CN116285969A - Super-bright green carbon quantum dot fluorescent probe, preparation method thereof and application thereof in DNA imaging - Google Patents
Super-bright green carbon quantum dot fluorescent probe, preparation method thereof and application thereof in DNA imaging Download PDFInfo
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/0883—Arsenides; Nitrides; Phosphides
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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Abstract
The invention discloses an ultra-bright green carbon quantum dot fluorescent probe, a preparation method thereof and application thereof in DNA imaging. Adding dilute hydrochloric acid into aqueous solution of m-phenylenediamine, performing hydrothermal reaction to obtain green solution containing carbon dots, and purifying by adopting a column chromatography method to obtain the ultra-bright green carbon quantum dot fluorescent probe. The green fluorescent carbon dot prepared by the invention has the advantages of good water solubility, good dispersibility, high biocompatibility and the like, the yield is up to 31%, and the absolute quantum efficiency of green fluorescence is up to 64.20%. In addition, when the carbon dot is used as a fluorescent probe, the fluorescent probe has the targeting fluorescent imaging capability on HeLa cell nuclei at the end of mitosis, can specifically image DNA, and has the application potential of monitoring cell separation cycle.
Description
Technical Field
The invention belongs to the field of preparation of nano materials, and relates to an ultra-bright green carbon quantum dot fluorescent probe, a preparation method thereof and application thereof in DNA imaging.
Background
The carbon quantum dots (hereinafter referred to as carbon dots) are novel carbon-based nano materials with fluorescence property, have the advantages of high luminous efficiency, adjustable luminous wave band, simple preparation, low price, good biocompatibility and the like, and have been widely paid attention at home and abroad. However, for the preparation of carbon dots with ultra-high quantum efficiency, the existing preparation methods are still lacking. Carbon dots with ultra-high quantum efficiency can be used in bioimaging, light Emitting Diode (LEDs) applications to reduce cytotoxicity by reducing drug delivery concentration, and to highlight LEDs at lower concentrations to avoid the problem of aggregation fluorescence quenching (ACQ) of carbon dots in a dispersion medium due to higher concentrations.
The current preparation method of the carbon dot with ultrahigh quantum efficiency mainly comprises a microwave method, a hydrothermal method and the like. M-phenylenediamine is widely used to synthesize carbon dots with green fluorescence. For example, in document 1 (ACS appl. Nano mate. 2021,4,8,8500-8510), m-phenylenediamine and urea are used as raw materials to prepare carbon dots by a hydrothermal method, but the separation and purification method is too complicated, and a plurality of eluents are needed to be used for eluting in steps to obtain green fluorescent carbon dots. Document 2 (Chinese Chemical Letters (2021) 3927-3930) uses dithiosalicylic acid and metaphenylene diamine to prepare a carbon dot having aggregation-induced fluorescence, but the carbon dot is poorly water-soluble and unsuitable for cell or DNA imaging.
Disclosure of Invention
The invention aims to provide an ultra-bright green carbon quantum dot (G-CDs) fluorescent probe, a preparation method thereof and application thereof in DNA imaging.
The technical scheme for realizing the purpose of the invention is as follows:
the preparation method of the ultra-bright green carbon quantum dot fluorescent probe comprises the following steps:
(1) Preparation of carbon dots: adding 5% diluted hydrochloric acid into m-phenylenediamine aqueous solution according to the volume ratio of water to diluted hydrochloric acid of 8-11:4, then placing the mixed solution into a reaction kettle, performing hydrothermal reaction at 160-200 ℃, and cooling to room temperature after the reaction is finished to obtain a carbon-point-containing green solution;
(2) Purification of carbon dots: purifying the green solution containing carbon dots by adopting a column chromatography, sequentially carrying out gradient elution by using DCM/methanol mixed eluents with the volume ratio of DCM to methanol of 10:1, 8:1 and 5:1, collecting the eluted solution, and drying to obtain the ultra-bright green carbon quantum dot fluorescent probe.
Preferably, in the step (1), the volume ratio of water to dilute hydrochloric acid is 8:4.
Preferably, in the step (1), the proportion relation of the m-phenylenediamine, the water and the dilute hydrochloric acid is 216:18-22:8-12, and the ratio of mg to mL is that of mL.
Preferably, in step (1), the hydrothermal reaction temperature is 180 ℃.
Preferably, in step (1), the hydrothermal reaction time is 2 hours.
Preferably, in the step (2), the elution time of the DCM/methanol mixed eluent with the volume ratio of DCM to methanol being 10:1 is 15 minutes, the elution time of the DCM/methanol mixed eluent with the volume ratio of DCM to methanol being 8:1 is 10 minutes, and the elution time of the DCM/methanol mixed eluent with the volume ratio of DCM to methanol being 5:1 is 10 minutes.
Compared with the prior art, the invention has the following advantages:
(1) The preparation method is simple, only m-phenylenediamine is used as a carbon source, a proper amount of diluted hydrochloric acid is added, a hydrothermal method is adopted to synthesize the green fluorescent carbon dots, a column chromatography is adopted to purify the green fluorescent carbon dots, and a specific elution mode and eluent are selected.
(2) The green fluorescent carbon dot prepared by the invention has the advantages of good water solubility, good dispersibility, high biocompatibility and the like, the yield is up to 31%, and the absolute quantum efficiency of green fluorescence is up to 64.20%. In addition, when the carbon dot is used as a fluorescent probe, the fluorescent probe has the targeting fluorescent imaging capability on HeLa cell nuclei at the end of mitosis, can specifically image DNA, and has the application potential of monitoring cell separation cycle.
