CN115894624B - A method for detecting gold nanoclusters and chlortetracycline hydrochloride using polypeptides as ligands - Google Patents

Abstract

The invention provides a method for detecting gold nanoclusters with polypeptides as ligands and chlortetracycline hydrochloride. In the gold nanoclusters with polypeptides as ligands, the sequence of the polypeptides is CCYFFKGGaa; the polypeptides are used as ligands. The bulk gold nanoclusters can be applied to the detection of chlortetracycline hydrochloride and the detection of chlortetracycline hydrochloride in river or lake water. The gold nanoclusters with polypeptides as ligands prepared by the present invention are added to the sample liquid to be tested, and the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting for 10-20 minutes in a 180rpm shaker at a temperature of 37-50°C , detect fluorescence changes, observe the fluorescence intensity at 420nm and 695nm, and have specificity for the detection of chlortetracycline hydrochloride; the detection method of the present invention has high detection sensitivity, strong anti-interference ability, low detection limit, and high detection curve correlation coefficient. It has good reproducibility in lake water detection and recovery experiments, and has good practical value in the detection of chlortetracycline hydrochloride.

Description

A method for detecting gold nanoclusters and chlortetracycline hydrochloride using polypeptides as ligands

Technical field

The invention belongs to the field of nanomaterials and relates to a gold nanocluster and a preparation method thereof. In particular, it relates to a polypeptide and a gold nanocluster prepared with the polypeptide as a ligand and a method for detecting chlortetracycline hydrochloride using the gold nanocluster.

Background technique

Due to the extensive use of antibiotics in breeding and animal husbandry, the problem of water environment pollution has become increasingly serious and has become one of the current international research hotspots. At present, research on the toxicity of antibiotics to aquatic organisms mainly focuses on bacteria, algae and crustaceans. Tetracycline antibiotics are the most widely used antibiotics in aquaculture and animal husbandry. Chlortetracycline hydrochloride is one of the tetracycline antibiotics. It is a low-toxic substance to Daphnia magna and a toxic substance to zebrafish and crucian carp. In the natural environment, daphnia and fish form an important food chain in the water environment, and pollutants are accumulated in high-trophic-level organisms through the food chain, which will pose a greater health threat to humans at the highest level of the food chain.

Current detection methods for chlortetracycline hydrochloride include high performance liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, capillary electrophoresis, spectrophotometer detection, electrochemical immunoassay, fluorescence single peak detection, etc. Traditional Chromatographic analysis methods require expensive experimental equipment or complex pretreatment processes, and have slow detection speeds and low detection sensitivity; fluorescence single-peak detection methods have low specificity, unstable detection results, and poor anti-interference ability.

Contents of the invention

In order to solve the above technical problems, the present invention provides a polypeptide, a gold nanocluster prepared with the polypeptide as a ligand and its application. The gold nanocluster is used to detect chlortetracycline hydrochloride.

The invention provides a polypeptide for preparing gold nanoclusters for detecting chlortetracycline hydrochloride, and the sequence is CCYFFKGGAA.

The invention provides a gold nanocluster prepared with a polypeptide as a ligand, which is prepared by the following method:

Prepare 2mM peptide solution and 2mM chloroauric acid solution;

Synthesis of AuNCs: Add 2mM peptide solution and 2mM chloroauric acid solution to the reaction vessel. The volume ratio of the peptide solution and chloroauric acid solution is 1:1; then immediately add NaOH solution to pH 14 to obtain a peptide-based formulation. solid gold nanoclusters.

The polypeptide is designed with a modular strategy, and the sequence of the polypeptide is CCYFFKGGAA; among them, CCY is a gold cluster stable reduction module, and FFK is a functional regulation module.

The gold nanoclusters with polypeptides as ligands prepared by the present invention are used in the detection of chlortetracycline hydrochloride.

The gold nanoclusters with polypeptides as ligands prepared by the present invention are used for the detection of chlortetracycline hydrochloride in river water or lake water.

The invention provides a detection method for chlortetracycline hydrochloride (CTC), which includes the following steps:

The gold nanoclusters with polypeptides as ligands prepared in the present invention are added to the sample liquid to be tested, and the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting for 10-20 minutes in a shaker at 180 rpm at a temperature of 37-50°C , detect fluorescence changes and observe the fluorescence intensity at 420nm and 695nm.

