AIE nanoparticle, preparation method and application thereof, and related product

Abstract

The invention provides AIE nanoparticles, a preparation method and application thereof, and a related product, and belongs to the technical field of nano sensing. The AIE nano particles provided by the invention are prepared from AIE515 and a blood coagulation factor Xa substrate S-2765. The preparation method of the AIE nanoparticles is simple, mild in reaction condition, low in cost and easy for batch preparation. When the prepared sensor is added into an aqueous solution, a fluorescence signal can be instantly obtained, and compared with traditional Chinese medicine active substance screening and existing Xa detection, rapid detection and screening can be achieved at the same time through the method.

Description

Technical Field

[0001] The present invention belongs to the field of nanosensing technology and relates to an AIE nanoparticle and a preparation method, application and related products thereof, and specifically relates to a preparation method of a fluorescence sensor based on coagulation factor Xa and the application of the fluorescence sensor in monitoring coagulation factor Xa and screening of anti-Xa inhibitors in Chuanxiong. Background Art

[0002] Coagulopathy is a serious and common complication in critically ill patients, which can lead to multiple organ dysfunction syndrome (MODS) and is associated with worse prognosis. Coagulopathy can be defined as any alteration or disorder in hemostasis that causes bleeding, coagulation, or both due to a variety of causes. Examples include sepsis-induced coagulopathy (SIC) and trauma-induced coagulopathy (TIC). In severe cases, these coagulopathy can progress to disseminated intravascular coagulation (DIC), leading to MODS.

[0003] Prothrombin is a key zymogen in the coagulation process and can be converted into thrombin by the prothrombinase complex, which is composed of coagulation factor Xa (FXa), cofactor Va (FVa) and phospholipids. Coagulation factor Xa is a vitamin K-dependent serine protease and one of the key factors in blood coagulation. The main form of coagulation factor Xa has a molecular weight of about 43kDa and contains two peptide chains of 27kDa and 16kDa connected by disulfide bonds. It plays a central role in activating the complex coagulation cascade reaction and is therefore considered to be the target of antithrombotic agents.

[0004] Chinese patent application 202311349277.0 provides a composition, a detection reagent, a detection kit and an application in the preparation of a method for detecting the activity of coagulation factor X. The composition includes: plasma without coagulation factor X and a buffer system. The technology provides a kit for detecting the activity of factor X by coagulation method, which has the characteristics of simple operation, high sensitivity, good repeatability, good stability and strong market competitive advantage. However, the technology fails to provide a method for detecting coagulation factor X by using aggregation-induced fluorescent nanoparticles.

[0005] Chinese patent application 202210662738.9 provides an anti-Xa activity assay kit with exogenously added antithrombin. The assay kit is a fully liquid reagent, which includes a first reagent, a second reagent and a third reagent; the first reagent, with a total volume of 1L, includes the following components: 10-30g of a first salt ion, 56-130g of a first stabilizer, 0.1-1g of a first preservative, a first buffer solution with a content of 50-100mmol, and an antithrombin content of 3000-5000IU; and the pH value of the first reagent is 7.2-7.6; the first stabilizer includes a polyhydroxy alcohol, a sugar, a first surfactant and a protein; The mass ratio of hydroxyl alcohol: sugar: first surfactant: protein is: (3.5-6.5): (1-3): (0.1-0.5): (1-3); the first surfactant is Tween-20; the second reagent, based on its total volume of 1L, includes the following components: 5-15g of second salt ions, 67-165g of second stabilizer, 0.1-1g of second preservative, a second buffer with a content of 50-100mmol, and a coagulation factor with a content of 1000-5000IU; and the second buffer has a The pH value is 7.8-8.2; the second stabilizer includes a polyhydric alcohol, a second saccharide, an amino acid, a second surfactant and a protein; the mass ratio of the polyhydric alcohol: the second saccharide: the amino acid: the second surfactant: the protein is: (3.5-6.5): (1.1-3.5): (1-3): (0.1-0.5): (1-3); the second surfactant is Tween-20; the second saccharide includes sucrose and dextran sulfate; the third reagent, based on its total volume of 1L, includes the following components: 5-1 5g of a third salt ion, 46-100g of a third stabilizer, 0.1-1g of a third preservative, a third buffer solution of 50-100mmol, and a chromogenic substrate of 0.5-1.5mmol; the pH value of the third buffer solution is 7.8-8.2; the third stabilizer comprises polyhydroxy, sugar and a third surfactant; the mass ratio of polyhydroxy alcohol: sugar: third surfactant is: (3-5): (1-3): (0.6-2); the third surfactant comprises Tween-20 and PVP. However, the technology fails to provide a method for detecting coagulation factor X using aggregation-induced fluorescent nanoparticles.

