Detection method of reduced glutathione
阅读说明：本技术 一种还原型谷胱甘肽的检测方法 (Detection method of reduced glutathione ) 是由 赵玉芬 王敏凝 蔡华欢 李福来 于 2019-09-24 设计创作，主要内容包括：一种还原型谷胱甘肽的检测方法,涉及谷胱甘肽。提供简单有效、检测灵敏度高的一种还原型谷胱甘肽的检测方法。以合成的含二硫键的双官能团有机小分子作为连接分子,构建了表面修饰荧光分子的纳米金探针。结合荧光共振能量转移FRET技术和还原型谷胱甘肽能够还原二硫键的性质,实现对还原型谷胱甘肽的检测。与现有方法相比,检测方法简单有效,对还原型谷胱甘肽的检测灵敏度高,所用的试剂都廉价易得,且绿色环保。(The detection method of reduced glutathione relates to glutathione, provides a simple, effective and high detection sensitivity reduced glutathione detection method, takes the synthesized bifunctional organic micromolecules containing disulfide bonds as connecting molecules, constructs a nano-gold probe with a surface modified fluorescent molecule, combines the Fluorescence Resonance Energy Transfer (FRET) technology and the property that the reduced glutathione can reduce the disulfide bonds, and realizes the detection of the reduced glutathione.)
1, kinds of reduced glutathione detection method, characterized by comprising the following steps:
1) with 2-aminoethanethiol (C)2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) as starting material in H2O2Under the participation of ammonia water, double-functional-group organic small molecules (Linker) containing disulfide bonds and with amino and carboxyl at two ends are synthesized, then Fluorescein Isothiocyanate (FITC) and the double-functional-group organic small molecules (Linker) react in an organic solvent to generate Linker-FITC, and redundant reaction solvent is removed through centrifugal concentration;
2) preparing gold nanoparticles, adding Polylysine (epsilon-Polylysine, PLL), standing overnight for reaction, removing free Polylysine through centrifugation, and adding water for re-suspension to obtain gold nanoparticles (Au @ PLL) with Polylysine modified on the surface;
3) activating carboxyl on Linker-FITC through reaction of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS), further performing condensation reaction with amino on Au @ PLL, removing unreacted (Linker-FITC) in a system through centrifugation, and re-dispersing precipitates with ultrapure water to obtain a gold nano-probe (Au @ PLL/Linker-FITC) for detection;
4) after GSH with different concentration gradients reacts with the nanogold probe in a constant-temperature water bath, 100mL of reaction system is added into a 96-hole enzyme label plate for fluorescence spectrum detection, a GSH standard curve is obtained by determination, and the detection limit is 1 nM.
2. The method for detecting reduced glutathione according to claim 1, wherein in step 1), the structural formula of the difunctional organic small molecule (Linker) containing disulfide bond is as follows:
3. the method for detecting reduced glutathione according to claim 1, wherein in step 1), the 2-aminoethanethiol (C) is detected2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) is 1: 1;
the equivalent ratio of Fluorescein Isothiocyanate (FITC) to bifunctional organic small molecule (Linker) is 1.2: 1.
4. The method for detecting reduced glutathione according to claim 1, wherein in step 1), the organic solvent is a mixed solution of methanol and triethylamine, and the equivalent ratio of methanol to triethylamine is 100: 1;
the reaction temperature of the reaction can be 35-40 ℃, the reaction time can be 6-8 h, and the centrifugal concentration condition is 40 ℃ and 2 h.
5. The method for detecting reduced glutathione according to claim 1, wherein in step 2), the preparation of gold nanoparticles is performed by citrate reduction, which comprises the steps of adding 100mL of ultrapure water and magnetite to a round-bottomed flask, heating in an oil bath to 120 ℃, adding 1mL of 1% chloroauric acid solution under the slightly boiling state, stirring by magnetic force, stirring uniformly, rapidly adding 750 μ L of 57mg/mL sodium citrate solution after the solution is slightly boiling, reacting for 20min while maintaining the temperature at about 120 ℃, observing that the solution color gradually changes into deep wine red, and stopping heating to complete the synthesis of gold nanoparticles.
