Thrombin activity detection kit and application thereof

Abstract

The invention discloses a thrombin activity detection kit and application thereof, and belongs to the technical field of biology. The unique characteristics of aggregation-induced emission (AIE) molecules are utilized, and the thrombin activity is monitored by detecting the change of luminophores in a blood coagulation system by virtue of the reaction process that fibrinogen is hydrolyzed by thrombin to generate fibrin monomers and the monomers are further self-polymerized into fibrin aggregates. Based on the principle, a thrombin activity detection method is established, a method for screening, separating and evaluating drugs containing potential thrombin inhibitor active components by taking immobilized thrombin as a medium is established, the thrombin inhibitor can be successfully screened out, and the inhibition effect of the thrombin inhibitor can be determined. A new method is provided for rapid detection of thrombin activity, and a new strategy is also provided for screening and evaluation of thrombin inhibitors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a thrombin activity detection kit and application thereof. BACKGROUND

[0002] With the increasing incidence of cardiovascular diseases year by year, the regulation mechanism of key proteins in the coagulation cascade becomes the research focus. Thrombin, as an indispensable key enzyme in the coagulation pathway, plays a "double-edged sword" role in the process of physiological hemostasis and pathological thrombosis, and its abnormal activity increase is an important reason for the occurrence and development of thrombotic diseases. In addition, fibrinogen is another key protein in the coagulation process, which is a large molecular glycoprotein with high content in plasma, and circulates in the blood in a soluble form. When the blood vessel wall is damaged, the coagulation cascade is activated, and thrombin rapidly cleaves fibrinogen to convert it into fibrin monomers, which form fibrin aggregates through non-covalent interactions. This biological aggregate can accelerate the growth and solidification of thrombus, threatening the health of the cardiovascular system. Traditional detection methods, including coagulation four items, imaging techniques, etc., can provide macroscopic coagulation indicators, but it is difficult to realize in situ and dynamic visualization monitoring of the coagulation process. Therefore, it is of great significance to monitor fibrin aggregation and find active substances that affect aggregation. SUMMARY

[0003] The purpose of the present application is to provide a thrombin activity detection kit and its application to solve the problems existing in the prior art. The present application utilizes the unique properties of aggregation-induced emission (AIE) molecules, and through the reaction process of thrombin hydrolyzing fibrinogen to produce fibrin monomers, and the monomers further self-polymerizing into fibrin aggregates, the change of luminophore in the coagulation system is detected to monitor the activity of thrombin. Based on this principle, the present application establishes a method for detecting the activity of thrombin, and a method for screening, separating and evaluating drugs containing potential thrombin inhibitor active ingredients using immobilized thrombin as a medium, which can successfully screen thrombin inhibitors and determine their inhibitory effect. The present application provides a new method for rapid detection of thrombin activity, and also provides a new strategy for screening and evaluation of thrombin inhibitors.

[0004] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0005] The present application provides a thrombin activity detection kit, which comprises fibrinogen and 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine.

[0006] The present application also provides the application of the above-mentioned thrombin activity detection kit in detecting the activity of thrombin for non-diagnostic purposes.

[0007] The application further provides a method for using the thrombin activity detection kit, wherein the sample to be detected is mixed with a fibrinogen solution and a 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution, and then is allowed to react for 15-25 min, after which the reaction system is imaged under ultraviolet light, the fluorescence area of the image is detected, and the activity of thrombin in the sample to be detected is calculated by substituting the standard curve prepared from standard thrombin.

[0008] Optionally, the fibrinogen solution is prepared from normal saline and has a concentration of 0.5-2 mg / mL; and the 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution is prepared from water and has a concentration of 80-120 μg / mL.

[0009] Optionally, the volume ratio of the sample to be detected, the fibrinogen solution and the 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution is 1:10:1.

[0010] The application further provides a kit for screening thrombin inhibitors, which comprises immobilized enzymes, fibrinogen and 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine.

[0011] The immobilized enzymes are obtained by loading thrombin on carbon nanotubes.

[0012] The application further provides the use of the kit in screening thrombin inhibitors or evaluating the inhibitory effect of thrombin inhibitors.

