CN115894624B - Gold nanocluster with polypeptide as ligand and detection method of aureomycin hydrochloride - Google Patents
Gold nanocluster with polypeptide as ligand and detection method of aureomycin hydrochloride Download PDFInfo
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- CN115894624B CN115894624B CN202211715289.6A CN202211715289A CN115894624B CN 115894624 B CN115894624 B CN 115894624B CN 202211715289 A CN202211715289 A CN 202211715289A CN 115894624 B CN115894624 B CN 115894624B
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a gold nanocluster with a polypeptide as a ligand and a detection method of aureomycin hydrochloride, wherein in the gold nanocluster with the polypeptide as the ligand, the sequence of the polypeptide is CCYFFKGGaa; the gold nanocluster with the polypeptide as the ligand can be applied to the detection of the chlortetracycline hydrochloride in river water or lake water. Adding the gold nanoclusters prepared by the method and taking the polypeptide as a ligand into a sample liquid to be detected, wherein the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting for 10-20min at 37-50 ℃ in a shaking table at 180rpm, detecting fluorescence change, observing fluorescence intensity at 420nm and 695nm, and having specificity for detecting aureomycin hydrochloride; the detection method has the advantages of high detection sensitivity, strong anti-interference capability, low detection limit, high correlation coefficient of a detection curve, good reproducibility in a lake water detection and recovery experiment and good practical value in the detection of aureomycin hydrochloride.
Description
Technical Field
The invention belongs to the field of nano materials, relates to a gold nanocluster and a preparation method thereof, and in particular relates to a polypeptide, a gold nanocluster prepared by taking the polypeptide as a ligand and a detection method for chlortetracycline hydrochloride by using the gold nanocluster.
Background
The problem of water environment pollution caused by the large amount of antibiotics in the breeding industry and the animal husbandry is becoming serious, and the antibiotics become one of the current international research hotspots. Currently, toxicity studies on antibiotics on aquatic organisms are mainly focused on bacteria, algae and crustaceans. Tetracyclines are the most widely used class of antibiotics in the aquaculture and animal husbandry. The aureomycin hydrochloride belongs to one of tetracycline antibiotics, belongs to low-toxicity substances for daphnia magna, and belongs to toxic substances for zebra fish and crucian. In natural environments, daphnia and fish constitute an important food chain in an aqueous environment, while contaminants are enriched in high nutrient organisms through the food chain, which would pose a greater health threat to humans at the highest level of the food chain.
The existing detection method of the chlortetracycline hydrochloride comprises a high performance liquid chromatography-mass spectrometry, an enzyme-linked immunosorbent assay, a capillary electrophoresis method, a spectrophotometry detection method, an electrochemical immunoassay method, a fluorescence unimodal detection method and the like, and the traditional chromatographic analysis method needs expensive experimental equipment or complex pretreatment process, and has low detection speed and low detection sensitivity; the fluorescent single-peak detection method has low specificity, unstable detection result and poor anti-interference capability.
Disclosure of Invention
In order to solve the technical problems, the invention provides a polypeptide, a gold nanocluster prepared by taking the polypeptide as a ligand and application thereof, and the gold nanocluster is used for detecting aureomycin hydrochloride.
The invention provides a polypeptide for preparing gold nanoclusters for detecting aureomycin hydrochloride, which has a sequence of CCYFFKGGAA.
The gold nanoclusters prepared by using the polypeptide as a ligand are prepared by the following method:
preparing a 2mM polypeptide solution and a 2mM chloroauric acid solution;
synthesis of AuNCs: adding a 2mM polypeptide solution and a 2mM chloroauric acid solution into a reaction vessel, wherein the volume ratio of the polypeptide solution to the chloroauric acid solution is 1:1; immediately thereafter, naOH solution was added to pH 14 to obtain gold nanoclusters with polypeptide as ligand.
The polypeptide is designed by a modularized strategy, and the sequence of the polypeptide is CCYFFKGGAA; wherein CCY is a gold cluster stable reduction module, and FFK is a function control module.
