A composite solid biological preparation based on self-assembled crystalline materials, and its preparation and application

Abstract

The present invention aims at the shortcomings of traditional liquid biological preparations and creatively uses self-assembled crystalline materials to prepare composite solid biological preparations. Self-assembled crystalline materials can protect biological molecules from interference from the external environment, improve storage stability, and maintain the activity of biological molecules to the maximum extent. At the same time, the prepared biological preparations can release biological molecules under mild conditions, and the released biological molecules can still maintain their original activity. The method of the present invention has the advantages of simple preparation method, low storage cost, high stability, and fast release rate, and will not affect the activity of biological molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of selective separation and immobilization of biological preparations, and in particular to a self-assembled crystalline material composite solid biological preparation with high biocompatibility and a preparation method thereof, as well as applications of the composite solid biological preparation in different fields. Background Art

[0002] There are various biological agents in nature, including biological macromolecules such as proteins, carbohydrates, lipids, and nucleic acids, as well as some biological small molecules. Biological agents have great application value. For example, fields such as cell culture, fermentation, chemical industry, and food cannot do without biological agents. However, biological agents are usually produced by living organisms and are difficult to obtain through artificial synthesis, especially biological agents in liquid state. Their shortcomings such as difficulty in storage, easy inactivation, and sensitivity to the environment limit their industrial application.

[0003] The immobilization technology of biological agents has opened up broad prospects for their industrial applications, such as the immobilization of enzyme molecules and antibody molecules. This technology improves the stability of biological agents and provides the possibility for large-scale industrial applications. The immobilization of biological agents has always been a research hotspot for many scientific researchers.

[0004] As a newly emerging self-assembled crystalline material in recent years (such as metal-organic framework materials, MOFs; hydrogen-bonded organic framework materials, HOFs; covalent organic framework materials, COFs), it has the advantages of high specific surface area, adjustable pore size, diverse structure, easy modification, excellent thermal stability and chemical stability, etc., and its application in the fields of gas adsorption, gas separation, sensing and catalysis has developed rapidly. At present, there is no immobilization technology for complex liquid biological product systems. In view of the shortcomings of complex biological product systems such as difficulty in storage and transportation, difficulty in preparation, and poor selectivity, the present invention creatively uses self-assembled crystalline materials with high biocompatibility to prepare solid preparations for complex liquid biological product systems.

[0005] This method designs the ligands of self-assembled crystalline materials or adds auxiliary factors to enable them to have stronger interactions with biological macromolecules, which can greatly promote their enrichment efficiency. It also has a fast solidification speed and mild synthesis conditions (such as room temperature and aqueous solution), which has a protective effect on biological agents. At the same time, it greatly reduces the cost of storage and transportation of biological agents and expands the application scenarios of biological agents. Summary of the invention

[0006] The present invention aims at the shortcomings of traditional biological preparations and innovatively utilizes self-assembled crystalline materials to prepare new solid biological preparations. Self-assembled crystalline materials can protect biological preparations from interference from the external environment, improve storage stability, and maintain the activity of biological preparations to the maximum extent. At the same time, the prepared biological preparations can be released and restored to a liquid state under the conditions of a suitable solvent, and the biological preparations after release can still maintain the original activity, and the self-assembled crystalline materials can be separated from the system with an extremely high separation rate. The biological preparations prepared by the method of the present invention have the advantages of simple preparation method, low storage cost, high stability, and fast release rate, and will not affect the activity of the biological preparations. In addition, different self-assembled crystalline materials can be used to extract and solidify specific active ingredients in the preparations, and selectively prepare them into solid biological preparations.

[0007] The biological preparation of the present invention refers to a biological product processed from effective antigen components of microorganisms and their metabolites, animal toxins, human or animal blood or tissues, etc. Biological preparations usually require cold chain transportation and low-temperature storage, are sensitive to the external environment, and are easily inactivated. The present invention creatively prepares them into composite solid biological preparations, thereby improving the stability of biological preparations.

[0008] At present, the biological preparations with the greatest application value are serum, antibodies, enzyme solutions, hormones, biological drugs, etc., and their storage and transportation costs are extremely high. Therefore, preparing them into solid preparations can enhance the stability of biological preparations and greatly reduce storage and transportation costs.

