CN114836196A

CN114836196A - Multicolor luminous nano material for biomedical dynamic imaging and preparation method thereof

Info

Publication number: CN114836196A
Application number: CN202210319551.9A
Authority: CN
Inventors: 刘国锋; 王学娟
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2022-08-02
Anticipated expiration: 2042-03-29
Also published as: CN114836196B

Abstract

The invention discloses a multicolor luminous nano material for biomedical dynamic imaging and a preparation method thereof, the multicolor luminous nano material is an Au (I) -BSA nano material formed by gold and bovine serum albumin, the microscopic morphology of the Au (I) -BSA nano particles is spherical or spheroidal, and the average particle size of the Au (I) -BSA nano particles is 26.2 +/-0.27 nm; the nano material provided by the invention has good biocompatibility and fluorescence and phosphorescence double-mode light-emitting characteristics depending on excitation wavelength, and can realize multicolor adjustable light emission; in addition, the invention adopts a one-pot synthesis method to prepare the nano material with good biocompatibility and applies the nano material to colorful biological imaging, and has the advantages of simple and convenient operation, easy control of reaction conditions, interference resistance, no cytotoxicity and photobleaching property.

Description

Multicolor luminous nano material for biomedical dynamic imaging and preparation method thereof

Technical Field

The invention relates to the technical field of nano materials, in particular to a multicolor luminous nano material for biomedical dynamic imaging and a method thereof.

Background

The excitation wavelength dependent luminescent material has potential application prospects in the aspects of biological imaging, display and dynamic anti-counterfeiting, wherein a multicolor biological imaging material with good biocompatibility draws great attention of scientists. In general, the Ex-De luminescence property of a molecular material can be obtained from a composite material composed of a plurality of luminescent components. However, this method has problems of poor stability, poor reproducibility, phase separation, color aging, and the like. Therefore, it is desirable to construct a single component system that can exhibit Ex-De luminescence simultaneously, overcoming the disadvantages of color separation and self-absorption. In recent years, research on Ex-De luminescent materials has focused mainly on metal complexes and nanostructures such as nanocrystals, carbon quantum dots, and silicon quantum dots. For transition metal complexes, the binding of metal ions to organic ligands is generally favorable for intersystem crossing of electrons and generation of phosphorescence, but this requires strong designability of the ligand structure and high professional requirements for designers. For carbon and silicon quantum dots, studies show that the lack of long-term biosafety can cause massive aggregation in cells and in vivo, and limit the application of the carbon and silicon quantum dots in cellular and biological multicolor imaging. Therefore, the significance of constructing a novel Ex-De luminescent material with good biocompatibility, no cytotoxicity and long-term biological safety by adopting the biological source protein molecules is great.

With the rapid development of life analytical chemistry, the demand of people for simultaneous analysis of multiple components in cells is increasing day by day, and multicolor cell dynamic imaging methods are receiving more and more attention due to the characteristics of intuition, visualization, interference resistance, multi-component analysis and the like. The development of a new multicolor imaging method and an imaging probe has important application value for researching the vital medical problems of intracellular substance interaction, intracellular communication and the like and realizing more accurate disease diagnosis and treatment.

In summary, the prior art is still lacking a safe and multicolor luminescent nano-material for biomedical dynamic imaging and a preparation method thereof.

Disclosure of Invention

The invention provides a green, safe and multicolor luminous nano material capable of multicolor dynamic luminescence and used for biomedical dynamic imaging and a preparation method thereof.

To achieve the purpose, the invention provides the following technical scheme:

in a first aspect of the present invention, a multicolor luminescent nano material for biomedical dynamic imaging is provided, the multicolor luminescent nano material is an au (i) -BSA nano material formed by gold and bovine serum albumin, the micro-morphology of the au (i) -BSA nanoparticles is spherical or quasi-spherical, and the average particle size of the au (i) -BSA nanoparticles is 26.2 ± 0.27 nm.

Preferably, the au (i) -BSA nanoparticles have a dispersion coefficient PDI of 0.17 in an aqueous solution.

In a second aspect of the present invention, there is provided a use of the multicolor luminescent nano material of the present invention in biomedical dynamic imaging.

Preferably, the multicolor luminous nano material is Au (I) -BSA nano material formed by gold and bovine serum albumin, the micro-morphology of the Au (I) -BSA nano particles is spherical or spheroidal, and the average particle size of the Au (I) -BSA nano particles is 26.2 +/-0.27 nm.

In a third aspect of the invention, there is provided a method of imaging a cell, comprising the steps of: the Au (I) -BSA nanoparticle solution and the cells are incubated for 3-8 h, the culture solution is removed and washed by PBS, and the cells are observed under an inverted microscope or a Confocal fluorescence microscope for imaging.