[0013] The application further provides a method for evaluating the inhibitory effect of thrombin inhibitors by using the kit, wherein the immobilized enzymes are mixed with the inhibitor to be detected and are allowed to incubate for 20-40 min, and then are mixed with a fibrinogen solution and a 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution, and are allowed to react for 15-25 min, after which the reaction system is imaged under ultraviolet light, the fluorescence area of the image is detected, and the activity of thrombin in the incubated carbon nanotubes is calculated by substituting the standard curve prepared from standard thrombin, and the inhibitory effect of the inhibitor to be detected is analyzed by comparing the activity of thrombin in the initial carbon nanotubes.

[0014] The application further provides a method for screening thrombin inhibitors by using the kit, wherein the immobilized enzymes are mixed with the active substance to be detected and are allowed to incubate for 20-40 min, and then are subjected to solid-liquid separation, and the obtained solid component is eluted with an acetonitrile aqueous solution, and the eluate is collected; the active monomer component in the eluate is identified by using a high-performance liquid chromatograph, and the inhibitory effect of the active monomer component is evaluated by using the above method.

[0015] The application discloses the following technical effects:

[0016] The present application utilizes the unique characteristics of the aggregation-induced emission (AIE) molecule, and detects the change of the luminescent body in the coagulation system by detecting the change of the luminescent body in the coagulation system, to monitor the thrombin activity. The potential thrombin inhibitor can inhibit the thrombin activity, resulting in a decrease in the luminescent product, and thus the sample containing the potential component can be screened. Based on this principle, the present application establishes a thrombin activity detection method, and establishes a method for screening, separating and evaluating the drugs containing the potential thrombin inhibitor active component by using immobilized thrombin as a medium, which can successfully screen the thrombin inhibitor and determine the inhibition effect.

[0017] The present application provides a thrombin activity determination method which is simple to operate and high in accuracy, provides a new method for rapid detection of thrombin activity, and provides a new strategy for screening and evaluation of thrombin inhibitors. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0019] Figure 1 Fig. 1 is a thrombin activity detection result graph; A is a thrombin activity monitoring image; B is a thrombin activity standard curve;

[0020] Figure 2 Fig. 2 is a specificity detection result of the thrombin activity detection method; A is an enzyme activity monitoring image; B is a fluorescence area size statistical graph; a-j are thrombin, alpha-glucosidase, beta-galactosidase, beta-lactamase, bovine serum albumin, lysozyme, acetaldehyde dehydrogenase, horseradish peroxidase, glucose oxidase and acetylcholinesterase;

[0021] Figure 3 Fig. 3 is an effectiveness detection result of the thrombin activity detection method; A is a monitoring image of the inhibition of thrombin activity by argatroban; B is a statistical graph of the inhibition of thrombin activity by argatroban; C is a monitoring image of the inhibition of thrombin activity by florfenicol, zanamivir and clavulanic acid potassium; D is a statistical graph of the inhibition of thrombin activity by florfenicol, zanamivir and clavulanic acid potassium;

[0022] Figure 4For solidification of enzyme structure characterization results and experimental results on drug adsorption; A is a TEM image; B is an infrared analysis image before and after enzyme immobilization; C is an XPS total spectrum image before and after enzyme immobilization; D-F are peak fitting images of the solidified enzyme; G-J are HPLC result images of argatroban, florfenicol, zanamivir, and potassium clavulanate, respectively, curve a represents before adsorption, and curve b represents after adsorption;

[0023] Figure 5 For thrombin inhibitor screening results; A is a schematic diagram of the thrombin inhibitor screening process; B is the thrombin inhibitor screening results, 1-21 are blank, magnolia, dragon's blood, dangshen, rhizoma atractylodis, epimedium, liver joy capsule, perilla, psoralea, licorice, radix polygoni multiflori, aconite, polygala, cassia seed, V-C yinqiao tablet, compound lobelia tablet, liver Su capsule, xiasangju granules, complex stomach powder capsule, and pulse network granules; C is a statistical diagram of the inhibition effect of thrombin inhibitors;

