A cysteine ​​detection method based on silver ions and gold nanoparticles

Abstract

The present invention provides a cysteine ​​detection method based on silver ions and gold nanoparticles, comprising the following steps: step 1, mixing an EDTA solution and a HAuCl4 solution to obtain a first mixed solution, taking the supernatant for standby use, and obtaining an AuNPs solution; step 2, mixing a histidine solution and a HAuCl4 solution to obtain a second mixed solution, and stirring to obtain an AuNCs solution; step 3, mixing the AuNPs solution and the AuNCs solution to form an AuNPs-AuNCs mixed solution; step 4, mixing the Ag-containing solution and the AuNCs solution to obtain an AuNPs-AuNCs mixed solution; and step 5, mixing the Ag-containing solution and the AuNCs solution to obtain an AuNPs-AuNCs mixed solution. + The cysteine ​​solution to be tested is added to the solution, and then added to the AuNPs-AuNCs mixed solution to obtain a third mixed solution, which is allowed to stand and the color and / or absorbance value are observed. This method utilizes the fact that cysteine ​​has a thiol group and can react with Ag. + The combined properties can realize the qualitative or quantitative detection of cysteine ​​with high detection accuracy and convenient and fast operation.

Description

Technical Field

[0001] The invention belongs to the technical field of cysteine ​​material detection, and particularly relates to a cysteine ​​detection method based on silver ions and gold nanoparticles. Background Art

[0002] Among the 20 essential amino acids, cysteine ​​is the only one containing a sulfhydryl group (-SH). Due to the reducing properties of sulfhydryl groups, cysteine ​​is widely used in various fields, particularly medicine. As an important endogenous small molecule thiol, cysteine ​​participates in numerous physiological processes and is crucial for regulating intracellular redox levels and synthesizing bioactive substances.

[0003] Cysteine ​​participates in numerous physiological processes. For example, it maintains the activity of sulfhydryl enzymes in skin keratin and replenishes sulfhydryl groups, ensuring normal skin metabolism. Cysteine ​​also has a detoxifying effect on many harmful substances and toxins. Serum cysteine ​​levels are directly or indirectly associated with a variety of diseases, including arteriosclerosis, cardiovascular disease, hypertension, neurological disorders, diabetes, malignant tumors, pregnancy-related disorders, eye diseases, and osteoporosis. Therefore, accurate, rapid, and highly selective cysteine ​​detection plays a crucial role in life science research and clinical diagnosis.

[0004] Although various chemical analysis, electrochemical, spectroscopic, and chromatographic methods are currently available for cysteine ​​testing, each has its own shortcomings. For example, chemical complexometric titration has difficulty detecting trace amounts of the substance; electrochemical analysis, while highly sensitive, suffers from poor selectivity; and high-performance liquid chromatography (HPLC) suffers from expensive instrumentation, high operating costs, and complex operational requirements. Colorimetric methods are particularly suitable for on-site testing due to their simplicity, rapidity, good specificity, and high sensitivity. Therefore, developing a simple, rapid, and highly sensitive colorimetric method for on-site cysteine ​​testing is of great significance for disease diagnosis. Summary of the Invention

[0005] The purpose of the present invention is to provide a cysteine ​​detection method based on silver ions and gold nanoparticles to solve the problems of poor sensitivity, high running costs and complex operation of current trace detection methods for cysteine.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for detecting cysteine ​​based on silver ions and gold nanoparticles, the method comprising the following steps:

[0008] Step 1, mixing an EDTA solution and a HAuCl4 solution to obtain a first mixed solution, taking the supernatant for later use, and obtaining a gold nanoparticle solution, i.e., an AuNPs solution;

[0009] Step 2: mixing the histidine solution and the HAuCl4 solution to obtain a second mixed solution, and stirring to obtain a gold cluster solution, i.e., an AuNCs solution;

[0010] Step 3, mixing the AuNPs solution and the AuNCs solution to form an AuNPs-AuNCs mixed solution;

[0011] Step 4: + The cysteine ​​solution to be tested is added to the solution, and then added to the AuNPs-AuNCs mixed solution to obtain a third mixed solution, which is allowed to stand at room temperature, and the color and / or absorbance value are observed.

