Tetraphenyl ethene carboxyl derivatives, process for their preparation and use thereof

Abstract

This invention discloses a tetraphenylethylene carboxylate derivative, its preparation method, and its applications, belonging to the technical fields of organic synthesis and electrochemiluminescence materials. The chemical formula of the derivative is TPED-(COOH). x In this invention, TPE is a tetraphenylethylene core; D is a linking group selected from single bonds, phenylene, biphenylene, or terphenylene; and x is an integer selected from 1, 2, 4, or 8. This invention prepares these compounds via Suzuki coupling and hydrolysis reactions. Experiments show that these derivatives can serve as co-reactant-free electrochemiluminescent materials, generating electrochemiluminescent signals via anodic potential scanning without the need for external co-reactants. The luminescence efficiency is related to the number of carboxyl groups, substitution positions, and molecular conjugation structure. This invention also provides an electrochemiluminescent immunosensor constructed based on these derivatives and its application in biosensoring. This invention provides a new option for the research and application of co-reactant-free ECL materials.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis and electrochemiluminescence materials technology, specifically relating to tetraphenylethylene carboxyl derivatives and their preparation methods and applications. Background Technology

[0002] Electrochemiluminescence (ECL) is an analytical technique that combines electrochemical excitation with luminescence detection. It boasts advantages such as high sensitivity, low background signal, and strong controllability, and is widely used in fields such as biological detection, environmental monitoring, and food safety inspection. Traditional ECL systems mostly rely on the synergistic effect of added co-reactants (such as tripropylamine and potassium persulfate) with the luminescent material to generate a luminescent signal. However, the efficiency of this intermolecular electron transfer is limited by the heterogeneous distribution of the co-reactant and the luminescent material, resulting in poor signal stability and high cost. Furthermore, many highly efficient co-reactants are biotoxic, limiting their application in biological systems.

[0003] Aggregation-induced emission (AIE) materials have attracted much attention due to their significantly enhanced fluorescence in the aggregated state. Tetraphenylene oxide (TPE), as a classic AIE molecule, is easily modified in structure and serves as an important framework for constructing high-performance luminescent materials. In recent years, the discovery of aggregation-induced electrochemiluminescence (AIECL) has provided new ideas for the development of novel ECL materials. However, most of the existing TPE-based ECL materials still rely on co-reactants to generate detectable signals, and their application scenarios are relatively limited. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides tetraphenylethylene carboxyl derivatives, their preparation methods, and applications, thereby providing TPE-based ECL materials that do not require external co-reactants, have high luminescence efficiency, and whose performance can be controlled through molecular design. Furthermore, it establishes an efficient preparation method for these materials, which has significant application value.

[0005] In a first aspect, the present invention relates to a tetraphenylethylene carboxyl derivative, wherein the derivative has the chemical formula TPED-(COOH). x Wherein, TPE is the tetraphenylethylene core; D is the linking group, selected from single bonds, phenylene, biphenylene, or terphenylene; x is an integer selected from 1, 2, 4, or 8.

[0006] The derivative is selected from at least one of the following structural formulas: .

[0007] Secondly, the method for preparing the tetraphenylethylene carboxyl derivative of the present invention includes the following steps: S1. A tetraphenylene precursor containing a brominated group is coupled with an arylboronic acid or borate ester containing a carboxylic acid ester group via a Suzuki coupling reaction in a mixed solvent of tetrahydrofuran and water in the presence of Pd(PPh3)4 and K2CO3 to obtain a carboxylic acid ester intermediate. S2. The carboxylic acid ester intermediate is hydrolyzed in the presence of sodium hydroxide, and then acidified with hydrochloric acid to obtain the target product.

[0008] Thirdly, the present invention provides the application of the tetraphenylethylene carboxyl derivative as a co-reactant-free electrochemiluminescent material.

[0009] The application includes the following steps: A solution containing tetraphenylethylene carboxyl derivatives is dropped onto the surface of the working electrode, dried to form a modification layer, and the working electrode is constructed. A three-electrode system is constructed by placing the working electrode, the reference electrode, and the counter electrode in an electrolyte solution; Without any added co-reactants, applying a voltage triggers the tetraphenylethylene carboxyl derivative to generate an electrochemiluminescent signal.

