Fluorescent sensor nano-particles based on thrombin and preparation method, related product and application of fluorescent sensor nano-particles

Abstract

The invention provides fluorescence sensor nanoparticles based on thrombin, a preparation method of the fluorescence sensor nanoparticles, a thrombin activity detection method, an anticoagulant active substance in ligusticum wallichii and a screening method of the anticoagulant active substance, and belongs to the technical field of nanometer fluorescence sensors and traditional Chinese medicine active ingredients. The fluorescence sensor nano particles are prepared by crosslinking and assembling AIE515 and a thrombin substrate S-2238. When the thrombin exists, the thrombin cuts off the thrombin substrate S-2238, so that the nanoparticles of the fluorescence sensor are decomposed, and the aggregation-induced fluorescence intensity is reduced. The fluorescence sensor nanoparticles provided by the invention can realize rapid detection of thrombin activity, and also can realize rapid screening of anticoagulant components in traditional Chinese medicine ligusticum wallichii.

Description

Technical Field

[0001] The invention belongs to the technical field of nano fluorescent sensors and active ingredients of traditional Chinese medicines, and relates to a fluorescent sensor nanoparticle based on thrombin and a preparation method thereof, a method for detecting thrombin activity, and anticoagulant active substances in chuanxiong and a method for screening thereof. Background Art

[0002] Chuanxiong is a herbal medicine widely used in traditional Chinese medicine, mainly used to promote blood circulation and remove blood stasis. Its unique bioactive ingredients, such as chuanxiongzine, ferulic acid, sodium ferulate, etc., have the effect of promoting blood circulation and removing blood stasis, can dilate coronary arteries, increase coronary flow, and relieve angina pectoris. They are the focus of modern drug research in terms of anticoagulation and antithrombosis. More importantly, component chemistry is now crucial for understanding the structure and function of various compounds in Chuanxiong. By analyzing a certain type of component in Chuanxiong, researchers can greatly facilitate the functional identification of Chuanxiong, such as chuanxiongzine can promote blood circulation and remove blood stasis, and dilate blood vessels. However, as far as Chuanxiong alone is concerned, there are dozens of components in it, which greatly limits the analysis of the anticoagulant function of Chuanxiong. Therefore, a new method is urgently needed to quickly screen out the anticoagulant components in Chuanxiong.

[0003] The current method for screening thrombin inhibitors includes combinatorial chemistry, Chinese medicine spectrum-effect correlation method, affinity ultrafiltration method, serum pharmacology and pharmacology combined method, molecular biochromatography, network pharmacology, etc. However, there is no method that can simultaneously realize the detection of thrombin and the screening of anticoagulant active substances. Therefore, it is urgent to establish a fast, economical and convenient method to detect thrombin activity and screen thrombin inhibitors. The present invention is a fluorescent sensor based on thrombin, which can be used for rapid screening of anticoagulant active components in Chinese medicine, which greatly facilitates the screening of anticoagulant active components in Chuanxiong.

[0004] Thrombin, also known as human α-thrombin (factor IIa), is a globular trypsin-like serine protease with a size of approximately 37 kilodaltons (KD) and is present in the common coagulation pathway. Thrombin is a key protein in hemostasis and thrombosis. When the body is injured, thrombin will quickly reach the damaged site, activate coagulation factors, and transform the blood from a liquid state to a coagulated state, thereby stopping bleeding and reducing blood loss. If the coagulation system is overactivated, resulting in a continuous increase in the activity of thrombin in the body, thrombin will convert fibrinogen into fibrin. After fibrin is activated, it can form fibrin polymers and aggregate at the broken blood vessels, ultimately increasing the risk of thrombosis. Thrombin also activates factor XIII, which then cross-links fibrin to strengthen the formed clot. Thrombin plays an important role in vascular injury and various pathological activities, such as thrombotic diseases, atherosclerosis, and tumors. At the same time, thrombin can serve as a major target for regulating inflammation, atherosclerosis, and thrombosis. In recent years, with the rapid development of biotechnology, sensors based on specific biomarkers have been widely used in the rapid screening of active ingredients in traditional Chinese medicine. Among them, thrombin, as an important blood coagulation factor, plays a particularly critical role in the screening of anticoagulant activity.

[0005] In this context, the development of a thrombin-based fluorescent sensor is of great significance for the rapid screening of anticoagulant active components in the various components of Chuanxiong. Combining the specific recognition ability of thrombin and the high sensitivity of fluorescence technology, this sensor can achieve efficient and accurate detection of potential anticoagulant active components in the various components of Chuanxiong. This not only helps to deeply understand the "blood circulation and stasis removal" mechanism of Chuanxiong, but also provides a scientific basis for the clinical application of its medicinal ingredients.

[0006] Chinese patent application 202210931602.3 provides a fluorescent sensor for detecting thrombin activity and thrombin inhibitor activity, and its preparation method and application. The fluorescent sensor includes nanoparticles obtained by cross-linking an aggregation-induced luminescence probe with a thrombin chromogenic substrate. The fluorescent sensor provided by this technology is simple to prepare, has mild reaction conditions, and is low in cost. It is easy to prepare in batches. It can be used to detect both thrombin activity and thrombin inhibitor activity, and can also be used for the screening of anticoagulant components in drugs. It has the advantages of low cost, simplicity, rapidity and high efficiency.

