Preparation method and application of co-reactive ligand-protected high-efficiency electrochemiluminescent gold nanocluster probes

Abstract

This invention belongs to the field of analytical chemistry and relates to the preparation and sensing applications of highly efficient electrochemiluminescent gold nanoclusters protected by co-reactive ligands. This invention synthesizes a class of gold nanoclusters protected by co-reactive ligands, whose ligand molecular backbone includes terminal thiol groups as anchoring groups and a tertiary amine structure. Using co-reactive ligands as stabilizers, a one-pot method is employed to synthesize highly efficient electrochemiluminescent gold nanoclusters. This method not only stabilizes the gold nanoclusters, preventing them from agglomerating and losing their optical properties, but also utilizes the tertiary amine structure to function as a co-reactant. This allows the synthesized gold nanoclusters to generate highly efficient anodic near-infrared electrochemiluminescence without the participation of exogenous co-reactants, providing an opportunity for the preparation of highly sensitive and convenient sensors, high-brightness organic light-emitting diodes, and deep tissue imaging research. Simultaneously, it enables highly sensitive, highly specific, and convenient detection of carboxylesterases, providing a new method for the early clinical diagnosis of hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of analytical chemistry, and relates to the preparation of a new type of electrochemiluminescence sensor probe, in particular to the preparation of a co-reactive ligand-protected high-efficiency electrochemiluminescence gold nanocluster probe and its sensing application. BACKGROUND

[0002] Chemiluminescence immunoassay technology is a new emerging in vitro immunodiagnosis mainstream technology, which has rapidly replaced traditional enzyme-linked immunoassay, fluorescence immunoassay and radioimmunoassay methods with its high sensitivity, good specificity, high degree of automation, precision and accuracy. This technology is widely used in clinical detection of tumor markers, cytokines, various hormones, prenatal examination of infant defects and almost all immunodiagnosis projects. The domestic chemiluminescence market size reached nearly 40 billion yuan in 2022. Electrochemiluminescence (ECL) technology is a new type of chemiluminescence phenomenon generated by high-energy electron transfer reaction triggered by electrode surface voltage, which has the advantages of convenient and low-cost equipment, strong controllability, high sensitivity, low background signal and wide dynamic response range, providing strong support for ultra-sensitive immunoassay technology. The performance (such as sensitivity, reproducibility and practicability) of ECL immunosensor is closely related to the characteristics (such as ECL efficiency, stability and biocompatibility) of ECL luminophore. Although the classic trispyridine ruthenium and luminol ECL luminophores have high ECL efficiency, they have the defects of high biological toxicity, difficulty in immobilization and labeling, fast signal decay and difficulty in regulation, which greatly affect the biocompatibility, applicability and reproducibility of the sensor, and affect its performance in clinical immunodetection. Gold nanoclusters (Au NCs) are composed of gold atomic nucleus and surface protective organic ligand layer, and have the advantages of good biocompatibility and low toxicity, good film-forming property and small size for easy labeling, strong light stability and easy functional regulation, and are considered as a new ideal ECL luminophore with great application potential. Therefore, the ECL system with gold nanoclusters as the luminophore provides a good opportunity for early immunodiagnosis technology. However, compared with traditional ECL luminophores, the current gold nanocluster system has the defect of low ECL efficiency, which is difficult to meet the high-sensitivity detection requirements of trace biomarkers in clinical practice.

