Fluorescence detection method and kit for detecting multiple metal ions

Abstract

The invention discloses a fluorescence detection method and a kit for detecting various metal ions. Aminated mesoporous silicon spheres induce aggregation of gold nanoclusters to enhance emission, carbon quantum dots are matched to construct the ratiometric fluorescent probe in an aqueous solution under a mild condition, and various metal ions are detected through the ratiometric fluorescent probe, so that preparation of the high-performance ratiometric fluorescent probe is realized. The detection method is easy to operate, high in detection efficiency, good in detection sensitivity and extremely wide in linear range, and a new method is provided for biomedical detection and environmental pollutant detection.

Description

Technical Field

[0001] This application relates to the field of substance detection, and in particular to a fluorescence detection method and kit for detecting multiple metal ions. Background Art

[0002] The concentration of metal ions is closely related to human health. Ag + , Zn 2+ , Cu 2+ are not only essential elements for the human body, but also closely related to all fields of human life. However, excessive heavy metal ions will cause irreversible damage to the human body and the environment. At the same time, heavy metals cannot be biodegraded, and the enrichment coefficients of heavy metal elements in polluted environment by animals and plants range from several times to hundreds of thousands of times, which will further affect biological health and environmental problems. Therefore, it is of great significance to develop a sensor that is fast, has a large detection range, and can be used for visual detection of multiple ions. Fluorescence detection has been widely used in the detection of trace heavy metal ions due to its simple operation, high sensitivity, excellent visualization effect, etc., but the research on high-concentration detection is still relatively scarce at present. More importantly, since it is difficult for the same probe to detect multiple substances through different sensing mechanisms, it has not been possible to distinguish and quantify Ag + , Zn 2+ , Cu 2+ by a single probe at present.

[0003] Some studies believe that embedding AuNCs in materials with nano- or micro-scale pore sizes can utilize the spatial confinement effect to achieve AIE luminescence. The commonly used material is zeolitic imidazolate framework material (ZIF), which is formed by the combination of metal ions and organic ligands and has an ordered pore structure, which can provide good physical protection for AuNCs and is an ideal candidate material for the immobilization of AuNCs. However, encapsulating AuNCs into ZIF requires a complex operation process, and the construction of some AuNCs-based ratio fluorescence will also inhibit the fluorescence signal of the single probe. At the same time, the high crystallinity of ZIF itself and its poor dispersibility in solvents cannot be ignored. As a porous material with good biocompatibility, mesoporous silica spheres have an adjustable framework structure, a large specific surface area, adjustable pore size and shape. These characteristics can provide good morphological control and multifunctionalization for AuNCs, and are expected to improve the fluorescence performance of gold nanoclusters through the spatial confinement effect, providing recyclability and high sensitivity for sensing and detection applications. Although a small number of studies have reported some applications of MSN-encapsulated AuNCs in sensing and imaging, most of them are carried out through complex embedding or strict calcination, or by aggregating AuNCs in an organic medium first, or simply mixing multiple probes. These studies do not fully utilize the material properties of MSN, nor do they study the luminescence and sensing mechanisms after the combination of AuNCs.

[0004] Therefore, it is highly necessary to develop a new type of visual detection sensing system with simple operation, high sensitivity, and a wide detection range for the differentiation and quantification of various metal ions. Summary of the Invention

[0005] In view of the above problems, the present invention proposes a fluorescence detection method for detecting various metal ions. By inducing the aggregation-enhanced emission of gold nanoclusters with amino-functionalized mesoporous silica spheres, a ratio fluorescence probe is constructed in aqueous solution and under mild conditions in cooperation with carbon quantum dots. The various metal ions are detected through the ratio fluorescence probe. The steps include: Prepare amino-functionalized mesoporous silica spheres. Provide mesoporous silica spheres, disperse them in an ethanol solution of 3-aminopropyltriethoxysilane, and quickly add ammonia water and stir. After centrifugation and washing, amino-functionalized mesoporous silica spheres are obtained; Prepare the first complex. Disperse the amino-functionalized mesoporous silica spheres in the carbon quantum dot precursor solution, obtain the in-situ growth product of carbon quantum dots inside the amino-functionalized mesoporous silica spheres, and perform centrifugation and washing to obtain the precipitate as the first complex; Prepare the ratio fluorescence probe solution. Disperse the first complex in the gold nanocluster dispersion and stir to react. Obtain the precipitate after the anchoring of gold nanoclusters outside the mesoporous silica spheres. After centrifugation and washing the precipitate, disperse it in an aqueous solution to obtain the ratio fluorescence probe solution; Add the metal ion solution to be detected into the ratio fluorescence probe solution and incubate to obtain the detection mixture; Collect the fluorescence emission peak intensity of the detection mixture through a fluorescence spectrometer, and calculate the concentration of the metal ion solution to be detected according to the peak intensity.