[0006] 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. However, this technology has failed to realize the preparation of a dual-function "on-off-on" sensor and its application in the monitoring of coagulation factor Xa and the screening of anti-Xa inhibitors in Chuanxiong.

[0007] However, the prior art has not yet provided a dual-function "on-off-on" sensor, a preparation method, and its application in monitoring coagulation factor Xa and screening of anti-Xa inhibitors in Chuanxiong. In view of this, in view of the problem that the prior art has not yet provided a method for preparing a dual-function "on-off-on" sensor and its application in the monitoring of coagulation factor Xa and the screening of anti-Xa inhibitors in Chuanxiong, the purpose of the present invention is to provide a sensing nanoparticle and its preparation method, application, and related products.

[0008] The inventive idea of ​​the present invention is: AIE 515 Aggregation-induced fluorescent nanoparticles (AIE nanoparticles) were synthesized by bonding and self-assembling with the coagulation factor Xa substrate S-2765 using glutaraldehyde. The end group of the coagulation factor Xa substrate S-2765 has a group that responds to coagulation factor Xa. When there is no coagulation factor, the AIE nanoparticles aggregate and induce fluorescence (on state); when coagulation factor Xa is present and active, the group that responds to coagulation factor Xa on the AIE nanoparticles falls off, making the aggregation state of the nanoparticles dispersed, and the aggregation-induced fluorescence intensity decreases (off state); at this time, if a coagulation factor Xa inhibitor is added, the activity of coagulation factor Xa decreases, the group that responds to coagulation factor Xa on the AIE nanoparticles falls off less, and the aggregation-induced fluorescence intensity increases (on state). The above principle can be used to screen coagulation factor Xa inhibitors and evaluate their inhibitory power.

[0009] In order to achieve the above-mentioned object of the invention, on the one hand, the present invention provides an AIE nanoparticle, wherein the AIE nanoparticle is composed of AIE 515 and coagulation factor Xa substrate S-2765.

[0010] The AIE 515 The molecular formula is as follows:

[0011] Preferably, the AIE nanoparticles are composed of AIE 515 It is prepared by bonding and self-assembly with coagulation factor Xa substrate S-2765.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned AIE nanoparticles, comprising the following steps: Mix water with the coagulation factor Xa substrate S-2765, then add tetrahydrofuran, glutaraldehyde, and finally AIE 515 , obtain a mixed solution, stir to react, and keep it still in the dark to obtain AIE nanoparticles.

[0013] Preferably, the stirring reaction speed is 100-400 rpm, and the stirring reaction time is 0.25-1.5 h; the coagulation factor Xa substrate S-2765 and the AIE 515 The mass ratio is 0.1:0.25-2.

[0014] More preferably, the stirring reaction speed is 100-250 rpm, and the stirring reaction time is 0.25-0.75 h; the coagulation factor Xa substrate S-2765 and the AIE 515 The mass ratio is 0.1:0.25-1.

[0015] Preferably, the stirring reaction is carried out at a speed of 250 rpm, and the coagulation factor Xa substrate S-2765 and the AIE 515 The mass ratio is 0.1:0.25.

[0016] In another aspect, the present invention provides a coagulation factor Xa detection reagent, comprising the above-mentioned AIE nanoparticles or the AIE nanoparticles prepared by the above-mentioned preparation method.

[0017] In yet another aspect, the present invention provides a coagulation factor Xa detection kit, comprising the above-mentioned coagulation factor Xa detection reagent.

[0018] In another aspect, the present invention provides an anticoagulant factor Xa component screening reagent, comprising the above-mentioned AIE nanoparticles or the AIE nanoparticles prepared by the above-mentioned preparation method.

[0019] In yet another aspect, the present invention provides a screening kit for anti-coagulation factor Xa components, comprising the above-mentioned anti-coagulation factor Xa component screening reagent.

[0020] In another aspect, the present invention provides use of the above-mentioned AIE nanoparticles or the AIE nanoparticles prepared by the above-mentioned preparation method in the detection of coagulation factor Xa.

[0021] In yet another aspect, the present invention provides the use of the above-mentioned AIE nanoparticles or the AIE nanoparticles prepared by the above-mentioned preparation method in the screening of anticoagulant factor Xa components.