[0003] So far, a variety of new principles and methods for enhancing the ECL efficiency of gold nanoclusters have been reported. According to the different ECL enhancement mechanisms, the following categories are mainly included: (1) introducing a co-reactive promoter to catalyze the generation of a large number of active co-reactive intermediates, thereby improving the interaction efficiency of the co-reactive radicals and the active intermediates of gold nanoclusters; (2) adding an additional electroactive agent or using a valence state regulation strategy to increase the accumulation of active intermediates of gold nanoclusters to improve the interaction efficiency with the co-reactive intermediates; (3) covalently coupling the co-reactive agent and the ligand of gold nanoclusters to shorten the ECL charge transfer path, reduce the inactivation of active intermediates due to side reactions and long diffusion time, and improve the ECL efficiency of gold nanoclusters; (4) using host-guest recognition, aggregation induction and coordination induction strategies to inhibit the vibration and rotation of ligands, reduce non-radiative transitions, improve the radiation transition efficiency of the excited state of gold nanoclusters, and further improve the ECL efficiency of gold nanoclusters. Although the above-mentioned ECL enhancement strategies have greatly promoted the application of gold nanoclusters in the field of early clinical diagnosis, these methods are based on the additional improvement of the synthesized gold nanoclusters. The multi-component ECL system leads to complex and time-consuming operation and is easily disturbed by external factors, which reduces the simplicity and reproducibility of the constructed sensor and greatly limits the practical sensing application of gold nanocluster ECL sensors. Therefore, directly solving the problem of low ECL efficiency of gold nanoclusters through the structural design of gold nanoclusters themselves to realize the construction of a single-component gold nanocluster system is an effective means to solve the above-mentioned problems and develop immunodiagnosis. SUMMARY

[0004] To solve the above technical problems, the present application provides a preparation method of a co-reactive ligand-protected high-efficiency electrochemiluminescence gold nanocluster probe and its sensing application.

[0005] The technical scheme of the present application is as follows:

[0006] The present application provides a design and synthesis method of a co-reactive ligand, the ligand having a thiol group as an anchor group and a tertiary amine as a molecular functional skeleton, and the structure of the co-reactive ligand is selected from any one of the following structures:

[0007] .

[0008] The synthesis path of the above-mentioned co-reactive ligand is as follows:

[0009] ;

[0010] In the formula, R is selected from the following groups 、 、 、 、 、 、 , 、 、 、 and of any one of claims 1-4.

[0011] The synthesis comprises the following steps: dissolving R-H and cyclothioethane in dichloromethane, stirring for 2h under nitrogen protection, rotary evaporation, vacuum distillation to obtain a colorless liquid, i.e. the co-reactive ligand.

[0012] Specifically, in the synthesis of the co-reactive ligand of the structure C1, C1 needs to be pretreated by solidification into a solid in a nitrogen bath; and after rotary evaporation, sodium ascorbate is added for filtration and then vacuum distillation.

[0013] The application also provides a synthesis method for synthesizing high-efficiency co-reactive ligand-protected gold nanoclusters by using the above ligand, and the synthesis path is as shown in Figure 3 , which comprises the following steps:

[0014] (1) respectively, the co-reactive ligand is added dropwise into the gold salt solution at 25 ℃, mixed thoroughly, then sodium bicarbonate aqueous solution is added to make the system in an alkaline environment, heated to 80 ℃, and stirred for 18 hours;

[0015] (2) the solution obtained in step 1) is dialyzed, freeze-dried, and extracted with isopropanol to obtain.

[0016] The gold salt is chloroauric acid.

[0017] Specifically, the pH of the alkaline environment in the above step (1) is 8.0.

[0018] Further, in the above step (2), the dialysis bag specification is MWCO 3500D.

[0019] Further, in the above step (3), the extraction solution is isopropanol.

[0020] The application also provides an application of the high-efficiency near-infrared electrochemiluminescence gold nanocluster probe in constructing a monovalent electrochemiluminescence carboxyl esterase detection system, which provides a new method for the early clinical diagnosis of hepatocellular carcinoma.

[0021] The application has the following beneficial effects:

[0022] 1. The application designs to synthesize a kind of co-reactive ligand protected gold nanoclusters, and the ligand molecular skeleton thereof includes end group mercapto as anchoring group and tertiary amine structure.Further, using it as ligand, high electric chemical luminescence gold nanocluster probe is synthesized by one-pot method.This kind of ligand not only can stabilize gold nanoclusters and avoid its agglomeration, and its tertiary amine structure has the function of co-reactive reagent, so that the synthesized gold nanoclusters can generate high-efficiency near-infrared anodic electrochemiluminescence without external co-reactive reagent, which provides opportunity for the preparation of high-sensitivity, one-convenient sensor, high-brightness organic light emitting diode and tissue deep imaging research.

