CN113234023B

CN113234023B - Pyrenyl benzimidazole fluorescent probe and preparation method and application thereof

Info

Publication number: CN113234023B
Application number: CN202110460745.6A
Authority: CN
Inventors: 聂海亮; 梁晓萌; 闫宏远; 白洁; 张国伟; 杨晓溪
Original assignee: Hebei University
Current assignee: Hebei University
Priority date: 2021-04-27
Filing date: 2021-04-27
Publication date: 2022-05-17
Anticipated expiration: 2041-04-27
Also published as: CN113234023A

Abstract

The invention provides a pyrenyl benzimidazole fluorescent probe and a preparation method and application thereof, wherein the fluorescent probe has a structure shown in a formula (I), and the preparation process is as follows: (1) adding 1, 2-dimethyl benzimidazole and (3-bromopropyl) dimethyl ammonium bromide into acetonitrile, heating and refluxing, and separating to obtain a compound A after the reaction is finished; (2) heating and refluxing the compound A and 1-pyrene formaldehyde in ethanol, and separating and purifying to obtain the fluorescent probe with the structure shown in the formula (I). The fluorescent probe can be used for detecting heparin, and has the characteristics of simple synthesis, good water solubility, long luminescence wavelength, high sensitivity to heparin, strong selectivity, dual-mode response and wide application range.

Description

Pyrenyl benzimidazole fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical detection, and particularly relates to a pyrenyl benzimidazole fluorescent probe, and a preparation method and application thereof.

Background

Heparin is a highly sulfated linear glycosaminoglycan, mainly composed of iduronic acid, glucuronic acid and glucosamine, containing a large number of sulfonate and carboxylate groups, and is known as the highest negative charge density biomacromolecule. It is ubiquitous in organs such as lung, heart, liver, pancreas and blood, and plays a crucial role in controlling immune defense mechanism, cell growth and differentiation, lipid transport and metabolism, and preventing blood clot formation. Heparin can enhance the inhibition effect of antithrombin on thrombin and other blood coagulation factors, relieves and inhibits the formation of thrombus, and is the most widely used anticoagulant medicament in clinic at present. However, heparin also has potential risks during use, and excessive ingestion may cause side effects such as bleeding, thrombocytopenia, inhibition of cell proliferation, and the like. In cardiovascular surgery, the dosage of heparin is controlled at 2-8U/mL (17-67 μ M), and in the postoperative care process, the dosage is controlled at 0.2-1.2U/mL (1.7-10 μ M). Therefore, establishing a simple and efficient analysis method to accurately determine the heparin content in blood has important significance for guaranteeing the medication safety of heparin.

Currently, heparin content is generally clinically determined by measuring Activated Clotting Time (ACT), activated partial thromboplastin time (ACTT), and anti-factor Xa activity. These methods usually require expensive equipment, skilled technicians and complicated, time-consuming and costly operations. In addition, the methods are indirect methods for detecting heparin, the selectivity is poor, the sensitivity is low, the detection result is easily influenced by the factors of the organism, and the actual application effect is seriously weakened. With the development of science and technology, a variety of new methods for detecting heparin, such as surface enhanced raman spectroscopy, spectrophotometry, electrochemical methods, high performance liquid chromatography, capillary electrophoresis, fluorescence analysis, etc., have been developed. Among them, the fluorescence analysis method has been widely paid attention to due to its characteristics of simple operation, short time consumption, high sensitivity, and good selectivity.

To date, researchers have reported numerous fluorescent probes for heparin detection. However, most of these fluorescent probes use traditional fluorescent dyes (rhodamine, fluorescein, pyrene, etc.) as luminescent groups, and have the defects of aggregation-induced quenching (ACQ), low sensitivity and poor stability, which severely limits their practical application range. In recent years, although heparin fluorescent probes based on aggregation-induced emission (AIE) dyes have been reported, these fluorescent probes still have the disadvantages of difficult synthesis, poor water solubility, short emission wavelength, low sensitivity, single-mode response and the like, and cannot realize colorimetric/fluorescent dual-mode detection of heparin at the same time.

Disclosure of Invention

The invention aims to provide a pyrenyl benzimidazole fluorescent probe and a preparation method and application thereof, and aims to solve the problems that the fluorescent probe in the prior art is difficult to synthesize, low in sensitivity, incapable of realizing colorimetric/fluorescent dual-mode detection of heparin and the like.