The gold nanoclusters prepared by the method and using the polypeptide as a ligand are applied to detection of aureomycin hydrochloride.
The gold nanoclusters prepared by the method and using the polypeptide as a ligand are applied to detection of chlortetracycline hydrochloride in river water or lake water.
The invention provides a detection method of chlortetracycline hydrochloride (CTC), which comprises the following steps:
adding the gold nanoclusters prepared by the method and taking the polypeptide as a ligand into a sample liquid to be detected, wherein the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting for 10-20min at 37-50 ℃ in a shaker at 180rpm, the fluorescence change is detected, and the fluorescence intensities at 420nm and 695nm are observed.
The invention has the beneficial effects that:
according to the invention, a modularized strategy is adopted to design and synthesize a polypeptide sequence, and the polypeptide is used as a ligand to prepare the gold nanocluster, and the quantitative analysis and detection of the aureomycin hydrochloride are realized by utilizing the fluorescence property change and the emission spectrum deviation property of the gold nanocluster and combining the relationship between the fluorescence bimodal ratio and the aureomycin hydrochloride concentration.
Compared with the traditional chromatographic analysis method, the detection method for the chlortetracycline hydrochloride has the advantages of no need of expensive experimental equipment, simplicity and rapidness; compared with the traditional fluorescence unimodal detection method, the bimodal detection method has higher specificity and more stable detection result; the self-calibration effect between the two fluorescence intensities can in this way effectively eliminate fluctuations in detection and background interference. In addition, the method has high detection sensitivity, strong anti-interference capability, low detection limit, high correlation coefficient of a detection curve and good reproducibility in a lake water detection and recovery experiment, so that the method has good practical value in the detection of aureomycin hydrochloride.
Drawings
FIG. 1 is a schematic view of fluorescence properties of gold nanoclusters of the present invention using polypeptides as ligands;
FIG. 2 is a graph showing fluorescence intensities of different concentrations of gold nanoclusters in the detection method of the present invention;
FIG. 3 is a second schematic diagram of fluorescence intensities of different concentrations of gold nanoclusters in the detection method of the present invention;
FIG. 4 is a schematic diagram showing fluorescence intensities at different reaction temperatures in the detection method of the present invention;
FIG. 5 is a second schematic diagram of fluorescence intensities at different reaction temperatures in the detection method of the present invention;
FIG. 6 is a schematic diagram of fluorescence intensity at different reaction times in the detection method of the present invention;
FIG. 7 is a graph showing fluorescence intensity of different CTC detection concentrations according to the present invention;
FIG. 8 is a schematic diagram of the linear relationship of the detection method of the present invention;
FIG. 9 is a graph showing the effect of different interfering ions on fluorescence intensity of detection effect according to the present invention;
FIG. 10 is a graph showing the effect of different interfering substances on fluorescence intensity of detection effect.
Detailed Description
The embodiment provides a polypeptide for preparing gold nanoclusters for detecting aureomycin hydrochloride, and the sequence is CCYFFKGGAA.
The gold nanoclusters prepared by using the polypeptide as a ligand are prepared by the following method:
synthesis of the polypeptide:
the polypeptide is synthesized by using a solid-phase synthesis method, and the polypeptide is designed by a modularized strategy, and the sequence of the polypeptide is CCYFFKGGAA. The specific implementation method is as follows:
(1) Soaking activation of resin
The resin was added to the polypeptide synthesis tube, 10mL of Dichloromethane (DCM) was added around the tube wall, and the synthesis tube was sealed and placed in a laboratory fume hood for overnight soak to fully activate the resin.
(2) Fmoc protecting group deprotection of resin
After the resin is soaked overnight, the DCM solution in the polypeptide synthesis tube is removed, fmoc group deprotection reagent is added, the polypeptide synthesis tube is obliquely placed in a constant temperature shaking table at 25 ℃, the polypeptide synthesis tube is taken out after shaking for 30min at constant speed, and the deprotection reagent in the polypeptide synthesis tube is removed. About 6mL of DCM, isopropanol and DMF were added to the polypeptide synthesis tubes, respectively, and each reagent was soaked for 2 minutes, after which the reagents were removed by vacuum pump filtration under reduced pressure, ensuring that the resin was adequately washed.