[0023] 2. The co-reactive ligand designed and synthesized in the application not only can be used as a protective agent for gold nanoclusters, but also has co-reactive function, effectively shortens electron transfer distance, and can realize high-efficiency near-infrared electrochemiluminescence without introducing external co-reactive reagent or further modification, which provides a platform for one-electrochemiluminescence system construction, high-brightness organic light emitting diode and super-resolution electrochemiluminescence imaging research.

[0024] 3. The high-efficiency co-reactive ligand protected gold nanocluster of the application takes carboxylesterase as target and acetic acid-1-naphthyl ester as catalytic substrate, and based on 2-diethylaminoethanethiol protected gold nanocluster, a one-quenching type sensor is constructed to realize high-sensitivity, high-specificity and convenient detection of carboxylesterase, which provides a new method for early clinical diagnosis of hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0026] Figure 1 It is a general structure of co-reactive gold nanocluster and its ligand.

[0027] Figure 2 It is a design diagram of co-reactive ligand structure.

[0028] Figure 3 It is a synthesis route of co-reactive ligand protected gold nanocluster.

[0029] Figure 4 It is the nuclear magnetic hydrogen spectrum of 2-diethylaminoethanethiol (DEA) prepared in Example 1.

[0030] Figure 5 It is the nuclear magnetic carbon spectrum of DEA prepared in Example 1.

[0031] Figure 6High resolution transmission electron microscopy image of an example of DEA-Au NCs prepared for Example 1.

[0032] Figure 7 UV-Vis spectrum of an example of DEA-Au NCs prepared for Example 1.

[0033] Figure 8 Fourier transform infrared spectrum of an example of DEA-Au NCs prepared for Example 1.

[0034] Figure 9 X-ray photoelectron spectrum of an example of DEA-Au NCs prepared for Example 1.

[0035] Figure 10 X-ray photoelectron spectrum of Au 4f of an example of DEA-Au NCs prepared for Example 1.

[0036] Figure 11 X-ray photoelectron spectrum of S 2p of an example of DEA-Au NCs prepared for Example 1.

[0037] Figure 12 Fluorescence spectrum of an example of DEA-Au NCs prepared for Example 1.

[0038] Figure 13 Electrochemiluminescence spectrum of an example of DEA-Au NCs prepared for Example 1.

[0039] Figure 14 Comparison of electrochemiluminescence performance of an example of DEA-Au NCs prepared for Example 1 and existing classic gold nanoclusters.

[0040] Figure 15 Electrochemiluminescence intensity of DEA-Au NCs of the present application in different active carboxyl esterase.

[0041] Figure 16 Working curve of DEA-Au NCs of the present application for detecting different active carboxyl esterase.

[0042] Figure 17 Anti-interference detection experiment of DEA-Au NCs of the present application. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0044] Unless otherwise specified, all raw materials used in the following embodiments are ordinary commercially available products.

[0045] Example 1

[0046] A method for synthesizing highly efficient co-reactive gold nanoclusters includes the following steps:

[0047] (1) (0.1 mol) solidifies into a solid in a nitrogen bath, then dissolves in 100 mL of DCM. Add 0.15 mol of cyclothioethane to 50 mL of DCM solution. After mixing, the reaction solution is stirred at room temperature for 2 h under nitrogen protection. After rotary evaporation, sodium ascorbate is added, filtered, concentrated, and vacuum distilled to obtain a colorless liquid, namely the co-reactive ligand 2-diethylaminoethanethiol, abbreviated as DEA.

[0048] (2) 0.029 mmol of the co-reactive ligand was dissolved in 0.2 mL of 0.1 M NaHCO3 aqueous solution and then added dropwise to 1 mL of chloroauric acid (0.029 mmol) aqueous solution at 25 °C. After thorough mixing, 4.15 mL of 0.1 M NaHCO3 aqueous solution was added. The reaction was heated to 80 °C and stirred continuously at 500 rpm for 18 h to obtain co-reactive gold nanoclusters.

[0049] (3) The solution obtained in step (2) was dialyzed in deionized water (MWCO 3500D) to remove excess reactants, and after freeze-drying, it was extracted with isopropanol to obtain Au NCs, namely DEA-Au NCs.

