A fluorescence sensing method for detecting vitamin B1 based on single-stranded DNA gold nanoclusters

Abstract

The present invention discloses a method for detecting vitamin B1 using fluorescence sensing using single-stranded DNA gold nanoclusters. Using gold nanoclusters synthesized using single-stranded A30-ssDNA as a template as fluorescent probes, this method enables label-free, rapid, highly sensitive, and highly specific quantitative detection of vitamin B1 content in complex systems. Specifically, gold nanoclusters synthesized using single-stranded A30-ssDNA as a template serve as fluorescent probes, specifically detecting vitamin B1 content in a solution through changes in fluorescence intensity. This method is simple, rapid, and possesses a wide linear range, a low limit of detection, good sensitivity, and specificity, and has promising application prospects in food, pharmaceuticals, and other areas.

Description

Technical Field

[0001] The present invention relates to a method for detecting vitamin B1, and in particular to a method for detecting vitamin B1 based on a fluorescence sensing method using single-stranded DNA gold nanoclusters, belonging to the field of biological detection technology. Background Art

[0002] Vitamin B1 (VB1), also known as thiamine, consists of an aminopyrimidine ring and a thiazole ring connected by a methylene group, with methyl and hydroxyethyl side chains on each ring, respectively. VB1 is an essential water-soluble vitamin and natural nutrient found in dairy products, with an average daily intake of 0.5 to 1 mg. It plays a vital role in the metabolism of carbohydrates, amino acids, and lipids in living systems. Furthermore, as a biologically and pharmaceutically important compound, VB1 is essential for maintaining the normal function of the heart, nervous system, and cardiovascular system. VB1 deficiency can have numerous adverse effects on humans, most notably leading to beriberi (Wernicke-Korsakoff syndrome). Therefore, VB1 is primarily used clinically to treat beriberi and various polyneuritises.

[0003] Currently, a variety of methods have been developed to measure VB1, including thin-layer chromatography, capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), spectrophotometry, and fluorescence spectroscopy. However, complex instrumentation and sample pretreatment often limit the application of these methods. Some methods also exhibit narrow linear ranges and are susceptible to interference from coexisting ions. Therefore, the development of a simple, sensitive, low-cost, efficient, and selective method for the detection of VB1 remains of great significance. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a method for detecting vitamin B1 by fluorescence sensing based on single-stranded DNA gold nanoclusters. Gold nanoclusters synthesized using single-stranded A30-ssDNA as a template are used as fluorescent probes. Based on the fact that VB1 can induce fluorescence quenching of fluorescent gold nanoclusters, a fluorescence sensing method for detecting VB1 is constructed.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] 1. A method for preparing a fluorescent probe, comprising uniformly mixing a DNA solution, a sodium citrate solution, a chloroauric acid solution, and ultrapure water to obtain a mixed solution, and then heating and incubating the mixture to obtain a fluorescent gold nanocluster solution, namely the fluorescent probe; wherein the DNA solution is obtained by dissolving single-stranded A30-ssDNA in ultrapure water, and the nucleotide sequence of the single-stranded A30-ssDNA is shown in SEQ ID NO. 1.

[0007] 5′-AAA AAAAAAAAAAAAAAAAAA AAAAAAAAA-3′, as shown in SEQ ID NO. 1, purified by HPLC.

[0008] Preferably, the volume ratio of DNA solution, sodium citrate solution, chloroauric acid solution and ultrapure water is 5:5:3:37, and the concentrations of DNA solution, sodium citrate solution and chloroauric acid solution are 2 μmol / L, 50 mmol / L (pH=6.0) and 1 mmol / L, respectively.

[0009] Preferably, the process conditions for heating and incubating are: incubating at 90° C. for 30 minutes.

[0010] 2. A fluorescent probe obtained by the above preparation method.

[0011] 3. Application of the above fluorescent probe in the quantitative detection of vitamin B1.

[0012] 4. Application of the above fluorescent probe in the quantitative detection of vitamin B1.

[0013] 5. A method for detecting vitamin B1 based on fluorescence sensing of single-stranded DNA gold nanoclusters, comprising adding VB1 solution to a centrifuge tube containing the aforementioned fluorescent probe, mixing and incubating, and using a fluorescence spectrophotometer to measure the fluorescence intensity before and after the addition of the VB1 solution to determine the VB1 content in the solution.

[0014] Preferably, PB buffer (phosphate buffer) with a pH of 6.5 is also added to the centrifuge tube, the volume ratio of VB1 solution, fluorescent probe, and PB buffer is 3:1:1, and the concentration of VB1 solution is in the range of 0.01 to 1 μmol / L.

[0015] Preferably, the process conditions for mixed incubation are: incubation at room temperature (25° C.) for 10 minutes.