[0010] The working electrode is a glassy carbon electrode, a gold electrode, or a platinum electrode; the reference electrode is an Ag / AgCl electrode; the counter electrode is a platinum electrode; the electrolyte solution is a phosphate buffer solution; and the voltage is applied by cyclic voltammetry.

[0011] The pH value of the phosphate buffer solution is 7.0-9.0; the potential range of the cyclic voltammetry scan is 0-1.6V.

[0012] Fourthly, the present invention also provides a method for constructing an electrochemiluminescent immunosensor based on tetraphenylethylene carboxyl derivatives, comprising the following steps: S1. Preparation of tetraphenylethylene carboxyl derivative-antibody conjugate: Tetraphenylethylene carboxyl derivative was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then coupled with the antibody of the target analyte. After purification, tetraphenylethylene carboxyl derivative-antibody conjugate was obtained. S2. Constructing the sensing interface: Pre-treat the working electrode and fix the antigen of the target analyte on its surface; S3. Assemble the immunosensor: Incubate the working electrode treated in S2 with the tetraphenylethylene carboxyl derivative-antibody conjugate prepared in S1. The conjugate is captured onto the electrode surface through antigen-antibody specific binding. After cleaning, the electrochemiluminescent immunosensor is obtained.

[0013] The working electrode in S2 is a glassy carbon electrode, and the pretreatment includes polishing, cleaning, and electrodeposition of a gold nanolayer on its surface.

[0014] Fourthly, the present invention also provides the application of an electrochemiluminescent immunosensor based on tetraphenylethylene carboxyl derivatives in the detection of manganese superoxide dismutase; By using an immunosensor as the working electrode in a three-electrode system, quantitative or qualitative analysis of manganese superoxide dismutase can be achieved by detecting the intensity of its electrochemiluminescence signal.

[0015] The beneficial effects of this invention are: Compared with existing technologies, the derivatives of this invention, through the introduction of carboxyl groups, can generate electrochemiluminescence signals without the addition of co-reactants, simplifying the detection system and avoiding the biotoxicity problems that co-reactants may cause. By controlling the number, substitution position, and conjugated structure of the carboxyl groups, the luminescence efficiency can be adjusted. Experimental results show that increasing the number of carboxyl groups and expanding the conjugated system is beneficial to improving ECL intensity, with H4TCTPE exhibiting superior luminescence efficiency compared to the traditional ruthenium bipyridine system. The carboxyl groups on these derivative molecules can serve as both luminescent functional groups and reaction sites for coupling with biomolecules, facilitating the construction of ECL biosensors suitable for fields such as biological detection and environmental monitoring. Furthermore, this ECL system can obtain detectable signals on various working electrodes, including glassy carbon, gold, and platinum, demonstrating good electrode compatibility. This invention provides a new option for the research and application of co-reactant-free ECL materials. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments are briefly introduced below.

[0017] Figure 1 It is TPE-BP-COOH(d6-DMSO) 1 H NMR spectrum.

[0018] Figure 2 It is TPE-BP-COOH(d6-DMSO) 13 C NMR spectrum.

[0019] Figure 3 This is the mass spectrum of TPE-BP-COOH.

[0020] Figure 4 This is an ECL intensity diagram of 10 μL of 1 mmol / L TPE-BP-COOH, TPE-(BP-COOH)2, H4TCTPE, H4ETBAC, H4TCBPE, H4TCPE, and H8ETTB on a glassy carbon electrode.

[0021] Figure 5 It is the pH effect on TPED-(COOH) x The effect diagram of the ECL signal.

[0022] Figure 6 It is TPED-(COOH) x The lowest unoccupied molecular orbital (LUMO), the highest occupied molecular orbital (HOMO), and the contribution diagram of excited state orbital transitions.

[0023] Figure 7 It is (AF)TPED-(COOH) x Normalized FL and ECL spectra.

