A fluorescence / electrochemical dual-mode probe for detecting copper ions, its preparation method and application

Abstract

This invention belongs to the field of organic probe molecular analysis and detection technology, and discloses a fluorescence / electrochemical dual-mode probe for detecting copper ions (Cu2+), its preparation method, and its application. The probe's molecular structure is as follows: Under argon protection and an ice-salt bath, halogenated hydroxyl chloride and pyridinecarboxyl chloride hydrochloride undergo an esterification reaction in anhydrous dichloromethane solvent catalyzed by triethylamine. The target product is obtained after extraction, drying, vacuum concentration, and column chromatography purification. This probe combines "on-screen" fluorescence detection with ratiometric electrochemical detection, enabling highly sensitive and selective quantitative analysis of free Cu2+ in complex biological samples such as dialysis fluid from Alzheimer's disease (AD) model mice. Compared to existing detection technologies, this probe has advantages such as high selectivity, low detection limit, good stability, and accurate dual-signal cross-validation results, providing a reliable tool for the accurate detection of Cu2+ in biological samples.

Description

Technical Field

[0001] This invention relates to the field of organic probe technology for analytical detection, specifically to a Cu probe that integrates "on" fluorescence and ratiometric electrochemical dual modes. 2+ The detection probe was applied to complex biological samples, such as the dialysis fluid of the cerebral cortex of Alzheimer's disease (AD) model mice, to detect free Cu. 2+ Quantitative analysis provides technical support for research on the pathogenesis of neurodegenerative diseases and evaluation of treatment efficacy. Background Technology

[0002] Copper ions (Cu) 2+ Cu is an essential trace metal ion in mammals, participating in various key physiological processes such as mitochondrial energy metabolism, superoxide dismutase-mediated antioxidant defense, and neural regulation. 2+ It has redox activity, and it is active in Cu 2+ / Cu + The cycle between them can catalyze Fenton-like reactions to produce reactive oxygen species (ROS), when Cu in the body 2+ When the steady-state equilibrium is disrupted, it can trigger oxidative damage to biological macromolecules, while Cu... 2+ It can specifically bind to histidine residues of β-amyloid protein (Aβ), promoting Aβ misfolding and aggregation, and is one of the core pathogenic factors of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease.

[0003] Achieving free Cu in biological samples 2+ Highly sensitive and selective quantitative detection is of great significance for elucidating the pathogenesis of Alzheimer's disease (AD) and evaluating the efficacy of related treatments. Existing Cu 2+ The detection methods have many limitations: inductively coupled plasma mass spectrometry and atomic absorption spectrometry are highly sensitive, but require destructive sample pretreatment, are expensive, and lack spatiotemporal resolution; traditional fluorescent probes for Cu 2+ Cu has poor selectivity and is easily interfered with by other metal ions. 2+ Paramagnetism can easily lead to fluorescence quenching; single-signal output electrochemical methods are easily affected by electrode preparation and environmental fluctuations, limiting detection accuracy; although fluorescence-electrochemical dual-mode sensors are a development trend, traditional designs often use dual-probe systems, which have problems such as cumbersome synthesis and signal deviation.

[0004] Therefore, there is an urgent need in this field to develop a single-probe-based, highly stable, selective, and accurate fluorescence / electrochemical dual-mode Cu... 2+ Detection materials for Cu in complex biological samples 2+ Precise quantification. Summary of the Invention

[0005] In view of the current state of technology, the purpose of this invention is to provide a method for detecting Cu. 2+ The fluorescent / electrochemical dual-mode probe (RP), its preparation method and application, solve the problems of poor selectivity, low sensitivity and susceptibility to interference of single signals in existing detection methods.

[0006] To achieve the above objectives, the present invention designs a single-probe dual-mode sensing platform, using Re as the dual-signal reporting unit and pyridine carboxylate groups as Cu. 2+ Response unit, utilizing Cu 2+ The specific catalytic hydrolysis of the pyridine carboxylate ester bond releases free halogenated esters, enabling synergistic detection by "on-hook" fluorescence and ratiometric electrochemistry. Combined with dual-mode signal cross-validation, interference is effectively eliminated, providing a method for detecting Cu in complex biological samples. 2+ It provides an ideal solution for accurate detection.

