A method and application for detecting heparin sodium based on a water-soluble cationic AIE fluorescent molecule

Abstract

The present invention discloses the application of a water-soluble and positively charged fluorescent probe TPA-3Py for the detection of sodium heparin. TPA-3Py is obtained by covalently connecting three methylated vinylpyridines to the three arms of a triphenylamine molecule respectively. Heparin can induce the aggregation-induced emission effect of this fluorescent molecule. With the increase of the heparin content, the fluorescence intensity increases significantly, showing a good fluorescence turn-on response, overcoming the deficiency that the fluorescence probe quenches due to aggregation during the detection of heparin in the prior art. When a 10 μM aqueous solution of TPA-3Py is used for the detection of heparin, a stable fluorescence intensity is achieved within 5 minutes, the linear range is 0 - 6.14 mg / L (R<supgt;2< / supgt> = 0.9938), the lowest detection limit is 49.53 ng / mL, and the quantum yield reaches 4.85%. For the detection of sodium heparin, the TPA-3Py probe adopted in the present invention overcomes the quenching phenomenon caused by aggregation and exhibits a significant AIE effect. The fluorescent probe TPA-3Py for the detection of sodium heparin in the present invention has high sensitivity, high fluorescence intensity, rapid response and stable signal. The fluorescent molecular structure of TPA-3Py is simple, and the detection method is simple and easy to implement, which is worthy of popularization and application in the determination of the sodium heparin content.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, particularly relates to the field of heparin sodium detection. Specifically, it discovers that a water-soluble, positively charged TPA-3Py molecule can aggregate after binding to negatively charged heparin sodium, resulting in a significant enhancement of fluorescence. The invention utilizes this phenomenon for the detection of heparin sodium. Background Art

[0002] As an anticoagulant widely used in clinical practice, heparin sodium is used to prevent thrombosis and other diseases during surgery and treatment. However, during the clinical use of heparin sodium, there are side effects such as bleeding. Therefore, quantitative monitoring of heparin sodium is very necessary. Heparin sodium is a type of linear glycosaminoglycan with the most negative charges, and its negative charges come from three sulfonic acid groups and one carboxylic acid group on the main disaccharide unit. Fluorescent probes for detecting heparin sodium usually electrostatically adsorb negatively charged heparin sodium molecules through their positive charges, resulting in probe aggregation and enhanced fluorescence signals. After traditional fluorescent probes bind to heparin sodium, due to aggregation, they cause the ACQ phenomenon, thus reducing their detection ability. AIE molecules are almost non-fluorescent in the solution state but become strongly luminescent when induced to aggregate by analytes, fundamentally solving the ACQ problem and being a powerful tool for current analytical detection research. Triphenylamine is a commonly used AIE core structure. After covalently connecting three methylated vinylpyridines to the three arms of the triphenylamine molecule, the TPA-3Py molecule is obtained. This molecule has strong water solubility and a positive charge. The present invention discovers that negatively charged long-chain heparin sodium can bind to TPA-3Py, aggregate, and restrict its intramolecular movement, causing its "turn-on" fluorescence response. The present invention utilizes this phenomenon to develop the application of the TPA-3Py molecule for the detection of heparin sodium.

[0003] Referring to relevant literature, there is currently no relevant report on the method of using the TPA-3Py molecule to detect the content of heparin sodium. Summary of the Invention

[0004] The present invention intends to provide a fluorescent probe that can overcome the deficiency of the quenching (ACQ) phenomenon caused by aggregation in the performance of existing technologies for heparin detection, and develop its application for the detection of heparin sodium using an aggregation-induced emission fluorescent probe.

[0005] Based on the above purpose, the technical solution involved in the present invention is as follows:

[0006] 1) Select an AIE fluorescent molecule: The AIE fluorescent molecule selected in the present invention is the TPA-3Py molecule, which is obtained by covalently connecting three methylated vinylpyridines to the three arms of the triphenylamine molecule respectively. It has water solubility and a positive charge. The specific structural formula of the TPA-3Py fluorescent molecule selected in the present invention is as follows:

[0007]

[0008] 2) Investigate the AIE property of TPA-3Py: The fluorescence of this molecule is very weak in aqueous solution, but as the ethanol content in the solution gradually increases, the fluorescence intensity gradually increases, demonstrating the AIE effect of TPA-3Py.

