CN114921245B - Near-infrared carbon dot and preparation method and application thereof - Google Patents
Near-infrared carbon dot and preparation method and application thereof Download PDFInfo
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
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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
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Abstract
The invention discloses a near-infrared carbon dot, a preparation method and application thereof, wherein the chemical structure of the near-infrared carbon dot is provided with a conjugated domain formed by fusion of conjugated molecules, the conjugated domain is also chemically bonded with amido, and the carbon dot is provided with a near-infrared main absorption peak and a main emission peak. The preparation method comprises the following steps: the conjugated molecule and the cosolvent with amide groups are fused under the action of solvothermal. Through solvothermal reaction, conjugated molecules can fuse to form a carbon core with an enlarged conjugated domain, and react with a cosolvent with an amide group to bond the amide group, resulting in a carbon dot with near-infrared absorption/emission characteristics. The water-soluble polymer molecular chain is further subjected to solvothermal reaction with carbon dots, and the water solubility, stability and near infrared fluorescence quantum efficiency of the carbon dots are improved. The preparation raw materials of the near-infrared carbon dot can be widely selected, the preparation process is simple, the carbon dot is not easy to quench in aqueous solution, has higher fluorescence quantum yield, and is suitable for near-infrared biological imaging.
Description
Technical Field
The invention relates to the technical field of carbon nano materials, in particular to a near infrared carbon dot and a preparation method and application thereof.
Background
Traditional biological imaging is mainly focused on the visible light wave band (400-700 nm), and in the wave band, biological tissues have serious absorption and scattering on excitation light and emission light, so that the penetration depth of the tissues is not deep; and larger autofluorescence is generated, so that the signal to noise ratio is low, and the biological imaging is not facilitated. The near infrared band (NIR, 700-1700 nm) is widely used for biological imaging due to its deeper penetration into tissue, low self-absorption and scattering by tissue, low autofluorescence, and the like, as compared to the visible band.
Carbon Dots (CDs) were first discovered in 2004 as a zero-dimensional photoluminescent carbon nanomaterial with a size less than 10 nm. The preparation method is simple, raw materials are easy to obtain, the cost is low, the biocompatibility is good, the color is adjustable, and the method is widely applied to the fields of biological imaging and the like. However, the absorption and emission of carbon dots reported so far are mostly in the visible region, which is disadvantageous for bioimaging applications. Although few carbon dots which absorb and emit light in the near infrared region using near infrared dyes as raw materials are reported, the raw materials are very expensive, which is unfavorable for mass production, and the quantum yield in aqueous solution is not high, which is unfavorable for biological imaging. Meanwhile, carbon dots which are reported individually can realize high-efficiency near-infrared absorption and emission in an organic solvent, but near-infrared fluorescence is quenched in an aqueous solution, and the biological environment is water environment, so that the method is not suitable for near-infrared biological imaging.
Therefore, there is an urgent need to develop a carbon dot having a main absorption peak and a main emission peak in an aqueous solution and having a high fluorescence quantum yield under near infrared light excitation.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a near infrared carbon dot, a preparation method and application thereof, so as to solve the technical problems.
The invention is realized in the following way:
in a first aspect, the present invention provides a near infrared carbon dot having a chemical structure comprising a conjugated domain formed by fusion of conjugated molecules, the conjugated domain further being chemically bound to an amide group, and the carbon dot having a near infrared main absorption peak and a main emission peak.
Optionally, the conjugated molecule is polycyclic aromatic hydrocarbon or derivative thereof, the conjugated molecule is at least one selected from biphenyl and biphenyl, polybiphenyl aliphatic hydrocarbon and polycyclic aromatic hydrocarbon, optionally, the conjugated molecule is at least one selected from perylene, pyrene, indene, fluorene, acenaphthene, naphthalene, acenaphthylene, anthracene, butryene, pentalene and phenanthrene and corresponding derivatives thereof, optionally, the conjugated molecule is 3,4,9, 10-perylenetetracarboxylic dianhydride.
Optionally, the surface of the carbon dot is further modified with a water-soluble polymer molecular chain, and optionally, the water-soluble polymer molecular chain is polyethyleneimine.
In a second aspect, the present invention also provides a method for preparing the near infrared carbon dot, which includes: the conjugated molecule and the cosolvent with amide groups are fused under the action of solvothermal.
In a third aspect, the invention also provides application of the near infrared carbon dots in near infrared encryption, near infrared anti-counterfeiting, dye or biological imaging, and the application is not aimed at diagnosis and treatment of diseases.
