Light-operated electrochemical molecular probe, preparation method thereof and application of light-operated electrochemical molecular probe in detection of copper ions

Abstract

The invention belongs to the technical field of organic probe molecule analysis and detection, and discloses a light-operated electrochemical molecular probe (TPMP), a preparation method thereof and application of the light-operated electrochemical molecular probe in detection of copper ions (Cu < 2 + >). The molecular structural formula of the probe is as follows: hydroquinone and 2-nitrobenzyl bromide react to prepare a compound 1, and then the compound 1 and pyridine formyl chloride hydrochloride are subjected to esterification reaction under the catalysis of triethylamine to obtain a target product. The probe realizes light-operated open-type electrochemical detection of Cu < 2 + > based on a sequential response mechanism activated by Cu < 2 + > specific recognized-365 nm ultraviolet light, has no electrochemical signal in darkness, quantitatively releases electroactive substances after light activation and generates significant response, has a detection limit as low as 30 nM, has ultrahigh selectivity on Cu < 2 + >, and can effectively resist biological matrix interference. The fluorescent probe is applied to quantitative detection of free Cu < 2 + > in a hippocampal dialysate of an AD (Alzheimer's disease) model mouse.

Description

Technical Field

[0001] This invention relates to the field of organic probe technology for analytical detection, specifically to a photocontrolled electrochemical molecular probe (TPMP) and its preparation method, which is applied to the detection of free copper ions (Cu) in complex biological samples such as dialysis fluid from the hippocampus of Alzheimer's disease (AD) model mice. 2+ The highly selective and sensitive quantitative analysis provides technical support for the study of the pathogenesis of neurodegenerative diseases and the evaluation of treatment effects. Background Technology

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide, characterized by progressive memory loss, cognitive decline, and neuronal damage. Numerous studies have shown that copper ions (Cu) in the brain... 2+ Homeostasis imbalance is one of the core pathogenic factors of AD: excessive Cu 2+ It can bind to β-amyloid protein, promoting its misfolding and aggregation to form neurotoxic oligomers and fibers; at the same time, Cu 2+ It can catalyze the Fenton reaction to produce reactive oxygen species, triggering oxidative stress, lipid peroxidation, and neuronal apoptosis. Therefore, it can achieve the goal of intracellular Cu in the brain. 2+ Precise and spatiotemporally controllable detection is of great significance for elucidating the pathogenesis of Alzheimer's disease and developing effective diagnostic and treatment strategies.

[0003] Existing Cu 2+ Among detection methods, electrochemical methods have attracted much attention due to their advantages such as high sensitivity, fast response, low cost, and easy miniaturization. However, traditional electrochemical probes for Cu... 2+ Its poor selectivity, susceptibility to interference from non-target substances in complex biological matrices, and lack of temporal and dimensional control over the detection process limit its application in in vivo analysis.

[0004] Optically controlled molecular probes, by introducing photolytic groups, enable precise temporal and dimensional control of the detection process: the probe's recognition or signal generation capability is "locked" in the dark and "unlocked" only under specific wavelengths of light, effectively avoiding interference from non-target substances in the biological environment, thus offering unique advantages in biological sample analysis. Currently reported optically controlled probes mostly focus on fluorescence detection; optically controlled molecular probes for electrochemical detection are rarely reported, and there are no probes applicable to Cu in the brains of AD model mice. 2+ The photocontrolled electrochemical probe for detection.

[0005] Therefore, there is an urgent need in this field to develop a highly selective, highly sensitive, and real-time scalable photo-controlled electrochemical Cu 2+ Detection probes for Cu in complex biological samples 2+ Precise quantification. Summary of the Invention

[0006] In view of the current state of technology, the purpose of this invention is to provide a photocontrolled electrochemical molecular probe (TPMP) and its preparation method and application, thereby solving the problem of traditional electrochemical probes being incompatible with Cu. 2+ Problems include poor selectivity, lack of time-space control, and significant interference from biological matrices.