Preferably, cell imaging is performed dynamically under red, green, blue, yellow and cyan light channels of an inverted microscope or a Confocal fluorescence microscope.

Preferably, the Au (I) -BSA nanoparticle solution is Au (I) -BSA nanoparticle aqueous solution dispersed in Phosphate Buffered Saline (PBS) at a concentration of 0.5-10 mg/mL.

Preferably, the Au (I) -BSA nano material is formed by gold and bovine serum albumin, the micro-morphology of the Au (I) -BSA nano particles is spherical or spheroidal, and the average particle size of the Au (I) -BSA nano particles is 26.2 +/-0.27 nm.

In a fourth aspect of the present invention, there is provided a method for preparing a multicolor luminescent nano material for biomedical dynamic imaging, comprising the following steps: .

S1, mixing BSA with HAuCl ₄ ^. 3H ₂ Fully and uniformly mixing O to obtain a compound of BSA and Au;

s2, adding a proper amount of NaOH aqueous solution, adjusting the pH value of the reaction solution, controlling the reaction conditions in the air atmosphere, observing the color change, raising the reaction temperature, and continuing to react for a period of time to obtain a crude product;

s3, centrifuging and filtering the obtained crude product, repeating for multiple times to obtain Au (I) -BSA nano particles, namely the multicolor luminescent nano material for biomedical dynamic imaging.

Preferably, in step S1, HAuCl ₄ ^. 3H ₂ The molar ratio of O to BSA is 30:1-40: 1.

Preferably, in step S1, HAuCl ₄ ^. 3H ₂ The molar ratio of O to BSA was 36: 1.

Preferably, in step S1, the total volume of the mixed solution is 1-8 mL, and the stirring rate is 800-1500 r/min.

Preferably, in step S2, the pH of the reaction solution is adjusted to 8 to 10.

Preferably, in step S2, the reaction conditions are 50-90 ℃ and 1-6 h.

Preferably, in step S3, a 220 μm filter membrane is used for filtration, and the centrifugation speed is 8000r/min-15000 r/min.

Compared with the prior art, the invention has the beneficial effects and remarkable progresses that: according to the Au (I) -BSA nano-particles and the method provided by the invention, biological macromolecular BSA is used as a ligand and a reducing agent, Au (III) (3-valent Au) is reduced in an air environment by raising the temperature under the alkalescent condition to obtain the Au (I) -BSA nano-particles, the method is simple and convenient to operate, the reaction condition is easy to control, and the prepared Au (I) -BSA nano-particles are uniform in size, have good biocompatibility and have the luminescence characteristic of excitation wavelength dependence, and are free from cytotoxicity and photobleaching when being applied to multicolor bioimaging.

Drawings

To more clearly illustrate the technical solution of the present invention, the drawings required for the embodiment of the present invention will be briefly described below.

It should be apparent that the drawings in the following description are only drawings of some embodiments of the invention, and that other drawings can be obtained by those skilled in the art without inventive exercise, and the other drawings also belong to the drawings required by the embodiments of the invention.

FIG. 1 is a Transmission Electron Microscope (TEM) photograph of various sets of nanomaterials of example 1 of the present invention;

FIG. 2 is a diagram of different sets of nanomaterials of example 2 of the present invention under fluorescent and UV lamps;

FIG. 3 is a diagram of Dynamic Light Scattering (DLS) of Au (I) -BSA nanomaterial of example 3 of the present invention;

FIG. 4 is a diagram of the photoluminescence dependence of the excitation wavelength of the Au (I) -BSA nano material of example 4 of the present invention;

FIG. 5 is a dynamic image of the cellular color of the Au (I) -BSA nanomaterial of example 4 of the present invention.

Detailed Description

In order to make the objects, technical solutions, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

It is to be understood that all of the described embodiments are merely some, and not all, embodiments of the invention; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It is to be understood that:

in the present invention, the term "nanomaterial" refers to a material having at least one dimension in a three-dimensional space in the nanometer size (1-100nm) or composed of them as a basic unit, which corresponds to a dimension of about 10 to 1000 atoms closely arranged together.

The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

It should be further noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments.

The technical means of the present invention will be described in detail below with specific examples.

Example 1 raw material screening

HAuCl was prepared as shown in Table 1 below ₄ ^. 3H ₂ O was tested with BSA starting material.

