Preparation of an oligonucleotide chain functionalized sandwich-like composite material and its application in a chemiluminescence sensor

Abstract

This invention discloses the preparation of an oligonucleotide chain-functionalized sandwich-like composite material and its application in a chemiluminescence sensor. The main technical features are: the preparation of Au / ZIF-8 and β-cyclodextrin / iron tetroxide@carbon nanotube composite materials, and modification of the surfaces of both with thrombin aptamers and their complementary chains. The two materials are then combined through base pairing to obtain a functionalized sandwich-like composite material with high specificity for recognizing and separating thrombin molecules. The preparation process is simple, the conditions are easy to control, and the production cost is low. This invention also provides a novel method for detecting thrombin. Applying the prepared oligonucleotide chain-functionalized sandwich-like composite material to a chemiluminescence sensor for thrombin detection demonstrates advantages such as high sensitivity, good selectivity, convenient operation, and simple instrumentation. Furthermore, it has been successfully used for the detection of thrombin in human serum samples, exhibiting high accuracy and precision, thus providing possibilities for application to the detection of more practical samples and possessing significant biological significance for human health.

Description

Technical Field

[0001] This invention relates to the preparation of a functionalized sandwich-like composite material and its application in a chemiluminescence sensor, belonging to the field of photochemical sensor technology. Specifically, it relates to the preparation of aptamer-functionalized Au / ZIF-8 and aptamer-complementary chain-functionalized β-cyclodextrin / iron tetroxide@carbon nanotube composite materials, and utilizes the complementary base pairing effect to prepare an aptamer-functionalized sandwich-like composite material and apply it to the detection of thrombin using a chemiluminescence sensor. Background Technology

[0002] Carbon nanotubes (CNTs) are three-dimensional (3D) hollow tubular carbon materials composed of carbon atoms. Based on the number of wall layers, they can be classified into single-walled, double-walled, and multi-walled carbon nanotubes. They possess advantages such as small size, large specific surface area, and layered hollow structure. As a relatively new type of carbon material, carbon nanotubes have wide applications in medicine, sensing, and energy storage. Furthermore, compared to conventional adsorbent materials such as activated carbon, the surface of carbon nanotubes is easier to modify. Magnetization technology, as a rapid and effective separation technique for magnetic materials, has gradually attracted the attention of many researchers and is used in medicine, analytical chemistry, mining, and environmental technologies. The main advantage of this technology is its ability to process large numbers of samples in a short time without generating pollution. Magnetizing double-walled carbon nanotubes combines the high adsorption capacity of carbon nanotubes with the easy separation properties of magnetic materials, making it better suited for the efficient separation of target analytes.

[0003] Thrombin (THR) is a serine protease produced in vascular or non-vascular tissues, playing a crucial role in blood clotting. Thrombin converts fibrinogen into fibrin, significantly promoting the occurrence and development of arteriosclerosis. It is commonly used to control capillary and venous bleeding, or as an adhesive fixative in skin and tissue grafts. Imbalances in thrombin levels can lead to bleeding or thrombotic diseases. Thrombin also serves as a biomarker for the diagnosis and treatment of cardiovascular diseases, tumor cell growth, inflammation, and coagulation disorders. Therefore, sensitive detection of thrombin is particularly important for human health. Currently, commonly used methods for thrombin detection include fluorescence methods, colorimetric methods, electrochemical methods, and electrochemiluminescence methods. However, these methods each have their own limitations, such as poor reproducibility, complex operation, and narrow linear range. Therefore, there is an urgent need to establish a highly sensitive and selective method for thrombin detection.

[0004] Flow injection-chemiluminescence (CI) combines the advantages of automated sample injection and ease of operation of CI with the high sensitivity, simple instrumentation, wide linear range, fast analysis speed, and pollution-free nature of chemiluminescence analysis, making it widely used in analytical fields. However, chemiluminescence methods have a significant drawback—poor selectivity—because various coexisting substances can easily cause changes in chemiluminescence intensity. This drawback can be overcome by introducing specific recognition materials, such as molecularly imprinted materials, antigen-antibody materials, and aptamer materials.

