Open tubular capillary electrochromatography drug screening method based on metal-organic framework material in-situ encapsulated biomacromolecules and application of open tubular capillary electrochromatography drug screening method

Abstract

The invention belongs to the technical field of analytical chemistry, separation science and drug screening, and particularly relates to an open tubular capillary electrochromatography (OT-CEC) screening method based on metal-organic framework materials (MOFs) in-situ encapsulated biomacromolecules and application of the open tubular capillary electrochromatography (OT-CEC) screening method. Introducing a surface active group containing sulfydryl, amino or carboxyl into the inner wall of the capillary tube; a precursor solution containing biomacromolecules is added into the activated capillary tube, so that the biomacromolecules and the surface groups are subjected to adsorption, complexation or covalent binding; a metal ion solution and an organic ligand solution are continuously added into the capillary tube, so that the metal-organic framework material grows in situ on the inner wall of the capillary tube, the biomacromolecule is encapsulated in the MOFs structure, and the biomacromolecule (at) MOFs (at) capillary tube stationary phase is formed; and separating a compound to be detected under the OT-ACEC condition by utilizing the formed biomacromolecule (at) MOFs (at) capillary tube, and screening candidate drugs based on the affinity interaction between the compound to be detected and the biomacromolecule.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of analytical chemistry, separation science and drug screening, and particularly relates to an open-tubular capillary electrochromatography (OT-CEC) screening method based on in-situ encapsulation of biological macromolecules by metal-organic framework materials (MOFs) and application thereof. BACKGROUND

[0002] THR is a key serine protease in the process of blood coagulation "cascade" reaction, which controls the coagulation cascade and regulates the process of thrombus formation, and has become a key target in the research and development of antithrombotic drugs. In order to prevent and treat thrombotic diseases, inhibiting the activity of THR is considered as an efficient treatment strategy. THR inhibitors, by directly or indirectly controlling the activity of THR, effectively block thrombus formation. Indirect inhibitors, such as heparin and vitamin K antagonists, indirectly affect the generation and function of THR by activating the substances combined with THR or inhibiting the synthesis of coagulation factors; while direct inhibitors, such as hirudin, argatroban, etc., directly inhibit the activity of THR by specifically binding with THR. Compared with indirect inhibitors, direct THR inhibitors do not require antithrombin cofactors, reducing the occurrence of side effects and interference from other factors, and showing more superior pharmacological and pharmacokinetic properties. Although some direct THR inhibitors have been applied in clinic, these drugs may cause serious complications such as bleeding and allergy.

[0003] The existing research provides an OT-ACEC drug screening platform for immobilizing proteins, but the selected target is not the real target. In addition, the physical force is relatively weak, and its binding stability needs to be improved. Furthermore, the method is limited by the MOFs that have been constructed for immobilization of biological macromolecules. The pore size, shape and stability of MOFs limit the type, size and application range of biological macromolecules that can be immobilized. More importantly, most of the structure of the biological macromolecules in the method is directly exposed, which is easy to denature under external stimulation. The defects such as multiple preparation process steps, complex operation and time-consuming cannot be ignored. Therefore, it is still necessary to further explore the OT-ACEC method for immobilizing target proteins with more superior MOFs. SUMMARY

[0004] To solve the above technical problems, the present application provides an open-tubular capillary electrochromatography (OT-CEC) drug screening method based on in-situ encapsulation of biological macromolecules by metal-organic framework materials (MOFs), comprising the following steps:

[0005] (1) introducing a surface active group containing thiol, amino or carboxyl group to the inner wall of a capillary;

[0006] (2) adding a precursor solution containing biological macromolecules into the activated capillary, so that the biological macromolecules are adsorbed, complexed or covalently bound with the surface group;

[0007] (3) continuously adding metal ion solution and organic ligand solution into the capillary, growing metal-organic framework material in-situ on the inner wall of the capillary and encapsulating the biomacromolecule into the MOFs structure, thereby forming a biomacromolecule@MOFs capillary stationary phase;

[0008] (4) separating the test compound under the OT-ACEC condition by using the formed biomacromolecule@MOFs capillary, and realizing the screening of candidate drugs based on the affinity between the test compound and the biomacromolecule;

[0009] The biomacromolecule includes protein, enzyme, polypeptide, nucleic acid or a complex thereof.

[0010] As an embodiment of the present application, the MOFs are selected from ZIF-, UiO-, MIL-, HKUST-, PCN- or other metal-organic framework materials with microporous / mesoporous structure.

[0011] As an embodiment of the present application, the MOFs are ZIF-8 and the biomacromolecule is thrombin.

