Polythiophene derivative fluorescent probe, preparation method thereof and application of polythiophene derivative fluorescent probe in detection of biological mercaptan

Abstract

The invention discloses a preparation method of a polythiophene derivative fluorescent probe and application of the polythiophene derivative fluorescent probe in detection of biological mercaptan, and belongs to the technical field of fluorescent sensing detection. The method comprises the following steps: firstly, performing fluorescence quenching on a polythiophene derivative by utilizing paramagnetism of Cu < 2 + >, and capturing Cu < 2 + > by virtue of stronger nucleophilic ability between biological mercaptan and Cu < 2 + >, so that the polythiophene derivative is separated from a complex and recovers a free state, thereby realizing opening of a fluorescence signal. Compared with a traditional small-molecule fluorescent probe, the synthesized polythiophene derivative fluorescent probe has higher stability and biocompatibility due to the introduction of a polymer skeleton and the characteristic that a side chain can be modified.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence sensing detection, and particularly relates to a polythiophene derivative fluorescent probe, a preparation method thereof, and an application thereof in detecting biological thiols. Background Art

[0002] Biothiols, a class of endogenous active molecules containing sulfhydryl groups, primarily include cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH). They play a vital role in maintaining physiological processes. Therefore, the development of highly selective and precise quantitative detection technologies for biothiols has important clinical and scientific value for disease prevention and diagnosis. With the deepening of life science research and the development of emerging technologies such as nanotechnology and biotechnology, the research of fluorescent probes has entered a new stage of development. The application of fluorescent probe technology to the detection of biothiols has important implications for the prevention, diagnosis, and treatment of diseases such as cancer and Alzheimer's disease.

[0003] Polythiophene is a water-soluble conjugated polymer that has attracted widespread attention due to its excellent optical properties, good photostability, unique conformational changes, good biocompatibility, and easy side chain modification. These characteristics make polythiophene an ideal fluorescent probe material.

[0004] Currently, research on polythiophene derivatives as fluorescent probes for detecting biothiols is based on the conjugated structure and easily modifiable side chains of polythiophene. Through structural design and performance optimization, functional fluorescent probes with high selectivity for biothiols and adjustable properties are designed and synthesized. Polythiophene derivative fluorescent probes for biothiols have broad application prospects in cell imaging, medical diagnosis, environmental monitoring, and food safety.

[0005] In terms of design and synthesis, based on the coordination mechanism, Cu 2+ The design is based on a polythiophene conjugated backbone and incorporates functional groups that specifically recognize metal ions to form a complex. The complex specifically recognizes biothiols, and through the electron delocalization characteristics of the π-π conjugated system and the fluorescence "off-on" mode, the fluorescence signal changes after the biothiol reacts with the probe system, thereby achieving the purpose of biothiol detection. Summary of the Invention

[0006] The purpose of the present invention is to construct a highly sensitive and highly selective polythiophene derivative thiol fluorescent probe, introduce specific groups that coordinate with metal ions and improve the water solubility of the polythiophene derivative, so as to achieve efficient binding and response of the probe to biological thiols.

[0007] The technical solution adopted by the present invention is: a polythiophene derivative fluorescent probe, wherein the detection principle of the probe is as follows: first, Cu 2+Paramagnetism makes polythiophene derivatives fluorescence quenching, and then through the biological thiol and Cu 2+ Between the stronger nucleophilic ability to snatch Cu 2+ Make polythiophene derivatives out of complex free state, so as to realize the opening of fluorescence signal; The structure of the polythiophene derivative is:

[0008]

[0009] m, n represent the relative number of two structure units in the brackets, respectively, S, O, N, Br - , HN, N + Are chemical elements, sulfur, oxygen, nitrogen, bromide, imine, nitrogen ion.