[0007] Chinese patent application 202310139744.0 provides a long-wavelength AIE array sensor, a preparation method and application thereof. The TPPESA array sensor designed by this technology can accurately identify and detect glycosaminoglycans with a concentration as low as 1 μg / mL. In terms of detecting glycosaminoglycan mixtures (impurities), the array sensor of this technology can detect pollutants in samples with a content as low as 2%. The detection operation is simple, fast and efficient. The detection of a variety of carbohydrate compounds can be achieved: the fluorescent sensor array constructed by this technology can be used for quantitative, accurate and rapid detection of the four common glycosaminoglycans in water samples and serum environments. In addition, the fluorescent sensor array of the present invention does not produce any harmful byproducts when used for the detection of carbohydrate compounds, and there is no secondary pollution.

[0008] Chinese patent application 202311037568.6 provides an AIE probe 935 covalently linked to a D-type heparin sodium binding peptide, and is used for the detection of heparin sodium. 935 is obtained by covalently linking a D-type amino acid to TPE-COOH (tetraphenylethylene-carboxyl) through a solubilizing group AEEA. Heparin sodium can induce 935 to produce an AIE effect. 935 has a good linear relationship with 0-6.1 μg / mL heparin sodium (R2=0.9917), and the minimum detection limit is 17.7 ng / mL. 935 has the advantages of high stability, good water solubility, high sensitivity, low cost, and simple operation. Compared with L-type TPE-1 (L-type heparin sodium binding peptide AG73 covalently bound to TPE-COOH), the water solubility and protease stability of D-type 935 are significantly enhanced, and it is worthy of promotion in the determination of heparin sodium content.

[0009] However, the prior art has not provided a thrombin-based fluorescent sensor nanoparticle with better detection effect, nor has it provided its application in screening anticoagulant active components in Chuanxiong medicines. Summary of the invention

[0010] In view of this, in order to solve the above-mentioned bottleneck problems that cannot be solved by the prior art, the purpose of the present invention is to provide a thrombin-based fluorescent sensor nanoparticle and a preparation method thereof, a method for detecting thrombin activity, and an anticoagulant active substance in Chuanxiong and a method for screening thereof.

[0011] To achieve the above-mentioned purpose, the present invention provides a thrombin-based fluorescent sensor nanoparticle, wherein the fluorescent sensor nanoparticle is composed of AIE 515 and thrombin substrate S-2238.

[0012] The AIE 515 The chemical structure is as follows: .

[0013] Preferably, the fluorescent sensor nanoparticles are composed of AIE 515 It is prepared by cross-linking and assembling with thrombin substrate S-2238.

[0014] In another aspect, the present invention provides a method for preparing the above-mentioned fluorescent sensor nanoparticles, comprising the following steps: Mix water with thrombin substrate S-2238, then add tetrahydrofuran, glutaraldehyde, and finally AIE 515 , obtain a mixed solution, stir to react, and keep it still in the dark to obtain fluorescent sensor nanoparticles.

[0015] Preferably, the rotation speed of the stirring reaction is 200-600 rpm.

[0016] More preferably, the rotation speed of the stirring reaction is 400-600 rpm.

[0017] More preferably, the rotation speed of the stirring reaction is 400 rpm.

[0018] Preferably, the stirring reaction time is 0.5-1.5h.

[0019] More preferably, the stirring reaction time is 0.5-1.0 h.

[0020] More preferably, the stirring reaction time is 1.0 h.

[0021] Preferably, the concentration of the thrombin substrate S-2238 in the mixed solution is 0.25-2 mg / mL.

[0022] More preferably, the concentration of the thrombin substrate S-2238 in the mixed solution is 1-1.15 mg / mL.

[0023] As an embodiment of the present invention, the concentration of the thrombin substrate S-2238 in the mixed solution is 1 mg / mL.

[0024] As an embodiment of the present invention, the concentration of the thrombin substrate S-2238 in the mixed solution is 1.17 mg / mL.

[0025] Preferably, and as a specific example of the present invention, the water, the thrombin substrate S-2238, the tetrahydrofuran, the glutaraldehyde and the AIE 515 The dosage ratio is: 2.3mL: 0.35mL (10mg / mL): 0.3mL: 0.01mL: 0.05mL (10mg / mL). After the tetrahydrofuran evaporates, water is added to 3mL, and the final AIENP concentration is 0.17mg / mL.

[0026] Preferably, the time of standing in dark place is 10-20 hours, more preferably 12 hours. The purpose of standing in dark place is to volatilize tetrahydrofuran. After the tetrahydrofuran volatilizes, the fluorescent sensor nanoparticles are obtained.

[0027] In yet another aspect, the present invention provides a kit for detecting thrombin activity, comprising the above-mentioned fluorescent sensor nanoparticles or the fluorescent sensor nanoparticles prepared by the above-mentioned preparation method.

[0028] Preferably, the kit further comprises a container, including but not limited to a well plate, a cuvette, a test tube, a dropper, and the like.

[0029] Preferably, the kit further comprises a thrombin standard.

[0030] Preferably, the kit further comprises a light-proof packaging box.

[0031] Preferably, the kit further comprises a solvent. The solvent includes but is not limited to water, sodium chloride aqueous solution, buffered aqueous solution, etc. More preferably, the buffered aqueous solution is PBS (phosphate buffered saline) with a pH value of 7.0. More preferably, the concentration of the buffered aqueous solution is 1× concentration (i.e. 0.01 mol / L).