[0009] In one aspect, the present invention provides a composite solid biological agent based on self-assembled crystalline materials, comprising a biological agent and a porous framework material. The biological agent is in liquid state at room temperature and pressure, or exists in the form of a solution.

[0010] Preferably, the porous framework material encapsulates and fixes active ingredients of biological agents.

[0011] Preferably, the biological preparations of the present invention include serum, antibodies, enzyme solutions, hormones, biological drugs, etc.

[0012] Preferably, the porous framework materials include metal-organic frameworks (MOFs), hydrogen-bonded organic frameworks (HOFs) and covalent organic frameworks (COFs).

[0013] Metal-Organic Frameworks (MOFs), also known as porous coordination polymers (PSPs), are porous crystalline materials formed by metal ions or ion clusters and organic ligands through coordination bonds. This type of material has the characteristics of high specific surface area, designable structure, and controllable pores.

[0014] Hydrogen-bonded organic frameworks (HOFs) are a type of ordered framework materials that are constructed solely by organic building blocks through intermolecular hydrogen bonding. This type of material not only has the characteristics of high specific surface area, designable structure and adjustable pores, but also has the unique advantages of mild synthesis conditions, solvent processability and easy regeneration.

[0015] Covalent-Organic Frameworks (COFs) are crystalline materials with ordered porous structures formed by covalently linking light elements such as C, O, N, and B through thermodynamically controlled reversible polymerization. COFs have the advantages of high specific surface area, diverse structures, and easy functionalization. COFs have attracted widespread attention in recent years. COFs have great application potential in the immobilization of biological agents due to their high chemical stability and adjustable functions.

[0016] Generally, metal organic framework materials, hydrogen bond organic framework materials and covalent organic framework materials can fix biological agents or biological agents through four methods, namely adsorption method, cross-linking method, covalent method and in-situ encapsulation method. In addition, metal organic framework materials, hydrogen bond organic framework materials and covalent organic framework materials can be designed to have the characteristics of selective adsorption or in-situ encapsulation.

[0017] Preferably, the present invention uses an in-situ encapsulation method to fix the biological agent. First, this method is simple to operate and has a fast synthesis speed. In addition, this method has no size requirements for the biological agent to be fixed, and can fix a wide variety of biological agents.

[0018] Preferably, the porous framework material has a pore size of 0.1-10 nm, and / or has good structural tailorability and functional adjustability.

[0019] Preferably, the porous framework material can be synthesized under mild conditions in room temperature and aqueous phase.

[0020] Preferably, the metal organic framework material is ZIF-8, ZIF-90, UTSA-280, ZPF-2, or MDP-Zn.

[0021] Preferably, the hydrogen bonding framework organic material is HOF-1, HOF-2, HOF-5, HOF-10, HOF-8, SOF-1, HOF-11, HOF-TCBP, SOF-7, HOF-GS-10, HOF-GS-11.

[0022] Preferably, the covalent organic framework material is COF-1, COF-2, COF-42-B, TAPB-BTCA-COF, TZ-BTCA-COF, HZ-BTCA-COF, TPA-DMTP, TPB-DMTP, or TPT-DMTP.

[0023] Preferably, the serum is bovine serum, horse serum, rabbit serum, chicken serum, pig serum, other animal serum, synthetic serum substitute, etc.

[0024] Preferably, the bovine serum is fetal bovine serum, calf serum, newborn calf serum, or adult bovine serum.

[0025] Further preferably, the fetal bovine serum is dialyzed fetal bovine serum, natural low IgG fetal bovine serum, stem cell culture fetal bovine serum, special purpose fetal bovine serum, activated carbon / glucose treated fetal bovine serum.

[0026] Further preferably, the calf serum is fortified calf serum or iron-supplemented calf serum.

[0027] On the other hand, the present invention provides a method for preparing a composite solid biological preparation based on a self-assembled crystalline material, which is prepared by an in-situ encapsulation method, an adsorption method, a cross-linking method or an embedding method, and specifically comprises the following steps:

[0028] 1) Using porous framework materials to immobilize active ingredients of biological agents;

[0029] 2) solid-liquid separation after the reaction is completed;

[0030] 3) After drying, a composite solid biological preparation is obtained.

[0031] Preferably, step 1) is carried out under normal temperature and pressure.

[0032] Preferably, step 1) is carried out in a solvent of water or an organic solvent.

