CN111286323B

CN111286323B - Fluorescent carbon dot capable of self-targeting cell nucleus and preparation method and application thereof

Info

Publication number: CN111286323B
Application number: CN202010177125.7A
Authority: CN
Inventors: 郭建花; 路雯婧; 郭忠慧; 张慧林; 双少敏; 董川
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2021-07-02
Anticipated expiration: 2040-03-13
Also published as: CN111286323A

Abstract

The invention discloses a self-targeting cell nucleus fluorescent carbon dot and a preparation method and application thereof, wherein the carbon dot is prepared by taking copper chloride and tryptophan as raw materials through a one-step hydrothermal synthesis method; the carbon dots are zero-dimensional carbon nano-materials, have the diameter of about 3.3-7.6nm and can emit bright blue fluorescence. The preparation method is simple and convenient, and the prepared carbon dots have good water solubility and stable fluorescence. Particularly, the carbon dots prepared by the method do not need additional modification, can target cell nucleus in cell imaging, and have sensitive response to intracellular pH. The application is that the fluorescent probe can enter cells and cell nucleuses rapidly without being marked as a fluorescent probe, and the fluctuation of pH in acidic living cells is monitored in real time.

Description

Fluorescent carbon dot capable of self-targeting cell nucleus and preparation method and application thereof

Technical Field

The invention relates to preparation and application of a fluorescent carbon dot, in particular to a fluorescent carbon dot for self-targeting cell nucleus and a preparation method and application thereof.

Background

The nucleus is the major organelle of a eukaryotic cell that maintains genetic integrity and regulates cell function, and contains a large amount of genetic material, i.e., DNA, inside. The nucleus, which is often the ultimate target for treating diseases after crossing a range of biological barriers, controls cellular processes by regulating gene expression and exchanging proteins with the cytoplasm. The human nuclear genome has 3.2 × 10⁹Base pairs, 2% of which are about 30000 genes. If 30000 genes are mutated, various gene-related diseases may occur. At present, it has been found that the change of the cell nucleus is closely related to the canceration state of the tumor cell. Therefore, the cell nucleus targeted fluorescence imaging plays an important role in disease diagnosis, cell nucleus tracking, cell state detection and the like.

pH is one of the most important parameters in the cell, and maintaining pH homeostasis is the basis for all organisms to perform normal vital activities. Abnormally small changes in intracellular pH can disrupt the acid-base balance of the physiological environment, leading to disturbances in normal cellular activities. Accompanying cardiopulmonary and nervous system diseases, such as silicosis, cancer and Alzheimer's disease. Measuring intracellular pH changes is of great importance for a better understanding of cellular function and may provide a critical aid for early disease diagnosis. Therefore, it is essential to develop a simple method for accurately and sensitively monitoring pH in a biological environment.

Fluorescence analysis methods are widely used due to the advantages of high sensitivity, non-invasiveness, rapid response, easy operation, real-time monitoring and the like. Carbon Dots (CDs) as a novel material have the advantages of small size, good photoluminescence performance, good biocompatibility, good optical stability, simple synthesis and the like, and have been well applied to the fields of biochemical sensing, biological imaging, environmental analysis and the like as fluorescent probes. However, the controllable preparation of fluorescent carbon dots with self-targeting organelles using simple preparation methods and their application to pH detection remain challenging.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a fluorescent carbon dot with excellent structural properties and a preparation method thereof.

The invention provides a preparation method of a self-targeting cell nucleus carbon dot, which comprises the following steps:

1) placing copper chloride and tryptophan into a glass beaker, adding deionized water, fully stirring, and ultrasonically dissolving for 10-15 minutes, wherein the mass ratio of the copper chloride to the tryptophan is as follows: 0.05-0.18: 0.01-0.15;

2) transferring the solution into a hydrothermal reaction kettle, placing the hydrothermal reaction kettle into an oven, and reacting for 3-5 hours at 180 ℃;

3) standing the hydrothermal reaction kettle after the reaction is stopped, cooling the hydrothermal reaction kettle to room temperature, filtering to remove insoluble substances to obtain a dark yellow solution, and filtering the dark yellow solution by using a 0.22 mu m filter membrane to obtain a pure carbon dot solution;

4) and freeze-drying the carbon dot solution to obtain a target product.

The preferable mass ratio of the copper chloride to the tryptophan is as follows: 0.15-0.18:0.08-0.12.

