A method for rapid adsorption of biomolecules onto cationic covalent organic frameworks using electrostatic interactions

Abstract

The present invention aims at the shortcomings of traditional physical adsorption immobilized enzymes, and provides a method for realizing rapid adsorption of biomolecules to cationic covalent organic frameworks by electrostatic interaction, which belongs to the technical field of immobilized enzymes. With the help of electrostatic action, the present invention realizes the surface adsorption of large-sized biomolecules on small-pore COFs and is not easy to fall off. First, a solvothermal method is used to prepare a cationic covalent organic framework as a carrier material, and then biomolecules with a low isoelectric point are selected, and the preparation of a biomolecule-COFs biocatalytic system is realized by an impregnation method. On the one hand, COFs can prevent biomolecules from being inactivated under high temperature or acid-base conditions to a certain extent, and on the other hand, they can be recycled after being combined with COFs. After the biomolecules are inactivated, some surfactants or high-concentration salt solutions can be used to elute the enzymes on the surface of COFs to realize the reuse of the carrier material.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and in particular relates to a method for realizing rapid adsorption of biomolecules onto a cationic covalent organic framework by utilizing electrostatic interaction. Background Art

[0002] Enzymes are non-toxic, environmentally friendly biomacromolecule catalysts with excellent properties such as high efficiency, regioselectivity and stereoselectivity, and can achieve high-speed conversion of complex biological reactions. Usually, enzymatic reactions need to be carried out under relatively mild conditions (such as room temperature, normal pressure and near physiological pH) to show high catalytic activity, and free enzymes after use are difficult to recover, which greatly limits the application of enzymes. However, the emergence of immobilized enzyme technology has greatly reversed this situation and improved the instability and reusability of enzymes.

[0003] Traditional physical adsorption methods for enzyme immobilization rely more on intermolecular van der Waals forces, π-π interactions, and hydrogen bonds, which not only require the enzyme size to match the pore size of the carrier material, but also cause the enzyme to leak easily. This greatly limits the choice of immobilized carriers, and it is difficult to reuse the carrier material because the enzyme cannot be completely eluted after entering the carrier pores.

[0004] Covalent organic frameworks (COFs) are porous crystalline polymers composed of organic units or molecules through covalent bonds. Due to their high specific area, high chemical stability, high porosity, and adjustable pores, they are very suitable for applications in catalysis, energy storage, separation, sensors, and other fields. In fact, due to their adjustable properties, they have become ideal host materials for improving enzyme stability and reusability. However, most enzymes are large in size, and it is difficult to find COFs with matching pore sizes. Therefore, the development of new enzyme immobilization technologies is of great significance. Summary of the invention

[0005] The present invention aims at the shortcomings of traditional physical adsorption immobilized enzymes, and proposes to utilize the strong electrostatic effect generated by the opposite charge on the surface of the enzyme and the surface charge of the COFs, so as to achieve the adsorption of large-sized enzymes on the surface of the cationic covalent organic framework. The method has a wide range of applications, does not need to consider the size of the enzyme, can ensure the conformational freedom of the enzyme as much as possible to maintain high enzyme activity, and improves the instability and recyclability of the enzyme. It is also one of the simplest and most economical methods for immobilizing enzymes.

[0006] The present invention adopts the following technical solution:

[0007] A method for realizing rapid adsorption of biomolecules onto a cationic covalent organic framework by utilizing electrostatic interaction, using the cationic covalent organic framework as a carrier, and the biomolecules have a negative charge on the surface in a buffer solution, specifically comprising the following steps;

[0008] The first step is the preparation of cationic covalent organic framework;

[0009] The second step is the immobilization of biomolecules. The cationic covalent organic framework prepared in the first step is immersed in a buffer solution, ultrasonically dispersed, added with biomolecule solution, and placed on a shaker for adsorption. After the adsorption is completed, it is washed with water three times, collected by centrifugation, and vacuum dried at 25°C overnight. The pH of the buffer solution is greater than the isoelectric point of the biomolecule.