[0006] In one embodiment, the average particle size of the mesoporous silica spheres is 50-200 nm, and the average pore size is 2-10 nm.

[0007] In one embodiment, the mass concentration of the ethanol solution of 3-aminopropyltriethoxysilane is 5%-20%.

[0008] In one embodiment, carbon quantum dots are synthesized inside the mesoporous silica spheres, and the fluorescence emission peak position is different from that of the gold nanoclusters.

[0009] In one embodiment, the gold nanoclusters are prepared with reduced glutathione as the thiol ligand and reducing agent, have AIE properties, and have distinguishable fluorescence responses to metal ions (Ag + , Zn 2+ , Cu 2+ ).

[0010] In one embodiment, during the process of adding the metal ion solution to be detected into the ratio fluorescence probe solution and incubating to obtain the detection mixture, the concentration of the ratio fluorescence probe solution is 1-5 mg / mL.

[0011] In one embodiment, the incubation time in the process of adding the metal ion solution to be measured into the ratio fluorescence probe solution and incubating to obtain the mixture to be detected is 5 - 20 minutes.

[0012] On the other hand, the present invention also provides a kit for detecting metal ions, including: mesoporous silica spheres, aminopropyltriethoxysilane, ethanol, ammonia water, carbon quantum dot precursor solution, and gold nanocluster dispersion. The kit can detect each metal ion by the above-mentioned fluorescence detection method for detecting multiple metal ions.

[0013] In one embodiment, the average particle size of the mesoporous silica spheres is 50 - 200 nm, and the average pore size is 2 - 10 nm; the concentration of the ethanol solution of aminopropyltriethoxysilane is 5% - 20%; the carbon quantum dots are synthesized inside the mesoporous silica spheres, and the position of the fluorescence emission peak is different from that of the gold nanoclusters; the gold nanoclusters are prepared with reduced glutathione as the thiol ligand and reducing agent, have AIE characteristics and have distinguishable fluorescence responses to metal ions (Ag + ,Zn 2+ ,Cu 2+ ).

[0014] The above detection method and the corresponding kit utilize high-performance ratio fluorescence probes to realize the identification and quantitative detection of multiple metal ions based on different sensing mechanisms, and have good visualization effects. At the same time, the detection method is convenient to operate, has high detection efficiency, good detection sensitivity, and an extremely wide detection range, providing a new method for biomedical detection and environmental pollutant detection, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following further details the specific embodiments of the present invention with reference to the drawings: Figure 1 Shows the step flow chart of the metal ion detection method in the embodiment of the present invention; Figure 2 Shows the spectra and linear relationships of the fluorescence method for detecting three metal ions in the embodiment of the present invention; Figure 3 Shows the specificity data of the metal ion detection in the example of the present invention; Figure 4 Shows the spectra and linear relationships of using gold nanoclusters to detect metal ions in the comparative example of the present invention; Figure 5 Shows the spectra and linear relationships of using the mesoporous silica sphere-gold nanocluster composite to detect metal ions in the comparative example of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] To clarify the present invention in more detail, the technical solutions of the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings.

[0017] Referring to Figures 1-5 , in the present application, a fluorescence detection method for detecting multiple metal ions is proposed.

[0018] The present application combines supramolecular self-assembly with the AIE effect, enabling the preparation and application of luminescent metal nanocluster materials with complex structures, diverse functions, and good optical properties. The main innovation of the present application is to propose a novel triggering strategy based on MSN to induce the aggregation-induced emission (AIEE) of AuNCs, and achieve the efficient self-assembly of multiple fluorescent probes (AuNCs and BQDs) in aqueous solution under mild conditions based on electrostatic adsorption. The constructed ratio fluorescence probe MSN@BQDs-AuNCs not only increases the quantum yields of the single probes (AuNCs and BQDs) by about 3 times each, but its porous characteristics can also adsorb and enrich analytes, amplifying the analysis signal. The excellent optical properties of MSN@BQDs-AuNCs are verified by sensitive metal ion sensing. Compared with free AuNCs, the composite probe has the advantages of expanding the detection range by 1000 times and reducing the detection limit to 1 / 100, achieving ultrasensitive and visual quantitative detection of high-concentration Ag + , Zn 2+ , Cu 2+ .