TABLE 1

The experimental procedure was as follows:

1.1 reaction of BSA with HAuCl ₄ ^. 3H ₂ O to prepare mother liquor with a certain concentration, and BSA and HAuCl are added into groups 1-13 according to the proportion in the table 1 ₄ ^. 3H ₂ O is fully stirred and uniformly mixed in a 25mL single-mouth bottle to obtain a compound of BSA and Au; the total volume of the reaction solution is 1-8 mL, and the stirring speed is 800-;

1.2, adding a proper amount of NaOH aqueous solution into a reaction bottle, adjusting the pH value of the reaction to 10, controlling the reaction conditions in the air atmosphere, observing the color change, raising the reaction temperature and continuing the reaction for a period of time to obtain a crude product. A color change, i.e., a change in the color of the reaction liquid from light yellow to black red, was observed; raising the reaction temperature, adjusting the temperature of the reaction liquid to be within the range of 50-90 ℃, continuously reacting for a period of time, and reacting for 2-6 h at high temperature.

1.3, centrifuging the obtained crude product by a centrifuge (the centrifugation speed is 8000-; au (I) -BSA nanoparticles of groups 1-13 can be obtained.

1.4, the Au (I) -BSA nanoparticle stock solution prepared in the groups 1-13 is diluted by 1000 times, a drop of acetone is firstly dropped on a 400-mesh copper net, and then the diluted nanoparticle solution is absorbed by a 10-mu L liquid-transferring gun and is vertically dropped on the copper net. After the solvent was completely volatilized, the mixture was dried overnight in a vacuum oven at 50 ℃. The next day, the samples were taken out and photographed under a transmission electron microscope.

Results of the experiment

As can be seen by the TEM images of groups 1-13, when HAuCl is present ₄ ^. 3H ₂ When the molar mass ratio of O to BSA is less than 30:1 or greater than 40:1, nanoclusters having a particle size of less than 2nm or nanocubes having a diameter of greater than 800nm are formed. Only when HAuCl is present ₄ ^. 3H ₂ The molar mass ratio of O to BSA is between 30:1 and 40:1, in particular when HAuCl ₄ ^. 3H ₂ When the molar mass ratio of O to BSA is 36:1, nanoparticles having a uniform particle diameter and excitation wavelength dependence are formed. Due to the large amount of data, only the results of group 5, group 9 and group 12 are disclosed in the present invention, as shown in fig. 1 (group 5 on the left, group 12 on the middle and group 9 on the right), it can be seen that only the au (i) -BSA nanoparticles of group 9 have spherical or spheroidal micro-morphology, uniform size, and an average particle diameter of 26.2 ± 0.27 nm.

Example 2 pH screening

Experimental methods

2.1 reaction of BSA with HAuCl ₄ ^. 3H ₂ Preparing mother liquor with a certain concentration from O, adding BSA and HAuCl ₄ ^. 3H ₂ O (according to the proportion of the group 9) is fully stirred and uniformly mixed in a 25mL single-mouth bottle to obtain a BSA-Au compound; the total volume of the reaction solution is 1-8 mL, and the stirring speed is 800-.

2.2, adding a proper amount of NaOH aqueous solution into the reaction bottle, adjusting the pH value of the reaction (the pH value is adjusted as shown in table 2), controlling the reaction conditions in the air atmosphere, observing the color change, raising the reaction temperature and continuing to react for a period of time to obtain a crude product; a color change, i.e., a change in the color of the reaction liquid from light yellow to black red, was observed. Raising the reaction temperature, adjusting the temperature of the reaction liquid to be within the range of 50-90 ℃, continuously reacting for a period of time, and reacting for 2-6 h at high temperature.

2.3, centrifuging the obtained crude product by a centrifuge (the centrifugation rate is 8000-.

2.4, observing the Au (I) -BSA nanoparticle aqueous solution of the groups 14-25 under a fluorescent lamp and a UV lamp.

TABLE 2

Grouping	pH value of the mixed solution
		Group 14	1
Group 15	2
		Group 16	3
Group 17	4
		Group 18	5
Group 19	6
		Group 20	7
Group 21	8
		Group 22	9
Group 23	10
		Group 24	11
Group 25	12

Results of the experiment

As can be seen from groups 14-25, the pH of the solution is important for the synthesis of nanoparticles with excitation wavelength dependence. When the pH is less than 8, the pH is close to the isoelectric point of BSA, so that serious coagulation is caused; when the pH is more than 10, HAuCl is greatly accelerated in a strongly alkaline solution ₄ ^. 3H ₂ The reduction of O produces a product which emits relatively weak light and has no excitation wavelength dependence. Only when the pH of the mixed solution is between 8 and 10, HAuCl can be used ₄ ^. 3H ₂ The reduction rate of O is moderate, and nano particles with excitation wavelength dependent luminescence property are generated. Due to the large amount of data, only the results of group 20, group 22, and group 24 are disclosed in the present invention, as shown in FIG. 2.