[0012] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, the detection method further comprises:

[0013] Step 5: Compare the absorbance value obtained in step 4 with the absorbance value-cysteine ​​standard curve to obtain the concentration of the cysteine ​​solution to be tested.

[0014] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, the steps of step 1 are specifically as follows:

[0015] The first mixed solution was stirred at 20-40° C. for 30 min, and then centrifuged to obtain a supernatant for later use.

[0016] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, the steps of step 2 are specifically as follows:

[0017] The second mixed solution was stirred at room temperature for 1-2 hours to obtain a gold cluster solution;

[0018] The histidine is L-histidine, D-histidine or racemic histidine or a mixture of L- and D-histidine in any proportion.

[0019] In the above-described method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, in step 1, the molar concentration ratio of HAuCl4 to EDTA in the first mixed solution is 1:7 to 1:10;

[0020] The concentration of HAuCl4 in the first mixed solution is 1 to 50 mM, and the concentration of EDTA is 7 to 500 mM;

[0021] Preferably, in step 1, the concentration of the AuNPs solution in terms of Au is 0.02 to 1.0 g / L.

[0022] In the above-described method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, in step 2, the molar concentration ratio of HAuCl4 to histidine in the second mixed solution is 1:20 to 1:50;

[0023] The concentration of HAuCl4 in the second mixed solution is 1 to 50 mM, and the concentration of histidine is 0.02 to 2.5 M;

[0024] Preferably, in step 2, the concentration of AuNCs calculated as Au is 0.01 to 1.0 g / L.

[0025] In the above-described method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, in step 4, the final concentration of AuNPs in the third mixed solution is 43 mg / L, and the final concentration of AuNCs is 47-141 mg / L;

[0026] In step 4, the concentration ratio of AuNPs to AuNCs in the third mixed solution is 1.2:1 to 0.4:1.

[0027] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, in step 4, the Ag in the third mixed solution is + The final concentration of is 10 μM to 1 mM, and the final concentration of cysteine ​​is 0 to 100 μM.

[0028] Preferably, in step 4, water is added to dilute the solution before forming the third mixed solution, and the Ag content in the third mixed solution obtained after dilution is + The final concentration of is 10 μM to 1 mM, and the final concentration of cysteine ​​is 0 to 100 μM.

[0029] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, the final concentration of cysteine ​​in the third mixed solution is 2 to 6 μM.

[0030] In the above-mentioned method for detecting cysteine ​​based on silver ions and gold nanoparticles, preferably, the absorbance value is the absorbance at 520 nm measured by ultraviolet-visible absorption method.

[0031] Beneficial effects:

[0032] The present invention provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles. The method utilizes the fact that cysteine ​​has a sulfhydryl group and can react with Ag. + The nature of the binding allows for qualitative or quantitative detection of cysteine.

[0033] This method creatively provides a mixed solution of AuNPs-AuNCs. EDTA reduces chloroauric acid to obtain a red AuNP solution, and histidine reduces chloroauric acid to obtain a colorless AuNCs solution. Neither of the two solutions alone can be used in the presence of Ag. + The mixed solution of the two can change color under the action of Ag + After the addition of cysteine, the Ag + The binding prevents the aggregation and discoloration of AuNPs, causing the mixed solution to change color. By observing the color, qualitative analysis of cysteine ​​can be performed. Within a certain range of cysteine ​​concentrations, the absorbance at 520 nm in the mixed solution exhibits a linear change. By measuring the absorbance at this point in the test solution and comparing it with a standard curve, quantitative analysis of the cysteine ​​solution can be achieved.

[0034] The detection method is simple to operate. It only requires the preparation of AuNPs solution, AuNCs solution, Ag + The solution is mixed with the cysteine ​​sample to be tested, and allowed to stand for 1 hour before colorimetric inspection with the naked eye. For precise content determination, the simple UV-Vis method can be used for quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings and the accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. Among them:

[0036] Figure 1 Ag in the embodiment of the present invention + Schematic diagram of cysteine ​​detection after adding AuNPs-AuNCs mixed solution;

[0037] Figure 2 The UV-Vis absorption spectra of AuNPs in the third mixed solution of Example 1 of the present invention at different cysteine ​​concentrations;