[0007] To achieve the above objectives, the present invention designs a photocontrolled electrochemical molecular probe that integrates a 2-nitrobenzyl photolytic group and a pyridine carboxylate Cu. 2+ The recognition group and the hydroquinone (HQ) electroactive reporter group form a single molecular backbone, utilizing Cu 2+ A specific recognition-UV light activation sequential response mechanism enables Cu 2+ Photocontrolled electrochemical detection: Cu in the dark 2+ The specific coordination catalytic hydrolysis with the pyridine carboxylate group generates a stable intermediate that retains the photolytic group, with the electroactive site still locked and no electrochemical signal. Upon irradiation with 365 nm ultraviolet light, the 2-nitrobenzyl photolytic group in the intermediate undergoes irreversible cleavage, quantitatively releasing free electroactive hydroquinone, producing a significant on-type electrochemical response, and realizing Cu… 2+ High selectivity and high sensitivity detection.

[0008] The specific technical solution is as follows: (1) Design of photocontrolled electrochemical molecular probes (TPMP)

[0009] The core design of this invention is: to incorporate 2-nitrobenzyl photolytic groups and pyridine carboxylate Cu... 2+ The recognition group is covalently linked to the HQ electroactive group to construct a single-molecule photocontrolled electrochemical probe TPMP. The design is based on the following: Photolysis unit: 2-nitrobenzyl is preferred as the photolysis group. This group is structurally stable in the dark and can undergo irreversible cleavage under 365 nm ultraviolet light irradiation, realizing the "lock-unlock" regulation of the probe's electroactive sites and providing a basis for the spatiotemporal control of the detection process; Recognition unit: preferably pyridine carboxylate group is Cu 2+ The specific response unit, the pyridine N and carbonyl O in the pyridine carboxylate group, can react with Cu 2+ Specific coordination occurs, enhancing the electrophilicity of the ester carbonyl group, thereby catalyzing the hydrolysis of the ester bond. This response mechanism is active group sensing, which can effectively avoid cross-reaction with other metal ions and greatly improve detection selectivity. Electroactive reporter unit: HQ is preferred as the electroactive reporter group. HQ has excellent electrochemical oxidation activity and can generate a distinctive oxidation peak on the surface of carbon nanotube modified carbon fiber electrode (CFME / CNT). The oxidation peak current has a good linear relationship with the concentration, which meets the requirements of quantitative detection.

[0010] The molecular structure of the photocontrolled electrochemical molecular probe (TPMP) is as follows:

[0011] (2) Preparation method of photocontrolled electrochemical molecular probe (TPMP) a. Reaction raw materials

[0012] Hydroquinone, 2-nitrobenzyl bromide, anhydrous potassium carbonate, triethylamine, pyridine carboxyl chloride hydrochloride, anhydrous acetone, anhydrous dichloromethane, saturated ammonium chloride solution, saturated brine, anhydrous sodium sulfate, petroleum ether, ethyl acetate, and methanol—all raw materials are of analytical grade and require no further purification; they can be used directly in the reaction.

[0013] b. Reaction process Reaction route: S1: Synthesis of Compound 1

[0014] Hydroquinone and 2-nitrobenzyl bromide were dissolved in anhydrous acetone under a nitrogen atmosphere, and anhydrous potassium carbonate was added. The mixture was stirred at room temperature. After the reaction was completed, the solid impurities were removed by filtration. The filtrate was concentrated under reduced pressure, and the crude product was purified to obtain a pale yellow solid compound 1. S2: Synthesis of the probe TPMP

[0015] Compound 1 was dissolved in anhydrous dichloromethane under argon protection, cooled in an ice-salt bath, and triethylamine was added. A solution of pyridine carboxyl chloride hydrochloride in anhydrous dichloromethane was added dropwise with stirring. After the addition was completed, stirring was continued. The mixture was then naturally heated to room temperature and stirred to react. After the reaction was completed, the reaction was quenched, and the organic phase was separated after standing. The product was washed, dried, and concentrated under reduced pressure to obtain the crude product. After purification, the probe TPMP was obtained.