[0007] The specific technical solution is as follows: (1) Design of dual-mode probe (RP)

[0008] The core design of this invention is to esterify the hydroxyl group of halogen with pyridine carboxylic acid to construct a halogen-pyridine carboxylic acid ester conjugate (RP). The design is based on the following: Signal reporting unit: Re is the preferred dual-signal reporting unit. Re has excellent fluorescence characteristics (emission peak at 594 nm when excitation wavelength is 532 nm) and also has clear electrochemical oxidation behavior (characteristic oxidation peak at -0.28 V), which can simultaneously meet the requirements of fluorescence and electrochemical detection. After esterification, the fluorescence of Re is quenched due to the increased gap between molecular orbital energy levels, and the electrochemical signal is also shifted (characteristic oxidation peak at -0.17 V), laying the foundation for signal "on".

[0009] Response unit: preferably pyridine carboxylate group as 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, which in turn catalyzes the hydrolysis of the ester bond, releasing free halogen and restoring its fluorescence and electrochemical activity. This response mechanism is an active group sensing mechanism, which can effectively avoid cross-reactions with other metal ions and significantly improve detection selectivity.

[0010] The molecular structure of the dual-mode probe (RP) is as follows:

[0011] (2) Preparation method of dual-mode probe (RP) a. Reaction raw materials

[0012] The following raw materials were used: Re, pyridine carboxyl chloride hydrochloride (PC), triethylamine (Et3N), anhydrous dichloromethane (DCM), ice-cold 0.1 M hydrochloric acid, saturated sodium bicarbonate solution, saturated saline solution, and anhydrous sodium sulfate. All raw materials were of analytical grade and did not require further purification; they could be used directly in the reaction. b. Reaction process

[0013] S1: Dissolve the test halothane in anhydrous dichloromethane under argon protection, cool it in an ice-salt bath, add triethylamine dropwise, and stir continuously. S2: Pyridine carboxyl chloride hydrochloride was slowly added to the solution obtained in S1, and the reaction was stirred under ice-salt bath conditions. The reaction progress was monitored by thin-layer chromatography (TLC). S3: After the reaction is complete, the reaction is extinguished, and the organic phase is separated after standing. The organic phase is washed, dried, and concentrated under reduced pressure to remove the solvent, yielding a crude product. The crude product is purified to obtain a red solid powder RP.

[0014] (3) Cu based on RP 2+ Dual-mode detection method This invention utilizes the structural characteristics of RP probes to construct a dual-mode detection system that combines "on" fluorescence detection with ratiometric electrochemical detection. The two detection methods share similar reaction conditions, enabling cross-validation of detection results and significantly improving detection accuracy.

[0015] 1. "Open-type" fluorescence detection method ① Solution preparation

[0016] S1: Prepare 20 mM HEPES buffer (pH 7.4, containing 2% acetonitrile, v / v). This buffer is the optimal substrate for detection and can improve probe solubility and reaction efficiency. S2: Dissolve RP 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 8.0 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 steps

[0017] S1: Take equal volumes of RP 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 37°C water bath for 5 minutes to ensure Cu 2+ The catalytic ester bond hydrolysis reaction proceeds completely; S2: Using a fluorescence spectrophotometer, set the excitation wavelength to 532 nm, and the excitation and emission slit widths to 5 nm. Scan the emission spectrum in the range of 550~800 nm, and focus on recording the fluorescence intensity at 594 nm (F1). S3: Cu-free 2+ The probe solution was used as a blank control, and the blank fluorescence intensity (F0) was detected and recorded following the same procedure. S4: Cu 2+ A standard curve was plotted with concentration on the x-axis and (F1-F0) on the y-axis. The linear equation for the low concentration range (40 nM~1.0 μM) was F1–F0 = 0.19459c + 1.95858 (R²). 2 =0.998), and the detection limit is calculated to be 19 nM according to the formula LOD=3σ / slope (σ is the standard deviation of the blank signal); S5: 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