Beneficial effects of the present invention:

The present invention adopts a modular strategy to design and synthesize polypeptide sequences, and uses polypeptides as ligands to prepare gold nanoclusters. It utilizes the changes in fluorescence properties of gold nanoclusters and the offset properties of emission spectra, and combines the ratio of fluorescence double peaks with the concentration of chlortetracycline hydrochloride. relationship to achieve quantitative analysis and detection of chlortetracycline hydrochloride.

The detection method of chlortetracycline hydrochloride provided by the present invention has the specificity of detecting chlortetracycline hydrochloride. Compared with the traditional chromatographic analysis method, it does not require expensive experimental equipment and has the advantages of simplicity and speed; it is comparable to the traditional fluorescence single peak detection method. Compared with the double-peak detection method, the specificity is higher and the detection results are more stable; the self-calibration effect between the two fluorescence intensities can effectively eliminate the fluctuation of detection interference and background interference in this way. In addition, this method has high detection sensitivity, strong anti-interference ability, low detection limit, high detection curve correlation coefficient, and good reproducibility in lake water detection and recovery experiments. Therefore, this method has good performance in the detection of chlortetracycline hydrochloride. practical value.

Description of the drawings

Figure 1 is a schematic diagram of the fluorescence properties of gold nanoclusters using polypeptides as ligands according to the present invention;

Figure 2 is a schematic diagram of the fluorescence intensity of different concentrations of gold nanoclusters in the detection method of the present invention;

Figure 3 is a schematic diagram 2 of the fluorescence intensity of different concentrations of gold nanoclusters in the detection method of the present invention;

Figure 4 is a schematic diagram of fluorescence intensity at different reaction temperatures in the detection method of the present invention;

Figure 5 is a schematic diagram 2 of fluorescence intensity at different reaction temperatures in the detection method of the present invention;

Figure 6 is a schematic diagram of fluorescence intensity at different reaction times in the detection method of the present invention;

Figure 7 is a schematic diagram of fluorescence intensity at different CTC detection concentrations according to the present invention;

Figure 8 is a schematic diagram of the linear relationship of the detection method of the present invention;

Figure 9 is a schematic diagram of the influence of different interfering ions on the fluorescence intensity of the detection effect according to the present invention;

Figure 10 is a schematic diagram of the influence of different interfering substances on the fluorescence intensity of the detection effect of the present invention.

Detailed ways

This embodiment provides a polypeptide for preparing gold nanoclusters for detecting chlortetracycline hydrochloride, with the sequence CCYFFKGGAA.

This example provides a gold nanocluster prepared with a polypeptide as a ligand, prepared by the following method:

Synthesis of peptides:

The polypeptide was synthesized using a solid-phase synthesis method. The polypeptide was designed with a modular strategy. The sequence of the polypeptide is CCYFFKGGAA. The specific implementation methods are as follows:

(1) Soaking activation of resin

Add the resin into the peptide synthesis tube, add 10 mL of dichloromethane (DCM) around the tube wall, seal the synthesis tube and place it in a laboratory fume hood to soak overnight to fully activate the resin.

(2) Deprotection of the Fmoc protecting group of the resin

After the resin is soaked overnight, remove the DCM solution from the peptide synthesis tube, add the Fmoc group deprotection reagent, place the peptide synthesis tube at an angle in a constant temperature shaker at 25°C, shake at a constant speed for 30 minutes, take it out, and remove the deprotection reagent from the peptide synthesis tube. Protection reagent removal. Add approximately 6 mL of DCM, isopropanol, and DMF to the peptide synthesis tube in order, soak each reagent for 2 minutes, and then use a vacuum pump to remove the reagents by vacuum filtration to ensure that the resin can be fully washed.

(3) Ninhydrin method to detect the deprotection of the Fmoc group of the resin

Take a trace amount of resin from the synthesis tube and transfer it to a 0.15mL centrifuge tube, add solution A and solution B to it, heat at a constant temperature of 100°C for 5 minutes and then take it out. If all the resin in the centrifuge tube turns blue-purple, it proves that Fmoc on the resin The group is completely deprotected. Solution A is water and ninhydrin dissolved in absolute ethanol, and solution B is molten phenol dissolved in absolute ethanol, the same below.

(4) Activation of amino acids protected by Fmoc group

According to the molecular weight of the amino acid to be connected, weigh out 1.2 mmol of the Fmoc group-protected amino acid and place it in a 10 mL centrifuge tube. Add 2.4 mL of HBTU, 2.4 mL of HOBT and 330 μL of DIEA to it in sequence, and shake with a vortex oscillator. The EP tube promotes the dissolution of amino acids and leaves it for 10 minutes to fully activate.