[0038] Figure 3 The color change of the third mixed solution of Example 1 of the present invention at different cysteine ​​concentrations;

[0039] Figure 4 The standard curve of cysteine ​​concentration versus absorbance at 520 nm in Example 1 of the present invention;

[0040] Figure 5 The UV-visible absorption spectra and solution colors of the interaction between different solutions and AuNPs in Experimental Example 1 of the present invention are shown in Figure 2. Figure 5 A is the UV-visible absorption spectra of the interaction between different solutions and AuNPs; Figure 5B From left to right, the solutions are AuNPs solution, AuNPs+Ag + solution, AuNPs+AuNCs+Ag + solution, AuNPs+AuNCs solution, AuNCs solution);

[0041] Figure 6 TEM spectra and solution colors of different solutions of Experimental Example 2 of the present invention ( Figure 6 A is the TEM spectrum of AuNPs solution; Figure 6 B is AuNPs+AuNCs+Ag + TEM spectrum of the solution; Figure 6 C is AuNPs+AuNCs+Cys+Ag + TEM spectrum of the solution; Figure 6 The solutions from left to right in D are AuNPs+AuNCs+Ag + solution, AuNPs+AuNCs+Cys+Ag + solution, AuNPs solution). DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention are within the scope of protection of the present invention.

[0043] The present invention will be described in detail below with reference to the embodiments. It should be noted that the embodiments and features of the embodiments of the present invention can be combined with each other without conflict.

[0044] The present invention provides a cysteine ​​detection method based on silver ions and gold nanoparticles, which is mainly based on the method of producing color changes and UV-Vis absorption curve changes caused by the aggregation of gold nanoparticles (AuNPs) and is used for the qualitative and quantitative determination of cysteine ​​content.

[0045] The basic principle of the present invention is that ethylenediaminetetraacetic acid (EDTA) reduces chloroauric acid (HAuCl4) to generate a red gold nanoparticle (AuNPs) solution with a maximum absorbance at 520nm; histidine reduces chloroauric acid to generate a colorless gold cluster (AuNCs) solution. + Ag is present, the solution turns blue, and the absorbance at 520 nm decreases. + The characteristics of the combination, free Ag in the mixed solution +The amount of cysteine ​​is reduced, thereby reducing the aggregation degree of AuNPs. The color of the mixed solution changes significantly from blue to red as the cysteine ​​content changes (the content remains unchanged and increases). The absorbance value at 520nm changes linearly within a certain range, and a clear absorption peak appears at 650nm. The cysteine ​​content is determined based on the color change of the mixed solution and the change of the UV-visible absorption curve. When the cysteine ​​concentration in the mixed solution is in the range of 2 to 6 μM, there is a linear relationship between the cysteine ​​concentration and the absorbance value at 520nm, thereby achieving quantitative detection of the cysteine ​​concentration. The Ag of the present invention + The principle diagram of cysteine ​​detection after adding AuNPs-AuNCs mixed solution is shown in the figure. Figure 1 shown.

[0046] In addition to using EDTA as a reducing agent, there are also methods for preparing AuNPs solutions using reducing agents such as citric acid. Similarly, in addition to using histidine, bovine serum albumin (BSA) can also be used to prepare AuNCs. However, after mixing AuNPs and AuNCs prepared with other reducing agents, the + After adding AuNPs-AuNCs mixed solution, no AuNPs aggregation phenomenon is induced, that is, no obvious color change and absorbance curve change. + Mediated cysteine ​​detection.

[0047] The present invention provides a mixed solution containing AuNPs and AuNCs. The solution is prepared by using HAuCl4 as a gold source and EDTA as a reducing agent to prepare an AuNPs solution; using HAuCl4 as a gold source and histidine as a reducing agent to prepare an AuNCs solution; the above two solutions are mixed in a certain proportion to obtain a mixed solution of AuNPs and AuNCs. AuNCs improve the AuNPs' ability to absorb Ag. + Sensitivity, Ag + The present invention also provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles, wherein the reagent required by the method is the AuNPs-AuNCs mixed solution of the above technical solution.