The technical scheme adopted by the invention for realizing the purpose is as follows: a pyrenyl benzimidazole fluorescent probe (PYNN for short) has a structure shown in a formula (I):

the preparation method of the fluorescent probe comprises the following steps:

(1) adding 1, 2-dimethylbenzimidazole and (3-bromopropyl) dimethylammonium bromide into acetonitrile, heating and refluxing, and separating to obtain a compound A:

(2) heating and refluxing the compound A and 1-pyrene formaldehyde in ethanol, and separating and purifying to obtain the fluorescent probe with the structure shown in the formula (I).

In the step (1), the molar ratio of the 1, 2-dimethylbenzimidazole to the (3-bromopropyl) dimethylammonium bromide is 1: 1.

In the step (1), the reaction temperature is 70-95 ℃, and the reaction time is 8-16 h.

In the step (1), the separation step is as follows: after the reaction, the reaction solution was concentrated to 1/2 in the original volume, and the product was obtained by filtration after precipitation of a solid.

In the step (2), the molar ratio of the compound A to the 1-pyrene formaldehyde is 1: 1.

In the step (2), the reaction temperature is 70-95 ℃, and the reaction time is 24-48 h.

In the step (2), piperazine is added as a catalyst, and the molar ratio of the compound A to the catalyst is 1: 2.

In the step (2), the separation and purification steps are as follows: and after the reaction is finished, removing the solvent from the reaction liquid by rotary evaporation, using a dichloromethane/methanol mixed solvent as an eluent for the obtained crude product, passing through a silica gel chromatographic column, removing the solvent from the column-passing liquid by rotary evaporation, and drying in vacuum to obtain a pure product of the fluorescent probe, wherein the dichloromethane/methanol volume ratio is 10: 1.

The reaction formula of step (1) of the present invention is:

the reaction formula of step (2) of the present invention is:

the fluorescent probe is applied to detecting heparin.

The invention has the beneficial effects that:

(1) simple synthesis

The fluorescent probe prepared by the invention has the advantages of easily available raw materials, simple synthesis steps, easy separation and purification, suitability for batch production and contribution to commercial popularization and application.

(2) Long luminescence wavelength and AIE property

The main luminescent region of PYNN is 550-750nm, the maximum emission wavelength is 580nm, and the interference of serum background fluorescence can be effectively reduced. PYNN has typical AIE properties, can emit high-intensity fluorescence in an aggregation state, improves the sensitivity of the method, and has the detection limit of 4.5-12.1 ng/mL.

(3) Colorimetric/fluorescent dual mode response

After the PYNN is combined with the heparin, the color of the solution is changed from colorless to yellow, the fluorescence is obviously enhanced, and the colorimetric and fluorescent dual-mode detection of the heparin can be realized simultaneously.

(4) High response speed, good selectivity and strong stability

The response process of PYNN to heparin can be completed within 2min, the complex of the PYNN and the heparin keeps stable within 5h, and the detection process is not interfered by other substances (such as ions, thiol, polysaccharide and amino acid).

(5) Wide application range

PYNN is suitable for detection of heparin in various serum matrixes such as human serum, rabbit serum, bovine serum and the like.

Drawings

FIG. 1 is a PYNN of¹H NMR spectrum.

FIG. 2 is a PYNN of¹³C NMR spectrum.

FIG. 3 is a fluorescence spectrum of PYNN in a toluene-tetrahydrofuran mixed solution with a toluene volume fraction of 0 to 100%.

Figure 4 is the trend of PYNN absorption spectra as a function of heparin concentration.

FIG. 5 is a plot of the fluorescence spectrum of PYNN as a function of heparin concentration.

Figure 6 is the response rate of PYNN to heparin.

Figure 7 is a fluorescence spectrum of PYNN after addition of heparin and other analytes.

FIG. 8 is a graph of PYNN and its complex with heparin showing the change in fluorescence intensity at 580nm over time.

FIG. 9 is a graph of the response of PYNN to heparin in human serum. Wherein (a) is the fluorescence responsiveness of PYNN in human serum to heparin; (B) is a linear relationship between the fluorescence intensity of PYNN at 580nm and heparin concentration in human serum.