[0050] The NMR and CMR spectra of 2-diethylaminoethanethiol are shown in [reference needed]. Figures 3-5 The specific data is shown below. 1 H NMR (500MHz, Chloroform- d ) 8 3.10 (d, J = 77.1Hz, 6H), 2.93 (q, J = 8.4 Hz, 2H), 2.19 (t, J = 8.7 Hz.1H), 1.33 (t, J = 7.3 Hz, 6H. 13 C NMR (126 MHz, Choroform- d ) δ 54.79, 47.08, 18.69, 8.70.

[0051] High-resolution transmission electron microscopy (HRTEM) images show that the prepared DEA-Au NCs exhibit good dispersion, with an average size of 2.0 nm and a lattice spacing of 0.22 nm, corresponding to the (111) facets of the face-centered cubic (fcc) structure of Au (as shown in the image). Figure 6 (As shown).

[0052] The UV-Vis spectrophotometer showed that the prepared DEA-Au NCs did not exhibit the characteristic surface plasmon resonance absorption peak of Au nanoparticles near 520 nm, indicating that the synthesized material contained almost no Au nanoparticles (e.g., Figure 7 (As shown).

[0053] Fourier transform infrared (FTIR) spectroscopy revealed a similar framework between DEA-Au NCs and DEA, indicating successful assembly of DEA into DEA-Au NCs. Furthermore, the -SH content was observed at 2592 cm⁻¹ in the FTIR spectra. −1 The disappearance of tensile vibration indicates successful assembly of DEA-Au NCs (e.g., Figure 8 (As shown).

[0054] X-ray photoelectron spectroscopy (XPS) spectra show that DEA-Au NCs are composed of four elements: C, N, Au, and S. The characteristic binding energy of S 2p 3 / 2 is 162.6 eV, indicating that the ligand is bound to the Au nucleus via an Au-S bond. Furthermore, the binding energy of Au 4f 7 / 2 in DEA-Au NCs is 84.2 eV, indicating that Au... 0 and Au I State coexistence. (e.g.) Figures 9-11 (As shown).

[0055] The superimposed fluorescence and electrochemiluminescence spectra show that the maximum emission spectra of the synthesized DEA-Au NCs are both 830 nm, indicating that the synthesized gold nanoclusters are nucleoluminescent (Nucleoluminescent). Figure 12 (As shown in Figure 13).

[0056] Compared with classic glutathione-protected gold nanoclusters (GSH-Au NCs), 6-aza-2-thiothymidine-protected gold nanoclusters (ATT-Au NCs), and bovine serum albumin-protected gold nanoclusters (BSA-Au NCs), the co-reactive ligand 2-diethylaminoethanethiol-protected gold nanoclusters exhibit the highest ECL efficiency, which is 212 times that of ATT-Au NCs, 48 ​​times that of BSA-Au NCs, and 13 times that of GSH-Au NCs (as shown in Figure 14).

[0057] Example 2

[0058] A method for synthesizing a high-efficiency co-reactive gold nanocluster, comprising the following steps:

[0059] (1) Dissolve 0.1 mol of the co-reactive ligand in 100 mL of DCM, add 12 g of thiacyclohexane (0.2 mol) to the 50 mL DCM solution, mix, and then stir the reaction solution at room temperature under nitrogen protection for 2 h, concentrate by rotary evaporation, and vacuum distill to obtain a colorless liquid, i.e., the co-reactive ligand. (0.1 mol) is dissolved in 100 mL of DCM, 12 g of thiacyclohexane (0.2 mol) is added to the 50 mL DCM solution, after mixing, the reaction solution is stirred at room temperature under nitrogen protection for 2 h, concentrated by rotary evaporation, and vacuum distilled to obtain a colorless liquid, i.e., the co-reactive ligand.

[0060] (2) Dissolve 0.029 mmol of the co-reactive ligand and 1.68 mg of sodium bicarbonate (0.02 mmol) in 0.2 mL of water, then add to 1 mL of chloroauric acid (0.029 mmol) solution at 25°C, mix thoroughly, and then add 4.15 mL of water solution containing 34.86 mg of sodium bicarbonate (0.415 mmol). Heat the reaction to 80°C, continuously stir at 500 rpm for 18 h, and obtain the co-reactive gold nanocluster.