[0024] Figure 8 It is TPED-(COOH) x The luminescence mechanism and the construction process of the SOD2 immune sensor are shown in the diagram. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0026] Example 1 Synthesis of TPE-BP-COOH 822 mg of TPE-Br was dissolved in a degassed mixed solvent (tetrahydrofuran / water, 50 mL / 20 mL). Then, 4-ethoxycarbonylphenylboronic acid (427 mg), K₂CO₃ (552 mg), and Pd(PPh₃)₄ (232 mg) were added to the reaction flask under a nitrogen atmosphere. The reaction was stirred overnight at 110 °C. The reaction progress was monitored by thin-layer chromatography until completion. After removing the tetrahydrofuran under vacuum, the organic compound was extracted from water with dichloromethane. After removing the dichloromethane under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane / hexane = 1 / 4, v / v) to give compound TPE-Et.

[0027] TPE-Et (480 mg) was dissolved in a degassed mixed solvent (tetrahydrofuran / water, 10 mL / 10 mL). Then, 120 mg of sodium hydroxide was added to the reaction flask under a nitrogen atmosphere. The resulting solution was stirred at 90 °C for 12 hours under a nitrogen atmosphere. The reaction progress was monitored by thin-layer chromatography until completion. Tetrahydrofuran was removed from the mixture by rotary evaporation, and the mixture was filtered to obtain a crude solid product. This product was washed with deionized water and dried under vacuum. The crude product was purified by silica gel column chromatography (dichloromethane / hexane = 1 / 1, v / v) to give compound TPE-BP-COONa. TPE-BP-COONa was dissolved in dichloroethane, and 0.1 mol / L hydrochloric acid solution was added to adjust the pH to acidic. The resulting pale yellow solid was collected and washed with deionized water to obtain TPE-BP-COOH.

[0028] The product obtained in this embodiment was characterized structurally, and the results are as follows: 1 H NMR (600 MHz, DMSO-d6) δ 12.93 (s, 1H), 7.98 (t, J = 8.2 Hz, 2H), 7.75 (dd, J = 16.1, 8.0 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.17 – 6.98 (m,17H). (eg Figure 1 (As shown).

[0029] 13 C NMR (151 MHz, DMSO) δ 167.04, 143.43, 143.21, 143.03, 141.02, 139.93, 136.67, 131.37, 130.67, 130.60, 129.87, 129.68, 129.49, 127.91, 127.85, 127.76, 126.69, 126.59, 126.54, 126.42, 126.17, 120.63, 60.66, 14.72 (e.g.) Figure 2 (As shown).

[0030] Mass spectrometry (MS) analysis results are consistent with the target molecular weight (e.g.) Figure 3 (As shown). Characterization data confirmed that the obtained product was the target compound TPE-BP-COOH.

[0031] Example 2: Preparation of a series of tetraphenylethylene carboxyl derivatives Referring to the synthetic strategy of Example 1, other tetraphenylethylene carboxyl derivatives can be prepared by selecting a suitable bromotetraphenylene precursor and coupling it with arylboronic acids / boronic esters of different substitutions via Suzuki coupling, followed by hydrolysis and acidification. Specifically: (1) Synthesis of compound TPE-(BP-COOH)2 Using 1,2-bis(4-bromophenyl)-1,2-diphenylethylene as a raw material, a Suzuki coupling reaction was carried out with 4-ethoxycarbonylphenylboronic acid to obtain a tetraphenylethylene-bis(biphenylcarboxylate) intermediate; the intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product TPE-(BP-COOH)2.

[0032] (2) Synthesis of compound H4TCTPE Using tetra(4-bromophenyl)ethylene as a starting material, a Suzuki coupling reaction was carried out with 4-ethoxycarbonylphenylboronic acid to obtain a tetraphenylethylene-tetra(phenylcarboxylic acid ester) intermediate; the intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product H4TCTPE (4,4,4,4-(ethylene-1,1,2,2-tetramethylene)tetraphenyl([1,1′:4′,1′′-triphenyl]-4-carboxylic acid)).