[0032] In another aspect, the present invention provides a method for detecting thrombin activity using the fluorescent sensor nanoparticles / fluorescent sensor nanoparticles prepared by the preparation method / the kit, comprising the following steps: S1. dissolving thrombin in a buffer solution to obtain a thrombin solution; S2. Add the thrombin solution obtained in step S1 to the well plate, incubate once, add the fluorescent sensor nanoparticles, incubate twice, add water, incubate three times, and measure the fluorescence intensity.

[0033] In another aspect, the present invention provides a method for screening anticoagulant components using the fluorescent sensor nanoparticles / fluorescent sensor nanoparticles prepared by the preparation method / the kit, comprising the following steps: T1, measure the blank fluorescence intensity A 1 ; T2. Determine the fluorescence intensity A of the thrombin control group 2 ; T3. Co-incubate the anticoagulant component with thrombin and measure the fluorescence intensity A after co-incubation 3 ; T4. Determine the basal fluorescence intensity A of the anticoagulant component 4 ; T5, A obtained according to step T1 1 , A obtained in step T2 2 , A obtained in step T3 3and A obtained in step T4 4 , judge A 3 -A 4 ∈[A 2 , A 1 ] is established, if established, then the anticoagulant component has an inhibitory effect on thrombin; T6, using the A obtained in step T1 1 , A obtained in step T2 2 , A obtained in step T3 3 and A obtained in step T4 4 The thrombin inhibition rate was calculated, and the calculation formula of the thrombin inhibition rate was as follows: Thrombin inhibition rate = (A 3 -A 4 )÷A 2 ×100%.

[0034] Preferably, step T1 comprises the following steps: adding a buffer solution to the well plate, incubating once, adding fluorescent sensor nanoparticles, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the blank fluorescence intensity A 1 .

[0035] Preferably, step T2 comprises the following steps: adding thrombin solution to the well plate, incubating once, adding fluorescent sensor nanoparticles, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the fluorescence intensity A of the thrombin control group. 2 .

[0036] Preferably, step T3 comprises the following steps: adding thrombin solution to the well plate, incubating once, adding anticoagulant components, incubating twice, adding fluorescent sensor nanoparticles, incubating three times, adding water, incubating four times, measuring the fluorescence intensity, and obtaining the fluorescence intensity A after the co-incubation. 3 .

[0037] Preferably, step T4 comprises the following steps: adding a buffer solution to the well plate, incubating once, adding an anticoagulant component, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the base fluorescence intensity A of the anticoagulant component. 4 .

[0038] In another aspect, the present invention provides an anticoagulant active substance in Ligusticum chuanxiong screened by the above method, wherein the anticoagulant active substance in Ligusticum chuanxiong is extracted from Ligusticum chuanxiong medicinal liquid, and the inhibition rate of the anticoagulant active substance in Ligusticum chuanxiong on thrombin is greater than 10%.

[0039] Preferably, the method for preparing the Ligusticum chuanxiong medicinal solution comprises the following steps: (1) Crush Chuanxiong, extract with ethanol, and concentrate under reduced pressure to obtain an ethanol extract; (2) resuspending the alcohol extract obtained in step (1) in water, extracting with petroleum ether and ethyl acetate respectively, to obtain a petroleum ether layer and an ethyl acetate layer respectively; (3) The petroleum ether layer and the ethyl acetate layer obtained in step (2) are subjected to column chromatography respectively, and the eluted portion obtained is the Ligusticum chuanxiong medicinal solution.

[0040] More preferably, the method for preparing the Ligusticum chuanxiong medicinal solution comprises the following steps: (1) Take 10 kg of dried rhizomes of Chuanxiong, crush them, and extract them with 25 L of 95% ethanol for 4 times, each time for 48 h. Combine the 4 extracts and concentrate them under reduced pressure to obtain the ethanol extract.

[0041] (2) The alcohol extract was resuspended in water, and extracted with petroleum ether, ethyl acetate, and n-butanol in turn to obtain a petroleum ether layer, an ethyl acetate layer, and an n-butanol layer, respectively.

[0042] (3) The petroleum ether layer and the ethyl acetate layer are separated and eluted using open silica gel column chromatography to obtain different components of Chuanxiong medicinal liquid.

[0043] In another aspect, the present invention provides the use of the above-mentioned fluorescent sensor nanoparticles, preparation methods, and kits in screening anticoagulant active substances in Chuanxiong.

[0044] Compared with the prior art, the present invention has the following beneficial effects: (1) Wide range of applications: The present invention can not only be used to quickly screen the anticoagulant active components in the components of the traditional Chinese medicine Chuanxiong, but also be used to detect thrombin activity. This versatility gives it a wide range of application prospects in biomedical research and the modernization of traditional Chinese medicine.

[0045] (2) Simple preparation process and high safety: The sensor preparation process of the present invention does not require complex instruments and cumbersome steps, which greatly simplifies the preparation process and makes it more suitable for large-scale application. The present invention does not require dangerous conditions such as high temperature and high pressure during the detection process, and the reaction conditions are mild. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 3 is a graph of the fluorescence intensity of nanoparticles at different stirring speeds in Example 1, wherein the curves in the direction of the arrows represent rotation speeds of 400 rpm, 500 rpm, 600 rpm, 300 rpm and 200 rpm, respectively.

[0047] Figure 2 3 is a graph of the fluorescence intensity of the nanoparticles at different concentrations of the thrombin substrate S-2238 in Example 2, wherein the curves in the direction of the arrows represent the concentrations of the thrombin substrate S-2238 as 1.0, 2.0, 0.5, 0.25, and 1.5 mg / mL, respectively.

