A long-wavelength AIE array sensor and its preparation method and application

Abstract

The present invention discloses a luminescent molecule based on a tetrakis(4-pyridylphenyl)ethylene core structure, a long-wavelength AIE array sensor, and a preparation method and application thereof. The TPPESA array sensor designed by the present invention can accurately identify and detect glycosaminoglycan with a concentration as low as 1 μg / mL. In terms of detecting a glycosaminoglycan mixture (impurity), the array sensor of the present invention can detect contaminants with a minimum content of 2% in a sample. The detection operation is simple, fast, and efficient. It can realize the detection of a variety of saccharide compounds: the fluorescence sensing array constructed by the present invention can be used for the quantitative, accurate, and rapid detection of common 4 glycosaminoglycans in a water sample and a serum environment. In addition, when the fluorescence sensing array of the present invention is used for detecting saccharide compounds, no harmful by-products are generated and there is no secondary pollution.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a long-wavelength AIE array sensor, a preparation method thereof, and an application thereof. Background Art

[0002] Glycosaminoglycan (GAG) is a general term for a class of linear polysaccharides with various biological activities. The most clinically used glycosaminoglycans include heparin, chondroitin sulfate, and sodium hyaluronate, etc. Heparin, chondroitin sulfate, and sodium hyaluronate have similar molecular structures but greatly different biological activities. For example, heparin is a widely used anticoagulant in clinics and can also be used for antithrombosis and the treatment of cardiovascular diseases; chondroitin sulfate is often used for the treatment of arthritis; hyaluronic acid is widely used in ophthalmic surgeries and skin care products. Different glycosaminoglycans have different biological activities, but their chemical structures are very similar and extremely complex, making it difficult to conveniently distinguish them technically, thus posing potential safety hazards in the clinical use of glycosaminoglycans. Therefore, developing convenient and reliable analysis techniques for the distinction and quantitative detection of glycosaminoglycans has great research significance for ensuring the safety of their drug use. [1-4] 。

[0003] Currently, one technique is to develop specific fluorescent probes for glycosaminoglycan detection. For example, the heparin-specific BODIPY-based pyridinium salt C10-Py constructed by the research group of Professor Kim at Kyung Hee University in South Korea + probe [5] ; the double-charged cationic fluorescent probe PYPN synthesized by the research group of Professor Yan at Hebei University based on the pyrene-pyridine skeleton [6] ; the research group of Professor Tian at Hubei Minzu University combined chitosan quaternary ammonium salt and the luminophore TPE-COOH with AIE effect through electrostatic complexation to construct a complex for rapid detection of heparin, etc. [7] From the research on these latest specific heparin fluorescent probes, it can be seen that due to the overly complex structure of glycosaminoglycans and the lack of suitable recognition sites, almost all existing specific fluorescent probes can only utilize the characteristic that heparin has the largest negative charge density and achieve specific detection of it through electrostatic interaction, but cannot be used for the detection of other glycosaminoglycans.

[0004] Another method is to construct an array sensor with multiple recognition units and achieve the discrimination detection of various glycosaminoglycans through pattern recognition. Compared with specific fluorescent probes, the design and construction of an array sensor do not require specific recognition units. [8], The use of fluorescence array sensors for the differential detection of glycosaminoglycans such as heparin can exactly solve the problem of difficult design of specific recognition units. However, the research on array sensors for the differential detection of glycosaminoglycans in the world is still in its infancy, and there are many deficiencies in technology. In this regard, our research group once used poly(diallyldimethylammonium chloride) (PDDA) as a supramolecular host and tetraphenylethylene carboxylic acid (TPE) as a guest. By subtly controlling the aggregation state of TPE on PDDA, a PDDA-TPE fluorescence array sensor based on different TPE loading amounts was constructed, realizing the pattern recognition of heparin, hyaluronic acid, chondroitin sulfate, chitosan, dextran sulfate, and chondroitin persulfate. [9] . A ratio sensor array Py-PP based on pyrene-porphyrin supramolecular complex was constructed. By regulating the FRET energy transfer efficiency between pyrene and porphyrin, it was successfully used for the detection of trace glycosaminoglycan pollutants in heparin and the differential detection of different glycosaminoglycans.

[10] . Although the existing glycosaminoglycan array sensors have achieved the differential detection of different glycosaminoglycans to a certain extent, the existing technology still has deficiencies such as short fluorescence detection wavelength, low sensitivity, and high preparation cost.

[0005] References:

[0006] [1] Elci S.G., Moyano D.F., Rana S., Tong G.Y., Phillips R.L., Bunz U.H.F., Rotello V.M. Recognition of glycosaminoglycan chemical patterns using an unbiased sensor array. Chemical science 2013, 4(5): 2076 - 2080.

[0007] [2] Francoia J.P., Vial L.A. KISS (keep it simple, sensor) array for glycosaminoglycans. Chemical Communications 2015, 51(99): 17544 - 17547.

[0008] [3] Jagt R.B.C., Gomez-Biagi R.F., Nitz M.P. Pattern-Based Recognition of Heparin Contaminants by an Array of Self-Assembling Fluorescent Receptors. Angew. Chem.-Int. Edit. 2009, 48(11): 1995-1997.

[0009] [4] Jia D., Yang C., Zhang W., Ding Y. Dyes inspired sensor arrays for discrimination of glycosaminoglycans. Dyes and Pigments 2021, 190: 109266.

[0010] [5] Kim D., Lee U., Bouffard J., Kim Y. Glycosaminoglycan-Induced Emissive J-Aggregate Formation in a meso-Ester BODIPY Dye. Advanced Optical Materials 2020, 8(14): 1902161.

[0011] [6] He Z., Nie H., Cui J., Zhang X., Yang X., Li C., Yan H. An electrostatically regulated organic self-assembly for rapid and sensitive detection of heparin in serum. Analytical methods: advancing methods and applications 2021, 13(32): 3620-3626.

[0012] [7] Liu W., Guan L.X., Wang T.F., Tan Z.W. hitosan Group Aggregation Induced Luminescence Complex for Heparin Detection. Fine Chemical Industry 2022, 39(09): 1805-1812.

