CN114689557A - Preparation method of chicken protein protected gold-copper nanocluster and application of chicken protein protected gold-copper nanocluster in mercury ion detection - Google Patents
Preparation method of chicken protein protected gold-copper nanocluster and application of chicken protein protected gold-copper nanocluster in mercury ion detection Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The invention relates to the technical field of gold-copper nanocluster synthesis, in particular to a preparation method of chicken protein protected gold-copper nanoclusters and application of the chicken protein protected gold-copper nanoclusters in mercury ion detection, and the method comprises the following specific steps: s1, weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding chloroauric acid for uniformly mixing, adding a copper sulfate solution, uniformly mixing and stirring for 2 minutes to obtain a solution A; s2, adding a 1M sodium hydroxide solution into the solution A obtained in the step S1, adjusting the pH of the solution to 9-13, and heating the solution in a water bath for 3-10 hours to obtain a solution B; s3, storing the solution B obtained in the step 2 at 4 ℃ to obtain the chicken protein-gold copper nanocluster. The invention adopts a one-step synthesis method to prepare the chicken protein-gold copper nanocluster in the aqueous solution, and has unique photophysical characteristics, simple preparation, low toxicity and good stability; the method has the advantages of rapidness and simplicity in mercury ion detection, high detection sensitivity and low detection limit, 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-copper nanocluster synthesis, in particular to a preparation method of a chicken protein protected gold-copper nanocluster and application of the chicken protein protected gold-copper nanocluster in mercury ion detection.
Background
The egg white is a high-viscosity solution containing rich protein and a small amount of acetic acid and lysozyme, contains more than forty proteins, such as ovalbumin, ovotransferrin, ovomucin, ovomucoid, and albumin, and has the functions of moistening lung, relieving sore throat, clearing away heat and toxic materials, enhancing skin immunity, etc. Ovalbumin contains sufficient amino acid residues, wherein cysteine and histidine residues can be used as protective agents for metal ions, and tyrosine residues can be used as reducing agents for reducing noble metal ions under alkaline conditions. In addition, the chicken protein has the characteristics of low price, practicability, environmental friendliness and the like, and is an ideal candidate material for synthesizing the metal nanocluster.
The metal nanocluster is a fluorescent nanomaterial with the diameter of less than 2nm and composed of several to hundreds of atoms, and has unique optical, physical, electrochemical and catalytic properties. The protein contains a plurality of active sites such as thiol, amino, carboxyl and hydroxyl, can protect and reduce metal ions, is usually used as a stabilizer and a reducing agent to act on a metal center, has the characteristics of excellent biocompatibility, good chemical stability and the like, and is a template material commonly used for preparing metal nanoclusters. The prepared metal nanocluster has the advantages of high fluorescence intensity, large Stokes displacement, low toxicity, good stability, simple and easy operation of a synthetic process and the like, and has special potential in identifying and quantifying micro-volume samples due to the characteristics of the molecules.
Heavy metal pollution is one of the problems which cause high attention and urgent solution in all countries, wherein mercury ion pollution is easy to accumulate in organisms due to high toxicity and wide existence in water, soil and food, so that brain, kidney, nerve and endocrine system damage is caused, and serious influence is caused on human health and natural environment, so that the detection of mercury ions is an important step of environmental and health monitoring. The traditional detection method for mercury ions has the limitations of complex sample pretreatment, high detection cost, long time consumption and the like, so that a quick and efficient method for accurately detecting mercury ions in a sample is urgently needed to be developed.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a preparation method of chicken protein protected gold-copper nanoclusters and application thereof in mercury ion detection.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of chicken protein protection gold-copper nanoclusters comprises the following specific steps:
s1, weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding chloroauric acid for uniformly mixing, adding a copper sulfate solution, uniformly mixing and stirring for 2 minutes to obtain a solution A;
s2, adding a 1M sodium hydroxide solution into the solution A obtained in the step S1, adjusting the pH of the solution to 9-13, and heating the solution in a water bath for 3-10 hours to obtain a solution B;
s3, storing the solution B obtained in the step 2 at 4 ℃ to obtain the chicken protein-gold copper nanocluster.