[0005] In this patent, Au / ZIF-8 and β-cyclodextrin / ferric oxide@carbon nanotube composite materials were prepared. Thrombin aptamers and their complementary chains were modified onto the surfaces of both materials, respectively. The two materials were then bonded together using the base complementarity principle to form a composite material, resulting in a sandwich-like composite material that is easy to separate, exhibits excellent immobilization properties, and possesses high specificity for recognition. This functionalized three-dimensional graphene composite material was combined with flow injection-chemiluminescence technology to construct a highly sensitive and selective chemiluminescent sensor for thrombin detection, thus inventing a simple, highly sensitive, and selective new method for thrombin detection. Summary of the Invention

[0006] One of the objectives of this invention is to provide a method for preparing oligonucleotide chain functionalized sandwich-like composite materials. The method mainly involves preparing Au / ZIF-8 and β-cyclodextrin / iron tetroxide@carbon nanotube composite materials, and modifying the surfaces of both materials with thrombin aptamers and complementary chains of the aptamers. The two materials are then linked together through base complementary pairing to obtain oligonucleotide chain functionalized sandwich-like composite materials with high specific recognition ability for thrombin molecules.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] (1) Preparation of aptamer-functionalized Au / ZIF-8: Au was fixed on the surface of ZIF-8 by the citric acid reduction of chloroauric acid method. 0.15~0.20 g of ZIF-8 was weighed into 25 mL of anhydrous ethanol, and 200~500 μL of 0.1 mol / L chloroauric acid solution was added. After magnetic stirring for 10~15 min, 0.01~0.05 g of sodium citrate was added, and magnetic stirring was continued for 10~15 min. The product was centrifuged at 10000 r / min for 6 min. After centrifugation, the product was vacuum dried at 70˚C. The dried product was placed in a crucible and calcined in a muffle furnace at 500˚C for 5~6 h to obtain Au / ZIF-8. 0.01~0.05 g of Au / ZIF-8 was weighed and added into 5 mL of 0.02 mol / L phosphate buffer (pH=7.4), and then 10~30 nmol The thrombin aptamer was added to the centrifuge tube, the centrifuge tube was shaken for 15-25 min, and then incubated at room temperature for 24-36 h; finally, it was centrifuged at 10000 r / min for 6 min, the supernatant was removed, and the aptamer-functionalized Au / ZIF-8 was obtained.

[0009] (2) Preparation of β-cyclodextrin / Fe3O4@carbon nanotube composite material functionalized with aptamer complementary chain: β-cyclodextrin / Fe3O4 was prepared by crosslinking β-cyclodextrin with epichlorohydrin; then, the surface of β-cyclodextrin / Fe3O4 was aminated with (3-aminopropyl)triethoxysilane; then, the carboxyl groups of carbon nanotubes were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and β-cyclodextrin / Fe3O4@carbon nanotubes were obtained by amide reaction; 0.05~0.15 g of β-cyclodextrin / Fe3O4@carbon nanotubes were weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH=7.4), and then 10~30 nmol of thrombin aptamer complementary chain was added to the centrifuge tube; the centrifuge tube was shaken for 15~25 min, and then incubated at 20˚C for 36 hours. h; Finally, centrifuge at 10000 r / min for 6 min, remove the supernatant, and obtain the aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube composite material;

[0010] (3) Preparation of oligonucleotide chain functionalized sandwich composite material: Transfer 0.2~0.5 mL of 2 mg / mL aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube solution into a 50 mL volumetric flask, add 1.0~6.0 mg of aptamer functionalized Au / ZIF-8 to the volumetric flask, and make up the volume with 0.02 mol / L phosphate buffer (pH=7.4); shake at room temperature for 1~3 h, and store in a refrigerator at 6℃ for later use.