(3) Fmoc group deprotection of detection resin by ninhydrin method
And taking out the micro-resin from the synthesis tube, transferring the micro-resin into a 0.15mL centrifuge tube, adding the solution A and the solution B into the centrifuge tube, heating the centrifuge tube at the constant temperature of 100 ℃ for 5min, and taking out the centrifuge tube, wherein if the resin in the centrifuge tube is completely bluish purple, the Fmoc groups on the resin are proved to be completely deprotected. Solution A is water and ninhydrin in absolute ethanol, and solution B is molten phenol in absolute ethanol, the same applies below.
(4) Fmoc group protected amino acid activation
According to the molecular weight of the amino acid to be connected, 1.2mmol of Fmoc group protected amino acid is weighed out and placed in a 10mL centrifuge tube, 2.4mL of HBTU,2.4mL of HOBT and 330 mu L of DIEA are sequentially added into the centrifuge tube, the EP tube is vibrated by a vortex oscillator to promote the dissolution of the amino acid, and the mixture is left stand for 10min to enable the amino acid to be completely activated.
(5) Ligation of amino acids
The fully activated amino acid was first added to the resin-containing polypeptide synthesis tube, then 12mL of DCM was added, and finally 3mL of xylene was added to fully suspend the resin. And (3) obliquely placing the polypeptide synthesis tube in a constant-temperature shaking table at 25 ℃, shaking at a constant speed for 3.5 hours, taking out, and removing the solution in the polypeptide synthesis tube by vacuum pump decompression and suction filtration. About 6mL of DMF, isopropanol and DCM were added sequentially to the polypeptide synthesis tube, after which the reagents were removed by suction filtration under reduced pressure using a vacuum pump, each reagent was added repeatedly for 2min each time of soaking, ensuring that the resin was adequately washed.
(6) Ninhydrin method for detecting amino acid connection
Taking out a trace amount of resin from a synthesis tube, transferring the resin into a 0.15mL centrifuge tube, respectively adding the solution A and the solution B, heating at the constant temperature of 100 ℃ for 5min, and taking out the resin, and if the resin in the centrifuge tube is transparent yellow, proving that the amino acid is completely connected, and then the connection of the next amino acid can be performed; if a portion of the resin is light blue, it is indicated that the amino acid is not completely linked, and steps (4) - (6) are repeated to religate the amino acid.
(7) Fmoc protecting group deprotection of amino acids
And adding 16mL of Fmoc group deprotection reagent into the polypeptide synthesis tube, obliquely placing the polypeptide synthesis tube in a constant temperature shaking table at 25 ℃, taking out after shaking at constant speed for 30min, and removing the deprotection reagent. The resin was washed by sequentially adding about 6mL of DCM, isopropanol and DMF, each reagent was repeated twice, each time for 2min.
(8) Fmoc group deprotection for detecting amino acid by ninhydrin method
Taking out a trace amount of resin from a synthesis tube, transferring the resin into a 0.15mL centrifuge tube, adding 50 mu L of solution A and 50 mu L of solution B into the centrifuge tube, heating the mixture at a constant temperature of 100 ℃ for 5min, taking out the mixture, and if the resin in the centrifuge tube is completely blue-purple, proving that Fmoc groups on the resin are completely deprotected; if a portion of the resin is clear yellow, this indicates that the Fmoc group of this amino acid is not completely deprotected, and it is necessary to reprotect it.
Repeating the steps (4) - (8) to connect the next amino acid, and connecting the amino acids in the order from right to left.
And (3) polypeptide shearing:
(1) Adding a shearing solution into the polypeptide synthesis tube after vacuum drying: trifluoroacetic acid, TIS, ultrapure water, total 20mL.
