A method for detecting heavy metal ions based on colorimetric sensor array regulated by nanozyme activity

Abstract

The present invention discloses a method for detecting heavy metal ions by a colorimetric sensor array based on nanozyme activity regulation. Three gold nanoclusters protected by three different small molecule ligands are used as three sensing elements, namely His-AuNCs, GSH-AuNCs, and MPA-AuNCs. The colorimetric sensor array has high sensitivity and shows good recognition ability for five common heavy metal ions at concentrations as low as 0.5μM. It is also very sensitive to mixtures of multiple heavy metal ions and may be suitable for the analysis of complex components. The five heavy metal ions were distinguished with actual samples, proving the applicability of the sensor in actual sample analysis, and has potential application prospects in water quality evaluation and environmental protection.

Description

Technical Field

[0001] The invention relates to a method for detecting heavy metal ions by a colorimetric sensor array based on nanozyme activity regulation. Background Art

[0002] With the rapid development of industry and the exploitation of resources, heavy metals are used in many fields, but a large amount of water containing heavy metal ions is continuously discharged into the ecosystem, which causes serious pollution to the environment. In addition, heavy metal ions can enter the human body through the food chain or direct contact, and accumulate in the body, causing damage to the nervous, blood and urinary systems, and in the most serious cases even leading to death. Therefore, the establishment of a simple, rapid, low-cost, high-throughput heavy metal ion detection technology is of great significance for ecological environmental protection and human health and safety.

[0003] At present, common analytical techniques for heavy metal ion detection are usually based on spectroscopic methods, including atomic absorption spectroscopy, ion coupling, plasma spectroscopy, inductively coupled plasma emission spectroscopy, X-ray fluorescence spectroscopy, and inductively coupled plasma mass spectrometry. Although these spectroscopic techniques have excellent sensitivity and accuracy, they also have their own shortcomings, such as relying on complex sample pretreatment procedures, expensive large instruments, and long detection time, which limits their application in field testing and resource-limited environments. In recent years, many heavy metal ion detection methods have been developed by combining fluorescence, colorimetry, electrochemistry and other detection technologies. Most of them are based on the "lock and key" principle. These highly specific detection methods can usually only detect a single heavy metal ion. However, in actual environments such as water environments, multiple heavy metal ions in water samples need to be detected simultaneously. Therefore, it is necessary to develop simple, fast and efficient sensing strategies to identify multiple heavy metal ions. Summary of the invention

[0004] In order to solve the problems in the prior art, the present invention provides a method for detecting heavy metal ions based on a colorimetric sensor array regulated by nanozyme activity.

[0005] The sensor array is an array-based sensing platform that uses several recognition elements, each of which exhibits different affinities for each analyte. One substance acts on multiple elements to varying degrees, and one element will also respond to multiple substances. The array sensor can generate different cross-reaction signals for each analyte, forming a unique "fingerprint" for each target analyte. With the continuous development of machine learning, more and more pattern recognition algorithms are being developed for result analysis, thereby achieving multiple detection.

[0006] The present invention uses three different ligand-protected gold nanoclusters (AuNCs) with peroxidase-like activity as three sensing elements to construct a colorimetric sensing array to detect five heavy metal ions (Hg 2+ , Pb 2+ , Cu 2+ 、Cd 2+ 、Co 2+ ). The surfaces of three gold nanoclusters with excellent peroxidase-like properties are modified with different ligands, providing a rich coordination environment. Due to the different binding affinities between the five heavy metal ions and the three gold nanoclusters, the enzyme-like activities of the three gold nanoclusters are regulated to different degrees, causing changes in the absorption signal of the substrate UV-visible absorption spectrum, thereby obtaining different colorimetric responses of the five heavy metal ions. The combination of the colorimetric responses of the three sensing elements for each heavy metal ion will produce a unique pattern. The pattern recognition algorithm LDA can be used to reduce the dimensionality of the huge spectral data, realize the differential detection of multiple heavy metal ions and their mixed solutions, and provide a new method for the rapid identification and detection of multiple heavy metal ions in water.

[0007] Unless otherwise specified, all parts described in the present invention are parts by weight and all percentages described are mass percentages.

