MOF (Metal Organic Framework) sensitization glucose detection system as well as preparation method and application thereof

Abstract

The invention discloses a preparation method of an MOF glucose detection system, ZIF is used as a metal organic framework, glucose oxidase (GOx) is loaded in the ZIF, the preparation method comprises the following steps: S1, water is added into a reaction bottle in advance, then metal ions, enzyme and organic ligands with the molar ratio of (100-1500): (1-10): (1-150) are added into the reaction bottle in advance, and the reaction bottle is filled with water; the method comprises the following steps: S1, adding 0.1-0.5% (by weight) of ammonia water into a reaction bottle, S2, mixing and reacting at room temperature for more than 6 hours, and S3, centrifuging by a centrifugal machine, and removing supernate to obtain solid precipitate of the GOx-MOF material loaded with glucose oxidase. The invention also discloses an application of the MOF glucose detection system in saliva glucose content detection. The method can be applied to glucose detection of oral saliva, and even if the glucose content is extremely low, the glucose concentration in the saliva can be efficiently and accurately detected.

Description

Technical Field

[0001] The present invention belongs to the fields of organic synthetic material preparation technology, electrochemical sensing technology and biomedical technology application, and specifically relates to a MOF-sensitized glucose detection system and a preparation method and application thereof. Background Art

[0002] Diabetes is a common chronic disease. Traditional methods of testing blood sugar require blood sampling, which takes a long time to test, causes trauma and pain to the human body, and easily increases infection and transmission routes. Some existing electronic blood sugar testers still fall into the category of invasive testing, and are complicated to operate and inconvenient to use.

[0003] Saliva is an oral fluid that contains a variety of substances, including inorganic ions, small organic molecules, lipids, proteins, and various hormones. Saliva is easy to collect and non-invasive, and can be used as a biological sample for research and clinical practice. In both type 1 diabetes and type 2 diabetes, salivary glucose levels can reflect blood sugar levels. Therefore, diabetes can be monitored by detecting low concentrations of glucose in saliva. The normal glucose concentration in human blood is 2-30mmol / L, and the glucose concentration in saliva is 0.025-0.1mmol / L. The glucose content in the saliva of diabetic patients is significantly increased, generally greater than 0.4mmol / L.

[0004] Therefore, it is feasible to indirectly detect the corresponding blood sugar concentration of the patient by detecting the saliva glucose concentration of the patient. However, the glucose concentration in oral saliva is very low, and how to efficiently and accurately detect the glucose concentration in saliva is a technical problem that needs to be solved urgently.

[0005] Metal organic frameworks (MOFs) are combined together through the interaction of metal ions and organic ligands. They play an important role in the biomedical field due to their large specific surface area, excellent thermal stability, variable functional groups and uniformly adjustable pore size.

[0006] Zeolitic Imidazolate Framework (ZIF) is an important type of MOF, formed by molecular self-assembly and bridging organic linkers between metal nodes. They have a tetrahedral three-dimensional network structure, similar to zeolite in structure, using zinc (Zn2+) or cobalt (Co2+) to replace the silicon in zeolite, and using imidazole ligands to replace the oxygen bridges in zeolite. ZIF has some functions of molecular sieves: ① The pore size is uniform and adjustable, and it has molecular sieving function; ② Ion exchange and selective adsorption function; ③ High temperature stability and selective catalytic performance, which can be used as a membrane reactor to separate the product while catalyzing the reaction process, realizing separation and catalysis integration; ④ The structure types are diverse, and it is an ideal separation and catalytic material.

[0007] Enzymes are an extremely important class of biological catalysts. Due to the action of enzymes, chemical reactions in organisms can be carried out efficiently and specifically under extremely mild conditions. Their efficiency, specificity and selectivity far exceed those of artificial catalysts. However, the inherent fragility of enzymes, such as being extremely susceptible to temperature and pH fluctuations, and having low tolerance to most organic solvents and small molecule inhibitors, severely limits the effectiveness of enzyme systems in actual operations.

[0008] The method of loading enzymes on MOF nanomaterials can be surface biocoupling method, pore permeation method or in situ encapsulation method. The details are as follows:

[0009] 1) Surface bioconjugation methods can be divided into physical adsorption, covalent bond connection and coordination bond connection according to different attachment processes and mechanisms.

[0010] ① Physical adsorption mainly relies on weak interactions to fix biomolecules on MOFs, such as van der Waals forces, hydrogen bonds, π-π interactions, electrostatic interactions, or hydrophobic interactions.

