Preparation method of sulfydryl gold nano-cluster with captopril providing ligand and application of sulfydryl gold nano-cluster in electrochemical luminescence detection

Abstract

The invention relates to a preparation method of a sulfydryl gold nano-cluster with captopril providing a ligand and application of the sulfydryl gold nano-cluster in electrochemical luminescence detection, and belongs to the technical field of electrochemical luminescence detection. The invention develops and verifies that the gold nanocluster of the sulfydryl ligand is used as an electrochemical luminescence material, has good water solubility, and can react with a co-reactant of tripropylamine (TPrA) and cephalosporin to achieve an efficient electrochemical luminescence behavior. On one hand, the problems that a traditional electrochemical luminescence material is poor in water solubility and a reaction tank is not easy to clean are solved, and on the other hand, an electrochemical luminescence detection technology is applied to trace detection of ceftriaxone sodium for the first time. The ceftriaxone sodium is detected according to the acceleration effect of ceftriaxone sodium with different concentrations on electron transfer in the electrochemical luminescence process and different response intensities of electrochemical luminescence signals.

Description

Technical Field

[0001] The invention relates to a method for preparing a thiol-based nanocluster of a captopril-provided ligand and its application in electrochemiluminescence detection, belonging to the technical field of electrochemiluminescence detection. Background Art

[0002] Ceftriaxone is widely used to treat lower respiratory tract infections, urinary tract infections, gonorrhea, and surgical infections caused by susceptible pathogens. However, people are often unaware that cephalosporin residues or contamination may pose significant risks to human health, especially allergic reactions, antibiotic resistance, and intestinal flora imbalance. Therefore, the development of a real-time, sensitive, and simple method for the detection of cephalosporin antibiotics will play a vital role in safeguarding human health.

[0003] Common methods for detecting antibiotics are high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and spectrophotometry. These methods have shown their advantages and are suitable for different types of analysis, depending on the nature of the sample, but none of these techniques can meet the needs of real-time detection, high sensitivity, and simple operation.

[0004] The basic principle of electrochemiluminescence (ECL) is that the luminescent material and the co-reactant produce a redox reaction under electrical stimulation to generate unstable excited state substances, which then produce light radiation during the process of returning to the stable ground state. It is often used in immunoassays and used together with specific antigen-antibody interactions and magnetic beads to achieve extremely high sensitivity and excellent selectivity.

[0005] Traditional ECL materials generally have the following two disadvantages: (1) The luminescent material has uneven particle size and poor water solubility, which may lead to decreased stability and inconsistent luminescence efficiency; (2) After the luminescent material, co-reactant and co-reactant promoter form a luminescence system, there are problems such as long electron transfer path, large energy loss and inconvenient operation. There is an urgent need to construct a highly stable and highly water-soluble luminescent material to improve the luminescence efficiency and stability in order to achieve high-sensitivity detection.

[0006] According to recent reports, accelerating the redox process in electrochemiluminescence, improving the efficiency of electron transfer, and reducing the quenching side reactions caused by oxidized molecules can effectively enhance the electrochemiluminescence signal. Experimental verification shows that ceftriaxone sodium can be used as a reducing and antioxidant co-reactant in the electrochemiluminescence reaction to enhance the luminescence signal and obtain label-free, sensitive detection results. Summary of the invention

[0007] In order to solve the above-mentioned problems existing in electrochemical detection, the present invention provides a method for preparing thiol-based gold nanoclusters providing ligands for captopril and its application in electrochemiluminescence detection.