[0022] Preferably, the application comprises the following steps: S1, incubate the AIE nanoparticles with Tris and ultrapure water, and measure the fluorescence intensity A0; S2, incubate the AIE nanoparticles with Tris, ultrapure water, and coagulation factor Xa, and measure the fluorescence intensity A1; S3, incubating the AIE nanoparticles with Tris, ultrapure water, coagulation factor Xa, and anticoagulation factor Xa components, and measuring the fluorescence intensity A2; S4. Calculate the coagulation factor Xa inhibition rate according to A0 obtained in step S1, A1 obtained in step S2, and A2 obtained in step S3.

[0023] Tris is the abbreviation of the chemical substance trishydroxymethylaminomethane.

[0024] More preferably, in S4, the calculation formula is as follows: coagulation factor Xa inhibition rate = (A2-A1) ÷ (A0-A1).

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a fluorescence sensor and a preparation method thereof. The preparation method of the sensor of the present invention is simple, the reaction conditions are mild, and the cost is low and it is easy to prepare in batches. The sensor prepared by the present invention is added to an aqueous solution to instantly obtain a fluorescence signal. Compared with the traditional screening of active substances in traditional Chinese medicines and the existing detection of Xa, this method can simultaneously achieve rapid detection and screening, that is, the fluorescence can be quickly detected after adding the sensor, which is simple, fast and accurate. More importantly, the application range of the present invention is extremely wide, and it can not only be used to monitor the size of Xa activity, but also can be used for the screening research of active ingredients in traditional Chinese medicines. In addition, the instantaneous response and simple preparation of the sensor constructed by the present invention are a new tool and new method for the application of probes in the modernization of traditional Chinese medicine.

[0026] The present invention prepares AIENP by a cross-linking method, and quenches fluorescence by a specific hydrolysis reaction between Xa and a substrate to form an AIENP fluorescent sensor. The sensor and the anticoagulant substance produce an inhibitory effect and generate a fluorescent signal. When there is an anticoagulant active substance, the AIENP fluorescent sensor will generate different fluorescent signals to reflect the strength of the anticoagulant activity. The sensor prepared by the present invention has broad application prospects in Xa activity monitoring and screening of active ingredients of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 For AIE 515 Fluorescence intensity diagram in mixed solutions of water and tetrahydrofuran with different volume ratios.

[0028] Figure 2 For different AIE 515 Fluorescence spectra of AIE nanoparticles synthesized by mass ratio of Xa substrate to Xa substrate. From top to bottom, the five curves represent the Xa substrate and AIE 515 Fluorescence spectrum curves of AIE nanoparticles synthesized at mass ratios of 0.5:0.1, 1.0:0.1, 0.25:0.1, 1.5:0.1, and 2.0:0.1.

[0029] Figure 3 Fluorescence spectra of AIE nanoparticles synthesized with different stirring times (0.25h, 0.5h, 0.75h, 1.00h, 1.50h).

[0030] Figure 4 The fluorescence spectra of AIE nanoparticles synthesized at different stirring speeds. From top to bottom, the five curves represent the fluorescence spectra of AIE nanoparticles synthesized at stirring speeds of 250 rpm, 200 rpm, 100 rpm, 300 rpm, and 400 rpm, respectively.

[0031] Figure 5 This is a curve diagram showing the effect of Tris concentration on the fluorescence sensor system of AIENP obtained in Example 5.

[0032] Figure 6 This is the transmission electron image obtained in Example 6.

[0033] Figure 7 This is a diagram of the hydrated particle size of the sensor obtained in Example 6.

[0034] Figure 8 This is the Zeta potential diagram of the sensor obtained in Example 6.

[0035] Fig. 9 This is a screening diagram for the anticoagulant active substances in Ligusticum chuanxiong obtained in Example 7.

[0036] Fig.10 This is a comparison chart of the anticoagulant activity of 9 standard products obtained in Example 8, wherein the numbers on the horizontal axis represent the following meanings: 1: ferulic acid, 2: ligusticolide A, 3: ligusticolide H, 4: ligusticolide I, 5: scopoletin, 6: ligustilide, 7: isochlorogenic acid, 8: angelica lactone, 9: chlorogenic acid. DETAILED DESCRIPTION

[0037] 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.

[0038] 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.