The carbon dots prepared by the method have stable fluorescence property, emit bright blue fluorescence under the irradiation of an ultraviolet lamp, and have good water solubility and dispersibility. The carbon dot is simple and convenient to prepare by a one-step hydrothermal synthesis method, and complicated sample pretreatment and purification processes are not needed. In addition, the carbon dots do not need additional modification, have an autonomous nucleus targeting imaging function, and are applied to detection of pH of acidic living cells.

The carbon dots prepared by the method are used as a nuclear targeting imaging fluorescent probe. Dissolving the obtained carbon dots in BR buffer solution, adding the BR buffer solution into a culture solution of HeLa cells, and culturing for 0.5-1 hour to obtain a cell nucleus targeted imaging fluorescent probe cell culture solution with the carbon dot concentration of 0.3-1 mg/L; adding the diluted acridine orange dye solution into a culture solution of HeLa cells, and culturing for 0.5-1 hour; adding the obtained carbon dots with the concentration of 0.3-1 mg/L and the diluted acridine orange dye solution into a culture solution of the HeLa cell, co-culturing for 0.5-1 h, and observing the imaging state of the HeLa cell through a laser confocal microscope to obtain a fluorescence photo of the nuclear imaging of the HeLa cell.

The obtained carbon dot solid was dissolved in a BR buffer solution and added to a culture solution of HeLa cells, co-cultured for 20 to 40 minutes, and then the morphology of HeLa cells was observed by a confocal laser microscope. When the carbon dot is at pH 2.0, the cell shows bright blue fluorescence; and the blue fluorescence of the carbon spot gradually decreases with increasing pH. The carbon dots can be used as detection reagents for monitoring the change of pH in acidic living cells.

The invention has the following beneficial technical effects:

(1) the carbon dot preparation method is simple, a one-step synthesis method is adopted, complicated sample pretreatment and purification processes are not needed, surface passivating agent treatment or modification is not needed, and the preparation process is energy-saving and time-saving.

(2) The carbon dots provided by the invention have good solubility and dispersibility in aqueous solution; the optical property is stable under long-time illumination and high ionic strength.

(3) The carbon dots provided by the invention have a nuclear self-targeting function, and provide a more selectable tool for imaging the nucleus of the biological cell.

(4) The carbon point has high sensitivity to pH value, can be used as a fluorescent probe for detecting the change of the pH value, and is used for monitoring the change of the pH value in the acidic living cells.

Drawings

FIG. 1 is a transmission electron micrograph of a carbon dot prepared in example 1;

FIG. 2 is an ultraviolet absorption spectrum and a fluorescence excitation-emission spectrum of the carbon dot prepared in example 1;

FIG. 3 is an XPS spectrum of carbon dots prepared in example 1;

FIG. 4 is an infrared spectrum of a carbon dot prepared in example 1, in which the abscissa is a detection wavelength and the ordinate is a transmittance;

FIG. 5 is a graph showing fluorescence spectra of carbon dots prepared in example 1 in BR buffer solutions at different pH values;

fig. 6 is a pH titration curve of fluorescence intensity of the carbon dots prepared in example 1 (pH 1.0 to 8.0);

FIG. 7 is a confocal display of laser imaging of the carbon dots prepared in example 1 as nuclear-targeted fluorescent probes into viable cell nuclei;

fig. 8 is a graph showing confocal laser scanning of the carbon dots prepared in example 1 into living cells under the conditions of pH 2.0, pH 4.0 and pH 6.0, respectively.

Detailed Description

The present invention will be described in detail with reference to the following examples and the accompanying drawings, wherein the examples show detailed embodiments and specific operation procedures, but the scope of the present invention is not limited to the following examples.

Example 1

The preparation method of the carbon dots comprises the following steps:

1) respectively weighing 0.17g of copper chloride and 0.1g of tryptophan, placing the copper chloride and the tryptophan into a glass beaker, adding 20mL of deionized water, fully stirring, and ultrasonically dissolving for 15 minutes;

2) transferring the reaction mixture into a hydrothermal reaction kettle, placing the hydrothermal reaction kettle into an oven, and reacting for 3 hours at 180 ℃;

3) standing and cooling to room temperature after the reaction is stopped, filtering, removing insoluble substances to obtain a dark yellow solution, and filtering with a 0.22 mu m filter membrane to obtain a pure carbon dot aqueous solution;

4) and freeze-drying the carbon dot aqueous solution to obtain a carbon dot solid.