[0010] Furthermore, the cationic covalent organic framework in the first step includes EB-COFs, Im-COF-Br, PyTTA-BFBIm-iCOF and Py-BPy 2+ -Any one of COF.

[0011] Furthermore, the biomolecule in the second step includes any one of proteins, amino acids and nucleic acids having a low isoelectric point.

[0012] Furthermore, the buffer in the second step includes any one of a phosphate buffer (pH 6-8), an acetic acid-sodium acetate buffer (pH 3.5-5), a sodium dihydrogen phosphate-sodium citrate buffer (pH 2-8) and a sodium carbonate-sodium bicarbonate buffer (pH 9-11).

[0013] Furthermore, the protein includes any one of bovine serum albumin (BSA), lipase (BCL), glucose oxidase (GOx) and alcohol dehydrogenase (ADH).

[0014] Furthermore, the amino acid includes any one of aspartic acid (Asp), glutamic acid (Glu) and tryptophan (Trp).

[0015] Furthermore, the nucleic acid includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0016] The principle of the present invention is as follows:

[0017] The isoelectric point is the pH value when the surface of a biomolecule is uncharged, and is an important indicator for measuring the surface charge of an enzyme. When the pH of the solution is higher than the isoelectric point of the enzyme, the surface of the enzyme carries a large amount of negative charge, and there is a strong electrostatic interaction with the positively charged material, which will facilitate the enzyme to be tightly adsorbed to the surface of the material.

[0018] In the present invention, enzymes, as natural catalysts, have some outstanding advantages, such as high efficiency, which can increase the reaction rate by millions of times; specificity, which only catalyzes specific reactions or produces specific configurations; and green and pollution-free. Immobilization technology can improve the chemical and thermal stability of enzymes, as well as better reusability, improve economic benefits, and promote the industrialization of enzymes.

[0019] Covalent organic frameworks (COFs) are a new type of porous framework material that has been very popular in recent years. They have regular pores, easily regulated structures, stability and good biocompatibility, making them an ideal carrier for enzyme fixation.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The method of the present invention has a certain universality, and there is no need to consider whether the size of the enzyme matches the pore size of COFs. By adjusting the pH value of the buffer, some enzymes are made to carry an appropriate amount of negative charge on the surface, and are tightly adsorbed to the surface or pores of the cationic covalent organic framework with the help of electrostatic action, thereby protecting the enzyme and improving the reusability of the enzyme.

[0022] 2. The present invention is applicable to most biomolecules (provided that the isoelectric point is low), is not limited by the size of the biomolecules, and can easily and quickly form a biomolecule-COFs biocatalytic system.

[0023] 3. The adsorbed biomolecules can be completely eluted from the COFs surface under the action of certain surfactants, thus achieving the reuse of the materials.

[0024] 4. The method of the present invention is used to prepare a biomolecule-COFs biocatalytic system, and the prepared biocatalytic system is used for enzyme catalysis, storage or transportation of biomolecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the curve of the adsorption amount of BSA on the surface of EB-COFs changing with time.

[0026] Figure 2 The zeta potential diagrams of EB-COFs, BSA and BSA / EB-COFs.

[0027] Figure 3 This is the curve of the adsorption amount of BCL on the surface of EB-COFs changing with time.

[0028] Figure 4 The zeta potential diagrams of EB-COFs, BCL and BCL / EB-COFs.

[0029] Figure 5 SEM images (2 μm) of EB-COFs (a) and BCL / EB-COFs (b and c).

[0030] Figure 6 Thermal stabilities of BCL and BCL / EB-COFs at different temperatures.

[0031] Figure 7The stability of BCL and BCL / EB-COFs in different pH buffers.

[0032] Figure 8 Test of the cycling stability of BCL / EB-COFs.