FIG. 10 is a graph of PYNN response to heparin in bovine serum. Wherein (a) is the fluorescent responsiveness of PYNN to heparin in bovine serum; (B) is a linear relationship between the fluorescence intensity of PYNN at 580nm and heparin concentration in bovine serum.

FIG. 11 is a graph of the response of PYNN to heparin in rabbit serum. Wherein (a) is the fluorescent responsiveness of PYNN to heparin in rabbit serum; (B) is a linear relationship between the fluorescence intensity of PYNN at 580nm in rabbit serum and heparin concentration.

Detailed Description

The invention is further illustrated by the following examples, which are given by way of illustration only and are not intended to limit the scope of the invention in any way.

Example 1 Synthesis of PYNN

(1) Synthesis of Compound A

A50 mL round bottom flask was charged with 0.44g (3mmol) of 1, 2-dimethylbenzimidazole and 0.78g (3mmol) of (3-bromopropyl) dimethylammonium bromide, 25mL of acetonitrile, heated to 90 ℃ with stirring, and refluxed for 8 h. Then, the reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure to 1/2 in the original volume to precipitate a white solid, which was then subjected to suction filtration under reduced pressure to obtain Compound A, which was then subjected to the next reaction.

(2) Synthesis of Compound PYNN

A150 mL round-bottomed flask was charged with 0.23g (1mmol) of 1-pyrenecarboxaldehyde, 0.41g (1mmol) of the compound A, 0.17g (2mmol) of piperazine, and 80mL of ethanol, and the reaction was terminated after stirring, heating to 90 ℃ and refluxing for 24 hours. Then, the reaction liquid is cooled to room temperature, the solvent is removed by rotary evaporation, the crude product is subjected to silica gel chromatographic column chromatography by using dichloromethane/methanol (V/V, 10:1) as eluent, the solvent is removed by rotary evaporation after column chromatography, and the pure product of PYNN is obtained by vacuum drying.

It is composed of¹The H NMR spectrum is shown in figure 1: (500MHz, Methanol-d4) δ 9.00(d, J ═ 16.3Hz,1H),8.79(d, J ═ 8.2Hz,1H), 8.72(d, J ═ 9.3Hz,1H),8.38(d, J ═ 8.2Hz,1H),8.33(dd, J ═ 8.6,3.1Hz,3H),8.25(d, J ═ 8.9Hz,1H),8.19(d, J ═ 8.9Hz,1H), 8.16-8.06 (m,2H), 8.06-7.95 (m,1H), 7.81-7.74 (m,2H), 7.68(d, J ═ 16.3Hz,1H),4.85(d, J ═ 7.0, 2H), 4.33.3H (m, 3H), 3.33, 3H, 13H), 13.55 (d, J ═ 8.2H, 1H).

It is composed of¹³The C NMR spectrum is shown in FIG. 2: (126MHz, Methanol-d4) delta 148.84,145.29,132.87,131.30,131.05, 130.61,130.11,129.14,129.08,127.54,127.02,126.91,126.43,126.31,126.02,125.16,124.52, 124.13,122.10,112.98,112.45,108.39,62.68,52.43,42.34,32.89,22.87.

Example 2 AIE Properties of PYNN

40. mu.L of PYNN stock solution (500. mu.M in concentration) prepared in example 1 was taken and added to a 2mL EP tube, and then toluene and tetrahydrofuran (V) were added to the tube in different volume fractions_Toluene：V_{Tetrahydrofuran (THF)}0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, 10: 0). The total volume of the solution was 1mL, and the final concentration of PYNN was 20. mu.M. The above solution was left at room temperature for 30min, and the fluorescence spectrum (excitation wavelength 325nm) was measured.

FIG. 3 shows the fluorescence spectrum of PYNN in a toluene-tetrahydrofuran mixture with a toluene volume fraction of 0-100%. As shown in fig. 3, as the volume fraction of toluene increased from 0 to 100%, the solubility of PYNN in the mixed solvent gradually decreased, and the fluorescence intensity gradually increased, indicating that PYNN has typical AIE properties.