[0018] The fluorescence detection mode exhibits excellent sensitivity and selectivity: the detection limit is as low as 19 nM, which can meet the requirements for low concentrations of free Cu in biological samples. 2+ The testing needs for K; + Na + Ca 2+ Mg 2+ Fe 3+ Zn 2+ Other metal ions show no obvious response, and even in the presence of 10 times the concentration of interfering ions, the fluorescence intensity change is <5%, demonstrating good selectivity and effectively avoiding interference from biological matrices. The detection signal is stable in the pH range of 5.5 to 8.5, matching the physiological pH of biological samples, and the reaction reaches equilibrium in just 5 minutes, resulting in high detection efficiency.

[0019] 2. Ratio-type electrochemical detection method ① Electrode preparation and pretreatment

[0020] S1: Pretreatment of carbon fiber microelectrode (CFME): The bare CFME is polished with alumina powder, ultrasonically cleaned with ultrapure water, and dried with nitrogen in sequence for later use; S2: Preparation of CFME / CNT modified electrode: Single-walled carbon nanotubes were dispersed in ethanol and sonicated for 30 min to form a uniform dispersion (concentration 0.5 mg / L). -1 ), 5 μL of dispersion was drop-coated onto the pretreated CFME surface using the dip-coating method, and then allowed to air dry for later use; carbon nanotube modification can increase the specific surface area of ​​the electrode, improve the conductivity and electrocatalytic performance of the electrode, and promote electron transfer between the probe and the electrode. S3: Electrode Activation: Before use, perform cyclic voltammetry (CV) scans on the CFME / CNT modified electrode in 20 mM HEPES buffer (pH 7.4), with a scan range of -0.5 to 0.1 V and a scan rate of 50 mV s. -1 Activation is completed after scanning 20 times. ② Solution preparation and incubation

[0021] The buffer used was consistent with that used for fluorescence detection, namely 20 mM HEPES buffer (pH 7.4), to ensure uniform detection conditions; 10 μM RP was reacted with different concentrations (0~4.0 μM) of Cu. 2+ The standard solution and the sample to be tested are mixed in this buffer solution and incubated at 37°C for 5 min to ensure that the probe and Cu are in contact. 2+ Fully react. ③ Testing steps

[0022] S1: Immerse the activated CFME / CNT electrode into the probe-Cu 2+ In the mixed solution, adsorption was carried out at room temperature for 5 min to allow the reaction products to be fully adsorbed onto the electrode surface; after removing the electrode, it was rinsed with HEPES buffer for 1 min to remove unadsorbed substances from the electrode surface and avoid interference with the detection signal. S2: Electrochemical detection is performed using a three-electrode system, with the working electrode being the CFME / CNT (i.e., CFME / CNT / RP or CFME / CNT / RP+Cu) after adsorption of the product. 2+ The reference electrode was an Ag / AgCl electrode saturated with KCl, and the counter electrode was a platinum wire; the test was performed using differential pulse voltammetry (DPV) on an electrochemical workstation. S3: DPV test parameter settings: potential range -0.55~0.05V, pulse amplitude 50 mV, pulse width 50 ms; record the current values ​​j at -0.17 V (characteristic oxidation peak of RP) and -0.28 V (characteristic oxidation peak of free Re) during the test. -0.17V and j -0.28V ; S4: Calculate the current ratio of the two characteristic peaks (j -0.28V / j -0.17V ), with Cu 2+ A standard curve was plotted with concentration on the x-axis and current ratio on the y-axis. The linear regression equation for the low concentration range (40 nM–1.0 μM) was j1-j0 = 0.00333c -1.22221 (R²). 2 =0.99158), the calculated detection limit is 7.2 nM; S5: 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. ④ Detection performance

[0023] The electrochemical detection mode features high sensitivity, high stability, and strong practicality: the detection limit is as low as 7.2 nM, which is superior to fluorescence detection, and it can achieve ultra-trace Cu detection. 2+ Quantitative analysis was performed; the characteristic peak potential difference between RP and free Re reached 0.11 V, with no signal overlap, eliminating the need for complex peak segmentation and simplifying data calculation; the electrode exhibited excellent stability, with peak current retention >60% after 500 consecutive CV scans, and a relative standard deviation (RSD) of 3.2% after 30 days of storage at room temperature; electrodes prepared in different batches were used to detect the same concentration of Cu. 2+ The reproducibility RSD is 4.2%, which meets the requirements for batch detection; it has excellent selectivity for common metal interfering ions, and the change in current ratio is <5% in the presence of interfering ions.

