CN109211862B - Preparation method and application of red fluorescent copper nanocluster probe - Google Patents
Preparation method and application of red fluorescent copper nanocluster probe Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a red fluorescent copper nanocluster probe, wherein dopamine is used as a protective agent and a reducing agent and is prepared by a one-pot method; firstly, preparing 10-20 mmol/L dopamine, adding 15-40 mmol/L copper nitrate solution into the dopamine solution under continuous stirring, and continuously stirring to fully and uniformly mix the dopamine solution and the copper nitrate solution, wherein the mass ratio of the copper nitrate to the dopamine is 3-16: 4; heating and stirring the obtained mixed solution, and standing at room temperature; and centrifuging to finally obtain the red fluorescent copper nanocluster probe solution. The method of the invention prepares the copper nanocluster with uniform size, good stability and red fluorescence; the prepared red fluorescent copper nanocluster probe is good in water solubility and strong in stability, and can be applied to detection of Human Serum Albumin (HSA).
Description
Technical Field
The invention relates to a preparation method and application of a red fluorescent copper nanocluster probe, and belongs to the field of preparation of fluorescent nanomaterials.
Background
The metal nanocluster is an ultra-small nanoparticle consisting of dozens of atoms and having a metal core size of less than 2 nm. As a novel fluorescent nanoprobe, the metal nanoclusters have unique electrical, physical and optical properties, and have been intensively reported in recent years for detecting various targets. One of the obvious characteristics of the metal nanocluster is that the metal nanocluster is strong in photoluminescence, small in size, non-toxic, good in water solubility, large in Stocks displacement and strong in photobleaching resistance, so that the metal nanocluster has attracted extensive interest of researchers. Copper is cheaper than gold and silver. Therefore, the copper nanoclusters gradually become an important component in the metal nanomaterials and are widely applied to the research fields of chemical analysis, biosensing, biological imaging, ion detection and the like.
Currently, most of the synthesized copper nanoclusters emit blue light under excitation of ultraviolet light. In the aspect of analytical detection and biology, the red fluorescent copper nanoclusters are more attractive, so that interference of autofluorescence of some organisms can be avoided.
Bio-small molecules such as glutathione, cysteine, dodecanethiol, and the like are used as excellent stabilizers for synthesizing the copper nanoclusters. The detection of the enzyme half-lactase can be carried out in the literature (position-drive luminescence self-associated dots of coater nanocrusters with aggregation-induced emission for B-lacosation activity monitoring, Y.Y. Huang, H.Feng, W.D. Liu, S.S. Zhang, C. Tang, J.R. Chen, Z.S. Qian, J.mater. chem. B2017, 5, 5120-loop 5127) by synthesizing copper nanoclusters using glutathione, a commonly used biomolecule. However, the synthesis process of this method is somewhat cumbersome. The Copper nanoclusters synthesized in the literature (cooper nanocrusters as an on-off-on fluorescent probe for ascorbic acid, H.B. Rao, H.W. Ge, Z.W. Lu, W.Liu, Z.Q. Chen, Z.Y. Zhang, X.X. Wang, P.Zou, Y.Y. Wang, H.He, X.Y. Zeng, Microchim Acta 2016, 183, 1651-1657) are used for the detection of ascorbic acid and are successfully applied to the detection of the recovery in actual biological samples such as cucumber, broccoli and balsam pear, however, the synthesis process is somewhat cumbersome and the use of blue-fluorescent Copper nanoclusters for biological detection presents a certain interference.
Disclosure of Invention
The invention aims to provide a preparation method and application of a red fluorescent copper nanocluster probe, the preparation method is simple, one-step synthesis is realized, the reaction conditions are mild, and the obtained red fluorescent copper nanocluster probe can avoid interference of autofluorescence of organisms and can be used for detection of HSA.
Due to the high surface area of the metal nanoclusters, the valence bonds of outer atoms are highly unsaturated, so that the surface free energy of the metal nanoclusters is high, and the metal nanoclusters have the tendency of self-aggregation and growth. Therefore, in the preparation process of the stable metal nanocluster, a protective agent is usually required to be added to reduce the surface free energy so as to prepare the nanocluster with uniform size and good stability. The method for preparing the copper nanocluster capable of emitting red fluorescence by using dopamine as a protective agent and a reducing agent is simple to operate, low in cost, wide and easily available in raw materials and good in repeatability.
