CN110963911A

CN110963911A - AIE fluorescent probe for heparin detection and pH response, synthetic method and application

Info

Publication number: CN110963911A
Application number: CN201911131348.3A
Authority: CN
Inventors: 张岩; 姜睿
Original assignee: Chongqing Normal University
Current assignee: Chongqing Normal University
Priority date: 2019-11-19
Filing date: 2019-11-19
Publication date: 2020-04-07
Anticipated expiration: 2039-11-19
Also published as: CN110963911B

Abstract

The invention relates to the field of organic luminescent materials, and particularly discloses an AIE fluorescent probe for heparin detection and pH response, a synthesis method and application, wherein the fluorescent probe with AIE property is BDA-4COOH, the fluorescent probe can sensitively perform fluorescent response to different pH values, and the fluorescence intensity shows obvious change and has extremely high sensitivity, so that the fluorescent probe has high application value; the fluorescent probe BDA-4COOH has strong binding capacity to protamine under certain conditions, and the heparin can generate antagonistic action with the protamine, so that the anisotropic response to the heparin is realized. The AIE fluorescent probe synthesized by the invention has larger modification space, can be subjected to group modification, and greatly improves the possibility of applying the probe to the inside of a living body.

Description

AIE fluorescent probe for heparin detection and pH response, synthetic method and application

Technical Field

The invention relates to the field of organic luminescent materials, in particular to preparation of an AIE fluorescent probe for pH response and heparin detection and research on luminescent properties.

Background

The fluorescence sensor has the advantages of outstanding sensitivity, high detection speed, high selectivity, convenience in operation and the like, and is widely applied to the fields of biomedicine, environmental monitoring and the like at present. Among many fluorescent sensing materials, fluorescent probes based on small organic molecules have the advantages of simple synthesis, low cost, easy operation and the like, and are favored by many scholars.

Organic fluorescent molecules are widely applied in the field of fluorescence sensing at present and are important fluorescence sensing materials. However, conventional organic fluorescent probes (such as fluorescein, rhodamine, and cyanine probes) generally have strong fluorescence emission only in dilute solution, and fluorescence quenching occurs at high concentration or aggregation state. Meanwhile, the Stokes shift of the traditional fluorescent probe is usually small and is often interfered by a complex environment. Therefore, it is necessary to synthesize an organic fluorescent probe with high sensitivity, good selectivity and excellent photophysical properties to overcome the Aggregation quenched Quenching (ACQ) effect. Because of the unique luminescence property of the AIE (aggregation induced emission) molecule in an aggregation state, a brand new idea is opened for solving the aggregation quenching problem of the traditional fluorescent molecule and preparing the Turn-On type fluorescent sensor. Due to the distorted molecular configuration in the aggregate (solid state), AIE molecules typically have large Stokes shifts. Meanwhile, researches show that the photobleaching resistance of molecules in an aggregation state is obviously stronger than that of a single-molecule state. These features and advantages all provide advantages for improving the sensitivity of the AIE fluorescence sensor and reducing background noise.

pH-responsive fluorescent molecules typically contain a large number of weak electrolyte groups that are susceptible to protonation or hydrolysis, such as pyridine, amino, and carboxyl groups. These groups can bind or release hydrogen ions under conditions of pH change, which can affect the solubility of the fluorescent molecule in the solvent, particularly in more polar solvents.

The heparin has strong negative charges, has complex and wide biological functions, can generate antagonism with protamine, can increase the activity of the lipoprotein, and effectively prevent and treat atherosclerosis. Heparin plays an important role in the field of medicine. The traditional methods for detecting heparin, such as spectrophotometry and capillary electrophoresis, have certain defects, such as poor sensitivity, long detection time, even certain toxicity of part of medicines, complex experimental operation and the like.

Disclosure of Invention

In order to overcome the defects, the invention provides an AIE fluorescent probe for heparin detection and pH response, a synthesis method and application. The invention relates the change of solubility caused by electrolyte groups and the unique luminescence property of AIE molecules to design a pH response type AIE fluorescent probe. In the invention, the heparin is detected by using a compound formed by the fluorescent probe and the protamine, and the aggregation state of the fluorescent probe is changed by utilizing the electrostatic effect so as to realize the detection of the heparin through the AIE property of molecules.