[0013] [8]Ma S.D., Chen Y.L., Feng J., Liu J.J., Zuo X.W., Chen X.G. One-Step Synthesis of Water-Dispersible and Biocompatible Silicon Nanoparticles for Selective Heparin Sensing and Cell Imaging. Analytical chemistry 2016, 88(21): 10474-10481.

[0014] [9]Yang Z., Fan X., Cheng W., Ding Y., Zhang W. AIE Nanoassemblies for Discrimination of Glycosaminoglycans and Heparin Quality Control. Analytical chemistry 2019, 91(15): 10295-10301.

[0015]

[10] Hou M., Fan L., Fan X., Liang X., Zhang W., Ding Y. Pyrene-porphyrin based ratiometric fluorescent sensor array for discrimination of glycosaminoglycans. Anal Chim Acta 2021, 1141: 214-220. Summary of the Invention

[0016] Object of the Invention: The technical problem to be solved by the present invention is to provide a luminescent molecule based on tetrakis(4-pyridylphenyl)ethylene.

[0017] The technical problem to be solved by the present invention is to provide a preparation method of the luminescent molecule.

[0018] The technical problem to be solved by the present invention is to provide a long-wavelength AIE array sensor and a method for constructing the same.

[0019] Another technical problem to be solved by the present invention is to provide the applications of the luminescent molecule and the long-wavelength AIE array sensor in the detection of glycosaminoglycans.

[0020] The last technical problem to be solved by the present invention is to provide a detection method for different types and / or different concentrations of glycosaminoglycans.

[0021] Technical solution: To solve the above technical problems, the present invention provides a luminescent molecule based on a tetrakis(4-pyridylphenyl)ethylene core structure, and the structural formula of the luminescent molecule is as follows:

[0022] wherein R includes one or more of the following.

[0023] Among them, the structural formula of the luminescent molecule specifically includes:

[0024]

[0025] The present invention also includes a preparation method of the luminescent molecule, which includes the following steps: reacting tetrakis(4-pyridylphenyl)ethylene with 4-bromomethylphenylboronic acid, iodomethane, and iodoheptane in DMF respectively to obtain.

[0026] The present invention also includes a long-wavelength AIE array sensor, and the long-wavelength AIE array sensor includes the luminescent molecule.

[0027] Among them, the long-wavelength AIE array sensor includes three sensing units: TPPEBA, TPPEMe, and TPPEC7.

[0028] The present invention also includes a preparation method of the long-wavelength AIE array sensor, which includes the following steps:

[0029] 1) Preparation of TPPEBA: Weigh the raw materials TPPE and 4-bromomethylphenylboronic acid, dissolve them in dry DMF, and heat the solution under nitrogen protection until the raw materials disappear. Spin dry the solvent, add dichloromethane and ultrasonicate, filter with acetonitrile to remove impurities, and collect the bright yellow solid product to obtain TPPEBA;

[0030] 2) Preparation of TPPEMe: Add the raw materials TPPE and excessive iodomethane to the reactor, use DMF as the solvent, react in an oil bath, filter, and wash the filter cake with dichloromethane to obtain the product TPPEMe;

[0031] 3) Preparation of TPPEC7: Add TPPE and excessive iodoheptane to the reactor, use DMF as the solvent, react overnight, filter, spin off the solvent, wash the product with dichloromethane, and filter to obtain the product TPPEC7;

[0032] 4) Construct an array sensor with the obtained TPPEBA, TPPEMe, and TPPEC7.

[0033] The present invention also includes the application of the luminescent molecule and the long-wavelength AIE array sensor in the detection of glycosaminoglycan.

[0034] Among them, the glycosaminoglycan includes one or more of heparin, chondroitin sulfate, hyaluronic acid, and dextran sulfate.

[0035] The present invention also includes a method for detecting different types and / or different concentrations of glycosaminoglycans, and the detection method includes the following steps:

[0036] 1) Construct an array sensor TPPESA with TPPEBA, TPPEMe, and TPPEC7;

[0037] 2) Preparation of the glycosaminoglycan sample solution to be measured: Prepare heparin sodium stock solution, chondroitin sulfate stock solution, sodium hyaluronate stock solution, and dextran sulfate stock solution;

[0038] Preferably, the glycosaminoglycan sample solution to be measured further includes heparin sodium stock solution with chondroitin sulfate impurities, heparin sodium stock solution with hyaluronic acid impurities, and heparin sodium stock solution with dextran sulfate impurities; Preferably, the glycosaminoglycan sample solution to be measured further includes heparin sodium stock solution containing serum, chondroitin sulfate stock solution containing serum, sodium hyaluronate stock solution containing serum, and dextran sulfate stock solution containing serum;

[0039] 3) Fluorescence response test of the array sensor constructed in step 1) for the glycosaminoglycans in step 2), and obtain the concentration of the glycosaminoglycan to be measured through the relationship between the fluorescence response degree of the glycosaminoglycan and the concentration of the glycosaminoglycan.

[0040] Among them, the construction of the array sensor in step 1) specifically includes the following steps:

[0041] S1) Arbitrarily select a 4-row × 3-column area on the 96-well plate;

[0042] S2) Add PBS buffer solution to each well in the above 4-row × 3-column area;

[0043] S3) Add TPPEBA stock solution to each well in the first column of the 4-row × 3-column area in the previous step;

[0044] S4) Add TPPEMe stock solution to each well in the second column of the 4-row × 3-column area in the previous step;

[0045] S5) Add TPPEC7 stock solution to each well in the third column of the 4-row × 3-column area in the previous step to complete the construction of the array sensor;

[0046] Preferably, the concentration of each glycosaminoglycan in the heparin sodium stock solution, chondroitin sulfate stock solution, sodium hyaluronate stock solution, dextran sulfate stock solution, heparin sodium stock solution containing chondroitin sulfate impurities, heparin sodium stock solution containing hyaluronic acid impurities, heparin sodium stock solution containing dextran sulfate impurities, heparin sodium stock solution containing serum, chondroitin sulfate stock solution containing serum, sodium hyaluronate stock solution containing serum, and dextran sulfate stock solution containing serum described in step 2) is 0.1-200 μg / mL;

[0047] Preferably, the specific steps of step 3) are as follows:

[0048] 3.1) Add a certain volume of the glycosaminoglycan stock solution to be tested to each well in the 4-row × 3-column area of the array sensor TPPESA, and calculate the actual concentration of glycosaminoglycan in each well after dilution;

[0049] 3.2) Measure the fluorescence intensity of each well in the above 4-row × 3-column area. The excitation wavelength of the first column is 365 nm, and the detection wavelength is 554 nm; the excitation wavelength of the second column is 418 nm, and the detection wavelength is 552 nm; the excitation wavelength of the third column is 393 nm, and the detection wavelength is 544 nm, to obtain a 4-row × 3-column data array of the tested glycosaminoglycan;

[0050] 3.3) Use the obtained glycosaminoglycan sample matrix data to perform data processing through linear discriminant analysis to obtain the detection result of the glycosaminoglycan to be tested in the array sensor.