6. The method for detecting reduced glutathione according to claim 1, wherein in step 2), the gold nanoparticles have a particle size of 17-18 nm, the polylysine is added in an amount of 0.1% by volume, and the water is ultrapure water.
7. The method for detecting reduced glutathione according to claim 1, wherein in step 2), the reaction volume ratio of the gold nanoparticles to the polylysine is 1: 3-5, and the centrifugation conditions are 5000-6000 rpm for 20 min.
8. The method for detecting reduced glutathione according to claim 1, wherein in step 3), the equivalent ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxythiosuccinimide (sulfo-NHS) in the reaction is 1: 1, the activation condition is a rotation reaction at room temperature for 15-30 min, the condensation reaction temperature can be 4 ℃, and the reaction time can be 1-2 h.
9. The method for detecting reduced glutathione according to claim 1, wherein in step 4), the concentration range of GSH with different concentration gradients is 10 mM-50 nM, and the volume ratio of the GSH with different concentration gradients to the nanogold probe solution is 1: 50, so that the concentration range of GSH in the final reaction system is 200 mM-1 nM.
10. The method for detecting reduced glutathione according to claim 1, wherein the temperature of the thermostatic water bath in step 4) is 37 ℃ and the reaction time is 2 h.
Technical Field
The invention relates to glutathione, in particular to a method for detecting kinds of reduced glutathione.
Background
Glutathione (glutathione, r-glutamyl cysteine + glycine, GSH) is the most abundant non-protein thiol in cells, and is the small molecule thiol most studied at present at pan, most of glutathione in cells (more than 95%) exists in reduced GSH, and the small part is oxidized GSSG, abnormal change of reduced glutathione content in organisms has close relation with the occurrence of a plurality of diseases such as Alzheimer disease, cystic fibrosis, liver injury, and the like.
The High Performance Liquid Chromatography (HPLC) and the Capillary Electrophoresis (CE) have high sensitivity to GSH detection, but have long time consumption and requirements on instruments, the Surface Enhanced Raman (SERS) method and the colorimetric method have short detection time, but have low sensitivity to GSH detection, and the detection limit is similar to the mu M level.
Chinese patent CN201610764935.6 discloses methods for detecting reduced glutathione with high sensitivity based on electrochemical probes, rare earth cerium (IV) is used as an electrochemical probe, redox reaction is carried out based on Ce (IV) and GSH, and the Ce (IV) is converted into Ce (III) with change of electrochemical signals, so that methods for detecting reduced glutathione by electrochemical sensor technology are established, the method is completed in CHI660D electrochemical workstation, the working electrode is a gold electrode, the counter electrode is a platinum electrode, the reference electrode is a silver/silver chloride electrode, the electrochemical test is a differential pulse method, and the experimental conditions are that the supporting electrolyte is 1.0mol/L Na2SO4The pH value of the solution is 6, and the testing temperature is 25 ℃; the change in the current value was measured by the DPV method. The current value and the added GSH concentration are in linear relation, the detection concentration of the GSH is obtained by calculation, and the detection limit is 0.05 nmol.L-1。
Chinese patent CN201310449795.X discloses a method for detecting reduced glutathione, which comprises diluting a solution containing gold nanoparticles and a chloroauric acid solution with buffer solutions respectively, mixing, adding a surfactant solution, mixing, adding a solution to be detected, standing for 5-10 min, and adding H2O2Forming a reaction system by the aqueous solution, starting the reaction, detecting the ultraviolet visible absorption spectrum of the obtained product system after the reaction is finished, and qualitatively judging the solution to be detected according to the absorbance change at the position of 520nm in the ultraviolet visible absorption spectrum of the detected product systemAnd (4) quantitatively detecting the content of the reduced glutathione in the solution to be detected through a determined linear regression equation if the solution contains the reduced glutathione.