[0033] (3) Synthesis of compound H4TCBPE Using tetra(4-bromophenyl)ethylene as a starting material, a Suzuki coupling reaction was carried out with 4-ethoxycarbonylbiphenylboronic acid to obtain a tetraphenylethylene-tetra(biphenylcarboxylic acid ester) intermediate; the intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product H4TCBPE (4′-[1,2,2-tris(4′-carboxy[1,1′-biphenyl]-4-yl)vinyl][1,1′-biphenyl]-4-carboxylic acid).

[0034] (4) Synthesis of compound H4TCPE Using tetra(4-bromophenyl)ethylene as a raw material, a Suzuki coupling reaction was carried out with 4-ethoxycarbonylphenylboronic acid to obtain a tetraphenylphenyl-tetra(phenylcarboxylic acid ester) intermediate; the intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product H4TCPE (1,1,2,2-tetra(4-carboxyphenyl)ethylene).

[0035] Note: The structural difference between H4TCPE and H4TCTPE lies in the different connecting groups. In the former, the carboxyl group is directly connected to the benzene ring, while in the latter, the carboxyl group is connected through a biphenyl group.

[0036] (5) Synthesis of compound H8ETTB Using tetra(4-bromophenyl)ethylene as a starting material, a Suzuki coupling reaction was carried out with 3,5-diethoxycarbonylphenylboronic acid to obtain a tetrastyrene-octa(phthalate) intermediate; this intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product H8ETTB (4′,4′′′,4′′′′′,4′′′′′′′-(ethylene-1,1,2,2-tetramethylene)tetra([1,1′-biphenyl]-3,5-dicarboxylic acid)).

[0037] (6) Synthesis of compound H4ETBAC Starting with tetra(4-bromophenyl)ethylene, a Suzuki coupling reaction was carried out with 3-ethoxycarbonylphenylboronic acid to obtain a tetraphenylethylene-tetra(me-phenylcarboxylic acid ester) intermediate; the intermediate was hydrolyzed in the presence of sodium hydroxide and then acidified with hydrochloric acid to obtain the target product H4ETBAC (4,4′,4′′,4′′′-tetra(3-carboxyphenyl)tetraphenyl).

[0038] Example 3: ECL performance test of tetraphenylethylene carboxylated derivatives without co-reactants 1. Electrochemiluminescence properties of tetraphenylethylene with carboxyl groups (1) TPE-BP-COOH, TPE-(BP-COOH)2, H4TCTPE, H4ETBAC, H4TCBPE, H4TCPE, and H8ETTB were selected as luminescent materials and dissolved in deionized water to prepare a solution of 1×10 -6 mol / L ~ 1×10 -3 0.1 mol / L solution. Preparation of phosphate buffer: Weigh 3.4 g of potassium dihydrogen phosphate and 5.7 g of dipotassium hydrogen phosphate, and sonicate them into 250 mL volumetric flasks to obtain 0.1 mol / L solutions. Prepare 0.1 mol / L phosphate buffer solutions (PBS) with different pH values ​​using different volumes of potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions.

[0039] Glassy carbon, gold, and platinum electrodes were used as working electrodes. Al₂O₃ powder was used to polish the surface of musk bark until a mirror-smooth finish was achieved. The surface was then ultrasonically cleaned 2-3 times with deionized water and anhydrous ethanol. 10 μL of a 1×10⁻⁶ solution was then dropped onto the surface of each working electrode after nitrogen drying. -3Solutions of TPE-BP-COOH, TPE-(BP-COOH)2, H4TCTPE, H4ETBAC, H4TCBPE, H4TCPE, and H8ETTB at 1 mmol / L were prepared and the electrodes were dried before use. 15 mL of PBS solution was added to a 30 mL quartz electrolytic cell. Electrochemiluminescence signals were detected in a three-electrode system using Ag / AgCl as the reference electrode, a platinum column electrode as the counter electrode, and a glassy carbon electrode as the working electrode. At the same concentration, the ECL intensity order of 1 mmol / L TPE-BP-COOH, TPE-(BP-COOH)2, H4TCTPE, H4ETBAC, H4TCBPE, H4TCPE, and H8ETTB was as follows: Figure 4 As shown, the ECL signal gradually strengthens with the increase of the number of carboxyl groups on the tetraphenylethylene derivative. The position of the carboxyl groups also affects the self-ECL of the tetraphenylethylene derivative; the ECL at the para position is significantly stronger than that at the meta position. The ECL intensity gradually increases with the increase of the amount of benzoic acid on the TPE. Furthermore, the π-electron conjugation range on the tetraphenylethylene also affects the ECL signal; the ECL signal gradually strengthens with the increase of the π-conjugation system.