[0016] (3) Cu based on TPMP 2+ Photocontrolled electrochemical detection method This invention utilizes the structural characteristics of TPMP probes to construct Cu 2+ A specific recognition-UV-activated photocontrolled electrochemical detection system effectively eliminates biological matrix interference through a sequential response mechanism, achieving Cu... 2+ High selectivity and high sensitivity for quantitative detection. 1. Preparation before testing

[0017] ① Preparation and pretreatment of modified electrodes S1: Pretreatment of carbon fiber microelectrode (CFME): The bare CFME was polished to a mirror finish with 0.05 μm alumina powder, ultrasonically cleaned with ultrapure water 3 times (5 min each time), and dried with nitrogen gas for later use. S2: Preparation of CFME / CNT modified electrode: Single-walled carbon nanotubes were dispersed in anhydrous ethanol and sonicated for 30 min to form a uniform 0.5 mg L⁻¹ electrode. -1 The dispersion was drop-coated onto the pretreated CFME surface using a dip-coating method and then air-dried in a clean environment to obtain a CFME / CNT modified electrode. Carbon nanotube modification can increase the specific surface area of ​​the electrode, improve its conductivity and electrocatalytic performance, and promote electron transfer between the electroactive material and the electrode. S3: Electrode Activation: Before use, perform cyclic voltammetry (CV) scans on the CFME / CNT modified electrode in 0.1M HEPES buffer (pH 7.4), with a scan range of -0.3 to 0.3 V and a scan rate of 50 mV s. -1 Activation is completed after scanning 20 times. ② Preparation of the detection system

[0018] S1: Prepare 0.1 M HEPES buffer (pH 7.4). This buffer is the optimal substrate for detection, which can improve probe solubility and reaction efficiency, and at the same time match the physiological pH of biological samples. S2: Dissolve TPMP in the above HEPES buffer to prepare a 10 μM probe working solution, ensuring that the probe is uniformly dispersed; S3: Cu 2+ The standards are dissolved in the same HEPES buffer to prepare a series of standard solutions with concentrations ranging from 0 to 10 μM, which are used to construct a standard curve. The test samples need to be appropriately diluted with the same buffer to ensure that the detection concentration is within the range of the standard curve. 2. Testing Procedures

[0019] S1: Sample incubation Take equal volumes of TPMP probe working solution and react them with Cu of different concentrations. 2+ The standard solution and the sample to be tested were mixed and incubated in a constant temperature water bath at 37 ± 0.5 ℃ for 10 min to ensure Cu 2+ It fully coordinates with the pyridine carboxylate group and catalyzes the hydrolysis of the ester bond to generate a stable photoresponsive intermediate.

[0020] S2: Photoactivation Process The incubated mixture was irradiated under 365 nm ultraviolet light for 300 s (irradiance 10 mW cm⁻¹). -2 This process causes irreversible cleavage of the 2-nitrobenzyl photolysis group in the intermediate, quantitatively releasing free electroactive HQ. This step is crucial for signal “activation,” and 300 s is the optimal time for the photolysis reaction, ensuring its complete completion.

[0021] S3: Electrochemical detection The activated CFME / CNT modified electrode was immersed in the photoactivated mixture, and differential pulse voltammetry (DPV) was performed using a three-electrode system. Working electrode: CFME / CNT modified electrode; Reference electrode: Ag / AgCl electrode saturated with KCl; Counter electrode: Platinum wire electrode; Test parameters: potential range -0.3~0.3 V, pulse amplitude 50 mV, pulse width 50 ms; Record the characteristic oxidation peak current of HQ at 0.036 V, in Cu-free conditions. 2+ The probe solution was used as a blank control, and the blank peak current was detected and recorded following the same procedure.