[0019] The fluorescence detection method of the present application generally includes the following steps: providing mesoporous silica spheres (MSN), dispersing them in an ethanol solution of aminopropyltriethoxysilane and quickly adding ammonia water and stirring, and obtaining amino-functionalized mesoporous silica spheres after centrifuging and washing multiple times; in this embodiment, the preparation process of amino-functionalized mesoporous silica spheres is a commonly used method in the art, and the mesoporous silica spheres (MSN) and the ethanol solution of aminopropyltriethoxysilane are common materials, and their concentrations and ratios are not specially restricted. For example, take 0.6 g of MSN, disperse it in 7.5 mL of aminopropyltriethoxysilane / anhydrous ethanol (10%), quickly add 600 μL of ammonia water and stir evenly, continuously stir at room temperature for 12 h, after the reaction is completed, alternately centrifuge and wash three times with ethanol and ultrapure water, and the precipitate is amino-functionalized mesoporous silica spheres (MSN-NH2), which are placed in an oven at 60 °C for drying and then reserved. The above 0.6 g of MSN can be 0.4 g, 0.5 g, 0.7 g, 0.8 g, etc., and 7.5 mL of aminopropyltriethoxysilane / anhydrous ethanol (10%) can also be 6 mL, 10 mL, etc.

[0020] This application provides a carbon quantum dot precursor solution. The amino-functionalized mesoporous silica spheres are dispersed in the carbon quantum dot precursor solution to obtain the in-situ growth product of carbon quantum dots inside the amino-functionalized mesoporous silica spheres, followed by centrifugation and washing to obtain a precipitate as the first composite. Among them, carbon quantum dots (CQDs, in this application, blue carbon quantum dots are used, abbreviated as BQDs), also known as carbon dots or carbon nanodots, are a class of zero-dimensional carbon nanomaterials with remarkable fluorescence properties, which are composed of ultra-fine, dispersed, quasi-spherical carbon nanoparticles with a size below 10 nm. In this application, carbon quantum dots and gold nanoclusters with fluorescence emission peaks at different wavelengths are selected as fluorescence probes. Especially by introducing carbon quantum dots with different fluorescence colors, while eliminating background interference through self-calibration of double fluorescence emission peaks, a better visualization effect can also be presented. The main purpose of dispersing the amino-functionalized mesoporous silica spheres in the carbon quantum dot precursor solution in this application is to implant carbon quantum dots into the mesoporous silica spheres to form fluorescent mesoporous silica spheres. The preparation process and the proportion of each material of BQDs in this application are as follows: Dissolve 6 g of citric acid monohydrate and 12 mL of diethylenetriamine in 60 mL of ultrapure water. Using the hydrothermal method, put it into a reaction kettle and react at 200 °C for 6 h. After cooling, dialyze it for 1 - 3 days using a dialysis bag with a molecular weight cut-off (MWCO) of 1000 Da, and store the purified BQDs in a refrigerator at 4 °C for later use.

[0021] The preparation principle of the first composite MSN@BQDs in this application is as follows: Using the amino-functionalized mesoporous silica sphere MSN-NH2 as the reaction center provider, BQDs grow in-situ in the pores. The preparation process and the proportion of each material are as follows: Take 0.6 g of MSN-NH2, disperse it in 60 mL of ultrapure water, add 6 g of citric acid monohydrate and 12 mL of diethylenetriamine, after ultrasonic dispersion, put it into a reaction kettle and react at 200 °C for 6 h. Centrifuge and wash alternately with alcohol and water until there is no obvious fluorescence in the supernatant. The precipitate is MSN-NH2@BQDs (abbreviated as MSN@BQDs), and dry it in an oven at 60 °C for later use.

[0022] The gold nanoclusters (GSH-AuNCs) mentioned in this application are glutathione (GSH) reduced gold nanoclusters (Goldnanoclusters, AuNCs), which are synthesized with reduced glutathione as the thiol ligand and reducing agent. The preparation process and the proportion of each material are as follows: Mix freshly prepared GSH (6 mL, 25 mM), chloroauric acid HAuCl4 (4 mL, 25 mM) and 40 mL of ultrapure water evenly, stir rapidly at room temperature until colorless, and then stir gently at 70 °C for 24 h until the solution turns light yellow. Dialyze it for 1 - 3 days using a dialysis bag with a MWCO of 3000 Da, and store the purified AuNCs in a refrigerator at 4 °C for later use.

[0023] The preparation process and proportion of each material of the MSN-AuNCs in this application are as follows: 100 mg of amino-rich mesoporous silica spheres (MSN-NH2) were dispersed in 8 mL of purified AuNCs solution and stirred gently at room temperature for 3 hours. The positively charged MSN-NH2 can self-assemble with the negatively charged AuNCs based on electrostatic adsorption, and the AuNCs will be evenly anchored on the surface of MSN-NH2 to form MSN-NH2-AuNCs (abbreviated as MSN-AuNCs). Ultrapure water was used for centrifugal washing 3 times to remove free AuNCs, and the precipitate was placed in an oven for drying and then used, and an orange-red product was obtained under a 365nm fluorescent lamp. The preparation process of MSN@BQDs-AuNCs is similar to that of MSN-AuNCs. In order to construct ratio fluorescence, 0.8 to 1.2 times the mass fraction of MSN@BQDs is used to replace MSN-NH2 and self-assemble with AuNCs. After stirring for 3 hours to fully anchor the AuNCs, it is washed three times by centrifugation with ultrapure water, dried in an oven, and placed in a 4°C refrigerator for use. The product that appears purple under a 365nm fluorescent light is obtained.