[0051] Principle of the present invention: The present invention obtains a three-component sensing unit through the supramolecular assembly of fluorescent dye molecules. Different sensing units have differential fluorescence response signals to the detected glycosaminoglycans, so as to obtain high-dimensional detection data and obtain specific patterns of four glycosaminoglycans. On this basis, linear discriminant analysis is used to reduce the dimension of the high-dimensional data, so as to more intuitively distinguish and detect the types and concentrations of glycosaminoglycans. Specifically, the present invention designs and synthesizes three corresponding long-wavelength aggregation-induced emission molecules TPPEBA, TPPEMe, and TPPEC7 by introducing arylboronic acid, methyl, and n-heptyl substituents into the tetrakis(4-pyridylphenyl)ethylene parent body. On this basis, an array sensor based on TPPEBA, TPPEMe, and TPPEC7 sensing units is designed and constructed to obtain high-dimensional detection signals of glycosaminoglycans, and to achieve rapid, accurate, and reliable detection of heparin, chondroitin sulfate, hyaluronic acid, and dextran sulfate in a pattern recognition manner. This sensing array can be used for the differential detection and quantitative analysis of glycosaminoglycans in water samples and serum samples.

[0052] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0053] 1. The detection reagent is easy to prepare and has low cost: The TPPESA array sensor designed in the present invention only needs to synthesize a small amount of 3 fluorescent dye molecules and prepare a stock solution with the same concentration in water or serum. This method is convenient to use, has low cost, does not require large and expensive instruments, and can be used for on-site instant detection.

[0054] 2. High sensitivity of fluorescence detection: The present invention can accurately identify and detect glycosaminoglycans with a concentration as low as 1 μg / mL. In terms of detecting glycosaminoglycan mixtures (impurities), the array sensor of the present invention can detect samples containing as low as 2% pollutants.

[0055] 3. Simple and fast detection operation: The detection process only needs to simply prepare the specified solution according to the steps and read the data on an enzyme-linked immunosorbent assay (ELISA) reader to analyze and obtain the final result, which is convenient and fast. The ELISA reader reading only takes about 1 minute, with high speed and efficiency.

[0056] 4. Detection of multiple saccharide compounds is achieved: The supramolecular fluorescence sensing array constructed in the present invention can be used for quantitative, accurate, and rapid detection of 4 common glycosaminoglycans in water samples and serum environments. In addition, when the fluorescence sensing array of the present invention is used for detecting saccharide compounds, no harmful by-products are generated and there is no secondary pollution.

[0057] In summary, the detection cost and detection performance of the fluorescence sensing array of the present invention are significantly better than the analytical detection methods relying on large instruments. Brief Description of the Drawings

[0058] Figure 1 For the synthesis of TPPEBA, TPPEMe and TPPEC7 molecules;

[0059] Figure 2 For 1 1H NMR spectrum (DMSO-d6) of TPPEBA;

[0060] Figure 3 For 13 13C NMR spectrum (DMSO-d6) of TPPEBA;

[0061] Figure 4 For the HRMS spectrum of TPPEBA;

[0062] Figure 5 For 1 1H NMR spectrum (DMSO-d6) of TPPEMe;

[0063] Figure 6 For 13 13C NMR spectrum (DMSO-d6) of TPPEMe;

[0064] Figure 7HRMS spectrum of TPPEMe;

[0065] Figure 8 For TPPEC7's 1 HNMR spectrum (DMSO-d6);

[0066] Figure 9 For TPPEC7's 13 C NMR spectrum (DMSO-d6);

[0067] Figure 10 HRMS spectrum of TPPEC7;

[0068] Figure 11 a-d) Fluorescence titration curves of TPPEBA (10 μM) in PBS (10 mM, pH = 7.40) solution with polysaccharides, a) Hep b) Chs c) HA d) DS, λex = 365 nm

[0069] Figure 12 a-d) Fluorescence titration curves of TPPEMe (10 μM) in PBS (10 mM, pH = 7.40) solution with polysaccharides, a) Hep b) Chs c) HA d) DS, λex = 418 nm;

[0070] Figure 13 a-d) Fluorescence titration curves of TPPEC7 (10 μM) in PBS (10 mM, pH = 7.40) solution with polysaccharides, a) Hep b) Chs c) HA d) DS, λex = 393 nm;

[0071] Figure 14 a) Fluorescence response patterns (fingerprint spectra) of various glycosaminoglycans generated by the sensor array, each error bar represents the standard deviation of four parallel tests; b) Heat map obtained from the fluorescence reaction patterns of the glycosaminoglycans to be measured;

[0072] Figure 15 LDA maps of the first two factors of the response patterns generated by the sensor array for each glycosaminoglycan in PBS (10 mM, pH = 7.40) solution, with concentrations of a) 0.1 μg / mL; b) 1 μg / mL; c) 5 μg / mL; d) 10 μg / mL; e) 100 μg / mL; f) 200 μg / mL;

[0073] Figure 16 For the array sensor to distinguish glycosaminoglycans with different concentrations in PBS (10 mM, pH = 7.40) solution, a) Hep b) Chs c) HA d) DS;

[0074] Figure 17LDA scatter plots for the array sensor to distinguish different ratios of polysaccharide mixtures (with a fixed total concentration of 10 μg / mL) in PBS (10 mM, pH = 7.40) solution, a) Hep / Chs mixture, b) Hep / HA mixture, c) Hep / DS mixture;

[0075] Figure 18 LDA maps of the first two factors of the response patterns generated by the sensor array for each glycosaminoglycan in 10% serum, with concentrations of a) 0.1 μg / mL; b) 1 μg / mL; c) 5 μg / mL; d) 10 μg / mL; e) 100 μg / mL; f) 200 μg / mL;

[0076] Figure 19 For the array sensor to distinguish glycosaminoglycans with different concentrations in 10% serum, a) Hep b) Chs c) HA d) DS;

[0077] Figure 20 LDA scatter plots for the array sensor to distinguish different ratios of polysaccharide mixtures (with a fixed total concentration of 10 μg / mL) in 10% serum, a) Hep / Chs mixture, b) Hep / HA mixture, c) Hep / DS mixture;

[0078] Figure 21 Schematic diagram of the fluorescence sensing array for testing the fluorescence of glycosaminoglycans. Detailed implementation mode

[0079] The technical solution of the present invention will be described in detail below, but the protection scope of the present invention is not limited to the described embodiments.