[0008] In order to achieve the above object, the technical features of the present invention are as follows:

[0009] A method for detecting heavy metal ions based on a nanozyme colorimetric sensor array, comprising the steps of preparing a sensor element, constructing a nanozyme colorimetric sensor array, identifying and detecting five kinds of heavy metal ions, and constructing a heavy metal ion map;

[0010] The preparation of the sensor element includes the preparation of histidine-gold nanoclusters (His-AuNCs), the preparation of glutathione-gold nanoclusters (GSH-AuNCs) and the preparation of mercaptopropionic acid-gold nanoclusters (MPA-AuNCs);

[0011] The preparation steps of the histidine-gold nanocluster (His-AuNCs) solution are as follows:

[0012] A HAuCl4·3H2O aqueous solution and a histidine (His) aqueous solution were mixed, and the mixture was incubated at room temperature for 2-3 hours. The color of the mixture changed from light yellow to light brown. When the solution was irradiated with 365nm ultraviolet light, strong blue-green light was emitted. The obtained His-AuNCs solution was centrifuged at 10000-12000rpm / min for 10-12min and then filtered using a 0.22μm microporous filter membrane to remove by-products with larger particle sizes. Subsequently, ultrafiltration was performed at 3500-4000rpm / min using an ultrafiltration tube with a molecular cutoff of 3KDa to eliminate residual unreacted reagents to obtain a histidine-gold nanocluster (His-AuNCs) solution.

[0013] The preparation steps of the glutathione-gold nanocluster (GSH-AuNCs) solution are as follows:

[0014] The HAuCl4·3H2O aqueous solution was added to the glutathione (GSH) aqueous solution, and the reaction mixture was heated to 90-95°C for 6.5-7 hours under gentle stirring (500-600rpm / min) to obtain a yellow GSH-AuNCs aqueous solution. When the solution was irradiated with 365nm ultraviolet light, it emitted strong orange fluorescence. The obtained GSH-AuNCs solution was centrifuged at 10000-12000rpm / min for 10-12min, and then filtered with a 0.22μm microporous filter membrane to remove byproducts with larger particle sizes, and then ultrafiltered at 3500-4000rpm / min using an ultrafiltration tube with a molecular cutoff of 3KDa to eliminate residual unreacted reagents to obtain a glutathione-gold nanocluster (GSH-AuNCs) solution;

[0015] The preparation steps of the mercaptopropionic acid-gold nanoclusters (MPA-AuNCs) solution are as follows:

[0016] HAuCl4·3H2O and mercaptopropionic acid (MPA) aqueous solution were added to deionized water and stirred at room temperature for 5-8 minutes. Then, NaOH aqueous solution was added to the reaction solution, followed by NaBH4 solution. The entire reaction was carried out in the dark. After stirring at room temperature for 3-4 hours, MPA-AuNCs were collected. The final reaction solution was aged at 4-6°C for 12-13 hours. The obtained MPA-AuNCs solution was centrifuged at 10000-12000 rpm / min for 10-12 minutes and then filtered using a 0.22 μm microporous filter membrane to remove larger particle size by-products. Subsequently, ultrafiltration was performed at 3500-4000 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents to obtain mercaptopropionic acid-gold nanoclusters (MPA-AuNCs) solution.

[0017] The nanozyme colorimetric sensor array is constructed and five heavy metal ions are identified and detected as follows: a gold nanocluster solution, a heavy metal ion solution, an acetic acid-sodium acetate buffer, H2O2 and 3,3',5,5'-tetramethylbenzidine (TMB) are sequentially added to a centrifuge tube, and then the mixed solution is incubated at 25-28°C for 30-35 minutes, and then H2SO4 is added to terminate the reaction. The ultraviolet-visible absorption spectrum of the system is detected by an ELISA instrument to obtain a training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions.

[0018] The heavy metal ion atlas is constructed by using the software SPSSStatistics 22 to perform data processing on the obtained training data matrix to achieve the distinction and quantification of five heavy metal ions.

