Synthesis and Application of Aggregation-Induced Emission Gold Nanoclusters

Abstract

The invention discloses a preparation method of aggregation-induced luminescence gold nanoclusters and its effect on Ag + , Hg 2+ , H2O2, GSH detection, including the synthesis of dual-ligand aggregation-induced emission gold nanoclusters, and their application in fluorescence detection of silver ions and mercury ions in aqueous solution, and continuous colorimetric detection of hydrogen peroxide and glutathione, and the invention of a material that can be used for colorimetric-fluorescence dual-mode multi-target detection. The probe completes the simultaneous detection of these two metal ions based on the aggregation-induced emission mechanism. The gold nanoclusters of the present invention also have excellent peroxidase-like activity and can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine. Glutathione, as a strong reducing agent, can decolorize the system and remove free radicals generated during the reaction, thereby realizing continuous colorimetric detection of hydrogen peroxide and glutathione. The invention realizes dual-modal multi-target detection and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano material synthesis and detection, and specifically relates to the synthesis and application of aggregation-induced emission (AIE) type gold nanoclusters. Background Art

[0002] Gold nanoclusters are usually composed of several to dozens of atoms, with a core size of less than 2 nm. As the size is reduced to close to the Fermi wavelength of electrons between metal atoms and nanoparticles, metal nanoclusters show a strong quantum size effect. There are many methods for synthesizing gold nanoclusters. The synthesis method usually selects proteins, peptides, thiols, polymers, etc. as templates or protective agents to synthesize fluorescent nanoclusters with different emission wavelengths. Generally, metal nanoclusters have strong fluorescence emission, stable fluorescence and adjustable fluorescence wavelength. They can be used as fluorescent probes for environmental analysis and sensing, including analysis, detection and imaging research of heavy metals, biomolecules, etc. [Jin RC, Zeng CJ, Zhou M, Chen YX. Atomically Precise colloidalmetal nanoclusters and nanoparticles: fundamentals and opportunities. Chemical Reviews, 2016, 116(18): 10346-10413] Gold nanoclusters have attracted extensive attention in the fields of pollutant monitoring, catalysis, cell imaging and bioanalytical sensing due to their advantages such as simple preparation method, large Stokes shift, good water solubility, low toxicity and good biocompatibility. However, most gold nanoclusters have aggregation-induced quenching (ACQ) effect, which makes gold nanoclusters often show low quantum yield and stability due to the energy elimination effect of ligand motion and self-quenching effect, which greatly limits the application of gold nanoclusters in chemical sensing. Therefore, it is necessary to develop a new strategy to improve the luminescence and stability of gold nanoclusters. Due to the universal influence of ACQ phenomenon, the research on aggregation-induced emission (AIE) type gold nanoclusters is still relatively small. AIE theory provides a new approach to solve the problem of aggregation-induced quenching of fluorescent gold nanoclusters, and compared with the closed-type sensors with ACQ characteristics, AIE-type open-type fluorescence sensors have higher sensitivity and selectivity. [Zhang, L.; Xu, Y.; Xu, J.; Zhang, H.; Zhao, T.; Jia, L. Intelligent multicolor nano-sensor based on nontoxic dual fluoroprobe and MOFs for colorful consecutive detection of Hg 2+and cysteine. J. Hazard. Mater. 2022,430, 128478]. In addition, the new concept of “single sensor multiple analytes”, namely “multi-sensor detection probe”, has attracted widespread interest among researchers. The ability to detect multiple targets simultaneously can shorten the analysis processing time and reduce costs. Therefore, it is very innovative to design a fluorescent probe based on AIE theory to achieve interference-free simultaneous detection of different analytes by converting between aggregated and dispersed states.

[0003] Toxic heavy metals (such as mercury, silver, etc.) can cause serious diseases and environmental pollution problems, posing a serious threat to human health and the ecosystem. In particular, silver ions (Ag + ) and mercury ions (Hg 2+ ) is widely distributed, easily accumulates in the food chain and is difficult to be biodegraded, so it is of great significance to track its concentration in natural water bodies. + Exposure can cause a variety of diseases, such as silver poisoning, skin and liver damage, stomach pain, slow growth, heart enlargement, etc. [An, J.; Chen, R.; Chen, M. An Ultrasensitive turn-on ratiometric fluorescent probes for detection of Ag + Based on carbon dots / SiO2 and gold nanoclusters. Sens. Actuators B Chem.2021, 329, 129097. Mercury is considered to be more dangerous because all forms of mercury, including elemental and ionic mercury, are ultimately converted into methylmercury by bacterial activity and then enriched in animals through the food chain. Hg 2+ It can cause disorders of the nervous and immune systems, irreversible damage to the lungs, kidneys, and brain, and even induce terrible diseases such as acrodynia (pink disease), Hunter-Russell syndrome, and Minamata disease [Cheng, Z.; Wei, J.; Gu, L.; Zou, L.; Wang, T.; Chen, L.; Li, Y.; Yang, Y. DNAzyme-based biosensors for mercury (Ⅱ) detection: Rational construction, advances and perspectives. J. Hazard. Mater. 2022, 431,128606.]. In view of this, effective monitoring and accurate detection of Ag in the environment is necessary. + and Hg 2+It is of great significance to prevent heavy metals from affecting human health.

