阅读说明：本技术 一种用于滴眼液质量检测的荧光分子及其制备和应用 (Fluorescent molecule for eye drop quality detection and preparation and application thereof ) 是由 徐灵峰 吴魁 韩润林 刘利民 刘荫秋 刘诚 黄艳蓉 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种用于滴眼液质量检测的荧光分子及其制备和应用,该荧光分子具有如下结构：本发明制备的荧光分子4-(((4’-(二苯胺基)-3-羟基-[1,1’-联苯]-4-基)亚甲基)氨基)苯甲腈(DHBMAB),具有分子内质子转移(ESIPT)效应和聚集诱导发光(AIE)特性,能够强化平面共轭,有利于荧光发射红移,同时在高浓度使用下,越聚集越发光,荧光强度更强,可视化程度更高,检测效果更佳,并且检测过程快速、高效、便捷、且用量低。(The invention discloses a fluorescent molecule for eye drop quality detection and preparation and application thereof, wherein the fluorescent molecule has the following structure: fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl) prepared by the invention]-4-yl) methylene) amino) benzonitrile (DHBMAB), which has intramolecular proton transfer (ESIPT) effect and Aggregation Induced Emission (AIE) characteristics, can enhance planar conjugation, is favorable for red shift of fluorescence emission, and simultaneously has stronger fluorescence intensity and higher visualization degree as more aggregation and more emission are used at high concentration,the detection effect is better, and the detection process is quick, efficient, convenient and low in dosage.)

1. A fluorescent molecule for quality detection of eye drops is characterized by having the following structural formula:

。

2. the method for preparing a fluorescent molecule for quality control of eye drops according to claim 1, comprising the steps of:

(1) dissolving 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde in ethanol, and uniformly stirring by ultrasonic waves to obtain a solution 1;

(2) dissolving p-aminobenzonitrile in ethanol, and uniformly stirring by ultrasonic to obtain a solution 2;

(3) and mixing the solution 1 and the solution 2, stirring uniformly, heating for reaction, and then separating and purifying to obtain orange powder, namely the fluorescent molecule for eye drop quality detection.

3. The method for preparing a fluorescent molecule used for eye drop quality detection according to claim 2, wherein the molar ratio of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-carbaldehyde in step (1) to p-aminobenzonitrile in step (2) is 1 (1-10).

4. The method for preparing a fluorescent molecule used for eye drop quality detection according to claim 2, wherein the concentration of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-carbaldehyde in the step (1) is 0.1M to 10M; the concentration of the p-aminobenzonitrile in the step (2) is 0.1M-100M.

5. The method for preparing a fluorescent molecule used for quality control of eye drops according to claim 2, wherein the heating temperature in step (3) is 30 ℃ to 78 ℃, and the heating reaction time is 0.5 h to 36 h.

6. The method for preparing a fluorescent molecule used for quality control of eye drops according to claim 2, wherein the separation and purification process in step (3) comprises: removing organic solvent, washing precipitate with ethanol for 2-3 times, and drying.

7. Use of the fluorescent molecule of claim 1 for eye drop quality detection.

8. Use of the fluorescent molecule according to claim 7 for the detection of eye drops quality, for the detection of eye drops deterioration.

9. Use of the fluorescent molecule of claim 7 or 8 in the quality control of eye drops, wherein the substance of the fluorescent molecule is present in a concentration of 1-100 μ M.

Technical Field

The invention relates to the technical field of drug detection, in particular to a fluorescent molecule for eye drop quality detection and preparation and application thereof.

Background

Eye drops, also known as "eye drops", are one of the most common agents in ophthalmic diseases. The eye drops are external medicines with short shelf life, are inevitably exposed to the air after being unsealed, are easy to deteriorate, are generally clinically recommended to be used up within 4 weeks, and are very easy to induce allergic and toxic reactions of eyes after being dripped into the eye drops, so that the eye drops are extremely harmful to the eye health of people. The development of preservative removal for eye drops is promoted in the industry, so that the quality safety detection of eye drops which are extremely easy to deteriorate becomes extremely important.

The fluorescent molecule is an organic micromolecule device with a molecular structure containing various freely rotatable aromatic rings and a conjugated structure. The small molecule device can rotate freely in a solution with low viscosity and shows very weak fluorescence, but when the small molecule device is in a solution with high viscosity, the free rotation is blocked, so that excited state energy originally dissipated through machinery is dissipated through radiation transition and returns to a ground state, and a fluorescence signal is released. The magnitude of the internal viscosity is reflected in conjunction with the change in the fluorescence signal. Fluorescent molecules have been used in the biological field as a rapid, efficient and convenient means for visualization of fluorescent imaging, but few fluorescent molecules have been developed for use in drug quality safety detection.