[0011] Another objective of this invention is to apply the oligonucleotide chain-functionalized sandwich-like composite material to a chemiluminescent sensor for the detection of thrombin. When thrombin is present, it specifically recognizes and binds to its aptamer, releasing Au / ZIF-8. The released Au / ZIF-8 catalyzes the luminol-hydrogen peroxide chemiluminescence system, causing a change in chemiluminescence intensity, thus enabling the detection of thrombin. This chemiluminescent sensor for thrombin detection is characterized by high sensitivity, good selectivity, convenient operation, and simple instrumentation. In the construction of this chemiluminescent sensor, the immobilization performance of aptamer-functionalized β-cyclodextrin / iron tetroxide@carbon nanotubes on aptamer-functionalized Au / ZIF-8 was studied, chemiluminescence conditions were optimized, working curves were plotted, and anti-interference ability was investigated. Finally, it was applied to the detection of thrombin in human serum samples.

[0012] The advantages and effects of this invention are:

[0013] (1) In this invention, aptamer-functionalized Au / ZIF-8 and aptamer-complementary chain-functionalized β-cyclodextrin / Fe3O4@carbon nanotubes were prepared and linked together by base complementary pairing to form a sandwich structure. Au / ZIF-8 and β-cyclodextrin / Fe3O4@carbon nanotubes have advantages such as large specific surface area, rich functional groups and ionic bonds, which provide a large number of interaction sites for the aptamer and make it possible to prepare sandwich-shaped composite materials. The saturated fixation amount of aptamer-functionalized Au / ZIF-8 by aptamer-complementary chain-functionalized β-cyclodextrin / Fe3O4@carbon nanotubes is 2.5 mg / mg.

[0014] (2) The present invention prepares a novel oligonucleotide chain functionalized sandwich composite material. The preparation process is simple and the conditions are easy to control, which can achieve accurate and rapid separation of thrombin.

[0015] (3) The novel oligonucleotide chain functionalized sandwich composite material prepared in this invention is applied to the detection of thrombin by a chemiluminescent sensor. The sensor exhibits a wide linear range and a low detection limit for thrombin detection. It also shows high accuracy and precision in the detection of thrombin in human serum samples, which has important biological significance in disease diagnosis and human health. Implementation Example 1

[0016] (1) Preparation of aptamer-functionalized Au / ZIF-8: Au was immobilized on the surface of ZIF-8 by the citrate reduction of chloroauric acid. 0.15 g of ZIF-8 was weighed into 25 mL of anhydrous ethanol, and 200 μL of 0.1 mol / L chloroauric acid solution was added. After stirring magnetically for 10 min, 0.01 g of sodium citrate was added, and stirring was continued for another 10 min. The product was centrifuged at 10000 r / min for 6 min. After centrifugation, the product was vacuum dried at 70˚C. The dried product was placed in a crucible and calcined in a muffle furnace at 500˚C for 5 h to obtain Au / ZIF-8. 0.01 g of Au / ZIF-8 was weighed and added into 5 mL of 0.02 mol / L phosphate buffer (pH=7.4). Then, 10 nmol of thrombin aptamer was added to the centrifuge tube. The centrifuge tube was shaken for 15 min and then incubated at room temperature for 24 hours. h; Finally, centrifuge at 10000 r / min for 6 min, remove the supernatant, and obtain aptamer-functionalized Au / ZIF-8;

[0017] (2) Preparation of β-cyclodextrin / Fe3O4@carbon nanotube composite material functionalized with aptamer complementary chain: β-cyclodextrin / Fe3O4 was prepared by crosslinking β-cyclodextrin with epichlorohydrin; then, the surface of β-cyclodextrin / Fe3O4 was aminated with (3-aminopropyl)triethoxysilane; then, the carboxyl groups of carbon nanotubes were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and β-cyclodextrin / Fe3O4@carbon nanotubes were obtained by amide reaction; 0.05 g of β-cyclodextrin / Fe3O4@carbon nanotubes was weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH=7.4), and then 10 nmol of thrombin aptamer complementary chain was added to the centrifuge tube; the centrifuge tube was shaken for 15 min, and then incubated at 20˚C for 36 h; finally, it was incubated at 10000 Centrifuge at 6 r / min for 6 min, remove supernatant to obtain aptamer complementary chain functionalized β-cyclodextrin / iron tetroxide@carbon nanotube composite material;