[0010] A kind of polythiophene derivative fluorescence probe preparation method, polythiophene derivative is designed, by modifying its side chain, the specific steps are as follows:

[0011] S1, synthesis of 3-(4-methyl-3-thienyloxy) propyl trimethyl ammonium bromide M1

[0012] 3-(3-bromo) propoxy-4-methyl thiophene 600 mg, 2.55 mmol is completely dissolved in 10 mL tetrahydrofuran, 13 mL of 30% trimethylamine aqueous solution is added, stirring at room temperature for 24 h, the reaction is completed, the solvent is spin-dried, 30 mL of tetrahydrofuran is added to the reaction bottle and stirred for 40 min, white solid is precipitated, and the crude product is washed with tetrahydrofuran, finally the off-white solid, monomer 1, 615.68 mg, yield 82% is obtained;

[0013] S2, synthesis of 3-(4-methyl-3-thienyloxy) propyl-1, 4, 7, 10-tetraazacyclododecane M2

[0014] 3-(3-bromo) propoxy-4-methyl thiophene 600 mg, 2.55 mmol, 1, 4, 7, 10-tetraazacyclododecane 879.20 mg, 5.10 mmol and K2CO3, 3.53 g, 25.5 mmol are dissolved in 20 mL tetrahydrofuran, and the reaction is carried out at 55-60 DEG C for 48 h; After the reaction is completed, it is cooled to room temperature, K2CO3 is removed by suction filtration, the tetrahydrofuran in the filtrate is spin-dried, and yellow oil is obtained. The yellow oil is dissolved in 20 mL dichloromethane, extracted with water until the upper aqueous phase is clear, anhydrous magnesium sulfate is added to the extracted organic phase and dried for 10 h, suction filtered and spin-dried to obtain the crude product. After column chromatography purification, 3-(4-methylthiophene-3-yloxy) propyl-1, 4, 7, 10-tetraazacyclododecane, i.e. M2, 619 mg, yield 74.3% is obtained;

[0015] S3. Synthesis of polythiophene derivatives

[0016] M1, 90.12 mg, 0.31 mmol, M2, 100 mg, 0.31 mmol, and ferric chloride 298.6 mg, 1.84 mmol were dissolved in 20 mL of N,N-dimethylacetamide and reacted at 50°C under nitrogen for 48 h. After the reaction, the reaction solution was cooled to room temperature and concentrated by rotary evaporation to 2 mL. The solution was precipitated in 100 mL of methanol and filtered to obtain a solid. The filter cake was extracted with methanol, and the ferric chloride was removed from the extraction solution and then dried to obtain 54.96 mg of a polythiophene derivative with a yield of 29%.

[0017] Application of a polythiophene derivative fluorescent probe in detecting biothiols, wherein the probe introduces Cu 2+ The probe can be used to quantitatively detect Hcy in a physiological cell environment. 2+ The continuous detection of Hcy and Hcy, and the detection limit of Hcy is as low as 9.87nM. 2+ The detection and response of the complex to Hcy is suitable for a neutral to weakly alkaline environment with a pH of 7-10. 2+ After fluorescence quenching, the addition of Hcy restored the fluorescence intensity of the polythiophene derivative by 5 times, with a response time as low as 10 s and stable fluorescence intensity within 100 s.

[0018] Beneficial effects of the present invention:

[0019] (1) The polythiophene derivative fluorescent probe synthesized in the present invention can detect Hcy using the metal displacement principle, and the detection limit of Hcy is as low as 9.87 nM.

[0020] (2) The polythiophene derivative fluorescent probe synthesized in the present invention can show excellent selectivity and sensitivity to biothiols within the physiological pH range. 2+ After fluorescence quenching, the addition of Hcy restored the fluorescence intensity of the polythiophene derivative by 5 times, the response time was as low as 10 s, and the fluorescence intensity was basically stable within 100 s.

