Metal organic framework immobilized enzyme based on three-enzyme cascade, preparation method and application of metal organic framework immobilized enzyme in alpha-Amy activity detection

Abstract

The invention discloses a metal organic framework immobilized enzyme based on three-enzyme cascade, a preparation method and application of the metal organic framework immobilized enzyme in alpha-Amy activity detection, and belongs to the technical field of biological engineering. According to the invention, alpha-Glu is taken as a core catalytic element, GOx and HRP are subjected to cascade immobilization, a three-enzyme cascade metal organic framework immobilized enzyme is constructed, and quantitative detection of alpha-Amy activity is realized. According to the method, a forward correlation model between the alpha-Amy activity and the TMB oxidation degree is constructed through the ratio of 2-methylimidazole to zinc acetate, optimization of synthesis time, the concentration ratio of alpha-Glu, GOx and HRP, the incubation temperature, the pH value and other key parameters of a reaction system, and a strategy is provided for accurate detection of the alpha-Amy activity. The detection system has the advantages of simplicity and convenience in operation, rapidness in detection, low detection limit, excellent stability, high specificity and the like, and has an important application prospect in accurate detection of alpha-Amy activity and clinical diagnosis of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a metal-organic framework immobilized enzyme based on a three-enzyme cascade, its preparation method, and its application in α-Amy activity detection. Background Technology

[0002] Pancreatic fistula (PF) is a common and serious complication following pancreatic surgery, and it can also occur in clinical settings such as acute pancreatitis, pancreatic trauma, and biliary tract diseases. Pancreatic juice is rich in various digestive enzymes, and its persistent leakage can cause abdominal infection, abdominal abscess, hemorrhage, sepsis, and dysfunction of multiple organs, potentially threatening the patient's life in severe cases. Therefore, early identification, dynamic monitoring, and severity assessment of pancreatic fistula are of paramount clinical importance. Currently, α-amylase is widely recognized as a key biomarker for reflecting the state of pancreatic juice leakage. When pancreatic tissue is damaged or the integrity of the pancreatic duct is compromised, large amounts of α-amylase enter the abdominal drainage fluid, leading to a significant increase in α-amylase activity in the drainage fluid. Therefore, the detection of α-amylase activity can serve as an important diagnostic tool for pancreatic fistula. In the clinical diagnosis of pancreatic fistula, the diagnostic criteria proposed by the International Study Group for Pancreatic Surgery (ISGPS) state that on the third day after surgery, the activity of α-amylase in the abdominal drainage fluid exceeds three times the upper limit of normal serum α-amylase (425 U / L) at the same time, which is an important basis for the diagnosis of pancreatic fistula.

[0003] α-Amylase (α-Amy) is a class of glycoside hydrolases widely found in animals, plants, and microorganisms, playing a crucial role in carbohydrate metabolism. Its core function is to catalyze the cleavage of α-1,4-glycosidic bonds in starch, producing products such as maltose and maltotriose. Currently, methods for detecting α-Amy activity mainly include turbidimetric assays, immunoassays, electrochemical methods, and fluorescence / chemiluminescence substrate methods. The above methods all have different limitations: turbidimetric methods have weak specificity and limited detection range, making them unsuitable for precise quantification of α-Amy; immunoassay methods rely on the specific binding reaction of antigen and antibody to achieve detection, which has certain specificity advantages, but the detection process involves complex steps such as antigen incubation, antibody binding, washing, and color development, resulting in a long detection cycle and strong dependence on the detection instrument platform; electrochemical detection methods utilize the interaction between electroactive substances generated during enzymatic reactions and the electrode surface to convert enzyme activity signals into detectable electrical signals; however, proteins, electrolytes, and reducing small molecules present in biological samples may undergo non-specific adsorption with the electrode, thereby reducing the stability and reproducibility of the detection signal; fluorescence / chemiluminescence substrate methods utilize chemically modified fluorescent or luminescent substrates that release fluorescence or chemiluminescent products after hydrolysis. Although the detection sensitivity is high, the specific fluorescent or chemiluminescent substrates used have complex and expensive synthesis processes, and the detection process requires specialized equipment such as high-precision fluorescence spectrophotometers or chemiluminescence detectors, resulting in high overall detection costs and making large-scale application difficult. Therefore, there is an urgent need to develop an α-Amy activity detection scheme that is fast, stable, has strong anti-interference ability, and is easy to promote.

