CN109211862B - A kind of preparation method and application of red fluorescent copper nanocluster probe - Google Patents

Abstract

Translated fromChinese

本发明公开了一种红色荧光铜纳米团簇探针的制备方法及其应用，本发明是以多巴胺作为保护剂和还原剂，通过“一锅法”制备得到；首先配制浓度为10‑20 mmol/L的多巴胺，不断搅拌下，向多巴胺溶液中加入浓度为15‑40 mmol/L的硝酸铜溶液，继续搅拌使两者充分混匀，硝酸铜与多巴胺的物质的量比为3~16:4；将所得混合溶液加热搅拌，室温下静置；然后经过离心，最终得到红色荧光铜纳米团簇探针溶液。本发明方法制得大小均匀、稳定性好、红色荧光的铜纳米团簇；制得的红色荧光铜纳米团簇探针水溶性好、稳定性强，可应用于人血清白蛋白（HSA）的检测。

The invention discloses a preparation method and application of a red fluorescent copper nano-cluster probe. The invention uses dopamine as a protective agent and a reducing agent, and is prepared by a "one-pot method"; first, the preparation concentration is 10-20 mmol The dopamine of /L, under constant stirring, add the copper nitrate solution that concentration is 15-40 mmol/L in dopamine solution, continue to stir to make both fully mix, and the amount ratio of copper nitrate and dopamine substance is 3～16: 4. Heating and stirring the obtained mixed solution and standing at room temperature; and then centrifuging to finally obtain a red fluorescent copper nanocluster probe solution. The method of the invention prepares copper nano-clusters with uniform size, good stability and red fluorescence; the prepared red fluorescent copper nano-cluster probes have good water solubility and strong stability, and can be applied to the detection of human serum albumin (HSA). detection.

Description

Preparation method and application of red fluorescent copper nanocluster probe

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)

Translated fromChinese

1.一种红色荧光铜纳米团簇探针的制备方法，其特征在于：是以多巴胺作为保护剂和还原剂，通过“一锅法”制备得到；所述制备方法具体包括以下步骤：1. a preparation method of red fluorescent copper nanocluster probe is characterized in that: take dopamine as protective agent and reducing agent, prepare by " one-pot method "; Described preparation method specifically comprises the following steps:(1) 配制浓度为10-20 mmol/L 的多巴胺，不断搅拌下，向多巴胺溶液中加入浓度为15-40 mmol/L的硝酸铜溶液，继续搅拌使两者充分混匀；(1) To prepare dopamine with a concentration of 10-20 mmol/L, under constant stirring, add a copper nitrate solution with a concentration of 15-40 mmol/L to the dopamine solution, and continue stirring to fully mix the two;多巴胺溶液与硝酸铜溶液的体积比为1:1；The volume ratio of dopamine solution and copper nitrate solution is 1:1;(2) 将步骤(1)得到的混合溶液，取体积为2-4 mL在55-70℃下搅拌6-7 h，室温下静置12 h；(2) The mixed solution obtained in step (1) is taken into a volume of 2-4 mL, stirred at 55-70 °C for 6-7 h, and allowed to stand at room temperature for 12 h;(3) 将步骤(2)得到的混合溶液经过离心，最终得到红色荧光铜纳米团簇探针溶液。(3) centrifuging the mixed solution obtained in step (2) to finally obtain a red fluorescent copper nanocluster probe solution.2.根据权利要求1所述的红色荧光铜纳米团簇探针的制备方法，其特征在于：步骤 (2)中温度为65℃。2. The preparation method of red fluorescent copper nanocluster probe according to claim 1, is characterized in that: the temperature in step (2) is 65 ℃.3.根据权利要求1所述的红色荧光铜纳米团簇探针的制备方法，其特征在于：步骤 (3)中离心机转速为10000-13000 r /min；离心时间为10-15 min。3. The preparation method of red fluorescent copper nanocluster probe according to claim 1, is characterized in that: in step (3), the rotating speed of centrifuge is 10000-13000 r/min; the centrifugation time is 10-15 min.4.一种权利要求1~3任一项所述的制备方法制得的红色荧光铜纳米团簇探针。4. A red fluorescent copper nanocluster probe prepared by the preparation method according to any one of claims 1 to 3.5.一种权利要求4所述的红色荧光铜纳米团簇探针在人血清白蛋白检测中的应用。5. The application of the red fluorescent copper nanocluster probe of claim 4 in the detection of human serum albumin.6.根据权利要求5所述的应用，其特征在于：将红色荧光铜纳米团簇探针溶液加入到二次蒸馏水中稀释，取稀释后的红色荧光铜纳米团簇探针溶液100 μL加入到900 μL含不同离子或其他小分子的溶液中，固定激发波长为560 nm，在室温下0-10 min内进行荧光光谱检测，根据624 nm的荧光峰强度，进行检测。6. The application according to claim 5, characterized in that: adding the red fluorescent copper nanocluster probe solution to double distilled water for dilution, and taking 100 μL of the diluted red fluorescent copper nanocluster probe solution and adding it to the solution. In 900 μL of solutions containing different ions or other small molecules, the fixed excitation wavelength is 560 nm, and fluorescence spectrum detection is performed within 0-10 min at room temperature, and the detection is performed according to the fluorescence peak intensity at 624 nm.

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