[0021] (3) Compared with traditional small-molecule fluorescent probes, the polythiophene derivative fluorescent probe synthesized in this invention has higher stability and biocompatibility due to the introduction of a polymer backbone and the modifiable side chains. In the presence of other metal ions, the polythiophene derivative fluorescent probe can still recover fluorescence intensity by 5 times in response to biothiols. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Synthetic route of polythiophene derivatives;

[0023] (1) Tetrahydrofuran, 25°C, 24h; (2) FeCl3, N,N-dimethylacetamide, 55-60°C, 48h;

[0024] Figure 2 Monomer 1 in D2O 1 HNMR spectrum;

[0025] Figure 3 Single Body 2 in CDCl3 1 HNMR spectrum;

[0026] Figure 4 Polymer in D2O 1 HNMR spectrum;

[0027] Figure 5 (a) In Tris-HCl buffer solution (2 mM, pH 7.4), polymer-Cu 2+ The relative fluorescence intensity (F) of each test amino acid at 534 nm was i / F0);

[0028] (b) Polymer-Cu was synthesized in Tris-HCl buffer solution (2 mM, pH 7.4) in the presence of 500 μM of different amino acids. 2+ Fluorescence spectra of (100 μM);

[0029] Figure 6 (a) In Tris-HCl buffer solution (2 mM, pH 7.4), different concentrations of Hcy (0-3.2 equivalents) were added, and polymer-Cu 2+ Fluorescence spectra of (100 μM);

[0030] (b) At 534 nm (F i -F0) / F0 curve changes with Hcy concentration. F0 is polymer-Cu 2+ The emission intensity of the complex (100 μM), F i For polymer-Cu 2+ Emission intensity of the complex with the addition of different concentrations of Hcy, λex = 407 nm;

[0031] Figure 7 In Tris-HCl buffer solution (2 mM, pH = 7.4), polymer-Cu 2+ , polymers and polymer-Cu 2+ -The fluorescence intensity of Hcy at 534 nm changes with pH;

[0032] Figure 8 In Tris-HCl buffer solution (2 mM, pH = 7.4), Cu 2+Effect of response time on fluorescence intensity of polymer (100 μM) at 534 nm in the presence of BSA (20 μM) and Hcy (500 μM), λex= 407 nm;

[0033] Figure 9 Mechanism of fluorescence probe detection. DETAILED DESCRIPTION

[0034] Example 1

[0035] A polythiophene derivative fluorescence probe, first uses Cu 2+ Paramagnetism makes polythiophene derivative fluorescence quenching, and then through the stronger nucleophilic ability of biological thiol and Cu 2+ Between them snatch Cu 2+ Make polythiophene derivative out of complex and restore free state, so as to realize the opening of fluorescence signal; the structure of the polythiophene derivative is as follows:

[0036] m, n represent the relative number of two structure units in the brackets, respectively, S, O, N, Br - , HN, N + Are chemical elements, sulfur, oxygen, nitrogen, bromide, imine, nitrogen ion.

[0037] Preparation process of polythiophene derivative: design the structure of polythiophene derivative, realize the synthesis route of polythiophene derivative by modifying its side chain as Figure 1 Shown.

[0038] Synthesis of 3-(4-methyl-3-thienyloxy) propyl trimethyl ammonium bromide (M1)

[0039] Dissolve 3-(3-bromo) propoxy-4-methyl thiophene (600 mg, 2.55 mmol) in 10 mL of tetrahydrofuran, after it is completely dissolved, add 13 mL of 30% mass fraction of trimethylamine aqueous solution, stir at room temperature for 24 h. After the reaction is completed, spin dry the solvent, add 30 mL of tetrahydrofuran to the reaction bottle and stir for 40 min. A large amount of white solid is precipitated. Filter, wash the crude product with tetrahydrofuran for three to four times, and finally obtain a grayish white solid, which is monomer 1 (615.68 mg, yield 82%).