In order to achieve the purpose of the invention, the invention adopts the technical scheme that:

an AIE fluorescent probe for heparin detection and pH response, wherein the AIE fluorescent probe is BDA-4COOH, and the structural formula of the AIE fluorescent probe is as follows:

the fluorescent probe has good AIE property, and can be used in water and CH₃In the OH mixed solvent, the fluorescence intensity of BDA-4COOH is enhanced along with the increase of water content, and an emission peak appears at 520 nm.

BDA-4COOH at V_{Water (W)}：V_CH3OH9:1 and pH 1, the fluorescence emission wavelength is 520nm, and the fluorescence intensity gradually decreases with increasing pH.

The invention also provides a synthetic method of the AIE fluorescent probe for heparin detection and pH response, and the synthetic method of the fluorescent probe comprises the following steps:

1) reacting anthracene with paraformaldehyde in an environment filled with hydrochloric acid gas to obtain 9, 10-dichloromethylanthracene;

2) reacting the product obtained in the step 1) with triethyl sulfite to obtain phosphoylide, 10-bis (diethoxyphosphoric acid methyl) anthracene;

3) reacting the product obtained in the step 2) with 4, 4-dicyanobenzophenone under the catalysis of potassium tert-butoxide to obtain a compound BDA-4 CN;

4) and (3) hydrolyzing the product obtained in the step 3) with NaOH to obtain a crude product, and purifying the crude product to obtain the AIE fluorescent probe for heparin detection and pH response.

Further, in the step 4), the product obtained in the step 3) is added with NaOH and THF in the ratio of NaOH to THF to C₂H₅O₅＝1:2:3。

Further, in the step 4), the reaction temperature is 90 ℃ and the reaction time is 20 h.

The more specific synthesis method comprises the following steps:

preparation of 1.9, 10-Dichloromethyl

In a 1000mL three-necked flask, anthracene (a mmol), paraformaldehyde (b mmol), 50mL of HCl solution and 300mL of 1, 4-dioxane solution were sequentially added, and the mixture was stirred. Refluxing for 2h under the condition of introducing HCl gas, continuously separating out yellow solid in the reaction solution, stopping introducing HCl gas after 2h, and continuously refluxing the reaction solution for 3 h. Then, the mixture was cooled to room temperature, filtered, and the filter cake was rinsed twice with 1, 4-dioxane solution and once again with water to give a yellow-green solid with a yield of 80%.

Preparation of 10-bis (diethoxyphosphorylmethyl) anthracene:

9, 10-Dichloromethylanthracene (a mmol) and 30mL of a triethylphosphite solution were added to a reaction flask, and the reaction was refluxed for 12 hours with stirring. Cooling to room temperature, filtering, leaching the filter cake twice by using petroleum ether to obtain a yellow-green solid with the yield of 83 percent.

Preparation of BDA-4 CN:

under the protection of nitrogen, the compound 9, 10-bis (diethoxyphosphorylmethyl) anthracene (a mmol), potassium tert-butoxide (b mmol) and 100mL of anhydrous tetrahydrofuran solution are sequentially added into a 250mL dry two-necked bottle, and the two-necked bottle is placed in an ice water bath at 0 ℃ for cooling and stirring for later use. A solution (20mL) of the compound 4, 4-dicyanobenzophenone (c mmol) in anhydrous tetrahydrofuran was added dropwise to the above solution, and after completion of the addition, the mixture was stirred at room temperature for 12 hours. The mixture was then spun through a rotary evaporator, dissolved by adding 5mL of dichloromethane solution, added to 200mL of methanol solution, and filtered.

Preparation of BDA-4 COOH:

a mixed solution of BDA-4CN (a mmol), NaOH, THF and ethanol which are prepared according to a certain proportion is added into a 100mL dry round-bottom flask to react for 20 hours at 90 ℃. After the product is concentrated in vacuum, the product is acidified to pH 1 by HCl, an organic layer solution is extracted, and the product is concentrated to obtain the required product.

The invention also provides application of the AIE fluorescent probe in protamine quantitative detection and/or heparin quantitative detection.

The invention also provides the application of the AIE fluorescent probe in protamine quantitative detection, in V_{Water (W)}：V_CH3OH9:1 and pH 6, the fluorescence intensity of the AIE fluorescent probe increased with increasing protamine concentration.

The invention provides an application of an AIE fluorescent probe in heparin quantitative detection, namely V_{Water (W)}:V_CH3OHUnder the conditions of 9:1 and pH 6, the fluorescence intensity of the AIE fluorescent probe and protamine complex decreased with increasing heparin concentration.