[0024] (4) Cu in the cerebral cortex dialysate of AD model mice 2+ Detection applications The RP of this invention was applied to the free Cu in the dialysis fluid of the cerebral cortex of AD model mice. 2+ The dual-mode quantitative detection validated the applicability of the probe in complex biological samples, providing reliable data for the study of AD pathological mechanisms. ① Sample pretreatment

[0025] S1: Animal preparation: AD model mice (APP / PS1 double transgenic mice, 8 months old) and wild-type (WT) mice (same age) were selected as experimental subjects, with 3 mice in each group; S2: Microdialysis probe implantation: Mice were anesthetized with isoflurane (induction concentration 5%, maintenance concentration 2-3%), fixed in a stereotaxic instrument, and the skull was exposed. A hole was drilled at the cerebral cortex coordinates (anterior fontanelle AP: +1.0 mm, lateral ML: ±1.5 mm, depth DV: −1.8 mm) 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 20 mM HEPES buffer (pH 7.4) as the perfusion medium, the perfusion flow rate was controlled at 2 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 at 4℃ for 10 min to remove impurities and stored at -80℃ to avoid Cu. 2+ Loss. ② Dual-mode detection

[0026] The above-mentioned "on-off" fluorescence detection method and ratiometric electrochemical detection method were used respectively to detect Cu in the collected cortical dialysate. 2+Concentration detection: Mix 200 μL of dialysate with 1800 μL of 10 μM RP probe working solution, incubate at 37℃ for 5 min, and then perform fluorescence and electrochemical detection respectively. Calculate Cu based on their respective standard curves. 2+ Concentration. Detection results showed that Cu in the cerebral cortex dialysate of AD mice was measured by fluorescence method. 2+ The concentration was 3.60±0.32 μM in AD mice and 0.45±0.09 μM in WT mice; the electrochemical method measured 3.52±0.30 μM in AD mice and 0.44±0.10 μM in WT mice. The results of the two methods were highly consistent, and Cu in the brains of AD mice was... 2+ The concentration was approximately 8 times higher than in WT mice, and was associated with the progression of AD pathology. 2+ The mechanisms of homeostasis imbalance and increased oxidative stress levels are consistent.

[0027] ③ Recovery rate verification To verify the accuracy of the detection method, 0.3, 0.5, and 1.0 μM Cu were added to the cerebral cortex dialysate of AD mice, respectively. 2+ The standard was tested using the same detection steps, and the recovery rate was calculated: the recovery rate of the fluorescence method was 96.0±2.4%~102.0±2.9%, and the recovery rate of the electrochemical method was 98.0±4.0%~99.0±2.5%, both of which meet the accuracy requirements for biological sample detection, proving that the detection method of the present invention can effectively eliminate the interference of biological matrix and the detection results are reliable.