[0008] The first part of the present invention provides a method for preparing a gold nanocluster electrochemiluminescent material with uniform particle size and good water solubility using captopril as a thiol ligand, comprising the following steps:

[0009] (1) Soak the glassware used in the experiment in aqua regia to remove impurities and avoid failure of gold nanocluster synthesis due to incomplete containers;

[0010] (2) Add 16.6 ml of pure water to a beaker treated with aqua regia, place in an ice-water mixture, stir continuously, add dropwise 0.79 ml of HAuCl4 (1%) and 0.8 ml of captopril solution (50 mM), and white floccules, i.e., complexes, appear;

[0011] (3) Add 1.2 ml of NaOH solution (1 M) again, and the white floccules disappear quickly, and the solution becomes transparent. After 5 minutes, add 0.4 ml of NaBH4 solution (43 mg of NaBH4 solid dissolved in 10 ml of 0.2 M NaOH solution) and continue stirring in an ice-water bath for 3 h;

[0012] (4) After the reaction is completed, the gold nanocluster solution is purified by dialysis, and the pure lyophilized powder is obtained by freeze drying technology, which is the gold nanocluster electrochemiluminescent material, and is stored in a refrigerator at 4°C away from light.

[0013] The second part of the present invention provides a thiol ligand gold nanocluster and tripropylamine electrochemiluminescence system, based on a three-electrode system, to construct an aqueous electrochemiluminescence sensor and test the generated electrochemiluminescence signal, including:

[0014] (1) Place the reaction pool in pure water, ethanol, and pure water and ultrasonicate for 3 minutes in sequence to thoroughly clean the reaction pool;

[0015] (2) taking an appropriate amount of the pure freeze-dried powder of gold nanoclusters obtained in the first part, dissolving it in pure water, and adding it to the reaction cell, using a glassy carbon electrode as the working electrode, a platinum electrode as the auxiliary electrode, and Ag / AgCl as the reference electrode;

[0016] (3) Pre-treating the three electrodes, using alumina powder to grind and polish the surface of the glassy carbon electrode to a mirror surface, and washing the other two electrodes with pure water, ethanol, and pure water, respectively, and then inserting them into the reaction cell after drying;

[0017] (4) A tripropylamine solution of a certain concentration is then added, and the electrochemiluminescence signal generated in the reaction cell is detected using cyclic voltammetry.

[0018] The third part of the present invention provides the optimal concentration and dosage ratio conditions of the prepared thiol ligand gold nanoclusters and tripropylamine in the electrochemiluminescence process

[0019] The fourth part of the present invention is to apply the prepared electrochemiluminescent sensor to the high-sensitivity detection of ceftriaxone sodium under the best optimization conditions of the above exploration, and provide a simple method for detecting ceftriaxone sodium, comprising:

[0020] (1) Dissolve the powder of ceftriaxone sodium standard in pure water to obtain a standard solution with a concentration of 4 g / L, store it in a refrigerator at 4°C away from light, and save it for subsequent dilution and testing;

[0021] (2) diluting the 4 g / L ceftriaxone sodium standard solution in step (1) to concentrations of 4 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, etc.;

[0022] (3) Based on the electrochemiluminescence signal detection process in the second part, under the optimal detection conditions, use a pipette to draw different concentrations of ceftriaxone sodium standard solution, add it to the reaction pool, record the change value of the electrochemiluminescence signal in the reaction pool, and draw a standard curve of the ceftriaxone sodium concentration.

[0023] (4) Repeat the operation in step (3) for the ceftriaxone sodium solution of unknown concentration to be tested, and obtain the concentration of ceftriaxone sodium in the ceftriaxone sodium solution to be tested based on the standard curve drawn in step (3).

[0024] The beneficial effects of the present invention are as follows:

[0025] (1) Since the prepared captopril thiol ligand gold nanoclusters have good water solubility and stable performance, they overcome the disadvantage of traditional luminescent materials adhering to the surface of the reaction pool and being difficult to clean. Secondly, they also provide a stable and highly efficient electrochemiluminescent material for the aqueous electrochemiluminescence detection kit, breaking the limitations of traditional materials.

[0026] (2) The present invention provides a luminescence mechanism of gold nanoclusters (AuNCs) and tripropylamine (TPrA): the coreactant TPrA is oxidized to the intermediate TPrA during a positive potential scan. . + , and then lose protons to generate active free radicals TPrA . Then, the Au core of AuNCs is oxidized under electrical excitation, and electrons are quickly transferred through the Au-S bond to produce more AuNCs. . + , at this time, TPrA in the solution . + With AuNCs . + Reaction to generate excited state AuNCs * , and in AuNCs * Relaxation back to the ground state produces intense light emission, and the process is repeated.