[0019] A method for detecting heavy metal ions based on a nanozyme colorimetric sensor array comprises the following steps:

[0020] (1) Preparation of three sensing elements:

[0021] 1) Histidine-gold nanoclusters (His-AuNCs): 1 mL of HAuCl4·3H2O aqueous solution (10 mM) and 3 mL of histidine (His) aqueous solution (0.15 M) were mixed, and the mixture was incubated at room temperature for 2 hours. The color of the mixture changed from light yellow to light brown. When the solution was irradiated with 365 nm ultraviolet light, strong blue-green light was emitted. The obtained His-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and then filtered using a 0.22 μm microporous filter membrane to remove by-products with larger particle sizes. Subsequently, ultrafiltration was performed at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents. The obtained solution was stored in a dark place at 4°C before use.

[0022] 2) Glutathione-gold nanoclusters (GSH-AuNCs): HAuCl4·3H2O aqueous solution (4 mM, 10 mL) was added to glutathione (GSH) aqueous solution (6 mM, 10 mL). The reaction mixture was heated to 90°C for 6.5 hours under gentle stirring (500 rpm / min) to obtain a yellow GSH-AuNCs aqueous solution. When the solution was irradiated with 365 nm ultraviolet light, it emitted strong orange fluorescence. The obtained GSH-AuNCs solution was centrifuged at 10000 rpm / min for 10 min, and then filtered with a 0.22 μm microporous filter membrane to remove byproducts with larger particle sizes. Subsequently, ultrafiltration was performed at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and the obtained solution was stored in a dark place at 4°C before use.

[0023] 3) Mercaptopropionic acid-gold nanoclusters (MPA-AuNCs): 1 mL of HAuCl4·3H2O (20 mM) and 8 mL of mercaptopropionic acid (MPA) aqueous solution (5 mM) were added to 9.4 mL of deionized water and stirred at room temperature for 5 min, then 1.2 mL of NaOH aqueous solution (1 M) was added to the reaction solution, followed by 0.4 mL of NaBH4 solution; the entire reaction was carried out in the dark, and MPA-AuNCs were collected after stirring at room temperature for 3 hours, and the final reaction solution was aged at 4°C for 12 hours; the obtained MPA-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and filtered using a 0.22 μm microporous filter membrane to remove larger particle size byproducts, and then ultrafiltered at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and the obtained solution was stored in a dark place at 4°C before use;

[0024] (2) Construction of nanozyme colorimetric sensor array and identification and detection of five heavy metal ions:

[0025] 30 μL of gold nanocluster solution, 30 μL of heavy metal ion solution, 400 μL of acetic acid-sodium acetate buffer, 30 μL of H2O2 (1 M) and 10 μL of 3,3',5,5'-tetramethylbenzidine (TMB) (10 mM) were added to a centrifuge tube in sequence, and then the mixed solution was incubated at 25 °C for 30 min and 2 M H2SO4 was added to terminate the reaction. The UV-visible absorption spectrum of the system was detected by an ELISA instrument to obtain a training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions;

[0026] (3) Construction of heavy metal ion maps: The obtained training data matrix was processed using the software SPSS Statistics 22 to achieve the differentiation and quantification of the five heavy metal ions.

[0027] The NaBH4 solution was prepared by dissolving 43 mg of NaBH4 powder in 10 mL of 0.2 M NaOH solution.

[0028] The five heavy metal ions are Hg 2+ , Pb 2+ , Cu 2+ 、Cd 2+ and Co 2+ .

[0029] Beneficial effects of the present invention:

[0030] Compared with the prior art, the present invention uses three gold nanoclusters with enzyme-like activity as sensing elements for the first time, and prepares three gold nanoclusters protected by ligands histidine, glutathione, and mercaptopropionic acid. Since the rich ligands on the surface of the gold clusters provide a rich coordination environment, the five heavy metal ions can show different binding forces with the three gold nanoclusters, thereby regulating the enzyme-like activity of the gold nanoclusters to varying degrees, causing changes in the ultraviolet absorption spectrum absorption signal of the peroxidase substrate TMB, and using this as an output signal to obtain the unique spectral fingerprint data of each heavy metal ion, effectively distinguishing the different concentrations of the five heavy metal ions within a time limit. At the same time, the sensor array obtains a good linear relationship within a certain range for different metal ions, and successfully distinguishes mixed samples of mixed heavy metal ions of different proportions, and can also effectively identify heavy metal ions in actual milk samples. Compared with traditional heavy metal ion detection methods, the advantages of the present invention are simple operation, fast detection speed, high sensitivity, and high-throughput detection, thereby providing a new detection method for the simultaneous detection of multiple heavy metal ions.