Example 3 colorimetric/fluorescent Dual-mode response of PYNN to heparin

To a 2mL EP tube was added 40 μ L of the DMSO stock solution of PYNN prepared in example 1 (concentration of 500 μ M), followed by deionized water, 100 μ L of HEPES solution (pH 7, concentration of 100mM) and different volumes of heparin stock solution (concentration of 50 μ g/mL) in sequence, with a total volume of 1mL, a final concentration of PYNN of 20 μ M and a heparin concentration of 0-8 μ g/mL. The above solution was left at room temperature for 30min, and then the absorption spectrum and fluorescence spectrum (excitation wavelength 325nm) were measured.

FIG. 4 is an absorption spectrum of PYNN after the addition of different concentrations of heparin; FIG. 5 is a fluorescence spectrum of PYNN after addition of different concentrations of heparin. As shown in FIGS. 4 and 5, with increasing concentration of added heparin (0-8. mu.g/mL), the absorbance of PYNN at 280nm and 370nm gradually decreased, while the absorbance at 315nm and 425nm gradually increased, and the color of the solution changed from light yellow to dark yellow, which can be used for the colorimetric determination of heparin. The fluorescence of PYNN is gradually increased along with the increase of the concentration of heparin, and the PYNN can be used for the fluorescence measurement of the heparin. Thus, PYNN can be applied to colorimetric/fluorescent dual-mode detection of heparin.

Example 4 response rate of PYNN to heparin

100. mu.L of PYNN stock solution (500. mu.M in DMSO) prepared in example 1, 40. mu. L, HEPES in solution (pH 7, 100mM in DMSO) and different volumes of heparin stock solution (50. mu.g/mL in DMSO) were sequentially added to a four-sided transmission quartz cuvette and immediately placed in a spectrofluorometer for measurement in time fluorescence scanning mode. The excitation wavelength was set at 325nm, the emission wavelength at 580nm, and the time interval was 2S. PYNN final concentration of 20 u M, heparin final concentration is 2 g/mL and 5 g/mL respectively.

FIG. 6 is a graph of PYNN fluorescence intensity at 580nm as a function of time after the addition of 2. mu.g/mL and 5. mu.g/mL heparin. After the addition of heparin, the fluorescence intensity of PYNN at 580nm rapidly increased and reached a stable level within 2min, indicating that the response rate of PYNN to heparin is fast.

Example 5 selectivity of PYNN to heparin

Will carry outThe DMSO stock solution (concentration of 500 μ M) of PYNN prepared in example 1 was added to a 2mL EP tube in an amount of 40 μ L, and then deionized water, a HEPES solution (pH 7, concentration of 100mM), and stock solutions of various analytes were sequentially added to the tube. The total volume of the solution was 1mL, the final concentration of PYNN was 20. mu.M, and the final concentration of HEPES was 10 mM. The above solution was left at room temperature for 30min, and then the fluorescence spectrum (excitation wavelength 325nm) was measured. The analytes include: blank, 2 heparin (Hep, 8. mu.g/mL), 3SO₃ ^2-(1mM)，4PO₄ ^3-(1mM), 5 citric acid (CA, 1mM), 6 glucose (Glu, 1mM), 7 glutathione (GSH, 1mM), 8 cysteine (Cys, 1mM), 9 dextran (DS, 100. mu.g/mL), 10 adenosine triphosphate (ATP, 100. mu.g/mL), 11 bovine serum albumin (BSA, 100. mu.g/mL), 12 chondroitin sulfate (ChS, 100. mu.g/mL), 13 hyaluronic acid (HA, 100. mu.g/mL), 14K⁺(1mM)，15Mg²⁺(1mM)，16Na⁺(1mM)， 17Ca²⁺(1mM)。

FIG. 7 is a fluorescence spectrum of PYNN in the presence of various analytes. As shown in figure 7, the PYNN fluorescence intensity at 580nm increased by about 18-fold upon heparin addition, emitting bright orange fluorescence; whereas the fluorescence shows only a slight change after addition of HA, ChS and other analytes, the fluorescence remains weak. The results show that PYNN has good selectivity to heparin.

Example 6 PYNN and its stability with heparin complexes

To a 2mL EP tube, 40 μ L of the PYNN stock solution (concentration 500 μ M) prepared in example 1 was added, followed by deionized water, 100 μ L of a HEPES solution (pH 7, concentration 100mM), and 100 μ L of a heparin stock solution (concentration 50 μ g/mL) in this order. The total volume of the solution was 1mL, the final concentration of PYNN was 20. mu.M, the final concentration of HEPES was 10mM, and the final concentration of heparin was 5. mu.g/mL. The above solution was subjected to fluorometry (excitation wavelength 325nm) every 1 hour for a total of 5 hours.