[0048] Figure 3 It is a fluorescence intensity diagram of the nanoparticles at different synthesis times in Example 3, wherein the curves in the direction of the arrows represent 1.0 h, 0.5 h, 0.75 h, and 1.5 h, respectively.

[0049] Figure 4 This is a graph showing the effect of different thrombin concentrations (0-50 μg / mL) on the fluorescence intensity of nanoparticles in Example 4, wherein the curves in the direction of the arrows represent 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

[0050] Figure 5 This is a graph showing the effect of different thrombin concentrations (0, 50-300 μg / mL) on the fluorescence intensity of nanoparticles in Example 4.

[0051] Figure 6 3 is a fluorescence intensity graph of the AIENP-thrombin system treated with different concentrations of argatroban in Example 5, wherein the curves in the direction of the arrows represent argatroban concentrations of 75, 100, 25, 50, and 0 μg / mL, respectively.

[0052] Figure 7 This is a graph of the relative intensity of the emission wavelength of AIENP at 515 nm over 7 days in Example 6.

[0053] Figure 8 This is a bar graph of the screening inhibition rate of AIENP on the monomer anticoagulant components of the relevant components in Chuanxiong in Example 7, wherein the meanings represented by the numbers on the abscissa are as follows: 1: HLC-I-31C, 2: HLC-I-11H, 3: HLC-I-11K, 4: HLC-I-15C34A, 5: HLC-I-22A35F, 6: HLC-I-2-14E, 7: HLC-I-31A, 8: HLC-I-32D, 9: HLC-I-33I, 10: HLC-I-27E, 11: Ligusticum chuanxiong lactone I, 12: Angelica sinensis Lactone A, 13: Ligustrazine, 14: HLC-I-4a, 15: HLC-I-4b, 16: HLC-I-4c, 17: HLC-I-4h, 18: MLC-I-4e, 19 :HLC-I-4f, 20:HLC-I-4g, 21:HLC-I-5a, 22:HLC-I-5b, 23:HLC-I-5c, 24:HLC-1-5d, 25: HLC-I-5e, 26:HLC-I-5f, 27:HLC-I-5g, 28:HLC-1-5h, 29:HLC-I-7d, 30:HLC-I-8b, 31:H LC-I-12j, 32:HLC-I-12k, 33:HLC-I-12l, 34:HLC-I-12m, 35:HLC-I-12n, 36:HLC-I-10d , 37:HLC-II-5a, 38:HLC-II-5b, 39:HLC-II-5c, 40:HLC-I1-5d, 41:HLC-II-5e, 42:HLC- II-6a, 43: HLC-II-6d, 44: HLC-II-6e, 45: HLC-II-6f, 46: HLC-II-10d, 47: HLC-II-10e.

[0054] Fig. 9 This is a bar graph of the screening inhibition rate of the anticoagulant activity of 9 standard products in Chuanxiong by AIENP in Example 8, wherein the meanings represented by the numbers on the abscissa are as follows: 1: Ferulic acid, 2: Ligusticum chuanxiong lactone A, 3: Ligusticum chuanxiong lactone H, 4: Ligusticum chuanxiong lactone I, 5: Scopoletin, 6: Ligustilide, 7: Isochlorogenic acid, 8: Angelica lactone, 9: Chlorogenic acid.

[0055] Fig.10 These are transmission electron microscope photos of AIENP in Example 9 at different magnifications.

[0056] Fig.11 This is the hydrated particle size distribution diagram of AIENP in Example 9.

[0057] Fig.12 This is the Zeta potential curve of AIENP in Example 9. DETAILED DESCRIPTION

[0058] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0059] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0060] In the following examples, the fluorescence microplate reader used for reading fluorescence values ​​was Spark multifunctional microplate reader purchased from Tecan, Switzerland; the zeta potential was measured using a Malvern Panalytical instrument; the transmission electron microscope was used to measure PDA-PE at an accelerating voltage of 200 kV using a JEOL JEM-200CX instrument; thrombin was purchased from Shanghai MacLean Biochemical Co., Ltd.; the thrombin chromogenic substrate S-2238 was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; glutaraldehyde was purchased from Sigma, Germany; tetrahydrofuran (THF) was purchased from Beijing Bailingwei Technology Co., Ltd.; and phosphate buffer solution (PBS) was purchased from Wuhan Saiwei Biotechnology Co., Ltd.

[0061] Example 1 Investigate the effect of stirring speed (200rpm, 300rpm, 400rpm, 500rpm, 600rpm) on AIENP synthesis: (1) First, add 2300 μL of ultrapure water to the vial, then add 350 μL of 10 mg / mL thrombin substrate S-2238 aqueous solution. Next, add 300 μL of THF and 10 μL of glutaraldehyde to the solution to enhance the stability of the reaction mixture through cross-linking. Finally, slowly add 50 μL of AIE 515 Probe (10 mg / mL AIE 515 THF solution of the molecule, the same below).

[0062] (2) Stir at room temperature at 200 rpm, 300 rpm, 400 rpm, 500 rpm, or 600 rpm for 1 h.

[0063] (3) After the reaction is completed, the mixture is kept away from light for 12 h to allow THF to evaporate. After removing THF, AIENP is obtained.

[0064] (4) The fluorescence intensity of AIENP at different rotation speeds was measured using an ELISA reader.