[0004] Enzyme-catalyzed biosensors, using enzymes as their core recognition element, possess high activity and substrate specificity, and are now widely used for the qualitative and quantitative detection of various target substances. α-Glucosidase (α-Glu) is a typical glycosidic bond hydrolase whose core catalytic function is the cleavage of α-glycosidic bonds. It specifically hydrolyzes glucose molecules linked at the ends of oligosaccharide and disaccharide molecules by α-glycosidic bonds, thereby releasing free glucose. Glucose oxidase (GOx) is a type of oxidoreductase that uses glucose as a specific substrate to produce gluconolactone and hydrogen peroxide (H₂O₂). Horseradish peroxidase (HRP) is a commonly used signal amplification enzyme in the field of biochemical detection. It uses H₂O₂ produced by GOx catalysis as an electron acceptor to drive the oxidation of the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine (TMB), generating a blue oxidation product (oxTMB) with a characteristic absorption peak at 652 nm. Based on the cascade catalytic reaction mechanism of these three enzymes, a three-enzyme detection system with α-Glu catalysis as the core cascade and GOx and HRP in between is constructed, which is expected to open up new strategies for the detection of α-Amy activity. However, due to the short half-life and other defects of natural free enzymes, they often need to be prepared and used immediately in practical applications, which seriously limits the application of natural free enzymes in the detection field.

[0005] Metal-organic frameworks (MOFs) are a widely used class of immobilized enzyme carriers, consisting of three-dimensional materials with porous structures formed by coordination bonds between metal ions and organic ligands. By controlling the ratio of metal ions to organic ligands and the synthesis time, the pore size, morphology, and dimensions of the material can be manipulated. Based on these characteristics, MOFs can serve as immobilization carriers for the three-enzyme cascade of α-Glu, GOx, and HRP, effectively improving the storage stability of enzymes and the environmental adaptability of the detection system. Furthermore, these materials offer the dual advantages of rapid synthesis and mesoporous structure, showing great promise in the field of α-Amy activity detection. Summary of the Invention

[0006] This invention provides a metal-organic framework-immobilized enzyme based on a three-enzyme cascade, its preparation method, and its application in α-Amy activity detection, using a defective metal-organic framework as a carrier.

[0007] This invention uses α-Glu as the core catalytic element, immobilizing GOx and HRP in a cascade to construct a three-enzyme cascade metal-organic framework immobilized enzyme, and achieves efficient detection of α-Amy activity via colorimetric method. This detection system boasts advantages such as ease of operation, rapid detection, low detection limit, excellent stability, and high specificity, showing significant application potential in the accurate detection of α-Amy activity and the clinical diagnosis of related diseases.

[0008] Unless otherwise specified, all solutions involved in this invention are deionized aqueous solutions.

[0009] The present invention discloses a method for preparing a metal-organic framework immobilized enzyme based on a three-enzyme cascade. The method involves rapidly mixing 0.5–2 mL of a 70–90 mM 2-methylimidazole solution and 0.5–2 mL of a 15–25 mM zinc acetate solution in a reaction vessel. Immediately afterwards, 0.05–0.1 mL of a mixed solution of GOx and HRP (GOx concentration 0.5–0.6 mg / mL and HRP concentration 0.2–0.3 mg / mL) is added. The mixture is stirred at room temperature for 25–35 min to obtain a GOx & HRP immobilized enzyme solution. Subsequently, 0.06–0.09 mL of an α-Glu solution (5–7 mg / mL) is added, and the reaction is continued at room temperature with stirring for another 25–35 min. The metal-organic framework immobilized enzyme α-Glu@GOx&HRP with an outer layer of α-Glu and an inner layer of GOx and HRP was obtained by centrifugation at 14000~16000 rpm / min for 4~6 min at room temperature, and the precipitate was collected. The precipitate was resuspended in deionized water, washed and centrifuged 2~5 times. The last collected precipitate was freeze-dried to obtain metal-organic framework immobilized enzyme powder based on three enzyme cascades.

[0010] The metal-organic framework immobilized enzyme based on a three-enzyme cascade described in this invention is prepared by the above-described preparation method.

[0011] The metal-organic framework immobilized enzyme based on a three-enzyme cascade described in this invention can be applied to α-Amy activity detection. The specific steps are as follows:

[0012] (1) The metal-organic framework immobilized enzyme powder based on the three-enzyme cascade was resuspended in deionized water and mixed evenly to prepare an α-Glu@GOx&HRP immobilized enzyme solution with a concentration of 0.8~1.2 mg / mL;

[0013] (2) Dissolve α-Amy completely in phosphate buffer (10 mM, pH 6.9) to prepare various α-Amy standard solutions with activities of 25~1000 U / L (specific activities are 25, 50, 100, 200, 400, 600, 800, 1000 U / L).

[0014] (3) Add branched soluble starch extracted from plant starches such as corn, potato, and cassava to an 85-95 ℃ solution, stir until fully dissolved, and then cool to room temperature to obtain a starch working solution with a concentration of 4-6 mg / mL.

[0015] (4) Dissolve TMB powder completely in dimethyl sulfoxide to prepare a TMB working solution with a concentration of 15~25 mM;

[0016] (5) Add 100-150 μL of starch working solution prepared in step (3) to 600-650 μL of various α-Amy standard solutions prepared in step (2), mix evenly, and incubate at 35-40 ℃ for 15-25 min; then add 240-260 μL of acetate-sodium acetate buffer (1 M, pH 5.0), 40-60 μL of TMB working solution prepared in step (4), and 40-60 μL of α-Glu@GOx&HRP immobilized enzyme solution prepared in step (1), and continue incubating at 35-40 ℃ for 5-15 min; after incubation, take 200 μL of the obtained reaction solution and use a multi-functional microplate reader to detect the absorbance value of the reaction solution at 652 nm. Finally, complete the plotting of the linear fitting curve of "absorbance value at 652 nm and α-Amy activity" and the fitting of the linear regression equation.

