CN114570936A - Preparation method of glutathione-S-transferase-Au-Pt nanocluster and application of glutathione-S-transferase-Au-Pt nanocluster in aureomycin detection - Google Patents
Preparation method of glutathione-S-transferase-Au-Pt nanocluster and application of glutathione-S-transferase-Au-Pt nanocluster in aureomycin detection Download PDFInfo
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Abstract
The invention relates to the technical field of gold-platinum nanocluster synthesis, in particular to a preparation method of glutathione sulfurtransferase-gold-platinum nanoclusters and application thereof in aureomycin detection, which specifically comprises the following steps: glutathione-S-transferase is used as a template to prepare glutathione-S-transferase-gold platinum nanocluster stock solution under a specific pH condition, and dialysis purification is further carried out. Under the excitation wavelength of 375nm, the gold platinum nanocluster presents red fluorescence, and two emission peaks are respectively arranged at 470nm and 656 nm; the gold platinum nanoclusters as an effective environment-friendly ratiometric fluorescent probe can be used for detecting aureomycin (CTC) in a solution. The gold platinum nano cluster has unique photophysical characteristics, almost no toxicity, excellent biocompatibility, quick and simple detection of aureomycin and good detection sensitivity, and is an ideal fluorescent nano material applied to the fields of biology and medicine.
Description
Technical Field
The invention relates to the technical field of gold-platinum nanocluster synthesis, in particular to a preparation method of glutathione S-transferase-gold-platinum nanoclusters and application of the glutathione S-transferase-gold-platinum nanoclusters in aureomycin detection.
Background
Glutathione s-transferase is a multifunctional enzyme that is widely distributed throughout the body and can bind to cytoplasm or membrane, and plays an important role in the organism. Glutathione S-transferase can catalyze a large number of chemical reactions such as nucleophilic aromatic substitution, Michael addition and the like, and can generate a plurality of electrophilic substances by catalytic reduction of endogenous glutathione and exogenous compounds in organisms, thereby protecting cells from chemical substances such as drugs, endogenous peroxide radicals, toxic metabolites and the like. Glutathione S-transferase appears to be highly expressed in certain cancer cells compared to normal tissues, playing an important role in the development of chemotherapeutic drug resistance. Thus, glutathione S-transferase is an important biological enzyme that defends against reductive and toxic electrophiles produced by normal metabolic processes.
The wavelength of the metal nanocluster (the diameter is less than 2nm) is equivalent to the Fermi wavelength of electrons, the metal nanocluster has the property of similar molecules, can perform photoluminescence, and has the advantages of good stability, mild synthesis conditions, low toxicity, high biocompatibility and the like. The bimetal nano-cluster is more beneficial to improving the fluorescence intensity due to the addition of the second metal element. Because the protein has reducing capacity and rich binding sites and groups, the protein usually acts on a metal center in the form of a stabilizer and a reducing agent, the metal nano-cluster protected by the protein shows stronger fluorescence under the action of a proper ligand, the secondary structure and the biological function of the protein are generally not obviously changed, and the stable structure of the protein can ensure that the fluorescence performance of the protein is not quenched due to aggregation.
Chlortetracycline (CTC) is a broad-spectrum tetracycline antibiotic, can be widely applied to animal husbandry because of the effective inhibition of infection of gram-positive bacteria and gram-negative bacteria and low price, and can also be used as a growth promoter for animal feed; however, many side effects such as water and soil pollution and drug resistance of bacteria are caused by improper dosage. Aiming at the limitations of high cost, low sensitivity, poor selectivity, long time consumption and the like of the traditional detection method of aureomycin, the development of a quick, efficient and low-cost detection method is urgently needed. Therefore, it is important to develop new technologies such as fluorescence sensing to detect CTCs.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a preparation method of glutathione-S-transferase-Au-Pt nanoclusters and application thereof in aureomycin detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of glutathione-S-transferase-Au-Pt nanoclusters comprises the following specific steps:
s1, adding the chloroauric acid solution into the glutathione-S-transferase solution, uniformly mixing, adding the chloroplatinic acid solution, and uniformly mixing to obtain a solution A;
s2, adding a sodium hydroxide solution into the solution A obtained in the step S1, adjusting the pH of the solution to 11-13, and heating the solution for 1-9 hours under the condition of metal bath to obtain a solution B;
s3, dialyzing the solution B obtained in the step S2 in phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to remove redundant reactants, so that the protein is recovered to the natural state as far as possible, and storing the product at 4 ℃ to obtain the glutathione S-transferase-gold platinum nanoclusters.