In a fourth aspect, the present invention also provides a fluorescence imaging agent, which includes the near infrared carbon dot.
In a fifth aspect, the present invention also provides the use of the above-described fluorescence imaging agent in vivo single-photon or two-photon near infrared fluorescence imaging, and the use is not for the purpose of diagnosis and treatment of diseases.
The invention has the following beneficial effects: by solvothermal reaction, conjugated molecules can fuse to form a carbon core with an enlarged conjugated domain and react with a co-solvent with an amide group to bond the amide group, resulting in Carbon Dots (CDs) with near infrared absorption/emission characteristics. The preparation raw materials of the carbon dot can be widely selected, the cost can be controlled to be lower, the popularization and the application are easy, the carbon dot is not easy to quench in aqueous solution, the fluorescence quantum yield is higher, and the preparation method is suitable for near infrared biological imaging.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings to be used in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a process for preparing near infrared carbon dots according to one embodiment of the present invention;
FIG. 2 is a transmission electron microscope TEM image (a, b) and an atomic force microscope AFM image (c) of CDs of example 1 of the present invention;
FIG. 3 is (a) ultraviolet visible (UV-Vis) absorption spectra (tested in water or DMF solvent) of the CDs of example 1 and PEI-CDs of example 11 of the present invention; (b) Fluorescence spectra (tested in water or aqueous BSA solution or DMF solvent); excitation-emission three-dimensional spectral mapping of aqueous solutions of PEI-CDs (c) and (d) DMF solutions of example 11; (e) from left to right, in order: water, mouse serum, aqueous solutions of CDs of example 1, aqueous solutions of PEI-CDs of example 11; aqueous BSA solution of PEI-CDs of example 11; near infrared imaging pictures under a CCD camera equipped with a 750nm long pass filter of the mouse serum solution of PEI-CDs of example 11 and the DMF solution of PEI-CDs of example 11, excitation wavelength was 690nm;
FIG. 4 is an ultraviolet visible light absorption diagram of the reactant of comparative example 1 in water;
FIG. 5 is an ultraviolet visible light absorption diagram of the reactant of comparative example 2 in water;
FIG. 6 shows the two photon fluorescence emission spectra of PEI-CDs of example 11 of the present invention in the near infrared first region under different power of near infrared two-region laser excitation of (a) aqueous solution, (b) BSA aqueous solution and (c) DMF solution; (d) a graph of the log-log relationship between laser power and concentration;
FIG. 7 is a photograph taken in near infrared imaging and sunlight of an aqueous solution of PEI-CDs of example 11 of the present invention, which was coated on A4 paper for information encryption, the photograph being obtained on a CCD camera equipped with a 750nm long pass filter, the excitation wavelength being 690nm;
FIG. 8 is a cytotoxicity test of (a) PEI-CDs of example 11 of the present invention; (b) mouse organ metabolism experiments; (c) high resolution imaging of the intestinal tract of mice before and after lavage;
FIG. 9 is a two-photon imaging experiment of the ear of a mouse with an aqueous solution of PEI-CDs of example 11 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The invention provides a near infrared carbon dot, a preparation method and application thereof.
Some embodiments of the present invention provide a near infrared carbon dot whose chemical structure has a conjugated domain formed by fusion of conjugated molecules, the conjugated domain further having an amide group chemically bonded thereto, and the carbon dot having a near infrared main absorption peak and a main emission peak.
The inventor finds that the carbon point with the conjugated domain and the amide group chemically bonded on the conjugated domain has the near infrared absorption and emission characteristics to a great extent, is not easy to quench in the aqueous solution at the carbon point, and has higher fluorescence quantum yield. Therefore, the near infrared carbon points with the structural characteristics can be obtained by selecting different reaction raw materials, so that the sources of the raw materials are enlarged, the cost can be reduced, and the application of the near infrared carbon points is promoted.
Specifically, in some embodiments, the conjugated molecule is a large conjugated molecule, for example, the conjugated molecule may be a polycyclic aromatic hydrocarbon or a derivative thereof, and the conjugated molecule may be selected from at least one of biphenyls and biphenyls, a polyphenylarene, and a polycyclic aromatic hydrocarbon.
For example, in some embodiments, conjugated molecules include, but are not limited to, at least one of perylene, pyrene, indene, fluorene, acenaphthylene, naphthalene, acenaphthylene, anthracene, butryene, pentylene, and phenanthrene and their corresponding derivatives. That is, the conjugated molecule may be selected not only from one of the above substances alone but also from a combination of several substances, that is, a scheme of selecting or combining conjugated molecules satisfying the above conditions is within the above-mentioned protective range as long as the conjugated molecules can fuse to form conjugated domains and can satisfy the conditions having a near infrared main absorption peak and a main emission peak.