[0011] ②The mechanism of covalent connection is that biological molecules, under the action of functional molecules or catalysts, form peptide bonds with the amino or carboxyl groups on MOF to achieve connection. Among them, glutaraldehyde coupling based on Schiff base reaction is the most commonly used method for carrier-immobilized enzymes. One aldehyde group of the glutaraldehyde molecule reacts with the amino group of the enzyme molecule, and the other aldehyde group reacts with the amino group of MOF. In addition, click reaction can also be used for enzyme immobilization, which is achieved through the CXC bond formed between heteroatoms.

[0012] ③ Coordination connection: There are unsaturated coordinated metal positions on the outer surface of MOF crystals, which have the possibility of forming coordination bonds with amino acids and can achieve coordination connection.

[0013] 2) The pore permeation method combines biomolecules under mild conditions, which largely avoids the impact on the structure and activity of biomolecules. The rich pores of MOF also provide environmental protection for enzymes (such as organic solvents, extreme pH values, denaturants, high temperatures, etc.), thereby improving the tolerance of enzymes to the environment. In addition, the tunable pore size of MOF provides selectivity for substrate enzyme molecules, but this method requires greater spatial compatibility between MOF and the coated enzyme, so MOFs with larger pore sizes such as MIL-100, Nu-100X and IR MOF-74 are often used.

[0014] 3) The typical representative of the in situ encapsulation method is the one-pot method. In this method, MOF forms a protective shell around the enzyme, which can reduce the leaching of the enzyme in the MOF matrix in extremely harsh environments and maintain the enzyme activity. The one-pot method can be divided into coprecipitation and biomineralization according to the presence or absence of a coprecipitant. For the coprecipitation method, an auxiliary stabilizer is required to maintain the activity of the enzyme. The common coprecipitant is polyvinyl pyrrolidone (PVP); for biomineralization, the enzyme can be directly mixed with MOF to form a biocomposite material without the presence of a coprecipitant.

[0015] Studies have shown that biomacromolecules induce and regulate the growth of MOFs in aqueous solutions, while MOFs can act as a protective coating to inhibit the harmful effects of high temperature and proteolytic agents on encapsulated biomacromolecules and ensure their functionality under general working conditions. This in situ encapsulation method, known as "one-pot precipitation" or "bionic mineralization," allows microporous MOFs to crystallize around biomolecules. This approach has been used in biological systems, including enzymes, antibodies, nucleic acids, etc. Among them, the porous MOF exoskeleton can improve the stability of encapsulated enzymes through structural constraints, and can also selectively transport guest molecules through the microporous network accessible to the exoskeleton. The symbiotic strengthening effect between MOF materials and enzymes makes MOF immobilized enzymes have great potential in various fields.

[0016] Zeolitic imidazolate frameworks (ZIFs) composed of zinc and 2-methylimidazole (Hmim) are the most widely studied MOFs to date, due to their simple synthesis conditions and compatibility with bio-antibodies. For example, ZIF-8, due to its microporosity (pore size is usually less than 2nm), makes the compactness of the structure restriction conducive to the stability of encapsulated biomacromolecules. However, enzymes encapsulated in ZIFs usually show low activity, which is far from satisfactory due to the limited mass diffusion rate. The inherent micropores restrict the free access of large matrices to the micropores and cannot enter the active sites of the bioactive ZIF composites.

[0017] In view of the above problems, there is an urgent need to develop materials that do not affect enzyme activity but have the functional ability to load enzymes for use in biochemical sensing, thereby enhancing detection performance, expanding detection limits, and improving signal effects. Summary of the invention

[0018] The technical problem to be solved by the present invention is to provide a sensitized glucose detection system that can quickly and accurately detect low-concentration glucose content.

[0019] To this end, the present invention provides a method for preparing a MOF glucose detection system, which uses a zeolitic imidazolate framework (ZIF) as a metal organic framework and loads glucose oxidase (GOx) in the ZIF. The preparation method comprises the following steps:

[0020] S1: Add water to the reaction bottle in advance, then add metal ions, enzymes, and organic ligands in a molar ratio of 100-1500:1-10:1-150, and 0.1%-0.5% (weight ratio) of ammonia water to the reaction bottle in sequence,

[0021] S2: Mixing reaction at room temperature for more than 6 hours,

[0022] S3: After centrifugation by a centrifuge, the supernatant is removed to obtain a solid precipitate of the GOx-MOF material loaded with glucose oxidase.

[0023] Preferably, in step S1, the water added to the reaction flask is 80-95% (by weight).