(2) And (3) obliquely placing the polypeptide synthesis tube in a constant-temperature shaking table at 25 ℃ for 2 hours, taking out the polypeptide synthesis tube, mounting the polypeptide synthesis tube on a pre-prepared suction filtration bottle filled with precooled anhydrous diethyl ether, allowing the shearing liquid to slowly flow into the suction filtration bottle until no liquid in the polypeptide synthesis tube is left, taking down the suction filtration bottle filled with crude peptide to separate out, sealing, and storing in a refrigerator at-20 ℃.
(3) And (3) adding half of the amount of the shearing liquid into the polypeptide synthesis tube again, obliquely placing the polypeptide synthesis tube into a constant-temperature shaking table at 25 ℃, shaking at a constant speed for 1h, taking out, and repeating the step (2).
(4) Standing for about 2-3h until all polypeptides are precipitated from pre-chilled anhydrous diethyl ether, filtering off the anhydrous diethyl ether with a G4 funnel, then scraping the crude peptide off the G4 funnel with a spoon, optionally rinsing the funnel with 50% acetonitrile/water, and then dissolving the crude peptide in a suitable amount of 50% acetonitrile/water in a lyophilization flask.
(5) Opening the cold trap for pre-cooling in advance for 2 hours, placing the freeze-drying bottle in a low-temperature cold trap groove for rotation, enabling the polypeptide solution to be frozen into ice in a spinning way until all the polypeptide solution is uniformly hung on the inner wall of the freeze-drying bottle, then freeze-drying the crude peptide completely by a freeze dryer by adopting a freeze drying method, finally forming crude peptide powder, weighing and recording the crude peptide, and then placing the crude peptide in a refrigerator at the temperature of minus 20 ℃ for standby.
Purification of the polypeptide:
(1) Crude peptide sample pretreatment
About 20mg of crude peptide powder is weighed by a 4mL centrifuge tube, 4mL of deionized water is added, and the mixture is placed on a vortex oscillator for shaking, so that the crude peptide powder is fully dissolved in water, and the dissolved polypeptide is filtered by an organic filter membrane with the aperture of 0.22 mu m to remove impurities.
(2) Crude peptide analysis
The method comprises the steps of analyzing a crude peptide sample by using a Shimadzu LC-20A semi-preparative reverse high performance liquid chromatography, setting a crude peptide analysis program, sucking 15 mu L of filtered crude peptide sample by using a microsyringe needle, wherein the peak with the strongest signal in the chromatogram is generally the peak time of a target polypeptide, and further optimizing the crude peptide analysis program and setting a proper polypeptide preparation program according to the peak time of the crude peptide sample.
(3) Polypeptide preparation
And (3) carrying out small-scale preparation on the crude peptide sample by using Shimadzu LC-20A semi-preparative reverse high performance liquid chromatography, opening a set polypeptide sample preparation program, sucking 4mL of filtered crude peptide sample by using a 5mL sample injection needle, injecting the sample into a pump, automatically executing the preparation program, and simultaneously opening a sample automatic collector to automatically collect the prepared mobile phase. And determining the peak time of the target polypeptide according to the chromatogram prepared by the polypeptide, finding out a corresponding tube, carrying out polypeptide analysis again, and comparing the peak time with the peak time of the previous crude peptide analysis, wherein if the peak time is consistent and other miscellaneous peaks are not present, the tube is proved to be the target polypeptide. All collecting pipes which contain the target polypeptide and are free of other impurities are determined and transferred into a freeze-drying bottle, the freeze-drying bottles are placed into a low-temperature cold trap groove to rotate into ice, then a freeze dryer is utilized to freeze-dry the pure peptide into pure peptide powder, and the pure peptide powder is placed into a refrigerator at the temperature of minus 20 ℃ for standby after weighing and recording.
And (3) identification:
detecting by a matrix-assisted laser analysis tandem time-of-flight mass spectrometer, and determining the molecular weight of the pure peptide so as to determine whether the prepared polypeptide is a target polypeptide.