Example 2

Characterizing the fluorescent carbon dots prepared in example 1 with a transmission electron micrograph of FIG. 1, the carbon dots appear as monodisperse spherical particles with a particle size of about 4.84 nm; the ultraviolet absorption spectrum and fluorescence excitation-emission spectrum of the synthesized carbon dot are shown in FIG. 2, and the absorption peak of the carbon dot is 278nm, and the optimal excitation wavelength and emission wavelength are 369 nm and 464 nm; the XPS spectrum of the synthesized carbon dots is shown in figure 3, and the carbon dots contain main elements of carbon, hydrogen, oxygen, nitrogen, copper and chlorine; the infrared spectrogram of the synthesized carbon point is shown in FIG. 4, which shows that the carbon point has a benzene ring structure and contains functional groups such as amino, carboxyl and the like on the surface; as shown in FIG. 5, the fluorescence of the carbon dots changes at different pH values, and the fluorescence intensity of the carbon dots gradually decreases from 1.0 to 8.0; as shown in fig. 6, the fluorescence intensity at the carbon point showed a double S-type curve at pH 1.0 to 8.0 and pKa of 2.49 and 6.21 in a pH titration curve (pH 1.0 to 8.0).

Example 3

The HeLa cells were incubated with the BR buffer solution with a carbon spot concentration of 0.7mg/mL, the diluted acridine orange stain solution, the BR buffer solution with a carbon spot concentration of 0.7mg/mL and the diluted acridine orange stain solution for 40 minutes, and morphology and fluorescence imaging phenomenon of the HeLa cells were observed by a confocal laser microscope to obtain a fluorescence photograph of nuclear imaging of the HeLa cells, as shown in FIG. 7. The first row of fig. 7 is, from left to right: (A) dark field (excitation 488nm) cytogram (green), (B) bright field, (C) bright field and dark field green overlay; the second row of fig. 7 is, from left to right: (D) dark field (excitation at 405nm) cytogram (blue), (E)) bright field, (F) bright field and dark field blue overlay; the third row of fig. 7 is, in order from left to right: (G) dark field (excitation 488nm) cytogram (green), (H) dark field (excitation 405nm) cytogram (blue), (I) bright field and dark field blue-green overlay. As can be seen in FIG. 7, HeLa cells incubated with fluorescent carbon spots overlapped well with the nuclear commercial fluorescent probe acridine orange staining. According to the results of the co-localization experiments, it was shown that most of the fluorescent carbon dots can be efficiently localized to the nucleus during endocytosis of live HeLa cells. The carbon dots have the property of self-targeting cell nucleus, and the fluorescent carbon dots can rapidly enter the cell nucleus by observing through a laser confocal microscope, so that the application of the high-efficiency fluorescent carbon dots in cell nucleus targeting imaging is realized.

Example 4

The obtained carbon dots were dissolved in BR buffer solutions at pH 2.0, pH 4.0, and pH 6.0 to prepare 0.7mg/mL solutions, which were added to the culture medium of HeLa cells, respectively, and cultured for 30 minutes, and the imaging state of HeLa cells was observed by a confocal laser scanning microscope, as shown in the first column of fig. 8: fluorescence image of blue channel (405nm), second column: bright field image, third column: a superimposed image of the first column and the second column. When the carbon dot is at pH 2.0, the cell shows bright blue fluorescence; and the blue fluorescence of the carbon spot gradually decreases with increasing pH. The carbon dots can be used as detection reagents for monitoring the change of pH in acidic living cells.

Claims

1. A preparation method of a self-targeting cell nucleus fluorescent carbon dot is characterized by comprising the following steps:

4) and freeze-drying the carbon dot solution to obtain a target product.

2. The method for preparing a fluorescent carbon dot as claimed in claim 1, wherein the mass ratio of the copper chloride to the tryptophan is as follows: 0.15-0.18:0.08-0.12.

3. A self-targeting nuclear fluorescent carbon dot prepared by the method of claim 1 or 2.

4. Use of the fluorescent carbon dot of claim 3 for the preparation of a nuclear-targeted imaging fluorescent probe.

5. Use of a fluorescent carbon dot according to claim 3 in the preparation of a reagent for monitoring the pH of acidic living cells.