[0027] (3) This study provides a simple and highly sensitive method for the detection of ceftriaxone sodium. The presence of the chemical structure amino group in ceftriaxone sodium is used to accelerate the redox process in electrochemiluminescence, improve the electron transfer efficiency in the aqueous phase, and reduce the quenching reaction caused by oxidative molecules. It acts as a reducing and antioxidant co-reactant in the reaction system to enhance the effect of the electrochemiluminescence signal and obtain label-free and sensitive detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 TEM characterization of thiol-based nanoclusters providing ligands for captopril

[0029] Figure 2 This is the electrochemiluminescence signal test diagram of the electrochemiluminescence system of thiol ligand gold nanoclusters and tripropylamine

[0030] Figure 3 Ceftriaxone sodium concentration standard curve

[0031] Figure 4 is the electrochemiluminescence signal change corresponding to the change of ceftriaxone sodium concentration DETAILED DESCRIPTION

[0032] The following specific examples are listed to further illustrate the present invention. It should be noted that the following implementation cases are intended to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0033] The experimental methods mentioned in the examples are all routine laboratory methods unless otherwise specified, and the experimental materials used are all purchased from conventional biochemical reagent manufacturers unless otherwise specified.

[0034] Example 1: Preparation of gold nanocluster materials with thiol ligands provided by captopril

[0035] Pretreatment: All glassware and magnetic stirring bars used were soaked in aqua regia for 2-3 hours, then rinsed with plenty of ethanol and purified water;

[0036] 16.6 ml of pure water was added to the beaker treated with aqua regia, and the mixture was placed in an ice-water mixture and stirred continuously. 0.79 ml of HAuCl4 (1%) and 0.8 ml of captopril solution (50 mM) were added dropwise, and white floccules appeared, which was a complex. 1.2 ml of NaOH solution (1 M) was added dropwise again, and the white floccules disappeared rapidly, and the solution became transparent. Five minutes later, 0.4 ml of NaBH4 solution (43 mg of NaBH4 solid was dissolved in 10 ml of 0.2 M NaOH solution) was added dropwise, and stirring was continued for 3 h. During the stirring process, the solution gradually changed from colorless to brown-black. The above reaction process was carried out in an ice-water mixture, and the product generated by the reaction was dialyzed and purified using a dialysis bag. The process was carried out in a PBS solution, and the dialysate was replaced once every 4 h, overnight, and 2 h, respectively. After the dialysis was completed, a freeze dryer was used to obtain the dried captopril-protected gold nanocluster pure powder, which was stored in a 4 ° C refrigerator away from light.

[0037] Example 2: Electrochemiluminescence system of thiol ligand gold nanoclusters and tripropylamine

[0038] Pretreatment: Take an appropriate amount of the captopril-protected gold nanocluster pure dry powder prepared in Example 1, dissolve it in pure water, and obtain a gold nanocluster solution; place the reaction cell in pure water, ethanol, and pure water for ultrasonic treatment for 3 minutes in sequence to thoroughly clean the reaction cell; use a glassy carbon electrode as a working electrode, a platinum electrode as an auxiliary electrode, and Ag / AgCl as a reference electrode, pretreat the three electrodes, use aluminum oxide powder to grind and polish the surface of the glassy carbon electrode to a mirror surface, and wash the other two electrodes with pure water, ethanol, and pure water, respectively, dry them, and insert them into the reaction cell;

[0039] The electrochemiluminescence signal was measured by intensity method in combination with cyclic voltammetry, with a sampling rate of 10T / S, a scanning potential between 0.2-1.3V, a scanning rate of 0.1V / S, a sensitivity of 10-3, a voltage of the high-voltage multiplier tube of 900V, and the electrochemiluminescence signal was measured in the aqueous phase;

[0040] Use a pipette to draw 200 μL of the prepared gold nanocluster solution and add it to the reaction pool. Measure data without obvious light intensity peaks. Then use a pipette to draw 200 μL of a certain concentration of tripropylamine solution. Repeat the above operation and detect the electrochemiluminescence signal generated in the reaction pool. Obvious and regular light intensity peaks appear.