[0018] (3) Preparation of oligonucleotide chain functionalized sandwich composite material: Transfer 0.2 mL of 2 mg / mL aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube solution into a 50 mL volumetric flask, add 1.0 mg of aptamer functionalized Au / ZIF-8 to the volumetric flask, and make up to volume with 0.02 mol / L phosphate buffer (pH=7.4); shake at room temperature for 1 h, and store in a refrigerator at 6℃ for later use. Example 2

[0019] (1) Preparation of aptamer-functionalized Au / ZIF-8: Au was immobilized on the surface of ZIF-8 by the citrate reduction of chloroauric acid. 0.17 g of ZIF-8 was weighed into 25 mL of anhydrous ethanol, and 350 μL of 0.1 mol / L chloroauric acid solution was added. After magnetic stirring for 13 min, 0.03 g of sodium citrate was added, and magnetic stirring was continued for another 13 min. The product was centrifuged at 10000 r / min for 6 min. After centrifugation, the product was vacuum dried at 70˚C. The dried product was placed in a crucible and calcined in a muffle furnace at 500˚C for 5.5 h to obtain Au / ZIF-8. 0.03 g of Au / ZIF-8 was weighed and added into 5 mL of 0.02 mol / L phosphate buffer (pH=7.4). Then, 20 nmol of thrombin aptamer was added to the centrifuge tube. The centrifuge tube was shaken for 20 min and then incubated at room temperature for 30 min. h; Finally, centrifuge at 10000 r / min for 6 min, remove the supernatant, and obtain aptamer-functionalized Au / ZIF-8;

[0020] (2) Preparation of β-cyclodextrin / Fe3O4@carbon nanotube composite material functionalized with aptamer complementary chain: β-cyclodextrin / Fe3O4 was prepared by crosslinking β-cyclodextrin with epichlorohydrin; then, the surface of β-cyclodextrin / Fe3O4 was aminated with (3-aminopropyl)triethoxysilane; then, the carboxyl groups of carbon nanotubes were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and β-cyclodextrin / Fe3O4@carbon nanotubes were obtained by amide reaction; 0.02 g of β-cyclodextrin / Fe3O4@carbon nanotubes was weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH=7.4), and then 20 nmol of thrombin aptamer complementary chain was added to the centrifuge tube; the centrifuge tube was shaken for 20 min, and then incubated at 20˚C for 36 h; finally, it was incubated at 10000 Centrifuge at 6 r / min for 6 min, remove supernatant to obtain aptamer complementary chain functionalized β-cyclodextrin / iron tetroxide@carbon nanotube composite material;

[0021] (3) Preparation of oligonucleotide chain functionalized sandwich composite material: 0.3 mL of 2 mg / mL aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube solution was transferred into a 50 mL volumetric flask, and 3.5 mg of aptamer functionalized Au / ZIF-8 was added to the volumetric flask. The solution was then diluted with 0.02 mol / L phosphate buffer (pH=7.4). The solution was shaken at room temperature for 2 h and stored in a refrigerator at 6℃ for later use. Example 3

[0022] (1) Preparation of aptamer-functionalized Au / ZIF-8: Au was immobilized on the surface of ZIF-8 by the citrate reduction of chloroauric acid. 0.20 g of ZIF-8 was weighed into 25 mL of anhydrous ethanol, and 500 μL of 0.1 mol / L chloroauric acid solution was added. After magnetic stirring for 15 min, 0.05 g of sodium citrate was added, and magnetic stirring was continued for another 15 min. The product was centrifuged at 10000 r / min for 6 min. After centrifugation, the product was vacuum dried at 70˚C. The dried product was placed in a crucible and calcined in a muffle furnace at 500˚C for 6 h to obtain Au / ZIF-8. 0.05 g of Au / ZIF-8 was weighed and added into 5 mL of 0.02 mol / L phosphate buffer (pH=7.4). Then, 30 nmol of thrombin aptamer was added to the centrifuge tube. The centrifuge tube was shaken for 25 min and then incubated at room temperature for 36 hours. h; Finally, centrifuge at 10000 r / min for 6 min, remove the supernatant, and obtain aptamer-functionalized Au / ZIF-8;

