A method for preparing semi-encapsulated enzyme materials based on metal-organic frameworks

Abstract

This invention discloses a method for preparing a semi-encapsulated enzyme material based on a two-dimensional metal-organic framework (MOF) material, belonging to the field of immobilized enzyme methods. The invention first involves thoroughly mixing glucose oxidase (GOx) and imidazole-2-carboxaldehyde (ICA), which bind through hydrogen bonding to form an ICA-GOx complex. Subsequently, a Co(NO3)2·6H2O solution is added, and under magnetic stirring, a two-dimensional MOF material (GOx@ZIF-90(Co)) is formed through coordination self-assembly between the organic ligand and metal ions. The enzyme of this invention allows the enzyme portion to be exposed outside the carrier, thereby reducing mass transfer resistance between the enzyme and substrate and maintaining high enzyme activity. Simultaneously, the nano-confinement effect of the MOF material restricts the enzyme structure, thereby improving enzyme stability. This unique encapsulation method balances the competition between enzyme activity and stability, and has broad application prospects in analytical detection and other fields.

Description

Technical Field

[0001] This invention belongs to the field of immobilized enzyme system preparation technology, specifically involving the preparation of metal-organic framework semi-encapsulated enzyme material (GOx@ZIF-90(Co)) through in-situ encapsulation technology to achieve semi-encapsulation of enzymes. Background Technology

[0002] Currently, the chemical manufacturing industry is undergoing a critical period of transformation from traditional production methods to biotransformation processes. Compared with traditional production methods, biotransformation manufacturing processes can be carried out under mild temperature and pressure conditions, achieving high energy conversion efficiency during production. As a key component of biotransformation processes, enzymes possess highly efficient catalytic activity; however, due to their fragile structure, harsh production processes can lead to structural changes and loss of activity. Furthermore, existing enzyme preparations often face limitations such as poor practicality and reusability, and require the addition of protective agents during storage, resulting in reduced enzyme activity. These drawbacks severely limit the application of enzymes in industrial production. Immobilized enzymes have become an effective method to overcome the defects in enzyme catalysis. Immobilizing enzymes in solid supports can transform the enzyme catalysis process from homogeneous to heterogeneous, thereby enhancing enzyme stability and recyclability.

[0003] Numerous methods have been developed for immobilizing enzymes in solid supports. Enzyme immobilization methods can be categorized into surface immobilization and total encapsulation based on the enzyme's location within the solid support. Surface immobilization places the enzyme on the surface of the solid support, while total encapsulation focuses on embedding the enzyme inside the solid support. Both methods effectively immobilize enzymes, but a competition arises between enzyme activity and stability. Surface immobilization maintains high enzyme activity but exposes enzyme molecules to the solid support surface, failing to provide adequate protection. Total encapsulation provides comprehensive protection but increases mass transfer resistance between the enzyme and substrate, inhibiting enzyme activity. Many studies have focused on enhancing mass transfer between enzymes and substrates through pore engineering; however, pore engineering involves cumbersome steps, and excessively large pore structures can lead to enzyme leakage. Therefore, a novel strategy for immobilizing enzymes that provides effective protection while maintaining high enzyme activity is needed.

[0004] In immobilized enzyme catalytic systems, enzyme activity is highly dependent on the morphology and structure of the support. The mass transfer resistance between the enzyme and substrate primarily depends on the morphology of the solid support. Unlike existing bulky three-dimensional supports, two-dimensional materials offer advantages such as adjustable thickness, highly exposed active sites, and low mass transfer resistance. Inspired by these superior characteristics, adjusting the thickness of the enzyme support to make its size smaller than the minimum three-dimensional size of the enzyme enables semi-encapsulation of the enzyme, where a portion of the enzyme is encapsulated and exposed outside the support. This encapsulation method reduces mass transfer resistance between the enzyme and substrate, thus maintaining high enzyme activity, and utilizes the nano-confinement effect of the support to protect the enzyme and improve its stability. This encapsulation method balances the competition between enzyme activity and stability, but this superior enzyme immobilization strategy has not yet been fully developed. Summary of the Invention

[0005] The purpose of this invention is to provide a strategy for constructing semi-encapsulated enzymes based on two-dimensional metal-organic framework (MOF) materials. The thickness of the synthesized MOF material is smaller than the minimum three-dimensional size of the enzyme, resulting in partial exposure of the enzyme. This reduces mass transfer resistance between the enzyme and substrate while providing effective protection for the enzyme. The semi-encapsulated enzyme strategy based on MOF materials developed in this invention is simple to prepare under mild conditions and can maintain high enzyme activity while imparting extremely high stability. This invention uses glucose oxidase (GOx) as a model to demonstrate the superior performance of this semi-encapsulated enzyme system.