[0004] Hydrogen peroxide (H2O2) is an important player in clinical biochemistry, energy, food, pharmaceuticals, enzyme catalysis and environmental monitoring. Abnormal accumulation of H2O2 can cause oxidative stress and some serious diseases, such as cardiovascular disease, diabetes, cancer, Parkinson's disease and Alzheimer's disease[Wang, S.; Hu, Z.; Wei, Q.; Zhang, H.; Tang, W.; Sun, Y.; Duan, H.; Dai, Z.; Liu, Q.; Zheng, X. Diatomic active sites nanozymes:Enhanced peroxidase-like activity for dopamine and intracellular H2O2detection. Nano Res. 2022, 15, 4266-4273.] Glutathione (GSH) is an intracellular biothiol that plays a key role in maintaining the intracellular redox state, participating in intracellular signal transduction, immune response, gene transcription regulation, etc. [Lu, W.; Chen, S.; Zhang, H.; Qiu, J.; Liu, X. FeNC single atom nanozymes with dual enzyme-mimicking activities for colorimetric detection of hydrogen peroxide and glutathione. J. Materiomics 2022, 8(6), 1251-1259] Since there is an excess of GSH in tumor cells, GSH is also considered to be a tumor biomarker [Lu, W.; Chen,S.; Zhang, H.; Qiu, J.; Liu, X. FeNC single atom nanozymes with dual enzyme-mimicking activities for colorimetric detection of hydrogen peroxide andglutathione. J. Materiomics 2022, 8(6), 1251-1259.]. Considering the important role of H2O2 and GSH in human health monitoring, and the fact that the intracellular imbalance of H2O2 and GSH is closely related to major diseases, the quantitative analysis of H2O2 and GSH in the blood is crucial for clinical diagnosis. Summary of the invention

[0005] The purpose of the present invention is to provide the synthesis and application of aggregation-induced emission (AIE) type gold nanoclusters.

[0006] Aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs, which are prepared by the following method:

[0007] 1) Add 20 mL of 2.43 mM chloroauric acid solution to a container, then take 0-5 mL of 0.01-0.05 M NAC solution and 0-5 mL of 0.01 M GSH solution, and quickly add them to the chloroauric acid solution in sequence, and vigorously stir for 25-35 minutes at room temperature to mix; the molar ratio of chloroauric acid: NAC: GSH is 0.972: (1-5): 1;

[0008] 2) Reflux at 60-100 °C for 12-36 h to obtain a light yellow NAC / GSH-AuNCs aqueous solution, which was then cooled to room temperature;

[0009] The molar ratio of chloroauric acid: NAC: GSH is 0.972:1:1;

[0010] The amount of NAC solution added is 0.01 M, 5 mL;

[0011] The amount of GSH solution added is 5 mL;

[0012] The reflux temperature is 80° C. and the reflux time is 24 h.

[0013] Application of the aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs in detecting heavy metal ions and biological small molecules;

[0014] The heavy metal ion is Ag + and / or Hg 2+ ;

[0015] The biological small molecules are H2O2 and / or GSH.

[0016] Ag detection using aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs + and Hg 2+ The method comprises:

[0017] 1) Drawing of standard curve: Mix the NAC / GSH-AuNCs solution with an equal volume of ultrapure water, measure the fluorescence intensity value I0 of the blank group, and mix the NAC / GSH-AuNCs solution with different known concentrations of Ag. + and Hg 2+After the solutions were mixed, the fluorescence intensity values ​​I at 600 nm were measured and the corresponding relative fluorescence intensity values ​​I0 / I were calculated. The relative fluorescence intensity values ​​I0 / I and Ag were established. + and Hg 2+ Standard curve corresponding to the concentration;

[0018] 2) Fluorescence detection of Ag + : Take NAC / GSH-AuNCs solution and the Ag concentration to be tested + After the solutions are mixed, the fluorescence intensity value I is measured and the relative fluorescence intensity value I0 / I is calculated. The Ag content in the solution to be tested is obtained according to the above standard curve. + concentration;

[0019] 3) Fluorescence detection of Hg 2+ : Take NAC / GSH-AuNCs solution and Hg 2+ After the solutions are mixed, the fluorescence intensity value I is measured and the relative fluorescence intensity value I0 / I is calculated. The Hg content in the solution to be tested is obtained according to the standard curve. 2+ concentration;

[0020] In step 1), the different known concentrations of Ag + Ag in solution + The concentration range is 0-10 μM. + The applicable range of concentration is 0.001-0.100 μM;

[0021] In step 1), the Hg concentrations of different 2+ Hg in solution 2+ The concentration of Hg in the test solution is between 0-200 μM. 2+ Concentration ranged from 0 to 100 μM;

[0022] The NAC / GSH-AuNCs solution was + The reaction time is 0-6 min, preferably 1 min;

[0023] The NAC / GSH-AuNCs solution was 2+ The reaction time of the solution is 1-10 min, preferably 1 min;

[0024] The reactions were all carried out at room temperature.