[0080] Example 1 Preparation of TPPEBA

[0081] Weigh the raw materials tetra(4-pyridylphenyl)ethylene (TPPE) (515 mg, 0.8 mmol) and 4-bromomethylphenylboronic acid (1752.5 mg, 8 mmol) in a 150 mL Schlenk tube, dissolve them with 75 mL of dry DMF, and heat the solution to 80 °C under nitrogen protection until the raw materials disappear. Rotate to dry the solvent, add 50 mL of dichloromethane for ultrasonic treatment, and filter with acetonitrile to remove impurities. Collect the bright yellow solid product (1136.9 mg, yield: 75%). The characterization results of the bright yellow solid product are as follows:

[0082] 11H NMR (DMSO-d6, 500 MHz, ppm): δ 9.15 (d, J = 6.65 Hz, 8H), 8.44 (d, J = 6.71 Hz, 8H), 8.12 (s, 8H), 7.95 (d, J = 8.40 Hz, 8H), 7.79 (d, J = 8.03 Hz, 8H), 7.44 (d, J = 8.04 Hz, 8H), 7.31 (d, J = 8.35 Hz, 8H), 5.80 (s, 8H)( Figure 2 ). 13 13C NMR (DMSO-d6, 125 MHz, ppm): δ 154.51, 146.47, 145.31, 141.64, 136.64, 135.38, 134.51, 132.64, 132.52, 128.67, 128.10, 125.95, 125.17, 62.88( Figure 3 ). HRMS: observed 295.1269( Figure 4 ), calculated for C 74 H 64 B4N4O8 ([M] 4+ ): 295.1269.

[0083] Example 2 Preparation of TPPEMe

[0084] Add the raw material TPPE (322.2 mg, 0.5 mmol) and excessive methyl iodide (592.7 mg, 4.17 mmol) into a Schlenk tube. Use 50 mL of DMF as the solvent and react at 140 °C in an oil bath for 8 hours. Filter, and wash the filter cake with dichloromethane to obtain 0.5013 g of the product, with a yield of 41.5%. The characterization results of the product are as follows:

[0085] 1 1H NMR (DMSO-d6, 500 MHz, ppm): δ 8.95 (d, J = 6.98 Hz, 8H), 8.44 (d, J = 6.99 Hz, 8H), 7.99 (d, J = 8.52 Hz, 8H), 7.35 (d, J = 8.52 Hz, 8H), 4.29 (s, 12H)( Figure 5 ). 13 13C NMR (DMSO-d6, 125 MHz, ppm), δ 153.63, 146.41, 146.09, 141.53, 132.55, 128.51, 124.34, 47.68( Figure 6 ). HRMS( Figure 7 ): observed 175.0886, calculated for C 50 H44 N4([M] 4+ ):175.0886.

[0086] Preparation of Example 3 TPPEC7

[0087] Add TPPE (320 mg, 0.5 mmol) and excessive iodoheptane (1.2 g, 5.31 mmol) into a Schlenk tube. Use 50 mL of DMF as the solvent and react at 140 °C overnight. Filter, remove the solvent by rotary evaporation, wash the product with dichloromethane, and filter by suction to obtain 311.9 mg of the product with a yield of 40%. The characterization results of the product are as follows:

[0088] 1 H NMR (DMSO-d6, 500 MHz, ppm): δ 9.04 (d, J = 6.89 Hz, 8H), 8.44 (d, J = 6.60 Hz, 8H), 7.99 (d, J = 8.45 Hz, 8H), 7.35 (d, J = 8.30 Hz, 8H), 4.52 (t, J = 11.06 Hz, 8H), 1.87 (m, 8H), 1.24 (m, 32H), 0.81 (t, J = 10.20 Hz, 12H)( Figure 8 ). 13 C NMR (DMSO-d6, 125 MHz, ppm): δ 162.88, 153.99, 146.43, 145.21, 132.53, 128.58, 124.74, 60.40, 34.93, 31.31, 28.59, 25.86, 22.49, 14.44( Figure 9 ). HRMS( Figure 10 ):observed 259.1825, calculated for C 74 H 92 N4([M] 4+ ):259.1825.

[0089] Preparation of Example 4 TPPEBA, TPPEMe and TPPEC7 Sensors and Their Detection of Glycosaminoglycans

[0090] 1. Preparation of TPPEBA, TPPEMe and TPPEC7 Sensing Units

[0091] (1) Preparation of TPPEBA sensing unit: Dissolve the TPPEBA (1 mg) prepared in Example 1 in 6.67 mL of DMF to obtain a 1 mM stock solution;

[0092] (2) Preparation of TPPEMe sensing unit: Dissolve the TPPEMe (1 mg) prepared in Example 2 in 8.28 mL of DMF to obtain a 1 mM stock solution;

[0093] (3) Preparation of the TPPEC7 sensing unit: Dissolve TPPEC7 (1 mg) prepared in Example 3 in 6.48 mL of DMF to obtain a 1 mM stock solution.