[0033] Since MOFs, HOFs and COFs materials are designable, the preparation of composite solid biological agents can select suitable MOFs, HOFs and COFs materials for encapsulation according to the characteristics of different biological agents. The ligands of MOFs, HOFs and COFs materials can also be designed to enhance their affinity for biological agents and increase the loading rate of biological agents.

[0034] The MOFs, COFs and MOFs materials involved can load biological agents under mild conditions, and the structures of MOFs, COFs and MOFs materials can ensure that biological agents are not affected by temperature, solvents and shear forces, thereby improving the stability of biological agents. Similarly, biological agents can be released under mild conditions, and the release process has no effect on the activity of biological agents.

[0035] Preferably, the selected MOFs, COFs and MOFs have high biosafety, and the ligands and / or metal ions released during the release process can also be easily removed by ultrafiltration, dialysis, centrifugation, filtration or other separation methods without causing biological toxicity.

[0036] Preferably, MOFs, COFs and MOFs materials are more sensitive to specific environments and are easier to release.

[0037] Preferably, when the porous framework material is MOFs, for biological agents that are sensitive to metal ions, a biocompatible polymer can be added to protect the biological agents, thereby further increasing the scope of applicable biological agents.

[0038] Preferably, for complex biological agent systems, the affinity of MOFs, COFs and MOFs can be improved by selecting appropriate MOFs, COFs and MOFs, or modifying the ligands of MOFs, COFs and MOFs, so that specific biological agents can be selectively immobilized, achieving the effect of selective separation.

[0039] Preferably, this method is universal, with a wide variety of MOFs, COFs and MOFs materials, strong ligand modifiability, and simple synthesis methods, which can be freely selected according to actual needs to prepare different solid biological preparations for application in the fields of cell biology, tissue engineering, biochemistry, medicine, chemical industry, etc.

[0040] More preferably, the solid biological preparation can be used for cell culture, fermentation, pharmaceutical preparation, etc.

[0041] In another aspect, the present invention provides a use of the aforementioned composite solid biological preparation, comprising the following steps:

[0042] 1) Composite solid organisms release biological agents under specific conditions;

[0043] 2) After separating the porous framework material, a liquid biological agent or a biological agent solution is obtained;

[0044] 3) The released biological agent maintains its original activity and can be used for its original purpose.

[0045] Preferably, the specific environment is a milder condition, ie, pH < 7.5; more preferably, the specific environment is an aqueous solution with a pH of 4-7.5.

[0046] Preferably, the aqueous solution includes: HEPES buffer, MES buffer, Tris-hydrochloric acid buffer, acetic acid-sodium acetate buffer, phosphate buffer, citric acid-sodium citrate buffer, boric acid-borax buffer, and PBS buffer.

[0047] Preferably, the release rate of the active ingredient of the biological agent is greater than 70%; further preferably, the release rate of the active ingredient of the biological agent is greater than 80%; more preferably, the release rate of the active ingredient of the biological agent is greater than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Schematic diagram of the preparation of bioagent@MOFs via in situ encapsulation method.

[0049] Figure 2 :Preparation of fetal bovine serum @MOFs and horse serum @MOFs. Total protein standard curve measured by Folin-phenol total protein detection kit. Detection wavelength: 500nm

[0050] Figure 3 : PXRD of fetal bovine serum@MOFs and horse serum@MOFs.

[0051] Figure 4 : In situ encapsulation method, loading rate of MOFs materials for fetal bovine serum and horse serum components.

[0052] Figure 5 : Adsorption method, loading rate of COFs for fetal bovine serum and horse serum components.

[0053] Figure 6 : Cytotoxicity experiment of MOFs materials, MCF-7 cells were cultured with horse serum.

[0054] Figure 7 : After release, the residual experiment of MOFs material is carried out to detect whether there are metal ions after release and whether there are residual materials by PXRD.

[0055] Figure 8 :Release curve of MOFs material after loading with horse serum. Release environment: citric acid-sodium citrate buffer, pH=6.

[0056] Fig. 9 : Effect of horse serum released from composite materials on cell culture.

[0057] Fig.10 : Horse serum released from the composites was used in MTT assay of cell culture.

[0058] Fig.11 : Effect of horse serum released from composite materials on cell passaging.