[0028] Advantages of this invention: The fluorescent / electrochemical dual-mode probe RP provided by this invention utilizes the dual-signal characteristics of halogenated esters and the Cu of pyridine carboxylate groups. 2+ The specific catalytic hydrolysis response enables synergistic detection of "on" fluorescence and ratiometric electrochemistry, offering the following core advantages compared to existing technologies: 1. Single probe dual-signal design: Integrating fluorescence and electrochemical signals into a single probe simplifies the synthesis process, improves biocompatibility, achieves synchronous response of dual signals, and effectively avoids signal deviation problems in dual probe systems; 2. Extremely high selectivity: based on Cu 2+ The specific catalytic hydrolysis mechanism targeting the pyridine carboxyl ester group, rather than simple coordination, affects K. + Na + Ca 2+ Fe 3+ Zn 2+ It does not respond to common metal ions and has strong resistance to interference from biological matrix. 3. High detection sensitivity: The fluorescence detection limit is 19 nM, and the electrochemical detection limit is 7.2 nM, both far lower than that of endogenous free Cu in biological samples. 2+ The concentration is sufficient to meet trace Cu requirements.2+ The quantitative demand; 4. Good stability and practicality: The probe has a stable signal in the physiological pH range (5.5~8.5), the reaction reaches equilibrium in only 5 minutes, and the detection efficiency is high; the CFME / CNT modified electrode has excellent stability, can be reused, and has good reproducibility in batch preparation; 5. Accurate and reliable test results: Dual-mode signal cross-validation effectively eliminates systematic errors and environmental interference from single methods. The recovery rate of spiked biological samples is 96.0%~102.0%, which meets the accuracy requirements of biological detection. 6. Broad application prospects: Successfully applied to Cu in the dialysis fluid of AD model mouse cerebral cortex. 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 dual-mode detection of other metal ions or biomarkers.

[0029] 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

[0030] Figure 1 The probe RP of this invention 1 1H NMR spectrum (400 MHz, CDCl3); Figure 2 The probe RP of this invention 13 C10 NMR spectrum (100 MHz, CDCl3); Figure 3 This is the high-resolution mass spectrum (HRMS) of the probe RP of this invention. Figure 4 The probe RP of this invention is reacted with different concentrations of Cu 2+ Fluorescence spectrum and linear relationship of the reaction; Figure 5 The probe RP of this invention is reacted with different concentrations of Cu 2+ DPV and linear relationship diagram of the product modified on the CFME / CNT surface after reaction; Figure 6 This is a detection diagram of the fluorescence selectivity and anti-interference of the probe RP of the present invention; Figure 7 This is a detection graph showing the selectivity and anti-interference properties of the electrochemical detection system of this invention; Figure 8 Cu in the cerebral cortex dialysis fluid of AD model mice and wild-type (WT) mice 2+Dual-mode detection results for concentration (error bar ±SD, n=3, ***p<0.001). Detailed Implementation

[0031] 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 Probe RP

[0032] In a 100 mL round-bottom flask, under argon protection, 0.50 g (2.27 mmol) of halogenated benzoyl peroxide (RP) was dissolved in 25 mL of anhydrous dichloromethane and cooled in an ice-salt bath at -10 °C. Triethylamine (0.344 g, 3.40 mmol) was added dropwise using a syringe, and after stirring for 10 min, pyridinecarboxyl chloride hydrochloride (0.526 g, 2.72 mmol) was slowly added. The reaction was maintained at -10 °C with stirring for 2 h, and the reaction was monitored by TLC until completion. The reaction was quenched with 15 mL of ice-cold 0.1 M hydrochloric acid, and the organic phase was separated. The aqueous phase was extracted three times with dichloromethane, and the organic phases were combined. The organic phase was washed successively with saturated sodium bicarbonate and saturated brine, dried overnight with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. Purification was performed by silica gel column chromatography (dichloromethane / methanol = 200:1), and the target fraction was collected and evaporated to dryness to obtain a red solid RP with a yield of 65%. 1 H NMR, 13 The product was characterized by C1NMR and HRMS, and the data are as follows: 1 H NMR(400 MHz, CDCl3) δ: 8.89-8.87 (dd), 8.32-8.30 (d), 7.99-7.95 (td), 7.89-7.86(d), 7.65-7.60 (m),7.47-7.45 (d), 7.33-7.30 (d), 6.90-6.87(dd), 6.36-6.35(d); 13 C NMR (100 MHz, CDCl3) δ: 186.34, 163.17, 153.64, 150.31, 149.29,148.50, 146.62, 144.41, 137.43, 135.24, 134.83, 131.53, 131.29, 127.95,126.22, 119.49, 110.03, 107.32; HRMS (ESI) m / z: [M + H] + The measured value of 319.0722 is in high agreement with the theoretical value of 319.0719, confirming the successful synthesis of the probe.