[0022] S4: Quantitative Analysis With Cu 2+ A standard curve was plotted with concentration on the x-axis and sample peak current minus blank peak current (j1-j0) on the y-axis. The linear equation for the low concentration range (0.1~10 μM) was j1–j0(μA) = 0.021608C (μM) + 0.02993(R0). 2 =0.9914); based on the formula LOD=3σ / slope (σ is the standard deviation of the blank signal), the detection limit is calculated to be 30 nM; Perform the above steps on the sample to be tested, and calculate the Cu content in the sample based on the standard curve. 2+ The concentration. 3. Detection performance

[0023] The photocontrolled electrochemical detection system of this invention exhibits excellent selectivity, sensitivity, and stability: it is effective against K⁺, Na⁺, and Ca⁺. 2 + Mg 2+ Fe 3 ⁺、Zn 2+ Common metal ions and biomolecules show no significant response; even in the presence of 5 times the concentration of interfering ions, the peak current change is <5%, effectively avoiding interference from the biological matrix; the detection limit is as low as 30 nM, far below that of endogenous free Cu in biological samples. 2+ The concentration is sufficient to meet trace Cu requirements. 2+ The quantitative requirements are met; the CFME / CNT modified electrode exhibits high peak current retention after multiple consecutive measurements, and electrodes prepared in different batches can detect the same concentration of Cu. 2+ The relative standard deviation (RSD) is 3.2%, which meets the requirements of batch detection. The probe has no electrochemical signal in the dark and only responds after being irradiated with 365 nm ultraviolet light, realizing precise time and temperature control of the detection process and effectively avoiding non-specific interference in sample transportation and pretreatment.

[0024] (4) Cu in the dialysate of the hippocampus of AD model mice 2+ Detection applications The TPMP probe of this invention was applied to the free Cu in the dialysate of the hippocampus of AD model mice. 2+ The photocontrolled electrochemical quantitative detection verified the applicability of the probe in complex biological samples and provided reliable data for the study of AD pathological mechanisms. ① Sample pretreatment

[0025] S1: Animal preparation: 8-10 week old APP / PS1 double transgenic AD model mice and age-matched wild-type (WT) C57BL / 6J mice were selected as experimental subjects, with 3 mice in each group; S2: Microdialysis probe implantation: Mice were anesthetized with sodium pentobarbital (50 mg / kg, intraperitoneal injection), fixed in a stereotaxic instrument, and the skull was exposed. A hole was drilled in the hippocampus and a microdialysis probe (membrane length 2 mm, molecular weight cutoff 20 kDa) was implanted and fixed with dental cement. S3: Sample Collection: After probe implantation, mice were allowed to recover for 24 h. Using 0.1 M HEPES buffer (pH 7.4) as the perfusion medium, the perfusion flow rate was controlled at 1 μL / min. The dialysate from the first 30 min was discarded to maintain equilibration. Subsequently, dialysate was collected every 30 min and stored on ice. The collected dialysate was centrifuged at 10000 rpm and 4 ℃ for 10 min to remove impurities and stored at -80 ℃ to avoid Cu. 2+ Loss; dilute the dialysate 10 times with 0.1 M HEPES buffer (pH 7.4) before testing. ② Photocontrolled electrochemical detection

[0026] The diluted dialysate was mixed with an equal volume of 10 μM TPMP probe working solution, incubated at 37 °C for 10 min, and then irradiated with 365 nm UV light for 300 s. Electrochemical detection was then performed using the DPV method described above, and Cu was calculated based on the standard curve. 2+ concentration.

[0027] The test results showed that Cu in the dialysate of the hippocampus of AD model mice 2+ The concentration was 8.3±0.7 μM in WT mice and 3.4±0.8 μM in AD mice brains. 2+ The concentration was approximately 2.4 times higher than in WT mice, which is consistent with the Cu concentration during the AD pathological process. 2+ The mechanisms of homeostasis imbalance and increased oxidative stress levels are consistent. ③ Recovery rate verification

[0028] To verify the accuracy of the detection method, 1.0 μM and 3.0 μM Cu were added to the dialysate of AD and WT mice, respectively, in the hippocampus. 2+ The standard was tested using the same detection steps, and the recovery rate was calculated: the recovery rate was 97.0%~102.7%, and the relative standard deviation (RSD) was <4.0%, which meets the accuracy requirements for biological sample detection, proving that the detection method of the present invention can effectively eliminate biological matrix interference and the detection results are reliable. Advantages of this invention:

[0029] The photocontrolled electrochemical molecular probe TPMP provided by this invention utilizes Cu 2+ A specific recognition-UV light activation sequential response mechanism was implemented for Cu. 2+ The photo-controlled on-state electrochemical detection has the following core advantages compared to existing technologies: 1. Single probe with multiple functions: Integrating photolytic groups, recognition groups, and electroactive reporter groups into a single probe object simplifies the synthesis process, improves biocompatibility, and achieves integrated "recognition-activation-signal output", avoiding the signal deviation problem of multi-probe systems; 2. Ultra-high selectivity and anti-interference performance: Based on Cu 2+ The specific catalytic hydrolysis mechanism for pyridine carboxyl ester groups, rather than simple coordination, is unresponsive to common metal ions and biomolecules; at the same time, the photocontrolled sequential response mechanism effectively eliminates non-specific interference from complex biological matrices, significantly improving detection specificity. 3. High detection sensitivity: The detection limit is as low as 30 nM, which can meet the requirements for trace free Cu in biological samples. 2+ The quantitative detection needs; 4. Spatiotemporal controllability of the detection process: The electroactive sites of the probe are locked in the dark, with no electrochemical signal. The electroactive substances are released and a response is generated only under 365 nm ultraviolet light irradiation, realizing precise spatiotemporal control of the detection process, which is suitable for in situ analysis of living organisms. 5. Broad application prospects: Successfully applied to Cu in the brains of AD model mice. 2+ The detection of Cu provides a tool for studying the pathological mechanisms of neurodegenerative diseases, and can be extended to various samples such as body fluids and environmental water samples. 2+ The detection of this probe can be extended to the photocontrolled electrochemical detection of other metal ions or biomarkers.

[0030] The probe preparation method of this invention is simple and has a stable yield, and the detection method is easy to operate and has mild conditions, which has good application prospects in biomedical research, clinical diagnosis and treatment evaluation, environmental monitoring and other fields. Attached Figure Description

[0031] Figure 1Cu for the probe TPMP of this invention 2+ Schematic diagram of the response mechanism triggered by light; Figure 2 The proton NMR spectrum of the probe TPMP of this invention; Figure 3 The carbon NMR spectrum of the probe TPMP of this invention; Figure 4 This is a high-resolution mass spectrometry representation of the probe TPMP of this invention; Figure 5 The detection system of this invention is for Cu 2+ The response DPV curve and standard curve; Figure 6 Selectivity detection diagram and anti-interference detection diagram of the detection system of this invention; Detailed Implementation

[0032] To better illustrate the present invention, specific embodiments are given below. It should be noted that the following embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention. Example 1: Synthesis and characterization of compound 1 and probe TPMP

[0033] Synthesis of Compound 1: In a 100 mL round-bottom flask under a nitrogen atmosphere, HQ (1.10 g, 10.0 mmol) and 2-nitrobenzyl bromide (2.29 g, 11.0 mmol) were dissolved in 50 mL of anhydrous acetone, and anhydrous potassium carbonate (3.45 g, 25.0 mmol) was added. The mixture was stirred at room temperature for 16 h. After filtration, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1) to give compound 1 (2.12 g, yield 86%) as a pale yellow solid.

[0034] Synthesis of probe TPMP: Compound 1 (0.246 g, 1.0 mmol) was dissolved in 20 mL of anhydrous dichloromethane in a 100 mL round-bottom flask under argon protection. After cooling in an ice-salt bath at -10 °C, triethylamine (0.152 g, 1.5 mmol) was added. Then, 5 mL of anhydrous dichloromethane solution of pyridinecarboxyl chloride hydrochloride (0.369 g, 2.0 mmol) was added dropwise. The mixture was stirred at -10 °C for 4 h and then at room temperature for 8 h. The mixture was quenched with 10 mL of saturated ammonium chloride solution. The organic phase was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 100:1) to obtain yellow crystalline TPMP (0.32 g, 90% yield).