[0012] As an embodiment of the present application, the in-situ encapsulation reaction is carried out at 15-40℃, pH 6-12, and the molar ratio of metal ion to organic ligand is 1:5-1:20.

[0013] As an embodiment of the present application, the functional group-containing molecule for activating the capillary surface includes L-cysteine, dithioproprionic acid, lysine, glutamic acid, mercaptosilane or aminosilane reagent.

[0014] A MOFs composite compound for the method of the present application is THR@ZIF-8 capillary.

[0015] As an embodiment of the present application, the preparation method of the THR@ZIF-8 capillary is as follows:

[0016] (1) Activation of the capillary

[0017] The uncoated fused quartz capillary is sequentially washed with 1M NaOH, deionized water, 0.1M HCl and deionized water, and dried by nitrogen blowing for 15min for standby;

[0018] (2) Preparation of Cys-modified capillary

[0019] The activated capillary is continuously injected with an APTES ethanol solution; subsequently, the prepared capillary is washed with anhydrous ethanol to remove residual APTES and dried with nitrogen for 30 min; the APTES modified capillary is continuously injected with glutaraldehyde and a Cys aqueous solution in sequence; the Cys modified capillary is washed with deionized water and dried with nitrogen;

[0020] (3) Preparation of THR@ZIF-8@capillary

[0021] THR is dissolved in deionized water, then PVP is added, after ultrasonic dissolution, the Cys modified capillary is continuously injected, and finally zinc acetate and 2-methylimidazole are injected into the treated capillary in sequence; the two ends of the capillary are sealed, and the reaction is placed at room temperature; after the reaction is completed, the capillary is washed with a running buffer to obtain a THR@ZIF-8@capillary.

[0022] The application provides a capillary electrophoresis device for OT-ACEC analysis, and a MOFs composite compound is used as a capillary stationary phase in the device.

[0023] As an embodiment of the application, the compound is used for affinity-based drug screening, target protein binding research, inhibitor screening, ligand library screening or biological activity evaluation.

[0024] As an embodiment of the application, the compound is used to improve the structural stability and biological activity of biological macromolecules in high temperature, strong acid / strong base, dissociation agent and organic solvent.

[0025] By adopting the technical scheme, the application has the following beneficial effects:

[0026] The application provides a synthesis strategy of in-situ growth of Cys-induced MOFs and enzyme encapsulation in a capillary, and a novel OT-ACEC drug screening method of in-situ encapsulation of enzymes in MOFs is constructed. First, the stability and enzyme activity of THR@ZIF-8 are discussed. THR@ZIF-8 has higher stability, and ZIF-8 encapsulated THR can create a more favorable microenvironment for THR, inhibit the unfolding process of THR and maintain the natural conformation of THR. THR@ZIF-8@capillary has higher THR loading capacity, better enzyme activity and stability than an open-tube affinity capillary (THR / ZIF-8@capillary) prepared by a surface immobilization method, and can realize specific screening of THR inhibitors. The application innovates the construction of an immobilized biological macromolecule OT-ACEC method, and provides new possibilities for efficient immobilization of biological macromolecules in a capillary and development of a drug screening method.

[0027] The in-situ growth of MOFs on the inner wall of the capillary enables the direct coating of the pore structure of the MOFs with biomacromolecules, thereby forming a three-dimensional confined environment. Compared with the physical adsorption method, the amount of the fixed biomacromolecules is increased by 30-100%, and the amount of the biomacromolecules retained after high-salt flushing is significantly improved (the THR immobilization rate is increased from 41.38% to 9.09%, and the amount of the biomacromolecules retained by the in-situ encapsulation is 37.64%), which indicates that the encapsulation structure can effectively prevent the biomacromolecules from falling off or being eluted during operation.

[0028] After 20 continuous operations, the EOF fluctuation of the stationary phase of the application is minimal, and the enzyme activity is optimal, while the traditional adsorption method shows obvious falling off and drifting, which proves that the stationary phase of the application has excellent column stability and running resistance.

[0029] The interface-induced immobilization mechanism and the in-situ encapsulation process are independent of specific protein structures, and can be applied to any protein, nucleic acid, polypeptide, etc. as a target to achieve: direct inhibitor screening, target protein-small molecule binding research, ligand library affinity screening, biological activity evaluation, and has significant promotional value. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the following description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0031] Figure 1 SEM images of ZIF-8 (A) and THR@ZIF-8 (B); XRD spectra of ZIF-8 and THR@ZIF-8 (C); FT-IR spectra of THR, ZIF-8 and THR@ZIF-8 (D); Dark field images of E. RhB-labeled THR@ZIF-8 under an inverted fluorescence microscope;

[0032] Figure 2 SEM images of bare capillary (A, D), ZIF-8@capillary (B, E) and THR@ZIF-8@capillary (C, F);

[0033] Figure 3 FT-IR spectra of bare quartz plate, APTES@plate, Cys@plate and THR@ZIF-8@plate.