[0024] Figure 6 For the screening results of active substances in liver joy capsule; A is an HPLC analysis image of liver joy capsule, a is the initial extract of liver joy capsule, b is the unabsorbed liquid, and c is the eluent; B is a liquid chromatography-mass spectrometry analysis image of the eluent (c); C is an HPLC comparison image of the eluent (c) and oleanolic acid standard (d); D is a monitoring image of the inhibition of oleanolic acid on thrombin activity; E is a statistical diagram of the inhibition rate of oleanolic acid on thrombin;

[0025] Figure 7 For the molecular docking results of oleanolic acid and thrombin; A is a two-dimensional model of the interaction between oleanolic acid and thrombin; B is a three-dimensional model of the interaction between oleanolic acid and thrombin. DETAILED DESCRIPTION

[0026] The various illustrative embodiments of the present application will now be described in detail in connection with the following figures. This description is made for the purpose of demonstrating and describing particular embodiments of the present application and is not intended to limit the scope of the present application in any way. Rather, the following description is intended to provide an example of the various aspects, features, and embodiments of the present application.

[0027] It should be understood that the terms used in the present application are merely used to describe particular embodiments and are not intended to limit the present application. In addition, for numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated value or stated range, and between any other stated value or stated range, is also included within the present application. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0028] Unless otherwise indicated, all technical and scientific terms have the same meaning as those one of ordinary skill in the art of the disclosure would understand. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In case of conflict, the content of the present specification will control.

[0029] Many modifications and variations of the present disclosure described in the detailed description of the specification can be made by those of skill in the art without departing from the scope or spirit of the present disclosure. Other embodiments of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. The specification and examples of the present disclosure are illustrative only.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to permit but not limit the inclusion of elements or the number of elements, as well as the possibility that one or more other elements can be added or otherwise included.

[0031] The aggregation-induced emission (AIE) molecule TTVP (3,6-bis(benzothiophen-2-yl)pyrrolo[3,4-d]pyrimidine) in the following examples of the present disclosure is prepared by the following steps:

[0032] 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde (0.2 mM) and 1-(3-trimethylammoniopropyl)-4-methylpyridinium dibromide (0.2 mM) are dissolved in ethanol, a small amount of piperidine is added under nitrogen protection, and heated to reflux for 12 h. After cooling to room temperature, the solvent is removed by evaporation under reduced pressure to obtain a red-brown powder of TTVP.

[0033] Example 1

[0034] The present example provides a method for detecting thrombin activity, and the specific process is as follows:

[0035] Thrombin and fibrinogen are dissolved in physiological saline to 50 μg / mL and 1 mg / mL, respectively, and TTVP is dissolved in water to 100 μg / mL. The mixture is mixed in a ratio of 1:10:1, and after standing for 20 min, the fluorescence image of the reaction system is taken under 365 nm in the ultraviolet box. The image is analyzed using Photoshop software to detect the fluorescence area size of the biological aggregate.

[0036] The linear relationship between the fluorescence area of the bio-aggregates and the concentration of thrombin was analyzed using a gradient concentration (6.25, 12.5, 25, 50, 100 μg / mL) of thrombin solution. The results are shown in Figure 1 It can be seen that, in the range of 6.25-100 μg / mL, the fluorescence area of the bio-aggregates increases with the increase of the concentration of thrombin. There is a linear relationship between them, the regression equation is y = 63.8565x - 41.7797, R 2 = 0.9979.

[0037] Example 2

[0038] This example analyzes the specificity of the thrombin activity detection method of Example 1, the specific process is as follows:

[0039] In order to detect the specificity of the method, nine substances, α-glucosidase (b), β-galactosidase (c), β-lactamase (d), bovine serum albumin (e), lysozyme (f), acetaldehyde dehydrogenase (g), horseradish peroxidase (h), glucose oxidase (i) and acetylcholine esterase (j), were used instead of thrombin (a). The results are shown in Figure 2 . It can be seen that, in the detection system containing fibrinogen, only thrombin can produce fluorescent bio-aggregates.