[0025] The fluorescence detection conditions are as follows: excitation wavelength 380 nm, excitation slit and emission slit width 3 nm, and the emission intensity of NAC / GSH-AuNCs at 600 nm is recorded. + and Hg 2+ Perform quantitative analysis.

[0026] The method for detecting H2O2 using aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs comprises:

[0027] 1) First, a 30% H2O2 solution is diluted into H2O2 solutions of different concentrations, and then equal amounts of the H2O2 solutions of different concentrations are added to the same volume of blank solutions to obtain multiple groups of test solutions: wherein the blank solution contains ultrapure water, HAc-NaAc buffer solution, TMB and the gold nanoclusters NAC / GSH-AuNCs;

[0028] 2) After the system reacts at room temperature for 5 min, the absorbance of the test solution is measured respectively;

[0029] 3) Drawing of standard curve: With the concentration of H2O2 added to the test solution as the horizontal axis and the absorbance value at 652 nm as the vertical axis, linear fitting is performed to obtain the equation;

[0030] 4) Add the test solution containing unknown concentration of H2O2 to the blank solution, then measure the absorbance A. Substitute the absorbance value into the linear equation to calculate the concentration of H2O2 in the test solution;

[0031] The amount of the HAc-NaAc buffer solution is 250 μL, 0.2 M;

[0032] The amount of TMB used is 50 μL, 0-2.00 mM;

[0033] The amount of the gold nanoclusters NAC / GSH-AuNCs used is 0-200 μL, preferably 75 μL.

[0034] In the H2O2 solutions of different concentrations in step 1), the concentration of H2O2 is 0.03–1 mM, and the concentration of H2O2 in the test solution is 0.03-0.20 mM;

[0035] The pH of the HAc-NaAc buffer solution is 3.6-5.6, the concentration of the TMB is 0-2.0 mM, and the concentration of the NAC / GSH-AuNCs is 0-200 μL.

[0036] The method for detecting GSH using aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs comprises:

[0037] 1) First, prepare GSH solutions of different concentrations, and then add equal volumes of GSH solutions of different concentrations to the same volume of blank solutions to obtain multiple groups of test solutions: wherein the blank solution contains ultrapure water, HAc-NaAc buffer solution, TMB, H2O2 and the gold nanoclusters NAC / GSH-AuNCs;

[0038] 2) After the system reacts at room temperature for 5 min, the absorbance of the test solution is measured respectively;

[0039] 3) Drawing of standard curve: With the concentration of GSH added in the test solution as the horizontal axis and the absorbance value at 652 nm as the vertical axis, linear fitting is performed to obtain the equation;

[0040] 4) Add the test solution containing unknown concentration of GSH to the blank solution, then measure the absorbance A, and calculate the concentration of GSH in the test solution by substituting the absorbance value into the linear equation;

[0041] Step 1) The amount of HAc-NaAc buffer solution is 250 μL, 0.2 M; the amount of TMB is 50 μL, 1.80 mM; the amount of H2O2 is 50 μL, 1.50 mM;

[0042] The amount of the gold nanoclusters NAC / GSH-AuNCs used was 75 μL;

[0043] In step 1), the concentration of GSH in the GSH solutions of different concentrations is between 0.50 and 60 μM, and the concentration of H2O2 in the test solution is between 0.50 and 8.00 μM;

[0044] The pH of the HAc-NaAc buffer solution is 3.6-5.6, the TMB concentration is 0-2.0 mM, and the H2O2 concentration is 0-2.0 mM. Detailed description of the invention:

[0046] The purpose of the present invention is to provide an aggregation-induced emission type fluorescent gold nanocluster and a synthesis method thereof.

[0047] Another object of the present invention is to develop a multi-element fluorescence-colorimetric dual-mode probe that can detect and distinguish two heavy metal ions Ag. + and Hg 2+ Gold nanoclusters NAC / GSH-AuNCs can be used as fluorescent probes to detect Ag + and Hg 2+ When a certain concentration of Ag is added to the NAC / GSH-AuNCs probe solution, + When a certain concentration of Hg2+ When the fluorescence is significantly quenched, NAC / GSH-AuNCs can achieve Ag + and Hg 2+ The specific detection is different from other metal ions, and the two targets detected are Ag + and Hg 2+ The relative fluorescence intensity (I0 / I) of NAC / GSH-AuNCs fluorescent probe and Ag + and Hg 2+ There is a good linear relationship between the concentrations of the samples, and the linear relationship equation is used to quantitatively analyze the samples of unknown concentration.