The invention provides a red fluorescent copper nanocluster probe which is prepared by taking dopamine as a protective agent and a reducing agent through a one-pot method.
The invention provides a preparation method of a red fluorescent copper nanocluster probe, which comprises the following steps:
(1) preparing 10-20 mmol/L dopamine, adding 15-40 mmol/L copper nitrate solution into the dopamine solution under continuous stirring, and continuously stirring to fully and uniformly mix the dopamine solution and the copper nitrate solution, wherein the mass ratio of the copper nitrate to the dopamine is 3-16: 4;
(2) taking 2-4mL of the mixed solution obtained in the step (1), heating and stirring at 55-70 ℃ for 6-7 h, and standing at room temperature for 12 h;
(3) and (3) centrifuging the mixed solution obtained in the step (2) to finally obtain the red fluorescent copper nanocluster probe solution.
In the preparation method, the concentration of the dopamine solution in the step (1) is 10-20 mmol/L; the volume ratio of the dopamine solution to the copper nitrate solution is 1: 1;
the temperature in the step (2) is preferably 65 ℃, and the stirring time is preferably 6 h.
The rotating speed of the centrifugal machine in the step (3) is 10000-; the centrifugation time is 10-15 min.
The invention provides a red fluorescent copper nanocluster probe prepared by the preparation method.
The invention provides an application of the red fluorescent copper nanocluster probe in Human Serum Albumin (HSA) detection.
The specific application is as follows: adding the red fluorescent copper nanocluster probe solution into secondary distilled water for dilution, adding 100 mu L of the diluted red fluorescent copper nanocluster probe solution into 900 mu L of solution containing different ions or other small molecules, fixing the excitation wavelength to be 560nm, performing fluorescence spectrum detection within 0-10 min at room temperature, and performing detection according to the fluorescence peak intensity of about 624 nm.
The invention has the beneficial effects that:
(1) the dopamine is used as a template, so that the dopamine is green and environment-friendly, the preparation method is simple, and the cost is low.
(2) The prepared red fluorescent copper nanocluster probe is small in size, strong in light stability, small in toxic and side effects, good in water solubility and high in fluorescence intensity, and has wide application prospects in the fields of biological imaging, biological labeling and the like.
(3) The prepared fluorescent copper nanocluster probe has good red luminescence performance, and can avoid interference of autofluorescence of some organisms when used for actual detection.
Drawings
FIG. 1 is a schematic diagram of the mechanism of action of a red fluorescent copper nanocluster probe according to the present invention;
fig. 2 is a fluorescence-ultraviolet diagram of a fluorescent copper nanocluster probe solution prepared in example 1 of the present invention, in which a is an ultraviolet-visible absorption spectrum diagram and b is a fluorescence spectrum diagram;
fig. 3 is a graph showing the variation of fluorescence peak intensity when different ions and other small molecules are added to the red fluorescent copper nanocluster probe solution in example 3 of the present invention;
FIG. 4 is a graph showing the change in fluorescence peak intensity of a red fluorescent copper nanocluster probe solution according to the change in ionic strength (concentration of sodium chloride) in example 2 of the present invention;
FIG. 5 is a graph showing the change of fluorescence intensity of a red fluorescent copper nanocluster probe solution according to example 4 of the present invention with time;
FIG. 6 is a graph showing the change in fluorescence intensity of a red fluorescent copper nanocluster probe solution according to the increase in dopamine concentration in example 5 of the present invention;
fig. 7 is a linear relationship diagram of the fluorescence intensity of the red fluorescent copper nanocluster probe solution in example 5 of the present invention and dopamine solutions with different concentrations.
Detailed Description
The present invention is further illustrated by, but is not limited to, the following examples.
The invention uses dopamine as a template and a reducing agent, prepares a red fluorescent copper nanocluster probe solution by a one-pot method, and is used for detecting the dopamine in the solution. The invention is further illustrated by the following examples in connection with the accompanying drawings.