[0024] The metal ion solution to be tested is added to the ratio fluorescence probe solution for incubation to obtain a mixed solution to be tested; the fluorescence emission peak intensity of the mixed solution to be tested is collected by a fluorescence spectrometer, and the concentration of the metal ion solution to be tested is calculated according to the relevant peak intensity.

[0025] In this application, AuNCs can already recognize and respond to three kinds of ions, and MSN-AuNCs can obviously increase the quantum yield of AuNCs by three times. Its sensing performance (detection range and detection limit) can also be seen from the comparative example. On this basis, this application further considers that the ratio fluorescence probe has a larger color change range, and innovatively introduces blue BQDs into this system, and introduces this system by in-situ growth, thereby further expanding the detection range.

[0026] The present application measures the concentration of the metal ion solution by a ratio fluorescence probe solution generated by mixing the first complex with the gold nanocluster dispersion. During the mixing process of the first complex with the gold nanocluster dispersion, after the first complex is mixed with the gold nanoclusters, the gold nanoclusters will be anchored on the first complex, and finally dispersed after centrifugation and washing to obtain the ratio fluorescence probe solution. The mesoporous silicon spheres can produce a spatial confinement effect, which greatly improves the fluorescence performance of the gold nanoclusters themselves and the sensing performance of the metal ions to be measured, which is mainly manifested in the decrease of the detection limit and the expansion of the detection range by dozens of times, thereby improving the application range of the probe.

[0027] The visualization effect of the ratiometric fluorescence probe is closely related to the selection and ratio of the probes. After a large number of exploratory experiments, in order to obtain a good visualization effect, carbon quantum dots with fluorescence emission peaks at different wavelengths and gold nanoclusters were selected as fluorescence probes and self-assembled on mesoporous silica spheres. The unique AIE property of gold nanoclusters and their specific responses to different metal ions enable them to distinguish and quantify different metal ions. This property is further amplified after the introduction of mesoporous silica spheres, which is a macroscopic manifestation of the spatial confinement effect of mesoporous silica spheres on gold nanoclusters. The introduction of carbon quantum dots with different fluorescence colors can not only eliminate background interference through self-calibration of double fluorescence emission peaks but also present a better visualization effect, which is beneficial to the visual analysis of metal ions. Their growth inside the mesoporous silica spheres is more conducive to protecting the carbon quantum dots from contacting the outer metal ions, enabling them to serve as stable reference probes for ratiometric fluorescence sensing.

[0028] Exemplarily, the present invention uses a ratiometric fluorescence probe composed of blue carbon quantum dots with a fluorescence emission peak at 463 nm and orange gold nanoclusters with an AIE effect and a fluorescence emission peak at 610 nm to determine the detection concentration ranges for + Ag 2+ Zn 2+ Cu Disperse a certain amount of mesoporous silica spheres in an ethanol solution of 3-aminopropyltriethoxysilane, quickly add ammonia water and stir evenly, and continuously stir at room temperature for 10-14 h. After the reaction is completed, wash by centrifugation three times alternately with ethanol and ultrapure water to obtain amino-functionalized mesoporous silica spheres. Mix the carbon source and nitrogen source for preparing blue carbon quantum dots in a certain ratio, and then disperse the amino-functionalized mesoporous silica spheres in the carbon quantum dot precursor solution, and use a reaction kettle for hydrothermal reaction to in-situ grow quantum dots inside the mesoporous silica spheres. After cooling, wash by centrifugation with pure water multiple times until there is no obvious fluorescence in the supernatant, and the precipitate obtained is the first complex. Add the first complex to the prepared dispersion of orange gold nanoclusters with an AIE effect, stir and react to anchor the gold nanoclusters on the surface of the first complex. Then wash by centrifugation with pure water multiple times, and disperse the precipitate in an aqueous solution to obtain a ratiometric fluorescence probe solution.

[0029] Prepare aqueous solutions with different concentrations and different metal ions (Ag + Zn 2+ Cu 2+) solution as the metal ion solution to be detected. Take 900 μL of the ratio fluorescence probe solution into a centrifuge tube, add 100 μL of the above-mentioned metal ion solution at a certain concentration to it, and mix to form the mixture to be detected. After the metal ion fully reacts with the ratio fluorescence probe, transfer the mixture to be detected into a cuvette, collect the fluorescence spectrum from 445 - 800 nm through a fluorescence spectrometer, record the fluorescence emission peak intensity of the mixture to be detected, and calculate the concentration of the metal ion solution to be detected according to the intensity ratio of the emission peaks of carbon quantum dots and gold nanoclusters.