[0023] (2) Preparation of β-cyclodextrin / Fe3O4@carbon nanotube composite material functionalized with aptamer complementary chain: β-cyclodextrin / Fe3O4 was prepared by crosslinking β-cyclodextrin with epichlorohydrin; then, the surface of β-cyclodextrin / Fe3O4 was aminated with (3-aminopropyl)triethoxysilane; then, the carboxyl groups of carbon nanotubes were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and β-cyclodextrin / Fe3O4@carbon nanotubes were obtained by amide reaction; 0.15 g of β-cyclodextrin / Fe3O4@carbon nanotubes were weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH=7.4), and then 30 nmol of thrombin aptamer complementary chain was added to the centrifuge tube; the centrifuge tube was shaken for 25 min, and then incubated at 20˚C for 36 h; finally, it was incubated at 10000 Centrifuge at 6 r / min for 6 min, remove supernatant to obtain aptamer complementary chain functionalized β-cyclodextrin / iron tetroxide@carbon nanotube composite material;

[0024] (3) Preparation of oligonucleotide chain functionalized sandwich composite material: Transfer 0.5 mL of 2 mg / mL aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube solution into a 50 mL volumetric flask, add 6.0 mg of aptamer functionalized Au / ZIF-8 to the volumetric flask, and make up to volume with 0.02 mol / L phosphate buffer (pH=7.4); shake at room temperature for 3 h, and store in a refrigerator at 6℃ for later use. Example 4

[0025] A method for using oligonucleotide chain-functionalized sandwich-like composite materials in a chemiluminescence sensor to detect thrombin: This composite material is used in conjunction with flow injection-chemiluminescence technology. Different concentrations of thrombin induce varying degrees of change in chemiluminescence intensity, allowing for quantitative detection of thrombin. The construction process of this chemiluminescence sensor is as follows:

[0026] (1) Study on the immobilization performance of aptamer-complementary chain functionalized β-cyclodextrin / iron oxide@carbon nanotubes: Equal amounts of aptamer-complementary chain functionalized β-cyclodextrin / iron oxide@carbon nanotubes were accurately transferred into 50 mL colorimetric tubes, and appropriate amounts of aptamer-complementary chain functionalized Au / ZIF-8 solution of known concentration were added. The chemiluminescence intensity was measured by flow injection-chemiluminescence analyzer. Based on the concentration of aptamer-complementary chain functionalized Au / ZIF-8 solution when the chemiluminescence intensity suddenly changes, the saturated immobilization amount of aptamer-complementary chain functionalized β-cyclodextrin / iron oxide@carbon nanotubes on aptamer-complementary chain functionalized Au / ZIF-8 can be calculated. Q ;

[0027] (2) Optimization of chemiluminescence conditions: By controlling a single variable, the pump speed, concentration of luminol, hydrogen peroxide and sodium hydroxide were optimized. The optimal pump speed or concentration was obtained based on the pump speed or concentration at the point of highest chemiluminescence intensity.

[0028] (3) Plotting the working curve: Prepare a series of standard concentrations of thrombin solutions, add the same mass of oligonucleotide chain functionalized sandwich composite material to each solution, and under the optimal experimental conditions (i.e. the optimal pump speed, the optimal concentration of luminol, hydrogen peroxide and sodium hydroxide solution obtained from the experiment), measure the chemiluminescence intensity of the series of standard concentrations of thrombin, and plot the working curve with the concentration of thrombin as the abscissa and the chemiluminescence intensity as the ordinate.

[0029] (4) Anti-interference performance study: Na was studied respectively. + The interference of bovine serum albumin, dopamine, tryptophan, glucose, lysozyme and other substances on thrombin detection was investigated. Under optimal experimental conditions, the chemiluminescence intensity of thrombin standard solution in the presence of different interfering substances was measured to study the anti-interference ability of the sensor.

[0030] (5) Actual sample detection: Under optimal experimental conditions, the thrombin content in human serum samples was detected, and a spiked recovery experiment was performed to determine the thrombin content.