[0024] Preferably, the preparation method further comprises: S4: using water to make up the volume, and storing the obtained GOx-MOF material loaded with glucose oxidase to 2-8° C. for standby use, and the ratio of the volume of the water used for the make up to the total volume of the raw materials added to the reaction bottle in step S1 is 0.1-100.

[0025] Preferably, the metal ion is selected from the group consisting of alkali metals, alkaline earth metals, transition metals, Group IIIA metals, Group VIII metals, rare earth metals, metal complexes and metal clusters.

[0026] Preferably, the organic ligand is selected from the group consisting of nitrogen-containing organic ligands, carboxylate ligands, amino group-modified ligands, photosensitizer-complex-modified ligands, and electron-rich conjugated ligands.

[0027] Preferably, the centrifuge speed is 1000-30000 rpm.

[0028] Preferably, an auxiliary agent may be added in step S1.

[0029] Preferably, the adjuvant is a Bis-PEGn-Acid chain.

[0030] Preferably, the Bis-PEGn-Acid chain is Bis-PEG2-Acid, Bis-PEG4-Acid, or Bis-PEG8-Acid.

[0031] Preferably, the metal ion is a zinc derivative or an iron derivative.

[0032] Preferably, the metal ion is zinc sulfide, zinc chloride, zinc bromide, zinc oxide, zinc iodide, zinc sulfate, zinc acetate, zinc carbonate, zinc nitrate or a hydrate thereof.

[0033] Preferably, the organic ligand is polyaminoimidazole.

[0034] Preferably, the organic ligand is 2-methylimidazole, 2-imidazolecarboxaldehyde, imidazole-2-carboxylic acid, benzimidazole, 2-methylbenzimidazole, 1,2,4-triazole, 3-methyl-1H-1,2,4-triazole, 1,2,4-triazole-3-carboxylic acid, polyazole or a derivative thereof.

[0035] Preferably, the water is ultrapure water.

[0036] Preferably, the mixing is shaking or stirring.

[0037] According to another aspect of the present invention, there is provided an application of a MOF glucose detection system in detecting glucose content in saliva, wherein the MOF glucose detection system is prepared by the above preparation method.

[0038] The present invention uses a water-based solvent "one-pot precipitation" method to engineer MOF materials into an outer framework, so that the enzyme is encapsulated in it for protection from the influence of complex environments. The results of the present invention show that the porous MOF outer framework can not only improve the activity, stability and sensitivity of the encapsulated enzyme through structural constraints, so that it maintains its biocatalytic function, but also endows the MOF material with great potential for encapsulating enzymes in complex applications through the symbiotic strengthening effect between the MOF material and the enzyme, including enzyme carriers for biochemical sensing, biocatalysis and cancer treatment.

[0039] In particular, the MOF glucose detection system of the present invention can be applied to the glucose detection of oral saliva. Even if the glucose content is extremely low, the present invention can efficiently and accurately detect the glucose concentration in saliva. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1-4 It is a comparison chart comparing the glucose detection performance enhancement of GOx and GOx-MOF materials in various embodiments.

[0041] To facilitate understanding, identical reference numerals have been used, where possible, to designate identical elements that are common to the figures. It is contemplated that elements disclosed in one embodiment may be beneficially utilized on other embodiments without specific recitation. DETAILED DESCRIPTION

[0042] The present invention is a MOF-sensitized glucose detection system, which uses a zeolitic imidazolate framework (ZIF) as a metal organic framework and loads glucose oxidase (GOx) in the ZIF.

[0043] The MOF-sensitized glucose detection system can be selected from the following concentration ratio range: it is made of metal ions, enzymes and organic ligands with a molar ratio of 100-1500:1-10:10-150, and 0.1%-0.5% (weight ratio) ammonia water.

[0044] The specific preparation method comprises the following steps:

[0045] S1: Add water to the reaction bottle in advance, and add the above materials with appropriate molar ratios into the reaction bottle in order.

[0046] S2: Mix at room temperature (e.g., shake, stir or similar means) for more than 6 hours and observe the formation of precipitation.

[0047] S3: After centrifugation in a centrifuge (speed 1000-30000 rpm), the supernatant is removed to obtain a solid precipitate.

[0048] S4: Use water to make up to volume, and obtain the GOx-MOF material loaded with glucose oxidase and store it at 2-8°C for later use.

[0049] In the above preparation method, auxiliary agents, such as Bis-PEGn-Acid chains, may also be added to effectively improve the water solubility of the MOF material.