[0017] (6) Add 100-150 μL of starch working solution prepared in step (3) to 600-650 μL of α-Amy solution to be tested. After mixing evenly, continue to incubate at 35-40 °C for 15-25 min. Then add 240-260 μL of acetate-sodium acetate buffer (1 M, pH 5.0), 40-60 μL of TMB working solution prepared in step (4), and 40-60 μL of α-Glu@GOx&HRP immobilized enzyme solution prepared in step (1). Continue to incubate for 5-15 min. After incubation, take 200 μL of the obtained reaction solution and use a multi-functional microplate reader to detect the absorbance value of the reaction solution at 652 nm. Substitute the absorbance value into the linear regression equation obtained in step (5) to calculate the activity of α-Amy, thus completing the activity detection of the α-Amy solution to be tested.

[0018] The core principle of this invention is as follows: α-Amy, as a key hydrolase, can specifically catalyze the hydrolysis of α-1,4-glycosidic bonds in starch solution to produce maltose; α-Glu belongs to the glycosidic hydrolase family and its main function is to catalyze the hydrolysis of glucosinolate bonds in maltose to release glucose; subsequently, GOx catalyzes the generation of H2O2 using glucose as a substrate, and HRP can further utilize the generated H2O2 to catalyze the oxidation of the chromogenic substrate TMB to produce the blue oxidation product oxTMB, changing the solution color from colorless to blue. Finally, the change in absorbance value is detected by a multifunctional microplate reader to achieve quantitative analysis of the amount of H2O2 generated in the system, thereby realizing the quantitative detection of α-Amy activity. This invention constructs a positive correlation model between α-Amy activity and the degree of TMB oxidation (absorbance value) by optimizing the ratio of 2-methylimidazole and zinc acetate, the synthesis time, the concentration ratio of α-Glu, GOx and HRP, and the incubation temperature and pH value of the reaction system, providing a strategy for the accurate detection of α-Amy activity.

[0019] The method established in this invention can be used as a biocolorimetric sensor to detect α-Amy activity. By adding the test solution to the detection system and performing a hydrolysis reaction, the change in absorbance value of the system at a wavelength of 652 nm is measured. Then, based on the correlation model between absorbance value and α-Amy activity, the quantitative detection of α-Amy activity in the test solution can be achieved.

[0020] Compared with existing technologies, the three-enzyme cascade metal-organic framework immobilized enzyme α-Glu@GOx&HRP constructed by the in-situ encapsulation method in this invention exhibits significantly improved stability compared to free enzymes. Simultaneously, the colorimetric detection strategy based on this system enables rapid and sensitive detection of α-Amy activity. Compared with other existing α-Amy activity detection methods, the α-Glu@GOx&HRP immobilized enzyme colorimetric detection system constructed in this invention demonstrates significant advantages, including ease of operation, rapid detection, low cost, and strong anti-interference capabilities. Furthermore, this system meets the core stability requirements for long-distance transportation and long-term storage in practical applications, aligning with the application demands for rapid and sensitive detection of α-Amy activity in biochemical analysis and clinical diagnostics. This invention provides a sound strategy for α-Amy activity detection and has broad application prospects in clinical testing and biochemical analysis. Attached Figure Description

[0021] Figure 1 The image shows a scanning electron microscope (SEM) image of the α-Glu@GOx&HRP immobilized enzyme obtained in Example 1, with a scale bar of 1 μm.

[0022] Figure 2The images shown are laser confocal microscopy images of the RBITC red fluorescence (Fig. a), FITC green fluorescence (Fig. b), 7-OHCCA-SE-HRP blue fluorescence (Fig. c), and red-green-blue trifluorescence colocalization (Fig. d) of the RBITC-α-Glu@FITC-GOx&7-OHCCA-SE-HRP immobilized enzyme obtained in Example 1, with a scale bar of 10 μm.

[0023] Figure 3 The powder X-ray diffraction patterns of the α-Glu@GOx&HRP immobilized enzyme, ZIF-8, and aZIF prepared in Example 1 are shown. The horizontal axis represents the diffraction angle of the crystal, and the vertical axis represents the diffraction intensity.

[0024] Figure 4 The N2 adsorption-desorption curves of the α-Glu@GOx&HRP immobilized enzyme, ZIF-8 and aZIF prepared in Example 1 are shown. The horizontal axis represents relative pressure and the vertical axis represents the volume of gas adsorbed per gram of material.

[0025] Figure 5 The linear fitting curves between different active α-Amy values ​​and absorbance values ​​at 652 nm in Example 2 are shown. The horizontal axis represents the α-Amy activity, and the vertical axis represents the absorbance value at 652 nm wavelength.