[0033] Fig. 9 This is the change in the amount of BCL adsorbed by EB-COFs when they are reused. DETAILED DESCRIPTION

[0034] The present invention is described below in conjunction with examples, but is not intended to limit the present invention.

[0035] Example 1

[0036] The preparation of cationic covalent organic framework, the specific steps are as follows:

[0037] Preparation of EB-COFs: Ethidium bromide (EB, 0.3mmol, 118mg) and 2,4,6-triformylphloroglucinol (TFP, 0.2mmol, 42mg) were weighed and transferred to a glass tube, and 1mL of 1,4-dioxane, 1mL of mesitylene and 0.2mL of acetic acid aqueous solution (6.0 M) were added. Then, freeze-vacuum-thaw degassing cycles were performed three times, and the tube was sealed and placed in a 120℃ oven for reaction for 3 days. After cooling to room temperature, the product was washed 4 times with THF, then Soxhlet extracted with 200mL of methanol:THF (1:1) mixed solution for 12h, and finally dried in vacuum at 100℃ for 12h to obtain a red powder.

[0038] Preparation of Im-COF-Br: Weigh 1,3,5-tris[3-(4-formylbenzyl)-1H-imidazol-1-yl]benzene bromide (TIBBr, 0.08mmol, 70.17mg) and benzidine (BZ, 0.12mmol, 22.11mg) into a glass tube, and then add 1.6 mL of 1,4-dioxane, 0.4 mL of anhydrous methanol and 0.2 mL of acetic acid aqueous solution (3.0 M). Then, freeze-vacuum-thaw degassing cycles were performed three times, and the tube was sealed and placed in a 120℃ oven for 7 days. After cooling to room temperature, the product was washed with THF and acetone three times, and finally dried in vacuum at 120℃ for 12h to obtain a dark yellow powder.

[0039] Example 2

[0040] Immobilization of biomolecules: Weigh 2~10 mg of COFs and dissolve them in 2~10 mL of buffer solution for ultrasonic dispersion. Add 2~10 mL of biomolecule solution and place on a shaker at 25°C and 200 rpm for adsorption. Then, collect the material by centrifugation, wash it with high-purity water three times to remove the loosely adsorbed biomolecules, and finally centrifuge to obtain the material, and vacuum dry it at 25°C overnight. The biomolecules here include bovine serum albumin (BSA) and lipase (BCL). For BSA (attached Figure 1 ) and BCL (with Figure 3 ) The optimal immobilization time was 1h. The Zeta potential change of the material before and after the immobilization of biomolecules confirmed that BSA (attached Figure 2 ) and BCL (Attached Figure 4 ) adsorption.

[0041] Example 3

[0042] Amination experiments of BSA.

[0043] First, 2 mL of ethylenediamine (EDA) (0.268 mL, 4.01 mmol) solution was prepared in ultrapure water (MQ water), and the pH was adjusted to 4.5 with 6 M HCl. Then 20 mg of BSA and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC•HCl) (7.2 mg, 0.038 mmol) were added. The solution was stirred on ice for 120 min, washed by ultrafiltration and concentrated to obtain A-BSA.

[0044] As a necessary control experiment: BSA was aminated to increase its isoelectric point, so that it has a positive charge (12.0 mV) on the surface in the buffer solution, which repels the positive charge on the surface of EB-COFs, and tests whether A-BSA can be successfully adsorbed on the surface of EB-COFs. Immobilization was carried out according to the above method, and the A-BSA concentration of the solution before and after immobilization was detected by Bradford method. The results showed that no A-BSA was adsorbed on the surface of EB-COFs, proving the significant contribution of electrostatic effect to the adsorption on the surface of COFs.

[0045] Example 4

[0046] Detection of biomolecule activity.