[0030] As Figure 2 shown, it is the fluorescence change of the mixture to be detected after adding different metal ions. Figure 2 a - b shows that as the concentration of Ag + gradually increases, the fluorescence peak at 610 nm in the mixture to be detected gradually redshifts and the fluorescence increases. It can be seen from the inset that the mixture to be detected shows a trend of gradually changing from purple to pink. At the same time, it can be observed that the corresponding fluorescence signals at Ag + concentrations of 0 - 10 μM show a good linear relationship, and the linear regression equations are Y = 0.067C Ag+ + 0.495 and Y = 0.010C Ag+ + 0.559 respectively. Here, Y represents the ratio of the fluorescence peak intensity of gold nanoclusters to the fluorescence peak intensity of carbon quantum dots in the mixture to be detected, that is, I2 / I1, and C Ag+ represents the final concentration of Ag + in the solution (μM, where 1 μM = 1 μmol / L = 10^(-6) mol / L). After multiple experimental verifications, the detection limit of this detection method is as low as 0.0048 μM. Figure 2 c - d shows that as the concentration of Zn 2+ gradually increases, the fluorescence peak intensity at 610 nm in the mixture to be detected gradually increases. It can be seen from the inset that the mixture to be detected shows a trend of gradually changing from purple to light pink. At the same time, it can be observed that the corresponding fluorescence signals at Zn 2+ concentrations of 0 - 10000 μM show a good linear relationship, and the linear regression equations are Y = 0.010C Zn2+ + 0.466 and Y = 4.433C Zn2+ + 0.578 respectively. Here, C Zn2+ represents the final concentration of Zn 2+ in the solution (μM). After multiple experimental verifications, the detection limit of this detection method is as low as 0.0160 μM. Finally, Figure 2 e - f shows that as the concentration of Cu 2+As the concentration gradually increases, the fluorescence peak intensity at 610 nm in the test mixture gradually decreases to quenching. The inset shows the trend of the test mixture gradually changing from purple to light blue. 2+ The corresponding fluorescence signals at concentrations of 0-10 μM showed a good linear relationship, where the linear regression equations were Y=-0.115C Cu2+ +0.507 and Y=-0.028C Cu2+ + 0.412. Among them, C Cu2+ represents the Cu in solution 2+ After multiple experimental verifications, the detection limit of this detection method was as low as 0.0043 μM.

[0031] On the other hand, the present invention also proposes a kit for performing the above detection steps to detect multiple metal ions based on the above detection method for metal ions. The kit includes mesoporous silica spheres, aminopropyltriethoxysilane, ethanol, ammonia water, carbon quantum dot precursor solution, and gold nanocluster dispersion solution. The Ag nanocluster dispersion can be detected by using a simple spectrometer. + , Zn 2+ , Cu 2+ High-precision, visual fluorescence detection of three metal ions.

[0032] Correspondingly, the kit for metal ion detection is characterized in that the average particle size of the mesoporous silica spheres is 50-200 nm and the average pore size is 2-10 nm; the concentration of the aminopropyltriethoxysilane ethanol solution is 5%-20%; the carbon quantum dots can be synthesized inside the mesoporous silica spheres, and the fluorescence emission peak position is different from that of the gold nanoclusters; the gold nanoclusters are prepared using reduced glutathione as a thiol ligand and a reducing agent, have AIE properties and are resistant to metal ions (Ag + , Zn 2+ , Cu 2+ ) have distinguishable fluorescence responses.

[0033] Embodiment 1: Specific detection of metal ions: The present invention uses a detection system composed of blue carbon quantum dots and orange gold nanoclusters to evaluate the specific detection of metal ions. Take 900 μL of ratiometric fluorescent probe solution and 100 μL of 1 mM metal ion mixture (Ag + 、Zn 2 + , Cu 2+ 、Na + Mg 2+ , Ba 2+ , Ca 2+ , Fe 2+ , Fe3+ , Cd 2+ , K + ) to form the mixed solution to be detected, use a fluorescence spectrometer to collect the fluorescence spectrum in the range of 445 - 800 nm, record the fluorescence emission peak intensity of the mixed solution to be detected, and calculate the concentration of the metal ion solution to be detected according to the intensity ratio of the emission peaks of carbon quantum dots and gold nanoclusters for comparison. As can be observed from Figure 3 , relative to the blank group, the ratio of I2 / I1 increases significantly after the introduction of Ag + and Zn 2+ , while the introduction of Cu 2+ , Fe 2+ , Fe 3+ will cause a significant decrease in the intensity ratio, and at the same time, there is almost no difference between other metal ions and the blank group. Considering that Fe 2+ and Fe 3+ will cause a decrease in the fluorescence intensity of the first complex and affect the ion detection, so the ratio fluorescence detection of Fe 2+ and Fe 3+ was not carried out. It can be seen that the change in the fluorescence intensity ratio of this sensing system is the result of the specific interaction between metal ions and the ratio fluorescence probe, and it has strong specificity for three metal ions.