[0061] (3) The solution obtained in step (2) is dialyzed (MWCO 3500D) in deionized water to remove excess reactants, freeze-dried, and then extracted with isopropanol to obtain Au NCs.

[0062] Example 3

[0063] A method for synthesizing a high-efficiency co-reactive gold nanocluster, comprising the following steps:

[0064] (1) Dissolve 0.1 mol of the co-reactive ligand in 100 mL of DCM, add 12 g of thiacyclohexane (0.2 mol) to the 50 mL DCM solution, mix, and then stir the reaction solution at room temperature under nitrogen protection for 2 h, concentrate by rotary evaporation, and vacuum distill to obtain a colorless liquid, i.e., the co-reactive ligand. (0.1 mol) is dissolved in 100 mL of DCM, 12 g of thiacyclohexane (0.2 mol) is added to the 50 mL DCM solution, after mixing, the reaction solution is stirred at room temperature under nitrogen protection for 2 h, concentrated by rotary evaporation, and vacuum distilled to obtain a colorless liquid, i.e., the co-reactive ligand.

[0065] (2) Dissolve 0.029 mmol of the co-reactive ligand and 1.68 mg of sodium bicarbonate (0.02 mmol) in 0.2 mL of water, then add to 1 mL of chloroauric acid (0.029 mmol) solution at 25°C, mix thoroughly, and then add 4.15 mL of water solution containing 34.86 mg of sodium bicarbonate (0.415 mmol). Heat the reaction to 80°C, continuously stir at 500 rpm for 18 h, and obtain the co-reactive gold nanocluster.

[0066] (3) The solution obtained in step (2) is dialyzed (MWCO 3500D) in deionized water to remove excess reactants, freeze-dried, and then extracted with isopropanol to obtain Au NCs.

[0067] Application Example 1

[0068] Hepatocellular carcinoma (HCC) is the major type of primary liver cancer, and the 5-year survival rate is very low (<5%) due to the fact that most patients are diagnosed at an advanced stage. Non-invasive and accurate diagnosis is of great significance to improve the survival rate of patients with hepatocellular carcinoma. Although alpha-fetoprotein (AFP) is a typical serum biomarker for early diagnosis of hepatocellular carcinoma, it cannot effectively distinguish hepatocellular carcinoma from other liver diseases due to its limited sensitivity or specificity. Fortunately, carboxylesterase (CE, EC 3.1.1.1) is one of the most abundant serine hydrolases in human liver, which is responsible for various biological metabolisms in living organisms.

[0069] Studies have shown that the difference in carboxylesterase between hepatocellular carcinoma and cirrhosis is more significant than AFP. Therefore, carboxylesterase can be used as an effective serum biomarker for hepatocellular carcinoma. Currently, commercial kits have been used to determine carboxylesterase activity by colorimetric method, which has relatively low sensitivity. In recent years, various fluorescent probes have been designed for high-sensitivity detection of carboxylesterase activity, but these probes have low selectivity in complex biological matrices and are prone to photobleaching. As a powerful analytical technique, electrochemiluminescence has the outstanding advantages of near-zero background, easy operation, and high sensitivity, providing an opportunity for high-sensitivity and high-selectivity detection of carboxylesterase activity, and there is currently no example of the development of an electrochemiluminescence carboxylesterase sensor. Based on this, the present patent aims to use co-reactant ligand-protected high-efficiency gold nanoclusters as luminophores to construct a high-sensitivity and high-specificity carboxylesterase sensor.

[0070] Taking the DEA-Au NCs prepared in Example 1 as an example, they are applied to the detection of the activity of carboxylesterase, a biomarker of hepatocellular carcinoma:

[0071] The construction process of the sensor is as follows: a glassy carbon electrode with a diameter of 3 mm is polished with 0.3 µm and 0.05 µm aluminum oxide powder, and then ultrasonically cleaned with distilled water and ethanol. After the glassy carbon electrode is dried, 8 μL of the DEA-Au NCs prepared in Example 1 are dropped onto the surface of the glassy carbon electrode, and the electrode is placed at room temperature for several hours until it is completely dried.

