Gold nanoparticle-based sensor and its detection method for glutathione and its oxidation degree

Abstract

This case involves a gold nanoparticle-based sensor and a method for detecting glutathione and the degree of glutathione oxidation. The sensor utilizes MPGN as the sensing platform and sodium pyrenesulfonate as the fluorescence indicator. The sensor recovers fluorescence upon addition of glutathione, while addition of oxidized glutathione does not cause a change in fluorescence. Standard curves for varying glutathione concentrations and fluorescence intensities, as well as fluorescence working curves for varying glutathione / oxidized glutathione ratios, can be generated. This gold nanoparticle-based sensor enables quantitative detection of glutathione with a detection limit as low as 1.1 μM. Furthermore, working curves for varying glutathione / oxidized glutathione ratios and fluorescence intensity are constructed, enabling quantitative detection of the degree of glutathione oxidation in unknown samples. The development of this method has important practical implications for drug monitoring and evaluation in industrial production.

Description

Technical Field

[0001] The present invention relates to the field of fluorescence sensing technology, in particular to a sensor based on gold nanoparticles and a method for detecting glutathione and the degree of glutathione oxidation thereof. Background Art

[0002] Glutathione is an antioxidant that helps the body scavenge free radicals and peroxides. It has the potential to enhance biological antioxidant capacity, promote growth, and improve biological resistance to adverse environmental influences. Currently, due to its functionality in important biological processes, glutathione is widely used as a mainstay in pharmaceuticals to treat diseases such as liver and kidney damage. Generally speaking, the sulfhydryl group in the glutathione molecule is its active group. However, during the production, processing, transportation, and storage of glutathione-related drugs, glutathione is easily oxidized to form oxidized glutathione, which is affected by temperature, light, pH, and other conditions. This oxidizes the active sulfhydryl group to a disulfide bond, thereby reducing its drug activity and potentially causing toxic side effects in humans. Although various methods for glutathione detection exist, such as high-performance liquid chromatography, electrochemical methods, mass spectrometry, enzymatic cycling, colorimetry, flow cytometry, and high-performance capillary electrophoresis, insufficient attention has been paid to measuring the degree of glutathione inactivation—that is, determining the ratio of glutathione to oxidized glutathione in commercial products. Summary of the Invention

[0003] In order to address the shortcomings of the existing technology, the present invention uses gold nanoparticles modified with surface ligands as a sensing platform and sodium pyrenesulfonate as a fluorescent indicator to construct a method for detecting glutathione and a method for detecting the degree of glutathione oxidation.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] A gold nanoparticle sensor is obtained by using MPGN as a sensing platform and sodium pyrenesulfonate as a fluorescence indicator. The specific preparation process is as follows:

[0006] 1) Extracting an aqueous solution of HAuCl4 with tetraoctylammonium bromide in a toluene solution purged with N2, adding dioctylamine to the resulting organic solution, and then stirring under N2 atmosphere until the color fades;

[0007] 2) After the color fades, cool the solution to 0°C, then quickly add NaBH4 aqueous solution and continue stirring at 0°C for 1 to 2 hours;

[0008] 3) removing the aqueous layer from step 2), rapidly adding a methanol solution containing ligand C3, and stirring the resulting mixture at 0° C. for 2.5 to 3.5 hours; after stirring, evaporating the solvent, washing the crude product 3 to 5 times with methanol and petroleum ether, and drying to obtain surface ligand-modified gold nanoparticles, designated MPGN;

[0009] 4) preparing a solution of MPGN and sodium pyrenesulfonate at a concentration ratio of 100 μM:20 μM to obtain a sensor in a critical state of fluorescence quenching; wherein,

[0010] The structural formula of the ligand C3 is:

[0011] Furthermore, the molar ratio of HAuCl4, tetraoctylammonium bromide, dioctylamine, NaBH4, and ligand C3 is 1:2.5:20:10:2.

[0012] Furthermore, the preparation process of the ligand C3 is as follows:

[0013] 1,8-Dibromooctane was dissolved in acetone, and potassium thioacetate was added. The mixture was stirred overnight under nitrogen protection at room temperature. After the solvent evaporated, the pale yellow solid was dissolved in CH2Cl2 and extracted three times with ultrapure water. The organic phase was rotary evaporated to obtain C1.

[0014] Compound C1 was dissolved in 10 ml of AcN, and a 30% trimethylamine ethanol solution was added. The mixture was reacted in a sealed tube at 82°C overnight. After evaporation of the solvent, the crude product was purified by column chromatography to obtain white crystals.