[0040] Synthesis of 3-(4-methyl-3-thienyloxy) propyl-1,4,7,10-tetraazacyclododecane (M2)

[0041] Dissolve 3-(3-bromo)propoxy-4-methylthiophene (600 mg, 2.55 mmol), 1,4,7,10-tetraazacyclododecane (879.20 mg, 5.10 mmol), and K2CO3 (3.53 g, 25.5 mmol) in 20 mL of tetrahydrofuran and react at 55-60°C for 48 hours. After the reaction is completed, cool to room temperature and filter to remove the K2CO3. Remove the tetrahydrofuran from the filtrate by spin drying to obtain a yellow oil. Dissolve the yellow oil in 20 mL of dichloromethane and extract with water three to four times until the upper aqueous phase becomes clear. Add anhydrous magnesium sulfate to the extracted organic phase and dry for 10 hours. Filter and spin dry to obtain the crude product. The crude product was purified by column chromatography (silica gel, dichloromethane:methanol:triethylamine = 2:1:0.1 (v / v)) to give 3-(4-methylthiophen-3-yloxy)propyl-1,4,7,10-tetraazacyclododecane, M2 (619 mg, 74.3% yield).

[0042] Synthesis of polythiophene derivatives

[0043] 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide (M1) (90.12 mg, 0.31 mmol), 3-(4-methylthien-3-yloxy)propyl-1,4,7,10-tetraazacyclododecane (M2) (100 mg, 0.31 mmol), and ferric chloride (298.6 mg, 1.84 mmol) were dissolved in 20 mL of N,N-dimethylacetamide and reacted at 50°C under nitrogen for 48 hours. After the reaction, the mixture was cooled to room temperature and concentrated by rotary evaporation to 2 mL. The mixture was then settled in 100 mL of methanol and filtered to obtain a solid. The filter cake was extracted with methanol, and the ferric chloride was removed from the extraction solution, which was then dried by rotary evaporation to yield 54.96 mg of a polythiophene derivative in a 29% yield.

[0044] Sample preparation

[0045] A certain amount of polythiophene derivative was dissolved in deionized water to prepare a concentration of 5×10 -3 mol / L mother solution. Prepare the concentration of 5×10 -3 mol / L Cu 2+ solution, concentration of 5×10 -3 mol / L other amino acids Ser, Leu, Val, Lys, Ala, His, Tyr, Trp solution, concentration is 5×10 -3 mol / L biothiol Cys, Hcy, and GSH solutions are ready for use.

[0046] In this experiment, ultraviolet-visible spectroscopy (UV), nuclear magnetic resonance spectroscopy ( 1 HNMR) and fluorescence spectroscopy were used to systematically characterize the polythiophene derivative biothiol fluorescent probe.

[0047] Weigh an appropriate amount of the polythiophene derivative fluorescent probe mother solution into a volumetric flask, dissolve it in deionized water and methanol, and dilute it to a concentration of 100 μmol / L. The procedures for using deionized water and methanol solutions as solvents are the same as follows: Take a portion of the mother solution and prepare a series of sample solutions of different concentrations by gradient dilution. At the same time, prepare deionized water and methanol as blank controls. Add the blank control solution to a cuvette, place it in the instrument, scan the wavelength range of 300-600 nm, and record the blank spectrum to use as the background subtraction baseline. Then, add sample solutions of different concentrations to the cuvette in sequence and place it in the same position on the instrument. Repeat the measurement for each sample three times and take the average value to reduce the error.

[0048] Weigh 5 mg of polythiophene derivative fluorescent probe and dissolve it in 0.5 mL of heavy water (D2O). After the polythiophene derivative is completely dissolved, place the solution into an NMR tube.

[0049] Fluorescence spectrum test was performed with 407nm fluorescence as excitation wavelength, slit width of 10, and scanning in the range of 410-850nm. A concentration of 5×10 -3 mol / L polythiophene derivative solution 100μM was prepared into polythiophene derivative Tris-HCl buffer solution 2mM, pH 7.4, and placed in the instrument to scan and record the initial fluorescence spectrum. -3 mol / L Cu 2+ solution until the fluorescence spectrum no longer changes. 2+ After the solution completely quenched the fluorescence of the polythiophene derivative fluorescent probe solution, 500 μM of three biothiols (Cys, Hcy, GSH) and other types of amino acids were added to the polymer-Cu 2+ In the complex solution, the fluorescence recovery intensity was recorded. Homocysteine ​​(Hcy), which has the best fluorescence response among the three biothiols, was selected for performance testing.