[0065] like Figure 1 As shown, when the rotation speed is 400 rpm, the fluorescence intensity of AIENP is optimal.

[0066] Example 2 The effects of different concentrations of thrombin substrate S-2238 (0.25, 0.5, 1.0, 1.5, 2.0 mg / mL) on AIENP synthesis were investigated: (1) Take 5 identical vials, the concentrations of thrombin substrate S-2238 in these 5 vials are 0.25, 0.5, 1.0, 1.5, 2.0 mg / mL, respectively, and number them 1, 2, 3, 4, 5. First, add 1300, 1275, 1225, 1175, 1125 μL of ultrapure water to the vials in sequence. Then, add 25, 50, 100, 150, 200 μL of thrombin substrate S-2238 (10 mg / mL) to the vials in sequence in sequence.

[0067] Next, 150 μL of THF and 5 μL of glutaraldehyde were added to each bottle to enhance the stability of the reaction mixture through cross-linking. Finally, AIE 515 Probe 25 μL.

[0068] (2) Stir at 400 rpm for 1 h at room temperature.

[0069] (3) After the reaction is completed, the mixture is kept away from light for 12 h to allow THF to evaporate. After removing THF, AIENP is obtained.

[0070] (4) The fluorescence intensity of AIENP at different rotation speeds was measured using an ELISA reader.

[0071] like Figure 2 As shown, when the concentration of thrombin substrate S-2238 was 1.0 mg / mL, the fluorescence intensity of AIENP was optimal.

[0072] Example 3 Investigate the effects of different reaction times (0.5h, 0.75h, 1h, 1.5h) on the synthesis of AIENP: (1) First, add 2300 μL of ultrapure water to the vial, then add 350 μL of 10 mg / mL thrombin substrate S-2238 aqueous solution. Next, add 300 μL of THF and 10 μL of glutaraldehyde to the solution to enhance the stability of the reaction mixture through cross-linking. Finally, slowly add 50 μL of AIE 515 Probe.

[0073] (2) Stir at room temperature at 400 rpm for 0.5 h, 0.75 h, 1 h, and 1.5 h.

[0074] (3) After the reaction is completed, the mixture is kept away from light for 12 h to allow THF to evaporate. After removing THF, AIENP is obtained.

[0075] (4) The fluorescence intensity of AIENP at different rotation speeds was measured using an ELISA reader.

[0076] like Figure 3 As shown, the fluorescence intensity of AIENP is optimal when the stirring time is 1 h.

[0077] In the following Examples 4-9, the AIENP (AIENP solution) used is the AIENP prepared by the optimal method in Examples 1-3, and the specific preparation process is as follows: (1) First, add 2300 μL of ultrapure water to the vial, then add 350 μL of 10 mg / mL thrombin substrate S-2238 aqueous solution. Next, add 300 μL of THF and 10 μL of glutaraldehyde to the solution to enhance the stability of the reaction mixture through cross-linking. Finally, slowly add 50 μL of AIE 515 Probe.

[0078] (2) Stir at room temperature at 400 rpm for 1 h.

[0079] (3) After the reaction is completed, the mixture is kept away from light for 12 h to allow THF to evaporate. After removing THF, AIENP is obtained.

[0080] After testing, the obtained AIENP was an AIENP solution with a mass concentration of 0.17 mg / mL. The AIENP (0.17 mg / mL) solution was sealed and kept for future use.

[0081] Example 4 The effects of different thrombin concentrations (0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL) on the specific hydrolysis of AIENP were investigated: (1) Dissolve thrombin in PBS buffer (pH 7) and shake with a turbine to obtain a thrombin stock solution with a concentration of 0.5 mg / mL.

[0082] (2) Investigation of thrombin concentration range (0μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL): First, add 60, 58, 56, 54, 52, and 50μL PBS buffer to the 96-well plate, respectively. Then, add 0, 2, 4, 6, 8, and 10μL thrombin stock solution, incubate in a 37℃ shaker for 5 minutes, and take out immediately. Take out the 96-well plate, add 40μL AIENP solution to each of the above wells, and incubate in a 37℃ shaker for 20 minutes. Use an enzyme reader to measure the fluorescence intensity at different thrombin contents. The results are as follows: Figure 4 As shown: as the thrombin concentration continues to increase, the fluorescence intensity continues to decrease, and the decrease is relatively uniform.

[0083] (3) Investigation of thrombin concentration range (0μg / mL, 50μg / mL, 100μg / mL, 150μg / mL, 200μg / mL, 250μg / mL, 300μg / mL): First, add 60, 50, 40, 30, 20, 10, and 0μL PBS buffer to the 96-well plate, respectively. Then, add 0, 10, 20, 30, 40, 50, and 60μL thrombin stock solution, incubate in a 37°C shaker for 5 minutes, and take out immediately. Take out the 96-well plate, add 40μL AIENP solution to each of the above wells, and incubate in a 37°C shaker for 20 minutes. Use an enzyme reader to measure the fluorescence intensity at different thrombin contents. The results are as follows. Figure 5 As shown: When the thrombin concentration is 50-300 μg / mL, the fluorescence intensity does not change significantly, which is due to the re-aggregation of the poorly soluble AIE monomers. Therefore, it is determined that the best effect is achieved when the thrombin concentration is 50 μg / mL.

[0084] Example 5 The effects of different argatroban concentrations (0μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL) on fluorescence intensity were investigated.

[0085] Argatroban is an anticoagulant small molecule drug used here as a positive drug.