[0034] Figure 4 Stability test graphs of THR@ZIF-8 and THR / ZIF-8.

[0035] Figure 5A. Fluorescence spectra of THR and ZIF-8; B. Relative activity of THR and THR@ZIF-8 after incubation in high temperature, organic solvent, urea and trypsin for 1 h.

[0036] Figure 6 EOF of bare capillary, APTES@capillary and THR@ZIF-8@capillary; CE experimental conditions: running voltage was 20 kV; sample injection: 30 mbar 3 s; detection wavelength was 210 nm; temperature was 25 °C.

[0037] Figure 7 Enzymatic activity change (A) and running stability of THR@capillary, THR / ZIF-8@capillary and THR@ZIF-8@capillary in 30 days (B) ; CE experimental conditions: running voltage was 20 kV; sample injection: 30 mbar 3 s; detection wavelength was 405 nm (A) and 210 nm (B) ; temperature was 25 °C.

[0038] Figure 8 CE spectra of negative sample (warfarin 0.1 mM, A) and positive sample (argatroban 0.1 mM, B) in bare capillary, ZIF-8@capillary and THR@ZIF-8@capillary, respectively; CE experimental conditions: running voltage was 20 kV; sample injection: 30 mbar 3 s; detection wavelength was 210 nm (B) ; temperature was 25 °C. DETAILED DESCRIPTION

[0039] The application will be further explained in connection with the specific embodiments.

[0040] The application will be further explained in connection with the specific embodiments.

[0041] Raw material description:

[0042] Dimethyl sulfoxide (DMSO), sodium hydroxide (NaOH), hydrochloric acid (HCl), urea and tris(hydroxymethyl)aminomethane (Tris) were purchased from Chengdu Kolon Chemical Co., Ltd. Warfarin and N-N'-dimethylformamide (DMF) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Argatroban and cysteine (Cys) were purchased from Shanghai Meray Biotech Co., Ltd. Rhodamine B was purchased from Hebei Pinke Scientific Biotech Co., Ltd. 2-Methylimidazole was purchased from Shanghai Adamas Reagent Co., Ltd. Anhydrous zinc acetate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Trypsin and Coomassie brilliant blue G250 were purchased from Shanghai McLean Biochemical Science and Technology Co., Ltd. Polyvinylpyrrolidone (PVP) was purchased from Shanghai Xushuo Biotechnology Co., Ltd. Thrombin (THR) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. Thrombin chromogenic substrate S-2238 was purchased from Shanghai Guchen Biotechnology Co., Ltd. Fused silica capillary was purchased from Hebei Yongnian Ruiheng Chromatography Device Co., Ltd.

[0043] Example 1

[0044] The present embodiment provides a composite material for open-tube capillary electrochromatography drug screening method based on in-situ encapsulation of biological macromolecules by metal-organic framework materials, and the preparation method is as follows:

[0045] (1) Activation of capillary

[0046] The uncoated fused silica capillary (75 μm i.d. × 28.5 cm, effective length 20 cm) was successively washed with 1 M NaOH (30 min), deionized water (20 min), 0.1 M HCl (30 min), deionized water (20 min) for activation, and dried with nitrogen blowing for 15 min for standby.

[0047] (2) Preparation of Cys-modified capillary

[0048] The activated capillary was successively injected with APTES ethanol solution (10%, v / v) for 2 h. Subsequently, the prepared capillary was washed with anhydrous ethanol for 5 min to remove residual APTES and dried with nitrogen for 30 min. Then, the APTES-modified capillary was successively injected with glutaraldehyde (2%, v / v, pH 11) and Cys (2 mg·mL -1 ) aqueous solution for 2 h. Finally, the Cys-modified capillary was washed with deionized water for 10 min and dried with nitrogen for 30 min.

[0049] (3) Preparation of THR@ZIF-8@capillary

[0050] THR@ZIF-8@capillary was prepared as follows: 2 mg of PVP was dissolved in 1 mL of deionized water, and then 2 mg of THR was added. After ultrasonic dissolution for 30 s, it was continuously injected into the Cys-modified capillary for 60 min. Finally, 2 mL of zinc acetate (40 mM) and 2 mL of 2-methylimidazole (160 mM, pH = 10.5) were sequentially injected into the treated capillary. The two ends of the capillary were sealed, and the reaction was allowed to stand at room temperature for 4 h. After the reaction was completed, the capillary was washed with a buffer solution (pH = 7.4 30 mM Tris-HCl buffer solution) for 5 min to obtain the THR@ZIF-8@capillary.