[0050] In the above preparation method, in step S1, the water added to the reaction bottle is 80-95% (by weight), and in one embodiment, preferably 1-5 mL of ultrapure water.

[0051] In the above preparation method, in step S4, the ratio of the volume of the water used for the constant volume to the total volume of the raw materials added to the reaction flask in step S1 is 0.1-100. In one embodiment, 1-3 mL of ultrapure water is preferred.

[0052] The metal ions are selected from at least one of the following metals:

[0053] 1) Alkali metals:

[0054] Lithium (Li + ): lithium chloride, lithium fluoride, lithium carbonate, lithium sulfate, lithium nitrate, lithium phosphate, lithium bromide, lithium iodide, lithium acetate and their hydrates, etc.

[0055] Sodium (Na + ): sodium chloride, sodium fluoride, sodium carbonate, sodium sulfate, sodium nitrate, sodium phosphate, sodium bromide, sodium iodide, sodium acetate and its hydrate, etc.

[0056] Potassium (K + ): potassium chloride, potassium fluoride, potassium carbonate, potassium sulfate, potassium nitrate, potassium phosphate, potassium bromide, potassium iodide, potassium acetate and its hydrates, etc.

[0057] Rubidium (Rb + ): rubidium chloride, rubidium fluoride, rubidium carbonate, rubidium sulfate, rubidium nitrate, rubidium bromide, rubidium iodide and their hydrates, etc.

[0058] Cesium (Cs + ): cesium chloride, cesium fluoride, cesium carbonate, cesium sulfate, cesium nitrate, cesium bromide, cesium iodide and their hydrates, etc.

[0059] 2) Alkaline earth metals:

[0060] Calcium (Ca 2+ ): calcium chloride, calcium bromide, calcium oxide, calcium iodide, calcium sulfate, calcium acetate, calcium carbonate, calcium nitrate and its hydrate, etc.

[0061] Magnesium (Mg 2+ ): magnesium chloride, magnesium bromide, magnesium oxide, magnesium iodide, magnesium sulfate, magnesium acetate, magnesium carbonate, magnesium nitrate and their hydrates, etc.

[0062] Strontium (Sr 2+ ): strontium chloride, strontium bromide, strontium oxide, strontium iodide, strontium carbonate, strontium nitrate and their hydrates, etc.

[0063] Barium (Ba 2+ ): barium chloride, barium bromide, barium oxide, barium iodide, barium carbonate, barium nitrate and its hydrate, etc.

[0064] 3) Transition metals:

[0065] Manganese (Mn 2+ ): manganese sulfide, manganese chloride, manganese bromide, manganese fluoride, manganese iodide, manganese oxide, manganese acetate, manganese carbonate and its hydrate, etc.

[0066] Copper (Cu + / Cu 2+ ): copper sulfide, copper chloride, copper bromide, copper fluoride, copper iodide, copper oxide, copper acetate, copper carbonate and its hydrate, etc.

[0067] Zinc (Zn 2+ ): zinc sulfide, zinc chloride, zinc bromide, zinc oxide, zinc iodide, zinc sulfate, zinc acetate, zinc carbonate, zinc nitrate and its hydrate, etc.

[0068] Iron (Fe 2+ / Fe 3+ ): ferric chloride, ferric oxide, ferric sulfate, ferric sulfide, ferric citrate, ferric nitrate and their hydrates, etc.

[0069] Indium 3+ ): indium chloride, indium acetate, indium sulfate, indium nitrate, indium phosphide, indium bromide, indium oxide, indium iodide, indium fluoride and hydrates thereof, etc.

[0070] Thallium (Tl + ): thallium chloride, thallium acetate, thallium sulfate, thallium nitrate, thallium bromide, thallium oxide, thallium iodide and their hydrates, etc.

[0071] Palladium (Pd + ): palladium chloride, palladium acetate, palladium sulfate, palladium nitrate, palladium bromide, palladium oxide, palladium iodide and their hydrates, etc.

[0072] Silver (Ag + ): silver chloride, silver acetate, silver sulfate, silver nitrate, silver bromide, silver oxide, silver iodide, silver fluoride and its hydrate, etc.

[0073] Gold + ): Gold chloride, gold bromide, gold iodide and their hydrates, etc.

[0074] Zirconium (Zr 2+ / Zr 4+ ): zirconium oxide, zirconium chloride, zirconium fluoride, zirconium sulfate, zirconium acetate, zirconium hydrogen phosphate, zirconium dihydroxy carbonate, zirconium oxychloride and hydrates thereof, etc.