[0016] Preferably, the fluorescence intensity reduction value of the gold nanoclusters is linearly related to the concentration of VB1, and the linear regression equation is y=437.16x+18.034, wherein x is the concentration of VB1, y is the fluorescence intensity reduction value, and the linear regression coefficient R is 2 The detection limit was 0.9935 and 3 nmol / L.

[0017] Beneficial effects of the present invention:

[0018] This method uses gold nanoclusters synthesized using single-stranded A30-ssDNA as a fluorescent probe for label-free, rapid, highly sensitive, and highly specific quantitative detection of VB1 content in complex systems. Specifically, the gold nanoclusters synthesized using single-stranded A30-ssDNA as a fluorescent probe specifically detect VB1 content in a solution through changes in fluorescence intensity. This method is simple, rapid, and offers a wide linear range, a low limit of detection, and excellent sensitivity and specificity, promising promising applications in food, pharmaceuticals, and other fields. The details are as follows:

[0019] (1) The synthesized gold nanoclusters have stable optical properties. The synthesis method is simple, fast, low-cost, and the synthesized materials have good fluorescence properties.

[0020] (2) The synthesized gold nanoclusters with unique optical properties are used as fluorescent probes to sense and detect the content of VB1 with high specificity. The operation is simple and rapid, and the specific detection of VB1 can be directly achieved.

[0021] Gold nanoclusters, a new type of functional nanoparticle, have broad application prospects as fluorescent probes in biosensing due to their simple synthesis, excellent photostability, and good dispersibility in aqueous solutions. This paper uses gold nanoclusters as fluorescent probes to achieve label-free, rapid, and quantitative detection of VB1 in solution.

[0022] Fluorescence metal nanoanalysis methods have the advantages of simple operation, fast speed, high sensitivity and good selectivity, and have attracted widespread research interest among researchers.

[0023] (3) After adding VB1 into the gold nanocluster system, the fluorescence intensity of the gold nanoclusters decreased. This method can achieve quantitative detection of VB1 with a wide linear detection range and a low detection limit.

[0024] (4) The present invention can conveniently detect the VB1 content in pharmaceutical samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the preparation of gold nanoclusters.

[0026] Figure 2 The fluorescence excitation and emission spectra of gold nanoclusters synthesized using single-stranded A30-ssDNA as a template show that the maximum excitation wavelength is 290 nm and the maximum emission wavelength is 475 nm.

[0027] Figure 3 This is the fluorescence spectrum of fluorescent gold nanoclusters.

[0028] Figure 4This is an optimization diagram of different pH values ​​of the buffer solution for the gold nanocluster detection VB1 reaction system. The pH values ​​of the buffer solution are: 6.0, 6.5, 7.0, 7.5, and 8.0.

[0029] Figure 5 This is an optimization diagram of the gold nanocluster detection VB1 reaction system with different incubation times of adding VB1, and the incubation times are: 5, 10, 15, 20, and 25 minutes.

[0030] Figure 6 This is a linear graph of gold nanoclusters detecting VB1 in solution, with a linear range of 0.01 to 1 μmol / L and a detection limit of 3 nmol / L. Among them, A is the emission spectra of different concentrations of VB1, and the concentrations of VB1 are: 0, 0.01, 0.03, 0.05, 0.08, 0.25, 0.4, 0.6, 0.8, 1, 2, 4, and 5 μmol / L. B is the calibration curve of fluorescence intensity at 475 nm and VB1 concentration.

[0031] Figure 7 This is a graph showing the specific analysis results of the gold nanoclusters detecting VB1 in the solution. DETAILED DESCRIPTION

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0033] All reagents used in this study were of analytical grade and were from the following manufacturers: vitamins, trisodium citrate dihydrate, and HAuCl₄·3H₂O (analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); phosphate buffer (PB) was used throughout the experiment, and ultrapure water (conductivity >18.25 MΩ) was used. DNA sequence: A30-ssDNA: 5′-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′, HPLC-purified, Shanghai Shenggong Bioengineering Co., Ltd.

[0034] Example 1

[0035] like Figure 1 As shown, the preparation of gold nanoclusters synthesized using A30-ssDNA as a template was carried out according to the following steps:

[0036] (1) Preparation of 50 mmol / L sodium citrate solution with pH = 6.0: Dissolve 0.1470 g of trisodium citrate dihydrate in ultrapure water, then dilute to 10 mL with ultrapure water, adjust the pH to 6.0 and set aside.

[0037] (2) Preparation of 1 mmol / L chloroauric acid solution (HAuCl4): Add 0.0039 g of tetrachloroauric acid trihydrate to 10 mL with ultrapure water and store in the dark until ready for use.