[0048] The present invention also provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles, the detection method comprising the following steps:

[0049] First, it is explained that the EDTA solution, HAuCl4 solution, histidine solution, cysteine ​​solution and Ag used in the present invention are + Ethylenediaminetetraacetic acid tetrasodium salt (EDTA·4Na), HAuCl4, histidine, cysteine, and AgNO3 were dissolved in deionized water at room temperature, and the solutions were stirred until completely dissolved, thereby obtaining five solutions.

[0050] Step 1: Mix the EDTA solution and the HAuCl4 solution to obtain a first mixed solution, and take the supernatant for later use to obtain a gold nanoparticle solution, namely, an AuNPs solution.

[0051] In a specific embodiment of the present invention, the first mixed solution is stirred at 20-40° C. (eg, 25° C., 30° C., 35° C.) for 30 min, and then centrifuged at 10,000 rpm for 5 minutes to obtain a supernatant for later use.

[0052] In a specific embodiment of the present invention, in step 1, the molar concentration ratio of HAuCl4 to EDTA in the first mixed solution is 1:7 to 1:10 (e.g., 1:8, 1:8.5, 1:9, 1:9.5). The concentration of HAuCl4 in the first mixed solution is 1 to 50 mM (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM), and the corresponding concentration of EDTA is 7 to 500 mM (e.g., 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM, 400 mM).

[0053] In a specific embodiment of the present invention, in step 1, the concentration of the AuNPs solution is 0.02 to 1.0 g / L (e.g., 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, or 0.8 g / L) in terms of Au. If the concentration of AuNPs is too low, the sensitivity of detection will decrease, and the detection limit for cysteine ​​will increase. If the concentration is too high, the AuNPs will easily aggregate, causing the reaction to occur too quickly and making quantitative detection difficult.

[0054] Step 2: Mix the histidine solution and the HAuCl4 solution to obtain a second mixed solution, and stir to obtain a gold cluster solution, namely, an AuNCs solution.

[0055] In a specific embodiment of the present invention, the second mixed solution is stirred at room temperature for 1-2 hours to obtain a gold cluster solution;

[0056] Histidine is L-histidine, D-histidine or racemic histidine or a mixture of L- and D-histidine in any ratio.

[0057] In a specific embodiment of the present invention, in step 2, the molar concentration ratio of HAuCl4 to histidine in the second mixed solution is 1:20 to 1:50 (e.g., 1:30, 1:35, 1:40, 1:45); the concentration of HAuCl4 in the second mixed solution is 1 to 50 mM (e.g., 5 mM, 10 mM, 20 mM, 30 mM, 40 mM), and the concentration of histidine is 0.02 to 25 M (e.g., 0.05 M, 0.1 M, 0.5 M, 1 M, 5 M, 10 M, 15 M, 20 M).

[0058] In a specific embodiment of the present invention, in step 2, the concentration of AuNCs, calculated as Au, is 0.01 to 1.0 g / L (e.g., 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, or 0.8 g / L). A concentration of AuNCs that is too low can reduce detection sensitivity and increase the detection limit for cysteine, while a concentration that is too high can prematurely cause AuNC aggregation, making it impossible to detect the sample.

[0059] Step 3: Mix the AuNPs solution and the AuNCs solution to form an AuNPs-AuNCs mixed solution.

[0060] In a specific embodiment of the present invention, in step 4, the final concentration of AuNPs in the third mixed solution is 43 mg / L, and the final concentration of AuNCs is 47-141 mg / L (e.g., 50 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L).

[0061] In step 4, the concentration ratio of AuNPs to AuNCs in the third mixed solution is 1.2:1 to 0.4:1 (e.g., 0.5:1, 0.6:1, 0.8:1, 1.0:1). A high ratio, i.e., an excessively high content of AuNPs, can lead to aggregation and precipitation of the mixed solution. A low ratio, i.e., an excessively low content of AuNPs, can result in insufficient sensitivity.

[0062] Step 4: + The cysteine ​​solution to be tested is added to the solution, and then added to the AuNPs-AuNCs mixed solution to obtain a third mixed solution, which is allowed to stand at room temperature, and the color and / or absorbance value are observed.