Example 3 Au (I) -BSA nanoparticles

The experimental method comprises the following steps:

detection of dynamic light scattering, 30 μ L of the au (i) -BSA nanoparticle raw solution prepared in group 22 in example 2 was diluted to 3mL, and then placed in a dynamic light scattering instrument, and its hydrated particle size in an aqueous solution was measured.

And (3) test results:

analysis of the Dynamic Light Scattering (DLS) pattern (fig. 3) of the au (i) -BSA nanoparticles revealed that the hydrated particle size of the synthetic material was about 30.6nm, which is comparable to the TEM statistics. In addition, the nanoparticles proved to have good dispersibility in aqueous solutions, with a dispersion coefficient PDI of 0.17, suitable for cellular and bioimaging.

Example 4 luminescence of Au (I) -BSA nanoparticles

The experimental method comprises the following steps:

4.1, the Au (I) -BSA nanoparticle aqueous solution prepared in group 22 of example 2 was dispersed in Phosphate Buffered Saline (PBS) at a concentration of 0.5-10 mg/mL, and dispersed by sonication for 1min for the test of cell imaging.

4.2, the cells were evenly seeded in six well plates, 1.2X 10 per well ⁶ And (3) incubating Au (I) -BSA nanoparticle solutions with different concentrations with the cells for 3-8 h. Then, the culture medium was removed and washed three times with PBS, and observed under different fluorescence channels of an inverted microscope.

The experimental results are as follows:

from the excitation wavelength-dependent luminescence spectrum (fig. 4) of au (i) -BSA nanoparticles, it is understood that the photoluminescence spectrum of the solution changes with the change in the excitation wavelength, and the luminescence color changes accordingly. Can be used for multicolor imaging of cells.

As can be seen from the cell imaging graph (figure 5), the material shows obviously different luminescence in the living cells under different excitation wavelengths, and the material is proved to be capable of well entering the living cells and serving as a multicolor imaging agent of the cells.

In addition, the invention also carries out dynamic imaging under yellow light and cyan light channels, and the material provided by the invention shows obvious luminescence in living cells. In addition, the material of the present invention has fluorescence and phosphorescence dual-mode light emission characteristics depending on excitation wavelength.

During the description of the above description:

the description of the terms "this embodiment," "an embodiment of the invention," "as shown at … …," "further improved technical solution," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention; in this specification, the schematic representations of the terms used above are not necessarily for the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc., may be combined or brought together in any suitable manner in any one or more embodiments or examples; furthermore, those of ordinary skill in the art may combine or combine features of different embodiments or examples and features of different embodiments or examples described in this specification without undue conflict.

Finally, it should be noted that:

the above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same;

although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that various changes and modifications may be made, and equivalents may be substituted for elements thereof without departing from the scope of the embodiments of the present invention.

Claims

1. The multicolor luminous nano material for biomedical dynamic imaging is characterized in that the multicolor luminous nano material is an Au (I) -BSA nano material formed by gold and bovine serum albumin, the micro-morphology of the Au (I) -BSA nano particles is spherical or spheroidal, and the average particle size of the Au (I) -BSA nano particles is 26.2 +/-0.27 nm.

2. The multicolor luminescent nanomaterial for biomedical dynamic imaging according to claim 1, wherein the au (i) -BSA nanoparticles have a dispersion coefficient PDI of 0.17 in an aqueous solution.

3. Use of the multicolor luminescent nanomaterial of any of claims 1 to 2 in biomedical dynamic imaging.

4. A method of imaging a cell, comprising the steps of: incubating the Au (I) -BSA nanoparticle solution of any one of claims 1-2 with the cells for 3-8 h, removing the culture medium and washing with PBS, and observing the cells for imaging under an inverted microscope or a Confocal fluorescence microscope.

5. A method of cellular imaging as claimed in claim 4 wherein the cellular imaging is performed dynamically under the red, green, blue, yellow and cyan light channels of an inverted fluorescence microscope or a Confocal fluorescence microscope.

6. A preparation method of multicolor luminous nanometer material for biomedical dynamic imaging is characterized by comprising the following steps:

7. The method of claim 6, wherein in step S1, HAuCl is added to the solution ₄ ^. 3H ₂ The molar ratio of O to BSA is 30:1-40: 1.

8. The method of claim 7A preparation method of multicolor luminous nano material for biomedical dynamic imaging is characterized in that in step S1, HAuCl is added ₄ ^. 3H ₂ The molar ratio of O to BSA was 36: 1.

9. The method of claim 6, wherein in step S2, the pH of the reaction solution is adjusted to 8-10.

10. The method of claim 6, wherein in step S2, the reaction conditions are 50-90 ℃ for 1-6 h.