[0048] At the same time, it has the performance of nanozymes, which can catalyze H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to turn it blue, thereby achieving the purpose of colorimetric detection of H2O2, and based on this result, GSH in serum is detected by reduction mechanism. For the colorimetric detection of H2O2 and GSH, the inherent nanozyme activity of gold nanoclusters plays an important role. Through experimental studies, it was found that NAC / GSH-AuNCs has peroxidase mimetic activity. In the presence of H2O2, NAC / GSH-AuNCs can catalyze the decomposition of H2O2 to produce hydroxyl radicals (∙OH), thereby oxidizing the colorless TMB substrate into soluble oxTMB, and the corresponding solution color turns blue. When different concentrations of H2O2 are added to the above NAC / GSH-AuNCs-TMB system, the amount of blue oxTMB generated in the system will also be different, so the concentration of H2O2 can be quantitatively detected according to the colorimetric method. However, GSH, as a biothiol, has strong reducing properties and can inhibit the color development process, causing the NAC / GSH-AuNCs-TMB-H2O2 system to fade. With the addition of different concentrations of GSH, the degree of fading of the system is also different, so GSH can be qualitatively and quantitatively detected by colorimetry.

[0049] The aggregation-induced luminescence gold nanoclusters described in the present invention are gold nanoclusters in which N-acetyl-L-cysteine ​​and glutathione serve as both a reducing agent and a stabilizing agent.

[0050] Aggregation-induced emission gold nanoclusters can detect trace amounts of Ag in aqueous solutions using fluorescence mode + and Hg 2 + , the concentrations of H2O2 and GSH in aqueous solution samples can be detected by colorimetric mode.

[0051] The purpose of the present invention is to provide a method for simultaneously detecting Ag in aqueous solution. + and Hg 2+And a method for continuous colorimetric detection of H2O2 and GSH based on its peroxidase-like activity.

[0052] The present invention has the following beneficial effects:

[0053] 1. The NAC and GSH used in the present invention are dual-ligand protected aggregation-induced emission gold nanocluster materials. After optimization and regulation, gold nanoclusters with stable optical properties can be obtained, and can be used as multi-element detection probes to detect Ag. + , Hg 2+ and detection of biomolecules H2O2 and GSH;

[0054] 2. The present invention utilizes the metal-philic interaction between silver ions, mercury ions and aggregation-induced emission gold nanoclusters to adjust the aggregation state of gold nanocluster particles, thereby changing their emission intensity. Silver ions significantly enhance the fluorescence of gold nanoclusters, while mercury ions significantly reduce the fluorescence of gold nanoclusters. This fluorescence enhancement and quenching phenomenon can be used for the detection of silver ions and mercury ions.

[0055] 3. The invention can be used for Ag + , Hg 2+ The dual-mode detection method of H2O2 and GSH has good selectivity, high sensitivity and low detection limit, among which the detection limit of silver ions can be as low as 0.87 nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the synthesis and multiplex detection platform of aggregation-induced emission NAC / GSH-AuNCs;

[0057] Figure 2 UV-visible absorption spectra, normalized fluorescence excitation spectra, and emission spectra of NAC / GSH-AuNCs. The insets are photos of NAC / GSH-AuNCs under natural light and UV light, respectively.

[0058] Figure 3 Fluorescence spectra of NAC / GSH-AuNCs, NAC-AuNCs, and GSH-AuNCs;

[0059] Figure 4 TEM images of NAC / GSH-AuNCs;

[0060] Figure 5 TEM images of NAC-AuNCs and GSH-AuNCs;

[0061] Figure 6 NAC / GSH-AuNCs added Ag + Size distribution diagram of gold cluster aggregates formed before and after;

[0062] Figure 7 NAC / GSH-AuNCs added Ag + or Hg 2+ TEM image after

[0063] Figure 8 XPS spectra of NAC / GSH-AuNCs;

[0064] Fig. 9 The experimental results of selectivity and anti-interference of NAC / GSH-AuNCs as fluorescent probes;

[0065] Fig.10 Add different concentrations of (a) Ag + or (b) Hg 2+ Fluorescence emission spectra of NAC / GSH-AuNCs after treatment with different concentrations of (c) Ag + or (d) Hg 2+ and standard curve of relative fluorescence intensity (I0 / I);

[0066] Fig.11 UV-visible absorption spectra and corresponding photos in different reaction systems: TMB, TMB +H2O2, TMB+NAC / GSH-AuNCs, TMB+H2O2+ NAC / GSH-AuNCs, H2O2+ NAC / GSH-AuNCs;

[0067] Fig.12 Standard curve of different concentrations of H2O2 and absorbance value;

[0068] Fig.13 The selectivity experimental results of NAC / GSH-AuNCs-TMB-H2O2 system as a colorimetric probe;

[0069] Fig.14 Standard curve of different concentrations of GSH and absorbance values. DETAILED DESCRIPTION

