route of CeMOF-on-CuMOF and its sensing route for α-Glu are presented.

[0031] Figure 2 The images show TEM images and elemental distribution maps of CuMOF, CeMOF, and CeMOF-on-CuMOF; where (A) is a transmission electron microscope image of CuMOF. (B) is an elemental distribution map of CuMOF. (C) is a transmission electron microscope image of CeMOF and its lattice spacing. (D) is an elemental distribution map of CeMOF. (E) is a transmission electron microscope image of CeMOF-on-CuMOF. (F) is an elemental distribution map of CeMOF-on-CuMOF.

[0032] Figure 3 XRD patterns of CuMOF, CeMOF, and CeMOF-on-CuMOF are shown below; (A) X-ray diffraction patterns of CuMOF, CeMOF, and CeMOF-on-CuMOF. (B) Full-spectrum X-ray photoelectron spectroscopy of CeMOF-on-CuMOF. (C) X-ray photoelectron spectroscopy of the 3d orbitals of Ce in CeMOF-on-CuMOF.

[0033] Figure 4 Characterization of enzyme activities of CeMOF-on-CuMOF; including: (A) Characterization of laccase-like activities of CeMOF-on-CuMOF; (B) Comparison of laccase-like activities of CuMOF, CeMOF, and CeMOF-on-CuMOF; (C) Characterization of oxidase-like activities of CeMOF-on-CuMOF; (D) Comparison of oxidase-like activities of CuMOF, CeMOF, and CeMOF-on-CuMOF.

[0034] Figure 5This study validated the α-Glu detection method, including: (A) Feasibility verification of the colorimetric detection method; (B) Absorption spectra of the colorimetric sensing system at different α-Glu concentrations; (C) Linear relationship between the absorbance difference (A-A0) and the α-Glu concentration, where A and A0 are the UV absorption intensities at 510 nm of the Phenyl-α-Glc / 4-AAP / CeMOF-on-CuMOF detection system in the presence and absence of α-Glu, respectively; (D) Feasibility verification of the fluorescence detection method; (E) Emission spectra of the fluorescence sensing system at different α-Glu concentrations; and (F) Linear relationship between the fluorescence intensity ratio (I / I0) and the logarithm of the α-Glu concentration, where I and I0 are the fluorescence intensities at 585 nm of the AAG / AR / CeMOF-on-CuMOF detection system in the presence and absence of α-Glu, respectively.

[0035] Figure 6 The test is for the selectivity of the detection method; among which, (A) the selectivity of colorimetric detection; (B) the anti-interference ability of colorimetric detection; (C) the selectivity of fluorescence detection; and (D) the anti-interference ability of fluorescence detection. Detailed Implementation

[0036] It should be noted that the following detailed description is illustrative and intended to provide further explanation 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.

[0037] The present invention will be further described below with reference to the embodiments.

[0038] Example 1 CuMOF preparation method: 0.855 g of 4,4'-bipyridine was completely dispersed in 6 mL of ethanol, and 0.935 g of CuCl2·2H2O was completely dispersed in 30 mL of water. The two solutions were mixed and stirred vigorously at room temperature for 1 hour. The solution was centrifuged, washed several times with ethanol, and dried at 37 °C to obtain CuMOF.

[0039] CeMOF preparation method: 2 mL of 2-methylimidazole (2-MI) aqueous solution (2.5 M) was added to 20 mL of Ce(NH4)2·(NO3)6 aqueous solution (60 mM). The mixture was stirred at room temperature for 1 hour. Then, the mixture was centrifuged at 10,000 rpm for 10 minutes and washed three times with deionized water. Finally, the resulting solid was dried in an oven at 60 °C.

[0040] CeMOF-on-CuMOF preparation method: CeMOF-on-CuMOF is synthesized by in-situ growth of CeMOF on CuMOF, with PVP as the linker. Specifically: Figure 1 As shown, 80 mg of CuMOF was added to 40 mL of water and sonicated. Then, 80 mg of PVP was added and the mixture was stirred for 12 hours. To the above solution, 8 mL of 2-MI (105 mM) was added first, followed by 10 mL of Ce(NH4)2(NO3)6 (20 mM), and the mixture was stirred at room temperature for 1 hour. CeMOF-on-CuMOF was washed several times with deionized water, and the solid was dried at 37 °C.