[0026] Figure 6 This is a bar graph showing the specificity analysis results of the α-Amy colorimetric detection system for α-Glu@GOx & HRP immobilized enzyme in Example 3; the substances on the horizontal axis correspond to the control group, β-glucosidase, cytochrome C, cellulase, cutinase, lipase, urea, citric acid, glutamate, tryptophan, aspartic acid, glycine, and calcium ions (Ca). 2+ ), potassium ions (K) + Sodium ions (Na) + The horizontal axis represents the name of the added control or interfering substance, and the vertical axis represents the percentage of relative absorbance.

[0027] Figure 7This is a bar graph showing the anti-interference ability analysis results of the α-Amy colorimetric detection system for α-Glu@GOx&HRP immobilized enzyme in Example 4; the substances on the horizontal axis correspond to the control group, β-glucosidase, cytochrome C, cellulase, cutinase, lipase, urea, citric acid, glutamate, tryptophan, aspartic acid, glycine, and calcium ions (Ca). 2+ ), potassium ions (K) + Sodium ions (Na) + The horizontal axis represents the name of the added control or interfering substance, and the vertical axis represents the percentage of relative absorbance.

[0028] Figure 8 The bar graph shows the changes in enzyme activity of the α-Glu@GOx&HRP immobilized enzyme colorimetric detection system in Example 5 after storage at 4 ℃ for 1 day, 2 days, 5 days, 6 days, and 10 days. The horizontal axis represents the number of days of storage at 4 ℃, and the vertical axis represents the percentage of total activity of the three-enzyme cascade reaction. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of the invention.

[0030] Example 1: Preparation and characterization of α-Glu@GOx & HRP immobilized enzyme

[0031] The preparation process of defective aZIF is as follows: 1 mL of 2-methylimidazole solution (80 mM) and 1 mL of zinc acetate solution (20 mM) were added to a 5 mL glass bottle and mixed rapidly. After stirring at room temperature for 60 min, the defective aZIF sample was obtained. The prepared sample was centrifuged at room temperature (15000 rpm / min, 5 min), the white precipitate was collected, and the precipitate was resuspended in deionized water, washed, and centrifuged three times (15000 rpm / min, 5 min). The washed white precipitate was collected and freeze-dried for 12 h. Finally, 1.6 mg of defective aZIF powder was collected.

[0032] The preparation process of α-Glu@GOx&HRP immobilized enzyme is as follows: 1 mL of 2-methylimidazole solution (80 mM) and 1 mL of zinc acetate solution (20 mM) were added to a 5 mL glass bottle and quickly mixed. Immediately afterwards, 80 μL of a mixed solution of GOx (0.56 mg / mL) and HRP (0.24 mg / mL) was added to the reaction system. The mixture was stirred at room temperature for 30 min to obtain the GOx&HRP immobilized enzyme solution. 80 μL of α-Glu solution (6 mg / mL) was then added to the reaction system, and the mixture was stirred for 30 min to obtain the α-Glu@GOx&HRP immobilized enzyme. The prepared α-Glu@GOx&HRP immobilized enzyme was centrifuged at room temperature (15000 rpm / min, 5 min). The white precipitate was collected, resuspended in deionized water, washed, and centrifuged three times (15000 rpm / min, 5 min). The precipitate was collected and freeze-dried for 12 h to obtain 2.1 μL of the enzyme. mg of α-Glu@GOx & HRP immobilized enzyme powder, its SEM characterization is as follows: Figure 1 As shown.

[0033] The preparation methods for fluorescein-labeled RBITC-α-Glu, FITC-GOx, and 7-OHCCA-SE-HRP are as follows: 264 μL, 48 μL, and 180 μL of RBITC, FITC, and 7-OHCCA-SE solutions (1 mg / mL) dissolved in dimethyl sulfoxide were added to 2 mL of α-Glu (6 mg / mL), GOx (2 mg / mL), and HRP (2 mg / mL) solutions, respectively. The reaction system was stirred at 4 °C in the dark for 12 h. The collected solutions were then dialyzed against deionized water at 4 °C in the dark for 48 h (to a molecular weight of 3500 Da). The dialysate was replaced after 4 h, 12 h, and 24 h of dialyzing. Finally, samples without free fluorescein were collected and stored at 4 °C in the dark.

[0034] The preparation method of the fluorescein-labeled RBITC-α-Glu@FITC-GOx&7-OHCCA-SE-HRP@aZIF immobilized enzyme is the same as that of the α-Glu@GOx&HRP immobilized enzyme. The fluorescein-labeled immobilized enzyme was characterized using a laser confocal microscope (FV4000). The laser confocal microscopy characterization results are as follows: Figure 2 As shown, RBITC-α-Glu exhibits the largest red fluorescence range (Fig. a), while FITC-GOx (Fig. b) and 7-OHCCA-SE-HRP (Fig. c) show green and blue fluorescence at the same location, with smaller fluorescence ranges compared to the red fluorescence. These results indicate that the three-enzyme cascade immobilized enzyme α-Glu@GOx&HRP with a reasonable spatial distribution was successfully prepared.