[0075] 4) Group IIIA metals:

[0076] Aluminum 3+ ): aluminum oxide, aluminum chloride, aluminum fluoride, aluminum carbonate, aluminum sulfate, aluminum nitrate, aluminum phosphate, aluminum bromide, aluminum iodide, aluminum acetate and its hydrate, etc.

[0077] Gallium (Ga 3+ ): Gallium oxide, gallium sulfate, gallium nitrate, gallium bromide, gallium iodide and their hydrates, etc.

[0078] 5) Group VIII metals: Cobalt (Co 2+ ), cobalt sulfide, cobalt oxide, cobalt carbide, cobalt bromide, zinc chloride, cobalt acetate, cobalt carbonate, cobalt nitrate and their hydrates, etc.

[0079] 6) Rare earth metals: compounds of rare earth metals such as lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).

[0080] 7) Metal complexes: lithium cobalt oxide, lithium cobalt phosphate, potassium cobalt cyanide, strontium titanate, potassium chloroaurate, etc.

[0081] 8) Metal clusters: Zr6O4(OH)4, Zr6O4(OH)4(H2O)4, Fe3O(OH)3, etc.

[0082] Preferably, the metal ion is zinc (Zn 2+), for example, zinc sulfide, zinc chloride, zinc bromide, zinc oxide, zinc iodide, zinc sulfate, zinc acetate, zinc carbonate, zinc nitrate or a hydrate thereof can be used.

[0083] The organic ligand is selected from at least one of the organic ligands listed below:

[0084] 1) Nitrogen-containing organic ligands:

[0085] ① Pyrimidines: pyrimidine, 4-amino-2-hydroxypyrimidine, 2,4-dihydroxy-5-methylpyrimidine, 2,4-dihydroxypyrimidine, etc.

[0086] ②Porphyrins: porphyrin and its derivatives.

[0087] ③Phthalocyanines: phthalocyanine and its derivatives.

[0088] ④ Polyazoles: imidazoles (such as imidazole, 2-methylimidazole, 2-imidazolecarboxaldehyde, imidazole-2-carboxylic acid, benzimidazole, 2-methylbenzimidazole, 1,2,4-triazole, 3-methyl-1H-1,2,4-triazole, 1,2,4-triazole-3-carboxylic acid, etc.), polyazoles and their derivatives.

[0089] 2) Carboxylate ligands:

[0090] ① Aliphatic carboxylic acids: acetic acid, propionic acid, etc.

[0091] ② Aromatic carboxylic acids: including monocyclic and polycyclic aromatic carboxylic acids, such as benzoic acid, cinnamic acid, terephthalic acid, phthalic acid, pyromellitic acid, biphenyl-4,4'-dicarboxylic acid, biphenyl-3,3'-dicarboxylic acid, 2,6-naphthalene dicarboxylic acid, [1,1':4',1"-terphenyl]-4,4"-dicarboxylic acid, 2',5'-dimethyl-[1,1':4',1"-terphenyl]-4,4"-dicarboxylic acid, [1,1':4',1":4" ,1"'-tetraphenyl]-4,4"'-dicarboxylic acid, [1,1':4',1":4" ,1 ”'-tetraphenyl]-3,3”',5,5”'-tetracarboxylic acid, 1,3,5-tri(4-carboxyphenyl)benzene, 1,2,4,5-tetra(4-carboxyphenyl)benzene, 5'-(3,5-dicarboxyphenyl)-[1,1':3',1"-terphenyl]-3,3",5,5"-tetracarboxylic acid, 4,4-(anthracene-9,10-diyl)dibenzoic acid, 4,4',4",4"'-(pyrene-1,3,6,8-tetrayl)tetrabenzoic acid, 6,6',6",6"'-(pyrene-1,3,6,8-tetrayl)tetra(2-naphthoic acid), etc.

[0092] ③ Aromatic carboxylic acids containing additional functional groups: carboxylic acids containing nitrogen, oxygen, sulfur, bromine and other heteroatoms, such as 2-aminoterephthalic acid, 2,5-diaminobenzene-1,4-dicarboxylic acid, 2-bromoterephthalic acid, 4,4',4"-(benzene-1,3,5-triyltri(oxy))tribenzoic acid, 4,4'-(benzo[c][1,2,5]thiadiazole-4,7-diyl)dibenzoic acid, etc.; carboxylic acids containing unsaturated bonds, such as 4,4'-[1,4-phenylenebis(acetylene-2,1-diyl)]dibenzoic acid, 4,4',4",4'-(ethylene-1,1,2,2-tetrayl)tetrabenzoic acid, 4,4',4"-(benzene-1,3,5-triyltri(acetylene-2,1-diyl))tribenzoic acid, etc.