[0033] Example 2: Detection of Cu using fluorescence mode 2+ Performance verification Prepare 20 mM HEPES buffer (pH 7.4, containing 2% acetonitrile), prepare 10 μM RP probe working solution, and Cu2O2 solutions at concentrations of 0, 0.04, 0.1, 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0 μM. 2+ Standard solution; take equal volumes of probe working solution and react them with Cu at various concentrations. 2+ The standard solutions were mixed and incubated at 37 °C for 5 min; the fluorescence spectrophotometer was set to λ. ex = 532 nm, the emission spectrum from 550 to 800 nm was scanned, and the fluorescence intensity at 594 nm was recorded. The results show that with Cu 2+ With increasing concentration, the fluorescence intensity at 594 nm significantly increased. The linear equation for the low concentration range (40 nM~1.0 μM) was F1–F0 = 0.19459c + 1.95858 (R0). 2 =0.998), detection limit 594 nm; selectivity experiments show that only Cu 2+ It can induce a significant fluorescence response, and other metal ions (K) + Na + Ca 2+ (etc.) No obvious interference; anti-interference experiments show that the fluorescence intensity change is <5% in the presence of 10 times the concentration of interfering ions, proving that the mode has excellent selectivity and anti-interference ability.

[0034] Example 3: Electrochemical mode detection of Cu 2+ Performance verification Single-walled carbon nanotubes were dispersed in ethanol and sonicated for 30 min to obtain 0.5 mg L. -1 The dispersion was dip-coated onto the pretreated CFME surface and air-dried to obtain the CFME / CNT electrode; a 20 mM HEPES buffer (pH 7.4) was prepared, and 10 μM RP was reacted with 0, 0.04, 0.1, 0.5, 1.0, 2.0, 3.0, and 4.0 μM Cu, respectively. 2+ Mix and incubate at 37 ℃ for 5 min; immerse the CFME / CNT electrode in the mixture for 5 min for adsorption, rinse, and then perform DPV testing, with a potential range of -0.55~0.05 V. Results show that without Cu... 2+ At -0.17 V, a characteristic oxidation peak of RP appears; with the addition of Cu 2+ Afterwards, the peak current at -0.17 V decreases, and the characteristic oxidation peak of free Re appears at -0.28 V, and it increases with Cu. 2+ Concentration gradually increases; current ratio j -0.28V / j -0.17V With Cu2+ The concentration showed a good linear relationship, and the linear regression equation for the low concentration range (40 nM–1.0 μM) was j1-j0 = 0.00333c -1.22221 (R²). 2 =0.99158), detection limit 7.2 nM; to 5 μM Cu 2+ Adding 10 times the concentration of interfering ions to the solution resulted in a change of less than 5% in the current ratio, demonstrating the excellent selectivity of this mode.

[0035] Example 4: Cu in the cerebral cortex dialysis fluid of AD model mice 2+ Detection Three 8-month-old APP / PS1 double transgenic AD model mice and three 8-month-old WT mice were selected. After anesthesia, microdialysis probes were implanted into the cerebral cortex. After 24 h of recovery, the mice were perfused with 20 mM HEPES buffer at a flow rate of 2 μL / min. The dialysate was collected and stored at -80 ℃. 200 μL of the dialysate was mixed with 1800 μL of 10 μM RP probe working solution and incubated at 37 ℃ for 5 min. Fluorescence and electrochemical detection were then performed. The Cu content of the AD mice was measured by fluorescence. 2+ The concentration was 3.60±0.32 μM in AD mice and 0.45±0.09 μM in WT mice; the electrochemical method yielded 3.52±0.30 μM in AD mice and 0.44±0.10 μM in WT mice, with consistent results from both methods; 0.3, 0.5, and 1.0 μM Cu were added to the dialysate of AD mice. 2+ The standard samples showed recoveries of 96.0±2.4% to 102.0±2.9% by fluorescence method and 98.0±4.0% to 99.0±2.5% by electrochemical method, demonstrating the accuracy and reliability of the detection methods.