Therefore, the development of a fluorescent molecule capable of rapidly, efficiently and visually detecting the quality safety of eye drops is urgently needed, so that great significance is provided for quality control of medicines and effective identification of patients.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the primary object of the present invention is to provide a fluorescent molecule for quality detection of eye drops, which has AIE characteristics and ESIPT effect, can respond to the viscosity of eye drops, and further can monitor the quality safety of eye drops.

Another object of the present invention is to provide a method for preparing the above fluorescent molecule.

The invention further aims to provide the application of the fluorescent molecule in the quality detection of the eye drops.

The purpose of the invention is realized by the following technical scheme:

a fluorescent molecule for quality detection of eye drops is 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl)]-4-yl) methylene) amino) benzonitrile, DHBMAB for short, of formula C₃₂H₂₃N₃O, relative molecular weight of 465.1849, and the specific structural formula is shown in the following figure:

。

the invention provides a preparation method of the fluorescent molecule for eye drop quality detection, which comprises the following steps:

(1) dissolving 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde in ethanol, and uniformly stirring by ultrasonic waves to obtain a solution 1;

(2) dissolving p-aminobenzonitrile in ethanol, and uniformly stirring by ultrasonic to obtain a solution 2;

(3) and mixing the solution 1 and the solution 2, stirring uniformly, heating for reaction, and then separating and purifying to obtain orange powder, namely the fluorescent molecule for eye drop quality detection.

The reaction equation for the preparation is as follows:

preferably, the molar ratio of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-carbaldehyde in step 1) to p-aminobenzonitrile in step 2) is 1 (1-10).

Preferably, the concentration of the 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-carbaldehyde prepared in the step (1) is 0.1M-10M; the concentration of the p-aminobenzonitrile in the step (2) is 0.1M-100M.

Preferably, the heating temperature in the step (3) is 30-78 ℃, and the heating reaction time is 0.5-36 h.

Preferably, the separation and purification process in step (3) comprises: removing organic solvent, washing precipitate with ethanol for 2-3 times, and drying.

The invention also provides the application of the fluorescent molecule in the quality detection of the eye drops, and the fluorescent molecule is used for detecting the deterioration degree of the eye drops.

When the fluorescent molecule provided by the invention is used for eye drop quality detection, the fluorescent molecule is prepared into a monomolecular dispersion solution, namely a test mother solution; when testing, using a syringe to suck a certain dose of test mother liquor and injecting the test mother liquor into eye drops, and controlling the final quantity concentration of the fluorescent molecule substance to be 1-100 mu M; the quality detection of the eye drops is realized by dripping the fluorescent molecule into eye drops with different viscosities, wherein the eye drops used in the embodiment are polyvinyl alcohol eye drops; the eye drop deterioration degree detection is to add the fluorescent molecule into different eye drops, and then judge the deterioration degree of the eye drops along with the observation of the change of the fluorescence.

The fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl) provided by the invention]-4-yl) methylene) amino) benzonitrile (DHBMAB) of formula C₃₂H₂₃N₃O, relative molecular weight 465.1849. The fluorescent molecule DHBMAB is orange solid powder, and is easily dissolved in solvents such as ethyl acetate, acetonitrile, dichloromethane, N-dimethylformamide, dimethyl sulfoxide and the like. The compound has good light stability, no toxicity, good physical stability and stable structure, and is suitable for long-term storage. At the same time, the fluorescent molecule DHBMAB has intramolecular proton transfer (ESIPT) effect, so that Schiff base-C = N-structure which can rotate freely originally is locked by hydroxyl (as shown in figure 1)The rotation of the fluorescent material is inhibited, so that the fluorescent material is more favorable for releasing fluorescence, and the coplanar conjugated structure is strengthened, so that the fluorescence emission is red-shifted.

When the DHBMAB molecule is added into eye drops with different viscosities, the aromatic ring structure which can rotate freely in a good solvent is increased along with the viscosity of the eye drops, and the aromatic ring structure which can rotate freely is restricted along with the viscosity of the eye drops, so that the original excited state energy returns to the ground state through a non-radiative transition mode and a radiative transition mode, and then fluorescence is released, and the phenomenon of fluorescence enhancement occurs. The fluorescent molecule DHBMAB emits strong fluorescence at 365 nm of excitation wavelength and around 568 nm of wavelength, and the fluorescence intensity is gradually enhanced along with the increase of viscosity, so that the DHBMAB can be used for detecting the deterioration degree of eye drops. The specific action mechanism is shown in the attached figure 2.