[0094] 2. Preparation of the sample solution of the glycosaminoglycan to be measured

[0095] (1) Preparation of the heparin sodium (Hep) stock solution: Weigh 1 mg of heparin sodium and dissolve it in 5 mL of 10 mM PBS (pH 7.4) buffer solution to obtain a heparin sodium stock solution with a concentration of 0.2 mg / mL;

[0096] (2) Preparation of the chondroitin sulfate (Chs) stock solution: Weigh 1 mg of chondroitin sulfate and dissolve it in 5 mL of 10 mM PBS (pH 7.4) buffer solution to obtain a chondroitin sulfate stock solution with a concentration of 0.2 mg / mL;

[0097] (3) Preparation of the sodium hyaluronate (HA) stock solution: Weigh 1 mg of sodium hyaluronate and dissolve it in 5 mL of 10 mM PBS (pH 7.4) buffer solution to obtain a sodium hyaluronate stock solution with a concentration of 0.2 mg / mL;

[0098] (4) Preparation of the dextran sulfate (DS) stock solution: Weigh 1 mg of dextran sulfate and dissolve it in 5 mL of 10 mM PBS (pH 7.4) buffer solution to obtain a dextran sulfate stock solution with a concentration of 0.2 mg / mL.

[0099] 3. Fluorescence response tests of the three sensing units of TPPEBA, TPPEMe, and TPPEC7 to glycosaminoglycans

[0100] (1) Add 30 μL of the 1 mM TPPEBA probe stock solution (the concentration of the probe in the cuvette after dilution is 10 μM) to a cuvette containing 3 mL of pure PBS (10 mM, pH 7.4) buffer solution, and measure the fluorescence emission spectrum of the TPPEBA solution; add 30 μL of the heparin sodium stock solution prepared in step 2 to this solution and measure the fluorescence emission spectrum; repeat adding the heparin sodium stock solution (30 μL each time) and measure the fluorescence emission spectrum until the spectrum no longer changes significantly;

[0101] (2) Repeat the above operation, and use the corresponding stock solutions in step 2 to conduct titration experiments to obtain the fluorescence response spectra of the TPPEBA sensing unit to heparin sodium, chondroitin sulfate, sodium hyaluronate, and dextran sulfate respectively; Figure 11It can be seen that the fluorescence of TPPEBA itself is weak in PBS solvent. When sodium heparin, chondroitin sulfate, sodium hyaluronate, and dextran sulfate are added to its solution respectively, the fluorescence intensity of the solution is significantly enhanced, indicating that TPPEBA can effectively bind to the measured glycosaminoglycans and cause fluorescence enhancement to varying degrees.

[0102] (3) Repeat the above operations, and perform fluorescence titration using the stock solutions prepared in Step 1 and Step 2 to obtain the fluorescence response spectra of TPPEMe and TPPEC7 to heparin, chondroitin sulfate, sodium hyaluronate, and dextran sulfate respectively. Figure 12 It can be seen that TPPEMe has an obvious fluorescence enhancement response to sodium heparin, chondroitin sulfate, and dextran sulfate, and the response degrees are different, while the fluorescence enhancement response to sodium hyaluronate is relatively weak. Similarly, TPPEC7 also has an obvious fluorescence enhancement response to sodium heparin, chondroitin sulfate, and dextran sulfate, while the response to sodium hyaluronate is weak.

[0103] 4. Obtain the fluorescence response patterns of glycosaminoglycans using the three sensing units of TPPEBA, TPPEMe, and TPPEC7

[0104] Obtain the response patterns using the data in Step 3 (the saturation concentrations of the sensing units to each glycosaminoglycan), that is, the fluorescence intensities of the three sensing units to the four tested glycosaminoglycans at the maximum emission wavelength ( Figure 14 ). Figure 14 It can be seen that the three sensing units of TPPEBA, TPPEMe, and TPPEC7 all have obvious but different degrees of fluorescence responses to the four glycosaminoglycans of heparin, chondroitin sulfate, hyaluronic acid, and dextran sulfate. Using the three sensing units of TPPEBA, TPPEMe, and TPPEC7 as an array sensor can obtain cross-response signals of different glycosaminoglycans and achieve pattern recognition of heparin, chondroitin sulfate, hyaluronic acid, and dextran sulfate.

[0105] Example 5 Construction of the array sensor TPPESA

[0106] (1) Arbitrarily select a 4-row × 3-column area on the 96-well plate;

[0107] (2) Add 198 - x μL of PBS (pH 7.4, 10 mM) buffer solution to each well in the above 4-row × 3-column area (x is the volume of the subsequent glycosaminoglycan added: according to the total test volume of 200 μL, first calculate the required volume of the glycosaminoglycan stock solution based on the concentration of the glycosaminoglycan stock solution, and then make up to 200 μL with PBS buffer solution);

[0108] (3) Add 2 μL of TPPEBA stock solution to each well in the first column of the 4-row × 3-column area in the previous step;

[0109] (4) Add 2 μL of TPPEMe stock solution to each hole in the second column of the 4-row × 3-column area in the previous step;

[0110] (5) Add 2 μL of TPPEC7 stock solution to each hole in the third column of the 4-row × 3-column area in the previous step to complete the construction of the array sensor.

[0111] Example 6 Detection of Glycosaminoglycan by Array Sensor TPPESA

[0112] 1. The array sensor TPPESA obtains the fluorescence response data matrix of glycosaminoglycan

[0113] (1) Add 0.1 μL of the glycosaminoglycan stock solution to be measured (x = 0.1) to each hole in the 4-row × 3-column area of the array sensor TPPESA prepared in Example 5, and the actual concentration of glycosaminoglycan in each hole after dilution is 0.1 μg / mL (the total volume of the solution in each hole is 200 μL, take 0.1 μL of the glycosaminoglycan stock solution (the concentration of the glycosaminoglycan stock solution is 0.2 mg / mL) and dilute it to 200 μL, the concentration is 0.1 μg / mL);

[0114] (2) Add 1 μL of the glycosaminoglycan stock solution to be measured (x = 1) to each hole in the 4-row × 3-column area of the above array sensor TPPESA, and the actual concentration of glycosaminoglycan in each hole after dilution is 1 μg / mL;

[0115] (3) Add 5 μL of the glycosaminoglycan stock solution to be measured (x = 5) to each hole in the 4-row × 3-column area of the above array sensor TPPESA, and the actual concentration of glycosaminoglycan in each hole after dilution is 5 μg / mL;

[0116] (4) Add 10 μL of the glycosaminoglycan stock solution to be measured (x = 10) to each hole in the 4-row × 3-column area of the above array sensor TPPESA, and the actual concentration of glycosaminoglycan in each hole after dilution is 10 μg / mL;