[0006] This method first involves thoroughly mixing GOx and imidazole-2-carboxaldehyde (ICA), which provide hydrogen bonding interactions to form an ICA-GOx complex. Then, a Co(NO3)2·6H2O solution is added, and the mixture is stirred with a magnetic stirrer. Through coordination self-assembly between the organic ligand and the metal ions, a two-dimensional metal-organic framework material (ZIF-90(Co)) is formed to semi-encapsulate the enzyme.

[0007] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0008] A method for preparing a semi-encapsulated enzyme material based on a metal-organic framework, characterized by the following steps:

[0009] (1) Weigh 0.1537 g ICA into 4 mL of deionized water and sonicate until completely dissolved; weigh 0.0582 g Co(NO3)2·6H2O into 4 mL of deionized water and sonicate until completely dissolved; weigh 2 mg GOx for later use.

[0010] (2) Under magnetic stirring, 2 mg of GOx was added to the ICA solution in (1). After the ICA and GOx were thoroughly mixed, the Co(NO3)2·6H2O solution in (1) was added. At this time, the total volume of the reaction system was 8 mL. The reaction was carried out under magnetic stirring for 3 h. After the reaction was completed, the product was collected by centrifugation (10000 rpm, 5 min). The obtained GOx@ZIF-90(Co) was washed 3 times with deionized water (10000 rpm, 5 min). The final product was dispersed in 3.2 mL buffer to form a uniform dispersion and stored at 4 °C for later use.

[0011] (3) Further, the buffer mentioned in step (2) above is a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution, 100 mM, pH 7.0, and the solvents for other drugs are deionized water unless otherwise specified.

[0012] (4) Further, the molar ratio of Co(NO3)2·6H2O and ICA added in step (2) above is 8:1.

[0013] (5) Furthermore, the purpose of dispersing the product in 3.2 mL buffer to form a uniform dispersion in (2) above is to facilitate subsequent experimental operations such as activity testing.

[0014] (6) Furthermore, the prepared encapsulated enzyme material GOx@ZIF-90(Co) is a rectangular nanosheet structure with a thickness of 3-5 nm, which is smaller than the three-dimensional size of the enzyme.

[0015] (7) Furthermore, the encapsulated enzyme system can expose the enzyme portion to the outside of the carrier. Due to the superiority of this encapsulation method, the enzyme can maintain catalytic activity comparable to that of the natural enzyme and is superior to enzymes encapsulated by other methods. Moreover, due to the protective nature of the carrier, its stability is superior to that of enzymes encapsulated by other methods.

[0016] (8) Furthermore, the natural enzyme model in the semi-encapsulated enzyme system is GOx. This encapsulation method is highly operable and can be extended to other enzyme catalytic systems and cascade enzyme catalytic systems. Moreover, due to the high activity and stability of this catalytic system, this high-performance enzyme encapsulation technology has broad application prospects.

[0017] The advantages of this invention are:

[0018] (1) The innovative use of two-dimensional metal-organic framework materials for semi-encapsulation of enzymes enriches the strategies of enzyme encapsulation technology.

[0019] (2) It maintains the high activity of the natural enzyme. The thickness of the two-dimensional metal-organic framework material is smaller than the minimum three-dimensional size of the enzyme, which exposes part of the enzyme molecule to the outside of the carrier, thereby reducing the mass transfer resistance between the enzyme and the substrate. The catalytic efficiency of the enzyme is maintained, and it exhibits catalytic activity comparable to that of the natural enzyme.

[0020] (3) It effectively improves the stability and reusability of enzymes and reduces the cost of enzyme application. As an enzyme encapsulation carrier, two-dimensional metal-organic framework materials can partially immobilize enzymes, thereby maintaining the stability of enzyme conformation and improving the stability of the enzyme itself.

[0021] (4) The present invention uses GOx as a model enzyme, which can be widely applied to enzyme immobilization of different types of enzymes and multi-enzyme cascade catalytic systems. It is a general preparation strategy. Detailed Implementation

[0022] The present invention will be described in detail below with reference to the embodiments, but this does not constitute a limitation on the present invention.