In some embodiments, the conjugated molecule may be 3,4,9, 10-perylenetetracarboxylic dianhydride.
Further, in order to further improve the fluorescence quantum yield of the near infrared carbon dot in the aqueous solution, the inventor finds through a great deal of research and practice that the quenching of the luminescence of the carbon dot by the water molecule can be further effectively prevented by modifying the molecular chain of the water-soluble polymer to the surface of the carbon dot.
Thus, in some embodiments, the surface of the carbon dot is further modified with a water-soluble polymer molecular chain, in particular, the selection of the water-soluble polymer molecular chain includes, but is not limited to, polyethyleneimine.
Specifically, in some embodiments, the near infrared carbon point has an absorption band of 600-800 nm and a near infrared emission peak of 700-850 nm.
Further, in some embodiments, when the carbon dots are not modified with water-soluble polymer molecular chains, the aqueous solution of the near-infrared carbon dots has an absorption band at 600-800 nm, an absorption peak at 720nm, and an excitation independent emission peak at 745nm under excitation at 680-730 nm. When the carbon point is modified with a water-soluble polymer molecular chain, the water solution of the near infrared carbon point has an absorption band at 600-800 nm, and the absorption peak is 726nm; under 680nm-730nm excitation, the DMF solution with near infrared carbon point has absorption band of 600-800 nm, absorption peak of 742nm and excitation independent emission peak of 765nm under 680-730 nm excitation.
Some embodiments of the present invention also provide a method for preparing the near infrared carbon dot, which includes: the conjugated molecule and the cosolvent with amide groups are fused under the action of solvothermal.
Long-term studies by the inventors have creatively found during long-term studies and practices that in the solvothermal process for preparing carbon dots, the precursor is a molecule with a large conjugated domain that can fuse under solvothermal conditions to form near-infrared absorbing/emitting carbon dots with a large conjugated domain carbon core.
Specifically, in some embodiments, the method for preparing the near infrared carbon dot includes: after dispersing the conjugated molecule and the cosolvent with amide groups in a polar solvent, performing a first solvothermal reaction.
In some embodiments, the mass ratio of conjugated molecule to co-solvent is from 0.05 to 1:0.2 to 4.
In some embodiments, the polar solvent is a polar aprotic solvent including, but not limited to, at least one of N, N-dimethylformamide, dimethylsulfoxide, and N-methylpyrrolidone. For example, the polar aprotic solvent may be any one of N, N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone, or may be a mixture of two or three thereof, and the mixing ratio is not limited.
In some embodiments, the first solvothermal reaction is at a temperature of 140-200 ℃, e.g., 150 ℃, 155 ℃, 160 ℃, 165 ℃, 170 ℃, 175 ℃, 180 ℃, 185 ℃, 190 ℃, or 195 ℃, etc., and the reaction time is 3-24 hours, e.g., 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, etc.
Further, in some embodiments, after the first solvothermal reaction is completed, the reaction solution is centrifuged, and the supernatant is diluted and purified by first dialysis. Specifically, the rotational speed of centrifugation is 3000-15000 r/min, and the centrifugation time is 5-30 min; preferably, the dialysis bags used for the first dialysis purification have a molecular weight cut-off of 1000-5000 Da and a dialysis time of 12-24 hours.
Further, formation of a protective layer by the surface-modified polymer prevents fluorescence quenching of the carbon dots by water molecules, improves fluorescence intensity, and in some embodiments, the preparation method further comprises performing a secondary solvothermal reaction of the product obtained by the first solvothermal reaction and the molecular chain of the water-soluble polymer.
Specifically, in some embodiments, the mass ratio of the water-soluble polymer molecular chain to the conjugate molecule is from 0.2 to 1:0.05-1. The second solvothermal reaction is carried out at a temperature of 140 to 200 ℃, for example, 150 ℃, 155 ℃, 160 ℃, 165 ℃, 170 ℃, 175 ℃, 180 ℃, 185 ℃, 190 ℃, 195 ℃ or the like, and a reaction time of 3 to 24 hours, for example, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours or the like.
Further, in some embodiments, the product obtained by the second solvothermal reaction is diluted and then subjected to second dialysis and purification, and the molecular weight cut-off of a dialysis bag used for the second dialysis and purification is 5000-20000 Da; the dialysis time is 12-48 hours.