[0035] X-ray diffraction analysis of α-Glu@GOx&HRP immobilized enzymes was performed using a Bruker D3 Advance X-ray diffractometer. The results are as follows: Figure 3 As shown. Unlike the X-ray diffraction results of traditional ZIF-8, aZIF and α-Glu@GOx&HRP immobilized enzymes do not have obvious crystal diffraction peaks, indicating that the materials prepared in this invention are defective aZIF and α-Glu@GOx&HRP immobilized enzymes.

[0036] The N2 adsorption-desorption analysis of α-Glu@GOx&HRP immobilized enzymes was performed using the Canta Autosorb-iQ method, and the results are as follows: Figure 4 As shown in Table 1, the defective aZIF material is mesoporous compared to traditional ZIF-8 materials, and this pore size makes it easier to achieve mass transfer of the substrate.

[0037] Table 1: Aperture Range Data

[0038]

[0039] Example 2: Colorimetric detection of α-Amy enzymes with different activities based on α-Glu@GOx & HRP immobilized enzymes

[0040] (1) Take 1 mg of the α-Glu@GOx&HRP immobilized enzyme powder prepared in Example 1 and disperse it in 1 mL of deionized water to prepare an α-Glu@GOx&HRP immobilized enzyme solution with a concentration of 1 mg / mL;

[0041] (2) Preparation of α-Amy solutions with different activities: Weigh 2 mg of α-Amy powder and dissolve it completely in 10 mL of phosphate buffer (10 mM, pH 6.9) to prepare 10000 U / L α-Amy stock solution. Then, dilute the stock solution with phosphate buffer (10 mM, pH 6.9) to α-Amy standard solutions with activities of 25, 50, 100, 200, 400, 600, 800, and 1000 U / L.

[0042] (3) Preparation of starch working solution: Take 500 mg of amylopectin extracted from corn starch, stir and dissolve it in 100 mL of deionized water at 90 °C to ensure that the starch is completely gelatinized and without clumping, and then cool it naturally to room temperature to obtain a 5 mg / mL starch working solution.

[0043] (4) Preparation of TMB working solution: Take 19.2 mg of TMB powder, dissolve it in 4 mL of dimethyl sulfoxide to prepare a TMB working solution with a concentration of 20 mM, and store it in the dark at 4 °C.

[0044] (5) Detection of different activities of α-Amy based on colorimetric method of α-Glu@GOx&HRP@aZIF: First, add 625 μL of α-Amy standard solutions with different activities (25, 50, 100, 200, 400, 600, 800, 1000 U / L) to a 1.5 mL EP tube, then add 125 μL of starch working solution (5 mg / mL), mix evenly and incubate at 37 ℃ for 20 min; add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution (20 mM) and 50 μL of α-Glu@GOx&HRP immobilized enzyme solution (1 mg / mL) prepared in step (1) to the reaction system in sequence, mix evenly and continue to incubate at 37 ℃ for 10 min; immediately after incubation, take 200 μL of reaction solution and transfer it to a 96-well microplate, and measure 652 μL of the enzyme using a multi-functional microplate reader. The absorbance value at nm was used to plot the linear regression curve and fit the linear regression equation based on the measured absorbance value and different activities of α-Amy.

[0045] The limit of detection (LOD) is calculated using the following formula: LOD = 3.3σ / k

[0046] σ: The standard deviation of the absorbance values ​​obtained after measuring the blank sample three times when the α-Amy activity is zero;

[0047] k: The slope of the linear regression curve.

[0048] like Figure 5 As shown, the absorbance value measured at 652 nm showed a good linear regression relationship with the activity of α-Amy (y=0.00094x+0.09223, R0). 2=0.996), and its limit of detection (LOD) is 6.07 U / L. Based on the above curves, the α-Glu@GOx&HRP immobilized enzyme colorimetric method has a wide linear detection range of 0~1000 U / L, combining the core advantages of wide linear range and high sensitivity. From a clinical application perspective, the linear range of this system completely covers the physiological and pathological activity range (425 U / L) of α-Amy in human drained pancreatic juice, and the upper limit of the linear range can fully meet the quantitative requirements of moderate to severe abnormal activity, and can accurately capture early mild abnormal activity. In addition, this detection method does not require complex sample pretreatment, has mild reaction conditions, does not require complex and expensive detection instruments, and has the dual functions of visual screening and quantitative detection. The synergistic advantages of high sensitivity and wide linear range of this invention effectively solve the technical bottlenecks of insufficient sensitivity and narrow linear range in traditional detection methods. It enables accurate and rapid detection of α-Amy activity in human pancreatic fluid samples, providing reliable technical support for early warning and timely intervention of pancreatic fistula. It significantly improves the timeliness, accuracy and ease of operation of clinical diagnosis, and shows good potential for clinical translation.