[0063] In a specific embodiment of the present invention, in step 4, the Ag in the third mixed solution + The final concentration of silver ions is 10 μM to 1 mM. If the concentration of silver ions is too low, the AuNPs-AuNCs mixed solution will not turn blue. If the concentration is too high, it will cause the aggregation of AuNPs and the sensitivity will be reduced, affecting the detection of cysteine ​​(Cys). The final concentration of cysteine ​​is 0 to 100 μM (for example, 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM). The final concentration of cysteine ​​within this range can be beneficial for color comparison or absorbance value comparison.

[0064] Preferably, in step 4, water is added to dilute the solution before forming the third mixed solution, and the Ag content in the third mixed solution obtained after dilution is +The final concentration of is 10 μM to 1 mM (e.g., 100 μM, 200 μM, 400 μM, 600 μM, 800 μM), and the final concentration of cysteine ​​is 0 to 100 μM (e.g., 1 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM).

[0065] In a specific embodiment of the present invention, the final concentration of cysteine ​​in the third mixed solution is 2 to 6 μM (e.g., 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM). Within this range, the concentration of cysteine ​​is linearly related to the absorbance value and can be compared with a standard curve for quantitative detection of the cysteine ​​solution to be tested.

[0066] Step 5: Compare the absorbance value obtained in step 4 with the absorbance value-cysteine ​​standard curve to obtain the concentration of the cysteine ​​solution to be tested.

[0067] In a specific embodiment of the present invention, the absorbance value is the absorbance at 520 nm measured by ultraviolet-visible absorption method.

[0068] Example 1

[0069] This embodiment provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles, comprising the following steps:

[0070] Step 1: Mix the EDTA solution and the HAuCl4 solution to obtain a first mixed solution, stir the first mixed solution at 30°C for 30 minutes, and then centrifuge at 10,000 rpm for 5 minutes to obtain a supernatant for standby use, thereby obtaining a gold nanoparticle (AuNPs) solution.

[0071] In the first mixed solution, the concentration of HAuCl4 was 1.25 mM, the corresponding concentration of EDTA was 10 mM, and the concentration of the AuNPs solution in terms of Au was 0.25 g / L.

[0072] Step 2: Mix the L-histidine solution and the HAuCl4 solution to obtain a second mixed solution, and stir the second mixed solution at room temperature for 1 hour to obtain a gold cluster solution, namely, an AuNCs solution.

[0073] The molar concentration ratio of HAuCl4 to histidine in the second mixed solution was 1:30; the concentration of HAuCl4 in the second mixed solution was 2.9 mM, and the concentration of histidine was 0.086 M. The concentration of AuNCs in terms of Au was 0.47 g / L.

[0074] Step 3: Mix the AuNPs solution and the AuNCs solution to form an AuNPs-AuNCs mixed solution.

[0075] Step 4: Add 11 cysteine ​​solutions to the AgNO3 solution, and then add it to the AuNPs-AuNCs mixed solution to obtain a third mixed solution. Let it stand at room temperature, observe the color of each solution and measure the absorbance at 520nm. The final concentration of AuNPs in the third mixed solution is 43mg / L, the final concentration of AuNCs is 141mg / L, and the final concentration of AgNO3 is 43mg / L. + The final concentration of all was 10 μM, and the final concentration of cysteine ​​was 0 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM, respectively.

[0076] The color and absorbance values ​​of the 11 samples measured in this Example 1 are as follows: Figure 2 and Figure 3 As shown, Figure 2 The UV-Vis absorption spectra of AuNPs at different cysteine ​​concentrations in the mixed solution are shown in Figure 2. The absorbance at 520 nm increases with the increase of cysteine ​​concentration. There is a linear relationship between the absorbance value and the cysteine ​​concentration in the range of 2 to 6 μM. Figure 4 As shown, the regression equation is y = 43.27x + 0.6656, R 2 =0.9963.

[0077] Figure 3 The middle shows the third mixed solution. As the cysteine ​​concentration continues to increase, the solution color changes. From left to right, the cysteine ​​concentrations are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM, respectively. The solution on the far right is the AuNPs-AuNCs mixed solution.

[0078] Example 2

[0079] In this example, L-histidine in step 2 was replaced with D-histidine, and the other steps were the same as those in Example 1, which will not be repeated here.

[0080] The color of the third mixed solution prepared in this example was observed and the absorbance value was measured. The performance results and experimental data were the same as those in Example 1.