[0093] ④Mixed carboxylic acid ligands: combines multiple different types of carboxylate ligands.

[0094] 3) Other organic ligands: ligands modified with amino groups, ligands modified with photosensitizer complexes, electron-rich conjugated ligands, etc.

[0095] Preferably, the organic ligand is a polyazole: imidazole (such as imidazole, 2-methylimidazole, 2-imidazolecarboxaldehyde, imidazole-2-carboxylic acid, benzimidazole, 2-methylbenzimidazole, 1,2,4-triazole, 3-methyl-1H-1,2,4-triazole, 1,2,4-triazole-3-carboxylic acid, etc.), polyazole or its derivatives.

[0096] The enzyme may be selected from natural glucose oxidase (GOx) or directed-evolved glucose oxidase (GOx).

[0097] The specific embodiment of GOx-MOF is described below, with GOx-MOFN-M as the specific material name, wherein N refers to the number of the organic ligand, and M refers to the number of the auxiliary agent Bis-PEGn-Acid.

[0098] Bis-PEGn-Acid includes Bis-PEG2-Acid (numbered 1), Bis-PEG4-Acid (numbered 2), and Bis-PEG8-Acid (numbered 3), and no Bis-PEG-Acid is numbered 0.

[0099] Preferably, the organic ligand imidazoles include: 2-methylimidazole (numbered 1), 2-imidazole carboxaldehyde (numbered 2), 3-methyl-1H-1,2,4-triazole (numbered 3), 2-imidazole carboxylic acid (numbered 4), 2-aminoimidazole sulfate (numbered 5), 3-bromo-1,2,4-triazole (numbered 6), etc. Aromatic carboxylic acid: 2-aminoterephthalic acid (numbered 7).

[0100] Therefore, for example, GOx-MOF3-2 is a GOx-MOF material made using 3-methyl-1H-1,2,4-triazole as an organic ligand and Bis-PEG8-Acid as an auxiliary agent, and so on.

[0101] Embodiment 1:

[0102] The metal is a zinc derivative Zn(OAc)2·H2O, the organic ligand is an imidazole derivative 3-methyl-1H-1,2,4-triazole, and the Bis-PEGn-Acid chain is Bis-PEG2-Acid, Bis-PEG4-Acid, and Bis-PEG8-Acid (which can effectively improve the water solubility of MOF materials).

[0103] Add 1 mL of ultrapure water to a 5 mL EP tube, then add 25 uL of 0.5 M Zn(OAc)2·H2O, 50 uL of 0.05 M Bis-PEG2-Acid (or Bis-PEG4-Acid, or Bis-PEG8-Acid), 25 uL of 100 U / mL GOx, 50 uL of 0.5 M 3-methyl-1H-1,2,4-triazole solution, and finally add 25 uL of 10% ammonia water, cover the lid, and oscillate the reaction at room temperature for 6 hours.

[0104] After the reaction, the supernatant was removed after centrifugation at 8000 rpm, and then 1 mL of ultrapure water was added to wash three times to obtain a white solid precipitate. Finally, 1 mL of ultrapure water was added to make up the volume, and an ultrasonic machine was used to disperse the mixture evenly to obtain a series of GOx-MOF materials with different water solubility, which were stored at 2-8°C for later use.

[0105] Glucose detection experiment results: The TMB colorimetric method (detection principle: GOx oxidizes glucose to produce H2O2, causing the TMB molecules to be oxidized and exhibiting ultraviolet absorption at 450nm) was used to compare the GOx enzyme activity. The results showed that the absorbance of GOx after responding to glucose was 0.356; the absorbance of GOx-MOF3-2 after responding to glucose was 0.650; and the absorbance of GOx-MOF3-3 after responding to glucose was 0.771.

[0106] Results analysis: The glucose signal detected by the MOF-loaded GOx system is 1.8 times and 2.2 times that of the single GOx system without MOF loading, respectively. Figure 1 shown.

[0107] Embodiment 2:

[0108] The metal is a zinc derivative Zn(OAc)2·H2O, the organic ligand is an imidazole derivative 3-bromo-1,2,4-triazole, and the Bis-PEGn-Acid chain is Bis-PEG2-Acid, Bis-PEG4-Acid, and Bis-PEG8-Acid (which can effectively improve the water solubility of MOF materials).