[0059] Fig.12 : The protective property of the composite material against horse serum, and whether the horse serum released after treatment still has cell culture activity. DETAILED DESCRIPTION

[0060] Unless otherwise specified in the context of this application, the technical terms and abbreviations used in this application have the conventional meanings known to those skilled in the art; unless otherwise specified, the raw material compounds used in the following examples are commercially available.

[0061] According to the present invention, the synthesis of metal organic framework materials (MOFs), the synthesis of biological agents @MOFs, the characterization tests of various properties, and the research specific implementation methods of the activity test of biological agents are as follows. On the contrary, the following examples are only used to further explain and invent the present invention, and should not be regarded as limiting the scope of the present invention, which will only be limited by the claims.

[0062] Embodiment 1:

[0063] The preparation of biopharmaceutical@MOFs loaded by in-situ encapsulation method is as follows:

[0064] Preparation of solid preparation of biological agent @UTSA-280: Weigh 32 mg of sodium squarate trihydrate and dissolve it in 1 ml of deionized water. Weigh 236 mg of calcium nitrate tetrahydrate and dissolve it in 1 ml of deionized water. Dilute the biological agent solution by an appropriate multiple, take 2 ml into a test tube, and add 1 ml of the prepared sodium squarate solution and calcium nitrate solution respectively. Let it stand at room temperature for 1 hour to obtain a white precipitate. Collect the white precipitate by centrifugation at 4000r, wash it with water 3 times, freeze-dry it, and put it in a 4°C refrigerator for use. The biological agents here include fetal bovine serum and horse serum.

[0065] Preparation of solid preparation of biological agent @MDP-Zn: Weigh 60 mg of sodium dicyanamide and 31 mg of pyrazine and dissolve them in 1 ml of deionized water. Weigh 75 mg of zinc nitrate hexahydrate and dissolve them in 1 ml of deionized water. Dilute the biological agent solution by an appropriate multiple, take 2 ml into a test tube, and add 1 ml of the prepared sodium dicyanamide / pyrazine solution and zinc nitrate solution respectively. Let it stand at room temperature for 1 hour to obtain a white precipitate. Collect the white precipitate by centrifugation at 4000r, wash it with water 3 times, freeze-dry it, and put it in a 4°C refrigerator for use. The biological agents here include fetal bovine serum and horse serum.

[0066] Preparation of solid preparation of biological agent @ZPF-2: Weigh 45 mg of F-hydroxypyrimidine and dissolve it in 1 ml of deionized water. Weigh 38 mg of zinc nitrate hexahydrate and dissolve it in 1 ml of deionized water. Dilute the biological agent solution by an appropriate multiple, take 2 ml into a test tube, and add 1 ml of the prepared F-hydroxypyrimidine solution and zinc nitrate solution respectively. Let it stand at room temperature for 1 hour to obtain a white precipitate. Collect the white precipitate by centrifugation at 4000r, wash it with water 3 times, freeze-dry it, and put it in a 4°C refrigerator for use. The biological agents here include fetal bovine serum and horse serum.

[0067] The formation of MOFs material structure was proved by PXRD. Figure 3 .

[0068] The calculation of the loading rate of MOFs materials on biological agents is carried out in the following specific steps:

[0069] The Folin-phenol total protein assay was used to quantify the total protein in serum components before and after encapsulation.

[0070] 1) In situ encapsulation method: Take 1 ml of sample solution (containing about 20-250 μg of peptide or protein), add 5 ml of reagent A and mix well, let it stand at room temperature for 10 minutes, add 0.5 ml of reagent B (Folin-phenol), shake well immediately, and keep it at room temperature for 30 minutes; then compare the color at 500 nm, and use 1 ml of water instead of the sample as a blank control.

[0071] After determination, the Figure 2 The concentration of unknown samples can be found in the standard curve shown. Figure 4 To calculate the encapsulation efficiency (LE%) and loading (g / g), the calculation formula is as follows:

[0072]

[0073]

[0074] Embodiment 2:

[0075] The adsorption method was used to prepare biopharmaceuticals@COFs. The specific implementation steps are as follows:

[0076] TPA-DMTP: Weigh the ligand tris(4-aminophenyl)amine (TPA 11.6mg) and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP 11.7mg) separately and add them into a thick-walled heat-resistant glass tube, then add 0.2mL o-dichlorobenzene, 0.8mL n-butanol and 0.2mL, 3M acetic acid, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame. Put the sealed glass tube into a 120℃ oven for reaction for 3 days.