[0034] In summary, in the present application, the inventors proposed a fluorescence detection method and kit for detecting multiple metal ions. This detection method is convenient to operate, has high detection efficiency, good detection sensitivity, and an extremely wide detection range, providing a new method for biomedical detection and environmental pollutant detection, and having good application prospects.

[0035] Comparison 1: Using free gold nanoclusters for metal ion detection: The gold nanoclusters used in the present invention have the AIE effect and can produce different fluorescence responses to metal ions to be detected. Mix 900 μL of the gold nanocluster dispersion with 100 μL of metal ion (Ag + , Zn 2+ , Cu 2+ ) solutions with different concentrations. After sufficient reaction, transfer it into a cuvette, and collect the fluorescence spectrum in the range of 445 - 800 nm through a fluorescence spectrometer, and record the intensity of the fluorescence emission peak of the gold nanoclusters to calculate the concentration of the metal ion to be detected.

[0036] As Figure 4 shown, it is the fluorescence change situation after adding different metal ions to the gold nanocluster dispersion. Figure 4 a - b shows that as Ag +With the gradual increase in concentration, the fluorescence peak at 610 nm in the mixed solution gradually redshifts and the fluorescence intensity increases. As can be seen from the inset, the mixed solution shows a gradually increasing and reddening trend. At the same time, it can be observed that Ag + The corresponding fluorescence signals at concentrations of 0 - 1 μM show a good linear relationship, and the linear regression equations are Y = 24.633C Ag+ + 773.090. Here, Y represents the fluorescence peak intensity of the gold nanoclusters after the reaction, and C Ag+ represents the final concentration (μM) of Ag + in the solution. After multiple experimental verifications, the detection limit of this detection method is as low as 0.0902 μM. Figure 4 c - d show that, with the gradual increase in the concentration of Zn 2+ the fluorescence peak intensity at 610 nm in the mixed solution gradually decreases. As can be seen from the inset, the mixed solution to be detected shows a gradually darkening trend. At the same time, it can be observed that Zn 2+ The corresponding fluorescence signals at concentrations of 0.5 - 10 μM show a good linear relationship, and the linear regression equations are Y = -2.942C Zn2+ + 781.439. Here, C Zn2+ represents the final concentration (μM) of Zn 2+ in the solution. After multiple experimental verifications, the detection limit of this detection method is as low as 0.4447 μM. Finally, Figure 4 e - f show that, with the gradual increase in the concentration of Cu 2+ the fluorescence peak intensity at 610 nm in the mixed solution gradually decreases to quenching. As can be seen from the inset, the intensity of the mixed solution shows a gradually decreasing trend. At the same time, it can be observed that Cu 2+ The corresponding fluorescence signals at concentrations of 0.5 - 5 μM show a good linear relationship, and the linear regression equations are Y = -15.403C Cu2+ + 829.183. Here, C Cu2+ represents the final concentration (μM) of Cu 2+ in the solution. After multiple experimental verifications, the detection limit of this detection method is as low as 0.436 μM.

[0037] This comparative example shows that gold nanoclusters can detect three metal ions, but the detection sensitivity is poor and the detection range is also very small, making it difficult to meet the requirements of practical applications.

[0038] Comparison 2: Using mesoporous silica spheres - gold nanocluster composites for metal ion detection: To further compare the improvement effect of mesoporous silica spheres on the sensing performance of gold nanoclusters, the amino-functionalized mesoporous silica spheres were directly dispersed in the gold nanocluster dispersion and stirred for reaction. After centrifugation and washing, the mesoporous silica sphere-gold nanocluster complex was obtained and dispersed in an aqueous solution. 900 μL of the mesoporous silica sphere-gold nanocluster complex dispersion was mixed with 100 μL of metal ion (Ag + , Zn 2+ , Cu 2+ ) solutions with different concentrations. After sufficient reaction, the mixture was transferred to a cuvette, and the fluorescence spectrum in the range of 445 - 800 nm was collected by a fluorescence spectrometer. The intensity of the fluorescence emission peak of the gold nanoclusters was recorded to calculate the concentration of the metal ions to be detected.

