Preparation of Glutathione-Modified Silver Nanotriangles and Their Application in Acidic Amino Acids

Abstract

The present invention discloses a preparation method of glutathione (GSH)-modified silver nanoplates (AgNTs) and its application in detecting acidic amino acids; the present invention ingeniously overcomes the defect that GSH as an etchant for AgNTs will cause the transformation of AgNTs from triangles to discs, and proposes a post-modification technique, that is, by controlling the concentration of GSH, GSH is stably modified on AgNTs, thereby obtaining GSH-modified AgNTs; this technique can not only modify sufficient GSH on the surface of AgNTs, but also maintain the good triangular morphology of AgNTs; furthermore, the electrostatic attraction between GSH on the surface of AgNTs and acidic amino acids can cause the aggregation of AgNTs, and the degree of aggregation is proportional to the concentration of acidic amino acids, so as to achieve the purpose of detecting acidic amino acids.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a preparation method of glutathione (GSH)-modified silver nanoplates (AgNTs) and their application in the detection of acidic amino acids. Background Art

[0002] In recent years, AgNTs have attracted extensive attention due to their unique local surface plasmon resonance properties and high sensitivity to shape. The three tips of AgNTs are easily etched and blunted due to their high activity. As one of the etching agents, biothiols cause the etching and blunting of the tips through the interaction of forming Ag-S bonds between -SH and AgNTs. As a member of biothiols, glutathione can also etch AgNTs. In the present invention, by utilizing the special branched molecular structure of GSH, GSH can be stably modified on the surface of AgNTs by controlling the concentration of GSH, and AgNTs are relatively dispersed and maintain a complete triangular morphology.

[0003] As is well known, proteins in organisms are composed of 20 essential amino acids, so amino acids play a key role in life activities. Amino acids can be divided into neutral amino acids, acidic amino acids, and basic amino acids according to the different side chain R groups. Acidic amino acids are amino acids containing two carboxyl groups, including aspartic acid (Asp) and glutamic acid (Glu). Research shows that acidic amino acids play an important role in the nervous system and brain function of organisms, and are related to diseases such as Alzheimer's disease, Parkinson's syndrome, epilepsy, and brain injury. Therefore, the rapid and efficient detection of acidic amino acids has important clinical value and application prospects. Summary of the Invention

[0004] To solve the above problems, the present invention discloses a preparation method and application of GSH-modified AgNTs. The preparation method of GSH-modified AgNTs is simple and easy to operate, and has strong repeatability. GSH-modified AgNTs can rapidly detect acidic amino acids.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] An object of the present invention is to provide a preparation method of a GSH-modified AgNTs solution, comprising the following steps:

[0007] (1) Preparation of AgNTs: Under vigorous stirring, an aqueous silver nitrate solution, an aqueous sodium citrate solution, an aqueous polyvinylpyrrolidone solution, an aqueous hydrogen peroxide solution, and an aqueous sodium borohydride solution are successively added to ultrapure water, and the colorless solution turns light yellow; as the reaction proceeds, the solution gradually changes from light yellow to yellow, orange, red, purple, blue, and finally a blue-green AgNTs solution is obtained.

[0008] (2) Preparation of GSH-modified AgNTs: Add the AgNTs solution obtained in step (1) to GSH and mix evenly, then incubate at room temperature for 30 - 50 min to obtain the GSH-modified AgNTs solution.

[0009] Furthermore, in step (1), the molar ratio of silver nitrate, trisodium citrate, polyvinylpyrrolidone, hydrogen peroxide, and sodium borohydride is 1 - 3:25 - 35:2 - 4:1000:20 - 30.

[0010] Furthermore, in step (1), the concentration of the silver nitrate aqueous solution is 25 - 75 mmol / L, the concentration of the trisodium citrate aqueous solution is 90 - 130 mmol / L, the concentration of the polyvinylpyrrolidone aqueous solution is 10 - 25 mmol / L, the mass fraction of hydrogen peroxide is 30%, and the concentration of the sodium borohydride aqueous solution is 80 - 120 mmol / L.

[0011] Furthermore, in step (2), the concentration of the GSH aqueous solution is 1.9 - 2.1 mmol / L.

[0012] Furthermore, in step (2), the volume ratio of the AgNTs solution to the GSH aqueous solution is 1:0.8 - 1.2.

[0013] The preparation method of the GSH-modified AgNTs of the present invention is simple and easy to implement, and has strong repeatability; the product has a uniform size distribution and a uniform morphology.

[0014] Another object of the present invention is to provide the application of the above GSH-modified AgNTs solution in detecting acidic amino acids.

[0015] Furthermore, the acidic amino acids are aspartic acid and glutamic acid.