[0039] In the following embodiments, the fluorescence microplate reader used for reading fluorescence values ​​is Spark multifunctional microplate reader purchased from Tecan Company of Switzerland; the zeta potential is measured by Malvern Panalytical instrument; the transmission electron microscope is used to measure PDA-PE at an accelerating voltage of 200 kV using a JEOL JEM-200CX instrument; coagulation factor Xa is purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; coagulation factor Xa substrate S-2765 (Xa chromogenic substrate S-2765) is purchased from Beijing Asrel Biotechnology Co., Ltd.; glutaraldehyde is purchased from Sigma Company of Germany; tetrahydrofuran is purchased from Beijing Bailingwei Technology Co., Ltd.; Tris (trishydroxymethylaminomethane) is purchased from Beijing Solaibao Technology Co., Ltd.

[0040] Example 1 Aggregation-induced emission performance detection of AIE probes: (1) Preparation of AIE 515 Probe stock solution (97 μM): Accurately weigh 1 mg AIE 515 The probe was dissolved in tetrahydrofuran (THF) at a concentration of 0.05 mg / mL, sealed and stored in a refrigerator at 4 °C away from light.

[0041] (2) Take AIE 515The probe stock solution was placed in a 96-well plate (10 µL / well), and then 100 μL of a mixed solution of Milli-Q water and THF in different ratios was added in sequence (the volume ratios of water to THF were 1:1, 2:1, 5:1, 10:1, and 20:1, respectively).

[0042] (3) The 96-well plate was shaken at a constant temperature and the AIE was measured using an ELISA reader. 515 Fluorescence value of the probe in THF / water mixed solution.

[0043] Figure 1 Given AIE 515 Fluorescence intensity in different ratios of water and tetrahydrofuran solutions. When the proportion of water increases, AIE 515 Aggregation occurs and the fluorescence intensity increases, which indicates that AIE 515 The aggregation inducing effect is good.

[0044] Example 2 Coagulation factor Xa substrate S-2765 (hereinafter referred to as Xa substrate) and AIE 515 Effect of mass ratio on the synthesis of AIE nanoparticles: Accurately weigh 5 portions of Xa substrate with different mass ratios (1:10, 1:2.5, 1:15, 1:20) and dissolve them in 1 mL of water respectively.

[0045] Accurately weigh AIE 515 2 mg was dissolved in 1 mL THF and the mixture was completely dissolved by rotating with a turbine mixer for 5 min.

[0046] Take 50 µL of AIE 515 The probe THF solution was added to the aqueous solution of Xa substrate with different concentrations, and then 15 μL THF and 7 μL glutaraldehyde were added. The mixture was stirred at 230 rpm at room temperature for 30 min.

[0047] After the reaction was completed, the mixture was kept away from light for 12 hours to allow THF to evaporate. After THF was removed, AIE nanoparticles were obtained.

[0048] The experimental results are as follows Figure 2 As shown. Figure 2 It can be seen that Xa substrate and AIE 515 Too large or too small a ratio of will have a significant impact on the fluorescence intensity of AIE nanoparticles. 515 When the mass ratio of AIE nanoparticles to Xa substrate is 1:5, the fluorescence intensity of AIE nanoparticles is optimal. 515 When the mass ratio of the substrate to Xa was 1:10, 1:2.5, 1:15, and 1:20, respectively, the fluorescence intensity decreased.

[0049] Example 3 Effect of different reaction times (0.25 h, 0.5 h, 0.75 h, 1 h, 1.5 h) on the synthesis of AIE nanoparticles.

[0050] Accurately weigh 5 parts of Xa substrate with a mass ratio of 1:10 and dissolve them in water.

[0051] Accurately weigh AIE 515 2 mg was dissolved in 1 mL THF and the mixture was completely dissolved by rotating with a turbine mixer for 5 min.

[0052] Take 50 µL of AIE 515 The probe solution in THF (2 mg / mL) was added to the aqueous solution of Xa substrate, followed by the addition of 15 µL of THF and 7 µL of glutaraldehyde.

[0053] The mixture was stirred at 230 rpm at room temperature for 0.25 h, 0.5 h, 0.75 h, 1 h, and 1.5 h.

[0054] After the reaction was completed, the solution was kept away from light for 12 hours to evaporate THF. After THF was removed, AIE nanoparticles were obtained. The final solution was sealed and stored at 4 °C away from light.

[0055] The experimental results are as follows Figure 3 As shown. Figure 3 It can be seen that when the stirring time is 0.25h, 0.5h, and 0.75h, the fluorescence intensity of the AIE nanoparticles is strong and the difference is small. However, when the stirring time is 1h and 1.5h, the fluorescence intensity of the AIE nanoparticles is significantly weakened.