[0038] (3) Preparation of DNA solution: Dissolve 1OD of lyophilized DNA powder in 1375μL of ultrapure water to 2μmol / L, mix thoroughly and set aside.

[0039] (4) Preparation of gold nanoclusters: 100 μL DNA solution, 100 μL sodium citrate solution, 60 μL chloroauric acid solution and 740 μL ultrapure water were mixed to make the total volume of the system solution 1000 μL, and then the mixed solution was placed in a constant temperature water bath at 90°C and incubated for 30 minutes to obtain a fluorescent gold nanocluster solution. The prepared fluorescent gold nanocluster solution was placed in a refrigerator at 4°C for later use. Fluorescence excitation spectrum and emission spectrum scanning were performed on a fluorescence spectrophotometer ( Figure 2 ), indicating that the maximum excitation wavelength is 290 nm and the maximum emission wavelength is 475 nm.

[0040] Example 2

[0041] 1. The preparation of gold nanoclusters synthesized using A30-ssDNA as a template was performed as described in Example 1.

[0042] 2. Feasibility analysis of using gold nanoclusters as fluorescent probes to detect VB1 in solution is characterized by the following steps:

[0043] (1) Preparation of VB1 mother solution: Dissolve 0.0033 g of vitamin B1 in ultrapure water, and then dilute to 10 mL with ultrapure water to prepare 1 mmol / L VB1 mother solution for use.

[0044] (2) Take 100 μL of fluorescent gold nanocluster solution and measure its fluorescence intensity using a fluorescence spectrophotometer. Scan the emission spectrum under excitation at an excitation wavelength of 290 nm. The probe shows strong emission at 475 nm.

[0045] (3) Add 20 μL of fluorescent gold nanocluster solution, 60 μL of 1 μmol / L VB1 solution and 20 μL of PB buffer (pH = 6.5) to the centrifuge tube, mix well, and react at room temperature for 10 minutes. The fluorescence intensity is measured using a fluorescence spectrophotometer. The fluorescence intensity is significantly reduced at this time. The reduction in the fluorescence emission spectrum intensity can be used to prove the feasibility of gold nanoclusters as fluorescent probes for detecting VB1 ( Figure 3 ).

[0046] Example 3

[0047] 1. The preparation of gold nanoclusters synthesized using A30-ssDNA as a template was performed as described in Example 1.

[0048] 2. Optimization of the pH value of the buffer solution in the reaction system for detecting VB1 using gold nanoclusters as fluorescent probes is characterized by the following steps:

[0049] (1) Add 20 μL of PB buffer (pH 6.0-8.0) with different pH values, 20 μL of fluorescent gold nanoclusters and 60 μL of ultrapure water to a 100 μL reaction system and mix well. After reacting at room temperature for 10 minutes, the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results showed that ( Figure 4 The blue curve shows that the fluorescence intensity of the gold nanoclusters does not change significantly in the solution pH range of 6.0 to 8.0.

[0050] (2) Add 20 μL of PB buffer (pH 6.0-8.0) with different pH values, 20 μL of fluorescent gold nanoclusters and 60 μL of 1 μmol / L VB1 sample to a 100 μL reaction system and mix well. After reacting at room temperature for 10 minutes, the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results showed that ( Figure 4 The red curve shows that the fluorescence response reaches its lowest at pH 6.5. This is because VB1 is unstable in an alkaline environment and decomposes when pH>7. Therefore, pH=6.5 is selected as the optimal buffer solution pH value.

[0051] Example 4

[0052] 1. The preparation of gold nanoclusters synthesized using A30-ssDNA as a template was performed as described in Example 1.

[0053] 2. Optimization of the VB1 incubation time in a reaction system using gold nanoclusters as fluorescent probes to detect VB1 is characterized by the following steps:

[0054] (1) Add 20 μL of fluorescent gold nanoclusters, 20 μL of PB buffer (pH 6.5) and 60 μL of ultrapure water to a 100 μL reaction system and mix well. After reacting at room temperature for 5, 10, 15, 20 and 25 minutes, the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results showed that ( Figure 5 Blue curve), time has little effect on the fluorescence intensity of gold nanoclusters.

[0055] (2) Add 20 μL of fluorescent gold nanoclusters, 20 μL of PB buffer (pH 6.5) and 60 μL of 1 μmol / L VB1 sample to a 100 μL reaction system and mix well. After reacting at room temperature for 5, 10, 15, 20 and 25 minutes, the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results showed that ( Figure 5 The red curve shows that the fluorescence intensity of the gold nanoclusters basically does not change after incubation for 10 minutes, indicating that the fluorescence quenching process of the gold nanoclusters induced by VB1 is completed within 10 minutes. Therefore, 10 minutes is selected as the optimal incubation time.