[0049] Example 3: Specificity Study of α-Glu@GOx & HRP Immobilized Enzyme Colorimetric Detection System

[0050] First, add 625 μL of α-Amy solution with an activity of 1000 U / L to a 1.5 mL EP tube, or add interfering enzymes with a concentration of 0.1 mg / mL: β-glucosidase, cytochrome C, cellulase, keratinase, lipase; or small molecule metabolites with a concentration of 20 mM: urea, citrate, glutamate, tryptophan, aspartic acid, glycine; or physiological ions with a concentration of 20 mM: calcium ions, potassium ions, sodium ions; then add 125 μL of starch working solution (5 mg / mL) and incubate at 37 °C for 20 min; add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution (20 mM), and 50 μL of α-Glu@GOx&HRP immobilized enzyme solution (1 mg / mL) to the reaction system, mix well, and continue incubation at 37 °C for 10 min; immediately after incubation, take 200 μL of the reaction solution and detect 652. absorbance value at nm.

[0051] The activity of the untreated control group (containing only α-Amy solution) was defined as 100%, and its absorbance value at 652 nm was defined as A0. The absorbance value of the reaction solution of other treatment groups (containing β-glucosidase, etc., but not α-Amy solution) at 652 nm was defined as A0. x The formula for calculating relative intensity is (Ax / A0)×100%. For example Figure 6 As shown, the relative activity of the group with added α-Amy was significantly different from that of the group with other interfering substances, indicating that the α-Glu@GOx&HRP immobilized enzyme colorimetric assay system has good detection specificity. This specificity is mainly attributed to the excellent substrate specificity of α-Glu itself. The core advantage of this highly specific assay system lies in its ability to effectively avoid the influence of endogenous interfering substances in complex clinical samples (such as human pancreatic drainage), providing a reliable guarantee for the accurate quantification of α-Amy activity, which is of great significance in the clinical diagnosis of pancreatic fistula and related diseases.

[0052] Example 4: Evaluation of the anti-interference ability of the α-Glu@GOx&HRP immobilized enzyme colorimetric detection system

[0053] First, add 312.5 μL of α-Amy solution with an activity of 1000 U / L and 312.5 μL of an interfering enzyme (β-glucosidase, cytochrome C, cellulase, keratinase, lipase; or a small molecule metabolite at a concentration of 20 mM: urea, citrate, glutamic acid, tryptophan, aspartic acid, glycine) dissolved in phosphate buffer (10 mM, pH 6.9) at a concentration of 0.1 mg / mL, or 312.5 μL of a physiological ion solution (calcium ion, potassium ion, sodium ion) at a concentration of 20 mM to a 1.5 mL EP tube; then add 125 μL of starch working solution (5 mg / mL) and incubate at 37 ℃ for 20 min; finally, add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution, and 50 μL of α-Glu@GOx&HRP immobilized enzyme solution (1 M, pH 5) to the reaction system. After mixing thoroughly, the solution was incubated at 37 °C for 10 min. After incubation, 200 μL of the reaction solution was immediately taken to detect the absorbance at 652 nm.

[0054] The activity of the untreated control group (with α-Amy solution added) was defined as 100%, and its absorbance value at 652 nm was defined as A0. The absorbance value of the reaction solution of other treatment groups (with α-Amy solution added along with one of β-glucosidase, calcium ions, etc.) at 652 nm was defined as A0. x The formula for calculating relative activity is (A) x / A0)×100%. For example Figure 7As shown, the relative activity in the presence of interfering substances was not significantly different from that of the positive control group (the system containing only α-Amy), indicating that the α-Glu@GOx&HRP immobilized enzyme colorimetric assay system has good anti-interference ability. This assay system can accurately quantify α-Amy activity even in complex biological matrices, successfully avoiding the influence of endogenous interfering substances in clinical samples, and significantly improving the accuracy and reliability of the test results. This characteristic enables it to meet the stringent requirements of specificity, stability, and anti-interference in clinical sample testing, providing key technical support for the early and rapid diagnosis of pancreatic fistula and related diseases, and demonstrating broad application prospects in the field of clinical laboratory medicine.

[0055] Example 5: Evaluation of the storage stability of the α-Glu@GOx & HRP immobilized enzyme colorimetric assay system

[0056] To detect the overall activity changes of the α-Glu@GOx&HRP immobilized enzyme colorimetric assay system during long-term storage, the present invention stored a 1 mg / mL α-Glu@GOx&HRP immobilized enzyme solution at 4 ℃ for 10 days, and took samples on days 1, 2, 5, 6, and 10 for activity detection.

[0057] The detection method is as follows: Add 625 μL of phosphate buffer (10 mM, pH 6.9) to a 1.5 mL EP tube, then add 125 μL of maltose solution (5 mM), and incubate at 37 ℃ for 10 min. Finally, add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution, and 50 μL of the above-mentioned α-Glu@GOx&HRP immobilized enzyme solutions stored for different times to the reaction system, mix well, and continue to incubate at 37 ℃ for 10 min. Immediately after incubation, take 200 μL of the reaction solution and detect the absorbance value at 652 nm.