[0081] Example 3

[0082] In this example, L-histidine in step 2 was replaced with racemic histidine, and the other steps were the same as those in Example 1, which will not be described again.

[0083] The color of the third mixed solution prepared in this example was observed and the absorbance value was measured. The performance results and experimental data were the same as those in Example 1.

[0084] Example 4

[0085] In the first mixed solution in step 1 of this embodiment, the concentration of HAuCl4 is 1.5 mM, the corresponding concentration of EDTA is 12 mM, and the concentration of the AuNPs solution in terms of Au is 0.30 g / L.

[0086] In step 4, the third mixed solution was obtained after dilution with water, and the solution was allowed to stand at room temperature. The color of each solution was observed and the absorbance at 520 nm was measured. The final concentration of AuNPs in the third mixed solution after dilution with water was 43 mg / L, the final concentration of AuNCs was 141 mg / L, and the final concentration of Ag + The final concentration of each was 10 μM; other steps were the same as those in Example 1 and will not be repeated here.

[0087] The color of the third mixed solution prepared in this example was observed and the absorbance value was measured. The performance results and experimental data were the same as those in Example 1.

[0088] Example 5

[0089] In the first mixed solution in step 2 of this embodiment, the molar concentration ratio of HAuCl4 to histidine in the second mixed solution was 1:50; the concentration of HAuCl4 in the second mixed solution was 7 mM, and the concentration of histidine was 0.35 M. The concentration of AuNCs in terms of Au was 0.60 g / L.

[0090] In step 4, the third mixed solution was obtained after dilution with water, and the solution was allowed to stand at room temperature. The color of each solution was observed and the absorbance at 520 nm was measured. The final concentration of AuNPs in the third mixed solution after dilution with water was 43 mg / L, the final concentration of AuNCs was 141 mg / L, and the final concentration of Ag + The final concentration of each was 10 μM; other steps were the same as those in Example 1 and will not be repeated here.

[0091] The color of the third mixed solution prepared in this example was observed and the absorbance value was measured. The performance results and experimental data were the same as those in Example 1.

[0092] Example 6

[0093] This example provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles. Steps 1 to 3 are the same as those in Example 1. In step 4, the cysteine ​​solution to be tested (100 μM) is diluted 20 times with water, and then an AgNO3 solution is added. The solution is then added to the AuNPs-AuNCs mixed solution to obtain a third mixed solution. The solution is allowed to stand at room temperature, and the color of the solution is observed and the absorbance at 520 nm is measured. The final concentration of AuNPs in the third mixed solution is 43 mg / L, the final concentration of AuNCs is 141 mg / L, and the AgNO3 solution is 0.05. + The final concentration was 10 μM.

[0094] The absorbance value of the third mixed solution at 520 nm was determined to be 0.881. Compared with the standard curve in Example 1, the cysteine ​​concentration in the third mixed solution was 4.98 μM. The cysteine ​​solution to be tested was 99.6 μM, and the quantitative detection accuracy reached 99.6%.

[0095] Experimental Example 1

[0096] Ag + Induce aggregation of AuNPs-AuNCs mixed solution.

[0097] The following five solutions were prepared respectively: AuNPs solution, AuNCs solution, AuNPs+Ag + solution, AuNPs-AuNCs solution, AuNPs-AuNCs+Ag + The solutions were allowed to stand at room temperature for 1 hour, and then the UV-Vis absorption spectra of the five solutions were measured.

[0098] The concentration of AuNPs calculated as Au was 43 mg / L, and the concentration of AuNCs calculated as Au was 141 mg / L;

[0099] AuNPs+Ag + The final concentration of AuNPs in the mixed solution was 43 mg / L. + The final concentration was 10 μM.

[0100] AuNPs-AuNCs+Ag + The final concentration of AuNPs in the mixed solution was 43 mg / L, the final concentration of AuNCs in the mixed solution was 141 mg / L, and the + The final concentration was 10 μM.

[0101] Figure 5 A is the UV-visible absorption spectra of the interaction between different solutions and AuNPs; Figure 5 B From left to right, the solutions are AuNPs solution, AuNPs+Ag + solution, AuNPs+AuNCs+Ag + solution, AuNPs+AuNCs solution, and AuNCs solution.