[0035] Nuclear magnetic resonance spectroscopy (NMR) 1 H NMR, 13The structures of compound 1 and probe TPMP were verified by C10 NMR and high-resolution mass spectrometry (HRMS): Compound 1: 1 H NMR (400 MHz, CDCl3) δ: 9.02 (s, 1H), 8.11-8.09 (d,1H), 7.78 (s, 2H), 7.60 (d, 1H), 6.85-6.81 (dt, 2H), 6.71-6.69 (m, 2H), 5.34 (s,2H); 13 C NMR (100 MHz, CDCl3) δ: 152.24, 151.16, 147.95, 134.31, 133.42, 129.69, 129.41, 125.15, 116.34, 116.26, 67.46; HRMS (ESI) m / z: [M+H]⁺. Theoretical value is C 13 H 12 NO4⁺ 246.0766, the measured value is 246.0768, which is highly consistent with the theoretical value.

[0036] TPMP probe: 1 H NMR (400 MHz, CDCl3) δ: 8.85-8.83 (d, 1H), 8.28-8.26 (d,1H), 8.18-8.16 (d, 1H), 7.92-7.90 (d, 2H), 7.70 (m, 1H), 7.57-7.49 (m, 1H),7.22-7.20 (d, 2H), 7.05-7.03 (d, 2H), 5.49 (s, 2H); 13 C NMR (100 MHz, CDCl3) δ: 164.12, 155.99, 150.10, 147.40, 146.82, 144.94, 137.26, 134.11, 133.65, 128.51, 128.41, 127.46, 125.85, 125.01, 122.67, 115.60, 67.24; HRMS (ESI) m / z: [M+H]⁺ Theoretical value is C 19 H 15 The measured value of N2O5⁺ was 351.0981, which is in high agreement with the theoretical value, confirming that the product structure is correct.

[0037] Example 2: Photocontrolled electrochemical detection of Cu 2+ Performance verification Preparation of modified electrode: After CFME pretreatment, 5 μL of 0.5 mg L⁻¹ electrode material was dip-coated. -1 A CFME / CNT modified electrode was obtained by naturally air-drying a single-walled carbon nanotube ethanol dispersion. Preparation of the detection system: Prepare 0.1 M HEPES buffer (pH 7.4), prepare 10 μM TPMP probe working solution, and different concentrations of Cu. 2+ Standard solution; Incubation and photoactivation: Take equal volumes of probe working solution and react them with Cu at various concentrations. 2+ The standard solutions were mixed, incubated at 37 °C for 10 min, and then irradiated with 365 nm ultraviolet light for 300 s. Electrochemical detection: DPV was measured using a three-electrode system, and the oxidation peak current at 0.036 V was recorded. The results show that with Cu... 2+ With increasing concentration, the oxidation peak current significantly increased, and a good linear relationship was observed in the range of 0.1–10 μM (R0). 2 =0.9914), detection limit 30 nM; selectivity experiments showed that only Cu 2+ It can induce a significant electrochemical response, with no obvious interference from other metal ions and biomolecules; the anti-interference experiment shows that the peak current change is <5% in the presence of 5 times the concentration of interfering ions, proving that the system has excellent selectivity and anti-interference properties.