[0039] As Figure 5 shown, it is the fluorescence change of the gold nanocluster dispersion after adding different metal ions. Figure 5 a - b show that as the concentration of Ag + gradually increases, the fluorescence peak at 610 nm in the mixture gradually redshifts and the fluorescence increases. It can be seen from the inset that the trend of the mixture to be detected gradually strengthens and turns red. At the same time, it can be observed that the corresponding fluorescence signals with Ag + concentrations of 0 - 5 μM show a good linear relationship, and the linear regression equations are Y = 59.577C Ag+ + 697.949 and Y = 28.018C Ag+ + 729.829 respectively. Here, Y represents the fluorescence peak intensity of the gold nanoclusters after the reaction, and C Ag+ represents the final concentration (μM) of Ag + in the solution. After multiple experimental verifications, the detection limit of this detection method is as low as 0.0245 μM. Figure 5 c - d show that as the concentration of Zn 2+ gradually increases, the fluorescence peak intensity at 610 nm in the mixture gradually increases. It can be seen from the inset that the trend of the mixture to be detected gradually darkens. At the same time, it can be observed that the corresponding fluorescence signals with Zn 2+ concentrations of 0.25 - 100 μM show a good linear relationship, and the linear regression equation is Y = 3.634C Zn2+ + 699.857. Here, C Zn2+ represents the final concentration (μM) of Zn 2+ in the solution. After multiple experimental verifications, the detection limit of this detection method is as low as 0.2330 μM. Finally, Figure 5 e - f show that as the concentration of Cu 2+ gradually increases, the fluorescence peak intensity at 610 nm in the mixture gradually decreases to quenching. It can be seen from the inset that the trend of the mixture intensity gradually decreases. At the same time, it can be observed that Cu2+ The corresponding fluorescence signals at concentrations of 0.5-10 μM showed a good linear relationship, where the linear regression equations were Y=-32.185C Cu2+ +660.445. Among them, C Cu2+ represents the Cu in solution 2+ After multiple experimental verifications, the detection limit of this detection method was as low as 0.436 μM.

[0040] This comparison shows that mesoporous silicon spheres can produce a spatial confinement effect, which greatly improves the fluorescence performance of gold nanoclusters themselves and the sensing performance of metal ions to be detected, mainly manifested in the decrease of detection limit and the expansion of detection range by dozens of times, which increases the application range of probes. However, it can be seen that the detection effect of a single probe is still defective compared to the ratio fluorescence probe. The introduction of carbon quantum dots with different fluorescence emissions is conducive to the improvement of the optical and sensing performance of the fluorescent probe.

[0041] This application combines supramolecular self-assembly with the AIE effect to achieve the preparation and application of luminescent metal nanocluster materials with complex structures, diverse functions and good optical properties. Based on the above objectives, the present invention proposes a new triggering strategy based on MSN to induce aggregation enhanced emission (AIEE) of AuNCs, and realizes efficient self-assembly of multiple fluorescent probes (AuNCs and BQDs) in aqueous solution and mild conditions based on electrostatic adsorption. The constructed ratiometric fluorescent probe MSN@BQDs-AuNCs not only increases the quantum yield of single probes (AuNCs and BQDs) by about 3 times, but also its porous properties can adsorb and enrich analytes and amplify analytical signals. The excellent optical properties of MSN@BQDs-AuNCs are verified by sensitive metal ion sensing. Compared with free AuNCs, the composite probe has the advantages of expanding the detection range by 1000 times and reducing the detection limit to 1 / 100, realizing ultra-sensitive and visual quantitative detection of high-concentration Ag+, Zn2+, and Cu2+.

[0042] The gold nanoclusters in this application are orange under ultraviolet light and have an aggregation-induced enhancement effect. + , Zn 2+ , Cu 2+ ) have distinguishable fluorescence responses. Specifically, for Ag + It will produce fluorescence enhancement + emission peak red shift (brighter and redder), for Zn 2+ Only fluorescence enhancement (brightening) will occur for Cu 2+Only fluorescence quenching (darkening) occurs, and the effect becomes more obvious as the concentration of metal ions increases. To improve the detection sensitivity, the fluorescence intensity of the gold nanoclusters themselves can be increased first. After the mesoporous silica spheres are aminated (+), they are added to the gold nanoclusters (-) and stirred, and they will be coupled due to electrostatic interaction. After centrifugation and washing to remove the uncoupled gold nanoclusters, the precipitate is dispersed in water (the concentration of the dispersion in the example is 2.5 mg / mL), and it will be found that the fluorescence is significantly enhanced compared with the previous gold nanoclusters. When used for metal ion detection, it will be found that the detection concentration range for the three metal ions is expanded and the sensitivity is improved. To further optimize the visualization effect, it is thought to fix carbon quantum dots of different colors (blue in the example) inside the mesoporous silica spheres to form a ratio fluorescence sensor with the orange gold nanoclusters outside. Coincidentally, the aminated silica spheres (+) can just provide sites for the growth of the carbon quantum dots (-). Therefore, after the precursor for synthesizing the carbon quantum dots and the aminated mesoporous silica spheres are subjected to hydrothermal reaction together, and after centrifugation and washing many times to remove the unfixed carbon quantum dots, the remaining part is the aminated mesoporous silica spheres showing blue fluorescence, and there is no obvious fluorescence quenching for the three metal ions detected. After coupling the gold nanoclusters in the same way, a purple complex is formed, which is dispersed in an aqueous solution (the concentration of the dispersion in the example is 2.5 mg / mL). When used for metal ion detection, for Ag + there will be (purple-pink), for Zn 2+ it is (enhanced purple), for Cu 2+ it is (purple-blue), and at the same time the detection concentration range is further expanded and the sensitivity is increased again.