[0015] Compound C2 was dissolved in ethanol, 6M hydrochloric acid was added, and the mixed solution was reacted at 70-80°C in a nitrogen atmosphere for 2-4 hours. The white solid obtained after rotary evaporation of the solvent was C3.

[0016] The second object of the present invention is to provide a method for detecting glutathione based on the gold nanoparticle sensor as described above, comprising the following steps:

[0017] Glutathione in different proportions was added to the sensor solution, and the fluorescence intensity at an emission wavelength of 380 nm was detected with an excitation wavelength of 350 nm. A linear relationship between the fluorescence intensity and the glutathione concentration was plotted, and glutathione was quantitatively detected using this linear relationship.

[0018] Furthermore, during the detection, the final concentration of glutathione was 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 140 μM, 180 μM, 250 μM, and 350 μM.

[0019] The third object of the present invention is to provide a method for quantitatively detecting the degree of glutathione oxidation based on the gold nanoparticle sensor as described above, comprising the following steps:

[0020] a) preparing mixed solutions of glutathione / oxidized glutathione at different ratios, denoted as GSH / GSSG mixed solutions;

[0021] b) adding a mixed solution of GSH / GSSG dropwise to the sensor solution to a final concentration of 100 μM;

[0022] c) Using 350 nm as the excitation wavelength and detecting the fluorescence intensity at 380 nm as the emission wavelength, a standard curve was drawn with the fluorescence intensity of F / F0 as the ordinate and the molar ratio of GSH / GSSG as the abscissa. The degree of glutathione oxidation of unknown samples was quantitatively detected using this standard curve.

[0023] Furthermore, the molar ratio of GSH:GSSG in the GSH / GSSG mixed solution is 1:9, 3:7, 5:5, 7:3, 9:1.

[0024] The present invention successfully constructs a sensing strategy using surface ligand-modified gold nanoparticles (MPGN) as a sensing platform and sodium pyrenesulfonate as a fluorescent indicator. Specifically, based on the metal surface energy transfer between sodium pyrenesulfonate and gold nanoparticles, when sodium pyrenesulfonate binds to MPGN through electrostatic and hydrophobic interactions, its fluorescence is quenched. However, when glutathione (GSH) is added, glutathione disrupts the original microenvironment of the gold nanoparticle surface ligand, causing sodium pyrenesulfonate to be displaced from the gold nanoparticle surface into the bulk solution, restoring fluorescence. Therefore, a glutathione detection method can be established using fluorescence as the output signal. Furthermore, due to the lack of a sulfhydryl group, oxidized glutathione (GSSG) cannot cause a change in the fluorescence of sodium pyrenesulfonate, so this sensing platform can also accurately detect the degree of glutathione oxidation.

[0025] Compared with existing technologies, the present invention has the following advantages: based on surface ligand-modified gold nanoparticles, the fluorescence intensity of sodium pyrenesulfonate is used as the output signal to achieve quantitative detection of glutathione, with high sensitivity and a detection limit as low as 1.1 μM. Furthermore, based on the fact that oxidized glutathione does not cause a change in the fluorescence of sodium pyrenesulfonate, a working curve for different ratios of glutathione to oxidized glutathione and fluorescence intensity is constructed, enabling quantitative detection of the degree of glutathione oxidation in unknown samples. The development of this method has important practical significance for drug monitoring and evaluation in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the synthetic route diagram of ligand C3.

[0027] Figure 2 TEM image of MPGN and its particle size distribution diagram.

[0028] Figure 3 This is the H NMR spectrum of MPGN.

[0029] Figure 4(a) Fluorescence spectrum of sodium pyrenesulfonate added dropwise to MPGN (100 μM) and (b) dot-line graph of fluorescence intensity at 380 nm versus sodium pyrenesulfonate concentration.

[0030] Figure 5 (a) Fluorescence spectrum of GSH added to the sensor solution and (b) dot-line graph of F / F0 and GSH concentration at 380 nm.

[0031] Figure 6 This is the standard curve fitting diagram of F / F0 and GSH concentration at 380 nm when GSH was added dropwise to the sensor solution.

[0032] Figure 7 This is a comparison of F / F0 at 380 nm when GSH and different concentrations of amino acids and metal ions are added to the sensor solution.

[0033] Figure 8 (a) Fluorescence spectrum of GSSG added to the sensor solution and (b) dot-line graph of F / F0 and GSSG concentration at 380 nm.