[0102] Figure 5As can be seen from the figure, the AuNPs solution is red, and its maximum UV-visible absorption curve has a maximum absorption peak at 520nm. The AuNCs solution is colorless, and its maximum UV-visible absorption curve has a maximum absorption peak at 255nm. The AuNPs-AuNCs solution obtained by mixing the two has two absorption peaks at 520nm and 255nm. Its absorption curve is basically equivalent to the superposition of the absorption curves of the two individual solutions. The solution color is red, which is consistent with the color of the individual AuNPs solution. + After that, the solution is still red, and the maximum absorption peak is at 520nm, indicating that Ag + It cannot cause AuNPs to aggregate. When the AuNPs-AuNCs mixed solution and Ag + After the solution was mixed, the absorbance at 520 nm decreased rapidly, an absorption peak appeared at 650 nm, and the solution color turned blue. This indicated that the AuNPs-AuNCs mixed solution + Aggregation occurs under the action of

[0103] Experimental Example 2

[0104] Feasibility of AuNPs-AuNCs solution system for cysteine ​​detection.

[0105] Prepare three solutions:

[0106] (1) Ethylenediaminetetraacetic acid tetrasodium salt solution (EDTA·4Na) was mixed with HAuCl4 solution, stirred vigorously at 30°C for 30 minutes, and then centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected for later use, i.e., AuNPs solution (Solution A).

[0107] (2) The L-histidine solution was mixed with the HAuCl4 solution and stirred at room temperature for 1 hour to obtain the AuNCs solution.

[0108] The above two solutions were mixed to obtain an AuNPs-AuNCs mixed solution, which was then mixed with AgNO3 to obtain solution B. Solution B contained an AuNPs concentration of 43 mg / L, an AuNCs concentration of 141 mg / L, and an AgNO3 concentration of 1. + The concentration is 10 μM.

[0109] (3) The mixed solution of cysteine ​​solution and AgNO3 was added to the AuNPs-AuNCs mixed solution and allowed to stand at room temperature for 1 hour to obtain solution C. Solution C contained AuNPs concentration of 43 mg / L, AuNCs concentration of 141 mg / L, and AgNO3. + The concentration was 10 μM and the cysteine ​​concentration was 10 μM.

[0110] Transmission electron microscopy was performed on solution A, solution B, and solution C in the embodiment of the present invention, respectively. Figure 4As shown in the electron microscopy image, it can be seen that the AuNPs in solution A are well dispersed; in solution B, due to the addition of Ag + and AuNCs, causing the AuNPs to aggregate; the AuNPs in solution C did not aggregate because the thiol group in cysteine + Binding, blocking Ag + induced aggregation of AuNPs.

[0111] Figure 6 A is the TEM spectrum of AuNPs solution, Figure 6 B is AuNPs+AuNCs+Ag + TEM spectrum of the solution, Figure 6 C is AuNPs+AuNCs+cysteine ​​(Cys)+Ag + TEM spectrum of the solution, Figure 6 The solutions from left to right in D are AuNPs+AuNCs+Ag + Solution, AuNPs+AuNCs+cysteine ​​(Cys)+Ag + solution, AuNPs solution.

[0112] Comparative Example 1

[0113] The difference between this comparative example and Example 1 is that in step 1, citric acid solution and HAuCl4 solution are mixed to obtain a first mixed solution. The other steps and methods are the same as those in Example 1 and are not repeated here.

[0114] The third mixed solution obtained, namely AuNPs+AuNCs+cysteine ​​(Cys)+Ag + The color of the solution remains blue and does not change.

[0115] Comparative Example 2

[0116] The difference between this comparative example and Example 1 is that in step 2, a bovine serum albumin solution and a HAuCl4 solution are mixed to obtain a second mixed solution. The other steps and methods are the same as those in Example 1 and are not described again here.

[0117] The third mixed solution obtained, namely AuNPs+AuNCs+cysteine ​​(Cys)+Ag + The color of the solution remains blue and does not change.

[0118] In summary, the present invention provides a method for detecting cysteine ​​based on silver ions and gold nanoparticles. The method utilizes the sulfhydryl group of cysteine ​​to react with Ag. + The nature of the binding allows for qualitative or quantitative detection of cysteine.