[0077] TPB-DMTP: Weigh the ligand 1,3,5-tri(4-nitrophenyl)benzene (TPB 14.1mg) and 2,5-dimethoxybenzene-1,4-dicarboxaldehyde (DMTP 11.7mg) separately and add them into a thick-walled heat-resistant glass tube, then add 0.6mL o-dichlorobenzene, 0.6mL n-butanol and 0.25mL, 3M acetic acid, then quickly freeze in liquid nitrogen, evacuate, and then seal the tube with a hydrogen-oxygen machine flame. Put the sealed glass tube into a 120℃ oven for reaction for 3 days.

[0078] Solvent Activation of Covalent Organic Frameworks (COFs)

[0079] The obtained solid was washed with anhydrous tetrahydrofuran several times until the supernatant was colorless, and then Soxhlet extraction was performed with anhydrous tetrahydrofuran for 24 hours. The obtained solid was placed in a vacuum drying oven at 60° C. for activation overnight, and then the product was stored in a glove box for later use.

[0080] Preparation of biomolecules @COFs by adsorption method: 10 mg of COFs material was weighed, 5 mL of fetal bovine serum and horse serum were added, and the mixture was shaken at 37 °C. The supernatant was taken at regular intervals, and the amount of serum protein contained in the supernatant was quantified using the Folin-phenol total protein quantification method to calculate the amount of serum protein adsorbed by the material. The final adsorption amount Figure 5 shown.

[0081] Embodiment 3:

[0082] The cytotoxicity of the three MOFs was tested, and the specific implementation steps are as follows:

[0083] According to the concentration of the dissolved and released material, take a 1 mg / ml material solution and add 10% of the volume of the cell culture medium. Set the normal serum cultured cells as the control group and the basal medium cultured cells as the blank group. Observe the growth state of the cells for five days and make an MTT test to determine the adhesion. Figure 6 Cell growth curves are shown.

[0084] Embodiment 4:

[0085] After the release of bioagent@MOFs material, the residue of MOFs material is tested. The specific implementation steps are as follows:

[0086] Take 10 mg of biological preparation @MOFs material respectively, add 10 ml of citric acid-sodium citrate buffer (pH=6), place for 30 minutes to dissolve, use 3000kD ultrafiltration tube to ultrafilter, use 3000kD dialysis bag to dialyze at 4℃ for 24 hours, ultrafilter and concentrate again, freeze-dry, measure PXRD and EDX, PXRD shows no peak diffraction of MOFs material, EDX shows no metal ion residue, see attached Figure 7 .

[0087] Embodiment 5:

[0088] Release curves of three serum@MOFs materials. The specific implementation steps are as follows:

[0089] Take 2 mg of serum@UTSA-280, serum@MDP-Zn, and serum@ZPF-2 in a test tube, add 4 ml of citric acid-sodium citrate buffer (pH=6), and regularly take points to detect the serum component content in the supernatant. The method used is the Folin-phenol total protein detection method. The release curve of serum@MOFs is shown in the attached Figure 8 .

[0090] Embodiment 6:

[0091] The effect of the released serum on cell culture is as follows:

[0092] Prepare fetal bovine serum complete medium: take 400 μl of double antibody, 4 ml of FBS, 35.6 ml of DMEM basal medium, mix to prepare fetal bovine serum complete medium. Prepare horse serum complete medium: take 400 μl of double antibody, 4 ml of horse serum, 35.6 ml of DMEM basal medium, mix to prepare horse serum complete medium. Prepare MOFs release serum medium: take 400 μl of double antibody, 4 ml of release serum, 35.6 ml of DMEM basal medium, mix to prepare release serum complete medium. After MCF-7 cells are cultured to 70-80% density, use trypsin to digest for 3 minutes, add complete medium to terminate digestion, centrifuge and precipitate, add complete medium, blow to disperse the cells into cell suspension, count the cells, take an appropriate volume of cell suspension into the culture bottle, add 4 ml of complete medium, UTSA-280 release serum complete medium, MDP-Zn release serum complete medium, ZPF-2 release serum complete medium, culture the cells, and observe the cell growth status under a microscope (see attached) Fig. 9 , and MTT assay was performed as shown in the attached Fig.10 .