[0041] Example 3: Based on Example 2, the optimal detection conditions in the electrochemiluminescence process are provided

[0042] (1) Under uniform conditions, by setting different photomultiplier tube high voltage conditions: 100, 200, 300, 400, 500, 600, 700, 800, 900 V, and the other steps remain unchanged, repeat Example 2 to measure the electrochemical luminescence signal, preferably 900 V;

[0043] (2) Unified conditions: under the photomultiplier tube high voltage condition of 900 V determined in step (1), the concentration of tripropylamine remains fixed. In the steps of Example 2, only the concentration of the gold nanocluster solution is changed to 5 mg / mL, 10 mg / mL, 20 mg / mL, and 30 mg / mL. The measurement steps of Example 2 are strictly repeated, preferably 20 mg / mL.

[0044] (3) Under the conditions determined in step (1) and step (2), only the concentration of tripropylamine was changed to 0.05 M, 0.1 M, 0.2 M, 0.3 M, and 0.5 M, and the measurement steps of Example 2 were strictly repeated, preferably 0.2 M;

[0045] (4) Under the conditions determined in steps (1), (2) and (3), by changing the dosage ratio of the gold nanocluster solution to the tripropylamine concentration, that is, repeating the measurement of Example 2, adding the same dosage and concentration (200) of the gold nanocluster solution, adding different dosages of the tripropylamine solution: 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, and keeping the other conditions the same, measuring the ECL signal value, and the final ratio of the gold nanocluster solution to the tripropylamine solution is preferably 4:5.

[0046] Example 4: Detection of Ceftriaxone Sodium

[0047] Pretreatment: Place the reaction pool in pure water, ethanol, and pure water for ultrasonic treatment for 3 minutes in sequence to thoroughly clean the reaction pool; Use alumina powder to polish the surface of the glassy carbon electrode to a mirror surface, and wash the other two electrodes with pure water, ethanol, and pure water respectively, dry them, and then insert them into the reaction pool;

[0048] The ECL signal was measured using the intensity method in conjunction with cyclic voltammetry, with a sampling rate of 10T / S, a scanning potential between 0.2-1.3V, a scanning rate of 0.1V / S, and a sensitivity of 10 -3 , the voltage of the high voltage multiplier tube is set to 900V

[0049] (1) Dissolve the powder of ceftriaxone sodium standard in pure water to obtain a standard solution with a concentration of 4 g / L, store it in a refrigerator at 4°C away from light, and save it for subsequent dilution and testing;

[0050] (2) diluting the prepared 4 g / L ceftriaxone sodium standard solution to concentrations of 4 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, and 400 mg / L;

[0051] (3) Based on Example 3, under the optimal detection conditions, different concentrations of ceftriaxone sodium standard solutions were drawn using a pipette and added to the reaction pool. The change value of the electrochemiluminescence signal in the reaction pool was recorded to draw a standard curve of the concentration of ceftriaxone sodium. The ECL intensity was linearly related to the concentration of ceftriaxone sodium, R 2 It is 0.986, which has a good correlation.

[0052] (4) Repeat the operation in step (3) for the ceftriaxone sodium solution of unknown concentration to be tested, and obtain the concentration of ceftriaxone sodium in the ceftriaxone sodium solution to be tested based on the standard curve drawn in step (3).