[0117] (5) Add 100 μL of the glycosaminoglycan stock solution to be measured (x = 100) to each hole in the 4-row × 3-column area of the above array sensor TPPESA, and the actual concentration of glycosaminoglycan in each hole after dilution is 100 μg / mL;

[0118] (6) Add 198 μL of the glycosaminoglycan stock solution to be measured (x = 198) to each hole in the 4-row × 3-column area of the above array sensor TPPESA, and the actual concentration of glycosaminoglycan in each hole after dilution is approximately 200 μg / mL;

[0119] (7) Measure the fluorescence intensity of each well in the above 4-row × 3-column region (for the first column, the excitation wavelength is 365 nm and the detection wavelength is 554 nm; for the second column, the excitation wavelength is 418 nm and the detection wavelength is 552 nm; for the third column, the excitation wavelength is 393 nm and the detection wavelength is 544 nm), and obtain a 4-row × 3-column data array of the tested glycosaminoglycan.

[0120] (8) Process the obtained glycosaminoglycan sample matrix data through linear discriminant analysis to obtain the discrimination results of six different concentrations of glycosaminoglycan: 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 100 μg / mL, and 200 μg / mL. See Figure 15 , from Figure 15 It can be seen that starting from a concentration of 1 μg / mL, the 4 glycosaminoglycans are clearly divided into 4 different clusters, and there is no intersection or overlap between all clusters, indicating that our array sensor TPPESA has excellent performance in distinguishing these glycosaminoglycans, and the lowest discrimination concentration is 1 μg / mL.

[0121] 2. Concentration detection of glycosaminoglycan by array sensor TPPESA

[0122] (1) Add a certain volume (such as x = 0.1, 1, 5, 10, 100, 198 μL) of the glycosaminoglycan stock solution to be tested to each well in the 4-row × 3-column region of the above array sensor TPPESA, and calculate the actual concentration of glycosaminoglycan in each well after dilution (the stock solution concentration is 0.2 mg / mL, and the volume after dilution is 200 μL, and the diluted concentrations can be calculated as 0.1 μg / mL, 1 μg / mL, 5, 10, 100, 200 μg / mL);

[0123] (2) Measure the fluorescence intensity of each well in the above 4-row × 3-column region (for the first column, the excitation wavelength is 365 nm and the detection wavelength is 554 nm; for the second column, the excitation wavelength is 418 nm and the detection wavelength is 552 nm; for the third column, the excitation wavelength is 393 nm and the detection wavelength is 544 nm), and obtain a 4-row × 3-column data array of the tested glycosaminoglycan.

[0124] Using the obtained glycosaminoglycan sample matrix data for data processing through linear discriminant analysis, it can be seen that the array sensor TPPESA can be used for quantitative detection of the concentrations of heparin and dextran sulfate, and the clusters of different concentrations are separated from each other without overlap; while for chondroitin sulfate and hyaluronic acid, their concentrations of 100 and 200 μg / mL cannot be distinguished (the corresponding clusters overlap), but the concentrations below 100 μg / mL can be distinguished ( Figure 16 ). It shows that the array sensor TPPESA can be effectively used for the detection of the concentrations of the above four glycosaminoglycans.

[0125] Example 7 Detection of Glycosaminoglycan Analogue Impurities in Heparin by Array Sensor TPPESA

[0126] 1. Prepare a contaminated sodium heparin (Hep) stock solution: Weigh 9.8 mg of sodium heparin and 0.2 mg of chondroitin sulfate, and dissolve them in 50 mL of PBS (pH 7.4, 10 mM) buffer solution to obtain a sodium heparin stock solution with a concentration of 0.2 mg / mL containing 2% chondroitin sulfate impurities;

[0127] 2. Adopt a similar method to prepare sodium heparin stock solutions with concentrations of 0.2 mg / mL containing 10%, 30%, and 50% chondroitin sulfate impurities respectively;

[0128] 3. Adopt a similar method to prepare sodium heparin stock solutions with concentrations of 0.2 mg / mL containing 2%, 10%, 30%, and 50% hyaluronic acid impurities respectively;

[0129] 4. Adopt a similar method to prepare sodium heparin stock solutions with concentrations of 0.2 mg / mL containing 2%, 10%, 30%, and 50% dextran sulfate impurities respectively;

[0130] 5. Refer to the operation in Example 6, add 10 μL of the sodium heparin stock solution containing glycosaminoglycan impurities to be tested to each well in the 4-row × 3-column area of the array sensor TPPESA, so that the total concentration of glycosaminoglycans in the solution is 10 μg / mL;

[0131] 6. Refer to the operation in Example 6, add 10 μL of the pure sodium heparin stock solution to each well in the 4-row × 3-column area of the array sensor TPPESA, so that the concentration of sodium heparin in the solution is 10 μg / mL;

[0132] 7. Refer to the operation in Example 6, add 10 μL of the pure glycosaminoglycan stock solution of impurities to each well in the 4-row × 3-column area of the array sensor TPPESA, so that the concentration of the pure glycosaminoglycan of impurities in the solution is 10 μg / mL;

[0133] 8. Test the fluorescence intensity of each well in the above 4-row × 3-column area (the excitation wavelength of the first column is 365 nm, and the detection wavelength is 554 nm; the excitation wavelength of the second column is 418 nm, and the detection wavelength is 552 nm; the excitation wavelength of the third column is 393 nm, and the detection wavelength is 544 nm) to obtain a data array of the tested glycosaminoglycans.

[0134] Perform data processing on the obtained glycosaminoglycan sample matrix data through linear discriminant analysis to obtain the detection results of the array sensor for glycosaminoglycan impurities in heparin.

[0135] From Figure 17It can be seen that when the total concentration of heparin, chondroitin sulfate, sodium hyaluronate, and dextran sulfate is 10 μg / mL, the LDA spectrum shows that the pure heparin cluster and the sample cluster contaminated with chondroitin sulfate, sodium hyaluronate, and dextran sulfate can be clearly separated, and impurities as low as 2% can be detected.