[0040] Next, PBS solutions with pH values ​​of 5, 6.0, 7.0, 8.0, and 9.0 were prepared. 10 μL of 1 mmol / L TPE-BP-COOH, TPE-(BP-COOH)2, H4TCTPE, H4ETBAC, H4TCBPE, H4TCPE, and H8ETTB were added to a clean glassy carbon electrode and used as the working electrode. Electrochemiluminescence signals were detected in a three-electrode system using Ag / AgCl as the reference electrode and a platinum electrode as the counter electrode. The scan potential range for electrochemiluminescence and cyclic voltammetry was 0–1.6 V, and the scan rate was set to 100 mV / s. TPED-(COOH) at different pH values ​​was also analyzed. x Electrochemiluminescence such as Figure 5 As shown. From Figure 5 As can be seen from this, all TPED-(COOH) under alkaline conditions x A clear ECL signal can be obtained in both cases.

[0041] 2. Analysis and performance verification of the electrochemiluminescence self-luminescence mechanism After establishing TPED-(COOH) xFollowing the efficient self-ECL system, we attempted to elucidate its underlying mechanism. A key question was whether the luminescence process depended on common external co-reactants (such as dissolved oxygen or electrolyte components) or was driven by an intramolecular pathway involving the functional groups themselves. Through control experiments involving adjusting the oxygen and deionized water content, changing the electrolyte, and substituting the functional groups of the TPE derivative, we concluded that TPED-(COOH)... x The self-ECL originates from the intramolecular charge transfer between the carboxyl group and the TPE core.

[0042] To further elucidate the mechanism underlying the observed self-ECL performance trends, the key component TPED-(COOH) is analyzed. x Density functional theory (DFT) calculations were performed on the derivatives. The molecular ground-state geometry was optimized at the B3LYP / 6-31+G(d) basis set level, and the energy level difference (ΔE) between its frontier orbitals and the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) was analyzed, thus establishing the correlation between the molecular structure and self-ECL-related electronic properties. Figure 6 Frontier molecular orbital analysis reveals a consistent and physically significant pattern: for all derivatives, the electron cloud of the HOMO is primarily concentrated on the π-conjugated backbone of the central TPE; in contrast, the electron density of the LUMO is significantly delocalized to the carboxyl-substituted benzene ring region. This obvious spatial separation highlights the strong electron-withdrawing property of the carboxyl group, i.e., the carboxyl group preferentially stabilizes the LUMO energy level. With the increase in the number of para-substituted benzoic acid groups on the TPE benzene ring, the LUMO-HOMO energy level difference shows a continuous decreasing trend (from 4.41 eV to 3.46 eV). Similar patterns were observed in the H4ETBAC and H8ETTB derivatives: the increase in the number of meta-carboxyl groups on the conjugated benzene ring of the TPE derivatives is positively correlated with the narrowing of the energy level difference and the enhancement of the ECL signal. The underlying mechanism of this phenomenon is closely related to the electron-withdrawing effect of the carboxyl group: while lowering the LUMO energy level of the TPE molecule, the carboxyl group also plays a fine-tuning role in the HOMO energy level, ultimately narrowing the HOMO-LUMO energy level difference and thus improving electron transfer efficiency. Besides the number of carboxyl groups, their substitution position also significantly affects the molecular electronic structure. A direct comparison of isomers of tetracarboxylated derivatives reveals that the calculated ΔE for the para-substituted H4TCBPE is smaller than that for the meta-substituted H4ETBAC. This calculation provides a reasonable explanation for the higher electroluminescence efficiency of H4TCBPE at the electronic structure level; a smaller ΔE typically corresponds to a lower energy barrier, which facilitates the formation of more easily excited luminescent states.