[0038] Example 3: Cu in the dialysate of the hippocampus of AD model mice 2+ Detection Sample collection: Three APP / PS1 AD model mice and three WT mice aged 8-10 weeks were selected. After anesthesia, a microdialysis probe was implanted in the hippocampus. After 24 hours of recovery, the dialysate was collected, diluted 10 times, and used for later use. Photocontrolled electrochemical detection: An equal volume of diluted dialysate was mixed with 10 μM TPMP probe working solution, incubated at 37 ℃ for 10 min, irradiated with 365 nm UV light for 300 s, and then DPV was detected. Cu was calculated based on the standard curve. 2+ concentration; Recovery experiment: 1.0 μM and 3.0 μM Cu were added to the dialysate. 2+ The standard was tested and the recovery rate was calculated using the same procedure. Results showed that Cu in the hippocampus of AD mice... 2+ The concentration was 8.3 ± 0.7 μM for WT mice and 3.4 ± 0.8 μM for WT mice; the recovery rate was 97.0%–102.7%, and the RSD was < 4.0%, demonstrating that the detection method is accurate and reliable and applicable to Cu in complex biological samples. 2+ Quantitative detection.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A photocontrolled electrochemical molecular probe, characterized in that, The molecular structure of the probe is as follows: 。 2. The method for preparing the photocontrolled electrochemical molecular probe as described in claim 1, characterized in that, This can be achieved through the following steps: a. Synthesis of Compound 1: Hydroquinone and 2-nitrobenzyl bromide were dissolved in anhydrous acetone under a nitrogen atmosphere, and anhydrous potassium carbonate was added. The mixture was stirred at room temperature. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified to obtain Compound 1. b. Synthesis of probe TPMP: Under argon protection, compound 1 was dissolved in anhydrous dichloromethane, cooled in an ice-salt bath, and triethylamine was added. Then, an anhydrous dichloromethane solution of pyridinecarboxyl chloride hydrochloride was added dropwise. After stirring, the mixture was heated to room temperature and the reaction was continued. After the reaction was completed, the reaction was quenched, the organic phase was separated by standing, dried, concentrated under reduced pressure to obtain the crude product, and purified to obtain probe TPMP.

3. The method for preparing the photocontrolled electrochemical molecular probe according to claim 2, characterized in that, In step a, the molar ratio of hydroquinone, 2-nitrobenzyl bromide, and anhydrous potassium carbonate is 1:1.1:2.5; in step b, the molar ratio of compound 1, triethylamine, and pyridinecarboxyl chloride hydrochloride is 1:1.5:2.

0.

4. The photocontrolled electrochemical molecular probe as described in claim 1 in Cu 2+ Applications in light-controlled electrochemical detection.

5. The photocontrolled electrochemical molecular probe according to claim 4 in Cu 2+ Its application in light-controlled electrochemical detection is characterized by, It was used to add Cu to the dialysate of the hippocampus of Alzheimer's disease model mice. 2+ Quantitative detection of Cu in body fluids or environmental water samples 2+ Quantitative detection.

6. The photocontrolled electrochemical molecular probe as described in claim 5 in Cu 2+ Its application in light-controlled electrochemical detection is characterized by, Includes the following steps: a. Preparation of modified electrode: The carbon fiber microelectrode, referred to as CFME, was polished with alumina powder, ultrasonically cleaned with ultrapure water, and treated with nitrogen blowing. Single-walled carbon nanotubes were dispersed in ethanol and ultrasonically prepared to obtain a dispersion. The dispersion was then applied to the pretreated CFME surface using a dip-coating method and air-dried to obtain the CFME / CNT modified electrode. b. Preparation of the detection system: Using pH 7.4 buffer as a base, prepare the TPMP probe working solution and a series of Cu concentrations. 2 + Standard solution; The sample to be tested was diluted with the same buffer solution; c. Sample incubation and photoactivation: Take TPMP probe working solution and react it with Cu... 2+ The standard solution and the sample to be tested were mixed and incubated at a constant temperature, followed by irradiation with 365 nm ultraviolet light for 300 s; d. Electrochemical detection: A three-electrode system was used, with CFME / CNT as the working electrode, Ag / AgCl electrode saturated with KCl as the reference electrode, and platinum wire as the counter electrode. Differential pulse voltammetry was performed using an electrochemical workstation, and the characteristic oxidation peak current of hydroquinone at 0.036 V was recorded. e. Quantitative analysis: based on the oxidation peak current and Cu 2+ A standard curve was established to determine the linear relationship between Cu concentration and the sample concentration. 2+ Quantitative detection.

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