Preparing polypeptide solution:
taking a certain amount of polypeptide powder, and preparing a 2mM polypeptide solution by using distilled water of the chen type;
preparing chloroauric acid solution:
200uL of 50mM chloroauric acid mother solution is taken, 4800uL of dronic distilled water is added to prepare 2mM chloroauric acid solution;
synthesis of AuNCs:
taking a 1.5mL EP tube, adding 200uL of a 2mM polypeptide solution, adding 200uL of a 2mM chloroauric acid solution, immediately adding 10uL of a 2M NaOH solution, uniformly mixing, and detecting the pH of the mixed solution to be 14 by using pH test paper to obtain the gold nanocluster with the polypeptide as a ligand.
The fluorescence properties of the gold nanoclusters prepared in this example and using the polypeptides as ligands are shown in fig. 1, and the excitation wavelength is 515nm and the emission wavelength is 695nm.
The prepared gold nanoclusters with polypeptide as a ligand are applied to the detection of aureomycin hydrochloride, and the detection method comprises the following steps:
adding the gold nanoclusters prepared in the embodiment and taking the polypeptide as a ligand into a sample liquid to be detected, wherein the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting for 10-20min at 37-50 ℃ in a shaker at 180rpm, the fluorescence change is detected, and the fluorescence intensities at 420nm and 695nm are observed.
Effect comparison experiment:
according to the detection method, the detection effects under different reaction conditions are respectively compared:
a. AuNCs concentration comparison:
AuNCs final concentration was selected to be 0.05mM (CTC 190uL+AuNCs 10uL), 0.25mM (CTC 150uL+AuNCs 50uL), 0.5mM (CTC 100uL+AuNCs 100uL), 0.75mM (CTC 50uL+AuNCs 150uL), 0.95mM (CTC 10uL+AuNCs 190uL), and the resultant mixture was reacted with a CTC solution of 4mM final concentration at 37℃in a shaker at 180rpm for 10 minutes to detect a change in fluorescence. As shown in FIGS. 2 to 3, the fluorescence at 690nm completely disappears at a final concentration of AuNCs of 0.05mM, so that the concentration is preferably 0.25mM.
b. Reaction temperature contrast:
AuNCs with a final concentration of 0.25mM and CTC solutions with a final concentration of 4mM were selected and reacted in a shaker at 180rpm at a temperature of 4℃and 25℃and 37℃and 50℃for 10min, respectively, to detect a change in fluorescence. As shown in FIGS. 4 to 5, the fluorescence intensity is least affected by temperature at 37℃and therefore the temperature is preferably 37 ℃.
c. Reaction time comparison:
the AuNCs with a final concentration of 0.25mM and the CTC solution with a final concentration of 4mM were selected to react at 37℃in a shaker at 180rpm for 10min, 20min, 30min, 40min, 50min, respectively, and fluorescence changes were detected. As shown in FIG. 6, the final reaction result is substantially reached at 10 minutes of reaction, so the reaction time is at least 10 minutes.
CTC detection effect and limit:
solutions with final CTC concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0uM are prepared respectively, 150uL of CTC solution is taken and added with 50uL of AuNCs, and after mixing, the mixture is placed into a shaking table at 37 ℃ and 180rpm for reaction for 10min, and fluorescence emission under excitation of 370nm is detected. As shown in FIGS. 7-8, the fluorescence emission position of AuNCs changed after CTC addition, a new emission peak appeared at 420nm, the fluorescence intensity of the emission peak at 420nm increased continuously with increasing CTC concentration, and F420/F695 had a linear relationship with a detection limit of 10.3nmol.
Anti-interference experiment:
a. preparation of 2.1mM CuSO respectively 4 、MgSO 4 、MnCl 2 、NaCl、CaCO 3 、Ba(OH) 2 The effect on fluorescence intensity was detected by adding 10uL of the solution to 200uL of AuNCs and AuNCs+CTC, respectively. As shown in FIG. 9, cuSO was added to the AuNCs and AuNCs+CTC solutions, respectively 4 、MgSO 4 、MnCl 2 、NaCl、CaCO 3 、Ba(OH) 2 The plasma was found to have little effect on fluorescence intensity.
b. 0.341mM of CTC, tetracycline, doxycycline, oxytetracycline hydrochloride, vancomycin, ofloxacin, arginine, serine, cysteine, leucine, histidine, glutamic acid, isoleucine, lysine and the like are respectively prepared, and fluorescence changes are sequentially detected according to the detection method of the invention. As shown in fig. 10, the selection of other substances according to the CTC detection method found that no other substances except CTCs produced an emission peak at 420 nm.