[0050] Characterization of 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide (M1)

[0051] 3-(3-bromo)propoxy-4-methylthiophene reacts with trimethylamine aqueous solution in tetrahydrofuran to produce a thiophene derivative substituted with trimethylammonium bromide group, namely 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide. The results of H NMR spectrum processing are as follows Figure 2As shown, δ = 6.94 (s, 1H), 6.41 (s, 1H) correspond to hydrogen signals on the thiophene five-membered ring; δ = 1.99 (s, 3H) corresponds to hydrogen signals on the methyl group of the thiophene side group; δ = 4.07 (s, 2H), 3.45 (d, J = 16.8 Hz, 2H), 2.25–2.19 (m, 2H) correspond to hydrogen signals on the carbon group of the thiophene side group; and δ = 3.07 (s, 9H) corresponds to hydrogen signals on trimethylammonium bromide. Analysis of the position and area of ​​the characteristic peaks in the H NMR spectrum correspond to the structure of the target product, indicating that the monomer, 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide, was successfully prepared. Analysis of the H NMR spectrum results indicated a purity of 97.32%, which meets the required purity for polymer synthesis.

[0052] Characterization of 3-(4-methyl-3-thienyloxy)propyl-1,4,7,10-tetraazacyclododecane (M2)

[0053] 3-(3-bromo)propoxy-4-methylthiophene reacts with 1,4,7,10-tetraazacyclododecane in the presence of K2CO3 to generate a thiophene derivative substituted with 1,4,7,10-tetraazacyclododecane, namely 3-(4-methyl-3-thienyloxy)propyl

[0054] -1,4,7,10-tetraazacyclododecane. The results of H NMR spectrum processing are as follows Figure 3 The results show that δ = 6.80 (s, 1H) and 6.16 (s, 1H) correspond to hydrogen signals on the thiophene five-membered ring; δ = 2.09 (s, 3H) corresponds to hydrogen signals on the methyl group of the thiophene side group; δ = 4.03 (s, 2H) and 1.98 (s, 2H) correspond to hydrogen signals on the carbon of the thiophene side group; δ = 2.76 (s, 8H) and 2.66 (s, 4H) correspond to hydrogen signals on the carbon of the tetraazacyclic ring; δ = 2.56 (s, 6H) corresponds to hydrogen signals on the carbon of the thiophene side group and the carbon of the tetraazacyclic ring; and δ = 1.47 (s, 3H) corresponds to hydrogen signals on the nitrogen of the tetraazacyclic ring. Analysis of the position and area of ​​the characteristic peaks in the H NMR spectrum correspond to the structure of the target product, indicating that the monomer di-3-(4-methyl-3-thienyloxy)propyl-1,4,7,10-tetraazacyclododecane was successfully prepared. The purity of monomer 2 was calculated to be 96% by H NMR spectrum analysis, which has reached the monomer purity required for polymer synthesis.

[0055] Characterization of polythiophene derivatives

[0056] Using the ferric chloride oxidative polymerization method, equal amounts of 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide and 3-(4-methylthien-3-yloxy)propyl-1,4,7,10-tetraazacyclododecane were polymerized. The results of the H NMR spectrum are as follows: Figure 4 As shown, δ = 4.09 (s, 4H) corresponds to the hydrogen signals on the side chain carbons of monomers 1 and 2, δ = 3.71-3.30 (m, 9H) corresponds to the hydrogen signals of trimethyl nitrogen bromide of monomer 1, δ = 3.14 (d, J = 24.6 Hz, 18H) corresponds to the hydrogen signals on the side chain carbons and heterocyclic carbons of monomers 2, δ = 2.04 (d, J = 124.0 Hz, 10H) corresponds to the hydrogen signals on the side chain carbons and methyl groups of monomers 1 and 2, and δ = 1.27 (d, J = 22.7 Hz, 3H) corresponds to the hydrogen signal on the dimethyl group of monomers. Comparison of the positions and areas of the characteristic peaks in the H NMR spectrum of the polymer with those of the two monomers indicates that the polythiophene derivative was successfully prepared.