The invention also provides a heparin detection method of the AIE fluorescent probe, which comprises the steps of taking the methanol solution of the fluorescent probe, adding the protamine solution into the methanol solution after full oscillation, adding the solution to be detected into the methanol solution, and carrying out fluorescent detection after incubation culture.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a fluorescent probe BDA-4COOH with AIE properties, and experimental results show that the fluorescent probe can sensitively carry out fluorescent response to different pH values, and the fluorescence intensity shows obvious change and has extremely high sensitivity (the fluorescent probe is sensitive to pH change, particularly in an alkaline region), so the fluorescent probe has high application value; the fluorescent probe BDA-4COOH has strong binding capacity to protamine under certain conditions, and the heparin can generate antagonistic action with the protamine, so that the anisotropic response to the heparin is realized. The AIE fluorescent probe synthesized by the invention has larger modification space, can be subjected to group modification, and greatly improves the possibility of applying the probe to the inside of a living body.

Compared with the traditional AIE fluorescent probe, the BDA-4COOH disclosed by the invention has unique excitation wavelength and emission wavelength, for example, the emission wavelength of the fluorescent probe disclosed by the invention is 520 nm; has recognition function, and can be directly used for fluorescent labeling;

drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows nuclear magnetic H spectrum of a fluorescent probe (BDA-4COOH, the same applies below) having a structure according to an embodiment of the present invention;

FIG. 2 is a nuclear magnetic C spectrum of a fluorescent probe of formula I in an embodiment of the present invention;

FIG. 3 is a nuclear magnetic mass spectrum of a fluorescent probe of formula I according to an embodiment of the present invention;

FIG. 4 is an infrared spectrum of a fluorescent probe of formula I according to an embodiment of the present invention;

FIG. 5 AIE property fluorescence spectrum of the fluorescent probe of the present invention;

FIG. 6 shows fluorescence spectra of a fluorescent probe with a structure according to an embodiment of the present invention at different pH values;

FIG. 7 shows the fluorescent spectrum of protamine response of the fluorescent probe with a structural formula in the example of the present invention;

FIG. 8 shows a linear fluorescence spectrum of protamine as an example of the fluorescent probe with a structural formula;

FIG. 9 shows the fluorescence spectrum of heparin response of the structural fluorescent probe in the example of the present invention;

FIG. 10 shows heparin linear fluorescence spectrum of a structural fluorescent probe according to an embodiment of the present invention;

FIG. 11 shows heparin selectivity of the structural fluorescent probe in the example of the invention.

Detailed Description

The invention is further illustrated by the following figures and specific examples.

Example 1 Synthesis of 9, 10-Dichloromethylanthracene

In a 1000mL three-necked flask, anthracene (250mmol), paraformaldehyde (40g), 50mL of concentrated HCl solution and 350mL of 1, 4-dioxane solution were sequentially added, and stirred. Refluxing for 2h under the condition of introducing HCl gas, continuously separating out yellow solid in the reaction solution, stopping introducing HCl gas after 2h, and continuously refluxing the reaction solution for 3 h. Then, the mixture was cooled to room temperature, filtered, and the filter cake was rinsed twice with 1, 4-dioxane solution and once again with water to give a yellow-green solid with a yield of 85%.

Example 2 Synthesis of 10-bis (diethoxyphosphorylmethyl) anthracene

9, 10-Dichloromethylanthracene (20mmol) and 20mL of a triethylphosphite solution were added to a reaction flask, and the reaction was refluxed for 12 hours with stirring. Cooling to room temperature, filtering, leaching the filter cake twice by using petroleum ether to obtain a yellow green solid 10-di (diethoxyphosphoric acid methyl) anthracene with the yield of 83 percent. The reaction scheme is as follows:

example 3 BDA-4CN Synthesis

Under the protection of nitrogen, 10-bis (diethoxyphosphorylmethyl) anthracene (6mmol), potassium tert-butoxide (20mmol) and 100mL of anhydrous tetrahydrofuran solution are sequentially added into a 250mL dry two-necked bottle, and the two-necked bottle is placed in an ice water bath at 0 ℃ for cooling and stirring for later use. A solution (20mL) of the compound 4, 4-dicyanobenzophenone (15mmol) in anhydrous tetrahydrofuran was added dropwise to the above solution, and after completion of the addition, the mixture was stirred at room temperature for 12 hours. The mixture was then spun through a rotary evaporator, dissolved by adding 5mL of dichloromethane solution, added to 200mL of methanol solution, and filtered. The reaction scheme is as follows:

example 4 Synthesis of BDA-4COOH

A100 mL dry round-bottom flask was charged with the product (50mg, 2.1mmol) obtained above, a mixed solution of NaOH and THF and ethanol in a certain ratio (NaOH: THF: C)₂H₅O₅1:2:3), and reacting at 90 ℃ for 20 h. After the product was concentrated in vacuo, it was acidified to pH 1 with HCl, the organic layer solution was extracted and concentrated to give the desired final product BDA-4 COOH. The reaction process is as follows:

FIG. 1 shows nuclear magnetic H spectrum of a fluorescent probe (BDA-4COOH, the same applies below) having a structure according to an embodiment of the present invention; FIG. 2 is a nuclear magnetic C spectrum of a fluorescent probe of formula I in an embodiment of the present invention; FIG. 3 is a nuclear magnetic mass spectrum of a fluorescent probe of formula I according to an embodiment of the present invention; FIG. 4 is an infrared spectrum of a fluorescent probe of formula I structure in an embodiment of the present invention.

Example 5 AIE Effect of fluorescent probes

Preparing a fluorescent probe into 1mM methanol solution, respectively placing 0.2mL of the solution into 5mL glass bottles, respectively adding methanol solutions with different volumes, finally adding water until the total volume is 4mL, uniformly mixing to obtain mixed solutions with different water contents, and finally introducing the mixed solutions into a quartz cuvette one by one for fluorescence spectrum test. The experimental results found that the fluorescence intensity increased continuously with increasing water content, indicating that the solution had good AIE properties (figure 5). The AIE properties of the fluorescent probes of the invention: in water and CH₃In the mixed solvent of OH, when the proportion of poor solvent in the environment is gradually increased, the solubility of BDA-4COOH molecules in an environment system is gradually reduced, the molecules are aggregated, the rotation of single bonds in the molecules is limited, the vibration of the molecules is limited, and the channels of non-radiative transition are closed, and the energy is mainly radiated in a radiative mode. At this time, the fluorescence intensity of BDA-4COOH molecules is increased along with the increase of water content, and strong fluorescence is emitted at 520 nm.

Example 6 fluorescence emission Spectroscopy of fluorescent probes at different pH values

50 microliter of 0.5mg/mL methanol solution of the fluorescent probe is respectively added into 3mL solutions with different pH values, and the solutions are transferred to a quartz cuvette for fluorescent detection after being sufficiently shaken. Under the condition of an acid medium, the fluorescence probe is aggregated by itself when the solubility of the fluorescence probe is not high, and the aggregation enables the attenuation of non-radiative energy rotating in molecules to be inhibited, so that an aggregation-induced luminescence phenomenon is generated; under the condition of an alkaline medium, the carboxyl in the fluorescent molecular structure is subjected to proton dissociation, the probe is in an ionic state, the solubility is increased, the aggregation degree is reduced, and at the moment, the molecular excited state energy is mainlyTo diverge in a non-radiative transition leading to a decrease in fluorescence intensity (fig. 6). The fluorescent probe has higher sensitivity to the change of pH: BDA-4COOH at V_{Water (W)}:V_CH3OHThe fluorescence emission wavelength is 520nm under the conditions of 9:1 (volume ratio of water to methanol in methanol solution) and pH 1, and the fluorescence intensity gradually decreases with the increasing pH.

Example 7 fluorescent Probe protamine response

Adding 50 microliters of methanol solution of 3.5mg/mL fluorescent probe into 3mL of buffer solutions with different pH respectively, adding protamine solutions with different concentrations into the buffer solutions after fully shaking, and then carrying out fluorescence detection. Four carboxyl groups exist in the BDA-4COOH molecules to provide negative charges for the surfaces of the BDA-4COOH molecules, so that the BDA-4COOH molecules and protamine with positive charges in a solution generate electrostatic interaction and are mutually combined, the molecules are changed from a free state to an aggregation state to initiate fluorescence enhancement, and the 'turn-on' detection of the protamine is realized by utilizing the AIE properties of the molecules. The experimental result shows that the fluorescence intensity of the solution is continuously increased along with the continuous increase of the concentration of the protamine in the system. The detection limit was calculated from the experimental data to be 29.8ng/mL, with a linear range of 0.02-0.4. mu.g/mL (FIGS. 7-8). The fluorescent probe has higher sensitivity to protamine: at V_{Water (W)}:V_CH3OHUnder the conditions of 9:1 and pH 6, the fluorescence intensity of the probe increases with the increasing concentration of protamine;