Disclosure of Invention
The purpose of the present invention is to provide a simple and effective method for detecting reduced glutathione species with high detection sensitivity.
The invention comprises the following steps:
1) with 2-aminoethanethiol (C)2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) as starting material in H2O2Under the participation of ammonia water, double-functional-group organic small molecules (Linker) containing disulfide bonds and with amino and carboxyl at two ends are synthesized, then Fluorescein Isothiocyanate (FITC) and the double-functional-group organic small molecules (Linker) react in an organic solvent to generate Linker-FITC, and redundant reaction solvent is removed through centrifugal concentration;
2) preparing gold nanoparticles, adding Polylysine (epsilon-Polylysine, PLL), standing overnight for reaction, removing free Polylysine through centrifugation, and adding water for re-suspension to obtain gold nanoparticles (Au @ PLL) with Polylysine modified on the surface;
3) activating carboxyl on Linker-FITC through reaction of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS), further performing condensation reaction with amino on Au @ PLL, removing unreacted (Linker-FITC) in a system through centrifugation, and re-dispersing precipitates with ultrapure water to obtain a gold nano-probe (Au @ PLL/Linker-FITC) for detection;
4) after GSH with different concentration gradients reacts with the nanogold probe in a constant-temperature water bath, 100mL of reaction system is added into a 96-hole enzyme label plate for fluorescence spectrum detection, a GSH standard curve is obtained by determination, and the detection limit is 1 nM.
In the step 1), the structural formula of the bifunctional organic small molecule (Linker) containing the disulfide bond is as follows:
the 2-aminoethanethiol (C)2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) is 1: 1;
the equivalent ratio of Fluorescein Isothiocyanate (FITC) to bifunctional organic small molecule (Linker) is 1.2: 1;
the organic solvent can adopt a mixed solution of methanol and triethylamine, and the equivalent ratio of the methanol to the triethylamine is 100: 1;
the reaction temperature of the reaction can be 35-40 ℃, the reaction time can be 6-8 h, and the centrifugal concentration condition is 40 ℃ and 2 h.
In step 2), the gold nanoparticles can be prepared by a citrate reduction method, and the specific steps can be as follows: 100mL of ultrapure water and magnetite were added to the round bottom flask, the flask was heated in an oil bath to 120 ℃ and 1mL of a 1% chloroauric acid solution was added while slightly boiling, and magnetic stirring was turned on. Stirring uniformly, and quickly adding 750 mu L of 57mg/mL sodium citrate solution after the solution boils slightly; the temperature is maintained at about 120 ℃ for reaction for 20min, the solution is observed to slowly turn into dark wine red, and heating is stopped, thus completing the synthesis of the nano gold particles;
the particle size of the nano gold particles can be 17-18 nm; the polylysine can be added by 0.1 percent by volume; the water can adopt ultrapure water; the reaction volume ratio of the gold nanoparticles to the polylysine can be 1: 3-5. The centrifugation condition can be 5000-6000 rpm for 20 min.
In the step 3), the equivalent ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxythiosuccinimide (sulfo-NHS) in the reaction can be 1: 1, and the rotation reaction is carried out for 15-30 min at room temperature under the activation condition. The condensation reaction temperature can be 4 ℃, and the reaction time can be 1-2 h.
In the step 4), the concentration range of the GSH of the different concentration gradients is 10 mM-50 nM;
the volume ratio of the GSH with different concentration gradients to the nano-gold probe solution is 1: 50, so that the concentration range of the GSH in the final reaction system is 200 mM-1 nM;
the temperature of the constant-temperature water bath can be 37 ℃, and the reaction time can be 2 hours.
The invention relates to a novel method for detecting reduced Glutathione (GSH) by bifunctional small organic molecule-modified nanogold probes, namely, the synthesized bifunctional small organic molecules containing disulfide bonds are used as connecting molecules to construct the nanogold probes with surface-modified fluorescent molecules.