[0034] Figure 9 (a) Fluorescence spectra of F / F0 when different ratios of GSH / GSSG mixed solutions (total concentration maintained at 100 μM) were added to the sensor solution, and (b) histogram of F / F0 and different ratios of GSH / GSSG of 50 μM / 100 μM at 380 nm.

[0035] Figure 10 Figure 4 is the standard curve of different ratios of GSH / GSSG and fluorescence intensity F / F0.

[0036] Figure 11 This is the standard curve fitting diagram of F / F0 and GSH / GSSG ratio at 380nm.

[0037] Figure 12 TEM image and UV spectrum of the mixture of MPGN and GSH.

[0038] Figure 13 This is the infrared spectrum of GSH and MPGN / GSH mixed system.

[0039] Figure 14 Schematic diagram of MPGN sensing of GSH. DETAILED DESCRIPTION

[0040] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0042] Example 1: Synthesis of ligands for gold nanoparticles (synthetic pathways such as Figure 1 shown)

[0043] Synthesis of C1: 1,8-Dibromooctane (4.35 g, 15.99 mmol) was dissolved in acetone (40 ml), and potassium thioacetate (1.826 g, 15.99 mmol) was added. The mixture was stirred under nitrogen at room temperature overnight. After the solvent evaporated, the pale yellow solid was dissolved in CH2Cl2 and extracted three times with ultrapure water (40 ml). The organic phase was rotary evaporated to obtain C1.

[0044] Synthesis of C2: Compound C1 (0.5 g, 1.87 mmol) was dissolved in 10 ml of AcN, and 30% trimethylamine ethanol solution (0.35 g, 1.78 mmol) was added. The mixture was reacted in a sealed tube at 82°C overnight. After evaporation of the solvent, the crude product was purified by column chromatography (developing solvent: CH2Cl2 / CH3OH = 9 / 1) to obtain white crystals.

[0045] Synthesis of C3: Compound C2 (0.2 g, 0.62 mmol) was dissolved in 2 ml of ethanol, and 2 ml of 6 M hydrochloric acid was added. The mixed solution was reacted at 78°C under a nitrogen atmosphere for 3 h. The solvent was rotary evaporated to obtain a white solid, which was C3.

[0046] Example 2: Preparation of surface ligand-modified gold nanoparticles

[0047] 2 ml of HAuCl4 (50 mg, 0127 mmol, 1 equiv) aqueous solution was extracted with tetraoctylammonium bromide (0.175 g, 0.318 mmol, 2.5 equiv) in toluene (125 ml) purged with N2. Subsequently, dioctylamine (0.613 g, 2.539 mmol, 20 equiv) was added to the resulting orange-red organic solution. The above mixture was vigorously stirred for 1.5 h under N2 atmosphere. After the color of the mixture faded, the solution was cooled to 0°C, and then 1 ml (48 mg, 1.269 mmol, 10 equiv) of NaBH4 aqueous solution was quickly added. The solution quickly turned black and was stirred at 0°C for 1.5 h before the aqueous layer was removed. C3 (0.06 g, 0.254 mmol, 2 equiv) dissolved in 3 ml of methanol was quickly added to the above nanoparticles. The mixture was stirred at 0°C for 3 h, the solvent was evaporated, and the crude product was washed 5 times (methanol / petroleum ether). Figure 2 As shown in the figure, TEM and particle size distribution diagram show that gold nanoparticles with a particle size of 2.2 nm were successfully synthesized. Figure 3 The NMR spectrum showed that the surface of the gold nanoparticles was successfully modified with C3 ligands, and the gold nanoparticles after surface ligand modification were recorded as MPGN.

[0048] Example 3: Construction of MPGN / sodium pyrenesulfonate system

[0049] With 350 nm as the excitation wavelength and the fluorescence intensity at 380 nm as the emission wavelength, 3 ml of 100 μM MPGN solution was prepared, and then 10 mM sodium pyrenesulfonate solution was gradually added dropwise. Figure 4 As shown, at low concentrations, fluorescence is quenched. As the concentration of sodium pyrenesulfonate increases, the fluorescence intensity of the solution gradually increases. At a 20 μM concentration of sodium pyrenesulfonate in the mixed solution, fluorescence quenching reaches a critical state. This mixture, prepared by adding 3 mL of a 100 μM MPGN solution and 6 μL of a 10 mM sodium pyrenesulfonate solution, can be used as a sensor solution for GSH.