[0009] 3) Develop the application of TPA-3Py in heparin detection: As the heparin concentration increases, the fluorescence signal of TPA-3Py significantly enhances, and sodium heparin has a good fluorescence "turn-on" response to the TPA-3Py probe. Specifically, an aqueous solution of TPA-3Py with a concentration of 10 μM was used to detect sodium heparin. Heparin had 7 different concentrations from 0 - 12 mg / L. After standing for 15 min, the maximum fluorescence emission intensity was measured at an excitation wavelength of 462 nm, and the linear relationship between sodium heparin and the fluorescence intensity at the maximum emission wavelength was obtained. The linear relationship was good, the linear equation was y = 0.807x + 0.780, 2 R = 0.9938, and the linear range was 0 - 6.14 mg / L. The lowest detection limit was 49.53 ng / mL, and the quantum yield reached 4.85%.

[0010] The present invention has the following advantages:

[0011] For the detection of sodium heparin, the TPA-3Py probe exhibits a relatively wide linear range, a low lowest detection limit, and a high quantum yield. The fluorescence probe of the present invention has a rapid response, high fluorescence intensity, stable signal and high sensitivity. The fluorescence molecular structure is simple, the detection method is simple and easy to implement, and it is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Fluorescence intensity spectrogram of TPA-3Py (8 μM) at different ethanol concentrations (a), binding curve showing that the fluorescence intensity increases with the increase of ethanol content at the maximum excitation wavelength (b) λex(TPA-3Py) = 476 nm, slit 2.5 / 2.5 nm

[0013] Figure 2 Fluorescence spectrum (a) and linear relationship diagram (b) of TPA-3Py (10 μM) binding to different concentrations of heparin, λex(TPA-3Py) = 462 nm, slit 10 / 10 nm

[0014] Figure 3 Effect of reaction time of TPA-3Py (10 μM) with Hep (12 mg / mL), λex(TPA-3Py) = 462 nm, slit10 / 10 nm DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] Examples of Implementation

[0016] 1. Study the AIE properties of TPA-3Py

[0017] (1) Preparation of the TPA-3Py mother liquor

[0018] Accurately weigh 0.0015 g of TPA-3Py solid powder into a 25-mL volumetric flask. After dissolving it with appropriate deionized water, make up the volume to the calibration line. An AIE-3 solution with a concentration of 66.219 μM is obtained.

[0019] (2) Preparation of the TPA-3Py detection solution

[0020] Take 11 1.5-mL centrifuge tubes, add 0.181 mL of the TPA-3Py mother liquor, and then add anhydrous ethanol with volumes of 0, 0.139, 0.278, 0.417, 0.555, 0.694, 0.833, 0.972, 1.110, 1.250, and 1.319 mL, and make up the volume to 1.5 mL.

[0021] (3) Measurement of fluorescence intensity

[0022] Take the above-prepared solutions, with an excitation wavelength of 476 nm and a slit of 2.5 nm, and measure the fluorescence emission intensity. TPA-3Py is a water-soluble molecule, and its fluorescence in aqueous solution is very weak. As shown Figure 1 , as the ethanol component in the solution gradually increases, the fluorescence intensity gradually increases. It is proved that polymerization occurs after the addition of ethanol, and the TPA-3Py probe shows AIE properties.

[0023] 2. Detection ability of TPA-3Py for heparin

[0024] (1) Preparation of the heparin sodium mother liquor

[0025] Accurately weigh 0.0075 g of heparin sodium standard product (Yantai Dongcheng Pharmaceutical Group Co., Ltd.) into a 100-mL volumetric flask, add deionized water, dissolve it, and make up the volume to the calibration line to obtain a heparin sodium standard solution with a concentration of 75 mg / L.

[0026] (2) Preparation of the heparin detection solution

[0027] Take 7 1.5-mL centrifuge tubes, add 0.227 mL of the TPA-3Py mother liquor, and then add heparin sodium mother liquor with volumes of 0, 0.04, 0.08, 0.12, 0.16, 0.20, and 0.24 mL respectively. Finally, make up the volume to 1.5 mL with deionized water.

[0028] (3) Measurement of fluorescence intensity

[0029] After reacting the above-mentioned solution to be measured in the dark at room temperature for 5 minutes, measure the fluorescence intensity at an excitation wavelength of 462 nm, and the slit used for measurement is 10 nm.

[0030] As Figure 2 shown, the fluorescence of TPA-3Py molecules in aqueous solution is extremely weak. After adding heparin, the maximum emission wavelength of TPA-3Py (10 μM) shifts from 610 nm to 645 nm, showing a red shift, and the fluorescence intensity increases significantly. The fluorescence change indicates that the electrostatic interaction between negatively charged heparin and positively charged TPA-3Py molecules forms the TPA-3Py+Hep complex. The increased steric hindrance restricts the intramolecular rotation of the fluorescent molecules, thus increasing the fluorescence intensity.