[0093] Embodiment 6:

[0094] The effect of released serum on cell passage is Fig.11 , the specific implementation steps are as follows:

[0095] Suction 25cm 2Add 0.5 ml of digestion solution (trypsin) to the culture medium in the culture flask, gently shake the culture flask, digest at 37℃ for 2-3 minutes, and then place the culture flask under a microscope for observation. When the cytoplasm shrinks and the intercellular space increases, immediately add 2 ml of complete culture medium to terminate the digestion, and then use a pipette to repeatedly blow the cells on the flask wall to form a cell suspension after the cells detach from the flask wall. Count the cell suspension, and then according to the subculture concentration of 5×10 5 The cells were cultured using horse serum and the three types of serum after release, and the cell growth status was observed under a microscope.

[0096] Embodiment 7:

[0097] The protective effect of MOFs materials on biological agents Fig.12 , the specific implementation steps are as follows:

[0098] Thermal stability test of horse serum@MOFs material.

[0099] Horse serum@MOFs was placed at 60°C for 10 minutes, and then its activity was characterized according to standard methods. The results showed that after high temperature treatment, horse serum@MOFs can still promote cell growth after release. However, horse serum in liquid state is inactivated and denatured, and loses its activity.

[0100] Solvent stability test of horse serum@MOFs material.

[0101] The horse serum@MOFs material was soaked in acetone for 1 hour, and then its activity was characterized according to the above method. The results showed that after solvent treatment, the activity of the horse serum@MOFs material could still be maintained and it could still promote cell growth, while the horse serum in liquid state was inactivated and denatured and lost its activity.

[0102] Storage stability test of horse serum@MOFs material.

[0103] The horse serum@MOFs material was placed at room temperature for 10 days, and then its activity was characterized according to the above method. The results showed that after 10 days, the horse serum@MOFs material could still maintain its activity, while the horse serum in liquid state was inactivated and denatured, losing its activity.

Claims

1. A composite solid biological preparation based on self-assembled crystalline materials, characterized in that: The composite solid biological agent includes a biological agent and a porous framework material, wherein the porous framework material is a metal organic framework material; the biological agent is serum, and the biological agent is liquid at room temperature and pressure; the porous framework material uses an in-situ encapsulation method to fix the biological agent; and the metal organic framework material is UTSA-280.

2. The composite solid biological preparation according to claim 1, characterized in that: The serum is any one or more of bovine serum, horse serum, rabbit serum, chicken serum, pig serum, other animal serum, and synthetic serum substitutes.

3. The composite solid biological preparation according to claim 2, characterized in that: The bovine serum is any one or more of fetal bovine serum, calf serum, newborn bovine serum and adult bovine serum.

4. The composite solid biological preparation according to claim 3, characterized in that: The bovine serum is any one or more of dialyzed fetal bovine serum, natural low IgG fetal bovine serum, stem cell culture fetal bovine serum, special purpose fetal bovine serum, activated carbon / glucose treated fetal bovine serum, enhanced calf serum, and iron-supplemented calf serum.

5. The method for preparing the composite solid biological preparation according to any one of claims 1 to 4, characterized in that: The preparation is carried out by an in-situ encapsulation method, which specifically comprises the following steps: 1) Porous framework materials to fix active ingredients of biological agents; 2) solid-liquid separation after the reaction is completed; 3) After drying, a composite solid biological preparation is obtained.

6. The method for preparing the composite solid biological preparation according to claim 5, characterized in that: Step 1) is carried out at room temperature and pressure with water as the solvent.

7. Use of the composite solid biological preparation according to any one of claims 1 to 4 in the preparation of a preparation for cell culture, comprising the following steps: 1) The composite solid organism releases the biological agent under specific conditions; 2) After separating the porous framework material, a liquid biological agent is obtained; 3) The released biological agent maintains its original activity; The specific environment of step 1) is a solution condition with a pH value less than 7.

5.

8. The method of use according to claim 7, characterized in that: The solution is any one of HEPES buffer, MES buffer, Tris-hydrochloric acid buffer, acetic acid-sodium acetate buffer, phosphate buffer, citric acid-sodium citrate buffer, and boric acid-borax buffer.

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