[0047] Lipase (BCL) activity detection: The steps for detecting the activity of free and immobilized BCL are as follows: 0.1~1.0 g p-nitrophenyl palmitate ( p -NPP) dissolved in 100 ml ethanol as substrate. Determined by spectrophotometry p -NPP hydrolysis process p-nitrophenol ( p The increase in absorbance at 410 nm caused by the release of p-nitrophenol (NP).410 =15,000M −1 •cm −1 Under the experimental conditions, the enzyme activity unit was defined as the amount of enzyme activity that catalyzes the production of 1 μmol of p-nitrophenol per minute. p -NP) enzyme amount (U).

[0048] Example 5

[0049] Stability test of immobilized enzyme.

[0050] (1) Acid-base stability test of immobilized enzyme.

[0051] Free lipase (BCL) and immobilized lipase (BCL / EB-COFs) were placed under different pH conditions (2.0-10.0) for 1 h. The enzyme activity was then tested according to the above standard method. Figure 6 It was shown that in a pH = 3 buffer, BCL / EB-COFs could provide a certain degree of protection for BCL, with the remaining activity being 85% of the original activity, while that of free BCL was only 15%.

[0052] (2) Thermal stability test of immobilized enzyme.

[0053] Free lipase (BCL) and immobilized lipase (BCL / EB-COFs) were placed at different temperatures (30-80°C) for 1 h. The enzyme activity was then tested according to the above standard method. Figure 7 The results showed that when the temperature was higher than 60°C, the activities of BCL and BCL / EB-COFs began to decline. When the temperature reached 80°C, the residual relative activity of BCL was only 5%, while BCL / EB-COFs still retained 65% relative activity.

[0054] (3) Cyclic stability test of immobilized enzyme

[0055] When testing the cyclic stability of the material, after a catalytic reaction, the solid was collected by centrifugation and then directly used in the next catalytic reaction. However, the activity dropped sharply. After many experiments, it was finally determined that simple centrifugation could not completely recover the material, resulting in a decrease in catalytic activity. Therefore, the material was dissolved in a buffer solution and injected into a microporous filter membrane with a pore size of 0.45μm through a syringe to circumvent the problem of material loss. After 10 cycles of use, the relative activity still remained at 75% (see attached). Figure 8 ).

[0056] Example 7

[0057] The elution of immobilized enzyme enables the reuse of COFs.

[0058] Usually, with the help of some surfactants, the enzyme can be eluted from the surface of the material. Here, we used Triton X-100 solution (0.5%~10%) to achieve complete elution of lipase (BCL) on the surface of BCL / EB-COFs within 0.5~2h. Interestingly, Triton X-100 solution has an activation effect on lipase (BCL), and the specific activity of lipase (BCL) after desorption reaches 170% of the original. The lipase (BCL) incubated with Triton X-100 also shows the same activation situation. Moreover, the desorbed COFs can still reach 95% of the initial adsorption amount after repeated adsorption / desorption of lipase (BCL) 8 times (Appendix Fig. 9 ).

Claims

1. A method for achieving rapid adsorption of biomolecules onto a cationic covalent organic framework using electrostatic interactions, characterized in that: Using a cationic covalent organic framework as a carrier, the biomolecule has a negative charge on the surface in a buffer solution, which specifically includes the following steps; The first step is the preparation of cationic covalent organic framework; The second step is the immobilization of biomolecules. The cationic covalent organic framework prepared in the first step is immersed in a buffer solution, ultrasonically dispersed, and the biomolecule solution is added and placed on a shaker for adsorption. After the adsorption is completed, it is washed with water three times, collected by centrifugation, and vacuum dried at 25°C overnight. The pH of the buffer solution is greater than the isoelectric point of the biomolecule. The buffer in the second step includes any one of a phosphate buffer, an acetic acid-sodium acetate buffer, a sodium dihydrogen phosphate-sodium citrate buffer, and a sodium carbonate-sodium bicarbonate buffer; The cationic covalent organic framework in the first step is TFP-EB-COFs; In the second step, the biomolecule is lipase.

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