[0016] Furthermore, the method for detecting acidic amino acids by the glutathione-modified silver nanoplates is: Mix the GSH-modified AgNTs solution with the aspartic acid aqueous solution and the glutamic acid aqueous solution respectively for 10 - 20 min, and use a UV-visible spectrophotometer to measure and record the data of their absorption spectra.

[0017] Furthermore, the concentration of aspartic acid is 0.5 - 1.5 mmol / L, and the concentration of glutamic acid is 0.5 - 2 mmol / L.

[0018] Furthermore, the volume ratio of the GSH-modified AgNTs solution to the amino acid aqueous solution is 1 - 3:1.

[0019] The beneficial effects of the present invention are:

[0020] (1) The present invention develops a preparation method of GSH-modified AgNTs, which overcomes the defect that low-concentration GSH will etch AgNTs. By controlling the concentration of GSH, GSH-modified AgNTs with a triangular morphology stably maintained can be obtained. The preparation method is simple and highly reproducible.

[0021] (2) The present invention can detect acidic amino acids by directly adding an aqueous solution of acidic amino acids to the GSH-modified AgNTs and mixing them. The detection steps are simple and rapid.

[0022] (3) The present invention directly measures the mixture of GSH-modified AgNTs and an aqueous solution of acidic amino acids by a UV-visible spectrophotometer, and quantitative detection of acidic amino acids can be achieved by analyzing the change in the position of the absorption peak, without the need for other expensive instruments.

[0023] (4) Using acidic amino acids as detection samples, the present invention verifies the detection performance of the GSH-AgNTs described in the present invention. The GSH-AgNTs can quantitatively detect two acidic amino acids, Asp and Glu, within a certain range, indicating that the present invention has broad application prospects in the field of visual detection of acidic amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the TEM image of the GSH-modified AgNTs of the present invention;

[0025] Figure 2 is the UV-Vis image of the GSH-modified AgNTs of the present invention;

[0026] Figure 3 is the UV-Vis image A of the mixture of different amino acids added to the GSH-modified AgNTs of the present invention and the schematic diagram B of the absorbance ratio of different amino acids at 850 nm and 700 nm;

[0027] Figure 4 is the TEM image of the mixture of acidic amino acids added to the GSH-modified AgNTs of the present invention, A, Asp, B, Glu;

[0028] Figure 5 is the UV-Vis image A of the GSH-modified AgNTs with different concentrations of Asp added to the present invention and the linear relationship diagram B between c(Asp) and A 850 / 700 ;

[0029] Figure 6 is the UV-Vis image A of the GSH-modified AgNTs with different concentrations of Glu added to the present invention and the linear relationship diagram B between c(Glu) and A 850 / 700 ; DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further clarified below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0031] In the experimental methods described in the examples, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels or simply prepared through existing technologies.

[0032] Example 1

[0033] A preparation method of GSH-modified AgNTs includes the following steps:

[0034] (1) Preparation of AgNTs: Under vigorous stirring, 140 mL of water, 300 μL of silver nitrate aqueous solution (50 mmol / L), 3 mL of trisodium citrate aqueous solution (75 mmol / L), 1.2 mL of polyvinylpyrrolidone aqueous solution (17.5 mmol / L), and 720 μL of hydrogen peroxide (30%) were successively added to a 250 mL three-necked flask. After reacting for 30 min, 2 mL of sodium borohydride aqueous solution (100 mmol / L) was quickly added to the mixed solution, and the colorless solution turned light yellow. Reacting at 25 °C, the solution gradually changed from light yellow to yellow, orange, red, purple, blue, and finally a blue-green AgNTs solution was obtained.

[0035] (2) Preparation of GSH-modified AgNTs: 100 μL of the AgNTs solution obtained in step (1) was added to 100 μL of GSH aqueous solution and mixed evenly. The concentration of GSH was 2 mmol / L, and it was incubated at room temperature for 40 min to obtain a GSH-modified AgNTs solution, and the solution was blue-green. Then the GSH-modified AgNTs were characterized. As Figure 1 can be seen from the transmission electron microscope image of, the modified AgNTs still maintained a good triangular morphology and were evenly dispersed. From the Uv-Vis spectrum ([ Figure 2 ) it can also be seen that the GSH-modified silver nanoplates have three characteristic resonance absorption peaks, located at 330 nm, 475 nm, and 700 nm respectively. The UV-Vis spectrum of the GSH-modified AgNTs ([ Figure 2 ) also shows that a low concentration of GSH (50 μM) will cause the characteristic absorption peak of AgNTs to blue-shift, indicating that the silver nanoplates are etched, while a higher concentration of GSH (5 mM) will cause the characteristic absorption peak of AgNTs to weaken significantly. The characteristic absorption peak of the 2 mM GSH-modified AgNTs described in the present invention is consistent with that of AgNTs, indicating that the 2 mM GSH-modified AgNTs of the present invention have good morphological characteristics.