[0056] Example 4 Effect of stirring speed (100 rpm, 200 rpm, 250 rpm, 300 rpm, 400 rpm) on the synthesis of AIE nanoparticles.

[0057] Accurately weigh 5 parts of Xa substrate with a mass ratio of 1:10 and dissolve them in water.

[0058] Accurately weigh AIE 515 2 mg was dissolved in 1 mL THF and the mixture was completely dissolved by rotating with a turbine mixer for 5 min.

[0059] Take 50 µL of AIE 515 The probe solution in THF was added to the aqueous solution of Xa substrate, followed by the addition of 15 μL THF and 7 μL glutaraldehyde.

[0060] Stir at room temperature at 100 rpm, 200 rpm, 250 rpm, 300 rpm, and 400 rpm for 45 min.

[0061] After the reaction was completed, the mixture was kept away from light for 12 hours to allow THF to evaporate. After removing THF, AIE nanoparticles were obtained, sealed and stored in a refrigerator at 4 °C away from light for subsequent experiments. The experimental results are as follows Figure 4 As shown. Figure 4 It can be seen that when the rotation speed is 250 rpm, the fluorescence intensity of AIE nanoparticles is optimal.

[0062] Example 5 The influence of Tris (tris(hydroxymethylaminomethane)) concentration on the detection effect of fluorescence sensor detection system.

[0063] Accurately weigh 1 mg of substrate and dissolve it in 1 mL of Milli-Q water to prepare a substrate aqueous solution.

[0064] Accurately weigh 0.5 mg of Xa substrate and dissolve each portion in 1 mL of Tris buffer solution containing 0 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL. Mix well to prepare Xa solution.

[0065] (3) Take 10 µL of glutaraldehyde solvent and add it to 90 µL of Milli-Q water, diluting it 10 times.

[0066] (4) Accurately measure 6 portions (100 µL) of substrate aqueous solution and 6 portions (100 µL) of Xa solution and mix them evenly.

[0067] (5) After mixing, place in a 37°C oven and incubate for 30 min. Measure the absorbance.

[0068] like Figure 5 The results showed that the fluorescence intensity of AIE nanoparticles was optimal when the Tris concentration was 0.5 mg / mL.

[0069] Example 6 The AIE nanoparticles prepared in Example 5 were tested.

[0070] (1) The morphology of AIE nanoparticles was observed using a transmission electron microscope (TEM).

[0071] (2) Pipette 200 µL of the freshly prepared AIE nanoparticle solution and use a Malvern Zeta sizer-Nano Zinstrument laser particle size analyzer to measure the hydrated particle size of the nanoparticles.

[0072] (3) Pipette 200 μL of the freshly prepared AIE nanoparticle solution into the folded capillary potential sample cell and measure the Zeta potential using a Malvern Panalytical laser particle size analyzer.

[0073] like Figure 6 As shown in Figure 2, the AIE nanoparticles are single, uniformly dispersed spherical, with a particle size of about 50 nm, indicating the successful preparation of AIE nanoparticles. Figure 7 As shown in the figure, the potential of AIE nanoparticles is about -44.9 mV, which proves that AIE nanoparticles can exist stably in aqueous solution. Figure 8 As shown, the average hydrated particle size of AIE nanoparticles is about 280 nm.

[0074] Example 7 Screening of anticoagulant active substances from Ligusticum chuanxiong (1) Fluorescence measurement of blank group 70 μL of Tris buffer solution was added to a 96-well plate, incubated at 37°C for 5 min, and immediately removed. 10 μL of milliQ water was added to the 96-well plate, incubated at 37°C for 10 min, and the 96-well plate was removed. 20 μL of AIE nanoparticle solution was added in a dark oven, mixed evenly, and placed in an oven at 30°C for 20 min. After the reaction was completed, the fluorescence intensity was measured at the excitation and emission wavelengths at 37 °C (λEx / Em = 415 / 600 nm) using a microplate reader, and recorded as A0.

[0075] (2) Fluorescence measurement of Xa control group 60 μL Tris buffer solution was added to a 96-well plate; then, 10 μL Xa solution was added to the 96-well plate, incubated at 37°C for 5 min, immediately removed, 10 μL Milli-Q water was added, added to the 96-well plate, incubated at 37°C for 10 min, the 96-well plate was removed, 20 μL AIE nanoparticle solution was added in a dark oven, mixed evenly, placed in an oven at 30°C, and incubated for 20 min. After the reaction was completed, the fluorescence intensity was measured at the excitation and emission wavelengths at 37 °C (λEx / Em = 415 / 600 nm) using a microplate reader, recorded as A1.