Referring to fig. 1, some embodiments of the present invention also provide a specific preparation method of near infrared carbon dots, which includes: taking perylene derivative-3, 4,9, 10-perylene tetracarboxylic dianhydride (PTCDA) with a large conjugated domain as a raw material, and urea as a cosolvent to form near infrared carbon points (CDs) with enlarged conjugated structure carbon cores through fusion under solvothermal conditions; further modified with polyethyleneimine to obtain near-infrared carbon dots (PEI-CDs) with increased fluorescence quantum efficiency.
Specifically, the preparation method of the near infrared carbon dots comprises the following steps:
(1) 3,4,9, 10-perylene tetracarboxylic dianhydride (PTCDA) and urea are uniformly mixed into polar aprotic solvent through ultrasonic, then solvothermal reaction is carried out, centrifugation is carried out after the reaction is finished, the supernatant is diluted and then subjected to dialysis purification to obtain the near infrared Carbon Dots (CDs), and the aqueous solution of the CDs has an absorption band and an emission band in a near infrared region.
(2) Adding Polyethyleneimine (PEI) into the reaction liquid in the step (1), uniformly mixing, then carrying out solvothermal reaction again, diluting the reaction liquid, and then carrying out dialysis purification to obtain polyethyleneimine modified carbon points (PEI-CDs), wherein compared with the carbon points synthesized in the first step, the absorption peak is slightly red-shifted, and the emission peak intensity is enhanced.
The amount of 3,4,9, 10-perylene tetracarboxylic dianhydride is 0.05-1 g, and the amount of urea is 0.2-4 g; the amount of the polyethyleneimine in the step (2) is 0.2-1 g.
Some embodiments of the invention also provide for the use of near infrared carbon dots in any of the above embodiments in near infrared encryption, near infrared anti-counterfeit, dye or bioimaging.
Some embodiments of the invention also provide a fluorescence imaging agent comprising near infrared carbon dots in any of the above embodiments.
The invention also provides application of the fluorescent imaging agent in living body single-photon or two-photon near infrared fluorescent imaging.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a preparation method of near infrared carbon dots, which comprises the following steps:
0.2g of PTCDA and 1g of urea are dispersed in 20mL of DMF, and the mixed turbid liquid is placed in a high-pressure reaction kettle provided with a polytetrafluoroethylene liner, and then placed in a high-temperature oven for solvothermal reaction, wherein the reaction temperature is 160 ℃, and the reaction time is 5 hours. After the reaction is cooled to room temperature, taking out the reaction liquid for centrifugal separation, collecting supernatant fluid after the rotation speed of the centrifugal machine is 8000 revolutions per minute, diluting the supernatant fluid with deionized water, and then placing the diluted supernatant fluid into a dialysis bag for dialysis and purification, wherein the molecular weight cut-off of the dialysis bag is 5000Da, and the dialysis time is 24 hours. After the dialysis is completed, the solution in the dialysis bag is freeze-dried to obtain black solid, namely near infrared Carbon Dots (CDs).
Example 2
This example provides a process for the preparation of near infrared carbon dots, which differs from example 1 only in that the mass of raw material PTCDA is 0.2g and the mass of urea is 2g.
Example 3
This example provides a process for the preparation of carbon dots with near infrared absorption and emission properties in aqueous solution, differing from example 1 only in that the mass of raw material PTCDA is 0.5g and the mass of urea is 2g.
Example 4
This example provides a process for the preparation of near infrared carbon dots, which differs from example 1 only in that the mass of raw material PTCDA is 1g and the mass of urea is 1g.
Example 5
This example provides a process for the preparation of near infrared carbon dots, which differs from example 1 only in that the mass of the raw material PTCDA is 0.2g and that of the biuret is 1g.
Example 6
This example provides a method for preparing near infrared carbon dots, which differs from example 1 only in that the solvent is dimethyl sulfoxide (DMSO).
Example 7
This example provides a method for preparing near infrared carbon dots, which differs from example 1 only in that the reaction temperature is 140 ℃.
Example 8
This example provides a method for preparing near infrared carbon dots, which differs from example 1 only in that the reaction temperature is 180 ℃.
Example 9
This example provides a method for preparing near infrared carbon dots, which differs from example 1 only in that the reaction temperature is 200 ℃.
Example 10
This example provides a method for preparing near infrared carbon dots, which differs from example 1 only in that the reaction temperature is 220 ℃.