[0036] Example 2

[0037] Application of GSH-modified AgNTs in the detection of acidic amino acids:

[0038] Prepare aqueous solutions of Asp at concentrations of 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L, 1.25 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, aqueous solutions of Glu at concentrations of 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.75 mmol / L, 1 mmol / L, 1.25 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 4 mmol / L, and aqueous solutions of arginine, lysine, histidine, glycine, methionine, phenylalanine, cysteine, asparagine, and glutamine at a concentration of 2 mmol / L. Take 100 μL of the GSH-modified AgNTs solution and mix it with 50 μL of the aqueous amino acid solution including Asp and Glu respectively, add them into quartz cuvettes, and use a UV-visible spectrophotometer to measure and record the data of their absorption spectra. Compare the absorption spectra after the interaction between GSH-modified silver nanoplates and amino acids. If the absorption peak at 700 nm weakens and the absorption peak at 850 nm strengthens, it indicates that the sample to be tested contains acidic amino acids.

[0039] As Figure 3 shown, comparison of the absorption peaks obtained by mixing different amino acids including Asp and Glu with the GSH-modified AgNTs solution at the same concentration and the absorbance ratio at 850 nm and 700 nm. At the same concentration, after adding the aqueous solutions of arginine, lysine, histidine, glycine, methionine, phenylalanine, cysteine, asparagine, and glutamine into the GSH-modified AgNTs solution, the color of the solution did not change significantly and the absorption spectrum did not shift significantly. However, after adding the Asp and Glu solutions, the solution changed from blue-green to light green, and at the same time, the absorption spectrum shifted significantly. It can also be clearly seen from the schematic diagram of the absorbance ratio at 850 nm and 700 nm the specificity of GSH-modified AgNTs for acidic amino acids. TEM image of the mixed solution after adding GSH-modified AgNTs to the Asp and Glu solutions ( Figure 4 ), indicating that the shift of the absorption spectrum is due to the aggregation of GSH-modified AgNTs caused by Asp / Glu.

[0040] As Figure 5The UV-Vis spectra of GSH-modified AgNTs after adding Asp solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5 mM are shown respectively. As the concentration of Asp increases, the position of the maximum LSPR peak of AgNTs gradually redshifts and is accompanied by the solution color changing from blue-green to light green. The ratio A of the absorbances at 850 nm and 700 nm 850 / 700 is used as the characterization data of the aggregation degree. Taking c(Asp) as the abscissa and A 850 / 700nm as the ordinate for data analysis, the results show that in the range of Asp concentration from 0.5 to 1.5 mM, the aggregation degree is positively correlated with the concentration of Asp, and the linear equation between c(Asp) and A 850 / 700nm is y = 0.9386lgx - 2.155, and its linear correlation coefficient is 0.9958.

[0041] As Figure 6 shown respectively are the UV-Vis spectra of GSH-modified AgNTs after adding Glu solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 4 mM. As the concentration of Glu increases, the position of the maximum LSPR peak of AgNTs gradually redshifts and is accompanied by the solution color changing from blue-green to light green. The ratio A of the absorbances at 850 nm and 700 nm 850 / 700 is used as the characterization data of the aggregation degree. Taking c(Glu) as the abscissa and A 850 / 700nm as the ordinate for data analysis, the results show that in the range of Glu concentration from 0.5 to 2 mM, the aggregation degree is positively correlated with the concentration of Glu, and the linear equation between c(Glu) and A 850 / 700nm is y = 0.8104lgx - 1.802, and its linear correlation coefficient is 0.9887.

[0042] Example 3

[0043] Under vigorous stirring, 140 mL of water, 300 μL of silver nitrate aqueous solution, 3 mL of trisodium citrate aqueous solution, 1.2 mL of polyvinylpyrrolidone aqueous solution, and 720 μL of hydrogen peroxide (30%) were successively added to a 250 mL three-necked flask. After reacting for 30 min, 2 mL of sodium borohydride aqueous solution was quickly added to the mixed solution. The molar ratio of silver nitrate, trisodium citrate, polyvinylpyrrolidone, and sodium borohydride was 1:25:2:20, and the colorless solution turned light yellow. Reacting at 25 °C, the solution gradually changed from light yellow to yellow, orange, red, purple, blue, and finally blue-green AgNTs were obtained.

[0044] 100 μL of the obtained AgNTs were added to 100 μL of an aqueous GSH solution and mixed evenly. The concentration of GSH was 2 mmol / L. After incubation at room temperature for 40 min, GSH-modified AgNTs were obtained, and the solution was blue-green. The GSH-modified AgNTs solution was mixed with aqueous solutions of aspartic acid and glutamic acid respectively, with a volume ratio of 1:1, and were respectively added into quartz cuvettes. The absorption spectrum data were measured and recorded using a UV-visible spectrophotometer. By comparing the absorption spectra of GSH-modified silver nanoplates after reacting with amino acids, the absorption peak at 700 nm weakened and the absorption peak at 850 nm strengthened, indicating that the sample to be tested contained acidic amino acids.