[0040] Example 3

[0041] This example analyzes the effectiveness of the thrombin activity detection method of Example 1 by a positive drug (argatroban) and three negative drugs (florfenicol, zanamivir and clavulanate potassium), the specific process is as follows:

[0042] 0-10 μg / mL of argatroban was mixed with 50 μg / mL of thrombin at a ratio of 1:1 (v / v), and after standing for 30 min, 50 μL was mixed with 50 μL of TTVP (200 μg / mL) and 500 μL of fibrinogen (1 mg / mL), and continued to stand for 20 min. The aggregates were separated, the remaining liquid was removed, and the fluorescence image of the reaction system was taken in the ultraviolet box under 365 nm, and subsequent analysis was carried out. In addition, florfenicol, zanamivir and clavulanate potassium were selected as negative drugs, dissolved to 50 μg / mL, mixed with 50 μg / mL of thrombin at a ratio of 1:1 (v / v), and the subsequent operation was consistent with the positive drug. At the same time, a blank control group without adding any drug was set.

[0043] The results are shown in Figure 3 . It can be seen that, with the increase of the concentration of the positive drug argatroban, the thrombin activity is inhibited, and the fluorescence area of the bio-aggregates decreases; while the negative drugs florfenicol, zanamivir and clavulanate potassium have no effect on the thrombin activity, and the fluorescence area of the bio-aggregates is the same as the blank control.

[0044] Example 4

[0045] This example provides an immobilized enzyme for screening thrombin activity inhibitors. The specific process is as follows:

[0046] EDC, NHS and carbon nanotubes (MWCNT) were mixed in 5 mL of M ES (0.1 M) at a mass ratio of 15:18:10, and shaken for 1 h; a physiological saline solution containing 2 mg of thrombin was added to the reaction system, and shaking was continued for 2 h, and washing with deionized water was performed 3 times; the prepared immobilized enzyme was dispersed in physiological saline, and stored at -20 °C.

[0047] The surface morphology of MWCNT and immobilized enzyme was characterized using a transmission electron microscope (TEM) (FEI Talos F200x). The chemical composition of MWCNT and immobilized enzyme was analyzed using X-ray photoelectron spectroscopy (XPS) (ESCALAB 250Xi). The structural characteristics of the immobilized enzyme were analyzed using a Fourier infrared spectrometer (FT-IR) (Tensor 27). The results are shown in Figure 4 , Figure A is a transmission electron micrograph of the immobilized enzyme, and the tubular structure of the carbon nanotube can be clearly seen; Figure B shows curves a, b, and c, which represent carbon nanotubes, thrombin, and immobilized enzyme, respectively. Compared with carbon nanotubes, the immobilized enzyme has new characteristic absorption peaks at 624 cm -1 , 906 cm -1 , 1134 cm -1 , and 3294 cm -1 , wherein the appearance of 624 cm -1 and 906 cm -1 may be due to the bending vibration of C-H, 1134 cm -1 may be due to the stretching vibration of C-O, in addition, the peak at 3294 cm -1 is due to the stretching vibration of N-H or O-H, proving the successful combination of thrombin and carbon nanotubes; Figure C is the full spectrum analysis result of XPS, and Figures D-F are the peak fitting results of C, N, and O elements. The obvious enhancement of N element in the result of the immobilized enzyme proves that the enzyme is loaded on the carbon nanotube.

[0048] The application effect of the immobilized enzyme was verified, and the specific process is as follows:

[0049] Take 500 μL immobilized enzyme (100 μg / mL), remove the supernatant after mixing with 200 μL argatroban (50 μg / mL), adsorb for 30 min, separate the supernatant as unadsorbed liquid; similarly, take 500 μL immobilized enzyme (100 μg / mL), remove the supernatant after mixing with 200 μL negative drug (50 μg / mL), adsorb for 30 min, separate the supernatant as unadsorbed liquid. Finally, analyze the initial liquid and unadsorbed liquid of active substances using high performance liquid chromatograph.

[0050] Argatroban chromatographic conditions: chromatographic column: (Shim-pack GIS C18 (4.6 mm x 250 mm, 5 μm)), mobile phase: 0.1% phosphoric acid: methanol - 40:60, column temperature: 35°C, flow rate: 1 mL / min.