FIG. 8 is a graph of PYNN and its fluorescence intensity with heparin complex at 580nm as a function of time. As shown in fig. 8, the fluorescence intensity of PYNN and its complex with heparin hardly changed at 580nm with the lapse of the standing time, indicating that PYNN and its complex with heparin have good stability.

Example 7 Linear response Range of PYNN to heparin in human serum

40 μ L of the PYNN stock solution (concentration 500 μ M) prepared in example 1 was added to a 2mL EP tube, and then deionized water, a HEPES solution (pH 7, concentration 100mM), 100 μ L of human serum, and different volumes of heparin stock solutions (concentration 0.05mg/mL) were sequentially added to the tube. The total volume of the solution is 1mL, the final concentration of PYNN is 20 mu M, the final concentration of HEPES is 10mM, the volume fraction of human serum is 10%, and the final concentration of heparin is 0-10 mu g/mL. The above solution was left at room temperature for 30min, and then the fluorescence spectrum (excitation wavelength 325nm) was measured.

FIG. 9 (A) is the fluorescence spectrum of PYNN after addition of 0-10. mu.g/mL heparin; FIG. 9 (B) is a linear relationship between PYNN fluorescence intensity increase at 580nm and heparin concentration (0-6. mu.g/mL) after addition of 0-10. mu.g/mL heparin. As shown in FIG. 9 (A) and FIG. 9 (B), after sequentially adding 0-10. mu.g/mL of heparin to a HEPES solution containing 10% human serum, the fluorescence gradually increased, showing a highly sensitive response to heparin. The increase of the fluorescence intensity of PYNN at 580nm and the heparin concentration in the range of 0-6 mug/mL are in accordance with the linear equation: F-F₀＝189.8*[Hep](R²0.9972), the limit of detection is 6.0ng/mL, which indicates that PYNN has higher sensitivity to heparin and is suitable for detection of heparin in human serum.

Example 8 Linear response Range of PYNN to heparin in bovine serum

40 μ L of the PYNN stock solution (500 μ M in concentration) prepared in example 1 was added to a 2mL EP tube, and then deionized water, 100 μ L of the HEPES solution (pH 7, 100mM in concentration), 30 μ L of bovine serum, and different volumes of heparin stock solutions (0.05 mg/mL) were sequentially added to the tube, with a total solution volume of 1mL, a final PYNN concentration of 20 μ M, a final HEPES concentration of 10mM, a volume fraction of bovine serum of 3%, and a final heparin concentration of 0-10 μ g/mL. The above solution was left at room temperature for 30min, and then the fluorescence spectrum (excitation wavelength 325nm) was measured.

FIG. 10 (A) is the fluorescence spectrum of PYNN after addition of 0-10. mu.g/mL heparin; FIG. 10 (B) is a linear graph of PYNN fluorescence intensity increase at 580nm versus heparin concentration (0-8. mu.g/mL) after addition of 0-10. mu.g/mL heparin. As in FIG. 10 (A)) And (B) in FIG. 10, after sequentially adding 0-10. mu.g/mL of heparin to a HEPES solution containing 3% bovine serum, the fluorescence gradually increased, showing a highly sensitive response to heparin. The increase of the fluorescence intensity of PYNN at 580nm and the heparin concentration in the range of 0-8 mug/mL accord with the linear equation: F-F₀＝293.1*[Hep](R²0.9993), the detection limit is 4.5 ng/mL, which indicates that PYNN has higher sensitivity to heparin and is suitable for detecting heparin in bovine serum.

Example 9 Linear response Range of PYNN to heparin in Rabbit serum

40 μ L of the PYNN stock solution (500 μ M concentration) prepared in example 1 was added to a 2mL EP tube, and then deionized water, 100 μ L of HEPES solution (pH 7, 100mM concentration), 40 μ L of rabbit serum, and different volumes of heparin stock solution (50 μ g/mL concentration) were sequentially added to the tube, with a total solution volume of 1mL, a final PYNN concentration of 20 μ M, a final HEPES concentration of 10mM, a rabbit serum volume fraction of 4%, and a final heparin concentration of 0-10 μ g/mL. The above solution was left at room temperature for 30min, and then the fluorescence spectrum (excitation wavelength 325nm) was measured.