[0051] Comparative Example 1

[0052] The present comparative example provides a composite material for an open-tube capillary electrochromatography drug screening method based on in-situ encapsulation of biological macromolecules by metal-organic framework materials, which is a THR / ZIF-8@capillary, and the preparation method is as follows:

[0053] 2 mg·mL -1 THR was directly injected into the activated (reference content Example 1 "(1) Activation of the capillary") fused quartz capillary. After sealing the two ends of the capillary and allowing the reaction to stand at room temperature for 4 h, the capillary was washed with a buffer solution (pH = 7.4 30 mM Tris-HCl buffer solution) for 5 min to obtain the THR@capillary.

[0054] Comparative Example 2

[0055] The present comparative example provides a composite material for an open-tube capillary electrochromatography drug screening method based on in-situ encapsulation of biological macromolecules by metal-organic framework materials, which is a ZIF-8@capillary, and the preparation method is as follows:

[0056] Except that no THR was added, the rest was prepared according to the preparation steps of THR@ZIF-8@capillary (reference content Example 1 "(3) Preparation of THR@ZIF-8@capillary": 2 mg of PVP was dissolved in 1 mL of deionized water, and then 2 mg of THR was added. After ultrasonic dissolution for 30 s, it was continuously injected into the Cys-modified capillary for 60 min. Finally, 2 mL of zinc acetate (40 mM) and 2 mL of 2-methylimidazole (160 mM, pH = 10.5) were sequentially injected into the treated capillary. The two ends of the capillary were sealed, and the reaction was allowed to stand at room temperature for 4 h. After the reaction was completed, the capillary was washed with a buffer solution (pH = 7.4 30 mM Tris-HCl buffer solution) for 5 min to obtain the THR@ZIF-8@capillary.). A ZIF-8 coated capillary (ZIF-8@capillary) without THR encapsulation was prepared.

[0057] Comparative Example 3

[0058] The comparative example provides a composite material for open-tube capillary electrochromatography drug screening method based on in-situ encapsulation of biological macromolecules on metal-organic framework material, which is THR / ZIF-8@capillary, and the preparation method is as follows:

[0059] 160mM 2-methylimidazole and 40mM zinc acetate methanol solution were injected into the Cys modified capillary (reference content example 1 “(2) Preparation of Cys modified capillary”: after activation, the capillary was continuously injected with APTES ethanol solution (10%, v / v) for 2h. Subsequently, the prepared capillary was washed with anhydrous ethanol for 5min to remove residual APTES and dried with nitrogen for 30min. Then, the APTES modified capillary was continuously injected with glutaraldehyde (2%, v / v, pH 11) and Cys (2mg·mL-1) aqueous solution in turn for 2h. Finally, the Cys modified capillary was washed with deionized water for 10min, and dried with nitrogen for 30min. After that, both ends of the capillary were sealed, and after reaction at room temperature for 4h, the capillary was washed with methanol for 5min and dried with nitrogen for 30min. Finally, 2mg·mL - 1 THR, after reaction for 4h, pH=7.430mM Tris-HCl buffer solution was washed for 5min, and THR / ZIF-8@capillary was obtained.

[0060] In addition:

[0061] The preparation method of ZIF-8 and THR@ZIF-8 is as follows for subsequent testing:

[0062] (1) Preparation of ZIF-8

[0063] After mixing and stirring 10mL 2-methylimidazole (160mM) methanol solution and 10mL zinc acetate (40mM) methanol solution for 12h, the product was washed with methanol for 3 times, and ZIF-8 was obtained.

[0064] (2) Preparation of THR@ZIF-8

[0065] 2mg THR was dissolved in 1mL deionized water, then 2mg PVP was added. After stirring for 30s, 2mg Cys was added, and then the solution was stirred for another 30s. Finally, 2mL 2-methylimidazole (160mM, pH=10.5) and 2mL zinc acetate solution (40mM) were added respectively, and reacted at room temperature for 4h. After the reaction was completed, 430mM Tris-HCl buffer solution with pH=7 was used to wash 3 times, and THR@ZIF-8 was obtained.