[0043] Furthermore, as shown by the test results of derivatives such as H4TCPE, H4TCBPE, and H4TCTPE, expanding the range of π-conjugated systems in TPE molecules can effectively reduce TPED-(COOH). x The energy level difference is significantly reduced, resulting in a significantly enhanced electroluminescence effect. This phenomenon also indicates that the HOMO / LUMO energy levels are significantly modulated by the conjugation effect during ECL emission, especially the lower LUMO energy level, which helps to enhance the ECL signal. This provides new insights for understanding the luminescence mechanism and designing high-performance TPE-based luminescent materials. (For TPED-(COOH)) x Time-dependent density functional theory (TD-DFT) calculations confirmed that its excited states are essentially π-π. This transition is consistent with the intrinsic emission characteristics of TPE molecules.

[0044] It is particularly important to note that the electroluminescent excited state and the photoluminescent excited state of this system are fundamentally different. Comparative analysis of normalized fluorescence (FL) and ECL spectra shows that the ECL emission peaks of all derivatives exhibit a significant redshift and broadening. Figure 7 This characteristic clearly demonstrates that the ECL emission state and the photo-excited singlet excited state (S1) are not the same species. We attribute this redshift emission to an intramolecular electron transfer (ICT) process: the carboxyl radical generated by the electrochemical reaction acts on the oxidized TPE through intramolecular electron transfer, thereby forming a charge-transfer (CT) excited state. These CT excited states are usually associated with "surface state" transitions, with lower energies than the locally excited (LE) states corresponding to photoluminescence, and their emission spectra have a wider bandwidth. This spectral characteristic is highly consistent with our proposed radical annihilation luminescence pathway. Combining all the above experimental and computational results, we obtain TPED-(COOH). x The self-electrochemiluminescence mechanism, such as Figure 8 As shown.

[0045] Example 4: Constructing an electrochemiluminescence immunosensor based on H4TCTPE for sensitive detection of SOD2 The sensor construction steps are as follows Figure 8As shown, 1.0 mg of H4TCTPE, 100 μL of 400 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) solution, and 100 μL of 100 mmol / L N-hydroxysuccinimide (NHS) solution were mixed in 300 μL of PBS (pH 7.4) and activated at room temperature for 30 minutes. 500 μL of manganese superoxide dismutase (SOD2) antibody solution (Ab, 10 μg / mL) was added to the activated mixture, and the mixture was mixed and reacted with gentle shaking at 4 °C for 12 hours. After the reaction, the mixture was centrifuged at 12000 rpm for 15 minutes at 4 °C. The precipitate was washed twice with PBS (pH 7.4) to remove unreacted coupling reagents and free antibody. The resulting H4TCTPE-Ab conjugate was redispersed in 1.0 mL of PBS (pH 7.4) and stored at 4 °C protected from light.

[0046] A glassy carbon electrode (GCE) with a diameter of 4 mm was polished to a mirror finish on chamois leather using an alumina polishing powder slurry. It was then ultrasonically cleaned for 2 minutes each in ultrapure water and anhydrous ethanol, and dried under nitrogen. Using the treated GCE as the working electrode, a GCE / Au electrode was prepared by deposition in a solution containing 1% HAuCl4 at a constant potential of -0.2 V (vs. Ag / AgCl) for 15 s. 10 μL of SOD2 antigen solutions of different concentrations were added to the surface of the GCE / Au electrode, and it was incubated at 37 °C for 1 hour. The electrode surface was then gently washed three times with PBS (pH 7.4) to remove unbound antigen. Subsequently, 5 μL of 1% bovine serum albumin (BSA) solution was added to the sensing interface to block non-specific active sites for 10 min. Unreacted BSA was removed by rinsing the electrode with PBS, and 10 μL of H4TCTPE-Ab conjugate solution was added to its surface. The electrode was then incubated at 37 °C in the dark for 90 min. Finally, unbound H4TCTPE-Ab was rinsed off the electrode surface with PBS to obtain the GCE / Au / SOD2 / BSA / H4TCTPE-Ab immunosensor, which was then temporarily stored at 4 °C. A three-electrode system using Ag / AgCl as the reference electrode, a platinum column electrode as the counter electrode, and GCE / Au / SOD2 / BSA / H4TCTPE-Ab as the working electrode was subjected to cyclic voltammetry scanning from 0 V to +1.6 V at a scan rate of 0.1 V / s under a photomultiplier tube voltage setting of 800 V, with simultaneous recording of the ECL signal. A quantitative detection curve was constructed by comparing the ECL signal intensity of the sensor under different SOD2 concentrations.