[0086] (1) Dissolve argatroban in PBS buffer (pH 7) and shake with a turbine to obtain an argatroban stock solution with a concentration of 0.5 mg / mL.

[0087] (2) Investigation of the argatroban concentration range (0μg / mL, 25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL): First, add 40 μL of PBS buffer to each of the 5 wells of a 96-well plate. Then, add 10 μL of thrombin, incubate in a 37°C shaker for 5 min, and take it out immediately. Take out the 96-well plate, and add 0, 5, 10, 15, and 20 μL of argatroban stock solution to the above wells in turn, and incubate in a 37°C shaker for 10 min. Take out the 96-well plate, add 30 μL of AIENP (0.17 mg / mL) solution to each of the above wells, and incubate in a 37°C shaker for 15 min. Take out the 96-well plate, and add 20, 15, 10, 5, and 0 μL of ultrapure water to the above wells in turn, and incubate in a 37°C shaker for 5 min. Use an enzyme-labeled instrument to measure the fluorescence intensity at different argatroban contents. The results are as follows: Figure 6 After the positive drug argatroban was added to AIENP and thrombin, the fluorescence could be restored; and when the concentration of argatroban was 75 μg / mL, the fluorescence was the highest, which was 1.6449 times that when no positive drug was added.

[0088] Example 6 Investigating the fluorescence stability of AIE probes: The fluorescence intensity of the AIENP solution was measured by an ELISA instrument at the same time for 7 consecutive days. Figure 7 As shown, the AIENP was successfully prepared and has stability, that is, the fluorescence remained basically unchanged within seven days.

[0089] Example 7 Screening of anticoagulant active substances in various components of Chuanxiong: (1) Fluorescence measurement of blank group Add 50 μL of PBS buffer solution to the 96-well plate, incubate on a shaker at 37°C for 15 minutes, and remove immediately. Then add 30 μL of AIENP solution, incubate on a shaker at 37°C for 15 minutes, and remove the 96-well plate. Finally, add 20 μL of ultrapure water and incubate on a shaker at 37°C for 5 minutes. After the reaction is completed, use an ELISA reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), and record it as A 1 .

[0090] (2) Fluorescence measurement of thrombin control group Add 45μL PBS buffer solution to the 96-well plate, then add 15μL thrombin stock solution, incubate in a 37℃ shaker for 15min, and remove immediately. Then add 35μL AIENP solution, incubate in a 37℃ shaker for 15min, and remove the 96-well plate. Finally, add 15μL ultrapure water and incubate in a 37℃ shaker for 5min. After the reaction is completed, use a microplate reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), recorded as A 2 .

[0091] (3) Fluorescence determination of monomers of related components of Chuanxiong Add 35μL PBS buffer solution to a 96-well plate, then add 15μL thrombin stock solution, incubate in a 37°C shaker for 5 minutes, and take it out immediately. Then add 10μL of Chuanxiong medicinal solution, incubate in a 37°C shaker for 10 minutes, and take it out immediately. Then add 35μL AIENP solution, incubate in a 37°C shaker for 15 minutes, and take out the 96-well plate. Finally, add 5μL ultrapure water and incubate in a 37°C shaker for 5 minutes. After the reaction is completed, use an enzyme reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength of 37°C (Ex / Em=400 / 515nm), recorded as A 3 .

[0092] (4) Basal fluorescence determination of monomers of Chuanxiong-related components Add 35 μL of PBS buffer solution to a 96-well plate, incubate at 37°C on a shaker for 5 minutes, and remove immediately. Then add 10 μL of Chuanxiong medicinal solution, incubate at 37°C on a shaker for 25 minutes, and remove immediately. Finally, add 55 μL of ultrapure water and incubate at 37°C on a shaker for 5 minutes. After the reaction is completed, use an enzyme marker to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), recorded as A 4 .

[0093] The experimental results were plotted using origin2018 software. All experiments were repeated at least three times.

[0094] Inhibition rate calculation method: Thrombin inhibition rate = (A 3 -A 4 )÷A 2 ×100%,A 3 -A 4 ∈[A 2 , A 1 ], indicating that the component has inhibitory activity against thrombin. Figure 8 As shown, a total of 24 components showed relatively stable inhibitory activity against thrombin during the screening.

[0095] Wherein, the extraction method of the Ligusticum chuanxiong medicinal liquid is as follows: Dried rhizomes of Chuanxiong (10 kg) were crushed and extracted with 25 L 95% ethanol for four times, each time for 48 h. The four extracts were combined and concentrated under reduced pressure to obtain the ethanol extract.

[0096] Take the 95% ethanol extract of Chuanxiong and add water to 1.6L of suspension, extract with petroleum ether, ethyl acetate and n-butanol respectively, and obtain 316g of petroleum ether layer, 107g of ethyl acetate layer and 52g of n-butanol layer.