[0057] The H NMR spectrum of the polymer Figure 4 Characteristic peak analysis and quantitative calculations indicate that 1,4,7,10-tetraazacyclododecane-functionalized structural units account for approximately 50% of the total structural units. This indicates that the molar ratio of quaternary ammonium salt-containing structural units to 1,4,7,10-tetraazacyclododecane-containing structural units is 1:1. Furthermore, the polymer exhibits significant yellow fluorescence emission under fluorescence excitation, and the quaternary ammonium salt groups attached to the polythiophene side chains impart good water solubility and stability in aqueous systems.

[0058] Experimental studies have shown that all amino acids containing sulfhydryl groups can bind to Cu 2+ ion affinity to coordinate the Cu 2+ The detachment from the complex allows the probe to recover fluorescence. 2+ The spectral response of the complex to Ser, Leu, Val, Lys, Ala, His, Tyr, Trp, Cys, GSH and Hcy. The polymer-Cu 2+ Fluorescence spectrum of the complex. Figure 5 (a) (b) shows that when the polymer-Cu 2+ After adding 500μM Cys, GSH and other amino acids to the complex, the fluorescence spectrum changed very little or the fluorescence recovery intensity was not high. Only Hcy caused a significant spectral change, and the fluorescence intensity at 534nm increased significantly by about 5 times. The above experimental results show that polymer-Cu 2+ The complex can selectively recognize Hcy in aqueous solution and capture Cu through the sulfhydryl group in Hcy. 2+ , thereby restoring the polymer to a fluorescent state.

[0059] Figure 6(a) shows the polymer-Cu with Hcy content ranging from 0 to 500 μM in Tris-HCl buffer solution (2 mM, pH 7.4) at an excitation wavelength of 407 nm. 2+ Fluorescence titration spectra of the complex (100 μM). As the concentration of Hcy increases, the polymer probe is 2+ The quenched fluorescence gradually recovered, and the emission peak red-shifted from 510 nm to 534 nm. When the Hcy concentration reached 3.2 equivalents, the fluorescence intensity no longer changed significantly, indicating that the polymer-Cu 2+ The complex has been fully reacted. 2+ The fluorescence emission intensity of the complex system was measured to explore its correlation with the concentration of homocysteine ​​(Hcy). Figure 6 (b) shows that in the range of Hcy concentration 0-400 μM, polymer-Cu 2+ The fluorescence intensity change of the complex (F i -F0) / F0 shows a good linear response relationship with Hcy concentration. After linear fitting analysis, the correlation coefficient of the linear relationship reaches 0.968. Among them, F0 represents the polymer-Cu when no Hcy is added 2+ The initial fluorescence emission intensity of the complex, F i The fluorescence emission intensity of the complex after adding different concentrations of Hcy is shown in Figure 2. According to the results of fluorescence spectrum titration, the detection limit (LOD) of Hcy was determined to be 9.87nM using the standard 3σ / S method. 2+ With a lower detection limit, polythiophene derivatives were introduced as fluorescent probes into Cu 2+ The subsequent formation of a complex is beneficial for the quantitative detection of Hcy in a physiological cell environment.