[0045] Example 4

[0046] Under vigorous stirring, 140 mL of water, 300 μL of an aqueous silver nitrate solution, 3 mL of an aqueous trisodium citrate solution, 1.2 mL of an aqueous polyvinylpyrrolidone solution, and 720 μL of hydrogen peroxide (30%) were successively added to a 250 mL three-necked flask. After reacting for 30 min, 2 mL of an aqueous sodium borohydride solution was quickly added to the mixed solution. The molar ratio of silver nitrate, trisodium citrate, polyvinylpyrrolidone, and sodium borohydride was 3:35:4:30, and the colorless solution turned light yellow. The reaction was carried out at 25 °C, and the solution gradually changed from light yellow to yellow, orange, red, purple, blue, and finally blue-green AgNTs were obtained. 100 μL of the obtained AgNTs were added to 100 μL of an aqueous GSH solution and mixed evenly. The concentration of GSH was 2 mmol / L. After incubation at room temperature for 40 min, GSH-modified AgNTs were obtained, and the solution was blue-green. The GSH-modified AgNTs solution was mixed with aqueous solutions of aspartic acid and glutamic acid respectively, with a volume ratio of 3:1, and were respectively added into quartz cuvettes. The absorption spectrum data were measured and recorded using a UV-visible spectrophotometer. By comparing the absorption spectra of GSH-modified silver nanoplates after reacting with amino acids, the absorption peak at 700 nm weakened and the absorption peak at 850 nm strengthened, indicating that the sample to be tested contained acidic amino acids.

[0047] It should be noted that the above content only illustrates the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. For those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches all fall within the protection scope of the claims of the present invention.

Claims

1. A preparation method of a glutathione-modified silver nanorod solution, characterized in that, it includes the following steps: (1) Preparation of the silver nanorod solution: Under vigorous stirring, successively add an aqueous silver nitrate solution, an aqueous trisodium citrate solution, an aqueous polyvinylpyrrolidone solution, an aqueous hydrogen peroxide solution, and an aqueous sodium borohydride solution to ultrapure water. The colorless solution turns light yellow; as the reaction proceeds, the solution gradually changes from light yellow to yellow, orange, red, purple, blue, and finally a blue-green silver nanorod solution is obtained; (2) Preparation of the glutathione-modified silver nanorod solution: Add an aqueous glutathione solution to the silver nanorod solution obtained in step (1), mix evenly, and incubate at room temperature for 30 - 50 min to obtain a glutathione-modified silver nanorod solution; In step (2), the concentration of the aqueous glutathione solution is 1.9 - 2.1 mmol / L; In step (2), the volume ratio of the silver nanorod solution to the aqueous glutathione solution is 1:0.8 - 1.

2.

2. The preparation method of a glutathione-modified silver nanorod solution according to claim 1, characterized in that, in step (1), the molar ratio of silver nitrate, trisodium citrate, polyvinylpyrrolidone, hydrogen peroxide, and sodium borohydride is 1 - 3:25 - 35:2 - 4:1000:20 - 30.

3. The preparation method of a glutathione-modified silver nanorod solution according to claim 1, characterized in that, in step (1), the concentration of the aqueous silver nitrate solution is 25 - 75 mmol / L, the concentration of the aqueous trisodium citrate solution is 90 - 130 mmol / L, the concentration of the aqueous polyvinylpyrrolidone solution is 10 - 25 mmol / L, the mass fraction of hydrogen peroxide is 30%, and the concentration of the aqueous sodium borohydride solution is 80 - 120 mmol / L.

4. Application of a glutathione-modified silver nanorod solution prepared by the method according to any one of claims 1 - 3 in the detection of acidic amino acids.

5. The application of a glutathione-modified silver nanorod solution in the detection of acidic amino acids according to claim 4, characterized in that, the acidic amino acids are aspartic acid and glutamic acid; the method for the glutathione-modified silver nanorod solution to detect acidic amino acids is: Mix the glutathione-modified silver nanorod solution with an aqueous aspartic acid solution and an aqueous glutamic acid solution respectively for 10 - 20 min, and use a UV-visible spectrophotometer to measure and record the data of their absorption spectra.

6. The application of a glutathione-modified silver nanorod solution in the detection of acidic amino acids according to claim 5, characterized in that, the concentration of the aqueous aspartic acid solution is 0.5 - 1.5 mmol / L, and the concentration of the aqueous glutamic acid solution is 0.5 - 2 mmol / L.

7. The application of a glutathione-modified silver nanorod in the detection of acidic amino acids according to claim 5, characterized in that, The volume ratio of the glutathione-modified silver nanotriangle solution to the amino acid aqueous solution is 1-3:1.

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