Example 11
This example provides a method for preparing PEI-CDs with modified carbon dots by polyethyleneimine as described in example 1, comprising the following steps:
0.5g of Polyethylenimine (PEI) was dispersed in the centrifuged supernatant as described in example 1, and the mixed suspension was placed in a high-pressure reactor equipped with a polytetrafluoroethylene liner, and then placed in a high-temperature oven for solvothermal reaction at 160℃for 5 hours. After the reaction is cooled to room temperature, the reaction solution is taken out, diluted by deionized water and placed into a dialysis bag for dialysis and purification, the molecular weight cut-off of the dialysis bag is 20000Da, and the dialysis time is 24 hours. After the dialysis is completed, the solution in the dialysis bag is freeze-dried to obtain black solid, namely polyethyleneimine modified near infrared carbon dots (PEI-CDs).
Example 12
This example provides a process for the preparation of PEI-CDs modified with polyethyleneimine at carbon sites as described in example 1, example 11 differing only in that the mass of polyethyleneimine is 1g.
Example 13
This example provides a process for the preparation of PEI-CDs modified with polyethyleneimine at carbon sites as described in example 1, example 11 differing only in that the dialysis bag has a molecular weight cut-off of 8000Da and a dialysis time of 48 hours.
Comparative example 1
This comparative example provides a process for the preparation of carbon dots which differs from example 1 only in that PTCDA is used as a starting material, comprising specifically the following steps:
0.2g of PTCDA was dispersed in 20mL of DMF, and the mixed suspension was placed in a high-pressure reaction vessel equipped with a polytetrafluoroethylene liner, and then placed in a high-temperature oven for solvothermal reaction at 160℃for 5 hours. After the reaction is cooled to room temperature, taking out the reaction liquid for centrifugal separation, wherein the rotating speed of the centrifugal machine is 8000 revolutions per minute, collecting supernatant, diluting the supernatant with deionized water, and then placing the diluted supernatant into a dialysis bag for dialysis and purification, wherein the molecular weight cut-off of the dialysis bag is 5000Da, and the dialysis time is 12 hours. After the dialysis is completed, the solution in the dialysis bag is freeze-dried to obtain carbon dots (CDs-1).
Comparative example 2
This comparative example provides a method for preparing carbon dots, which differs from example 1 only in that water is used as the solvent, and specifically includes the following steps:
0.2g of PTCDA and 1g of urea are dispersed in 20mL of water, the mixed turbid liquid is placed into a high-pressure reaction kettle provided with a polytetrafluoroethylene liner, and then the mixture is placed into a high-temperature oven for solvothermal reaction, wherein the reaction temperature is 160 ℃, and the reaction time is 5 hours. After the reaction is cooled to room temperature, taking out the reaction liquid for centrifugal separation, collecting supernatant fluid after the rotation speed of the centrifugal machine is 8000 revolutions per minute, diluting the supernatant fluid with deionized water, and then placing the diluted supernatant fluid into a dialysis bag for dialysis and purification, wherein the molecular weight cut-off of the dialysis bag is 5000Da, and the dialysis time is 12 hours. After the dialysis is completed, the solution in the dialysis bag is freeze-dried to obtain carbon dots (CDs-2).
Experimental example 1
The morphology of the near infrared Carbon Dots (CDs) obtained in example 1 was subjected to Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM), and the results are shown in fig. 2. Fig. 2 is a TEM image and an AFM image of near infrared carbon dots in example 1 of the present invention. As can be seen from fig. 2, CDs are uniformly distributed with a particle size of 3.3±0.7nm, and a High Resolution Transmission Electron Microscope (HRTEM) image shows lattice fringes of 0.21nm due to the (100) crystal plane of graphene; AFM images showed CDs between 0.5-3.5nm in height and 1.0nm in average height, indicating that they consisted of few layers of graphene fragments.
Experimental example 2
Ultraviolet absorption tests were performed on the near infrared carbon points (CDs) obtained in example 1 and the polyethyleneimine modified carbon points (PEI-CDs) obtained in example 11, as well as the raw material PTCDA. As shown in fig. 3 (a), the DMF solution of the raw material PTCDA has absorption peaks only in the ultraviolet and visible light regions, the aqueous solution of CDs has a main absorption peak band in the near infrared I region, and the absorption peak is at 720nm; the absorption peak of the aqueous solution of PEI-CDs in the near infrared I region is at 726nm, and the absorption peak of the DMF solution of PEI-CDs in the near infrared I region is at 742nm. The carbon dots obtained in comparative example 1 were diluted with deionized water and then tested for uv-vis absorption spectrum, the results of which are shown in fig. 4, and the product of comparative example 1 was not significantly absorbed in the near infrared region, indicating the indispensable role of urea (including structurally similar or analogous species thereof, such as, but not limited to, biuret, etc.) in the preparation of a near infrared carbon dot by the macroconjugated molecule fusion strategy described in the embodiments of the present invention. The carbon dots obtained in comparative example 2 were diluted with deionized water, and then the ultraviolet-visible light absorption spectrum was measured, and the results are shown in fig. 5.