[0119] This method creatively provides a mixed solution of AuNPs-AuNCs. EDTA reduces chloroauric acid to obtain a red AuNP solution, and histidine reduces chloroauric acid to obtain a colorless AuNCs solution. Neither of the two solutions alone can be used in the presence of Ag. + The mixed solution of the two can change color under the action of Ag + After the addition of cysteine, the Ag + The binding prevents the aggregation and discoloration of AuNPs, causing the mixed solution to change color. By observing the color, qualitative analysis of cysteine ​​can be performed. Within a certain range of cysteine ​​concentrations, the absorbance at 520 nm in the mixed solution exhibits a linear change. By measuring the absorbance at this point in the test solution and comparing it with a standard curve, quantitative analysis of the cysteine ​​solution can be achieved.

[0120] The detection method is simple to operate. It only requires the preparation of AuNPs solution, AuNCs solution, Ag + The solution is mixed with the cysteine ​​sample to be tested, and allowed to stand for 1 hour before colorimetric inspection with the naked eye. For precise content determination, the simple UV-Vis method can be used for quantitative detection.

[0121] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for detecting cysteine ​​based on silver ions and gold nanoparticles, characterized in that: The detection method comprises the following steps: Step 1, mixing an EDTA solution and a HAuCl4 solution to obtain a first mixed solution, taking the supernatant for later use, and obtaining a gold nanoparticle solution, i.e., an AuNPs solution; Step 2: mixing the histidine solution and the HAuCl4 solution to obtain a second mixed solution, and stirring to obtain a gold cluster solution, i.e., an AuNCs solution; Step 3, mixing the AuNPs solution and the AuNCs solution to form an AuNPs-AuNCs mixed solution; Step 4: + Adding the cysteine ​​solution to be tested to the solution, and then adding it to the AuNPs-AuNCs mixed solution to obtain a third mixed solution, standing it at room temperature, and observing the color and / or measuring the absorbance value; In step 1, the molar concentration ratio of HAuCl4 to EDTA in the first mixed solution is 1:7 to 1:10; The concentration of HAuCl4 in the first mixed solution is 1~50mM, and the concentration of EDTA is 7~500mM; In step 1, the concentration of the AuNPs solution in terms of Au is 0.02–1.0 g / L; In step 2, the molar concentration ratio of HAuCl4 to histidine in the second mixed solution is 1:20 to 1:50; The concentration of HAuCl4 in the second mixed solution is 1~50mM, and the concentration of histidine is 0.02~2.5M; In step 2, the concentration of AuNCs calculated as Au is 0.01-1.0 g / L; In step 4, the Ag in the third mixed solution + The final concentration of is 10 μM~1 mM, and the final concentration of cysteine ​​is 0~100 μM; In step 4, water is added to dilute the solution before forming the third mixed solution. The Ag in the third mixed solution obtained after dilution is + The final concentration of is 10μM~1mM, and the final concentration of cysteine ​​is 0~100μM.

2. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1, wherein The detection method further comprises: Step 5: Compare the absorbance value obtained in step 4 with the absorbance value-cysteine ​​standard curve to obtain the concentration of the cysteine ​​solution to be tested.

3. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1 or 2, wherein: The steps of step 1 are specifically as follows: The first mixed solution was stirred at 20-40° C. for 30 min, and then centrifuged to obtain a supernatant for later use.

4. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1 or 2, wherein: The steps of step 2 are specifically as follows: The second mixed solution was stirred at room temperature for 1-2 hours to obtain a gold cluster solution; The histidine is L-histidine, D-histidine or racemic histidine or a mixture of L- and D-histidine in any proportion.

5. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1 or 2, wherein: In step 4, the final concentration of AuNPs in the third mixed solution is 43 mg / L, and the final concentration of AuNCs is 47-141 mg / L; In step 4, the concentration ratio of AuNPs to AuNCs in the third mixed solution is 1.2:1 to 0.4:

1.

6. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1, wherein: In the third mixed solution, the final concentration of cysteine ​​is 2-6 μM.

7. The method for detecting cysteine ​​based on silver ions and gold nanoparticles according to claim 1 or 2, wherein: The absorbance value is the absorbance at 520 nm measured by UV-visible absorption method.

Images