[0066] Test:

[0067] Test 1: characterization test

[0068] The morphology and size of ZIF-8 and THR@ZIF-8 were characterized by SEM. As shown in Figs. 1A and 1B, compared with ZIF-8, THR@ZIF-8 after encapsulating THR showed similar morphology with slightly smaller size, indicating that the presence of THR in the crystallization process would not affect the shape of ZIF-8 crystal, but the crystal size decreased slightly. This size difference can be attributed to the influence of THR on the size growth of ZIF-8 crystal. The similar XRD patterns of THR@ZIF-8 and ZIF-8 (Fig. 1C) indicated that the encapsulation of THR had no obvious effect on the crystal structure of ZIF-8. FT-IR spectra showed that the appearance of stretching vibration peaks of 2-methyl imidazole and skeleton vibration peaks of imidazole ring in ZIF-8 and characteristic peaks of THR in THR@ZIF-8 confirmed the successful preparation of THR@ZIF-8 composite (Fig. 1D). Fluorescence microscope images (Fig. 1E) showed that the fluorescence of ZIF-8 crystals was uniformly distributed, indicating that ZIF-8 contained uniformly distributed THR. The above results confirmed that THR was successfully encapsulated in ZIF-8, and the incorporation of THR did not affect the crystal structure of ZIF-8. Figure 1 Figure 1 Figure 1 Figure 1

[0069] The morphology of the inner wall of bare capillary, ZIF-8@capillary and THR@ZIF-8@capillary was characterized by SEM. As shown in Figs. 2A and 2B, the inner wall of bare capillary was smooth, while the inner wall of ZIF-8@capillary and THR@ZIF-8@capillary was rough and covered with crystals, which proved that ZIF-8 was successfully grown in situ on the inner wall of capillary. Subsequently, the content of relevant elements on ZIF-8@capillary and THR@ZIF-8@capillary was studied by EDS analysis. As shown in Table 1, due to the introduction of THR, THR@ZIF-8@capillary had higher content of C, O and N elements than ZIF-8@capillary. Figure 2

[0070] Table 1 Element content (%) of ZIF-8@capillary and THR@ZIF-8@capillary

[0071]

[0072] ​​​​​APTES@plate, Cys@plate and THR@ZIF-8@plate were prepared by the same method as APTES@capillary, Cys@capillary and THR@ZIF-8@capillary (the method refers to the contents of "(1)-(3)" in Example 1, and the bare capillary is replaced by bare quartz plate; first, the activated quartz plate was prepared, and the activated quartz plate was continuously injected into the APTES ethanol solution (10%, v / v) for 2 h. The activated quartz plate was continuously injected into the APTES ethanol solution (10%, v / v) for 2 h. Then, the prepared quartz plate was washed with anhydrous ethanol for 5 min to remove residual APTES and dried with nitrogen for 30 min to obtain APTES@plate; the obtained APTES@plate was sequentially injected into the APTES modified quartz plate with glutaraldehyde (2%, v / v, pH 11) and Cys (2 mg·mL-1) aqueous solution for 2 h. Finally, the Cys modified bare quartz plate was washed with deionized water for 10 min, and dried with nitrogen for 30 min to obtain Cys@plate; 2 mg of THR was dissolved in 1 mL of deionized water, then 2 mg of PVP was added, and after ultrasonic dissolution for 30 s, the Cys modified quartz plate was continuously injected for 60 min, and finally 2 mL of zinc acetate (40 mM) and 2 mL of 2-methylimidazole (160 mM, pH = 10.5) were sequentially injected into the treated quartz plate. The ends of the quartz plate were sealed, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, the quartz plate was washed with buffer solution (pH = 7.4 30 mM Tris-HCl buffer solution) for 5 min to obtain THR@ZIF-8@plate. ) and the bare quartz plate, APTES@plate, Cys@plate and THR@ZIF-8@plate were characterized by ATR-FT-IR. As shown in FIG. 8, in the FT-IR spectrum of APTES@plate, the characteristic absorption peaks appearing at 3300 cm-1, 1555 cm-1and 1484 cm-1are related to the stretching vibration of amine group. In the FT-IR spectrum of Cys@plate, the new absorption peaks near 1573 cm-1, 1613 cm-1and 1392 cm-1are respectively attributed to the C=N group generated by the Schiff base condensation reaction between amine group and aldehyde group and the asymmetric and symmetric stretching vibration of carboxyl group. In addition, 2551 cm-1 Figure 3 , 1555 cm -1 , 1484 cm -1 and 1392 cm -1 in the FT-IR spectrum of Cys@plate are related to the stretching vibration of amine group. In the FT-IR spectrum of Cys@plate, the new absorption peaks near 1573 cm -1 , 1613 cm -1 and 1392 cm -1 are respectively attributed to the C=N group generated by the Schiff base condensation reaction between amine group and aldehyde group and the asymmetric and symmetric stretching vibration of carboxyl group. In addition, 2551 cm -1The absorption band of Cys@APTES@plate is attributed to the -SH stretching vibration of Cys, which confirms that Cys is successfully grafted on APTES@plate. THR@ZIF-8@plate has the FT-IR waveband signals of ZIF-8 and THR, indicating that THR is successfully immobilized on the quartz plate through ZIF-8. The above results demonstrate the successful preparation of ZIF-8 in situ encapsulated THR capillary.