[0060] Optimization of detection conditions

[0061] Biothiols participate in important physiological processes in organisms, such as redox regulation and protein folding, and exist in complex environments. As an effective tool for detecting biothiols, the detection performance (sensitivity, selectivity, response time, etc.) of fluorescent probes is affected by a variety of experimental conditions. By optimizing detection conditions, the performance of the probes can be maximized, and accurate detection of biothiols can be achieved, providing reliable data support for disease diagnosis, drug development, and other aspects. Detection results are limited by a series of detection conditions, such as reaction time, reaction temperature, pH value, probe concentration, metal ions, and other biomolecules. The effects of various detection conditions on the detection of biothiols by fluorescent probes were studied, and the detection conditions were analyzed and optimized.

[0062] Effect of pH

[0063] In order to study the application effect of polymers in actual complex environments, the polymers and polymer-Cu 2+ The fluorescence response characteristics of the complex in the pH range of 5-11. Figure 7 It shows that in the range of pH=5-11, the polymer and polymer

[0064] -Cu 2 The fluorescence intensity of the complex is basically stable, which shows that the polymer has good stability in different acid and base environments and can react with Cu 2+ The experimental data show that pH has a great influence on the reaction between Hcy and Cu. 2+ When the system is in an acidic environment (pH = 5 or 6) or a strong alkaline condition (pH = 11), even if the polymer-Cu 2+ When 500 μM homocysteine ​​(Hcy) was added to the complex system, the fluorescence intensity still could not return to the level of the free polymer. This phenomenon may be due to the excessively acidic or alkaline pH conditions that destroyed the binding of Hcy and Cu. 2+ The ability to form stable complexes makes it difficult for the polymer to 2+ In sharp contrast, when the pH value of the system was maintained in the range of 7-10, after adding 500μM Hcy, the polymer-Cu 2+ The fluorescence intensity of the complex can be almost completely restored to the initial state of the polymer. 2+ The detection and response of the complex to Hcy is preferably carried out in a neutral to weakly alkaline environment of pH = 7-10.

[0065] Impact of response time

[0066] In order to study the effect of polymer on Cu 2+ and Hcy continuous detection response time, this stage of the experiment tested polymer, polymer-Cu 2+ and polymer-Cu 2+ -Hcy fluorescence intensity stability within 100s, such as Figure 8 The experimental results show that adding Cu 2+ After that, the fluorescence of the system is rapidly quenched in a very short time, and the fluorescence of the system turns to polymer-Cu 2+ After Hcy was added to the complex, the fluorescence intensity recovered rapidly. Kinetic monitoring showed that the fluorescence intensity after recovery remained relatively stable within 100s, demonstrating the rapid response ability and signal stability of the system to Hcy. Studies on the effect of response time showed that the polymer can be used as a fluorescent probe to quickly and continuously detect Cu in aqueous solution. 2+ and Hcy.

[0067] From the above experiments, it can be seen that in Tris-HCl buffer solution (2mM, pH=7.4), adding Cu 2+ , fluorescence quenching; to polymer-Cu 2+ After adding Hcy (500 μM) to the complex (100 μM), the fluorescence intensity can be restored to the same fluorescence intensity as the polymer (100 μM). According to the data analysis, it is inferred that the polymer can continuously detect Cu 2+ The mechanism of Hcy is as follows Figure 9 As shown. Cu was introduced into the polymer aqueous solution 2+ When the system is added with enough homocysteine ​​(Hcy), the sulfhydryl group (-SH) in the Hcy molecule reacts with the Cu 2+ Has a stronger affinity, promoting Cu 2+ From polymer-Cu 2+ The complex dissociates, the polymer molecules return to a freely dispersed state, and the fluorescence signal recovers. This process reveals the dynamic equilibrium mechanism of metal ion coordination and competitive binding with thiol groups, providing a theoretical basis for the recognition mechanism of probe molecules in response to biothiols.