[0070] Example 1 Preparation of Aggregation-Induced Emission Gold Nanoclusters NAC / GSH-AuNCs

[0071] 20 mL of 2.43 mM HAuCl4 (chloroauric acid) solution was added to a round-bottom flask, and then 5 mL of 0.01-0.05 M NAC (N-acetyl-L-cysteine) solution and 5 mL of 0.01 M GSH (glutathione) solution were added thereto in sequence, and the molar ratio of chloroauric acid: NAC: GSH was 0.972: (1-5): 1; the reaction was vigorously stirred at room temperature for 0.5 h, and then refluxed in an oil bath at 60-100 ° C for 12-36 h to obtain a light yellow NAC / GSH-AuNCs aqueous solution ( Figure 1 ).

[0072] Example 2 Preparation of Aggregation-Induced Emission Gold Nanoclusters NAC / GSH-AuNCs

[0073] Take 20 mL of 2.43 mM HAuCl4 (chloroauric acid) solution and add it into a round-bottom flask, then add 5 mL of 0.01 M NAC solution and 5 mL of 0.01 M GSH solution in sequence, fix the molar ratio of chloroauric acid: NAC: GSH at 0.972:1:1, stir vigorously at room temperature for 0.5 h, and then reflux in an oil bath at 80 °C for 24 h to obtain a light yellow NAC / GSH-AuNCs aqueous solution.

[0074] Example 3 Characterization of Aggregation-Induced Emission Gold Nanoclusters NAC / GSH-AuNCs

[0075] (1) UV-visible absorption spectrum

[0076] From the UV-visible absorption spectrum, it can be observed that NAC / GSH-AuNCs do not have an obvious surface plasmon resonance peak at 520 nm, indicating that there are no large gold nanoparticles in NAC / GSH-AuNCs ( Figure 2 ).

[0077] (2) Fluorescence spectrum

[0078] From the fluorescence emission spectrum of NAC / GSH-AuNCs, it can be seen that at the optimal excitation wavelength of 380 nm, the strongest emission peak is at 600 nm ( Figure 1 ), and it exhibits strong orange fluorescence under 365 nm ultraviolet light ( Figure 2 ).

[0079] In order to verify the protective effect of dual ligands NAC and GSH on NAC / GSH-AuNCs, fluorescence spectroscopy tests were performed on NAC / GSH-AuNCs and NAC-AuNCs and GSH-AuNCs synthesized with single ligands ( Figure 3 ). The results showed that the fluorescence emission of NAC / GSH-AuNCs was stronger than that of gold nanoclusters prepared by the other two single protective agents, which preliminarily proved that the protective effect of the dual ligands promoted the emission efficiency of gold nanoclusters.

[0080] (3) Transmission electron microscopy (TEM)

[0081] The NAC / GSH-AuNCs probe, NAC-AuNCs, GSH-AuNCs and NAC / GSH-AuNCs probe were added with Ag by TEM. + and Hg 2+The morphology was then characterized. Figure 4 TEM images of NAC / GSH-AuNCs, where Figures a and b show that the synthesized NAC / GSH-AuNCs have obvious aggregation and the size is about 26.33 nm ( Figure 6 a, n=62). Figure 4 c It can be seen that the NAC / GSH-AuNCs aggregates are composed of spherical NAC / GSH-AuNCs with an average particle size of about 1.80 nm (n = 100), verifying that the strong luminescence properties of NAC / GSH-AuNCs are due to the aggregation-induced emission mechanism. Figure 4 The high-resolution transmission image in d shows clear lattice fringes with lattice spacings of 0.235 nm, 0.200 nm, and 0.140 nm, corresponding to the (1 1 1), (20 0), and (2 2 0) planes of Au(0), respectively, confirming the synthesis of gold clusters. To further prove that the dual ligands NAC and GSH are jointly involved in the synthesis of gold clusters, TEM characterization of NAC-AuNCs and GSH-AuNCs synthesized with single ligands was also performed. Figure 5 It shows that GSH-AuNCs and NAC-AuNCs have the same spherical morphology. The average particle size of NAC-AuNCs is 1.70 nm, showing a certain degree of agglomeration. Compared with NAC / GSH-AuNCs, the distance between particles of NAC-AuNCs is larger and the particles are more dispersed ( Figure 5 a); GSH-AuNCs have good monodispersity and an average particle size of 5.60 nm ( Figure 5 b). The results show that the aggregation degree of the synthesized gold clusters directly affects their luminescence intensity. The addition of GSH ligands provides more thiol compounds for the synthesis of gold clusters, increases the content of Au(Ⅰ)-SR in the gold clusters, and further enhances the emission efficiency of NAC / GSH-AuNCs.