[0073] Test 2: Stability study

[0074] To test the stability of THR@ZIF-8, high-concentration salt solution was used to elute THR@ZIF-8, and the desorption of THR@ZIF-8 was determined and compared with that of the composite material (THR / ZIF-8) physically adsorbed with THR. (In the adsorption experiment, 10 mg of ZIF-8 was added to 2 mL of THR (2 mg·mL -1 ) at room temperature for 4 h, and then washed with a pH = 7.430 mM Tris-HCI buffer solution for 3 times to obtain THR / ZIF-8. The developer was prepared by using Coomassie brilliant blue G250, and the initial and final concentrations of THR in the supernatant were determined by Bradford assay to determine the THR adsorption amount.

[0075] In the desorption experiment, 10 mg of THR@ZIF-8 and THR / ZIF-8 was added to 2 mL of 2M NaCI Tris-HCI buffer solution (pH = 7.4, 30 mM) for 72 h. It was washed with a pH = 7.430 mM Tris-HCI buffer solution for 3 times. The initial and final concentrations of THR in the supernatant of THR@ZIF-8 and THR / ZIF-8 were determined. According to the different absorption values of the combination of different concentrations of THR and Coomassie brilliant blue at 595 nm, a standard curve was obtained. According to the standard curve, the THR concentrations before and after synthesis and adsorption of THR@ZIF-8 and THR / ZIF-8 and after desorption were determined. As shown in Figure 4 After desorption, the THR loading rate of THR / ZIF-8 decreased from 41.38% to 9.09%. This means that a large amount of THR is desorbed from the carrier ZIF-8, which is related to the weak physical adsorption binding force between THR and ZIF-8. For THR@ZIF-8, the THR loading rate decreased from 62.87% to 37.64%, which indicates that the tightly surrounding ZIF-8 layer can prevent THR from leaking during use, while the physically adsorbed THR is unstable and easy to fall off. Therefore, the in situ encapsulation method of MOFs has more stability advantages than the physical adsorption method of THR on the surface of MOFs.

[0076] Test 3: Study on the influence of enzyme conformation and activity

[0077] The conformation of THR in THR@ZIF-8 was studied by fluorescence spectroscopy. (1. The performance of immobilized THR was investigated; Trypsin (TRY) can hydrolyze the peptide chain in the protein, and can digest and dissolve denatured enzyme protein. Urea is a strong protein denaturant, which can destroy the internal hydrogen bonds and non-covalent bonds of the protein, destroy the secondary structure of the protein, and make the protein structure unfold. In addition, organic solvents and high temperature, etc. can also cause the enzyme conformation to change and become inactive or denatured. Therefore, whether ZIF-8 can improve the stability of THR under denaturing conditions was discussed. Free THR and THR@ZIF-8 were incubated in 50C, 70C, DMF, DMSO, 1mg·mL -1 urea and 1mg·mL -1 TRY for 1h, and then reacted with the chromogenic substrate S-2238 to determine the absorbance value of the product at 405nm to evaluate the enzyme activity. 2. Enzyme activity determination; the product pNP generated by the reaction of THR with the chromogenic substrate S-2238 has maximum absorption at 405nm, and the enzyme activity of THR@ZIF-8@capillary is determined according to the strength of the pNP absorption signal. As shown in Figure 5 A, free THR and THR@ZIF-8 have the same maximum fluorescence emission wavelength. The phenomenon that the maximum fluorescence emission wavelength of encapsulated THR does not shift indicates that THR can maintain its natural conformation in ZIF-8. Subsequently, the enzyme activity of THR@ZIF-8 under harsh conditions was explored. As shown in Figure 5 B, after Try treatment, the enzyme activity of free THR was retained by 9.53%, while when THR was encapsulated in MOFs, due to the protection of the MOFs layer, 81.98% of the original enzyme activity was retained. The difference in enzyme activity indicates that the micropores of ZIF-8 can exclude Try and protect THR from being hydrolyzed. After treatment with urea and heating, THR@ZIF-8 still maintains relatively high enzyme activity, indicating that under unfolding conditions, THR is strictly confined in the stable ZIF-8 channel, and its conformation is maintained. After treatment with organic solvents DMF or DMSO, the activity of free THR is only 4.64% and 5.89%. This phenomenon is caused by the denaturation of the enzyme by organic solvents by penetrating into the enzyme active site to change the local environment. While the enzyme activity of THR@ZIF-8 can be preserved by 46.07% and 64.78%, indicating that the encapsulation of ZIF-8 improves the resistance of THR to organic solvents. The above results show that the THR encapsulated by this method can retain a certain enzyme activity even when exposed to high temperature, denaturing environment, proteolytic environment, and organic solvents.