[0043] Finally, it should be noted that the above-described embodiments are only preferred examples for clearly illustrating the present invention, but this is not a limitation on the implementation of the present invention. Those of ordinary skill in the art should understand that the technical features in the above solutions can be combined arbitrarily, and other different forms of modifications or equivalent replacements of some technical features can be made on the basis of the above specific implementation manners. It is impossible to enumerate all the implementation manners here. Therefore, all any modifications, improvements, equivalent replacements, etc. derived from the technical solutions of the present invention within the spirit and principles of the present invention should be within the technical scope claimed by the present invention.

Claims

1. A fluorescence detection method for detecting multiple metal ions, characterized in that, Enhanced emission is achieved by the aggregation of gold nanoclusters induced by amino-functionalized mesoporous silica spheres, and a ratiometric fluorescence probe is constructed in aqueous solution and under mild conditions in combination with carbon quantum dots. A variety of metal ions are detected by the ratiometric fluorescence probe; The steps include: Prepare amino-functionalized mesoporous silica spheres. Provide mesoporous silica spheres, disperse them in an ethanol solution of 3-aminopropyltriethoxysilane and quickly add ammonia water and stir. After centrifugation and washing, amino-functionalized mesoporous silica spheres are obtained; Prepare the first complex. Disperse the amino-functionalized mesoporous silica spheres in the carbon quantum dot precursor solution, obtain the in-situ growth product of carbon quantum dots inside the amino-functionalized mesoporous silica spheres and carry out centrifugation and washing. The precipitate is the first complex; Prepare the ratiometric fluorescence probe solution. Disperse the first complex in the gold nanocluster dispersion and stir to react. Obtain the precipitate after the anchoring of gold nanoclusters outside the mesoporous silica spheres. After centrifugation and washing the precipitate, disperse it in an aqueous solution to obtain the ratiometric fluorescence probe solution; Add the metal ion solution to be measured into the ratiometric fluorescence probe solution and incubate to obtain the mixed solution to be detected; Collect the fluorescence emission peak intensity of the mixed solution to be detected by a fluorescence spectrometer, and calculate the concentration of the metal ion solution to be measured according to the peak intensity.

2. The fluorescence detection method for detecting multiple metal ions according to claim 1, wherein The average particle size of the mesoporous silica spheres is 50-200 nm, and the average pore size is 2-10 nm.

3. The fluorescence detection method for detecting multiple metal ions according to claim 1, characterized in that, The mass concentration of the ethanol solution of 3-aminopropyltriethoxysilane is 5%-20%.

4. The fluorescence detection method for detecting multiple metal ions according to claim 1, characterized in that, The carbon quantum dots are synthesized inside the mesoporous silica spheres, and the fluorescence emission peak position is different from that of the gold nanoclusters.

5. The fluorescence detection method for detecting multiple metal ions according to claim 1, wherein The gold nanoclusters are prepared with reduced glutathione as a thiol ligand and a reducing agent, have AIE characteristics and have distinguishable fluorescence responses to metal ions (Ag + , Zn 2 + , Cu 2+ ).

6. The fluorescence detection method for detecting multiple metal ions according to claim 1, wherein During the process of adding the metal ion solution to be measured into the ratiometric fluorescence probe solution and incubating to obtain the mixed solution to be detected, the concentration of the ratiometric fluorescence probe solution is 1-5 mg / mL.

7. The fluorescence detection method for detecting multiple metal ions according to claim 1, characterized in that, The incubation time during the process of adding the metal ion solution to be measured into the ratiometric fluorescence probe solution and incubating to obtain the mixed solution to be detected is 5-20 minutes.

8. A kit for detecting metal ions, characterized in that, Include: Mesoporous silica spheres, 3-aminopropyltriethoxysilane, ethanol, ammonia water, carbon quantum dot precursor solution, gold nanocluster dispersion. The kit is used to detect various metal ions by the method according to any one of claims 1-7.

9. A kit for detecting metal ions according to claim 8, characterized in that, The average particle size of the mesoporous silica spheres is 50 - 200 nm, and the average pore size is 2 - 10 nm; the concentration of the ethanol solution of 3-aminopropyltriethoxysilane is 5% - 20%; the carbon quantum dots are synthesized inside the mesoporous silica spheres, and the fluorescence emission peak position is different from that of the gold nanoclusters; the gold nanoclusters are prepared with reduced glutathione as the thiol ligand and reducing agent, have AIE properties and have distinguishable fluorescence responses to metal ions (Ag + , Zn 2+ , Cu 2+ ).

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