[0031] The present invention uses three gold nanoclusters that simulate the activity of peroxidase as sensing elements to detect five heavy metal ions. The design is simple, the materials involved can be synthesized under laboratory conditions, the operation is simple, the toxicity is low, and the environment is friendly. The sensor designed by the present invention is simple to operate, time-saving and efficient, with high sensitivity and rapid analysis of multiple targets. The nanozyme colorimetric sensor array prepared by the method of the present invention can provide a new method for the detection of heavy metal ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : The recognition results of the sensor of the present invention for 5 heavy metal ions (10 μM); wherein A represents the fingerprint spectrum of the 5 heavy metal ions, and B represents the LDA two-dimensional score map of the 5 heavy metal ions.

[0033] Figure 2 : The recognition results of the sensor of the present invention for five heavy metal ions at higher concentrations and lower concentrations; A represents the LDA two-dimensional score graph of the five heavy metal ions at 50 μM, B represents the LDA two-dimensional score graph of the five heavy metal ions at 25 μM, C represents the LDA two-dimensional score graph of the five heavy metal ions at 5 μM, and D represents the LDA two-dimensional score graph of the five heavy metal ions at 0.5 μM.

[0034] Figure 3 :The sensor of the present invention is effective for Hg 2+ Identification results at a certain ion concentration; A represents the LDA two-dimensional score graph, and B represents the linear relationship graph between ion concentration and the first discriminant factor.

[0035] Figure 4 :The sensor of the present invention is effective for Pb 2+Identification results at a certain ion concentration; A represents the LDA two-dimensional score graph, and B represents the linear relationship graph between ion concentration and the first discriminant factor.

[0036] Figure 5 :The sensor of the present invention is Cu 2+ Identification results at a certain ion concentration; A represents the LDA two-dimensional score graph, and B represents the linear relationship graph between ion concentration and the first discriminant factor.

[0037] Figure 6 :The sensor of the present invention is sensitive to Cd 2+ Identification results at a certain ion concentration; A represents the LDA two-dimensional score graph, and B represents the linear relationship graph between ion concentration and the first discriminant factor.

[0038] Figure 7 :The sensor of the present invention is for Co 2+ Identification results at a certain ion concentration; A represents the LDA two-dimensional score graph, and B represents the linear relationship graph between ion concentration and the first discriminant factor.

[0039] Figure 8 : LDA two-dimensional score diagram of the sensor of the present invention for a binary mixture of two heavy metal ions at a certain concentration.

[0040] Fig. 9 : LDA two-dimensional score diagram of the sensor of the present invention for five heavy metal ions in actual samples. DETAILED DESCRIPTION

[0041] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0042] The following examples are the main chemical reagents used in the examples of the present invention:

[0043] Tetrachloroauric acid (HAuCl4·3H2O) was purchased from Sigma-Aldrich (USA); histidine (His), glutathione (GSH), mercaptopropionic acid (MPA), mercuric nitrate, lead nitrate, copper nitrate, cadmium nitrate, cobalt nitrate; 3,3',5,5'-tetramethylbenzidine (TMB), hydrogen peroxide (H2O2), sodium hydroxide (NaOH), sodium borohydride (NaBH4), sulfuric acid (H2SO4), sodium acetate (trihydrate), glacial acetic acid and other reagents were all of analytical grade and purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

[0044] Example 1 Three kinds of gold nanoclusters were prepared according to the following steps:

[0045] (1) His-AuNCs: 1 mL of freshly prepared HAuCl4·3H2O aqueous solution (10 mM) and 3 mL of His (0.15 M) were mixed, and the mixture was incubated at room temperature for 2 h. The color of the mixture changed from light yellow to light brown. When the solution was irradiated with ultraviolet light at 365 nm, strong blue-green light was emitted. The obtained His-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and then filtered using a 0.22 μm microporous filter membrane to remove by-products with larger particle sizes, followed by ultrafiltration at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents. The obtained solution was stored in a dark place at 4°C before use.