[0136] Example 8 Detection of Glycosaminoglycans in 10% Serum Samples

[0137] 1. Preparation of the sample solution of the glycosaminoglycan to be measured

[0138] (1) Preparation of the heparin sodium (Hep) stock solution: Weigh 1 mg of heparin sodium and dissolve it in a mixture of 4.5 mL of 10 mM PBS (pH 7.4) buffer and 0.5 mL of fetal bovine serum to obtain a heparin sodium stock solution with a concentration of 0.2 mg / mL containing 10% serum;

[0139] (2) Preparation of the chondroitin sulfate (Chs) stock solution: Weigh 1 mg of chondroitin sulfate and dissolve it in a mixture of 4.5 mL of 10 mM PBS (pH 7.4) buffer and 0.5 mL of fetal bovine serum to obtain a chondroitin sulfate stock solution with a concentration of 0.2 mg / mL containing 10% serum;

[0140] (3) Preparation of the sodium hyaluronate (HA) stock solution: Weigh 1 mg of sodium hyaluronate and dissolve it in a mixture of 4.5 mL of 10 mM PBS (pH 7.4) buffer and 0.5 mL of fetal bovine serum to obtain a sodium hyaluronate stock solution with a concentration of 0.2 mg / mL containing 10% serum;

[0141] (4) Preparation of the dextran sulfate (DS) stock solution: Weigh 1 mg of dextran sulfate and dissolve it in a mixture of 4.5 mL of 10 mM PBS (pH 7.4) buffer and 0.5 mL of fetal bovine serum to obtain a dextran sulfate stock solution with a concentration of 0.2 mg / mL containing 10% serum.

[0142] 2. The array sensor TPPESA obtains the fluorescence response data matrix of glycosaminoglycans

[0143] Repeat the steps for constructing the fluorescence response data matrix of glycosaminoglycans by the array sensor TPPESA in Example 6 to obtain a 4-row × 3-column data array of glycosaminoglycans in 10% serum samples; perform data processing on the matrix data of glycosaminoglycans in the obtained 10% serum samples by linear discriminant analysis to obtain the discrimination results of six different concentrations of glycosaminoglycans: 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 100 μg / mL, and 200 μg / mL:

[0144] From Figure 18It can be seen that starting from a concentration of 1 μg / mL, the four glycosaminoglycans were clearly separated into four different clusters, and there was no crossover or overlap between all the clusters, indicating that our TPPESA array sensor has excellent performance in differentiating these glycosaminoglycans, with the lowest discrimination concentration being 1 μg / mL.

[0145] 3. Concentration of glycosaminoglycans detected by the TPPESA array sensor

[0146] Repeat the steps of detecting the concentration of glycosaminoglycans by the TPPESA array sensor in Example 6, and perform data processing on the obtained glycosaminoglycan sample matrix data through linear discriminant analysis:

[0147] From Figure 19 It can be seen that the TPPESA array sensor can be used for quantitative detection of the concentrations of heparin and dextran sulfate. The clusters of different concentrations are separated from each other without overlap; for chondroitin sulfate, its concentrations of 100 and 200 μg / mL cannot be distinguished, but concentrations below 100 μg / mL can be distinguished; for hyaluronic acid, its concentrations of 0.1 and 1 μg / mL cannot be distinguished, but concentrations above 1 μg / mL can be distinguished. This shows that the TPPESA array sensor can be effectively used for the detection of the concentrations of the four glycosaminoglycans.

[0148] 4. Detection of glycosaminoglycan analog impurities in heparin by the TPPESA array sensor

[0149] (1) Prepare a contaminated heparin sodium (Hep) stock solution: Weigh 9.9 mg of heparin sodium and 0.1 mg of chondroitin sulfate, and dissolve them in a mixture of 45 mL of PBS (pH 7.4, 10 mM) buffer solution and 5 mL of fetal bovine serum to obtain a heparin sodium stock solution with a concentration of 0.2 mg / mL containing 1% chondroitin sulfate impurity (serum content 10%);

[0150] (2) Adopt a similar method to prepare heparin sodium stock solutions with concentrations of 0.2 mg / mL containing 10%, 30%, and 50% chondroitin sulfate impurities (serum content 10%) respectively;

[0151] (3) Adopt a similar method to prepare heparin sodium stock solutions with concentrations of 0.2 mg / mL containing 1%, 10%, 30%, and 50% hyaluronic acid impurities (serum content 10%) respectively;

[0152] (4) Adopt a similar method to prepare heparin sodium stock solutions with concentrations of 0.2 mg / mL containing 1%, 10%, 30%, and 50% dextran sulfate impurities (serum content 10%) respectively;

[0153] (5) Repeat the operations in (5)-(8) of Example 7, and perform data processing on the obtained glycosaminoglycan sample matrix data through linear discriminant analysis to obtain the detection results of the glycosaminoglycan impurities in heparin by the array sensor.

[0154] It can be seen from Figure 20 that when the total concentration of heparin, chondroitin sulfate, sodium hyaluronate, and dextran sulfate is 10 μg / mL, the LDA spectrum shows that the pure heparin cluster and the sample clusters contaminated by chondroitin sulfate, sodium hyaluronate, and dextran sulfate can be clearly separated, and impurities as low as 1% can be detected.

[0155] 5. Identification of unknown glycosaminoglycans by the array sensor TPPESA

[0156] (1) Preparation of unknown glycosaminoglycan samples. Taking sodium heparin as an example: Weigh 1 mg of sodium heparin and dissolve it in a mixed solution of 4.5 mL of PBS (pH 7.4, 10 mM) and 0.5 mL of fetal bovine serum to obtain a 0.2 mg / mL sodium heparin stock solution containing 10% serum.

[0157] (2) Similarly, prepare 0.2 mg / mL stock solutions of chondroitin sulfate, hyaluronic acid, and dextran sulfate containing 10% serum respectively.

[0158] (3) Add 100 μL (x = 100) of the unknown glycosaminoglycan stock solutions in steps (1) and (2) to each well in the 4-row × 3-column area of the array sensor TPPESA constructed in Example 5, so that the actual concentration of glycosaminoglycan in each well after dilution is 100 μg / mL.

[0159] (4) Measure the fluorescence intensity of each well in the above 4-row × 3-column area (the excitation wavelength of the first column is 365 nm, and the detection wavelength is 554 nm; the excitation wavelength of the second column is 418 nm, and the detection wavelength is 552 nm; the excitation wavelength of the third column is 393 nm, and the detection wavelength is 544 nm) to obtain a 4-row × 3-column data array of the tested glycosaminoglycans.