[0041] Example 2 1. Characterization of CuMOF, CeMOF and CeMOF-on-CuMOF The morphologies of CuMOF, CeMOF, and CeMOF-on-CuMOF were investigated using transmission electron microscopy (TEM). Figure 2 As shown in Figure A, CuMOF exhibits a rectangular plate-like structure with uniformly distributed elements Cu, N, and C. Figure 2 B). CeMOFs exhibit uniform particles with regular lattice fringes, and the calculated average lattice fringe spacing is 0.29 nm ( Figure 2 C). Elements Ce, N, and C are uniformly dispersed on its surface ( Figure 2 D). TEM images from CeMOF-on-CuMOF ( Figure 2 E) As can be seen, tiny particles adhere to the CuMOF surface, further confirming the successful in-situ synthesis of CeMOF on the CuMOF surface with PVP assistance. Elements Ce, Cu, and N are uniformly distributed ( Figure 2 F).

[0042] like Figure 3 The XRD pattern of A shows that CeMOF-on-CuMOF, obtained by in-situ synthesis of CeMOF on CuMOF, combines the lattice characteristics of both CuMOF and CeMOF, indicating the successful synthesis of MOF-on-MOF nanozymes. XPS full-spectrum scanning results are shown below. Figure 3 As shown in B, the main elements in CeMOF-on-CuMOF are Ce, Cu, N, C and O. Figure 3 C shows the spectrum of the Ce 3d orbital, and the peaks at energies of 916.31, 907.2, 901.19, 897.86, 882.48, and 887.74 eV are attributed to Ce. 4+ The peaks at 903.86, 899.78, 881.29, and 884.87 eV are attributed to Ce. 3+ .

[0043] 2. Laccase-like and oxidase-like activities of CeMOF-on-CuMOF Enzyme activity assay: 20 µL CeMOF-on-CuMOF (1.2 mg / mL), 20 μL 4-AAP (5 mM), and 20 μL 2,4-DP (10 mM) were mixed, and then the volume was adjusted to 200 μL with Tris-HCl buffer (100 mM, pH = 6). The mixture was then incubated at 40 °C for 1 h, and the absorption spectrum was finally collected using a spectrophotometer.

[0044] Oxidase-like activity assay: 20 µL of CeMOF-on-CuMOF (1.2 mg / mL) was mixed with 20 μL of TMB (10 mM) in acetic acid buffer (100 mM, pH = 4), and the volume was adjusted to 200 µL. The mixture was then incubated at 40 °C for 40 min, and the absorption spectrum was collected using a spectrophotometer.

[0045] like Figure 4 As shown in Figure A, a significant absorption at 510 nm was observed in the 2,4-DP + 4-AAP system in the presence of CeMOF-on-CuMOF. This absorption peak originates from the coupling of 2,4-DP with 4-AAP after laccase-catalyzed oxidation, forming a red quinone imine product, which can serve as a characteristic signal of laccase activity. Under the same conditions, CeMOF-on-CuMOF exhibits stronger laccase activity than CuMOF and CeMOF alone. Figure 4 (B) confirmed the synergistic effect between CuMOF and CeMOF. Based on the TMB oxidation colorimetric mechanism, a significant absorption at 652 nm was observed in the presence of CeMOF-on-CuMOF, indicating that TMB is catalyzed by an oxidase-like enzyme to generate the blue oxTMB product, confirming that its oxidase-like activity can effectively catalyze TMB oxidation. Under the same conditions, the oxidase-like activity of CeMOF-on-CuMOF is higher than that of the single MOF material (B). Figure 4 (C and D), further confirming the enhancing effect of hybrid structure on enzyme activity.

[0046] 3. Colorimetric and fluorescence dual-mode detection of α-glucosidase The colorimetric detection of α-Glu is based on the laccase-like activity of CeMOF-on-CuMOF. When α-Glu hydrolyzes Phenyl-α-Glc to generate phenol, the phenol is oxidized in the presence of CeMOF-on-CuMOF, subsequently coupling with 4-AAP to form a red quinone imine dye, resulting in an increase in absorbance at 510 nm. Therefore, the absorbance value is expected to increase with increasing α-Glu concentration.

[0047] 3.1, Evaluate the feasibility of colorimetric detection of α-Glu: Colorimetric feasibility test method: Phenol solution (20 µL, 10 mM), 4-AAP solution (30 μL, 5 mM), phenol solution (20 µL, 10 mM) / 4-AAP solution (30 μL, 5 mM) mixture, Phenyl-α-Glc solution (40 µL, 40 mM) / 4-AAP solution (30 μL, 5 mM) mixture, α-Glu (10 µL, 2000 U / L) / Phenyl-α-Glc solution (40 µL, 40 mM) / 4-AAP solution (30 μL, 5 mM) mixture were mixed with Tris-HCl buffer (100 mM, pH 6.0) and brought to a final volume of 100 µL. The mixtures were then incubated at 37 °C for 40 min. The above mixture was then mixed with 50 μL of CeMOF-on-CuMOF material (160 µg / mL) and diluted to a final volume of 200 µL with Tris-HCl buffer (100 mM, pH = 6). The mixture was then incubated at 40 °C for 1 h, and the absorption spectrum was finally collected using a spectrophotometer.