[0068] The above are only preferred embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A polythiophene derivative fluorescent probe, characterized in that: The detection principle of the probe is as follows: First, Cu 2+ The paramagnetism quenches the fluorescence of polythiophene derivatives, and then the biothiol reacts with Cu 2+ The stronger nucleophilic ability between them captures Cu 2+ The polythiophene derivative is separated from the complex and restored to a free state, thereby realizing the opening of the fluorescent signal; the structural formula of the polythiophene derivative is: m, n represent the relative number of the two structural units in the brackets, S, O, N, Br - 、HN、N + They are chemical elements, sulfur, oxygen, nitrogen, bromide ion, imino group and nitrogen ion.

2. A method for preparing a polythiophene derivative fluorescent probe, characterized in that: The structure of polythiophene derivatives is designed by modifying their side chains. The specific steps are as follows: S1. Synthesis of 3-(4-methyl-3-thienyloxy)propyltrimethylammonium bromide M1 600 mg (2.55 mmol) of 3-(3-bromo)propoxy-4-methylthiophene was completely dissolved in 10 mL of tetrahydrofuran. 13 mL of a 30% (mass fraction) trimethylamine aqueous solution was added and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the solvent was dried and 30 mL of tetrahydrofuran was added to the reaction flask and stirred for 40 min. A white solid precipitated and was filtered. The crude product was rinsed with tetrahydrofuran to obtain an off-white solid, i.e., monomer 1, 615.68 mg, with a yield of 82%. S2. Synthesis of 3-(4-methyl-3-thienyloxy)propyl-1,4,7,10-tetraazacyclododecane M2 3-(3-bromo)propoxy-4-methylthiophene 600 mg, 2.55 mmol, 1,4,7,10-tetraazacyclododecane 879.20 mg, 5.10 mmol and K2CO3, 3.53 g, 25.5 mmol were dissolved in 20 mL of tetrahydrofuran and reacted at 55-60°C for 48 hours; after the reaction, the mixture was cooled to room temperature, filtered to remove K2CO3, and the tetrahydrofuran in the filtrate was dried to obtain a yellow oil, which was dissolved in 20 mL of dichloromethane and extracted with water until the upper aqueous phase became clear. Anhydrous magnesium sulfate was added to the extracted organic phase and dried for 10 hours, filtered, and dried to obtain a crude product. The crude product was purified by column chromatography to obtain 3-(4-methylthiophen-3-yloxy)propyl-1,4,7,10-tetraazacyclododecane, i.e., M2, 619 mg, with a yield of 74.3%; S3. Synthesis of polythiophene derivatives M1, 90.12 mg, 0.31 mmol, M2, 100 mg, 0.31 mmol, and ferric chloride 298.6 mg, 1.84 mmol were dissolved in 20 mL of N,N-dimethylacetamide and reacted at 50°C under nitrogen for 48 h. After the reaction, the reaction solution was cooled to room temperature and concentrated by rotary evaporation to 2 mL. The solution was precipitated in 100 mL of methanol and filtered to obtain a solid after standing. The filter cake was extracted with methanol, and the ferric chloride was removed from the extraction solution and then dried to obtain 54.96 mg of a polythiophene derivative with a yield of 29%.

3. Application of a polythiophene derivative fluorescent probe in the detection of biological thiols.

4. Use of a polythiophene derivative fluorescent probe in detecting biothiols according to claim 3, characterized in that: The probe introduces Cu 2+ The application of the formed complex in the quantitative detection of Hcy in physiological cell environment.

5. Use of a polythiophene derivative fluorescent probe in detecting biothiols according to claim 4, characterized in that: The probe uses the metal displacement principle to detect Hcy, and the detection limit of Hcy is as low as 9.87 nM.

6. Use of a polythiophene derivative fluorescent probe in detecting biothiols according to claim 5, characterized in that: Polythiophene derivative fluorescent probe-Cu 2+ The detection and response of the complex to Hcy is suitable for a neutral to weakly alkaline environment with a pH of 7-10. 2+ After fluorescence quenching, the addition of Hcy restored the fluorescence intensity of the polythiophene derivative by 5 times, with a response time as low as 10 s, and the fluorescence intensity was stable within 100 s.

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