[0050] Embodiment 4:

[0051] The prepared GSH and GSSG (10 mM) were added dropwise to the sensor solution, and the fluorescence intensity of GSH and GSSG at different concentrations at an emission wavelength of 380 nm was detected with an excitation wavelength of 350 nm. Figure 5 As shown, after the addition of GSH, the F / F0 fluorescence intensity gradually increased with the increase of concentration; Figure 7 As shown in the figure, after adding GSSG, the F / F0 fluorescence intensity remains unchanged as the concentration increases. Figure 5 The relationship curve between GSH concentration and fluorescence intensity F / F0 in the range of 0-350 μM was obtained, and the linear fitting diagram of GSH detection working curve was obtained ( Figure 6 ), the linear equation is: y=0.09294x+1.1041, and this linear equation can be used for the quantitative detection of GSH concentration in unknown samples.

[0052] Cysteine ​​(Cys), lysine (Lys), histidine (His), Arg (arginine), serine (Ser), glutamine (Gln), proline (Pro), Na + and Mg 2+ The results showed that when the concentrations of GSH and the above substances were all at 100 μM, the fluorescence change induced by GSH was much greater than that of other substances ( Figure 7 a). When the concentration of GSH was 100 μM and the concentration of other substances was 500 μM, although Cys showed a significant fluorescence enhancement ability, its increase was only 41% of that of GSH ( Figure 7 b). The above results show that the sensor has excellent selectivity.

[0053] In Examples 1-2 of this case, functional ligands are modified on the surface of gold nanoparticles to impart a positive charge to the surface of the gold nanoparticles. Simultaneously, the presence of the long alkyl chains of the ligands creates a hydrophobic microenvironment within the gold nanoparticles. In Example 3, an appropriate amount of sodium pyrenesulfonate is added to the surface-modified functionalized gold nanoparticles. Through electrostatic and hydrophobic interactions, the sodium pyrenesulfonate binds to the gold nanoparticles, quenching the fluorescence of the sodium pyrenesulfonate. Example 4 verifies the recovery of fluorescence upon addition of GSH or GSSG. The addition of GSH disrupts the surface microenvironment of the gold nanoparticles, displacing sodium pyrenesulfonate from the surface of the gold nanoparticles into the bulk solution, thereby restoring fluorescence. Using fluorescence as the output signal, quantitative detection of GSH can be achieved. However, GSSG, due to the lack of a thiol group, cannot restore fluorescence in the MPGN / sodium pyrenesulfonate system.

[0054] Based on the above research, this case can also achieve quantitative detection of the GSH oxidation degree of unknown samples.

[0055] Example 5:

[0056] Prepare 100 μM of GSH / GSSG mixed solutions with different ratios (molar ratio GSH:GSSG = 1:9, 3:7, 5:5, 7:3, 9:1); add GSH / GSSG mixed solutions with different ratios dropwise into the sensor solution, use 350 nm as the excitation wavelength, and detect the fluorescence intensity at the emission wavelength of 380 nm. Figure 9As shown in a, as the proportion of GSH increases, the degree of fluorescence recovery of the solution is also greater. With the fluorescence intensity F / F0 as the vertical axis and the GSH / GSSG molar ratio as the horizontal axis, a bar graph is drawn when the concentration of the GSH / GSSG mixed solution is 50μM and 100μM ( Figure 9 b), it can be found that the standard curve can be drawn at both concentrations. Take the data of GSH / GSSG mixed solution with a concentration of 100μM as an example to draw a dot-line graph ( Figure 10 ), and then the linear fitting diagram of GSH / GSSG ratio and F / F0 was obtained ( Figure 11 ), the linear equation for detection is: y = 2.22689x - 0.89502. This standard curve can be used to detect the oxidation degree of GSH in unknown samples.

[0057] Verification: titration was performed with a GSH / GSSG mixed solution at a concentration of 100 μM and a molar ratio of 4:6, and the fluorescence intensity was obtained on the standard curve ( Figure 10 Marking points and Figure 11 Mark point).