FIG. 11 (A) shows the fluorescence of PYNN after addition of 0-10. mu.g/mL heparin; FIG. 11 (B) is a linear relationship between PYNN fluorescence intensity increase at 580nm and heparin concentration (0-5.5. mu.g/mL) after addition of 0-10. mu.g/mL heparin. As shown in FIG. 11 (A) and FIG. 11 (B), after sequentially adding 0-10. mu.g/mL of heparin to HEPES solution containing 4% rabbit serum, the fluorescence gradually increased, showing a highly sensitive response to heparin. The increase in fluorescence intensity of PYNN at 580nm with heparin concentration (0-5.5. mu.g/mL) was in accordance with the linear equation: F-F₀＝108.3*[Hep](R²0.9994), 12.1ng/mL, and the result shows that PYNN has higher sensitivity to heparin and is suitable for detecting heparin in rabbit serum.

Example 10 Synthesis of PYNN

(1) Synthesis of Compound A

A50 mL round bottom flask was charged with 0.44g (3mmol) of 1, 2-dimethylbenzimidazole and 0.78g (3mmol) of (3-bromopropyl) dimethylammonium bromide, 25mL of acetonitrile, heated to 70 ℃ with stirring, and refluxed for 16 h. Then, the reaction solution was cooled to room temperature, and the reaction solution was concentrated under reduced pressure to 1/2 in the original volume to precipitate a white solid, which was then subjected to suction filtration under reduced pressure to obtain Compound A, which was then subjected to the next reaction.

(2) Synthesis of Compound PYNN

To a 150mL round-bottomed flask were added 0.23g (1mmol) of 1-pyrenecarboxaldehyde and 0.41g (1mmol) of the compound A0.17 g (2mmol), piperazine and 80mL of ethanol, and the mixture was stirred, heated to 70 ℃ and refluxed for 48 hours, whereupon the reaction was terminated. Then, the reaction liquid is cooled to room temperature, the solvent is removed by rotary evaporation, the crude product is subjected to silica gel chromatographic column chromatography by using dichloromethane/methanol (V/V, 10:1) as eluent, the solvent is removed by rotary evaporation after column chromatography, and the pure product of PYNN is obtained by vacuum drying. The resulting product was characterized and had similar properties to the compound of example 1.

Claims

1. A pyrenyl benzimidazole fluorescent probe is characterized by having a structure shown in a formula (I):

（Ⅰ）。

2. a method for preparing the fluorescent probe of claim 1, comprising the steps of:

；

(2) heating and refluxing the compound A and 1-pyrene formaldehyde in ethanol, and adding piperazine as a catalyst; separating and purifying to obtain the fluorescent probe with the structure shown in the formula (I).

3. The method according to claim 2, wherein in the step (1), the molar ratio of 1, 2-dimethylbenzimidazole to (3-bromopropyl) dimethylammonium bromide is 1: 1.

4. The preparation method according to claim 2, wherein in the step (1), the reaction temperature is 70-95 ℃ and the reaction time is 8-16 h.

5. The method according to claim 2, wherein in the step (1), the separation step is: after the reaction, the reaction solution was concentrated to 1/2 in the original volume, and the product was obtained by filtration after precipitation of a solid.

6. The method according to claim 2, wherein in the step (2), the molar ratio of the compound A to the 1-pyrenecarboxaldehyde is 1: 1.

7. The preparation method according to claim 2, wherein in the step (2), the reaction temperature is 70-95 ℃ and the reaction time is 24-48 h.

8. The process according to claim 2, wherein in the step (2), the molar ratio of the compound A to the catalyst is 1: 2.

9. The method according to claim 2, wherein in the step (2), the separation and purification step is: and after the reaction is finished, removing the solvent from the reaction liquid by rotary evaporation, using a dichloromethane/methanol mixed solvent as an eluent for the obtained crude product, passing through a silica gel chromatographic column, removing the solvent from the column-passing liquid by rotary evaporation, and drying in vacuum to obtain a pure product of the fluorescent probe, wherein the dichloromethane/methanol volume ratio is 10: 1.

10. Use of the fluorescent probe of claim 1 in the preparation of a reagent for detecting heparin.