(5)Connection of amino acids

First, add fully activated amino acids to the peptide synthesis tube containing the resin, then add 12 mL of DCM, and finally add 3 mL of xylene to completely suspend the resin. Place the peptide synthesis tube at an angle in a constant-temperature shaker at 25°C, shake at a constant speed for 3.5 hours, take it out, and use a vacuum pump to filter under reduced pressure to remove the solution in the peptide synthesis tube. Add about 6 mL of DMF, isopropyl alcohol and DCM to the peptide synthesis tube in sequence, and then use a vacuum pump to remove the reagents by vacuum filtration. Each reagent is added twice and soaked for 2 minutes each time to ensure that the resin can be fully washed.

(6) Ninhydrin method to detect the connection of amino acids

Take out a small amount of resin from the synthesis tube and transfer it to a 0.15mL centrifuge tube. Add solution A and solution B respectively, heat at a constant temperature of 100°C for 5 minutes and then take it out. If all the resin in the centrifuge tube turns bright yellow, it proves that the amino acids are completely connected. Connect the next amino acid; if part of the resin turns light blue, it means that the amino acid is not completely connected, and you need to repeat steps (4)-(6) to reconnect the amino acid.

(7) Deprotection of Fmoc protecting group of amino acids

Add 16 mL of Fmoc group deprotection reagent to the peptide synthesis tube, place the peptide synthesis tube at an angle in a constant temperature shaker at 25°C, shake at a constant speed for 30 minutes, then take it out and remove the deprotection reagent. Add about 6 mL of DCM, isopropyl alcohol and DMF in order to wash the resin. Repeat each reagent added twice and soak for 2 minutes each time.

(8) Ninhydrin method to detect deprotection of Fmoc group of amino acids

Take out a small amount of resin from the synthesis tube and transfer it to a 0.15mL centrifuge tube, add 50 μL each of solution A and solution B, heat at 100°C for 5 minutes and then take it out. If all the resin in the centrifuge tube turns blue-purple, it means that the resin has The Fmoc group of the amino acid is completely deprotected; if part of the resin is bright yellow, it means that the Fmoc group of the amino acid is not completely deprotected, and it needs to be deprotected again.

Repeat steps (4)-(8) to connect the next amino acid, and the amino acids are connected in order from right to left.

Cleavage of polypeptides:

(1) Add shear liquid: trifluoroacetic acid, TIS, and ultrapure water to the vacuum-dried peptide synthesis tube, totaling 20 mL.

(2) Place the peptide synthesis tube in a 25°C constant-temperature shaker at an angle, shake it at a constant speed for 2 hours, and then take it out. Install the peptide synthesis tube into a prepared filter bottle filled with pre-cooled anhydrous ether and let the shear liquid Slowly flow into the filtration flask until no liquid remains in the peptide synthesis tube. Remove the seal from the filtration flask containing the crude peptide and store it in a -20°C refrigerator.

(3) Add half the amount of the above-mentioned shear solution to the peptide synthesis tube again, place the peptide synthesis tube at an angle in a constant temperature shaker at 25°C, shake at a constant speed for 1 hour, take it out, and repeat step (2).

(4) Let it stand for about 2-3 hours until all the peptides precipitate from the pre-cooled anhydrous ether. Use a G4 funnel to filter to remove the anhydrous ether. Then use a spoon to scrape the crude peptide off the G4 funnel. You can also use 50% acetonitrile. /water to rinse the funnel, and then use an appropriate amount of 50% acetonitrile/water to dissolve the crude peptide in the freeze-drying bottle.

(5) Open the cold trap 2 hours in advance to pre-cool, place the freeze-drying bottle in the low-temperature cold trap tank and rotate it to spin-freeze the peptide solution into ice until all the peptide solution hangs evenly on the inner wall of the freeze-drying bottle, and then use The freeze-drying method uses a freeze dryer to freeze-dry the crude peptide completely, and finally forms crude peptide powder. The crude peptide is weighed and recorded and then stored in a -20°C refrigerator for later use.

Purification of peptides:

(1) Crude peptide sample pretreatment

Weigh about 20 mg of crude peptide powder in a 4 mL centrifuge tube, add 4 mL of deionized water, and shake on a vortex oscillator to fully dissolve the crude peptide powder into the water. The dissolved peptide needs to be filtered out with an organic filter membrane with a pore size of 0.22 μm. Impurities.