[0051] Florfenicol chromatographic conditions: chromatographic column: (Shim-pack GIS C18 (4.6 mm x 250 mm, 5 μm)), mobile phase: water: acetonitrile - 30:70, column temperature: 30°C, flow rate: 1 mL / min.

[0052] Zanamivir chromatographic conditions: chromatographic column: (Shim-pack GIS C18 (4.6 mm x 250 mm, 5 μm)), mobile phase: 0.1% phosphoric acid: methanol - 60:40, column temperature: 30°C, flow rate: 1 mL / min.

[0053] Potassium clavulanate chromatographic conditions: chromatographic column: (Shim-pack GIS C18 (4.6 mm x 250 mm, 5 μm)), mobile phase: 0.1% phosphoric acid: methanol - 45:55, column temperature: 30°C, flow rate: 1 mL / min.

[0054] The results are shown in G-J of Figure 4 The adsorption rate of immobilized enzyme to positive drugs is as high as 80%, while the adsorption rate to negative drugs is less than 10%, which proves that immobilized enzyme can effectively adsorb active ingredients.

[0055] Example 5

[0056] This example based on the immobilized enzyme prepared in Example 4, provides a kit for screening thrombin activity inhibitors, and a screening method of thrombin inhibitor active substances, and a method for evaluating the inhibitory effect of thrombin inhibitor active substances. The process of the screening method is as follows: Figure 5A. The 13 kinds of Chinese medicine extracts (Magnolia, Dragon's Blood, Codonopsis, Giant Knotweed, Fried Atractylodes, Herba Epimedii, Perilla, Psoralea, Licorice, Radix Polygoni Multiflori, Aconite, Polygala, Cassia Seed, numbered 2-14) and 7 kinds of compound preparations (Ganxile Capsule, Vitamin C Yinqiao Tablet, Compound Lobelia Capsule, Gansu Capsule, Xiasangju Granules, Fugan San Capsule, Mailuoning Granules, numbered 15-21) were used as test materials to screen active substances and evaluate the inhibitory effect of active substances. The specific process is as follows:

[0057] 1. Screening method of thrombin inhibitor active substances:

[0058] The Chinese medicine powder and compound preparation powder were extracted with anhydrous ethanol for 30 min, concentrated and dried, then redissolved to 500 μg / mL, mixed with immobilized enzyme (50 μg / mL) at a ratio of 1:1 (v / v) and incubated for 30 min. Then, 50 μL of the reacted solution was mixed with 50 μL TTVP (200 μg / mL) and 500 μL fibrinogen (1 mg / mL), and continued to stand for 20 min. Finally, the aggregates were separated, and whether the thrombin activity was inhibited was analyzed by taking pictures. The results are shown in Figure 5 B and C of FIG. 1, it can be seen that the Ganxile Capsule extract has a significant inhibitory effect on thrombin.

[0059] Next, the Ganxile Capsule extract was used as the active substance initial solution, 500 μL of immobilized enzyme (100 μg / mL) was taken, after removing the supernatant, it was mixed with 200 μL of Ganxile Capsule extract (1 mg / mL), adsorbed for 30 min, and the supernatant was separated as the unadsorbed liquid; the remaining solid was eluted with eluent (water:acetonitrile = 1:1, v / v) for 30 min, and the eluent was obtained by centrifugation. The active substance initial solution, unadsorbed liquid and eluent were analyzed by high performance liquid chromatograph, and the potential active monomers in the eluent were identified by liquid chromatography-mass spectrometry. The chromatographic conditions of Ganxile Capsule are as follows: chromatographic column: (Shim-pack GIS C18 (4.6 mm x 250 mm, 5 μm)), mobile phase: 0.1% phosphoric acid:methanol-45:55, column temperature: 30°C, flow rate: 1 mL / min.

[0060] The HPLC analysis image of Ganxile Capsule is shown in Figure 6 A of FIG. 2, a is the Ganxile Capsule extract initial solution, b is the unadsorbed liquid, and c is the eluent. Further analysis was performed on the eluent (c), and the results are shown in Figure 6 B of FIG. 3, it is speculated that the eluent contains oleanolic acid. Further comparative analysis of the eluent composition was performed using oleanolic acid standard, and the results are shown in Figure 6 C of FIG. 4, it can be determined that the eluent contains oleanolic acid.