[0082] Figure 7 a and Figure 7 b: Addition of Ag to NAC / GSH-AuNCs + After the transmission image, it can be observed that 10 μM Ag + Induced NAC / GSH-AuNCs to form larger and more aggregates, with a size of about 33.83 nm ( Figure 6 b, n=62), the average particle size of the small spherical NAC / GSH-AuNCs in the aggregates was about 1.72 nm (n=100). The results showed that the particle diameter did not change significantly, but the distance between the particles was significantly shortened, resulting in a tighter aggregation, which significantly enhanced the fluorescence of NAC / GSH-AuNCs, thus verifying the aggregation-induced emission effect. More interestingly, 100 μM Hg 2+The introduction of NAC / GSH-AuNCs will transform from agglomerated state to monodispersed state ( Figure 7 c and Figure 7 d), the average particle size is about 2.04 nm (n=100). The agglomeration state completely disappears, and the particle diameter remains basically unchanged, indicating that Hg 2+ The addition of only changes the distance between particles, making them more dispersed and eliminating the aggregation-induced quenching effect. + and Hg 2+ The fluorescence enhancement and quenching phenomena of NAC / GSH-AuNCs after introduction are consistent with the aggregation-induced emission mechanism.

[0083] The above spectra can prove the successful preparation of dual-ligand protected aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs and the effect of Ag on + and Hg 2+ The fluorescence response is based on the aggregation-induced emission mechanism, which indicates its application as a multiplex probe.

[0084] (4) X-ray photoelectron spectroscopy (XPS)

[0085] XPS of the test samples verified the valence state distribution of gold in NAC / GSH-AuNCs. Figure 8 The high-resolution XPS spectrum of Au shows two obvious peaks at 84.5 eV and 88.2 eV, which belong to Au 4f 7 / 2 and Au 4f 5 / 2 The energy difference of spin-orbit splitting is 3.7 eV, which is consistent with the results reported in the literature. Au(0) 4f 7 / 2 and Au(0) 4f 5 / 2 At 84.2 and 87.9 eV, Au(I) 4f 7 / 2 and Au(I) 4f 5 / 2 The ratio of Au(I) to Au(0) in NAC / GSH-AuNCs is 1.87 / 1, and the surface Au(I) content is high, which is consistent with the high content of metal oxidation states in aggregation-induced emission metal nanoclusters, and also confirms the origin of the aggregation-induced emission of gold clusters.

[0086] Example 4 Aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs as a fluorescent probe to detect Ag + and Hg 2+

[0087] Aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs were used as fluorescent probes to detect Ag + and Hg 2+When a certain concentration of Ag was added to the NAC / GSH-AuNCs probe solution, + When a certain concentration of Hg 2+ When the fluorescence is significantly quenched, NAC / GSH-AuNCs can achieve Ag + and Hg 2+ The specific detection is different from other metal ions, and the two targets detected are Ag + and Hg 2+ The relative fluorescence intensity (I0 / I) of NAC / GSH-AuNCs fluorescent probe and Ag + and Hg 2+ There is a good linear relationship between the concentrations of the samples, and the linear relationship equation is used to quantitatively analyze the samples of unknown concentration.

[0088] (1) Fluorescence detection of Ag + and Hg 2+ Selective evaluation

[0089] Since excellent selectivity and anti-interference ability are important indicators for evaluating the sensing performance of fluorescent probes, the present invention studies the NAC / GSH-AuNCs for Ag + and Hg 2+ The selectivity and anti-interference ability of the detection were studied ( Fig. 9 ). NAC / GSH-AuNCs probe and Ag + or Hg 2+ 100 µM Ca2+ was added to the solution. 2+ , Pb 2+ , Zn 2+ , Mn 2+ , Ba 2+ ,Cr 3+ , Ni 2+ , Mg 2+ , Cu 2+ , Fe 2+ , Co 2+ , Cd 2+ Solution, investigate the effect of other metal ions on the detection of Ag by NAC / GSH-AuNCs probe + or Hg 2+ The results show that although the concentration of coexisting metal ions is Ag + The fluorescence of the probe alone could not change significantly, but the presence of only 10 µM Ag + When the concentration of Hg 2+Leading to obvious fluorescence quenching, the probe quenching efficiency is as high as 63%, clearly indicating Hg 2+ The results showed that the NAC / GSH-AuNCs probe had excellent selectivity and anti-interference ability for Ag + and Hg 2+ The simultaneous detection of provides an “on” and “off” fluorescence analysis strategy.