[0047] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not describe all details exhaustively, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification.

Claims

1. A tetraphenylstilbene carboxy derivative, characterized in that, The derivative has a chemical formula of TPED-(COOH) x wherein TPE is a tetraphenylethylene core; D is a linking group selected from a single bond, a phenylene group, a biphenylene group, or a terphenylene group; and x is an integer selected from 1, 2, 4, or 8.

2. The tetraphenylstyrene carboxyl derivative according to claim 1, characterized by The derivative is selected from at least one of the following structural formulas: 。 3. Process for the preparation of tetraphenylstyrene carboxy derivatives as claimed in claim 1 or 2, characterized in that, Includes the following steps: S1. A tetraphenylene precursor containing a brominated group is coupled with an arylboronic acid or borate ester containing a carboxylic acid ester group via a Suzuki coupling reaction in a mixed solvent of tetrahydrofuran and water in the presence of Pd(PPh3)4 and K2CO3 to obtain a carboxylic acid ester intermediate. S2. The carboxylic acid ester intermediate is hydrolyzed in the presence of sodium hydroxide, and then acidified with hydrochloric acid to obtain the target product.

4. The application of the tetraphenylethylene carboxyl derivative according to claim 1 or 2 as a co-reactant-free electrochemiluminescent material.

5. Use according to claim 4, characterized in that, Includes the following steps: A solution containing tetraphenylethylene carboxyl derivatives is dropped onto the surface of the working electrode, dried to form a modification layer, and the working electrode is constructed. A three-electrode system is constructed by placing the working electrode, the reference electrode, and the counter electrode in an electrolyte solution; Without any added co-reactants, applying a voltage triggers the tetraphenylethylene carboxyl derivative to generate an electrochemiluminescent signal.

6. Use according to claim 5, characterized in that, The working electrode is a glassy carbon electrode, a gold electrode, or a platinum electrode; the reference electrode is an Ag / AgCl electrode; the counter electrode is a platinum electrode; the electrolyte solution is a phosphate buffer solution; and the voltage is applied by cyclic voltammetry.

7. Use according to claim 6, characterized in that, The pH value of the phosphate buffer solution is 7.0-9.0; the potential range of the cyclic voltammetry scan is 0-1.6V.

8. A method for constructing a tetraphenyl ethene carboxyl derivative-based electrochemiluminescence immunosensor, characterized in that, Includes the following steps: S1. Preparation of tetraphenylethylene carboxyl derivative-antibody conjugate: Tetraphenylethylene carboxyl derivative was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, and then coupled with the antibody of the target analyte. After purification, tetraphenylethylene carboxyl derivative-antibody conjugate was obtained. S2. Constructing the sensing interface: Pre-treat the working electrode and fix the antigen of the target analyte on its surface; S3. Assemble the immunosensor: Incubate the working electrode treated in S2 with the tetraphenylethylene carboxyl derivative-antibody conjugate prepared in S1. The conjugate is captured onto the electrode surface through antigen-antibody specific binding. After cleaning, the electrochemiluminescent immunosensor is obtained.

9. The method for constructing a tetraphenylstilbene carboxyl derivative-based electrochemiluminescent immunosensor according to claim 8, characterized by, The working electrode in S2 is a glassy carbon electrode, and the pretreatment includes polishing, cleaning, and electrodeposition of a gold nanolayer on its surface.

10. Application of an electrochemiluminescence immunosensor based on tetraphenylethylene carboxyl derivative in the detection of manganese superoxide dismutase: The immunosensor is used as the working electrode in a three-electrode system. The quantitative or qualitative analysis of manganese superoxide dismutase is achieved by detecting the intensity of its electrochemiluminescence signal.

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