The invention provides a fluorescent molecule (DHBMAB) for eye drop quality safety detection, which can gradually increase the fluorescence intensity along with the increase of viscosity, and realize in-situ, sensitive and efficient 'turn-on' type signal release and visual detection.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1) the fluorescent molecule (DHBMAB) provided by the invention has the characteristics of intramolecular proton transfer (ESIPT) effect and Aggregation Induced Emission (AIE), can strengthen coplanar conjugation, is favorable for fluorescence emission red shift, and has stronger fluorescence intensity, higher visualization degree, better detection effect, quick, efficient and convenient detection process and low dosage under the condition of high concentration use and more aggregation and more luminescence;

2) the fluorescent molecule (DHBMAB) provided by the invention can be used for detecting the deterioration degree of eye drops, the viscosity of the eye drops generally increases in the deterioration process, and the free-rotation aromatic ring structure of the DHBMAB in the environment with increased viscosity can be mechanically inhibited, so that the fluorescence is enhanced, and the DHBMAB is suitable for visual detection of the deterioration of the eye drops;

3) the fluorescent molecule (DHBMAB) provided by the invention has strong luminescence, the Stokes shift reaches 203 nm, the anti-interference performance is better, the light stability and the chemical structure stability are good, and the DHBMAB is suitable for being used in a complex solution environment containing a plurality of components in eye drops;

4) the fluorescent molecule (DHBMAB) provided by the invention is prepared by adopting a one-step method, has the characteristics of simplicity, high efficiency, high yield and environment-friendly process, is easy to carry out chemical design, is suitable for large-scale industrial production, does not have complex post-treatment steps and complex equipment, and is simple and easy to obtain raw materials and low in price.

Drawings

FIG. 1 is a schematic diagram of the intramolecular proton transfer (ESIPT) effect of a fluorescent molecule (DHBMAB) provided by the present invention;

FIG. 2 is a schematic diagram of the mechanism of applying the fluorescent molecule (DHBMAB) provided by the present invention to the detection of eye drop quality;

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-yl) methylene) amino) benzonitrile obtained in example 1;

FIG. 4 is a mass spectrum of the fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-yl) methylene) amino) benzonitrile obtained in example 1;

FIG. 5 is a graph of the fluorescence spectra of DHBMAB as described in example 4 in various volume ratios of mixed tetrahydrofuran/water solutions;

FIG. 6 is a graph showing the change in fluorescence intensity of DHBMAB in example 4 at 568 nm in various volume ratios of a tetrahydrofuran/water mixed solution;

FIG. 7 is a graph of the fluorescence spectra of DHBMAB in example 4 in varying proportions of glycerol/methanol mixed solutions in response to viscosity;

FIG. 8 is a graph of the fluorescence intensity of DHBMAB in different polyvinyl alcohol eye drops of example 5;

FIG. 9 is a graph showing the change in fluorescence intensity with the degree of deterioration of DHBMAB in different types of eye drops in example 5.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the embodiments of the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions and alterations are intended to be included in the scope of the present invention.

The following examples the chemical reaction formula for preparing fluorescent compounds for measuring the viscosity of wines is shown below:

。

example 1

A preparation method of fluorescent molecules for eye drop quality detection comprises the following steps:

1) 365 mg of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is dissolved in ethanol, and the solution is ultrasonically stirred uniformly, wherein the concentration of the 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is controlled to be 0.1M, so that a solution 1 is obtained;

2) dissolving 118 mg of p-aminobenzonitrile in ethanol, uniformly stirring by ultrasonic waves, and controlling the concentration of the p-aminobenzonitrile to be 0.1M to obtain a solution 2;

3) and mixing the solution 1 and the solution 2, stirring uniformly, heating to 30 ℃ for reaction for 36 h, removing the organic solvent, washing the precipitate with ethanol for 2-3 times, and drying to obtain orange yellow powder (330.28 mg, with the yield of 72%), namely the fluorescent molecule DHBMAB for eye drop quality detection.