[0046] (2) GSH-AuNCs: Freshly prepared HAuCl4·3H2O aqueous solution (4 mM, 10 mL) was added to GSH aqueous solution (6 mM, 10 mL). The reaction mixture was heated to 90 °C for 6.5 h under gentle stirring (500 rpm / min) to obtain a yellow GSH-AuNCs aqueous solution. When the solution was irradiated with 365 nm ultraviolet light, it emitted strong orange fluorescence. The obtained GSH-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and filtered using a 0.22 μm microporous filter membrane to remove larger particle size byproducts, and then ultrafiltered at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents. The obtained solution was stored in a dark place at 4 °C before use.

[0047] (3) MPA-AuNCs: A freshly prepared aqueous solution of 1 mL of HAuCl4·3H2O (20 mM) and 8 mL of MPA (5 mM) was added to 9.4 mL of deionized water and stirred at room temperature for 5 min. Then, 1.2 mL of NaOH aqueous solution (1 M) was added to the reaction solution, followed by 0.4 mL of NaBH4 solution (prepared by dissolving 43 mg of NaBH4 powder in 10 mL, 0.2 M NaOH solution). The entire reaction was carried out in the dark, and MPA-AuNCs were collected after stirring at room temperature for 3 h. The final reaction solution was aged at 4 °C for 12 h. The obtained MPA-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and then filtered using a 0.22 μm microporous filter membrane to remove larger particle size byproducts, followed by ultrafiltration at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents. The obtained solution was stored in a dark place at 4°C before use.

[0048] Example 2 Construction of nanozyme colorimetric sensor array and detection of heavy metal ions.

[0049] (1) Detection of five heavy metal ions at a certain concentration using sensors

[0050] The steps are as follows:

[0051] Prepare stock solutions of 5 heavy metal ions at 5 different concentrations (50μM, 25μM, 10μM, 5μM, 0.5μM). Add 30μL of 5 heavy metal ion solutions at the same concentration (with 30μL of deionized water as blank) to the centrifuge tubes containing 30ul AuNCs solution (His-AuNCs, GSH-AuNCs, MPA-AuNCs), and then add 400μL of acetic acid-sodium acetate buffer, 30μL of H2O2 (1M) and 10μL of TMB (10mM) to the EP tube and mix well. Repeat the experiment 5 times. After incubating the above mixed solution at 25℃ for 30min, add 2M H2SO4 to terminate the reaction. Subsequently, the UV-visible absorption spectrum of the system was detected by an ELISA instrument. A training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions was obtained.

[0052] (2) Detection of single heavy metal ions at different concentrations using nanozyme colorimetric array

[0053] In order to further verify the quantitative detection capability of the sensor, this experiment will verify the sensor array for Hg 2+ , Pb 2+ , Cu 2 + 、Cd 2+ 、Co 2+ The quantitative detection capability of the test sample is as follows:

[0054] Prepare 5 stock solutions of 5 appropriate concentrations of 5 heavy metal ions. Add 30μL of 5 different concentrations of the same heavy metal ion solution (with 30μL deionized water as blank) to the centrifuge tube containing 30ul AuNCs solution (His-AuNCs, GSH-AuNCs, MPA-AuNCs), then add 400μL acetic acid-sodium acetate buffer, 30μL H2O2 (1M) and 10μL TMB (10mM) to the EP tube and mix well. Repeat the experiment 5 times. After incubating the above mixed solution at 25℃ for 30min, add 2M H2SO4 to terminate the reaction. Subsequently, the UV-visible absorption spectrum of the system was detected by an ELISA instrument. A training data matrix of 3 sensor elements × 5 ion concentrations × 5 repetitions was obtained.