[0056] Example 5

[0057] 1. The preparation of gold nanoclusters synthesized using A30-ssDNA as a template was performed as described in Example 1.

[0058] 2. The content analysis of VB1 in the solution is detected using gold nanoclusters as fluorescent probes, which is characterized by the following steps:

[0059] 60 μL of VB1 samples of different concentrations, 20 μL of fluorescent gold nanoclusters and 20 μL of PB buffer (pH = 6.5) were added to a centrifuge tube and mixed evenly in a 100 μL reaction system. After reacting for 10 minutes at room temperature, the fluorescence intensity was measured using a fluorescence spectrophotometer. The experimental results showed that ( Figure 6 , where A is the emission spectra of different concentrations of VB1, and B is the calibration curve of fluorescence intensity at 475 nm and VB1 concentration. In the linear range of VB1 concentration of 0.01 to 1 μmol / L, the decrease in fluorescence intensity of gold nanoclusters is linearly related to the concentration of VB1. The linear regression equation is y=437.16x+18.034, and the linear regression coefficient R 2 The detection limit was 0.9935 and 3 nmol / L.

[0060] Example 6

[0061] 1. The preparation of gold nanoclusters synthesized using A30-ssDNA as a template was performed as described in Example 1.

[0062] 2. The configuration of VB1 solution refers to Example 2;

[0063] 3. Specific analysis of VB1 in solution using gold nanoclusters as fluorescent probes is characterized by the following steps:

[0064] (1) Vitamin B3, Vitamin B6, Vitamin B 12 And vitamin C solution preparation:

[0065] (2) Add 60 μL of vitamin B3 solution for specific analysis, 20 μL of fluorescent gold nanoclusters and 20 μL of PB buffer (pH = 6.5) to the centrifuge tube and mix them evenly. After reacting at room temperature for 10 minutes, the fluorescence intensity was measured using a fluorescence spectrophotometer. At this time, the fluorescence intensity of VB1 was significantly reduced, and the fluorescence intensity of other compounds did not change significantly compared with the blank control, which proved that the gold nanoclusters had good specificity as a fluorescent probe for detecting VB1 ( Figure 7 ).

[0066] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

[0067]

Claims

1. A method for detecting vitamin B1 based on fluorescence sensing of single-stranded DNA gold nanoclusters, characterized in that: Add VB1 solution to the centrifuge tube containing the fluorescent probe, and also add PB buffer (pH = 6.5) to the centrifuge tube, mix and incubate, and use a fluorescence spectrophotometer to measure the fluorescence intensity before and after the addition of VB1 solution to determine the VB1 content in the solution; The fluorescent probe is prepared as follows: a DNA solution, a sodium citrate solution, a chloroauric acid solution, and ultrapure water are uniformly mixed to obtain a mixed solution, and the mixed solution is heated and incubated to obtain a fluorescent gold nanocluster solution, which is the fluorescent probe; wherein the DNA solution is obtained by dissolving single-stranded A30-ssDNA in ultrapure water, and the nucleotide sequence of the single-stranded A30-ssDNA is shown in SEQ ID NO.

1.

2. The method according to claim 1, characterized in that The volume ratio of DNA solution, sodium citrate solution, chloroauric acid solution and ultrapure water is 5:5:3:37, and the concentrations of DNA solution, sodium citrate solution and chloroauric acid solution are 2 μmol / L, 50 mmol / L and 1 mmol / L, respectively.

3. The method according to claim 1, characterized in that The process conditions for heating incubation are: incubation at 90°C for 30 minutes.

4. The method according to claim 1, wherein the volume ratio of VB1 solution, fluorescent probe, and PB buffer is 3:1:1, and the concentration of VB1 solution is in the range of 0.01 to 1 μmol / L.

5. The method according to claim 1, wherein The process conditions for the mixed incubation are: incubation at room temperature for 10 minutes.

6. The method according to claim 1, characterized in that The decrease in fluorescence intensity of gold nanoclusters is linearly related to the concentration of VB1. The linear regression equation is y=437.16x+18.034, where x is the concentration of VB1, y is the decrease in fluorescence intensity, and the linear regression coefficient R 2 The detection limit was 0.9935 and 3 nmol / L.

7. Application of a fluorescent probe in the quantitative detection of vitamin B1, characterized in that: The fluorescent probe is prepared as follows: a DNA solution, a sodium citrate solution, a chloroauric acid solution, and ultrapure water are uniformly mixed to obtain a mixed solution, and the mixed solution is heated and incubated to obtain a fluorescent gold nanocluster solution, which is the fluorescent probe; wherein the DNA solution is obtained by dissolving single-stranded A30-ssDNA in ultrapure water, and the nucleotide sequence of the single-stranded A30-ssDNA is shown in SEQ ID NO.1.

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