[0090] (2) Fluorescence detection of Ag + and Hg 2+ Sensitivity assessment

[0091] Based on the fluorescence probe of NAC / GSH-AuNCs for Ag + and Hg 2+ The good detection selectivity of NAC / GSH-AuNCs for Ag + and Hg 2+ The sensitivity of the detection ( Fig.10 The test solutions with different concentrations were mixed with NAC / GSH-AuNCs in equal volumes. After 1 min of reaction, the fluorescence spectroscopy was used to monitor the effects of NAC / GSH-AuNCs on Ag with different concentrations. + (0-10 μM) and Hg 2+ The fluorescence response of 0-200 μM was measured to verify its sensitivity. Fig.10 a and Fig.10 As can be seen in b, with the increase of Ag + With the increase of the concentration, the fluorescence intensity at 600 nm gradually increased, accompanied by a slight red shift, which was consistent with the change of fluorescence intensity under ultraviolet light. The relative fluorescence intensity I0 / I showed a good linear relationship with the silver ion concentration in the range of 0.001-0.100 μM ( Fig.10 c), calculated according to the formula 3σ / K (σ is the standard deviation of the detection method for the blank solution, K is the slope of the linear equation of the detection method) + The detection limit can be as low as 0.87 nM. 2+ The sensitivity of Hg 2+ As the concentration increases, the fluorescence intensity at 600 nm continues to decrease, accompanied by a slight blue shift, which is consistent with the image of fluorescence intensity changes under ultraviolet light. In the range of 0.01-100 μM, the relative fluorescence intensity I0 / I and Hg 2 + The linear equation for the concentration ( Fig.10 d), calculated Hg 2+ The detection limit was 47.0 nM. Therefore, NAC / GSH-AuNCs can be used as a detector for Ag based on different response phenomena.+ and Hg 2+ A fluorescent probe that can detect Ag in aqueous solution + and Hg 2+ Simultaneous, multiple, fast, accurate, real-time and on-site measurement.

[0092] (3) Ag in actual water samples + and Hg 2+ Recovery analysis

[0093] The NAC / GSH-AuNCs prepared in Example 1 were used to detect the Ag in the actual water sample. + and Hg 2+ The sample needs to be pre-treated to remove solid impurities in the water. Using step (2) of Example 4 as a reference, the standard addition method was used to simulate the actual test. Different concentrations of Ag were added to the two water samples. + Standard solution (5.00, 8.00, 10.00 nM) and Hg 2+ Hg in standard solutions (0.50, 0.80, 1.00 μM) 2+ The standard solution was added to the NAC / GSH-AuNCs solution, and the reaction was carried out at room temperature for 1 min. The fluorescence intensity at 600 nm was measured with an excitation wavelength of 380 nm. The specific results of the sample recovery test (n=3) are shown in Tables 1 and 2.

[0094]

[0095]

[0096] Example 5 Study on the mimetic enzyme activity of NAC / GSH-AuNCs and detection of H2O2 and GSH

[0097] For the colorimetric detection of H2O2 and GSH, the inherent nanozyme activity of gold nanoclusters plays an important role. Through experimental studies, it was found that NAC / GSH-AuNCs have peroxidase mimetic activity. In the presence of H2O2, NAC / GSH-AuNCs can catalyze the decomposition of H2O2 to produce hydroxyl radicals (∙OH), thereby oxidizing the colorless TMB substrate to soluble oxTMB, and the corresponding solution color changes to blue. When different concentrations of H2O2 are added to the above NAC / GSH-AuNCs-TMB system, the amount of blue oxTMB generated in the system will also be different, so the concentration of H2O2 can be quantitatively detected according to the colorimetric method. However, GSH, as a biothiol, has strong reducing properties and can inhibit the color development process, causing the NAC / GSH-AuNCs-TMB-H2O2 system to fade. With the addition of different concentrations of GSH, the degree of fading of the system is also different, so GSH can be qualitatively and quantitatively detected by colorimetry.

[0098] (1) Peroxidase mimetic activity of NAC / GSH-AuNCs-TMB

[0099] In order to explore the enzyme-like catalytic activity of gold nanoclusters, the following control experiments were performed ( Fig.11 ). The results showed that only when TMB and H2O2 co-existed, NAC-GSH-AuNCs could catalyze H2O2 to oxidize the colorless TMB substrate to blue oxTMB, with a maximum absorbance at 652 nm. However, in the absence of H2O2 and NAC / GSH-AuNCs, no color reaction occurred, and no typical absorption peak appeared. The results showed that the enzyme-like catalytic activity of NAC-GSH-AuNCs was peroxidase-like activity, not oxidase-like activity.

[0100] (2) Sensitivity of NAC / GSH-AuNCs-TMB system as a colorimetric probe for detecting H2O2

[0101] When the NAC / GSH-AuNCs-TMB system was used as a colorimetric detection probe, H2O2 solutions of different concentrations (0.03-1.00 mM) were added to the colorimetric probe. After 5 min of reaction, the UV-visible absorption spectra of the samples were tested to verify the sensitivity of the probe. Fig.12 It can be seen that the absorbance of the system increases with the increase of H2O2 concentration, which is consistent with the color change under natural light. The absorbance value at 652 nm shows a good linear relationship with H2O2 in the linear range of 0.03 mM to 0.20 mM. According to the formula 3σ / K, the detection limit of H2O2 is calculated to be 2.9 μM ( Fig.12b). Therefore, the NAC / GSH-AuNCs-TMB system can be used as a colorimetric probe for detecting H2O2, which can quickly and accurately detect H2O2 in aqueous solution.