[0048] like Figure 5 As shown in Figure A, in the presence of phenol, the 4-AAP and CeMOF-on-CuMOF system exhibits significant absorption at 510 nm (blue curve); while in the presence of α-Glu, the Phenyl-α-Glc, 4-AAP and CeMOF-on-CuMOF system also exhibit significant absorption at 510 nm (purple curve), indicating that the substrate Phenyl-α-Glc is hydrolyzed to generate phenol, which is further oxidized and coupled with 4-AAP under the catalysis of CeMOF-on-CuMOF with laccase-like activity, consistent with the expected reaction mechanism, confirming the feasibility of colorimetric detection.

[0049] 3.2 Linearity and detection limit of colorimetric detection of α-Glu Linearity detection: Different concentrations of α-Glu (0-160 U / L), 40 μL of Phenyl-α-Glc (40 mM), and 20 μL of Tris-HCl buffer (100 mM, pH = 6) were mixed and reacted at 37 °C for 40 min. After the reaction was complete, 20 μL of Tris-HCl buffer (100 mM, pH = 6), 30 μL of 4-AAP (5 mM), and 50 μL of CeMOF-on-CuMOF material (160 µg / mL) were added sequentially, and the reaction was continued at 40 °C for 1 h. Subsequently, the absorption spectrum and absorbance value at 510 nm were measured. A standard working curve was plotted with α-Glu concentration on the x-axis and the absorbance difference A-A0 at 510 nm wavelength (A and A0 are the UV absorption intensities at 510 nm of the Phenyl-α-Glc / 4-AAP / CeMOF-on-CuMOF detection system in the presence and absence of α-Glu, respectively) on the y-axis.

[0050] Limit of Detection (LOD) Determination: Under the linear detection experimental conditions described above, a detection system without α-Glu (i.e., concentration of 0 U / L) was used as a blank solution. The determination was performed independently and repeatedly at least 11 times (n ≥ 11), and the absorbance value A0 at 510 nm was recorded for each determination. The standard deviation σ of the absorbance values ​​of this set of blank determinations was calculated. LOD was determined using the formula LOD = 3σ / k, where k is the slope of the standard working curve described above.

[0051] Within the α-Glu concentration range of 0.2-160 U / L, the absorbance increases with increasing enzyme concentration, following the functional relationship A-A0=0.0012[α-Glu] - 0.0019 ( Figure 5 The limit of detection (LOD) for the BC sample was 0.16 U / L.

[0052] 3.3, Evaluation of the feasibility of fluorescence detection of α-Glu H₂O solution (40 μL), AA solution (40 μL, 5 mM), AAG solution (40 μL, 5 mM), and α-Glu (10 µL, 2000 U / L) / AAG solution (40 μL, 5 mM) mixtures were each mixed with acetate buffer (100 mM, pH 5.0) and brought to a final volume of 100 µL. The mixtures were then incubated at 37 °C for 40 min. Subsequently, the mixtures were combined with 20 µL of LCeMOF-on-CuMOF (1.2 mg / mL) and 20 µL of AR (41.67 µg / mL), and brought to a final volume of 200 µL with acetate buffer (100 mM, pH = 5). Additionally, the 20 µL of AR (41.67 µg / mL) was brought to a final volume of 200 µL with acetate buffer (100 mM, pH = 5). The mixture was incubated at 40 °C for 40 min, and finally the fluorescence emission spectrum was collected using a spectrophotometer with an excitation wavelength of 525 nm.

[0053] Fluorescence detection is based on the OXD-like activity of CeMOF-on-CuMOF. In the presence of CeMOF-on-CuMOF, the substrate AR is oxidized to fluorescent oxAR; when α-Glu is present, the substrate AAG is hydrolyzed to reductive AA, which inhibits the oxidation process of AR, resulting in a decrease in fluorescence intensity at 585 nm with increasing α-Glu concentration. The feasibility of fluorescence detection is verified as follows: Figure 5 As shown in D: The substrate AR itself does not have fluorescence, but it is oxidized to fluorescent oxAR in the presence of CeMOF-on-CuMOF; when AA or AAG+α-Glu coexist, the fluorescence intensity of oxAR decreases significantly, proving that α-Glu can successfully catalyze the hydrolysis of AAG to produce reduced AA, and AA can effectively inhibit the oxidation of AR.