Recovery experiment:
the detection effect of the detection method in the actual sample is verified, lake water is selected, the lake water is centrifuged in a method of 10000rpm for 5min, the centrifuged sample is filtered by a 0.22uM filter membrane, CTC is added into the sample to prepare a CTC solution of 1uM, 2uM and 3uM, and the detection concentration of CTC in the sample is detected by the detection method.
As shown in the following table, the linear model constructed by the invention has a linear range of 0-8nmol/mL, a standard curve equation of y=0.448x+0.184 (y is the ratio of fluorescence intensity at 420nm to fluorescence intensity at 695nm, x is CTC concentration), and a correlation coefficient R 2 =0.999, the limit of detection was calculated as low as 10.3nmol, and the recovery obtained by the method was 96.8% -103.6%. The detection method has good detection accuracy and plays an important role in monitoring the problem of CTC residues in lake water.
Claims (6)
1. The preparation method of the polypeptide for detecting the gold nanoclusters of the aureomycin hydrochloride is characterized by comprising the following steps of: the polypeptide sequence is CCYFFKGGAA.
2. A preparation method of gold nanoclusters with polypeptides as ligands is characterized by comprising the following steps:
adding a 2mM polypeptide solution and a 2mM chloroauric acid solution into a reaction vessel, wherein the volume ratio of the polypeptide solution to the chloroauric acid solution is 1:1; immediately adding NaOH solution to pH 14 to obtain gold nanoclusters with polypeptide as ligand;
the sequence of the polypeptide is CCYFFKGGAA.
3. A gold nanocluster with polypeptide as ligand is characterized in that: is prepared by the preparation method of claim 2.
4. The use of a gold nanocluster with a polypeptide as a ligand according to claim 3, wherein: the kit is applied to detection of the aureomycin hydrochloride for non-diagnosis or treatment purposes.
5. The use of a gold nanocluster with a polypeptide as a ligand according to claim 3, wherein: the method is applied to detection of the chlortetracycline hydrochloride in river water or lake water.
6. A detection method for non-diagnostic or therapeutic purposes of aureomycin hydrochloride is characterized by comprising the following steps: the method comprises the following steps:
adding the gold nanocluster with the polypeptide as a ligand according to claim 3 into a sample liquid to be detected, wherein the final concentration of the gold nanocluster is not less than 0.25mM; after reacting for 10-20min at 37-50 ℃, the fluorescence change is detected, and the fluorescence intensities at 420nm and 695nm are observed.
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| CN113376130A (en) * | 2021-05-14 | 2021-09-10 | 南京师范大学 | Fluorescence open-type probe for detecting ampicillin residue and preparation method and application thereof |
| CN113390843A (en) * | 2021-06-16 | 2021-09-14 | 南通大学 | Preparation method of casein-gold nanocluster and application of casein-gold nanocluster in aureomycin detection |
| CN115452787A (en) * | 2022-09-22 | 2022-12-09 | 山东理工大学 | Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles |
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| CN113376130A (en) * | 2021-05-14 | 2021-09-10 | 南京师范大学 | Fluorescence open-type probe for detecting ampicillin residue and preparation method and application thereof |
| CN113390843A (en) * | 2021-06-16 | 2021-09-14 | 南通大学 | Preparation method of casein-gold nanocluster and application of casein-gold nanocluster in aureomycin detection |
| CN115452787A (en) * | 2022-09-22 | 2022-12-09 | 山东理工大学 | Method for measuring streptomycin in milk by using fluorescence sensor constructed by silver nanoclusters and gold palladium nanoparticles |
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