The product is characterized by hydrogen nuclear magnetic resonance spectroscopy,¹H NMR (400 MHz, CDCl₃)δ 8.65 (s, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.52 (dd, J33.4, 8.2 Hz, 3H), 7.41-7.21 (m, 9H), 7.20-7.06 (m, 7H), wherein the chemical shift at 8.65 ppm corresponds to the proton characteristic peak on Schiff base, the chemical shift at 7.75 ppm corresponds to the proton characteristic peak on benzene ring near aromatic cyano, the chemical shift at 7.52 ppm corresponds to the proton characteristic peak on partial benzene ring on salicylaldehyde and triphenylamine, the chemical shift at 7.41-7.21 ppm mainly comprises the proton characteristic peak on partial benzene ring on triphenylamine and aromatic cyano, the chemical shift at 7.20-7.06 ppm mainly comprises triphenylamine and the residual benzene ring on salicylaldehydeA sub-characteristic peak. In addition, the relative molecular mass was verified by high resolution mass spectrometry, MS (ESI): M/z 465.1849 [ M + H [)]⁺Its NMR spectrum is shown in FIG. 3, and its mass spectrum is shown in FIG. 4. The synthesized product can be determined to be target fluorescent molecule DHBMAB through the analysis of hydrogen nuclear magnetic resonance spectrum and mass spectrum.

Example 2

A preparation method of fluorescent molecules for eye drop quality detection comprises the following steps:

1) 365 mg of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is dissolved in ethanol, and the solution is ultrasonically stirred uniformly, wherein the concentration of the 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is controlled to be 1M, so that a solution 1 is obtained;

2) dissolving 590 mg of p-aminobenzonitrile in ethanol, uniformly stirring by ultrasonic, and controlling the concentration of the p-aminobenzonitrile to be 10M to obtain a solution 2;

3) and mixing the solution 1 and the solution 2, stirring uniformly, heating to 50 ℃ for reacting for 18 h, removing the organic solvent, washing and precipitating for 2-3 times by using ethanol, and drying to obtain orange yellow powder (367.50 mg, wherein the yield is 79%), namely the fluorescent molecule DHBMAB for eye drop quality detection.

The results of the characterization of the fluorescent molecule DHBMAB obtained in this example are the same as those of example 1, and reference is made to fig. 3 and 4.

Example 3

A preparation method of fluorescent molecules for eye drop quality detection comprises the following steps:

1) 365 mg of 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is dissolved in ethanol, and the solution is ultrasonically stirred uniformly, wherein the concentration of the 4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-formaldehyde is controlled to be 10M, so that a solution 1 is obtained;

2) dissolving 1180 mg of p-aminobenzonitrile in ethanol, uniformly stirring by ultrasonic, and controlling the concentration of the p-aminobenzonitrile to be 100M to obtain a solution 2;

3) and mixing the solution 1 and the solution 2, stirring uniformly, heating to 78 ℃ for reaction for 0.5 h, removing the organic solvent, washing and precipitating for 2-3 times by using ethanol, and drying to obtain orange yellow powder (390.76 mg, wherein the yield is 84%), namely the fluorescent molecule DHBMAB for eye drop viscosity detection.

The results of the characterization of the fluorescent molecule DHBMAB obtained in this example are the same as those of example 1, and reference is made to fig. 3 and 4.

Example 4

And (3) testing the optical performance of the fluorescent molecule (DHBMAB) for eye drop quality detection.

1) Aggregation-induced emission characterization of DHBMAB fluorescent molecules:

1.39 mg of the fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-yl) methylene) amino) benzonitrile (DHBMAB, prepared in example 1) was dissolved in tetrahydrofuran and formulated into a 1mM stock fluorescence assay solution. During testing, the fluorescent molecules are diluted to 10 mu M, the total volume of the testing system is kept at 3 mL, the fluorescent molecules are dropwise added into solutions with different tetrahydrofuran/water volume fractions (0% -90%), the testing is carried out at room temperature, the excitation wavelength is set to 365 nm, the measured fluorescence spectrum is shown in the attached figure 5, and the change rule of the corresponding fluorescence intensity along with the water volume fraction is shown in the attached figure 6. As can be seen from FIGS. 5 and 6, when the volume fraction of water is less than 40%, the fluorescence intensity of the solution is weak, indicating that the fluorescent compound DHBMAB can be well dissolved in the solution. However, with further increase of the water volume fraction, the fluorescence intensity of the test solution gradually increased, and when the water volume fraction reached 70%, the fluorescence intensity reached a maximum, which may be because the increase of the water volume fraction resulted in a gradual decrease of the solubility of the fluorescent compound DHBMAB in the solution, resulting in aggregation, resulting in the dissipation of the excited state energy by radiation transition instead of by a mechanical dissipation of the excited state energy, which was originally observed in the form of fluorescence. This fully characterizes aggregation-induced emission.