Preferably, in the S1, the concentration ratio of the chloroauric acid to the copper sulfate is 8:1, and the concentration of the chicken protein is 50mgml-1。
Preferably, in the S2, the pH value of the solution is adjusted to 13 by sodium hydroxide, the heating temperature is 50 ℃, and the reaction time is 10 hours.
The invention also provides application of the chicken protein-gold copper nano cluster obtained by the preparation method of the chicken protein protection gold copper nano cluster in mercury ion detection, the chicken protein-gold copper nano cluster is diluted by phosphate buffer solution, mercury ions with different concentrations are added and mixed uniformly, incubation is carried out at room temperature, under the condition of excitation wavelength, the fluorescence intensity of the chicken protein-gold copper nano cluster is gradually weakened along with the gradual increase of the concentration of the mercury ions, and the detection is realized.
Preferably, the ratio-based detection is achieved at 368nm excitation wavelength with incubation for 10 minutes at room temperature.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention takes the chicken protein as a template, adopts a one-step synthesis method to prepare the chicken protein-gold copper nanocluster in the aqueous solution, and has unique photophysical characteristics, low toxicity, good stability and excellent biocompatibility.
2. The chicken protein-gold copper nanocluster prepared by the invention is rapid and simple in mercury ion detection method and good in detection sensitivity, and is an ideal fluorescent nanomaterial applied to the fields of biology and medicine.
Drawings
FIG. 1 shows excitation and emission spectra of the chicken egg white-gold copper nanoclusters of the present invention;
FIG. 2 is a fluorescence spectrum of chicken protein-Au-Cu nanoclusters synthesized by different Au-Cu ratios according to the present invention;
FIG. 3 is a fluorescence spectrum of the chicken protein-gold copper nanoclusters synthesized by different pH values according to the present invention;
FIG. 4 is a fluorescence spectrum of chicken protein-gold copper nanoclusters synthesized by different reaction times according to the present invention;
FIG. 5 is a fluorescence spectrum of a chicken protein-gold copper nanocluster synthesized by different chicken protein concentrations according to the present invention;
FIG. 6 is a fluorescence spectrum of chicken protein-gold copper nanoclusters synthesized at different temperatures according to the present invention;
FIG. 7 is a fluorescence emission spectrum of chicken protein-Au-Cu nanoclusters after adding mercury ions of different concentrations according to the present invention;
FIG. 8 is a graph showing the linear relationship between the fluorescence intensity of mercury ions of different concentrations and the fluorescence intensity of chicken protein-gold copper nanoclusters in accordance with the present invention.
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.
Referring to fig. 1 to 8, in order to improve the luminescence property of the product, the following steps are included:
weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding 100 mu L of chloroauric acid solution for uniformly mixing, adding 50 mu L of copper sulfate solution for uniformly mixing, then violently stirring for 2 minutes, adding 1M sodium hydroxide solution into the solution, uniformly mixing to adjust the pH value of the solution, and heating in a water bath for a period of time; and detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. Changing the volume of the copper sulfate to adjust the concentration ratio of the chloroauric acid to the copper sulfate, and observing a fluorescence spectrum, wherein when the concentration ratio of the chloroauric acid to the copper sulfate is 8:1, the fluorescence intensity is strongest; therefore, the concentration ratio of the chloroauric acid to the copper sulfate is selected to be 8:1 as the optimal ratio for preparing the chicken protein-gold copper nanoclusters.
Weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding 100 mu L of chloroauric acid solution for uniformly mixing, adding 12.5 mu L of copper sulfate solution for uniformly mixing, violently stirring for 2 minutes, adding 10 mu L of sodium hydroxide solution with the concentration of 1M into the solution, uniformly mixing, adjusting the pH value of the solution to 9, and heating in a water bath for a period of time; and detecting the excitation spectrum and the emission spectrum of the product by using a fluorescence spectrometer. The volume of the sodium hydroxide is increased to change the pH value of the solution, the fluorescence spectra at different pH values are observed, the fluorescence intensity is gradually increased along with the increase of the pH value of the solution, and the fluorescence intensity is strongest when the pH value of the solution is 13; thus pH 13 was chosen as the optimal pH for the preparation of the chicken protein-gold copper nanoclusters.
Weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding 100 mu L of chloroauric acid solution for uniformly mixing, adding 12.5 mu L of copper sulfate solution for uniformly mixing, violently stirring for 2 minutes, adding 22 mu L of sodium hydroxide solution with the concentration of 1M into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath for 3 hours; the emission spectrum of the product was detected using a fluorescence spectrometer. Increasing the reaction time, observing the fluorescence spectra of different reaction times, and gradually increasing the fluorescence intensity along with the increase of the reaction time, wherein the fluorescence intensity reaches the strongest intensity in 10 hours of the reaction time; therefore, 10 hours was chosen as the optimal reaction time for the preparation of the chicken protein-gold copper nanoclusters.
Weighing 1mg of chicken protein, adding 100 mu L of water for dissolving, adding 100 mu L of chloroauric acid solution for uniformly mixing, adding 12.5 mu L of copper sulfate solution for uniformly mixing, violently stirring for 2 minutes, adding 22 mu L of sodium hydroxide solution with the concentration of 1M into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath for 10 hours; the emission spectrum of the product was detected using a fluorescence spectrometer. Increasing the concentration of chicken protein, observing fluorescence spectrum, and gradually increasing fluorescence intensity with increasing concentration of chicken protein, when 50mg ml is used-1The fluorescence intensity is strongest when the chicken protein is prepared; thus 50mg ml was selected-1The chicken protein is used as the optimal concentration for preparing the chicken protein-gold copper nano cluster.
Weighing 5mg of chicken protein, adding 100 mu L of water for dissolving, adding 100 mu L of chloroauric acid solution for uniformly mixing, adding 12.5 mu L of copper sulfate solution for vigorously stirring for 2 minutes, adding 22 mu L of sodium hydroxide solution with the concentration of 1M into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 20 ℃ for 10 hours; the emission spectrum of the product was detected using a fluorescence spectrometer. Increasing the heating temperature of the water bath, observing the fluorescence spectrum, gradually increasing the fluorescence intensity along with the increase of the water bath temperature, and when the reaction temperature is 50 ℃, the fluorescence intensity is optimal; therefore 50 ℃ was chosen as the optimal temperature for the preparation of the chicken protein-gold copper nanoclusters.
Detecting mercury ions by taking the chicken protein-gold copper nanocluster as a fluorescent probe:
diluting the chicken protein-gold copper nano-cluster by using a phosphate buffer solution, adding a certain amount of mercury ions, incubating the mixed solution for 10 minutes at room temperature, and gradually reducing the fluorescence intensity of the chicken protein-gold copper nano-cluster at 655nm along with the gradual increase of the concentration of the mercury ions under the condition of 368nm excitation wavelength.
Preparation and optimization of (I) chicken protein-gold copper nanocluster
The first embodiment is as follows: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example two: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 25 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example three: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 50 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example four: the chicken protein-gold copper nanoclusters obtained in the first embodiment, the second embodiment and the third embodiment are determined to have the strongest fluorescence emission intensity when the concentration ratio of chloroauric acid to copper sulfate is 8:1 by comparing the fluorescence intensity of the chicken protein-gold copper nanoclusters at an excitation wavelength of 368nm as shown in fig. 2.
Example five: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 10 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 9, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example six: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 16 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 11, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example seven: the chicken protein-gold copper nanoclusters obtained in the first embodiment, the fifth embodiment and the sixth embodiment are determined to have the strongest fluorescence emission intensity when the solution pH is 13 by comparing the fluorescence intensity of the chicken protein-gold copper nanoclusters at the excitation wavelength of 368nm as shown in FIG. 3.
Example eight: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 3 hours to obtain the chicken protein-gold copper nanocluster.
Example nine: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 6 hours to obtain the chicken protein-gold copper nanocluster.