[0055] (3) Using nanozyme colorimetric array to analyze different proportions of Hg 2+ and Cu 2+ Detection of binary mixtures

[0056] In order to further verify the multiple detection capabilities of the sensor array, the constructed sensor array was used to detect mixed samples of mixed heavy metal ions with different proportions. This experiment will distinguish mixed samples of heavy metal ions with different proportions and a total concentration of 10 μM. The operation steps are as follows:

[0057] Prepare 7 different ratios (Hg 2+ :Cu 2+ =100:0, 90:10, 75:25, 50:50, 25:75, 10:90, 0:100) 2+ and Cu 2+ Binary mixture stock solution. 30 μL of 7 different ratios of 2 heavy metal ion solutions were added to the centrifuge tubes containing 30 ul AuNCs solution (His-AuNCs, GSH-AuNCs, MPA-AuNCs) (30 μL deionized water was used as blank), and then 400 μL acetic acid-sodium acetate buffer, 30 μL H2O2 (1M) and 10 μL TMB (10mM) were added to the EP tube and mixed evenly. Repeat the experiment 5 times. After incubating the above mixed solution at 25°C for 30 minutes, 2M H2SO4 was added to terminate the reaction. Subsequently, the UV-visible absorption spectrum of the system was detected by an enzyme marker. A training data matrix of 3 sensor elements × 7 different ratios × 5 repetitions was obtained.

[0058] (4) Detection of five heavy metal ions in actual samples using nanozyme colorimetric array

[0059] In order to further verify the applicability of the multi-design sensor array in actual sample molecules, five heavy metal ions were added to the tap water samples for detection. The operation steps are as follows:

[0060] The tap water collected was filtered using a 0.22 μM microporous filter membrane as a solvent to prepare a stock solution of five heavy metal ions with a final concentration of 10 μM. 30 μL of five heavy metal ion solutions with tap water as solvent were added to the centrifuge tubes containing 30 ul AuNCs solution (His-AuNCs, GSH-AuNCs, MPA-AuNCs) (30 μL of deionized water as blank), and then 400 μL of acetic acid-sodium acetate buffer, 30 μL of H2O2 (1M) and 10 μL of TMB (10 mM) were added to the EP tube in sequence and mixed evenly. The experiment was repeated 5 times. After the above mixed solution was incubated at 25 ° C for 30 minutes, 2M H2SO4 was added to terminate the reaction. Subsequently, the UV-visible absorption spectrum of the system was detected by an ELISA instrument. A training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions was obtained.

[0061] Example 3 uses software SPSS Statistics 22 to perform data processing.

[0062] (1) Detection of heavy metal ions using nanozyme colorimetric array

[0063] When the final concentration of metal ions is 10 μM, the obtained training data matrix is ​​processed by software to obtain the recognition results of 5 heavy metal ions. Figure 1 As shown in the figure, Figure A is the fingerprint of 5 heavy metal ions, and Figure B is the LDA two-dimensional score diagram of 5 heavy metal ions. The first two typical discriminant factors are 81.8% and 18.9% respectively. It can be seen that the 5 heavy metal ions can be clearly distinguished, indicating that the constructed nanozyme colorimetric sensor array can successfully distinguish and identify the 5 heavy metal ions. Since the sensor array has a significant colorimetric response to 10μM heavy metal ions, its recognition ability for high and low concentrations of heavy metal ions was further evaluated. Figure 2 As shown in Figures A, B, C, and D, the sensor array exhibits good recognition performance for five heavy metal ions of 50 μM, 25 μM, 5 μM, and 0.5 μM, with a recognition accuracy of 100%, proving that the sensor array has high sensitivity in distinguishing the detection boxes for the five heavy metal ions studied.

[0064] (2) Quantitative detection of heavy metal ions using nanozyme colorimetric array

[0065] The training data matrix obtained at different concentrations of the five heavy metal ions was processed by software to obtain the LDA two-dimensional score graph of the five heavy metal ions within a certain concentration range and the linear relationship graph between the ion concentration and the first discriminant factor. Figures 3 to 7 As shown, the linear relationship between the heavy metal ion concentration and the first discrimination factor indicates that the interaction between the sensor element and the heavy metal ions is regular, uniform and stable within a certain concentration range. The larger the first discrimination factor, the stronger the interaction.