2) DHBMAB fluorescent molecule response to viscosity test:

glycerol with different volumes is added into methanol to prepare test systems with different glycerol volume fractions (0% -99%), the total volume is controlled to be 3 mL, the test is carried out at room temperature, the excitation wavelength is set to be 365 nm, and the rule of the change of the fluorescence intensity along with the viscosity is shown in figure 7. The viscosity of common organic solvents such as methanol is generally 0.6 cp (25 ℃) and the viscosity of glycerol is as high as 945.0 cp (25 ℃), and it can be seen from fig. 7 that when the volume fraction of methanol in the solution system is higher, i.e. the volume fraction of glycerol is lower than 50%, the fluorescence intensity is lower, but as the volume fraction of glycerol is further increased, i.e. the viscosity of the solution system is further increased, the fluorescence intensity also increases significantly, especially when the volume fraction of glycerol is increased to 99%, the fluorescence intensity reaches a maximum value at 568 nm, and compared with the solution system of glycerol with the volume fraction of lower than 50%, the fluorescence intensity increases significantly by more than 22 times.

Example 5

The fluorescent molecule (DHBMAB) is applied to eye drop quality detection.

(1) Fluorescence test of DHBMAB fluorescent molecule in polyvinyl alcohol eye drops:

specifically, the fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-yl) methylene) amino) benzonitrile (DHBMAB, prepared in example 1) was dissolved in 1 mL of dimethyl sulfoxide to prepare a 1mM stock solution to be tested. During testing, the DHBMAB mother solution is dripped into three different polyvinyl alcohol eye drops (comprising polyvinyl alcohol eye drops with alcoholysis degree of 78.5-81.5 mol%, polyvinyl alcohol eye drops with alcoholysis degree of 99.0-99.4 mol% and polyvinyl alcohol eye drops with alcoholysis degree of 97.5-99.0 mol% and 3 mL), the concentration of fluorescent molecular substances is 10 mu M, and the total volume of the testing system is kept at 3 mL. The measurement is carried out at room temperature, the excitation wavelength is set to 365 nm, and the fluorescence intensity of the three polyvinyl alcohol eye drops is shown in the attached figure 8. As can be seen from FIG. 8, the three eye drops also release fluorescence of different intensities due to their different viscosities. The fluorescent molecule DHBMAB can fully sense the viscosity change of the eye drops, and can perform better fluorescence detection on the viscosity atmosphere of eye drop solutions with different viscosities.

(2) Testing the safety of DHBMAB fluorescent molecules on the quality of eye drops:

specifically, the fluorescent molecule is dripped into three different types of eye drops (including compound asparticitin eye drops and lutein eye drops), the total test volume is controlled to be 3 mL, the concentration of the fluorescent molecule is 10 mu M, then the viscosity changes of the three types of eye drops in the air for 0 day, 10 days and 30 days are respectively tested, the test is carried out at room temperature, the excitation wavelength is set to be 365 nm, and the tested fluorescence intensity and the corresponding viscosity change are shown in figure 9. As can be seen from the attached figure 9, after the eye drops are unsealed, the viscosity of the eye drops is obviously increased from 0 day to 30 days along with the gradual increase of the standing time of the eye drops in the air, and correspondingly, the fluorescence intensity of the three eye drops is also obviously improved after the fluorescent molecule DHBMAB is added. The results show that the fluorescent molecule DHBMAB can effectively perform fluorescence tracking on the viscosity change of the eye drops, namely can effectively detect the deterioration degree of the eye drops, and has a certain monitoring effect on the quality safety of the eye drops.

The invention provides a fluorescent molecule 4- (((4'- (diphenylamino) -3-hydroxy- [1,1' -biphenyl ] -4-yl) methylene) amino) benzonitrile (DHBMAB), wherein the probe molecule has AIE characteristics and ESIPT effect, can sense the viscosity of eye drops, and can further detect the quality safety of the eye drops. The molecular structure of the fluorescent molecule contains a plurality of aromatic rings and other structures capable of rotating freely, the change of liquid viscosity can be sensed sufficiently, test results show that when the viscosity of a solvent environment is increased, energy in an excited state returns to a ground state in a radiation transition mode to release a fluorescent signal, the eye drops are easy to deteriorate after being unsealed, the viscosity is increased generally in the deterioration process, the free rotation of the aromatic rings and other structures in the molecular structure is limited, the deterioration degree of the eye drops can be judged in a fluorescence release mode, and a certain monitoring effect is achieved on the quality safety of the eye drops. Meanwhile, although the fluorescent molecule has a large conjugated structure, the fluorescent molecule has AIE characteristics and does not cause fluorescence quenching due to aggregation. In addition, the molecule is simple, efficient and quick to prepare, high in product yield, green and environment-friendly in process, and has the potential of large-scale production and preparation.