[0031] (4) Detection of linear relationship

[0032] Plot the relative fluorescence intensity (I / I0) at the maximum emission wavelength against the concentration of heparin to obtain the linear equation y = 0.807x + 0.780, (R 2 = 0.9938). The results prove that there is a good linear relationship between heparin concentration in the range of 0 - 6.14 mg / L and 10 μM TPA-3Py ( Figure 2 b).

[0033] 3. Influence of reaction time on fluorescence intensity

[0034] By measuring the fluorescence intensity at the maximum emission wavelength of the complex TPA-3Py (10 μM) and Hep (12 mg / mL) within 20 min, investigate the influence of reaction time ( Figure 3 ). The fluorescence intensity of this molecule shows no obvious fluctuation within 20 minutes and stabilizes at about 580. The reaction time for measuring AIE-3 is set to 5 minutes. The above results indicate that heparin can quickly bind with TPA-3Py to form a relatively stable complex.

[0035] 4. Determination of the limit of detection (LOD) of the TPA-3Py probe for detecting heparin

[0036] Prepare 10 blank solutions of TPA-3Py (without sodium heparin) and measure the fluorescence intensity of the blank samples. The obtained data is used to calculate the standard deviation in Excel. Prepare a series of TPA-3Py solutions with different concentrations of heparin added and measure the fluorescence intensity. Plot the change graph of the fluorescence intensity at the maximum emission wavelength at different heparin concentrations, and use the linear relationship graph of the relative fluorescence intensity of TPA-3Py against the heparin concentration to find the slope of the straight line and R 2 . Substitute the data used into the detection limit calculation formula LOD = 3δ / S.

[0037] The LOD of heparin was calculated according to the formula to be 49.53 ng / mL.

[0038] 5. Study the fluorescence quantum yield of TPA-3Py

[0039] Measure the fluorescence quantum yield of TPA-3Py with an FS5 fluorescence spectrophotometer. Instrument components need to be replaced during the measurement. Before measurement, first measure the absorbance of the sample with an ultraviolet spectrophotometer to ensure that the absorbance of the measured sample is less than 0.1. During measurement, adjust the slit to make the Emission signal value reach the maximum, and at the same time ensure that the Ex slit is 10 times that of the Em slit.

[0040] When there is no heparin, the quantum yield of TPA-3Py is 2.61%, and after adding 12 mg / L heparin, the quantum yield of TPA-3Py increases to 4.85%.

[0041] For the detection of sodium heparin, the TPA-3Py probe adopted in the present invention overcomes the ACQ phenomenon and exhibits a significant AIE effect. The fluorescence probe used in the present invention for the detection of sodium heparin has a rapid response, high sensitivity, high fluorescence intensity, and stable signals. The fluorescence molecular structure of TPA-3Py is simple, and the detection method is simple and easy to implement, which is worthy of popularization and application in the determination of sodium heparin content.

Claims

1. Application of a fluorescent probe TPA-3Py in the detection of heparin sodium, characterized in that: The probe has aggregation-induced emission (AIE) properties. The structural formula of the fluorescent probe TPA-3Py is as follows: When TPA-3Py detects heparin sodium, the fluorescence signal of TPA-3Py significantly increases with the increase of heparin sodium concentration, and heparin sodium has a good fluorescence "on" response to the TPA-3Py probe.

2. The use according to claim 1, characterized in that TPA-3Py has AIE properties: TPA-3Py is a water-soluble molecule with very weak fluorescence in aqueous solution, but as the poor solvent ethanol in the solution gradually increases, the fluorescence intensity increases significantly, and TPA-3Py undergoes AIE effect.

3. The use according to claim 1, characterized in that Sodium heparin can induce the AIE effect in TPA-3Py: the probe molecule is dissolved in an aqueous solution, and different concentrations of sodium heparin are added to obtain a test solution. The fluorescence emission spectrum is recorded using a fluorescence meter. The fluorescence intensity of the TPA-3Py aqueous solution is significantly enhanced with the addition of sodium heparin, indicating an AIE effect.

4. The use according to claim 1, characterized in that Heparin sodium was detected using a 10 μM TPA-3Py aqueous solution at seven different heparin concentrations ranging from 0 to 12 mg / L. After 15 minutes, the maximum fluorescence emission intensity was measured at an excitation wavelength of 462 nm, and a linear relationship between the heparin sodium concentration and the fluorescence intensity at the maximum emission wavelength was obtained.

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