[0031] Oligonucleotide chain-functionalized sandwich-like composite materials were applied to a chemiluminescence sensor for thrombin detection. The saturated fixation amount of aptamer-complementary chain-functionalized β-cyclodextrin / iron tetroxide@carbon nanotubes for aptamer-functionalized Au / ZIF-8 was 2.5 mg / mg. The optimal experimental conditions were: a main pump rate of 30 r / min, a secondary pump rate of 40 r / min, 0.01 mol / L NaOH, 0.25 mol / L H2O2, and 7.0 × 10⁻⁶ ppm. -4 mol / L luminol; the relationship between chemiluminescence intensity and thrombin concentration was measured as Δ I =15439.18+1141.40lg c ( R =0.9483), with a linear range of 1.5×10. -13 ~2.5×10 -10 mol / L, detection limit is 3.8×10 -15 The concentration was measured to be mol / L; the detection method also showed strong anti-interference ability; the recovery rate of serum sample detection was between 98.4% and 102.2%, and the relative standard deviation was less than 2.6%, indicating that the method has high accuracy and precision in detecting thrombin, which provides the possibility for application to more practical sample detection.

Claims

1. A method for preparing a functionalized sandwich-like composite material, characterized in that... This method has the following process steps: (1) Preparation of aptamer-functionalized Au / ZIF-8: Au was fixed on the surface of ZIF-8 by the citric acid reduction of chloroauric acid. 0.15-0.20 g of ZIF-8 was weighed into 25 mL of anhydrous ethanol, and 200-500 μL of 0.1 mol / L chloroauric acid solution was added. After magnetic stirring for 10-15 min, 0.01-0.05 g of sodium citrate was added, and magnetic stirring was continued for another 10-15 min. The product was centrifuged at 10000 r / min for 6 min. After centrifugation, the product was vacuum dried at 70 °C. The dried product was placed in a crucible and calcined in a muffle furnace at 500 °C for 5-6 h to obtain Au / ZIF-8. 0.01-0.05 g of the product was weighed into the ZIF-8 solution. Au / ZIF-8 was added to a centrifuge tube containing 5 mL of 0.02 mol / L phosphate buffer (pH 7.4), and then 10–30 nmol of thrombin aptamer was added to the tube. The tube was shaken for 15–25 min and then incubated at room temperature for 24–36 h. Finally, the tube was centrifuged at 10,000 rpm for 6 min, and the supernatant was removed to obtain aptamer-functionalized Au / ZIF-8. (2) Preparation of aptamer complementary chain functionalized β-cyclodextrin / ferric oxide@carbon nanotube composite material: β-cyclodextrin / ferric oxide was prepared by crosslinking β-cyclodextrin with epichlorohydrin; subsequently, the surface of β-cyclodextrin / ferric oxide was aminated with (3-aminopropyl)triethoxysilane; then, the carboxyl groups of carbon nanotubes were activated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, and β-cyclodextrin / ferric oxide@carbon nanotubes were obtained by amide reaction; 0.05-0.15 g of β-cyclodextrin / ferric oxide@carbon nanotubes was weighed and added to a solution containing 5 mL of... In a centrifuge tube containing 0.02 mol / L phosphate buffer (pH 7.4), 10–30 nmol of thrombin aptamer complementary strand was added. The centrifuge tube was shaken for 15–25 min, then incubated at 20 °C for 36 h. Finally, the tube was centrifuged at 10,000 r / min for 6 min, and the supernatant was removed to obtain the aptamer complementary strand functionalized β-cyclodextrin / iron tetroxide@carbon nanotube composite material. (3) Preparation of oligonucleotide chain functionalized sandwich composite material: Transfer 0.2-0.5 mL of 2 mg / mL aptamer complementary chain functionalized β-cyclodextrin / iron tetraoxide@carbon nanotube solution into a 50 mL volumetric flask, add 0.1-6.0 mg of aptamer functionalized Au / ZIF-8 to the volumetric flask, and make up the volume with 0.02 mol / L pH=7.4 phosphate buffer; shake at room temperature for 1-3 h, and store in a refrigerator at 6℃ for later use.