Example ten: example one, example eight, and example nine the resulting chicken protein-gold copper nanoclusters were compared at 368nm excitation wavelength to determine the intensity of fluorescence emission was the strongest when the reaction time was 10 hours, as shown in fig. 4.
Example eleven: weighing 4mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example twelve: weighing 3mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 50 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example thirteen: example one, example eleven and example twelve, the chicken protein-gold nanoclusters obtained in example one, example eleven and example twelve were determined by comparing their respective fluorescence emission intensities at an excitation wavelength of 368nm as shown in fig. 5 when the chicken protein concentration was 50mg ml-1Intensity of fluorescence emissionThe strongest.
Example fourteen: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 37 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example fifteen: weighing 5mg of chicken protein, and adding 100 mu L of water for dissolving; adding 100 mu L of chloroauric acid, mixing uniformly, adding 12.5 mu L of copper sulfate solution, mixing uniformly and stirring for 2 minutes; and secondly, adding 22 mu L of 1M sodium hydroxide solution into the solution, uniformly mixing, adjusting the pH value of the solution to 13, and heating in a water bath at 55 ℃ for 10 hours to obtain the chicken protein-gold copper nanocluster.
Example sixteen: in the chicken protein-gold copper nanoclusters obtained in the first embodiment, the fourteenth embodiment and the fifteenth embodiment, as shown in fig. 6, the fluorescence emission intensity is determined to be the strongest when the reaction temperature is 50 ℃ by comparing the fluorescence emission intensities of the chicken protein-gold copper nanoclusters at the excitation wavelength of 368 nm.
(II) detection of mercury ions by taking chicken protein-gold copper nanocluster as fluorescent probe
Example seventeen: diluting the chicken protein-gold copper nanocluster stock solution by using a phosphate buffer solution, adding a mercury ion solution with the final concentration of 5-50 mu M, incubating for 10 minutes at room temperature, and detecting the fluorescence spectrum under the excitation wavelength of 368nm by using a fluorescence spectrometer.
As can be seen from the above, at 368nm (E)x1) Under the excitation wavelength, the gold-copper nanocluster shows red fluorescence at 455nm (E)m1) And 655nm (E)m2) Has a fluorescence emission peak; the gold-copper nanocluster serving as an effective environment-friendly fluorescent probe can be used for detecting mercury ions (Hg) in solution2+)。
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 chicken protein protection gold-copper nanoclusters is characterized by comprising the following specific steps:
s1, weighing a certain amount of egg white, adding 100 mu L of water for dissolving, adding chloroauric acid for uniformly mixing, adding a copper sulfate solution, uniformly mixing and stirring for 2 minutes to obtain a solution A;
s2, adding a 1M sodium hydroxide solution into the solution A obtained in the step S1, adjusting the pH of the solution to 9-13, and heating the solution in a water bath for 3-10 hours to obtain a solution B;
s3, storing the solution B obtained in the step 2 at 4 ℃ to obtain the chicken protein-gold copper nanocluster.
2. The method of claim 1, wherein the concentration ratio of chloroauric acid to copper sulfate in S1 is 8:1, and the concentration of chicken protein is 50mg ml-1。
3. The method of claim 1, wherein in S2, pH of the solution is adjusted to 13 by sodium hydroxide, the heating temperature is 50 ℃, and the reaction time is 10 hours.
4. The application of the chicken protein-gold copper nanocluster obtained by the preparation method of chicken protein protection gold copper nanoclusters in mercury ion detection is characterized in that the chicken protein-gold copper nanoclusters are diluted by phosphate buffer solution, mercury ions with different concentrations are added and mixed uniformly, incubation is carried out at room temperature, and under the condition of excitation wavelength, the fluorescence intensity of the chicken protein-gold copper nanoclusters is gradually weakened along with the gradual increase of the concentration of the mercury ions, so that the detection is realized.
5. The application of the chicken protein protected gold-copper nanocluster in mercury ion detection according to claim 4, wherein the detection is realized under the condition of incubation for 10 minutes at room temperature and excitation wavelength of 368 nm.
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