Claims

1. A method for preparing thiol-based nanoclusters with captopril as ligand, characterized in that: The following steps are involved: (1) Soak the glassware used in the experiment in aqua regia to remove impurities and avoid failure of gold nanocluster synthesis due to incomplete containers; (2) Add 16.6 ml of pure water to a beaker treated with aqua regia, place in an ice-water mixture, stir continuously, add dropwise 0.79 ml of HAuCl4 (1%) and 0.8 ml of captopril solution (50 mM), and white floccules, i.e., complexes, appear; (3) Add 1.2 ml of NaOH solution (1 M) again, and the white floccules disappear quickly and the solution becomes transparent. After 5 minutes, add 0.4 ml of NaBH4 solution (43 mg of NaBH4 solid dissolved in 10 ml of 0.2 M NaOH solution) and continue stirring in an ice-water bath for 3 h; (4) After the reaction is completed, the gold nanocluster solution is purified by dialysis, and the pure lyophilized powder is obtained by freeze drying technology, which is the gold nanocluster electrochemiluminescent material, and is stored in a refrigerator at 4°C away from light.

2. The establishment of the electrochemiluminescence system of thiol ligand gold nanoclusters and tripropylamine as claimed in claim 1, characterized in that: include: The glassy carbon electrode was used as the working electrode, the platinum electrode as the auxiliary electrode, and the Ag / AgCl electrode as the reference electrode. The electrochemical luminescence signal was measured by intensity mode in combination with cyclic voltammetry. The sampling rate was 10T / S, the scanning potential was between 0.2 and 1.3V, the scanning rate was 0.1V / S, and the sensitivity was set to 10 -3 , the voltage of the high-voltage multiplier tube is set to 900V; Pretreatment: Place the reaction pool in pure water, ethanol, and pure water for ultrasonic treatment for 3 minutes in sequence to thoroughly clean the reaction pool; Use alumina powder to polish the surface of the glassy carbon electrode to a mirror surface, and wash the other two electrodes with pure water, ethanol, and pure water respectively, dry them, and then insert them into the reaction pool; Take the pure freeze-dried powder of gold nanoclusters as claimed in claim 1, dissolve it in pure water, and add it to the reaction tank; A tripropylamine solution of a certain concentration is then added, and the electrochemiluminescence signal generated in the reaction cell is detected using cyclic voltammetry.

3. The electrochemiluminescence test process of the thiol ligand gold nanoclusters prepared as claimed in claim 2 and tripropylamine is characterized in that: include: The concentration of the thiol-based nanocluster solution for preparing captopril as claimed in claim 1 is 5-30 mg / mL, preferably 20 mg / mL; The concentration of tripropylamine in the electrochemiluminescent system of thiol ligand gold nanoclusters and tripropylamine as claimed in claim 2 is 0.05M-0.5M, preferably 0.2M; The establishment of the electrochemiluminescence system of thiol ligand gold nanoclusters and tripropylamine as described in claim 2, the dosage ratio of the gold nanocluster solution to the tripropylamine concentration has an effect on the electrochemiluminescence efficiency, 200 μL of 20 mg / mL gold nanocluster solution is added, and different doses of tripropylamine solution 100 μL-450 μL are added, and the other conditions remain the same. The electrochemiluminescence signal value is measured, and the final ratio of the gold nanocluster solution to the tripropylamine solution is preferably 4:

5.

4. The electrochemiluminescence detection method according to claim 1-3 is used to detect ceftriaxone sodium, characterized in that: include: (1) Dissolve the powder of ceftriaxone sodium standard in pure water to obtain a standard solution with a concentration of 4 g / L, store it in a refrigerator at 4°C away from light, and save it for subsequent dilution and testing; (2) diluting the 4 g / L ceftriaxone sodium standard solution in step (1) to concentrations of 4 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, etc.; (3) Based on the electrochemiluminescence signal detection process of claims 2-3, a pipette is used to draw different concentrations of ceftriaxone sodium standard solutions, which are added to the reaction pool, and the change value of the electrochemiluminescence signal in the reaction pool is recorded to draw a standard curve of the concentration of ceftriaxone sodium. The electrochemiluminescence intensity is linearly related to the concentration of ceftriaxone sodium, R 2 is 0.986; (4) Repeat the operation in step (3) for the sample solution containing ceftriaxone sodium of unknown concentration to be tested, and obtain the concentration of ceftriaxone sodium in the ceftriaxone sodium solution to be tested based on the standard curve drawn in step (3).

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