[0078] Test 4: Investigation of EOF

[0079] EOF of the prepared capillary was evaluated using 0.01% DMSO as EOF marker. (μ eofAmong them, L eff The effective length of the capillary is 20 cm, and L represents the total length of the capillary (28.5 cm). V is the applied operating voltage (20.0 kV). t o (This refers to the migration time of EOF markers) Figure 6 As shown, among all capillaries, the bare capillary has the largest EOF value, while the APTES@capillary has the lowest and negative EOF value. This phenomenon may be attributed to the positively charged amino groups in the APTES coating and the masking of some silanol groups inside the capillary. After ZIF-8 was fixed to the inner wall of the capillary with Cys assistance, the EOF value of the resulting THR@ZIF-8@capillary was between that of the bare capillary and the APTES@capillary. This is because the ZIF-8 crystals masked the APTES coating, which also confirms the successful growth of ZIF-8 crystals on the inner wall of the capillary.

[0080] Formula (1)~(2):

[0081] Calculate the electroosmotic flow (EOF) and electrophoretic velocity (v) using the following formulas:

[0082]

[0083]

[0084] Where Leff is the effective length of the capillary (20 cm), L is the total length of the capillary (28.5 cm), V is the applied operating voltage (10.0 kV), and to is the migration time of the EOF marker. E t is the electrophoretic velocity of the analyte, and t is its migration time.

[0085] Test 5: Performance of THR@ZIF-8@capillary

[0086] The changes in enzyme activity of THR@ZIF-8@capillary, THR / ZIF-8@capillary, and THR@capillary over 30 days were compared and examined. (Enzyme activity assay: The product pNP generated by the reaction of THR with the chromogenic substrate S-2238 has a maximum absorption at 405 nm. The daily enzyme activity of THR@ZIF-8@capillary, THR / ZIF-8@capillary, and THR@capillary over 30 days was determined based on the intensity of the pNP absorption signal, and an activity change graph was plotted.) Figure 7It can be seen from A that, compared with THR / ZIF-8@capillary and THR@capillary, THR@ZIF-8@capillary has better enzyme activity stability, which benefits from the encapsulation of ZIF-8 to THR. Secondly, the EOF change of THR@ZIF-8@capillary, THR / ZIF-8@capillary and THR@capillary before and after 20 continuous runs was also investigated respectively. As shown in Figure 7 As shown in B, after 20 continuous runs, the THR coated in THR@capillary is easy to fall off, and the EOF change is large, while THR@ZIF-8@capillary has excellent column stability.

[0087] Test 6: Enzyme inhibitor screening test of THR@ZIF-8@capillary

[0088] (1) Affinity screening study

[0089] In order to investigate the affinity specificity of THR@ZIF-8@capillary for THR inhibitor screening, bare capillary and ZIF-8@capillary were selected as negative columns, and the chromatographic behaviors of negative sample warfarin and positive sample argatroban on the negative column and THR@ZIF-8@capillary were observed respectively. As shown in Figure 8As shown, there was no obvious change in the peak shape and peak height of warfarin in bare capillary, ZIF-8@capillary and THR@ZIF-8@capillary, which proved that there was no interaction between warfarin and THR. Agatroban was obviously retained on THR@ZIF-8@capillary, which indicated that there was interaction between THR@ZIF-8@capillary and agatroban. The specific binding between THR and agatroban in THR@ZIF-8@capillary prolonged the retention time of agatroban. From the peak shape, the peak time of warfarin was prolonged due to the decrease of EOF in ZIF-8@capillary and THR@ZIF-8@capillary, but there was no significant difference in the peak shape. Compared with the electrophoretic behavior of agatroban in ZIF-8@capillary, the peak height of agatroban decreased and the peak width increased in THR@ZIF-8@capillary, which indicated that the electrophoretic behavior of agatroban in THR@ZIF-8@capillary was caused by the interaction between agatroban and THR, rather than the non-specific interaction with ZIF-8 stationary phase. The differences in the peak shape, the change in the peak time and the unchanged peak area between warfarin and agatroban confirmed the specific interaction between THR and agatroban in THR@ZIF-8@capillary. Therefore, THR@ZIF-8@capillary can be used for affinity screening of direct inhibitors of THR. In this method, THR is immobilized on the inner wall of the capillary, so that the mobility of THR is 0, and the interaction system in which there is no difference between the mobility of free ligand and the mobility of complex can be evaluated by ACE.