Preferably, in the S1, the concentration ratio of the chloroauric acid to the chloroplatinic acid is 8:1, and the concentration of the glutathione S-transferase is 20mg ml-1。
Preferably, in the S2, the pH of the solution is adjusted to 11 by sodium hydroxide, the reaction is carried out for 5 hours, and the reaction temperature is 80 ℃.
The invention also provides application of the glutathione sulfurtransferase-gold platinum nanocluster obtained by the preparation method of the glutathione sulfurtransferase-gold platinum nanocluster in aureomycin detection, the glutathione sulfurtransferase-gold platinum nanocluster is diluted by phosphate buffer solution, aureomycin with different concentrations is added and uniformly mixed, the mixture is incubated at room temperature, the fluorescence intensity of the glutathione sulfurtransferase-gold platinum nanocluster is gradually enhanced along with the gradual increase of the aureomycin concentration under the condition of excitation wavelength, and the ratio type detection is realized.
Preferably, the detection is effected at an excitation wavelength of 375nm, with an incubation time of 5 minutes at room temperature.
Compared with the prior art, the invention has the following beneficial effects:
1. the glutathione-S-transferase-gold platinum nanocluster is prepared by using the glutathione-S-transferase as a template and adopting a one-step synthesis method, and has the advantages of unique photophysical characteristics, almost no toxicity, excellent biocompatibility and low cost.
2. The glutathione s-transferase-gold platinum nanocluster prepared by the invention is rapid and simple in method for detecting aureomycin and good in detection sensitivity, and is an ideal fluorescent nanomaterial applied to the fields of biology and medicine.
Drawings
FIG. 1 shows an excitation spectrum and an emission spectrum of glutathione S-transferase-Au-Pt nanoclusters prepared by the present invention;
FIG. 2 is a fluorescence spectrum of glutathione-S-transferase-Au-Pt nanoclusters prepared under different pH conditions according to the present invention;
FIG. 3 is a fluorescence spectrum of glutathione S-transferase-Au-Pt nanoclusters prepared by chloroauric acid and chloroplatinic acid in different proportions according to the present invention;
FIG. 4 is a fluorescence spectrum of glutathione S-transferase-Au-Pt nanoclusters prepared at different temperatures according to the present invention;
FIG. 5 is a fluorescence spectrum of glutathione S-transferase-Au-Pt nanoclusters prepared according to the present invention at different times;
FIG. 6 is a fluorescence spectrum of glutathione s-transferase-Au-Pt nanoclusters prepared by glutathione s-transferase of different concentrations according to the present invention;
FIG. 7 is a transmission electron micrograph and a size distribution of glutathione S-transferase-Au-Pt nanoclusters after the conditions of the present invention are optimized;
FIG. 8 is the fluorescence emission spectrum of glutathione S-transferase-Au-Pt nanocluster solution after adding aureomycin with different concentrations.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention is more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.
In order to improve the luminescence property of the product, the method comprises the following steps:
adding chloroauric acid solution into glutathione-S-transferase solution, mixing, adding chloroplatinic acid into the solution, and mixing; adjusting the pH value of the mixed solution to 11, and heating the metal bath of the mixed solution to obtain a product. And detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. The fluorescence spectrum was observed by changing the pH of the mixed solution, and the fluorescence intensity gradually decreased with decreasing pH, and was optimal when the pH of the solution was 11. Therefore, pH 11 was selected as the optimum pH condition for the preparation of glutathione S-transferase-Au-Pt nanoclusters.
Adding 50 mu L of 10mM chloroauric acid solution into 125 mu L glutathione-s-transferase solution, mixing uniformly, adding 16 mu L of 10mM chloroplatinic acid into the solution, and mixing uniformly; adjusting the pH value of the mixed solution to 11, and heating the mixed solution in a metal bath to obtain the product. The excitation spectrum and emission spectrum of the product were detected by fluorescence spectroscopy. The fluorescence spectrum was observed by changing the volume of chloroplatinic acid to change the concentration ratio of chloroauric acid to chloroplatinic acid, and the fluorescence intensity was optimum when the concentration ratio of chloroauric acid to chloroplatinic acid was 8: 1. Therefore, the concentration ratio of chloroauric acid to chloroplatinic acid is selected to be 8:1 as the optimal ratio for preparing the glutathione-S-transferase-gold-platinum nanoclusters.
Adding 50 μ L of 10mM chloroauric acid solution into 125 μ L glutathione s-transferase solution, mixing, adding 6.25 μ L10 mM chloroplatinic acid into the solution, and mixing; the pH of the mixed solution was adjusted to 11, and the mixed solution was heated in a metal bath for 1 hour to obtain a product. And detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. The reaction time is increased, the fluorescence spectrum is observed, the fluorescence intensity is gradually enhanced along with the increase of the reaction time, and the fluorescence intensity is optimal when the reaction time is 5 hours. Therefore 5 hours was chosen as the optimal time for the preparation of glutathione S-transferase-gold platinum nanoclusters.