[0061] 2. Method for evaluating inhibitory effect of thrombin inhibitor active substance:

[0062] Different concentrations of oleanolic acid (50, 80, 110, 140, 170, 200, 230, 260, 290, 320 μg / mL) were mixed with immobilized enzyme (50 μg / mL) at a ratio of 1:1 (v / v) and incubated for 30 min. Then, 50 μL of the reacted solution was mixed with 50 μL TTVP (200 μg / mL) and 500 μL fibrinogen (1 mg / mL), and continued to stand for 20 min. Finally, the inhibition of thrombin activity was analyzed by taking a photo. The results are shown in Figs. D and E. As can be seen, the inhibition rate of thrombin activity gradually increased with the increase of the concentration of oleanolic acid. Figure 6

[0063] Finally, molecular docking analysis was performed on oleanolic acid and thrombin. The crystal structure of thrombin was obtained from the PDB database (PDB ID: 1DWC), and the docking was preprocessed by the protein preparation wizard module in Discovery Studio software. The process included protein preprocessing, natural ligand state regeneration, hydrogen bond distribution optimization, and protein energy minimization. The binding site was determined based on the binding region of the ligand in the original co-crystal structure of the protein. The results are shown in Fig. F. As can be seen, oleanolic acid can form specific interactions with the residues GLN-38, GLU-39, TRP-141, and GLU-192 of the thrombin protein to inhibit the activity of thrombin. Figure 7

[0064] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope of the present application as defined by the claims.​​

Claims

1. A kit for detecting thrombin activity, characterized by, The thrombin activity detection kit comprises fibrinogen and 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine.

2. Use of the thrombin activity detection kit of claim 1 in detecting thrombin activity for non-diagnostic purposes.

3. A method of using the test kit for thrombin activity as claimed in claim 1, wherein, The sample to be detected is mixed with a fibrinogen solution and a 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution, and is allowed to stand for 15-25 min for reaction. After the reaction is completed, the image of the reaction system is taken under ultraviolet light, the fluorescent area of the image is detected, and the activity of thrombin in the sample to be detected is calculated by substituting the standard curve prepared from standard thrombin.

4. The method of use of claim 3, wherein, The fibrinogen solution is prepared from physiological saline with a concentration of 0.5-2 mg / mL; and the 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution is prepared from water with a concentration of 80-120 μg / mL.

5. The method of use of claim 3, wherein, The volume ratio of the sample to be detected, the fibrinogen solution and the 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution is 1:10:

1.

6. A kit for screening for thrombin inhibitors, characterized in that, The kit comprises an immobilized enzyme, fibrinogen and 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine. The immobilized enzyme is obtained by loading thrombin on carbon nanotubes.

7. Use of the kit of claim 6 in screening thrombin inhibitors or evaluating the inhibitory effect of thrombin inhibitors.

8. A method for evaluating the inhibitory effect of a thrombin inhibitor using the kit according to claim 6, characterized by, After the immobilized enzyme is mixed with the inhibitor to be detected and incubated for 20-40 min, the immobilized enzyme is mixed with a fibrinogen solution and a 3,6-bis(benzothiophene-2-yl) pyrrolo[3,4-d]pyrimidine solution, and is allowed to stand for 15-25 min for reaction. After the reaction is completed, the image of the reaction system is taken under ultraviolet light, the fluorescent area of the image is detected, and the activity of thrombin in the incubated carbon nanotubes is calculated by substituting the standard curve prepared from standard thrombin. The inhibitory effect of the inhibitor to be detected is analyzed by comparing the activity of thrombin in the initial carbon nanotubes.

9. A method for screening a thrombin inhibitor using the kit according to claim 6, characterized by, After the immobilized enzyme is mixed with the active substance to be detected and incubated for 20-40 min, solid-liquid separation is performed, and the obtained solid component is eluted with an acetonitrile aqueous solution, and the eluate is collected. The active monomer component in the eluate is identified by high performance liquid chromatography, and the inhibitory effect of the active monomer component is evaluated by the method of claim 8.

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