[0090] (2) Stability and reproducibility investigation

[0091] Based on the RSD values of the migration time, peak area, peak height and peak width of positive sample agatroban in THR@ZIF-8@capillary, the reproducibility and stability of THR@ZIF-8@capillary for THR inhibitor screening analysis were investigated. As shown in Table 2, agatroban had good inter-day, intra-day and inter-column RSD values on THR@ZIF-8@capillary, which indicated that the preparation method of THR@ZIF-8@capillary was reliable, and THR@ZIF-8@capillary had good reproducibility and stability for THR direct inhibitor screening analysis.

[0092] Table 2 Reproducibility and stability of THR@ZIF-8@capillary

[0093]

[0094] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for open-tube capillary electrochromatographic drug screening based on in-situ encapsulation of biomacromolecules using metal-organic framework materials, characterized in that, Includes the following steps: (1) Introduce surfactant groups containing thiol, amino or carboxyl groups into the inner wall of the capillary; (2) In the activated capillary, a precursor solution containing biomacromolecules is added to cause the biomacromolecules to adsorb, complex, or covalently bind with surface groups. (3) A metal ion solution and an organic ligand solution are continuously added into the capillary to allow the metal-organic framework material to grow in situ on the inner wall of the capillary and to encapsulate the biomacromolecules in the MOFs structure, thereby forming a biomacromolecule@MOFs@capillary stationary phase. (4) Using the formed biomacromolecules @MOFs@capillaries, the test compound is separated under OT-ACEC conditions, and the candidate drug is screened based on the affinity between the biomacromolecules. The biological macromolecules include: proteins, enzymes, polypeptides, nucleic acids, or complexes thereof.

2. The method as described in claim 1, characterized in that, The MOFs are selected from ZIF-type, UiO-type, MIL-type, HKUST-type, PCN-type, or other metal-organic framework materials with microporous / mesoporous structures.

3. The method as described in claim 1, characterized in that, The MOFs are ZIF-8, and the biomolecule is thrombin.

4. The method as described in claim 1, characterized in that, The in-situ encapsulation reaction is carried out at 15–40 °C and pH 6–12, with a molar ratio of metal ions to organic ligands of 1:5–1:

20.

5. The method as described in claim 1, characterized in that, Functional group molecules used for capillary surface activation include: L-cysteine, dithiopropionic acid, lysine, glutamic acid, mercaptosilane, or aminosilane reagents.

6. A MOFs composite compound for use in the method of claim 1, characterized in that, The composite compound is THR@ZIF-8@capillary.

7. A MOF complex compound for OT-ACEC drug screening according to claim 1, characterized in that, The preparation method of THR@ZIF-8@capillary is as follows: (1) Capillary activation The activated uncoated fused silica capillary was rinsed sequentially with 1M NaOH, deionized water, 0.1M HCl, and deionized water, and then dried by nitrogen blowing for 15 minutes. (2) Preparation of Cys-modified capillaries The activated capillary was continuously injected with an ethanol solution of APTES; then, the prepared capillary was rinsed with anhydrous ethanol to remove residual APTES and dried with nitrogen for 30 min; the APTES-modified capillary was then continuously injected with glutaraldehyde and Cys aqueous solution in sequence. The Cys-modified capillary was rinsed with deionized water and dried with nitrogen. (3) Preparation of THR@ZIF-8@capillary THR was dissolved in deionized water, then PVP was added, and after sonication, it was continuously injected into Cys-modified capillaries. Finally, zinc acetate and 2-methylimidazole were injected into the treated capillaries in sequence. The capillaries were sealed at both ends and allowed to stand at room temperature for reaction. After the reaction was completed, the capillaries were rinsed with running buffer to obtain THR@ZIF-8@capillaries.

8. A capillary electrophoresis apparatus for OT-ACEC analysis, characterized in that, The capillary stationary phase built into the device is the MOFs composite compound as described in claim 7.

9. The MOFs composite compound according to claim 7, characterized in that, The compounds are used for affinity-based drug screening, target protein binding studies, inhibitor screening, ligand library screening, or bioactivity assessment.

10. The MOFs composite compound according to claim 7, characterized in that, The compound is used to improve the structural stability and biological activity of biomolecules in high temperature, strong acid / base, dissociation agent, and organic solvent.

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