[0097] The petroleum ether layer was subjected to open silica gel column chromatography, and the gradient elution was petroleum ether-ethyl acetate (100:0→20:1→10:1→5:1→3:1→1:1) / dichloromethane-methanol (20:1→10:1→5:1) to obtain components HLC-Ⅰ-4a~HLC-Ⅰ-4h, HLC-Ⅰ-5a~HLC-Ⅰ-5h), among which HLC-Ⅰ-5d~HLC-Ⅰ-5h were eluted with dichloromethane-methanol. HLC-Ⅰ-4c (65 g) was subjected to MCI column, and the gradient elution was carried out with methanol-water to obtain components HLC-Ⅰ-7c, HLC-Ⅰ-7d, HLC-Ⅰ-7h, HLC-Ⅰ-7i, HLC-Ⅰ-8b. HLC-Ⅰ-4d (22.3 g) was subjected to MCI column, and the gradient elution was carried out with methanol-water to obtain components HLC-I-10a, HLC-Ⅰ-10d. HLC-Ⅰ-7c was purified by ODS column and gradient elution with methanol-water (0:100→100:0) to obtain components HLC-Ⅰ-12f, HLC-Ⅰ-12j~HLC-Ⅰ-12n, and HLC-Ⅰ-12p.

[0098] Component HLC-Ⅰ-12f was prepared by high performance liquid chromatography (40% acetonitrile-water) to obtain compounds HLC-I-31C and HLC-I-31A; component HLC-Ⅰ-12p was prepared by high performance liquid chromatography (26% acetonitrile-water) to obtain compound HLC-I-32D, and HLC-Ⅰ-7h was prepared by high performance liquid chromatography (69% methanol-water) to obtain compounds HLC-I-11H and HLC-I-11K; HLC-Ⅰ-7i was prepared by high performance liquid chromatography (66% acetonitrile-water) to obtain compounds HLC-I-15C and HLC-I-22A; HLC-I-10a was prepared by high performance liquid chromatography (77% acetonitrile-water) to obtain compound HLC-I-33I. The obtained ethyl acetate layer was dry loaded and subjected to open silica gel column chromatography, and gradient eluted with petroleum ether-ethyl acetate (100:0→20:1→10:1→5:1→3:1→1:1) / dichloromethane-methanol (20:1→10:1→5:1) to obtain components HLC-Ⅱ-5a~HLC-Ⅱ-5e, HLC-Ⅱ-5g, HLC-Ⅱ-6a, HLC-Ⅱ-6d, HLC-Ⅱ-6e, HLC-Ⅱ-6f. HLC-Ⅱ-5g was wet loaded on an ODS column and gradient eluted with methanol-water (0:100→100:0) to obtain components HLC-Ⅱ-10d, HLC-Ⅱ-10e, HLC-Ⅱ-11b. HLC-Ⅱ-11b was subjected to high performance liquid chromatography (30% acetonitrile-water) to obtain compound HLC-I-2-14E.

[0099] Example 8 Nine standard substances including ferulic acid, scopoletin A, scopoletin H, scopoletin I, ligustilide, isochlorogenic acid, angelica lactone and chlorogenic acid were investigated, and their anticoagulant activities were determined respectively.

[0100] (1) Fluorescence measurement of blank group Add 50 μL of PBS buffer solution to the 96-well plate, incubate on a shaker at 37°C for 15 minutes, and remove immediately. Then add 30 μL of AIENP solution, incubate on a shaker at 37°C for 15 minutes, and remove the 96-well plate. Finally, add 20 μL of ultrapure water and incubate on a shaker at 37°C for 5 minutes. After the reaction is completed, use an ELISA reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), and record it as A 1 .

[0101] (2) Fluorescence measurement of thrombin control group Add 45μL PBS buffer solution to the 96-well plate, then add 15μL thrombin stock solution, incubate in a 37℃ shaker for 15min, and remove immediately. Then add 35μL AIENP solution, incubate in a 37℃ shaker for 15min, and remove the 96-well plate. Finally, add 15μL ultrapure water and incubate in a 37℃ shaker for 5min. After the reaction is completed, use a microplate reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), recorded as A 2 .

[0102] (3) Fluorescence determination of different Chuanxiong standards Add 35 μL of PBS buffer solution to a 96-well plate, then add 15 μL of thrombin stock solution (0.5 mg / mL), incubate in a 37°C shaker for 5 minutes, and take it out immediately. Then add 10 μL of Chuanxiong standard product, incubate in a 37°C shaker for 10 minutes, and take it out immediately. Then add 35 μL of AIENP solution, incubate in a 37°C shaker for 15 minutes, and take out the 96-well plate. Finally, add 5 μL of ultrapure water and incubate in a 37°C shaker for 5 minutes. After the reaction is completed, use an enzyme reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength of 37°C (Ex / Em=400 / 515nm), recorded as A 3 .

[0103] (4) Determination of the basal fluorescence of different Chuanxiong standards Add 35 μL of PBS buffer solution to a 96-well plate, incubate at 37°C on a shaker for 5 minutes, and remove immediately. Then add 10 μL of Chuanxiong standard, incubate at 37°C on a shaker for 25 minutes, and remove immediately. Finally, add 55 μL of ultrapure water and incubate at 37°C on a shaker for 5 minutes. After the reaction is completed, use an enzyme reader to measure the fluorescence intensity at the excitation wavelength and emission wavelength at 37°C (Ex / Em=400 / 515nm), recorded as A 4 .

[0104] The experimental results were plotted using oringin2018 software. All experiments were repeated at least three times.

[0105] Inhibition rate calculation method: Thrombin inhibition rate = (A 3 -A 4 )÷A 2 ×100%,A 3 -A 4 ∈[A 2 , A 1 ], indicating that the component has inhibitory activity against thrombin. Fig. 9 As shown, a total of 8 components showed relatively stable inhibitory activity against thrombin during the screening.

[0106] Example 9 (1) The morphology of AIENP was observed using a transmission electron microscope (TEM). Fig.10 shown.