example 8 fluorescent Probe heparin response

50 mu L of methanol solution of 3.5mg/mL fluorescent probe is respectively added into 3mL of buffer solution with different pH values, 60 microliter of 0.1mg/mL protamine solution is added after full shaking, and heparin solution with different concentrations is added into the solution. The mixture was incubated for 30 minutes and finally subjected to fluorescence detection. The protamine egg can form stable salt with heparin to make heparin lose anticoagulation effect. Therefore, when heparin is added to the BDA-4COOH molecule and protamine complex system, the heparin peels the BDA-4COOH molecule from the protamine, and the state of the molecule changes from an aggregated state to a free state, resulting in a decrease in fluorescence intensity. The experimental results show thatThe concentration of heparin in the system is continuously increased, and the fluorescence intensity of the solution is continuously weakened. According to the experimental results, the detection limit of heparin is 37ng/mL, and the linear range is 0.08-8 mug/mL (figure 9-10). The fluorescent probe and protamine compound of the invention have higher sensitivity to heparin, and the sensitivity is V_{Water (W)}:V_CH3OHUnder the conditions of 9:1 and pH 6, the fluorescence intensity of the AIE fluorescent probe and protamine complex decreased with increasing heparin concentration.

Example 9 heparin selectivity of fluorescent probes

50 microliter of 3.5mg/mL methanol solution of the fluorescent probe is respectively added into 3mL of buffer solutions with different pH values, 60 microliter of 0.1mg/mL protamine solution is added after full shaking, 60 microliter of 1mg/mL different heparin structure analogues is added, the mixture is incubated for 30 minutes, and finally, fluorescence detection is carried out. Since these heparin analogues have relatively weak binding ability to protamine, the BDA-4COOH molecule cannot be effectively stripped from protamine. The experimental result proves that the fluorescent probe-protamine compound only has obvious fluorescent response to the heparin. (FIG. 11)

The examples described are illustrative of the invention and are not to be construed as limiting the invention, and any variations and modifications which come within the meaning and range of equivalency of the invention are to be considered within the scope of the invention.

Claims

1. An AIE fluorescent probe for heparin detection and pH response, which is characterized in that the AIE fluorescent probe is BDA-4COOH and has the following structural formula:

2. the AIE fluorescent probe for heparin detection and pH response of claim 1, wherein said fluorescent probe is in water and CH₃In the OH mixed solvent, the fluorescence intensity of BDA-4COOH is enhanced along with the increase of water content, and an emission peak appears at 520 nm.

3. The AIE fluorescent probe for heparin detection and pH response of claim 1, wherein BDA-4COOH is at V_{Water (W)}:V_CH3OHThe fluorescence emission wavelength was 520nm at 9:1 and pH 1, and the fluorescence intensity gradually decreased with increasing pH.

4. The method of claim 1, wherein the method of synthesizing AIE fluorescent probes for heparin detection and pH response comprises the steps of:

1) reacting anthracene with paraformaldehyde in the presence of hydrochloric acid gas to obtain 9, 10-dichloromethylAnthracene；

5. The method for synthesizing the AIE fluorescent probe for heparin detection and pH response according to claim 4, wherein in the step 4), the product obtained in the step 3) is added with NaOH, THF and the ratio of NaOH to THF to C₂H₅O₅＝1:2:3。

6. The method for synthesizing the AIE fluorescent probe for heparin detection and pH response according to claim 5, wherein the reaction temperature in the step 4) is 90 ℃ and the reaction time is 20 h.

7. Use of the AIE fluorescent probe for heparin detection and pH response according to claim 1 for protamine quantitative detection and/or heparin quantitative detection.

8. Use of the AIE fluorescent probe of claim 7 for quantitative protamine detection at V_{Water (W)}:V_CH3OHThe AIE fluorescent probe increased in fluorescence intensity with increasing protamine concentration under conditions of 9:1 and pH 6.

9. The use of the AIE fluorescent probe of claim 7 for the quantitative detection of heparin, wherein V is the number of V_{Water (W)}:V_CH3OHUnder the conditions of 9:1 and pH 6, the fluorescence intensity of the AIE fluorescent probe and protamine complex decreased with increasing heparin concentration.

10. The heparin detection method by using the AIE fluorescent probe as claimed in claim 1, wherein the fluorescent probe is taken from a methanol solution, the methanol solution is fully shaken, then a protamine solution is added, then a solution to be detected is added, and the fluorescence detection is performed after incubation culture.