[0054] 3.4 Linearity and detection limit of α-Glu by fluorescence method Linear detection: Different concentrations of α-Glu (0-200 U / L), 40 μL of AAG (5 mM), and acetic acid buffer solution (100 mM, pH 5.0) were mixed and reacted at 37 ℃ for 40 min. After the reaction was complete, 60 μL of acetic acid buffer solution (100 mM, pH = 4), 30 μL of 20 µL AR (41.67 µg / mL), and 20 μL of CeMOF-on-CuMOF material (1.2 mg / mL) were added sequentially, and the reaction was continued at 40 ℃ for 40 min. Subsequently, the fluorescence emission spectrum and fluorescence intensity at 585 nm were measured. A standard working curve was plotted with α-Glu concentration as the x-axis and the fluorescence intensity ratio at 585 nm (I / I0, where I and I0 are the fluorescence intensities at 585 nm in the presence and absence of α-Glu, respectively) as the y-axis.

[0055] Limit of Detection (LOD) Determination: Under the linear detection conditions described above, a detection system without α-Glu (i.e., a concentration of 0 U / L) was used as a blank solution. The assay was independently repeated at least 11 times (n ≥ 11), and the fluorescence intensity I0 at 585 nm was recorded for each measurement. The standard deviation σ of the absorbance values ​​from these blank measurements was calculated. LOD was determined using the formula LOD = 3σ / k, where k is the slope of the standard working curve described above. Fluorescence intensity decreases with increasing α-Glu concentration: in the range of 0.6-40 U / L ( Figure 5 The method detection limit (LOD) is 0.35 U / L. The linear relationship is I / I0 = -0.15 lg[α-Glu] + 0.88 within the range of 40-200 U / L.

[0056] 4. Evaluation of selectivity and anti-interference ability The selectivity and anti-interference ability of the method were evaluated by replacing α-glucosidase (α-Glu) in the sensing system with alkaline phosphatase, galactose oxidase, glucose oxidase, pepsin, D-galactose, MgCl2, and NH4SO4.

[0057] like Figure 6 As shown in Figure A, only α-Glu caused a significant change in absorbance, while the listed interfering substances only elicited a weak response. The anti-interference capability was evaluated by simultaneously introducing the listed interfering substances into the sensing system, such as... Figure 6As shown in Figure B, the signal difference produced when interfering substances coexisted was limited compared to α-Glu alone, indicating that these substances did not significantly interfere with the detection of α-Glu. Similarly, in fluorescence detection mode, only α-Glu caused a significant decrease in fluorescence intensity ( Figure 6 C), while the signal difference between α-Glu alone and that of interfering substances is small when they coexist (C). Figure 6 D). The above results confirm that the designed sensing strategy has good selectivity and anti-interference ability.

[0058] Example 3 α-glucosidase detection method based on CeMOF-on-CuMOF nanozymes The procedure for colorimetric and fluorescence dual-mode detection of α-Glu is as follows: For colorimetric detection, 40 μL of α-Glu (5–1000 U / L), 40 μL of Phenyl-α-Glc (40 mM), and 20 μL of Tris-HCl (100 mM, pH = 6) were mixed and reacted at 37 °C for 40 min. After the reaction was complete, 20 μL of Tris-HCl (100 mM, pH = 6), 30 μL of 4-AAP (5 mM), and 50 μL of CeMOF-on-CuMOF (160 µg / mL) were added sequentially, and the mixture was reacted at 40 °C for 1 h. The absorbance at 510 nm was then measured.

[0059] For fluorescence detection, 40 μL of α-Glu (0–1000 U / L) and 40 μL of AAG were mixed with 20 μL of acetate buffer (100 mM, pH = 5) and reacted at 37 °C for 40 min. Then, 60 μL of acetate buffer (100 mM, pH 4), 20 µL of CeMOF-on-CuMOF (1.2 mg / mL), and 20 µL of AR (41.67 µg / mL) were added sequentially. The reaction was further carried out at 40 °C in the dark for 40 min, and fluorescence at 585 nm was measured with an excitation wavelength of 525 nm.