Adding 50 μ L of 10mM chloroauric acid solution into 125 μ L glutathione s-transferase solution, mixing, adding 6.25 μ L10 mM chloroplatinic acid into the solution, and mixing; the pH of the mixed solution was adjusted to 11, and the mixed solution was heated in a metal bath at 37 ℃ for 5 hours to obtain a product. And detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. The reaction temperature is increased, the fluorescence spectrum is observed, the fluorescence intensity is gradually enhanced along with the increase of the reaction temperature, and the fluorescence intensity is optimal when the reaction temperature is 80 ℃. Therefore 80 ℃ was chosen as the optimal temperature for the preparation of glutathione S-transferase-Au-Pt nanoclusters.
mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 5mg ml-1Mixing the glutathione-S-transferase solution uniformly, adding 6.25 mu L of chloroplatinic acid with the concentration of 10mM into the solution, and mixing uniformly; the pH of the mixed solution is adjusted to 11, and the mixed solution is heated for 5 hours in a metal bath at 80 ℃ to obtain a product. And detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. Increasing glutathione-S-transferase concentration, observing fluorescence spectrum, and increasing fluorescence intensity with increasing glutathione-S-transferase concentration, when using 20mg ml-1The fluorescence intensity is optimal for glutathione S-transferase of (4); thus 20mg ml was selected-1Glutathione S-transferases asThe optimal concentration for preparing the glutathione-S-transferase-gold platinum nanocluster is achieved. The glutathione sulfurtransferase-gold platinum nanoclusters prepared after the conditions are optimized are uniform in size distribution, and the average particle size of the glutathione sulfurtransferase-gold platinum nanoclusters is about 1.7 nm.
And (2) detecting the aureomycin by taking the glutathione-S-transferase-gold platinum nanocluster as a fluorescent probe:
diluting the glutathione sulfurtransferase-aureoplatinum nanocluster by using a phosphate buffer solution, adding aureomycin with the concentration range of 5-70 mu M, incubating the mixed solution for 5 minutes at room temperature, and gradually increasing the fluorescence emission of the glutathione sulfurtransferase-aureoplatinum nanocluster at 475nm along with the gradual increase of the aureomycin concentration when the excitation wavelength is 375 nm.
Preparation and optimization of glutathione-S-transferase-gold platinum nanocluster
The first embodiment is as follows: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 3, heating the mixed solution in a metal bath at 80 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the glutathione-S-transferase-gold platinum nanocluster.
Example two: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 7, heating the mixed solution in a metal bath at 80 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example three: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting pH of the mixed solution to 11, heating the mixed solution at 80 deg.C in metal bath for 5 hr, and mixing with waterDialyzing the dialysis bag with the molecular interception amount of 12-14kDa in phosphate buffer for 24 hours to obtain the product glutathione-S-transferase-Au-Pt nanocluster.
Example four: the glutathione sulfurtransferase-gold platinum nanoclusters obtained in the first, second and third examples are determined to have the strongest fluorescence emission intensity at pH 11 by comparing fluorescence intensities at an excitation wavelength of 375nm as shown in fig. 2.
Example five: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 16 mu L of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 80 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example six: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 10 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 80 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example seven: in the glutathione-transferase-Au-Pt nanoclusters obtained in example three, example five and example six, as shown in FIG. 3, when the ratio of chloroauric acid to chloroplatinic acid is 8:1, the fluorescence emission intensity of the glutathione-transferase-Au-Pt nanoclusters is the strongest as determined by comparing the fluorescence intensity of the glutathione-transferase-Au-Pt nanoclusters at the excitation wavelength of 375 nm.
Example eight: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting pH of the mixed solution to 11, and subjecting the mixed solution to 37 deg.CHeating the mixture in a metal bath for 5 hours, and dialyzing the mixture in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain a product glutathione-S-transferase-gold platinum nanocluster.
Example nine: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 50 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular interception amount of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nano-cluster.
Example ten: in the glutathione S-transferase-Au-Pt nanoclusters obtained in example III, example eight and example nine, as shown in FIG. 4, the fluorescence emission intensity of the glutathione S-transferase-Au-Pt nanoclusters is determined to be the strongest when the reaction temperature is 80 ℃ by comparing the fluorescence intensities thereof at the excitation wavelength of 375 nm.