[0036] 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 fluorescence / electrochemical dual-mode probe, characterized in that, The molecular structure of the probe is as follows: 。 2. The method for preparing the fluorescence / electrochemical dual-mode probe as described in claim 1, characterized in that, This can be achieved through the following steps: a. Under argon protection, dissolve the halogenated amine in anhydrous dichloromethane, cool it in an ice-salt bath, and add triethylamine while stirring; b. Slowly add pyridinecarboxyl chloride hydrochloride to the solution obtained in step a, and stir the reaction under ice-salt bath conditions. Monitor the reaction progress by thin-layer chromatography. c. After the reaction is complete, the reaction is quenched, and the organic phase is separated after standing. The organic phase is washed, dried, and concentrated under reduced pressure to remove the solvent, yielding a crude product. The crude product is purified to obtain the target probe RP.

3. The method for preparing a fluorescence / electrochemical dual-mode probe according to claim 2, characterized in that, The molar ratio of the halogenated triethylamine and pyridinecarboxyl chloride hydrochloride is 1:1.5:1.

2.

4. The fluorescence / electrochemical dual-mode probe as described in claim 1 in Cu 2+ Applications in detection.

5. The fluorescence / electrochemical dual-mode probe as described in claim 4 in Cu 2+ Its application in detection is characterized by, It was used in the cerebral cortex dialysis fluid of Alzheimer's disease model mice. 2+ Quantitative detection of Cu in body fluids or environmental water samples 2+ Quantitative detection.

6. The fluorescence / electrochemical dual-mode probe as described in claim 4 in Cu 2+ Its application in detection is characterized by, The fluorescence detection method includes the following steps: a. Preparation of the detection system: Using pH 7.4 buffer as a base, prepare the RP probe working solution and a series of Cu concentrations. 2+ Standard solution; b. Sample incubation: Take the RP probe working solution and react it with Cu... 2+ The standard solution and the sample to be tested are mixed at a volume ratio and incubated at a constant temperature. c. Fluorescence detection: Using a fluorescence spectrophotometer, with the excitation wavelength set to 532 nm, the emission spectrum from 550 to 800 nm was scanned, and the fluorescence intensity F1 at 594 nm was recorded. (Note: The last part, "without Cu," appears to be an error and doesn't need a direct translation.) 2+ The probe solution was used as a blank control, and the blank fluorescence intensity F0 was recorded; d. Quantitative analysis: based on F1-F0 and Cu 2+ A standard curve was established to determine the linear relationship between Cu concentration and the sample concentration. 2+ Quantitative detection.

7. The fluorescence / electrochemical dual-mode probe as described in claim 4 in Cu 2+ Its application in ratiometric electrochemical detection is characterized by, Ratio-modulated electrochemical detection includes the following steps: a. Preparation of working electrode: Single-walled carbon nanotubes were dispersed in ethanol and sonicated. The carbon nanotube dispersion was then coated onto the surface of the pretreated carbon fiber microelectrode using a dip-coating method. The electrode was then air-dried to obtain a CFME / CNT modified electrode. b. Solution preparation and incubation: Using pH 7.4 HEPES buffer as a substrate, the RP probe was reacted with a series of Cu solutions of different concentrations. 2+ Standard solution / sample to be tested are mixed and incubated; c. Electrode adsorption: Immerse the CFME / CNT modified electrode in the above mixture for adsorption, then rinse with HEPES buffer to remove unadsorbed substances; d. Electrochemical detection: A three-electrode system is used, with CFME / CNT / RP or CFME / CNT / RP + Cu. 2+ The working electrode was an Ag / AgCl electrode saturated with KCl, the reference electrode was an Ag / AgCl electrode, and the counter electrode was a platinum wire. Differential pulse voltammetry was used for testing with an electrochemical workstation, and the characteristic oxidation peak current j of RP at -0.17 V was recorded. -0.17V The characteristic oxidation peak current of free halogenated trihalogen at -0.28 V. -0.28V ; e. Quantitative analysis: Calculate the current ratio j -0.28V / j -0.17V Based on this ratio 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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