[0109] Add 1 mL of ultrapure water to a 5 mL EP tube, then add 25 uL of 0.5 M Zn(OAc)2·H2O, 50 uL of 0.05 M Bis-PEG2-Acid (or Bis-PEG4-Acid, or Bis-PEG8-Acid), 25 uL of 100 U / mL GOx, 50 uL of 0.5 M 3-bromo-1,2,4-triazole solution, and finally add 25 uL of 10% ammonia water, cover the lid, and oscillate the reaction at room temperature for 6 hours.

[0110] After the reaction, the supernatant was removed after centrifugation at 8000 rpm, and then 1 mL of ultrapure water was added to wash three times to obtain a white solid precipitate. Finally, 1 mL of ultrapure water was added to make up the volume, and an ultrasonic machine was used to disperse the mixture evenly to obtain a series of GOx-MOF materials with different water solubility, which were stored at 2-8°C for later use.

[0111] Results of glucose detection experiment: The TMB colorimetric method (detection principle: H2O2 produced by GOx oxidation of glucose causes TMB molecules to be oxidized, resulting in ultraviolet absorption at 450nm) was used to compare the GOx enzyme activity. The results showed that the absorbance of GOx after responding to glucose was 0.356; the absorbance of GOx-MOF6-0 after responding to glucose was 0.637; the absorbance of GOx-MOF6-1 after responding to glucose was 0.643; the absorbance of GOx-MOF6-2 after responding to glucose was 0.605; and the absorbance of GOx-MOF6-3 after responding to glucose was 0.705.

[0112] Results: The glucose signals detected by the MOF-loaded GOx system were 1.8, 1.8, 1.7 and 2.0 times higher than those detected by the single GOx system without MOF loading. Figure 2 shown.

[0113] Embodiment 3:

[0114] The metal is an iron derivative FeCl3·6H2O, and the organic ligand is an aromatic carboxylic acid derivative 2-aminoterephthalic acid.

[0115] Add 1 mL of ultrapure water to a 5 mL EP tube, then add 25 uL of 0.5 M FeCl3·6H2O, 25 uL of 100 U / mL GOx, and 50 uL of 0.5 M 2-aminoterephthalic acid in sequence, cover the tube, and shake at room temperature for 6 hours.

[0116] After the reaction, the supernatant was removed after centrifugation at 8000 rpm, and then 1 mL of ultrapure water was added to wash three times to obtain a brown solid precipitate. Finally, 1 mL of ultrapure water was added to make up the volume, and an ultrasonic machine was used to disperse it evenly to obtain the GOx-MOF material, which was stored at 2-8°C for use.

[0117] Glucose detection experiment results: The TMB colorimetric method (detection principle: GOx oxidizes glucose to produce H2O2, causing the TMB molecules to be oxidized and exhibiting ultraviolet absorption at 450nm) was used to compare the GOx enzyme activity. The results showed that the absorbance of GOx after responding to glucose was 0.356; the absorbance of GOx-MOF7 after responding to glucose was 0.675.

[0118] Results: The glucose signal detected by the MOF-loaded GOx system is 1.9 times that of the single GOx system without MOF loading. Figure 3 shown.

[0119] Embodiment 4:

[0120] The metal is a zinc derivative Zn(OAc)2·H2O, the organic ligand is an imidazole derivative 2-methylimidazole, and the Bis-PEGn-Acid chain is Bis-PEG2-Acid, Bis-PEG4-Acid, and Bis-PEG8-Acid (which can effectively improve the water solubility of MOF materials).

[0121] Add 1 mL of ultrapure water to a 5 mL EP tube, then add 25 uL of 0.5 M Zn(OAc)2·H2O, 50 uL of 0.05 M Bis-PEG2-Acid (or Bis-PEG4-Acid, or Bis-PEG8-Acid), 25 uL of 100 U / mL GOx, and 50 uL of 0.5 M 2-methylimidazole solution in sequence, and finally add 25 uL of 10% ammonia water, cover the lid, and oscillate the reaction at room temperature for 6 hours.

[0122] After the reaction, the supernatant was removed after centrifugation at 8000 rpm, and then 1 mL of ultrapure water was added to wash three times to obtain a white solid precipitate. Finally, 1 mL of ultrapure water was added to make up the volume, and an ultrasonic machine was used to disperse it evenly to obtain the GOx-MOF material, which was stored at 2-8°C for use.