The near infrared fluorescence spectra of the aqueous solution of near infrared carbon point (CDs) obtained in example 1 and the aqueous solution of polyethyleneimine-modified carbon point (PEI-CDs), aqueous solution of Bovine Serum Albumin (BSA) and DMF solution under the excitation of 725nm light source (CDs and PEI-CDs are measured under the condition that the absorption of the CDs and PEI-CDs in the near infrared region are consistent; the concentration of PEI-CDs in different solvents is consistent). The results are shown in fig. 3 (b): before PEI modification, PLQY of CDs in an aqueous solution is 3.33%, after PEI modification, fluorescence of carbon dots is obviously enhanced, and PLQY of PEI-CDs in the aqueous solution is 5.33%; due to the water blocking effect of BSA, the fluorescence of PEI-CDs is further enhanced, and the PLQY of PEI-CDs in the BSA aqueous solution is improved to 8.28%. PLQY of PEI-CDs in DMF solution is as high as 18.80%.
The aqueous solution of polyethyleneimine modified carbon dots (PEI-CDs) and DMF solution obtained in example 11 were subjected to excitation-emission three-dimensional spectroscopy. As a result, as shown in FIGS. 3 (c) and (d), respectively, the aqueous solutions of PEI-CDs showed excitation independent emission with a luminescence center of 751nm; the DMF solution of PEI-CDs showed excitation independent emission with a luminescence center of 765nm.
Under 690nm excitation, water, mouse serum, aqueous solutions of CDs of example 1, aqueous solutions of PEI-CDs of example 11; aqueous BSA solution of PEI-CDs of example 11; the mouse serum solution of PEI-CDs of example 11 and the DMF solution of PEI-CDs of example 11 were subjected to near infrared imaging under a CCD camera equipped with a 750nm long pass filter. The results are shown in fig. 3 (e): the trend of the increase in fluorescence intensity was consistent with (b) in FIG. 3, and in particular, the increase in fluorescence intensity of the serum solution of PEI-CDs of example 11 was similar to or higher than that of the BSA aqueous solution of PEI-CDs, meaning that PEI-CDs of example 11 could bind to serum proteins in the blood of mice during application to mouse imaging, achieving near infrared fluorescence enhancement.
Experimental example 3
The aqueous solution of PEI-CDs, the aqueous solution of BSA and the solution of DMF of example 11 are subjected to two-photon emission spectrum under the excitation of near infrared two-area lasers with different powers, and the concentrations of the solutions are consistent and are all concentrated solutions. The results are shown in FIG. 6: all three solutions show emission in the near infrared first region under the excitation of the near infrared second region laser, and the power of the excitation light source is increased. The two-photon fluorescence intensity and the laser power show good linear relation, and the slopes of the two-photon fluorescence intensity and the laser power are close to 2, so that the two-photon excitation process is proved.
Experimental example 4
The aqueous solution of PEI-CDs of example 11 was applied for information encryption/security. As shown in FIG. 7, an aqueous solution of PEI-CDs was used as the encryption ink, and a stamp was printed on a blank A4 paper in the form of "Australian Tha ". Under sunlight and in bright field, the "Chamomile " word is invisible, and it is placed under a CCD camera equipped with a 750nm long pass filter, the "Chamomile " word is clearly visible; further, a layer of A4 paper is covered on the A4 paper, and the word of 'Australian Thymus-Chamomillae ' can still be recorded by a camera.
Experimental example 5
Cytotoxicity test was performed on different concentrations of PEI-CDs of example 11, and the results are shown in FIG. 8 (a), in which PEI-CDs show lower cytotoxicity even at higher concentrations; further, organ metabolism experiments were performed on aqueous solutions of PEI-CDs. 200. Mu.L of PEI-CDs aqueous solution with concentration of 15mg/mL was injected into mice through tail vein, and main organs of the mice at different times after injection were subjected to imaging analysis, and the result was mainly enriched into kidney and liver in 1.5 hours, followed by gradual decrease of fluorescence, and all discharged from the body for 24 hours, as shown in FIG. 8 (b).