[0058] The total activity of the three-step cascade reaction of the mixed solution of free α-Glu, GOx, and HRP (Free enzyme) stored for 1 day and the α-Glu@GOx&HRP immobilized enzyme prepared in Example 1 was defined as 100%, and the absorbance value of the reaction system detected at 652 nm was defined as A0. The absorbance value of the α-Glu@GOx&HRP immobilized enzyme reaction system after other different storage times was defined as A0. x The formula for calculating the relative total activity is (A) x / A0)×100%. For example Figure 8As shown, the α-Glu@GOx&HRP immobilized enzyme retained more than 60% of its initial activity after 10 days of storage at 4 ℃, while the free enzyme almost completely lost its activity after 10 days of storage under the same conditions. In summary, the α-Glu@GOx&HRP immobilized enzyme colorimetric detection system exhibits excellent storage stability. Compared to the easy inactivation of free enzymes, the high stability of this system overcomes the operational limitations of traditional detection methods that require the preparation and use of related solutions on the spot, improving the convenience and reproducibility of the detection process, and laying a solid foundation for its application in the early diagnosis of pancreatic fistula.

[0059] Example 6: Evaluation of the ability of the α-Glu@GOx&HRP immobilized enzyme colorimetric assay system for α-Amy activity determination using the standard addition method.

[0060] (1) Processing of true concentration samples: Two drainage fluids from different postoperative pancreatic fistula patients were centrifuged at 4 ℃ (6000 rpm, 2 min) as Sample 1 and Sample 2, respectively. The supernatant was collected and diluted 10 times for standard addition method detection.

[0061] (2) Preparation of α-Amy solutions with different activities: Weigh 2 mg of α-Amy powder and dissolve it in 10 mL of phosphate buffer (10 mM, pH 6.9) to prepare a stock solution with an activity of 10000 U / L. Then dilute α-Amy to make its activities 25, 50, 100, 200, 400, 600, 800 and 1000 U / L respectively.

[0062] (3) Preparation of the test sample solution: Weigh 200 mg of α-Amy powder and dissolve it in 100 mL of phosphate buffer (10 mM, pH 6.9), mix thoroughly, and prepare α-Amy stock solution with an activity of 10000 U / L; add 0 mL, 1 mL, and 2.25 mL of α-Amy stock solution to 9 mL of sample 1 and sample 2 in step (1), respectively, mix thoroughly, so that the activity of α-Amy in sample 1 and sample 2 solutions increases by 0, 100, and 200 U / L, respectively.

[0063] (4) Preparation of α-Glu@GOx&HRP immobilized enzyme solution: Weigh 1 mg of α-Glu@GOx&HRP immobilized enzyme powder, disperse it in 1 mL of deionized water, vortex and mix thoroughly to prepare an α-Glu@GOx&HRP immobilized enzyme solution with a concentration of 1 mg / mL.

[0064] (5) Preparation of TMB working solution: Dissolve 19.2 mg of TMB powder in 4 mL of dimethyl sulfoxide to prepare a TMB working solution with a concentration of 20 mM. Store at 4 °C in the dark.

[0065] (6) Preparation of starch working solution: Dissolve 500 mg of soluble starch in 100 mL of deionized water at 90 °C to ensure that the starch is completely gelatinized and free of lumps. Cool naturally to room temperature to obtain a 5 mg / mL starch working solution.

[0066] (7) Detection and analysis of α-Amy solutions with different activities based on the colorimetric detection system of α-Glu@GOx&HRP immobilized enzyme: First, add 625 μL of different α-Amy solutions (0, 25, 50, 100, 200, 400, 600, 800, 1000 U / L) prepared in step (2) to a 1.5 mL EP tube, then add 125 μL of starch working solution and incubate at 37 ℃ for 20 min; add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution and 50 μL of α-Glu@GOx&HRP immobilized enzyme solution to the system in sequence, mix well and continue to incubate at 37 ℃ for 10 min; immediately after incubation, transfer 200 μL of reaction solution to a 96-well microplate and measure 652 μL of α-Amy solution. The absorbance value at nm was used to plot linear regression curves and fit linear regression equations based on the measured absorbance value and different activities of α-Amy.

[0067] (8) Detection of α-Amy activity of the test sample based on the colorimetric detection system of α-Glu@GOx&HRP immobilized enzyme: First, add 625 μL of the unknown activity of the α-Amy solution prepared in step (3) to a 1.5 mL EP tube, followed by 125 μL of starch working solution, and incubate at 37 ℃ for 20 min; add 250 μL of acetate-sodium acetate buffer (1 M, pH 5), 50 μL of TMB working solution and 50 μL of α-Glu@GOx&HRP immobilized enzyme solution to the reaction system in sequence, mix well and continue to incubate at 37 ℃ for 10 min; after incubation, immediately transfer 200 μL of the reaction solution to a 96-well microplate and measure the absorbance at 652 nm.

[0068] (9) Based on the linear regression equation obtained in step (7), calculate the activity of α-Amy in all unknown sample solutions, and then calculate the spiked recovery rate of α-Amy activity of sample 1 and sample 2.