[0072] Take 3 mL of the above carboxylesterase phosphate buffer solution with different activities and 30 μL of substrate aceto-1-naphthyl ester with a concentration of 30 mM and add them to the electrolytic cell. The classic three-electrode system is used to detect the activity of carboxylesterase on the electrochemical luminescence instrument, in which the glassy carbon electrode is the working electrode, the platinum wire is the counter electrode, and the Ag / AgCl (saturated KCl solution) is the reference electrode.

[0073] Prepare carboxylesterase phosphate buffer solutions with activities of 1 × 10 -9 U / mL, 1 × 10 -8 U / mL, 1 × 10-7 U / mL, 1 x 10 -7 U / mL, 1 x 10 -6 U / mL, 1 x 10 -5 U / mL, 1 x 10 -4 U / mL, 1 x 10 -3 U / mL, 1 x 10 -2 U / mL, 1 x 10 -1 U / mL of carboxylesterase phosphate buffer solution.

[0074] The results show that with the increase of carboxylesterase activity, the kinetics of naphthol generated by hydrolysis of aceto-1-naphthyl ester is accelerated, and the electrochemiluminescence intensity is gradually weakened (see Figure 15 ).

[0075] The log C CE is taken as the abscissa, and the reduction value of the electrochemiluminescence intensity compared with the blank intensity is taken as the ordinate to draw a working curve (see Figure 16 ); Figure 16 The results show that the linear regression equation is: y=1743.88 x+15645.39. The log C CE has a good linear relationship with the reduction value of the electrochemiluminescence intensity compared with the blank intensity.

[0076] The glucose, bovine serum albumin and glucose oxidase phosphate buffer solution with a concentration of 1 μg / mL were prepared as interference substances for the selectivity study of the sensor, and the carboxylesterase phosphate buffer solution with a concentration of 10 ng / mL was used as a control.

[0077] Compared with the blank, the electrochemiluminescence intensity in 10 ng / mL carboxylesterase is significantly reduced, but the decrease in electrochemiluminescence intensity observed in ten times the concentration of the interference substance is negligible (see Figure 17 ), which reflects the strong specific response ability of the electrochemiluminescence sensing platform. Therefore, we use the co-reactive ligand-protected gold nanoclusters as the luminescent body and aceto-1-naphthyl ester as the catalytic substrate to construct a high-sensitivity and high-specificity carboxylesterase sensor, which provides a good platform for early diagnosis of hepatocellular carcinoma.

[0078] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing high-efficiency electrochemiluminescence gold nanoclusters probe with co-reactive ligand, characterized in that, The steps are: (1) adding a co-reactive ligand into an aqueous solution of gold salt, mixing uniformly, then adding an aqueous solution of sodium bicarbonate, and heating and stirring under alkaline conditions until the reaction is complete; (2) the solution after the reaction in step (1) is subjected to dialysis, freeze-drying, and isopropanol extraction to obtain the high-efficiency electrochemiluminescence gold nanocluster probe; The co-reactive ligand has the following general structure: ； R is selected from any one of the following groups , , , , , , , , , , and . 2.The method for preparing high-efficiency electrochemiluminescence gold nanoclusters probe according to claim 1, characterized in that: The molar ratio of the co-reactive ligand to gold salt in step (1) is 1:1; the gold salt is chloroauric acid; the heating and stirring conditions are a temperature of 80°C, a stirring speed of 500 rpm, a time of 18 h, and a pH of 8.0; and the dialysis bag used in step (2) has a MWCO of 3500D.

3. The high-efficiency electrochemiluminescence gold nanocluster probe prepared by the method of claim 1 or 2.

4. The high-efficiency electrochemiluminescence gold nanocluster probe of claim 3, for use in the preparation of a monomeric quenching type carboxylic acid esterase sensor with 1-naphthyl acetate as a catalytic substrate.

5. The high-efficiency electrochemiluminescence gold nanocluster probe of claim 4, for use in the preparation of a reagent for early diagnosis of hepatocellular carcinoma.

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