Compared with the prior art, the detection method is simple and effective, has high detection sensitivity on reduced glutathione, and the reagents used in the method are cheap and easily available, and are environment-friendly.
Drawings
FIG. 1 is a standard curve for detection of reduced glutathione GSH.
Detailed Description
The following examples are presented to illustrate the present invention in a further step with reference to the drawings.
The invention comprises the following steps:
1) with 2-aminoethanethiol (C)2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) as starting material in H2O2Under the participation of ammonia water, double-functional-group organic small molecules (Linker) containing disulfide bonds and with amino and carboxyl at two ends are synthesized, then Fluorescein Isothiocyanate (FITC) and the double-functional-group organic small molecules (Linker) react in an organic solvent to generate Linker-FITC, and redundant reaction solvent is removed through centrifugal concentration;
the structural formula of the bifunctional organic small molecule (Linker) containing the disulfide bond is as follows:
the 2-aminoethanethiol (C)2H7NS) and 3-mercaptopropionic acid (C)3H6O2S) is 1: 1; the equivalent ratio of Fluorescein Isothiocyanate (FITC) to bifunctional organic small molecule (Linker) is 1.2: 1; the organic matterThe solvent can adopt a mixed solution of methanol and triethylamine, and the equivalent ratio of the methanol to the triethylamine is 100: 1; the reaction temperature of the reaction can be 35-40 ℃, the reaction time can be 6-8 h, and the centrifugal concentration condition is 40 ℃ and 2 h.
2) Preparing gold nanoparticles, adding Polylysine (epsilon-Polylysine, PLL), standing overnight for reaction, removing free Polylysine through centrifugation, and adding water for re-suspension to obtain gold nanoparticles (Au @ PLL) with Polylysine modified on the surface; the preparation of the nano gold particles can adopt a citrate reduction method, and the specific steps can be as follows: 100mL of ultrapure water and magnetite were added to the round bottom flask, the flask was heated in an oil bath to 120 ℃ and 1mL of a 1% chloroauric acid solution was added while slightly boiling, and magnetic stirring was turned on. Stirring uniformly, and quickly adding 750 mu L of 57mg/mL sodium citrate solution after the solution boils slightly; the temperature is maintained at about 120 ℃ for reaction for 20min, the solution is observed to slowly turn into dark wine red, and heating is stopped, thus completing the synthesis of the nano gold particles; the particle size of the nano gold particles can be 17-18 nm; the polylysine can be added by 0.1 percent by volume; the water can adopt ultrapure water; the reaction volume ratio of the gold nanoparticles to the polylysine can be 1: 3-5. The centrifugation condition can be 5000-6000 rpm for 20 min.
3) Activating carboxyl on Linker-FITC through reaction of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (sulfo-NHS), further performing condensation reaction with amino on Au @ PLL, removing unreacted (Linker-FITC) in a system through centrifugation, and re-dispersing precipitates with ultrapure water to obtain a gold nano-probe (Au @ PLL/Linker-FITC) for detection; in the reaction, the equivalent ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxy-thiosuccinimide (sulfo-NHS) can be 1: 1, and the activation condition is that the rotation reaction is carried out for 15-30 min at room temperature.
The condensation reaction temperature can be 4 ℃, and the reaction time can be 1-2 h.
4) After GSH with different concentration gradients reacts with the nanogold probe in a constant-temperature water bath, 100mL of reaction system is added into a 96-hole enzyme label plate for fluorescence spectrum detection, a GSH standard curve is obtained by determination, and the detection limit is 1 nM. The GSH concentration range of the different concentration gradients is 10 mM-50 nM; the volume ratio of the GSH with different concentration gradients to the nano-gold probe solution is 1: 50, so that the concentration range of the GSH in the final reaction system is 200 mM-1 nM; the temperature of the constant-temperature water bath can be 37 ℃, and the reaction time can be 2 hours.