[0123] Glucose detection experiment results: The TMB colorimetric method (detection principle: GOx oxidizes glucose to produce H2O2, which causes the TMB molecules to be oxidized and exhibits ultraviolet absorption at 450nm) was used to compare the GOx enzyme activity. The results showed that the absorbance of GOx after responding to glucose was 0.086; the absorbance of GOx-MOF1-3 after responding to glucose was 0.536.

[0124] Results analysis: The glucose signal detected by the MOF-loaded GOx system is 5 times higher than that detected by the single GOx system without MOF loading. Figure 4 shown.

[0125] The MOF glucose detection system of the present invention can be applied to glucose content detection, especially to glucose content detection in oral saliva, and can be used as a glucose conversion layer coated on a biosensor to make an oral blood glucose meter.

[0126] Those skilled in the art will appreciate that the foregoing examples are illustrative and not restrictive. It will be apparent to those skilled in the art upon reading the specification and studying the drawings that all permutations, enhancements, equivalents, and improvements are included within the true spirit and scope of the present invention. Therefore, the appended claims are intended to include all such modifications, permutations, and equivalents that fall within the true spirit and scope of these teachings.

Claims

1. A method for preparing a MOF glucose detection system, characterized in that: Zeolitic Imidazolate Framework (ZIF) is used as a metal organic framework, and glucose oxidase (GOx) is loaded in the ZIF. The preparation method comprises the following steps: S1: Add water to the reaction bottle in advance, then add metal ions, enzymes, and organic ligands in a molar ratio of 100-1500:1-10:1-150, and 0.1%-0.5% (weight ratio) of ammonia water to the reaction bottle in sequence, S2: Mixing reaction at room temperature for more than 6 hours, S3: After centrifugation by a centrifuge, the supernatant is removed to obtain a solid precipitate of the GOx-MOF material loaded with glucose oxidase.

2. The method for preparing the MOF glucose detection system according to claim 1, characterized in that: In step S1, 80-95% (by weight) of water is added to the reaction flask.

3. The preparation method of the MOF glucose detection system according to claim 1 or 2, characterized in that: The preparation method further comprises: S4: The volume of the fixed volume water is then used to store the obtained GOx-MOF material loaded with glucose oxidase at 2-8° C. for later use. The ratio of the volume of the fixed volume water to the total volume of the raw materials added to the reaction bottle in step S1 is 0.1-100.

4. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: The metal ion is selected from the group consisting of alkali metals, alkaline earth metals, transition metals, Group IIIA metals, Group VIII metals, rare earth metals, metal complexes and metal clusters.

5. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: The organic ligand is selected from one of nitrogen-containing organic ligands, carboxylate ligands, amino group-modified ligands, photosensitizer-composite-modified ligands, and electron-rich conjugated ligands.

6. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: The centrifuge speed is 1000-30000 rpm.

7. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: An auxiliary agent may also be added in step S1.

8. The method for preparing the MOF glucose detection system according to claim 7, characterized in that: The auxiliary agent is a Bis-PEGn-Acid chain.

9. The method for preparing the MOF glucose detection system according to claim 8, characterized in that: The Bis-PEGn-Acid chain is Bis-PEG2-Acid, Bis-PEG4-Acid, or Bis-PEG8-Acid.

10. The method for preparing the MOF glucose detection system according to claim 4, characterized in that: The metal ion is a zinc derivative or an iron derivative.

11. The method for preparing the MOF glucose detection system according to claim 10, characterized in that: The metal ion is zinc sulfide, zinc chloride, zinc bromide, zinc oxide, zinc iodide, zinc sulfate, zinc acetate, zinc carbonate, zinc nitrate or a hydrate thereof.

12. The method for preparing the MOF glucose detection system according to claim 5, characterized in that: The organic ligand is polyaminoimidazole.

13. The method for preparing the MOF glucose detection system according to claim 12, characterized in that: The organic ligand is 2-methylimidazole, 2-imidazole carboxaldehyde, imidazole-2-carboxylic acid, benzimidazole, 2-methylbenzimidazole, 1,2,4-triazole, 3-methyl-1H-1,2,4-triazole, 1,2,4-triazole-3-carboxylic acid, polyazole or its derivatives.

14. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: The water is ultrapure water.

15. The method for preparing the MOF glucose detection system according to any one of claims 1 to 3, characterized in that: The mixing is shaking or stirring.

16. Application of a MOF glucose detection system in the detection of saliva glucose content, characterized in that: The MOF glucose detection system is prepared by the preparation method described in any one of claims 1-15.

Images