An in vivo imaging experiment of mice was performed on the aqueous solution of PEI-CDs of example 11 as a fluorescent reagent. 400. Mu.L of PEI-CDs in water was injected into the stomach of the mice by gavage, and near infrared imaging was performed on the mice before and after gavage under a CCD camera equipped with a 750nm long-pass filter. As a result, as shown in FIG. 8 (c), the intestinal tract of the mouse was clearly seen for a while after the gastric lavage, and the background of other tissues was low and the signal to noise ratio was high.
Experimental example 6
The mouse ear vascular biphoto imaging experiment was performed on the aqueous solution of PEI-CDs of example 11 as a fluorescent reagent.
200. Mu.L of PEI-CDs aqueous solution at a concentration of 15mg/mL was injected into mice via tail vein. Two-photon imaging was performed on mouse ear vessels under near infrared two-zone laser excitation. As a result, as shown in fig. 9, in the eighth second after injection, the ear blood vessel was visible, and the fluorescence intensity was maximized for fifteen seconds, so that the ear blood vessel was clearly visible.
In summary, the present invention provides a carbon dot with strong near infrared absorption and emission characteristics in aqueous solution, prepared by a molecular fusion strategy with large conjugated domains; the large conjugated molecule refers generally to molecules having a plurality of benzene rings and derivatives thereof, including but not limited to polycyclic aromatic hydrocarbons such as perylene, pyrene and the like and derivatives thereof. The method is characterized in that 3,4,9, 10-perylene tetracarboxylic dianhydride and urea serving as derivatives of perylene are used as raw materials, and near-infrared Carbon Dots (CDs) are prepared through a molecular fusion strategy of a large conjugated domain under the condition of a solvothermal method. Experimental results show that the aqueous solution and the organic solution of the CDs have near infrared main absorption peaks and main emission peaks at 720nm and 745 nm. Further, polyethyleneimine (PEI) and CDs are further reacted under solvothermal conditions to obtain polyethyleneimine modified carbon points (PEI-CDs), the water solubility and stability of the PEI-CDs are improved, and the absorption and emission of the PEI-CDs have slight red shift of 726nm and 751nm respectively. Under the excitation of near infrared light, PEI-CDs have very high near infrared fluorescence photon yield (3.3-18.8) in both aqueous solution and organic solvent. Further, the CDs are dissolved in an aqueous solution of Bovine Serum Albumin (BSA), and the fluorescence quantum yield in the near infrared region is further improved. The near infrared nature of PEI-CDs enables its application in information encryption/security. PEI-CDs have low cytotoxicity and high biocompatibility, and can be used as a fluorescent reagent for high-resolution living body imaging and two-photon ear imaging of mice.
In summary, the embodiment of the present invention mainly provides a near-infrared carbon dot having near-infrared absorption and emission characteristics in an aqueous solution and a preparation method thereof. The starting material 3,4,9, 10-perylenetetracarboxylic dianhydride (PTCDA) has a large conjugated domain, reacts with urea under solvothermal conditions, fuses to form a carbon core with an enlarged conjugated domain by a large conjugated molecule fusion strategy, generating Carbon Dots (CDs) with near infrared absorption/emission properties. The aqueous solution of CDs has an absorption band at 600-800 nm, and the absorption peak is 720nm; under 680nm-730nm excitation, the fluorescent quantum yield is 3.33% under excitation of 745nm excitation independent emission peak and near infrared one-region light source. The aqueous solution of the carbon point (PEI-CDs) modified by the polyethyleneimine has an absorption band at 600-800 nm and an absorption peak of 726nm; under 680nm-730nm excitation, the fluorescent quantum yield of the fluorescent dye has an excitation independence emission peak of 751nm, and the fluorescent quantum yield of the bovine serum albumin aqueous solution is up to 5.33 percent, and is up to 8.28 percent. The DMF solution of near infrared PEI-CDs has an absorption band at 600-800 nm and an absorption peak at 742nm; under 680nm-730nm excitation, the fluorescent quantum yield reaches 18.80% with 765nm excitation independent emission peak. Under the excitation of the near infrared-II region femtosecond laser, the near infrared-I region emission induced by two photons is provided, and the emission peak position of the near infrared-I region is within the spectral range of 700-900 nm. The polyethyleneimine modified carbon dots (PEI-CDs) have low cytotoxicity, high biocompatibility, good water solubility, simple synthesis and easy preparation.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (22)
1. A near-infrared carbon dot characterized by having a chemical structure with a conjugated domain formed by fusion of conjugated molecules, the conjugated domain further being chemically bonded with an amide group, and the near-infrared carbon dot having a near-infrared main absorption peak and a main emission peak; the absorption band of the near infrared carbon point is 600-800 nm, and the near infrared emission peak is 700-850 nm; the preparation method of the near infrared carbon dots comprises the following steps: the conjugated molecule and the cosolvent with amide groups are fused under the action of solvothermal.