[0102] (3) Selectivity of GSH detection based on the NAC / GSH-AuNCs-TMB-H2O2 system as a colorimetric probe

[0103] The NAC / GSH-AuNCs-TMB-H2O2 system was used as a colorimetric probe to detect GSH and its common biomolecules and cations in serum. Under the same conditions, 4 mM glutathione (GSH), bovine serum albumin (BSA), cysteine ​​(Cys), methionine (Met), ascorbic acid (AA), histidine (His), tryptophan (Trp), glutamic acid (Glu), 100 μM cation (K + , Ca 2+ 、Na + ) solution was added into the system to observe the experimental phenomena, and the UV-visible absorption spectrum was tested to further calculate the absorbance reduction efficiency of the sample solution. Fig.13 It shows that the efficiency of glutathione in reducing the absorbance of the solution is much higher than that of other analytes, because it has strong reducing properties, which reduces the oxidized oxTMB, causing its oxidized state to gradually change to colorless. The corresponding photos also show that only the addition of GSH can completely reduce the blue solution to colorless, indicating that the NAC / GSH-AuNCs-TMB-H2O2 system has good selectivity for the detection of GSH as a colorimetric probe, and the detection system can be further extended to a detection probe for GSH.

[0104] (4) Sensitivity of GSH detection based on the NAC / GSH-AuNCs-TMB-H2O2 system as a colorimetric probe

[0105] When the NAC / GSH-AuNCs-TMB-H2O2 system was used as a colorimetric detection probe, different concentrations of GSH solution (0.50 μM -60.00 μM) were added to the colorimetric probe. After 5 min of reaction, the sensitivity of the probe was verified based on the UV-visible absorption spectrum of the test sample. Fig.14It can be seen that the higher the concentration of added GSH, the lower the absorbance value of the system, which is the same as the fading of the solution under natural light. The absorbance value at 652 nm shows a good linear relationship with the GSH concentration in the range of 0.50-8.00 μM. According to the formula 3σ / K and the linear equation, the detection limit of GSH is calculated to be 0.22 μM. Therefore, the NAC / GSH-AuNCs-TMB-H2O2 system can be used as a colorimetric probe for the detection of GSH, which can realize the visual detection and quantitative analysis of GSH. The detection system can complete a series of tests and is expected to be applied to the monitoring of GSH in human serum.

[0106] (5) Recovery rate test of GSH in serum

[0107] The NAC / GSH-AuNCs prepared in Example 1 have peroxidase-like activity. According to step (4) of this example, a recovery test of GSH in serum was performed to verify its feasibility. The pretreatment process of human blood samples is as follows. The sample is centrifuged at 4500 r / min for 10 minutes, and the supernatant obtained is serum, which is diluted 100 times for use. Then, the 50 mM GSH stock solution is diluted with serum samples to 4.00, 8.00, and 10.00 μM, respectively, and then added to the NAC / GSH-AuNCs-TMB-H2O2 system, and the absorbance value is determined by the spike recovery test (n=3). Substitute Fig.14 The recovery rate was calculated by fitting the linear equation in b. The specific results are shown in Table 3. The results show that the NAC / GSH-AuNCs probe can not only detect H2O2, but also continuously detect GSH on this basis, and has good environmental adaptability.

[0108]

[0109] In summary, NAC / GSH-AuNCs can be used as a multi-element probe to complete the detection of Ag in the fluorescence mode. + and Hg 2+ It can detect H2O2 and GSH continuously in colorimetric mode by simulating the activity of peroxidase.

Claims

1. Aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs were prepared by the following method: 1) Add 20 mL of 2.43 mM chloroauric acid solution to a container, then take 0-5 mL of 0.01-0.05 M N-acetyl-L-cysteine ​​(NAC) solution and 0-5 mL of 0.01 M glutathione (GSH) solution, and quickly add them to the chloroauric acid solution in sequence, and vigorously stir for 25-35 minutes at room temperature to mix; the molar ratio of chloroauric acid: NAC: GSH is 0.972: (1-5): 1; 2) Reflux at 60-100 °C for 12-36 h to obtain a light yellow NAC / GSH-AuNCs aqueous solution, which was then cooled to room temperature.

2. The aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs according to claim 1, characterized in that: The molar ratio of chloroauric acid:NAC:GSH is 0.972:1:

1.

3. The aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs according to claim 2, characterized in that: The amount of NAC solution added is 0.01 M, 5 mL.

4. The aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs according to claim 3, characterized in that: The amount of GSH solution added is 5 mL.

5. The aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs according to claim 1, 2, 3 or 4, characterized in that: The reflux temperature is 80° C. and the reflux time is 24 h.

6. Application of the aggregation-induced emission gold nanoclusters NAC / GSH-AuNCs according to claim 1 in detecting heavy metal ions and bio-small molecules; the heavy metal ions are Ag + ; The biological small molecules are H2O2 and / or GSH.

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