[0160] (5) Use the obtained glycosaminoglycan sample matrix data as the test data set test data, and use the data matrix of 100 μg / mL glycosaminoglycans in Example 7 as the training data set training data, and perform data processing through linear discriminant analysis to obtain the pattern recognition results for the simulated unknown glycosaminoglycan samples.

[0161] Table 1 Identification table of 100 μg / mL unknown glycosaminoglycan samples using the TPPESA sensing array

[0162]

[0163] As can be seen from Table 1, the array sensor TPPESA can achieve 100% accurate recognition of two glycosaminoglycans, chondroitin sulfate and hyaluronic acid, which are unknown.

Claims

1. A long-wavelength AIE array sensor, characterized in that, The long-wavelength AIE array sensor includes three sensing units, namely TPPEBA, TPPEMe, and TPPEC7. The structural formulas of TPPEBA, TPPEMe, and TPPEC7 are as follows:

2. The preparation method of the long-wavelength AIE array sensor according to claim 1, characterized in that, The method includes the following steps: 1) Preparation of TPPEBA: Weigh the raw materials TPPE and 4-bromomethylphenylboronic acid, dissolve them in dry DMF, heat the solution under nitrogen protection until the raw materials disappear, spin-dry the solvent, add dichloromethane and sonicate, filter with acetonitrile to remove impurities, and collect the bright yellow solid product to obtain TPPEBA; 2) Preparation of TPPEMe: Add the raw materials TPPE and excessive methyl iodide to the reactor, use DMF as the solvent, react in an oil bath, filter, and wash the filter cake with dichloromethane to obtain the product TPPEMe; 3) Preparation of TPPEC7: Add TPPE and excessive iodoheptane to the reactor, use DMF as the solvent, react overnight, filter, spin off the solvent, wash the product with dichloromethane, and filter to obtain the product TPPEC7; 4) Construct an array sensor with the obtained TPPEBA, TPPEMe, and TPPEC7.

3. Application of the long-wavelength AIE array sensor according to claim 1 in the detection of glycosaminoglycans.

4. The application according to claim 3, characterized in that The glycosaminoglycans include one or more of heparin, chondroitin sulfate, hyaluronic acid, and dextran sulfate.

5. A method for detecting different types and / or different concentrations of glycosaminoglycans, characterized in that, The detection method includes the following steps: 1) Construct an array sensor TPPESA with TPPEBA, TPPEMe, and TPPEC7; 2) Preparation of the glycosaminoglycan sample solution to be measured: Prepare heparin sodium stock solution, chondroitin sulfate stock solution, sodium hyaluronate stock solution, and dextran sulfate stock solution; 3) Fluorescence response test of the array sensor constructed in step 1) for the glycosaminoglycans in step 2), and obtain the concentration of the glycosaminoglycan to be measured through the relationship between the fluorescence response degree of the glycosaminoglycan and its concentration.

6. The detection method for different types and / or different concentrations of glycosaminoglycans according to claim 5, wherein The glycosaminoglycan sample solution to be measured also includes heparin sodium stock solution with chondroitin sulfate impurities, heparin sodium stock solution with hyaluronic acid impurities, and heparin sodium stock solution with dextran sulfate impurities.

7. The detection method for different types and / or different concentrations of glycosaminoglycan according to claim 5, characterized in that, The glycosaminoglycan sample solution to be measured also includes heparin sodium stock solution containing serum, chondroitin sulfate stock solution containing serum, sodium hyaluronate stock solution containing serum, and dextran sulfate stock solution containing serum.

8. The detection method for different types and / or different concentrations of glycosaminoglycan according to claim 5, characterized in that, The construction of the array sensor in step 1) specifically includes the following steps: S1) Arbitrarily select a 4-row × 3-column area on the 96-well plate; S2) Add PBS buffer solution to each well in the above 4-row × 3-column area; S3) Add TPPEBA stock solution to each well in the first column of the 4-row × 3-column area in the previous step; S4) Add TPPEMe stock solution to each well in the second column of the 4-row × 3-column area in the previous step; S5) Add TPPEC7 stock solution to each well in the third column of the 4-row × 3-column area in the previous step to complete the construction of the array sensor.

9. The detection method for different types and / or different concentrations of glycosaminoglycans according to claim 5, characterized in that, In step 2), the concentration of each glycosaminoglycan in the heparin sodium stock solution, chondroitin sulfate stock solution, sodium hyaluronate stock solution, and dextran sulfate stock solution is 0.1 - 200 μg / mL.

10. The detection method for different types and / or different concentrations of glycosaminoglycan according to claim 6, characterized in that, The concentration of each glycosaminoglycan in the heparin sodium stock solution of chondroitin sulfate impurities, the heparin sodium stock solution of hyaluronic acid impurities, and the heparin sodium stock solution of dextran sulfate impurities is 0.1 - 200 μg / mL.

11. The detection method for different types and / or different concentrations of glycosaminoglycan according to claim 7, characterized in that, The concentration of each glycosaminoglycan in the heparin sodium stock solution containing serum, the chondroitin sulfate stock solution containing serum, the sodium hyaluronate stock solution containing serum, and the dextran sulfate stock solution containing serum is 0.1 - 200 μg / mL.

12. The detection method for different types and / or different concentrations of glycosaminoglycan according to claim 8, characterized in that, The steps of step 3) are as follows: 3.1) Add a certain volume of the glycosaminoglycan stock solution to be measured into each well in the 4-row × 3-column area of the array sensor TPPESA, and calculate the actual concentration of glycosaminoglycan in each well after dilution; 3.2) Measure the fluorescence intensity of each well in the above 4-row × 3-column area. The excitation wavelength of the first column is 365 nm, and the detection wavelength is 554 nm; the excitation wavelength of the second column is 418 nm, and the detection wavelength is 552 nm; the excitation wavelength of the third column is 393 nm, and the detection wavelength is 544 nm, to obtain a 4-row × 3-column data array of the measured glycosaminoglycan; 3.3) Use the obtained glycosaminoglycan sample matrix data for data processing through linear discriminant analysis to obtain the detection result of the glycosaminoglycan to be measured in the array sensor.

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