[0066] (3) Detection of mixed samples using nanozyme colorimetric array

[0067] The training data matrix was processed by the software to obtain Hg 2+ and Cu 2+ Recognition results of different mixing ratios. Figure 8 As shown, the LDA two-dimensional score graph shows that the first two typical discriminant factors are 48.4% and 39.3%, respectively. Binary mixtures of different proportions are well clustered into different groups, demonstrating the multiple recognition capability of the sensor array.

[0068] (4) Detection of actual samples using nanozyme colorimetric array:

[0069] After the training data matrix was processed by software, the identification results of 5 heavy metal ions in the actual sample were obtained. Fig. 9 As shown in the figure, the LDA two-dimensional score graph shows that the first two typical discriminant factors are , and the five different heavy metal ions are well clustered into five groups. Further blind sample experiments were carried out, and all blind samples were correctly identified with 100% accuracy. This shows the feasibility of developing a sensor array for actual environmental samples.

[0070] Table 1 Nanozyme colorimetric sensor array for measuring 10 heavy metal ion samples with a concentration of 10 μM in actual samples

[0071]

Claims

1. A method for detecting heavy metal ions based on a nanozyme colorimetric sensor array, comprising the steps of preparing a sensor element, constructing a nanozyme colorimetric sensor array, identifying and detecting five heavy metal ions, and constructing a heavy metal ion map; The preparation of the sensor element includes the preparation of histidine-gold nanoclusters His-AuNCs, the preparation of glutathione-gold nanoclusters GSH-AuNCs and the preparation of mercaptopropionic acid-gold nanoclusters MPA-AuNCs; The preparation steps of the histidine-gold nanocluster His-AuNCs solution are as follows: A HAuCl4·3H2O aqueous solution and a histidine His aqueous solution were mixed, and the mixture was incubated at room temperature for 2-3 hours. The color of the mixture changed from light yellow to light brown. When the solution was irradiated with 365 nm ultraviolet light, strong blue-green light was emitted. The obtained His-AuNCs solution was centrifuged at 10000-12000 rpm / min for 10-12 min, and then filtered using a 0.22 μm microporous filter membrane to remove by-products. Subsequently, ultrafiltration was performed at 3500-4000 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and a histidine-gold nanocluster His-AuNCs solution was obtained. The preparation steps of the glutathione-gold nanocluster GSH-AuNCs solution are as follows: The HAuCl4·3H2O aqueous solution was added to the glutathione GSH aqueous solution, stirred at 500-600 rpm / min, and the reaction mixture was heated to 90-95°C for 6.5-7 hours to obtain a yellow GSH-AuNCs aqueous solution. When the solution was irradiated with 365 nm ultraviolet light, it emitted strong orange fluorescence. The obtained GSH-AuNCs solution was centrifuged at 10000-12000 rpm / min for 10-12 min, and then filtered with a 0.22 μm microporous filter membrane to remove by-products, and then ultrafiltered at 3500-4000 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents to obtain a glutathione-gold nanocluster GSH-AuNCs solution; The preparation steps of the mercaptopropionic acid-gold nanocluster MPA-AuNCs solution are as follows: HAuCl4·3H2O and MPA-mercaptopropionic acid aqueous solution were added to deionized water and stirred at room temperature for 5-8 minutes. Then, NaOH aqueous solution was added to the reaction solution, followed by NaBH4 solution. The entire reaction was carried out in the dark. After stirring at room temperature for 3-4 hours, MPA-AuNCs were collected. The final reaction solution was aged at 4-6°C for 12-13 hours. The obtained MPA-AuNCs solution was centrifuged at 10000-12000 rpm / min for 10-12 minutes and then filtered using a 0.22 μm microporous filter membrane to remove by-products. Subsequently, ultrafiltration was performed at 3500-4000 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents to obtain a MPA-AuNCs solution of mercaptopropionic acid-gold nanoclusters.