(2) Crude peptide analysis

Use Shimadzu LC-20A semi-preparative reverse-phase high-performance liquid chromatography to analyze the crude peptide sample. Set the crude peptide analysis program and use a micro-injection needle to draw 15 μL of the filtered crude peptide sample. The signal in the chromatogram is the strongest. The peak is generally the peak elution time of the target peptide. Based on the peak elution time of the crude peptide sample, the crude peptide analysis program can be further optimized and the appropriate peptide preparation program can be set.

(3) Peptide preparation

Use Shimadzu LC-20A semi-preparative reverse-phase high-performance liquid chromatography to prepare crude peptide samples in small quantities. Open the set peptide sample preparation program, use a 5mL syringe to draw 4mL of filtered crude peptide sample and inject it into the pump. The intermediate and post-preparation procedures are automatically executed, and the automatic sample collector is opened at the same time to automatically collect the prepared mobile phase. According to the chromatogram prepared by the peptide, determine the peak time of the target peptide, and then find the corresponding tube and perform peptide analysis again. Compare it with the peak time of the previous crude peptide analysis. If the peak time is consistent and there are no other impurity peaks, , it proves that the tube is the desired target polypeptide. Determine all collection tubes containing the target polypeptide and without other impurities and transfer them to freeze-drying bottles. Place them in a low-temperature cold trap and spin them into ice. Then use a freeze-drying machine to freeze-dry the pure peptide into pure peptide powder. After weighing and recording Store in -20°C refrigerator for later use.

Identification:

Use matrix-assisted laser desorption tandem time-of-flight mass spectrometry for detection to determine the molecular weight of the pure peptide to determine whether the prepared peptide is the target peptide.

Preparation of peptide solution:

Take a certain amount of peptide powder and use Watsons distilled water to prepare a 2mM peptide solution;

Preparation of chloroauric acid solution:

Take 200uL of 50mM chloroauric acid stock solution and add 4800uL of Watson's distilled water to prepare a 2mM chloroauric acid solution;

Synthesis of AuNCs:

Take a 1.5mL EP tube, first add 200uL, 2mM peptide solution, then add 200uL, 2mM chloroauric acid solution, then immediately add 10uL, 2M NaOH solution, mix well and use pH test paper to check that the pH of the mixed solution is 14. Gold nanoclusters with polypeptides as ligands were obtained.

The fluorescence properties of the gold nanoclusters with polypeptides as ligands prepared in this example are shown in Figure 1, with an excitation wavelength of 515 nm and an emission wavelength of 695 nm.

In this example, the prepared gold nanoclusters with polypeptides as ligands are applied to the detection of chlortetracycline hydrochloride. The detection method is:

Add the gold nanoclusters with polypeptides as ligands prepared in this example to the sample liquid to be tested. The final concentration of the gold nanoclusters is not less than 0.25mM; react in a 180rpm shaker at a temperature of 37-50°C for 10-20 minutes. Finally, detect the fluorescence changes and observe the fluorescence intensity at 420nm and 695nm.

Effect comparison experiment:

According to the above detection methods, compare the detection effects under different reaction conditions:

a. AuNCs concentration comparison:

Select the Auncs final concentration of 0.05mm (CTC 190ul+Auncs 10ul), 0.25mm (CTC 150ul+Auncs 50ul), 0.5mm (CTC 100UL+Auncs 100UL), 0.75mm (CTC 50ul+Auncs 150ul), and 0.95. MM (CTC 10uL + AuNCs 190uL), react with a CTC solution with a final concentration of 4mM for 10min at 37°C in a shaker at 180rpm, and detect the fluorescence change. As shown in Figure 2-3, the fluorescence at 690nm completely disappears when the final concentration of AuNCs is 0.05mM, so the minimum concentration is preferably 0.25mM.

b. Comparison of reaction temperatures:

AuNCs with a final concentration of 0.25mM and CTC solution with a final concentration of 4mM were selected and reacted in a shaker at 180rpm for 10 minutes at temperatures of 4°C, 25°C, 37°C, and 50°C, respectively, and fluorescence changes were detected. As shown in Figure 4-5, under the condition of 37°C, the fluorescence intensity is least affected by temperature, so the temperature is preferably 37°C.

c. Reaction time comparison:

AuNCs with a final concentration of 0.25mM and CTC solution with a final concentration of 4mM were selected to react in a shaker at 180rpm at 37°C for 10min, 20min, 30min, 40min, and 50min respectively, and the fluorescence changes were detected. As shown in Figure 6, the final reaction result is basically reached after 10 minutes of reaction, so the reaction time is at least 10 minutes.