[0023] Example 1: Systematic investigation of the enzyme exposure state of semi-encapsulated enzymes (including material thickness test, protease treatment test, and enzyme elution test)

[0024] (1) Set the concentration of GOx@ZIF-90(Co) dispersion to 0.01 mg / mL, take 30 μL and drop it onto a single-sided polished silicon wafer. After the silicon wafer is naturally air-dried, the thickness of GOx@ZIF-90(Co) on the surface is measured by atomic force microscopy. The thickness of GOx@ZIF-90(Co) is 3-5 nm, which is smaller than the three-dimensional size of the GOx encapsulated inside, indicating that the enzyme can be partially exposed outside ZIF-90(Co).

[0025] (2) Mix the GOx@ZIF-90(Co) dispersion with an equal volume of trypsin solution and incubate at 37 °C for 30 min. After incubation, perform enzyme activity testing. Add 130 μL of 2,2'-adiazonobis-3-ethylbenzothiazoline-6-sulfonic acid solution (ABTS, 5 mM), 40 μL of horseradish peroxidase solution (HRP, 0.2 mg / mL), 3.7 mL of HEPES buffer, and 100 μL of glucose solution (GLU, 25 mM) to 30 μL of incubated GOx@ZIF-90(Co), and incubate in a constant temperature shaker at 37 °C for 10 min. After the reaction, the reaction solution was transferred to a cuvette, and its absorbance at 415 nm was measured using a UV-Vis spectrophotometer. The results showed that Try could affect the activity of GOx@ZIF-90(Co), indicating that GOx can be exposed on the surface of ZIF-90(Co) and can come into contact with the outside.

[0026] (3) GOx@ZIF-90(Co) was incubated with a certain amount of sodium dodecyl sulfate solution (SDS, 5 wt%) for 20 min. After incubation, it was centrifuged (10000 rpm, 5 min), and the supernatant was transferred to a cuvette. The emission spectrum at 275 nm excitation was measured using a fluorescence spectrophotometer. The results showed that GOx was immobilized by ZIF-90(Co), and no GOx was observed to be adsorbed on the carrier surface, indicating the successful construction of the semi-encapsulated system.

[0027] Example 2: Investigation of Enzymatic Activity and Reaction Kinetics of a Semi-Encapsulated Enzyme System

[0028] (1) The enzymatic activities of enzymes with different encapsulation forms and free enzymes were investigated. 130 μL of ABTS solution (5 mM), 40 μL of HRP solution (0.2 mg / mL), 3.7 mL of HEPES buffer, and 100 μL of GLU solution (25 mM) were added to 30 μL of GOx dispersions with different encapsulation forms (surface-fixed, fully encapsulated, and semi-encapsulated) and free GOx, respectively. The mixtures were incubated in a 37 °C incubator for 10 min. After the reaction, the reaction solution was transferred to a cuvette, and its absorbance at 415 nm was measured using UV-vis. The results showed that the semi-encapsulated GOx (GOx@ZIF-90(Co)) developed in this invention has highly efficient enzymatic activity, with catalytic activity significantly higher than that of fully encapsulated enzymes and close to that of surface-encapsulated enzymes and free enzymes.

[0029] (2) The reaction kinetics of GOx with different encapsulation forms and free GOx were investigated. The activity of GOx with different encapsulation forms (surface immobilization, full encapsulation, and semi-encapsulation) on the catalytic reaction of GLU at different concentrations was measured. Kinetic curves were fitted according to the Michaelis-Menten equation, and kinetic parameters were calculated. The results show that the Michaelis constant (K) of the semi-encapsulated GOx catalyzing GLU in this invention is [missing information]. m The catalytic efficiency was 4.58 mM, which is superior to that of fully encapsulated GOx (54.02 mM) and surface-fixed GOx (8.87 mM), and close to that of free GOx (5.05 mM). This indicates that it has superior catalytic efficiency compared to other encapsulation forms of GOx, and is close to that of free GOx.