2. The method according to claim 1, characterized in that: The nanozyme colorimetric sensor array is constructed and five heavy metal ions are identified and detected as follows: a gold nanocluster solution, a heavy metal ion solution, an acetic acid-sodium acetate buffer, H2O2 and 3,3',5,5'-tetramethylbenzidine are sequentially added to a centrifuge tube, and then the mixed solution is incubated at 25-28°C for 30-35 minutes, and then H2SO4 is added to terminate the reaction. The ultraviolet-visible absorption spectrum of the system is detected by an ELISA instrument to obtain a training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions.

3. The method according to claim 1, characterized in that: The heavy metal ion atlas is constructed by performing data processing on the obtained training data matrix using software SPSS Statistics 22 to achieve differentiation and quantification of five heavy metal ions.

4. The method according to any one of claims 1 to 3, characterized in that: The NaBH4 solution was prepared by dissolving 43 mg of NaBH4 powder in 10 mL of 0.2 M NaOH solution.

5. The method according to any one of claims 1 to 3, characterized in that: The five heavy metal ions are Hg 2+ , Pb 2+ , Cu 2 + 、Cd 2+ and Co 2+ .

6. A method for detecting heavy metal ions based on a nanozyme colorimetric sensor array, comprising the following steps: (1) Preparation of three sensing elements: 1) Histidine-gold nanoclusters His-AuNCs: 1 mL of 10 mM HAuCl4·3H2O aqueous solution and 3 mL of 0.15 M histidine His aqueous solution were mixed, and the mixture was incubated at room temperature for 2 hours. The color of the mixture changed from light yellow to light brown. When the solution was irradiated with 365 nm ultraviolet light, strong blue-green light was emitted. The obtained His-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and then filtered using a 0.22 μm microporous filter membrane to remove by-products. Subsequently, ultrafiltration was performed at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and the obtained solution was stored in a dark place at 4°C before use. 2) Glutathione-gold nanoclusters GSH-AuNCs: 10 mL of 4 mM HAuCl4·3H2O aqueous solution was added to 10 mL of 6 mM glutathione GSH aqueous solution, stirred at 500 rpm / min, and the reaction mixture was heated to 90°C for 6.5 hours to obtain a yellow GSH-AuNCs aqueous solution; when the solution was irradiated with 365 nm ultraviolet light, a strong orange fluorescence was emitted, and the obtained GSH-AuNCs solution was centrifuged at 10000 rpm / min for 10 min, and then filtered with a 0.22 μm microporous filter membrane to remove by-products, and then ultrafiltered at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and the obtained solution was stored in a dark place at 4°C before use; 3) Mercaptopropionic acid-gold nanoclusters MPA-AuNCs: 1 mL of 20 mM HAuCl4·3H2O and 8 mL of 5 mM mercaptopropionic acid MPA aqueous solution were added to 9.4 mL of deionized water and stirred at room temperature for 5 min, then 1.2 mL of 1 M NaOH aqueous solution was added to the reaction solution, followed by 0.4 mL of NaBH4 solution; the entire reaction was carried out in the dark, and MPA-AuNCs were collected after stirring at room temperature for 3 hours, and the final reaction solution was aged at 4°C for 12 hours; the obtained MPA-AuNCs solution was centrifuged at 10000 rpm / min for 10 min and filtered using a 0.22 μm microporous filter membrane to remove by-products, and then ultrafiltered at 3500 rpm / min using an ultrafiltration tube with a molecular cutoff of 3 KDa to eliminate residual unreacted reagents, and the obtained solution was stored in a dark place at 4°C before use; (2) Construction of nanozyme colorimetric sensor array and identification and detection of five heavy metal ions: 30 μL of gold nanocluster solution, 30 μL of heavy metal ion solution, 400 μL of acetic acid-sodium acetate buffer, 30 μL of 1 M H2O2 and 10 μL of 10 mM 3,3',5,5'-tetramethylbenzidine were added to the centrifuge tube in sequence, and then the mixed solution was incubated at 25 °C for 30 min and 2 M H2SO4 was added to terminate the reaction. The UV-visible absorption spectrum of the system was detected by an ELISA instrument to obtain a training data matrix of 3 sensor elements × 5 heavy metal ions × 5 repetitions; (3) Construction of heavy metal ion maps: The obtained training data matrix was processed using the software SPSS Statistics 22 to achieve the differentiation and quantification of the five heavy metal ions.

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