[0058] Example 6: Study on the detection mechanism of GSH by MPGN

[0059] like Figure 12 As shown in (a), the transmission electron microscopy image shows that the original 2nm MPGN aggregated after adding GSH, and its diameter reached about 50nm. Secondly, the ultraviolet spectrum ( Figure 12 b) It was found that in the presence of GSH, the maximum absorption wavelength of MPGN red-shifted from 518 nm to 525 nm, further proving that GSH induced the aggregation of nanoparticles. Figure 13 As shown in the figure, the infrared spectra of MPGN+GSH and GSH were detected. When MPGN exists in GSH, the infrared spectrum at 2500 cm -1 The signal corresponding to the thiol group at the end of the reaction disappears, indicating that -SH binds to MPGN. The above experimental results show that after GSH is added to the MPGN / sodium pyrenesulfonate system, the thiol group of GSH interacts with the MPGN surface, inducing MPGN to aggregate, disrupting its original surface microenvironment, and causing sodium pyrenesulfonate to be replaced into the bulk solution, thereby changing the fluorescence output signal. The sensing mechanism diagram is shown in the figure. Figure 14 shown.

[0060] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A gold nanoparticle sensor, characterized in that It is obtained by using MPGN as the sensing platform and sodium pyrenesulfonate as the fluorescence indicator. The specific preparation process is as follows: 1) Extracting an aqueous solution of HAuCl4 with tetraoctylammonium bromide in a toluene solution purged with N2, adding dioctylamine to the resulting organic solution, and then stirring under N2 atmosphere until the color fades; 2) After the color fades, cool the solution to 0°C, then quickly add NaBH4 aqueous solution and continue stirring at 0°C for 1-2 hours; 3) removing the aqueous layer from step 2) and rapidly adding a methanol solution containing ligand C3, and stirring the resulting mixture at 0°C for 2.5-3.5 hours. After stirring, evaporating the solvent, washing the crude product with methanol and petroleum ether 3-5 times, and drying to obtain surface ligand-modified gold nanoparticles, designated MPGN. 4) A solution of MPGN and sodium pyrenesulfonate at a concentration ratio of 100 μM:20 μM was prepared to obtain a sensor in a critical state of fluorescence quenching; wherein, The structural formula of the ligand C3 is: .

2. The gold nanoparticle sensor according to claim 1, wherein The molar ratio of HAuCl4, tetraoctylammonium bromide, dioctylamine, NaBH4 and ligand C3 is 1:2.5:20:10:

2.

3. The gold nanoparticle sensor according to claim 1, wherein The preparation process of the ligand C3 is as follows: 1,8-Dibromooctane was dissolved in acetone, and potassium thioacetate was added. The mixture was stirred overnight under nitrogen protection at room temperature. After the solvent evaporated, the pale yellow solid was dissolved in CH2Cl2 and extracted three times with ultrapure water. The organic phase was rotary evaporated to obtain C1. Compound C1 was dissolved in 10 ml of AcN, and 30% trimethylamine ethanol solution was added. The mixture was reacted in a sealed tube at 82°C overnight. After evaporation of the solvent, the crude product was purified by column chromatography to obtain white crystals. Compound C2 was dissolved in ethanol, 6 M hydrochloric acid was added, and the mixed solution was reacted at 70-80 °C under a nitrogen atmosphere for 2-4 h. The white solid obtained after rotary evaporation of the solvent was C3.

4. A method for detecting glutathione based on the gold nanoparticle sensor according to any one of claims 1 to 3, characterized in that: The steps include: Different proportions of glutathione were added to the sensor, and the fluorescence intensity at an emission wavelength of 380 nm was detected with an excitation wavelength of 350 nm. A linear relationship between the fluorescence intensity and the glutathione concentration was plotted, and glutathione was quantitatively detected using this linear relationship.

5. The method according to claim 4, wherein During the assay, the final concentrations of glutathione were 0 μM, 10 μM, 20 μM, 30 μM, 40 μM, 60 μM, 80 μM, 100 μM, 140 μM, 180 μM, 250 μM, and 350 μM.

6. A method for detecting the degree of glutathione oxidation based on the gold nanoparticle sensor according to any one of claims 1 to 3, characterized in that: The steps include: a) preparing mixed solutions of glutathione / oxidized glutathione at different ratios, denoted as GSH / GSSG mixed solutions; b) adding a mixed solution of GSH / GSSG dropwise to the sensor to a final concentration of 100 μM; c) Use 350 nm as the excitation wavelength and 380 nm as the emission wavelength to detect the fluorescence intensity. Draw a standard curve with the fluorescence intensity of F / F0 as the ordinate and the molar ratio of GSH / GSSG as the abscissa. Use this standard curve to quantitatively detect the degree of glutathione oxidation in unknown samples.

7. The method according to claim 6, wherein The molar ratio of GSH:GSSG in the GSH / GSSG mixed solution is 1:9, 3:7, 5:5, 7:3, 9:1.

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