[0107] (2) Take 200 μL of the newly prepared AIENP solution and use the Malvern Zetasizer - NanoZinstrument laser particle size analyzer to measure the hydrated particle size of the nanoparticles. Fig.11 As shown, the average hydrated particle size of AIENP is about 220 nm.

[0108] (3) Pipette 200 μL of the newly prepared AIENP solution and add it to the folded capillary potential sample cell. Use a Malvern Panalytical laser particle size analyzer to measure the Zeta potential. Fig.12 As shown, the potential of AIENP is around -46.2 mV, proving that AIENP can exist stably in aqueous solution.

[0109] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A thrombin-based fluorescent sensor nanoparticle, characterized in that: The fluorescent sensor nanoparticles are composed of AIE 515 and thrombin substrate S-2238.

2. The method for preparing the fluorescent sensor nanoparticles according to claim 1, characterized in that: The following steps are involved: Mix water with thrombin substrate S-2238, then add tetrahydrofuran, glutaraldehyde, and finally AIE 515 , obtain a mixed solution, stir to react, and keep it still in the dark to obtain fluorescent sensor nanoparticles.

3. The preparation method according to claim 2, characterized in that: The speed of the stirring reaction is 200-600 rpm, and the time of the stirring reaction is 0.5-1.5 h; The concentration of the thrombin substrate S-2238 in the mixed solution is 0.25-2 mg / mL.

4. A kit for detecting thrombin activity, characterized in that: The invention comprises the fluorescent sensor nanoparticles according to claim 1 or the fluorescent sensor nanoparticles prepared by the preparation method according to any one of claims 2 to 3.

5. A method for detecting thrombin activity using the fluorescent sensor nanoparticles according to claim 1 or the fluorescent sensor nanoparticles prepared by the preparation method according to any one of claims 2 to 3 or the kit according to claim 4, characterized in that: The following steps are involved: S1, dissolving thrombin in a buffer solution to obtain a thrombin solution; S2. Add the thrombin solution obtained in step S1 to the well plate, incubate once, add the fluorescent sensor nanoparticles, incubate twice, add water, incubate three times, and measure the fluorescence intensity.

6. A method for screening anticoagulant components using the fluorescent sensor nanoparticles according to claim 1 or the fluorescent sensor nanoparticles prepared by the preparation method according to any one of claims 2 to 3 or the kit according to claim 4, characterized in that: The following steps are involved: T1, measure the blank fluorescence intensity A1; T2, measure the fluorescence intensity A2 of the thrombin control group; T3, co-incubate the anticoagulant component with thrombin, and measure the fluorescence intensity A3 after co-incubation; T4, measure the basal fluorescence intensity A4 of the anticoagulant component; T5, judging whether A3-A4∈[A2, A1] holds according to A1 obtained in step T1, A2 obtained in step T2, A3 obtained in step T3 and A4 obtained in step T4. If so, the anticoagulant component has an inhibitory effect on thrombin; T6. Calculate the thrombin inhibition rate using A1 obtained in step T1, A2 obtained in step T2, A3 obtained in step T3, and A4 obtained in step T4. The calculation formula of the thrombin inhibition rate is as follows: Thrombin inhibition rate = (A3-A4) ÷ A2 × 100%.

7. The method according to claim 6, characterized in that Step T1 comprises the following steps: adding a buffer solution to the well plate, incubating once, adding fluorescent sensor nanoparticles, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the blank fluorescence intensity A1; Step T2 comprises the following steps: adding thrombin solution to the well plate, incubating once, adding fluorescent sensor nanoparticles, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the fluorescence intensity A2 of the thrombin control group; Step T3 comprises the following steps: adding thrombin solution to the well plate, incubating once, adding anticoagulant components, incubating twice, adding fluorescent sensor nanoparticles, incubating three times, adding water, incubating four times, measuring fluorescence intensity, and obtaining the fluorescence intensity A3 after co-incubation; Step T4 includes the following steps: adding a buffer solution to the well plate, incubating once, adding an anticoagulant component, incubating twice, adding water, incubating three times, measuring the fluorescence intensity, and obtaining the anticoagulant component base fluorescence intensity A4.

8. The anticoagulant active substance in Rhizoma Chuanxiong obtained by screening using the method described in any one of claims 6 to 7 is characterized in that: The anticoagulant active substance in the rhizoma chuanxiong is extracted from the rhizoma chuanxiong medicinal liquid, and the inhibition rate of the anticoagulant active substance in the rhizoma chuanxiong on thrombin is greater than 10%.

9. The anticoagulant active substance in Rhizoma Chuanxiong according to claim 8, characterized in that: The preparation method of the Ligusticum chuanxiong medicinal liquid comprises the following steps: (1) Crush Chuanxiong, extract with ethanol, and concentrate under reduced pressure to obtain an ethanol extract; (2) resuspending the alcohol extract obtained in step (1) in water, extracting with petroleum ether and ethyl acetate respectively, to obtain a petroleum ether layer and an ethyl acetate layer respectively; (3) The petroleum ether layer and the ethyl acetate layer obtained in step (2) are subjected to column chromatography respectively, and the eluted portion obtained is the Ligusticum chuanxiong medicinal solution.

10. Use of the fluorescent sensor nanoparticles according to claim 1, or the fluorescent sensor nanoparticles prepared by the preparation method according to any one of claims 2-3, or the kit according to claim 4, or the method according to any one of claims 6-7 in screening anticoagulant active substances in Chuanxiong.

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