CTC detection effect and detection limit:

Prepare solutions with final CTC concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0uM respectively. Take 150uL of the CTC solution and add 50uL of AuNCs. After mixing, place it in a shaker at 37°C and 180rpm to react for 10 minutes. Detect 370nm excitation. Fluorescence emission below. As shown in Figure 7-8, the fluorescence emission position of AuNCs changes after adding CTC, and a new emission peak appears at 420nm. As the CTC concentration increases, the fluorescence intensity of the emission peak at 420nm continues to increase, and There is a linear relationship between F420/F695, and the detection limit is 10.3nmol.

Anti-interference experiment:

a. Prepare 2.1mM CuSO ₄ , MgSO ₄ , MnCl ₂ , NaCl, CaCO ₃ , and Ba(OH) ₂ solutions respectively, and add 10uL to 200uL AuNCs and AuNCs+CTC to detect the effect on fluorescence intensity. As shown in Figure 9, CuSO ₄ , MgSO ₄ , MnCl ₂ , NaCl, CaCO ₃ , and Ba(OH) ₂ plasma were added to the AuNCs and AuNCs+CTC solutions respectively, and it was found that there was almost no effect on the fluorescence intensity.

b. Prepare 0.341mM CTC, tetracycline, doxycycline, oxytetracycline hydrochloride, vancomycin, ofloxacin, arginine, serine, cysteine, leucine, histidine, and glutamine respectively. Acid, isoleucine, lysine, etc., according to the detection method of the present invention, the fluorescence changes are detected sequentially. As shown in Figure 10, by selecting other substances and following the CTC detection method, it was found that no other substances except CTC would produce an emission peak at 420 nm.

Recycling experiment:

To verify the detection effect of the detection method of the present invention in actual samples, select lake water, centrifuge the lake water at 10,000 rpm for 5 minutes, pass the centrifuged sample through a 0.22uM filter membrane, add CTC to it, and prepare 1uM, 2uM, and 3uM CTC solution, and use the detection method of the present invention to detect the detection concentration of CTC in the sample.

As shown in the table below, the linear range of the linear model constructed by the present invention is 0-8nmol/mL, and the standard curve equation is y=0.448x+0.184 (y is the ratio of the fluorescence intensity at 420nm to the fluorescence intensity at 695nm, x is CTC concentration), the correlation coefficient R ² =0.999, the detection limit was calculated as low as 10.3 nmol, and the recovery rate obtained by the method was 96.8%-103.6%. The detection method of the present invention has good detection accuracy and plays an important role in monitoring the residual problem of CTC in lake water.

Claims (6)

1. A polypeptide for preparing gold nanoclusters for detecting chlortetracycline hydrochloride, characterized in that: the polypeptide sequence is CCYFFKGGAA. 2. A method for preparing gold nanoclusters with polypeptides as ligands, characterized by: Add 2mM peptide solution and 2mM chloroauric acid solution to the reaction vessel. The volume ratio of the peptide solution and chloroauric acid solution is 1:1; then immediately add NaOH solution to pH 14 to obtain gold nanoparticles with the peptide as the ligand. cluster; The sequence of the polypeptide is CCYFFKGGAA. 3. A gold nanocluster with a polypeptide as a ligand, characterized in that it is prepared by the preparation method of claim 2. 4. The application of gold nanoclusters with polypeptides as ligands according to claim 3, characterized in that it is applied to the detection of chlortetracycline hydrochloride for non-diagnostic or therapeutic purposes. 5. The application of gold nanoclusters with polypeptides as ligands according to claim 3, characterized in that it is applied to the detection of chlortetracycline hydrochloride in river water or lake water. 6. A non-diagnostic or therapeutic detection method for chlortetracycline hydrochloride, characterized in that it includes the following steps: Add the gold nanoclusters with polypeptides as ligands according to claim 3 into the sample liquid to be tested, and the final concentration of the gold nanoclusters is not less than 0.25mM; after reacting at 37-50°C for 10-20 minutes, the fluorescence is detected Change and observe the fluorescence intensity at 420nm and 695nm.