Example 1
Preparing a red fluorescent copper nanocluster probe by taking dopamine as a template:
(1) preparing 20 mmol/L dopamine, adding 25 mmol/L copper nitrate solution into the dopamine solution under continuous stirring, and continuously stirring to fully and uniformly mix the dopamine solution and the copper nitrate solution, wherein the volume ratio of the dopamine solution to the copper nitrate solution is 1: 1; the mass ratio of the copper nitrate to the dopamine is 5: 4;
(2) heating and stirring the mixed solution obtained in the step (1) for 6 hours at 65 ℃ with the volume of 2 mL, and standing for 12 hours at room temperature;
(3) and (3) centrifuging the mixed solution obtained in the step (2) to finally obtain the red fluorescent copper nanocluster probe solution.
The action mechanism of the prepared red fluorescent copper nanocluster probe is schematically shown in fig. 1.
The prepared fluorescent copper nanocluster probe solution is dark brown under the irradiation of a fluorescent lamp and red under the irradiation of a 365 nm ultraviolet lamp.
In addition, a fluorescence-ultraviolet diagram of the prepared fluorescent copper nanocluster probe solution is shown in fig. 2, which shows that the emission peak position of the prepared fluorescent copper nanocluster probe is about 624 nm under the condition that the fixed excitation wavelength is 560 nm.
Example 2
Experiment on influence of anions and cations and other small molecules on fluorescence peak intensity of the fluorescent copper nanocluster probe solution prepared in example 1:
using redistilled water and NaNO3、KNO3、Mg(NO3)2、Ca(NO3)2、Hg(NO3)2、Zn(NO3)2、Cu(NO3)2、Al(NO3)2、Co(NO3)2、Cr(NO3)2、KCl、KBr、KI、KAc、K2SO4GSH, Cys, Hcy and AA are respectively preparedThe concentration is 0.1 mol.L-1The solution of (1). The red fluorescent copper nanocluster probe solution prepared in example 1 was diluted 10 times, 100 μ L of the diluted red fluorescent copper nanocluster probe solution was added to 900 μ L of the above solution containing different ions or other small molecules, the fixed excitation wavelength was 560nm, fluorescence spectrum detection was performed at room temperature, and the influence of different ions or small molecules on the fluorescence peak intensity of the red fluorescent copper nanocluster probe solution was detected from the fluorescence peak intensity around 624 nm (fig. 3).
The effect of ions on the fluorescence peak intensity of the red fluorescent copper nanocluster probe solution is shown in fig. 4: under the excitation of 560nm, the fluorescence intensity F of the red fluorescent copper nanocluster probe solution containing ions or small molecules and the fluorescence peak intensity F of the red fluorescent copper nanocluster probe solution are measured0The ratio of (A) to (B) gives: the change of dopamine is the largest, and the change of other ions or small molecules is relatively small, so that the red fluorescent copper nanocluster probe solution prepared by the method can detect dopamine.
Example 3
Experiment on influence of ion intensity on fluorescence peak intensity of red fluorescent copper nanocluster probe solution prepared in example 1:
100. mu.L of the red fluorescent copper nanocluster probe solution prepared in example 1 was added to 900. mu.L of redistilled water, the fixed excitation wavelength was 560nm, sodium chloride solutions (0.04 to 0.22 mol/L) of different concentrations were added, and the influence of the ion intensity on the fluorescence peak intensity of the red fluorescent copper nanocluster probe solution was detected from the fluorescence peak intensity around 624 nm.
The effect of ion intensity on the fluorescence peak intensity of the fluorescent copper nanocluster probe solution is shown in fig. 3: under the excitation of 560nm, the fluorescence peak intensity of the red fluorescent copper nanocluster probe solution is basically unchanged in the range of sodium chloride solutions with different concentrations (0.04-0.22 mol/L), which shows that the red fluorescent copper nanocluster probe solution prepared by the invention has strong anti-ionic interference.