[0069] Table 2: Detection results of α-Amy activity in actual samples

[0070]

[0071] The standard addition method is a common testing method for verifying the accuracy of instruments and the precision of detection methods. It involves adding a quantitative standard solution with known activity to the sample to be tested, measuring the sample activity before and after addition, and calculating the difference in activity to evaluate the accuracy of the detection system. The results are shown in Table 1. The activities of unspiked sample 1 and sample 2 were 57.09 ± 0.69 and 234.20 ± 21.18 U / L, respectively. Based on the activity detection of the spiked samples, the spiked recovery rate of the colorimetric detection system for α-Glu@GOx&HRP immobilized enzyme ranged from 102.66% to 108.89%, with an RSD range of 0.93% to 9.05%, indicating good detection accuracy.

[0072] 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, or improvements 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 a metal-organic framework immobilized enzyme based on a three-enzyme cascade, characterized in that: Add 0.5–2 mL of 70–90 mM 2-methylimidazole solution and 0.5–2 mL of 15–25 mM zinc acetate solution to a reaction vessel and mix rapidly. Immediately add 0.05–0.1 mL of a mixed solution of GOx and HRP, where the concentration of GOx is 0.5–0.6 mg / mL and the concentration of HRP is 0.2–0.3 mg / mL. Stir the reaction at room temperature for 25–35 min to obtain a GOx & HRP immobilized enzyme solution. Subsequently, add 0.06–0.09 mL of 5–7 mg / mL α-Glu solution and continue stirring at room temperature for 25–35 min to obtain a metal-organic framework immobilized enzyme α-Glu@GOx & HRP with an outer layer of α-Glu and an inner layer of GOx and HRP. Centrifuge the metal-organic framework immobilized enzyme α-Glu@GOx & HRP at room temperature and 14,000–16,000 rpm for 4–6 seconds. min, collect the precipitate; wash the precipitate with deionized water and centrifuge 2-5 times, freeze-dry the last collected precipitate to obtain metal-organic framework immobilized enzyme powder based on three enzyme cascade.

2. A metal-organic framework immobilized enzyme based on a three-enzyme cascade, characterized in that: It is prepared by the preparation method described in claim 1.

3. The application of a metal-organic framework immobilized enzyme based on a three-enzyme cascade as described in claim 2 in the detection of α-Amy activity.

4. The application of a metal-organic framework immobilized enzyme based on a three-enzyme cascade in the detection of α-Amy activity, as described in claim 3, comprises the following steps: (1) The metal-organic framework immobilized enzyme powder based on the three-enzyme cascade was resuspended in deionized water and mixed evenly to prepare an α-Glu@GOx&HRP immobilized enzyme solution with a concentration of 0.8~1.2 mg / mL; (2) Dissolve α-Amy completely in 10 mM, pH 6.9 phosphate buffer to prepare various α-Amy standard solutions with an activity of 25~1000 U / L; (3) Add soluble starch to a solution at 85~95 ℃, stir until fully dissolved, and then cool to room temperature to obtain a starch working solution with a concentration of 4~6 mg / mL; (4) Dissolve TMB powder completely in dimethyl sulfoxide to prepare a TMB working solution with a concentration of 15~25 mM; (5) Add 100-150 μL of starch working solution prepared in step (3) to 600-650 μL of various α-Amy standard solutions prepared in step (2), mix evenly, and incubate at 35-40 ℃ for 15-25 min; then add 240-260 μL of 1M and pH 5.0 acetate-sodium acetate buffer, 40-60 μL of TMB working solution prepared in step (4) and 40-60 μL of α-Glu@GOx&HRP immobilized enzyme solution prepared in step (1), mix evenly, and continue to incubate at 35-40 ℃ for 5-15 min; after incubation, take 200 μL of the obtained reaction solution, detect the absorbance value of the reaction solution at 652 nm, and finally complete the plotting of the linear fitting curve of "absorbance value at 652 nm and α-Amy activity" and the fitting of the linear regression equation; (6) Add 100-150 μL of starch working solution prepared in step (3) to 600-650 μL of α-Amy solution to be tested. After mixing evenly, continue to incubate at 35-40 °C for 15-25 min. Then add 240-260 μL of 1 M acetate-sodium acetate buffer, 40-60 μL of TMB working solution prepared in step (4) and 40-60 μL of α-Glu@GOx&HRP immobilized enzyme solution prepared in step (1). Continue to incubate for 5-15 min. After incubation, take 200 μL of the obtained reaction solution and detect the absorbance value of the reaction solution at 652 nm. Substitute the absorbance value into the linear regression equation obtained in step (5) to calculate the activity of α-Amy, thus completing the activity detection of the α-Amy solution to be tested.

5. The application of a metal-organic framework immobilized enzyme based on a three-enzyme cascade in the detection of α-Amy activity as described in claim 4, characterized in that: In step (2), the activities of various α-Amy standard solutions are 25, 50, 100, 200, 400, 600, 800, and 1000 U / L, respectively.

6. The application of a metal-organic framework immobilized enzyme based on a three-enzyme cascade in the detection of α-Amy activity as described in claim 4, characterized in that: In step (3), the soluble starch is amylopectin extracted from corn, potato or cassava starch.

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