[0060] The detection principle is based on the enzymatic hydrolysis of phenyl-α-D-glucopyranoside (Phenyl-α-Glc) and 2-O-α-D-glucopyranoside-L-ascorbic acid (AAG). For example... Figure 1As shown, phenol, the reaction product from Phenyl-α-Glc, further serves as a substrate for CeMOF-on-CuMOF laccase activity, with absorbance at 510 nm increasing with increasing α-Glu concentration, thus enabling colorimetric detection. The AAG hydrolysis product, ascorbic acid (AA), inhibits the oxidation of 10-acetyl-3,7-dihydroxyphenazine (AR), leading to a reduction in the fluorescent product oxAR, thus enabling fluorescent detection. This work not only provides a powerful tool for α-Glu detection with great potential in clinical diagnostics but also establishes a general strategy for designing advanced dual-activity nanozymes for complex biosensing applications. The synthesized MOF-on-MOF structure exhibits higher activity than single MOFs, demonstrating the effectiveness of constructing MOF-on-MOFs for high-performance dual-activity nanozymes.

[0061] Example 4 1. Detection of α-Glu in human serum Human serum was precipitated with acetonitrile and centrifuged at 13,000 rpm for 20 minutes. The supernatant was then filtered through a 0.22 µm filter. The supernatant was diluted for later use. α-Glu activity in the human serum samples was measured using the procedure described in Example 3. Spiked serum samples were used as the analyte instead of pure α-Glu.

[0062] 2. Since α-Glu is an important indicator for disease diagnosis, this study applied the established colorimetric-fluorescence dual-mode detection method to the detection of α-Glu in human serum. As shown in Table 1, the recovery rate of the fluorescence sensing method ranged from 100.6% to 105.0%, with relative standard deviations (RSDs) all below 6.07%; the recovery rate of the colorimetric sensing method ranged from 97.2% to 119.1%, with a maximum RSD of 2.81%. These results demonstrate the good reliability of this dual-mode detection strategy in practical sample analysis.

[0063] Table 1. Results of colorimetric and fluorescence dual-mode detection of α-Glu in human serum.

[0064] 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 method for preparing CeMOF-on-CuMOF nanoszyme, characterized in that, CuMOF was first synthesized as the core and polyvinylpyrrolidone was connected for subsequent in-situ synthesis of CeMOF; then, Ce 4+ and 2-methylimidazole and stirred to form CeMOF on the surface of CuMOF through the bridging effect of polyvinylpyrrolidone.

2. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 1, characterized in that, Comprising the following steps: Step 1, CuMOF is added to water and mixed by ultrasonic; polyvinylpyrrolidone is added and stirred to prepare PVP@CuMOF; Step 2, 2-methylimidazole was added, followed by dropwise addition of Ce 4+ salt solution and stirred at room temperature to obtain CeMOF-on-CuMOF.

3. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 1, characterized in that, In step 1, the mass ratio of CuMOF to polyvinylpyrrolidone is 1:2-2:

1.

4. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 1, characterized in that, In step 1, CuMOF and polyvinylpyrrolidone are mixed and stirred for 10-14 hours.

5. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 1, characterized in that, In step 2, the Ce 4+ added as Ce(NH4)2(NO3)6.

6. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 5, characterized in that, In step 2, the molar ratio of 2-methylimidazole to Ce(NH4)2(NO3)6 is 105:20-105:

60.

7. The method for preparing CeMOF-on-CuMOF nanozymes according to claim 5, characterized in that, In step 2, the mass ratio of Ce(NH4)2(NO3)6 added to CuMOF in step 1 is 5:15-80.

8. A CeMOF-on-CuMOF nanozyme, characterized in that, CeMOF and CuMOF are connected by polyvinylpyrrolidone, and CuMOF sheets are the substrate, and CeMOF nanoparticles are loaded on the surface.

9. The application of the CeMOF-on-CuMOF nanoscale enzyme of claim 8 in the detection of α-glucosidase.

10. A method for detecting α-glucosidase, characterized by, The colorimetric and fluorescent dual-mode detection of α-glucosidase is as follows: For colorimetric detection, mix α-glucosidase, Phenyl-α-Glc and Tris-HCl, after the reaction is completed, add Tris-HCl, 4-aminoantipyrine and CeMOF-on-CuMOF of claim 9 in turn, after the reaction is completed, measure the absorbance at 510 nm; For fluorescent detection, mix α-glucosidase and 2-O-α-D-glucopyranoside-L-ascorbic acid with acetic acid buffer, after the reaction, add acetic acid buffer, CeMOF-on-CuMOF of claim 9 and 10-acetyl-3,7-dihydroxyphenoxazine in turn, avoid light reaction, measure the fluorescence at 585 nm, and the excitation light is 525 nm.

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