Example 4
Effect experiment of time on red fluorescent copper nanocluster probe solution prepared in example 1 after HSA addition:
after adding HSA to 100. mu.L of the red fluorescent copper nanocluster probe solution prepared in example 1, the solution was added to 900. mu.L of deionized water, the excitation wavelength was fixed at 560nm, fluorescence spectrum detection was performed at room temperature for 0 to 10 min, and the influence of the detection time on the fluorescence peak intensity of the fluorescent copper nanocluster probe solution was determined from the fluorescence peak intensity around 624 nm. The effect of time on the fluorescence intensity of the fluorescent copper nanocluster probe solution is shown in fig. 5: the fluorescence intensity of the fluorescent copper nanocluster probe remained substantially unchanged within 10 min.
Example 5
Experiment of detection of HSA by the red fluorescent copper nanocluster probe solution prepared in example 1:
the red fluorescent copper nanocluster probe solution prepared in example 1 was diluted 10 times, 100. mu.L of the diluted fluorescent copper nanocluster probe solution was taken and added to 900. mu.L of an HSA-containing solution, the excitation wavelength was fixed at 560nm, fluorescence spectrum detection was performed at room temperature, and the influence of HSA on the fluorescence intensity of the red fluorescent copper nanocluster probe solution was detected from the fluorescence intensity at about 624 nm.
The effect of HSA on the fluorescence intensity of the red fluorescent copper nanocluster probe solution is shown in fig. 6: under the excitation of 560nm, after HSA with different concentrations is added into the fluorescent copper nanocluster probe solution, the fluorescence intensity is gradually reduced, and finally the fluorescence peak basically tends to be smooth; wherein 0-200 μ g/mL is a fluorescence spectrogram of 0, 1, 5, 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200 μ g/mL HSA influencing the fluorescence intensity of the fluorescent copper nanocluster probe solution, which illustrates that the red fluorescent copper nanocluster probe solution prepared by the present invention can realize the detection of HSA.
In addition, the change in fluorescence intensity of the red fluorescent copper nanocluster probe solution prepared according to the present invention is linear with the concentration of HSA, and as shown in fig. 7, the fluorescence intensity of the copper nanocluster and the concentration of HSA are linear in two stages. As shown in FIG. 7 (a), the linear equation for HSA is F0/F=1.102+0.007C (R2= 0.991); as shown in FIG. 7 (b), the linear equation for HSA is F0/F=-7.910+0.095C (R2=0.970)。
Claims (6)
1. A preparation method of a red fluorescent copper nanocluster probe is characterized by comprising the following steps: the dopamine is used as a protective agent and a reducing agent and is prepared by a one-pot method; the preparation method specifically comprises the following steps:
(1) preparing 10-20 mmol/L dopamine, adding 15-40 mmol/L copper nitrate solution into dopamine solution under continuous stirring, and continuously stirring to mix the two solutions fully and uniformly;
the volume ratio of the dopamine solution to the copper nitrate solution is 1: 1;
(2) stirring the mixed solution obtained in the step (1) for 6-7 h at 55-70 ℃ by taking 2-4mL of the mixed solution, and standing for 12h at room temperature;
(3) and (3) centrifuging the mixed solution obtained in the step (2) to finally obtain the red fluorescent copper nanocluster probe solution.
2. The method for preparing a red fluorescent copper nanocluster probe according to claim 1, wherein: the temperature in step (2) was 65 ℃.
3. The method for preparing a red fluorescent copper nanocluster probe according to claim 1, wherein: the rotating speed of the centrifugal machine in the step (3) is 10000-; the centrifugation time is 10-15 min.
4. A red fluorescent copper nanocluster probe prepared by the preparation method of any one of claims 1 to 3.
5. Use of the red fluorescent copper nanocluster probe of claim 4 in human serum albumin detection.
6. Use according to claim 5, characterized in that: adding the red fluorescent copper nanocluster probe solution into secondary distilled water for dilution, adding 100 mu L of the diluted red fluorescent copper nanocluster probe solution into 900 mu L of solution containing different ions or other small molecules, fixing the excitation wavelength to be 560nm, performing fluorescence spectrum detection within 0-10 min at room temperature, and performing detection according to the fluorescence peak intensity of 624 nm.
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