[0030] Example 3: Stability and reusability of a semi-encapsulated enzyme system

[0031] (1) Organic solvent stability study: The organic solvent stability of GOx was evaluated by measuring the residual activity of GOx in different encapsulation forms and free GOx after incubation with organic solvents. 1 mL of GOx dispersions in different encapsulation forms and free GOx solutions were added to 1 mL of different organic solvents (N,N-dimethylformamide (DMF), methanol, acetonitrile (ACN), and tetrahydrofuran (THF)) or 1 mL of 3 M urea solution, respectively. The mixtures were incubated in a constant temperature shaker at 37 °C for 2 h. After incubation, 130 μL of ABTS solution (5 mM), 40 μL of HRP solution (0.2 mg / mL), 3.7 mL of HEPES buffer, 100 μL of GLU solution (25 mM) and 30 μL of enzyme solution were mixed and incubated in a constant temperature shaker at 37 °C for 10 min. The absorbance at 415 nm was then measured using UV-vis to measure the catalytic activity. The results show that the semi-encapsulated GOx prepared in this invention can better retain its catalytic activity after being treated in different organic solvents for 2 h.

[0032] (2) Thermal stability study: The thermal stability of GOx with different encapsulation forms and free GOx was evaluated by measuring the residual activity after heating at 60 °C for different times. 30 μL of GOx with different encapsulation forms and free GOx were placed in a water bath at 60 °C and heated for 1, 2, 3, 4, 5 and 6 h and then removed. After cooling to room temperature, 130 μL of ABTS solution (5 mM), 40 μL of HRP solution (0.2 mg / mL), 3.7 mL of HEPES buffer, and 100 μL of GLU solution (25 mM) were added and mixed. The mixture was incubated in a constant temperature shaker at 37 °C for 10 min, and then the absorbance at 415 nm was measured by UV-vis to measure its catalytic activity. The results showed that after heating at 60 °C for 6 h, the semi-encapsulated GOx prepared in this invention could retain 93.58% of its original activity, while the fully encapsulated GOx retained only 47.06% of its activity, the surface-fixed GOx retained only 31.98% of its activity, and the free GOx retained only 40.40% of its activity. This indicates that the semi-encapsulated GOx of this invention can better retain its catalytic activity after heat treatment.

[0033] (3) Storage stability study: The storage stability was evaluated by measuring the residual activity of GOx in different encapsulation forms and free GOx after storage at 4 °C for different times. GOx dispersions in different encapsulation forms and free GOx were placed in a refrigerator at 4 °C. 30 μL of each dispersion was taken out on days 1, 2, 7, 9, 14, and 20 of storage and mixed with 130 μL of ABTS solution (5 mM), 40 μL of HRP solution (0.2 mg / mL), 3.7 mL of HEPES buffer, and 100 μL of GLU solution (25 mM). The mixture was incubated in a 37 °C incubator for 10 min, and the absorbance at 415 nm was measured using UV-vis to assess its catalytic activity. The results showed that after 20 days of storage at 4 °C, the semi-encapsulated GOx prepared in this invention retained significantly higher activity than other enzyme materials.

[0034] (4) Reusability Assessment: The reusability of GOx in different encapsulation forms was evaluated by measuring its reusability after repeated recycling. 30 μL of GOx in different encapsulation forms was mixed with 130 μL of ABTS solution (5 mM), 40 μL of HRP solution (0.2 mg / mL), 3.7 mL of HEPES buffer, and 100 μL of GLU solution (25 mM). The mixture was incubated at 37 °C in a shaker for 10 min. The reaction solution was then centrifuged (10000 rpm, 5 min), and the supernatant was transferred to a cuvette. The absorbance was measured using UV-vis. The precipitate was washed once with HEPES buffer, and its activity was measured again. The results showed that after 6 reuses, the semi-encapsulated GOx prepared in this invention maintained 92.1% of its activity, significantly better than other enzyme materials, and free GOx did not exhibit reusability.

[0035] Example 4: A semi-encapsulated enzyme system was used to construct a wearable patch sensor for detecting glucose in sweat.

[0036] (1) Preparation of GOx-HRP@ZIF-90(Co): Weigh 0.1537 g ICA into 4 mL of deionized water and sonicate until completely dissolved; weigh 0.0582 g Co(NO3)2·6H2O into 4 mL of deionized water and sonicate until completely dissolved; weigh 1 mg GOx and 3 mg HRP for later use. Under magnetic stirring, add 1 mg GOx and 3 mg HRP to 4 mL of ICA solution, mix well, then add 4 mL of Co(NO3)2·6H2O solution and react for 3 h under magnetic stirring. After the reaction is complete, centrifuge (10000 rpm, 5 min) to collect the product, wash the precipitate three times with deionized water (10000 rpm, 5 min), and finally disperse the product in 4 mL of buffer to form a uniform dispersion and store at 4 °C for later use.