[0076] (3) Fluorescence measurement of the sample to be tested 60 μL Tris buffer solution was added to the 96-well plate; then, 10 μL Xa solution was added to the 96-well plate, incubated at 37°C for 5 min, and immediately removed, 10 μL of the sample to be tested was added to the 96-well plate, incubated at 37°C for 10 min, the 96-well plate was removed, 20 μL AIE nanoparticle solution was added to the plate in a dark environment, mixed evenly, and placed in an oven at 30°C for 20 min. After the reaction was completed, the fluorescence intensity was measured at the excitation and emission wavelengths at 37 °C (λEx / Em = 415 / 600 nm) using a microplate reader, and recorded as A2.

[0077] Inhibition rate calculation method: Inhibition rate (%) = (A2-A1) / (A0-A1) × 100%, (where A0 is the fluorescence intensity of the blank group, A1 is the fluorescence intensity of the Xa control group, and A2 is the fluorescence intensity of the sample to be tested). The experimental results were plotted using oringin2018 software to draw the fluorescence intensity-response curve. All experiments were repeated at least three times.

[0078] The above method was used to test the thrombin inhibition rate of 47 different extraction parts of Chuanxiong extract. The results are as follows: Fig. 9 After testing, a total of 13 extraction parts had significant thrombin inhibitory activity.

[0079] Example 8 Comparison of anticoagulant activity of nine standard components of Ligusticum chuanxiong Nine standard products including ferulic acid, ligusticolide A, ligusticolide H, scopoletin, ligusticolide I, ligustilide, isochlorogenic acid, angelica lactone, and chlorogenic acid were selected as characteristic components in Ligusticum chuanxiong to determine their anticoagulant activity. Fig.10 It can be seen that, except for scopoletin, the remaining eight components all have certain anticoagulant activity.

[0080] Examples 7 and 8 fully illustrate that the bifunctional sensing nanoparticles provided by the present invention can realize the rapid screening of anticoagulant active components / active sites in Chuanxiong.

[0081] 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. An AIE nanoparticle, characterized in that: The AIE nanoparticles are composed of AIE 515 and coagulation factor Xa substrate S-2765.

2. The method for preparing the AIE nanoparticles according to claim 1, characterized in that: The following steps are involved: Mix water with the coagulation factor Xa substrate S-2765, then add tetrahydrofuran, glutaraldehyde, and finally AIE 515 , obtain a mixed solution, stir to react, and keep it still in the dark to obtain AIE nanoparticles.

3. The preparation method according to claim 2, characterized in that: The stirring reaction speed is 100-400 rpm, and the stirring reaction time is 0.25-1.5 h; the coagulation factor Xa substrate S-2765 and the AIE 515 The mass ratio is 0.1:0.25-2.

4. A coagulation factor Xa detection reagent, characterized in that: The invention comprises the AIE nanoparticles according to claim 1 or the AIE nanoparticles prepared by the preparation method according to any one of claims 2 to 3.

5. A coagulation factor Xa detection kit, characterized in that: The invention comprises the coagulation factor Xa detection reagent as claimed in claim 4.

6. A reagent for screening anticoagulant factor Xa components, characterized in that: The invention comprises the AIE nanoparticles according to claim 1 or the AIE nanoparticles prepared by the preparation method according to any one of claims 2 to 3.

7. A kit for screening anticoagulant factor Xa components, characterized in that: The invention comprises the anti-coagulation factor Xa component screening reagent as described in claim 6.

8. Use of the AIE nanoparticles according to claim 1 or the AIE nanoparticles prepared by the preparation method according to any one of claims 2 to 3 in the detection of coagulation factor Xa.

9. Use of the AIE nanoparticles according to claim 1 or the AIE nanoparticles prepared by the preparation method according to any one of claims 2 to 3 in screening anticoagulant factor Xa components.

10. The use according to claim 9, characterized in that: The following steps are involved: S1, incubate the AIE nanoparticles with Tris and ultrapure water, and measure the fluorescence intensity A0; S2, incubate the AIE nanoparticles with Tris, ultrapure water, and coagulation factor Xa, and measure the fluorescence intensity A1; S3, incubating the AIE nanoparticles with Tris, ultrapure water, coagulation factor Xa, and anticoagulation factor Xa components, and measuring the fluorescence intensity A2; S4. Calculate the coagulation factor Xa inhibition rate according to A0 obtained in step S1, A1 obtained in step S2, and A2 obtained in step S3.

Images