2. The near infrared carbon dot of claim 1, wherein the conjugated molecule is selected from at least one of biphenyl and biphenyl, a polybiphenyl aliphatic hydrocarbon, and a polycyclic aromatic hydrocarbon.
3. The near infrared carbon dot of claim 1, wherein the conjugated molecule is selected from at least one of perylene, pyrene, indene, fluorene, acenaphthylene, naphthalene, acenaphthylene, anthracene, butyne, pentylene, and phenanthrene and derivatives thereof.
4. The near infrared carbon dot of claim 1, wherein the conjugated molecule is 3,4,9, 10-perylene tetracarboxylic dianhydride.
5. The near infrared carbon dot of claim 1, wherein the surface of the carbon dot is further modified with a molecular chain of a water-soluble polymer.
6. The near infrared carbon dot of claim 5, wherein the water-soluble polymer molecular chain is polyethyleneimine.
7. The near-infrared carbon dot of claim 1, wherein when the near-infrared carbon dot is not modified with a water-soluble polymer molecular chain, the aqueous solution of the near-infrared carbon dot has an absorption band at 600-800 nm, an absorption peak at 720nm, and an excitation independent emission peak at 745nm under excitation at 680-730 nm; when the near-infrared carbon points are modified with water-soluble polymer molecular chains, the aqueous solution of the near-infrared carbon points has an absorption band at 600-800 nm, and the absorption peak is 726nm; under excitation of 680nm-730nm, the DMF solution of the near-infrared carbon point has an absorption band at 600-800 nm, and has an excitation independent emission peak at 765nm under excitation of 680nm-730nm, wherein the absorption band is 742nm.
8. The method for preparing the near infrared carbon dot according to any one of claims 1 to 7, which is characterized by comprising the following steps: the conjugated molecule and the cosolvent with amide groups are fused under the action of solvothermal.
9. The method for preparing near infrared carbon dots according to claim 8, comprising: after dispersing the conjugated molecule and the cosolvent with amide groups in a polar solvent, performing a first solvothermal reaction.
10. The method for preparing near infrared carbon dots according to claim 9, wherein the mass ratio of the conjugated molecule to the cosolvent is 0.05-1: 0.2 to 4.
11. The method for preparing near infrared carbon dots according to claim 9, wherein the polar solvent is a polar aprotic solvent including at least one of N, N-dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone.
12. The method for preparing near infrared carbon dots according to claim 9, wherein the temperature of the first solvothermal reaction is 140-200 ℃ and the reaction time is 3-24 hours.
13. The method for preparing near infrared carbon dots according to claim 9, wherein after the first solvothermal reaction is completed, the reaction solution is centrifuged, and the supernatant is diluted with deionized water and subjected to first dialysis purification.
14. The method for preparing near infrared carbon dots according to claim 13, wherein the rotational speed of centrifugation is 3000-15000 r/min and the centrifugation time is 5-30 min.
15. The method for preparing near infrared carbon dots according to claim 13, wherein the dialysis bag used for the first dialysis purification has a molecular weight cut-off of 1000-5000 da and a dialysis time of 12-24 hours.
16. The method for preparing near infrared carbon dots according to claim 9, further comprising: and mixing the product obtained by the first solvothermal reaction with a water-soluble polymer molecular chain, and performing a second solvothermal reaction.
17. The method for preparing near infrared carbon dots according to claim 16, wherein the mass ratio of the water-soluble polymer molecular chain to the conjugated molecule is 0.2 to 1:0.05-1.
18. The method for preparing near infrared carbon dots according to claim 16, wherein the temperature of the second solvothermal reaction is 140-200 ℃ and the reaction time is 3-24 h.
19. The method for preparing near infrared carbon dots according to claim 16, wherein the product obtained by the second solvothermal reaction is diluted with deionized water and then subjected to second dialysis and purification, and a dialysis bag used for the second dialysis and purification has a molecular weight cutoff of 5000-20000 da; the dialysis time is 12-48 h.
20. The use of the near infrared carbon dot of any one of claims 1-7 in near infrared encryption, near infrared anti-counterfeit, dye or biological imaging, wherein the use is not for the diagnosis and treatment of disease.
21. A fluorescence imaging agent, comprising the near infrared carbon dot of any one of claims 1 to 7.
22. Use of the fluorescent imaging agent of claim 21 in vivo single-photon or two-photon near infrared fluorescent imaging, and said use is not for the diagnosis and treatment of diseases.
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