[0037] (2) Preparation of GLU sensing paper: Cut cellulose chromatography paper into discs with a diameter of 6 mm, add 10 μL of GOx-HRP@ZIF-90(Co), let it air dry naturally, then add 5 μL of ABTS solution (5 mM), and let it air dry naturally. Store the prepared GLU sensing paper discs at -20 °C for later use.

[0038] (3) Preparation of wearable patch sensor: Cellulose chromatographic paper was cut into cross-shaped fluid layers (5 mm wide). Medical double-sided tape was cut into 2.5 × 2.5 cm squares with sweat collection holes of 6 mm in diameter. Polydimethylsiloxane (PDMS) film was cut into 2.5 × 2.5 cm squares. The fluid layer was attached to the medical double-sided tape, and three GLU sensing paper pieces were attached on top of the fluid layer. The top layer was covered with the PDMS film to assemble the wearable patch sensor. The prepared wearable patch sensor was stored at -20 °C for later use.

[0039] (4) Construction of GLU detection standard curve: 40 μL of GLU solutions of different concentrations were added to the collection holes of the wearable patch. The solution diffused through the fluid layer to the GLU sensing paper. GOx-HRP@ZIF-90(Co) loaded on the GLU sensing paper catalyzed the oxidation of GLU and caused ABTS to develop color. After reacting for 10 min, the patch was photographed with a smartphone. The sensing area of ​​the patch was identified and analyzed using the "Photoshop" application. The color difference value (I) of the sensing area was calculated using the CIELAB color space. I represents the comparison of the color values ​​of the GLU sensing paper before and after detection. The calculation formula is:

[0040] Where L, a, and b represent the brightness, red-green coordinates, and yellow-blue coordinates of the GLU sensing paper before detection, respectively, and L', a', and b' represent the brightness, red-green coordinates, and yellow-blue coordinates of the GLU sensing paper after detection, respectively. The resulting GLU detection standard curve is I=4.31C. GLU +8.53, R 2 =0.9922. The linear range is 0.1-10 mM, and the detection limit is 0.04 mM.

[0041] (5) GLU selectivity test: 40 μL of solutions of GLU, urea, uric acid, ascorbic acid, lactose, fructose, maltose, and sucrose (all at a concentration of 10 mM) were added to the sweat collection pores of the wearable patch. After 10 min, the patch was photographed with a smartphone and the color difference value was analyzed. The results showed that the interfering substances had little effect on the color difference value of the sensing patch, while GLU enabled the sensing patch to exhibit a high color difference value, indicating that the prepared wearable patch sensor has high selectivity for GLU.

[0042] (6) Real Human Sweat Sample Detection: All volunteers fasted for 6 hours before the test. The sides of the volunteers' foreheads were cleaned with alcohol wipes, and then the prepared wearable patch sensor was attached. Volunteers sweated through running. The test was conducted twice: the first time was a 10-minute run on an empty stomach, followed by a 10-minute rest before the second test. Before the second test, 40 mL of 50% GLU oral solution was taken orally, followed by a 10-minute run. The patch was photographed with a smartphone, and color difference analysis was performed. The experimental results showed that the GLU concentration in the sweat of the four volunteers differed significantly before and after GLU intake, indicating that the GLU wearable patch sensor developed based on the semi-encapsulated immobilized enzyme system prepared in this invention has good application prospects.

Claims

1. A method for preparing a semi-encapsulated enzyme material based on a metal-organic framework, characterized in that, Includes the following steps: (1) Weigh 0.1537 g imidazole-2-carboxaldehyde (ICA) into 4 mL of deionized water and sonicate until completely dissolved; weigh 0.0582 g Co(NO3)2·6H2O into 4 mL of deionized water and sonicate until completely dissolved; weigh 2 mg glucose oxidase (GOx) for later use. (2) Under magnetic stirring, 2 mg of GOx was added to the ICA solution in (1), and after ICA and GOx were mixed thoroughly, the Co(NO3)2·6H2O solution in (1) was added; the reaction was carried out with magnetic stirring for 3 h; the obtained GOx@ZIF-90(Co) was washed with deionized water; the final product was dispersed in 3.2 mL buffer to form a uniform dispersion and stored at 4 °C for later use.

2. The method for preparing a semi-encapsulated enzyme material based on a metal-organic framework according to claim 1, characterized in that, The buffer is a 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer solution, 100 mM, pH 7.

0. Unless otherwise specified, the solvents for other drugs are deionized water.

3. Semi-encapsulated enzyme material based on metal-organic framework material prepared according to the method of any one of claims 1-2.

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