Example eleven: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 80 ℃ for 3 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example twelve: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 20mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 80 ℃ for 7 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example thirteen: the glutathione S-transferase-Au-Pt nanoclusters obtained in example III, example eleven and example twelve are compared with each other in fluorescence emission intensity at an excitation wavelength of 375nm to determine that the reaction time is 5 hours and the fluorescence emission intensity is strongest, as shown in FIG. 5.
Example fourteen: mu.L of 10mM chloroauric acid solution was added to 125. mu.L of 5mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 70 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example fifteen: 50. mu.L of a 10mM chloroauric acid solution was added to 125. mu.L of 10mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 70 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nanocluster.
Example sixteen: 50 μ L of 10mM chloroauric acid solution was added to 125 μ L of 15mg ml-1The glutathione-s-transferase solution is evenly mixed; then 6.25 mul of chloroplatinic acid with the concentration of 10mM is added into the solution and mixed evenly; adjusting the pH value of the mixed solution to 11, heating the mixed solution in a metal bath at 70 ℃ for 5 hours, and dialyzing the mixed solution in a phosphate buffer solution for 24 hours by using a dialysis bag with the molecular interception amount of 12-14kDa to obtain the product glutathione-S-transferase-gold platinum nano-cluster.
Example seventeen: in the glutathione S-transferase-Au-Pt nanoclusters obtained in example III, example fourteen, example fifteen and example sixteen, the glutathione S-transferase concentration was determined to be 20mg ml/ml by comparing the respective fluorescence emission intensities at the excitation wavelength of 375nm as shown in FIG. 6-1The fluorescence emission intensity is strongest.
(II) glutathione-S-transferase-gold platinum nanocluster as fluorescent probe for detecting aureomycin
Example eighteen: diluting glutathione sulfurtransferase-gold platinum nanocluster stock solution by using phosphate buffer solution, then adding aureomycin solution with the final concentration of 0-70 mu M, incubating for 10 minutes at room temperature, and detecting the fluorescence spectrum under the excitation wavelength of 375nm by using a fluorescence spectrometer.
The description and practice of the disclosure herein will be readily apparent to those skilled in the art from consideration of the specification and understanding, and may be modified and modified without departing from the principles of the disclosure. Therefore, modifications or improvements made without departing from the spirit of the invention should also be considered as the protection scope of the invention.
Claims (5)
1. A preparation method of glutathione-S-transferase-Au-Pt nanoclusters is characterized by comprising the following specific steps:
s1, adding the chloroauric acid solution into the glutathione-S-transferase solution, mixing uniformly, adding the chloroplatinic acid solution, and mixing uniformly to obtain a solution A;
s2, adding a sodium hydroxide solution into the solution A obtained in the step S1, adjusting the pH of the solution to 11-13, and heating the solution for 1-9 hours under the condition of metal bath to obtain a solution B;
s3, dialyzing the solution B obtained in the step S2 in phosphate buffer solution for 24 hours by using a dialysis bag with the molecular cut-off of 12-14kDa to remove redundant reactants, so that the protein is recovered to the natural state as far as possible, and storing the product at 4 ℃ to obtain the glutathione S-transferase-gold platinum nanoclusters.
2. The method for preparing the glutathione sulfurtransferase-platinic nanocluster of claim 1, wherein the concentration ratio of chloroauric acid to chloroplatinic acid in S1 is 8:1, and the concentration of glutathione sulfurtransferase is 20mg ml-1。
3. The method for preparing glutathione sulfurtransferase-Au-Pt nanoclusters of claim 1, wherein in S2, pH of the solution is adjusted to 11 by sodium hydroxide, and the reaction is performed for 5 hours at 80 ℃.
4. The application of the glutathione sulfurtransferase-gold platinum nanocluster obtained by the method for preparing the glutathione sulfurtransferase-gold platinum nanocluster according to any one of claims 1 to 3 in aureomycin detection is characterized in that the glutathione sulfurtransferase-gold platinum nanocluster is diluted by phosphate buffer, aureomycin with different concentrations is added and mixed uniformly, the mixture is incubated at room temperature, and the fluorescence intensity of the glutathione sulfurtransferase-gold platinum nanocluster is gradually enhanced along with the gradual increase of the aureomycin concentration under the excitation wavelength condition, so that the detection is realized.
5. The use of the glutathione S-transferase-Au-Pt nanoclusters of claim 4 in aureomycin assay, which is characterized in that the assay is carried out by incubating for 5 minutes at room temperature and under the condition of 375nm excitation wavelength.
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| CN115889757A (en) * | 2022-11-09 | 2023-04-04 | 南通大学 | Preparation method and application of bromelain-gold zinc nanocluster |
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| CN115889757A (en) * | 2022-11-09 | 2023-04-04 | 南通大学 | Preparation method and application of bromelain-gold zinc nanocluster |
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