Drawings
FIG. 1 is a graph of a G-CDs transmission electron microscope and a high resolution transmission electron microscope prepared in example 1.
FIG. 2 is an absorption spectrum of G-CDs prepared in example 1.
FIG. 3 is a fluorescence spectrum of G-CDss prepared in example 1.
FIG. 4 is a three-dimensional fluorescence spectrum of G-CDs prepared in example 1.
FIG. 5 is an absolute quantum efficiency plot of G-CDs prepared in example 1.
FIG. 6 is an X-ray photoelectron spectrum of G-CDs prepared in example 1.
FIG. 7 is a graph showing the cytotoxicity test results of G-CDs prepared in example 1.
FIG. 8 is a graph of nuclear-targeted imaging of HeLa cells by G-CDs prepared in example 1.
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings.
Example 1
216mg of m-phenylenediamine was dissolved in 20mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 10mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 180 ℃ and kept stand for 2 hours, and when the autoclave is cooled to room temperature, the green solution containing G-CDs is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and bright green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The yield of the prepared G-CDs is up to 31%, and the fluorescence quantum efficiency is up to 64.2%.
Example 2
216mg of m-phenylenediamine was dissolved in 20mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 10mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The yield of the prepared G-CDs was 27.6%, and the fluorescence quantum efficiency was 62.5%.
Example 3
216mg of m-phenylenediamine was dissolved in 20mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 10mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 200 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The yield of the prepared G-CDs was 29.1%, and the fluorescence quantum efficiency was 60.3%.
Example 4
216mg of m-phenylenediamine was dissolved in 18mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 12mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The yield of the prepared G-CDs was 26.9%, and the fluorescence quantum efficiency was 60.4%.
Example 5
216mg of m-phenylenediamine was dissolved in 22mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. Then 8mL of diluted hydrochloric acid (5%) was added and the mixed solution was transferred to a 50mL teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The yield of the prepared G-CDs was 26.5%, and the fluorescence quantum efficiency was 59.8%.
Comparative example 1
216mg of m-phenylenediamine was dissolved in 27mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. Then 3mL of diluted hydrochloric acid (5%) was added and the mixed solution was transferred to a 50mL teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by sequentially using DCM and methanol mixed eluents with the volume ratio of 10:1, 8:1 and 5:1, the elution time is sequentially 15min, 10min and 10min, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product G-CDs. The fluorescence quantum efficiency of the carbon dots prepared under the conditions is low, about 35%.
Comparative example 2
216mg of m-phenylenediamine was dissolved in 20mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 10mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by dialysis (molecular weight cut-off 3000). Transferring the solution in the dialysis bag to a rotary evaporator for drying to obtain the product carbon dot powder. The carbon dot prepared by the method has poor uniformity of particle size, and the emitted fluorescence spectrum is wider than pure green fluorescence.
Comparative example 3
216mg of m-phenylenediamine was dissolved in 30mL of deionized water. The solution was transferred to a 50mL teflon lined stainless steel autoclave by sonication for 10 minutes. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. The solution was purified by column chromatography. Gradient elution is carried out by using DCM and methanol mixed eluent with the volume ratio of DCM to methanol of 10:1, 8:1 and 5:1, and the green solution is collected and transferred to a rotary evaporator for drying, thus obtaining the product carbon dot powder. The carbon dot yield prepared under the condition is low, and is only about 10%.
Comparative example 4
216mg of m-phenylenediamine was dissolved in 20mL of deionized water. Ultrasound for 10 minutes, the mixture became clear. 10mL of diluted hydrochloric acid (5%) was then added and the mixed solution was transferred to a 50mL Teflon lined stainless steel autoclave. Then the electric heating blow-drying box is placed at 160 ℃, and kept stand for 2 hours, and when the autoclave is cooled to room temperature, a carbon dot solution is obtained. Purifying the solution by column chromatography, eluting with DCM/methanol mixed solution with volume ratio of 10:1, transferring the solution to rotary evaporator, and drying to obtain carbon dot powder. The purity of the carbon dots prepared under the conditions is low, resulting in a reduction of the quantum efficiency of green fluorescence, about 42%.
FIG. 1 is a Transmission Electron Microscope (TEM) photograph and a high-resolution transmission electron microscope (HRTEM) photograph of G-CDs prepared in example 1. As can be seen from FIG. 1, the G-CDs are spherical, have very good dispersibility in water, have uniform particle sizes, have a clear lattice structure, and have a single size of about 5nm.
FIG. 2 is a graph of the ultraviolet-visible absorption spectrum of G-CDs prepared in example 1, from which it can be seen that G-CDs have a distinct absorption band at 263nm and a broad absorption peak at 446nm caused by n-pi transitions.
FIG. 3 is a fluorescence spectrum of G-CDs prepared in example 1, and it can be seen from the graph that the G-CDs have an optimal emission peak at 510nm and an excitation peak at 450nm.
FIG. 4 is a three-dimensional fluorescence spectrum of G-CDs prepared in example 1, showing that the fluorescence emission peak of G-CDs is independent of the wavelength band of excitation light.
FIG. 5 is a graph for testing the absolute quantum efficiency of G-CDs prepared in example 1, which shows that the absolute quantum efficiency of G-CDs reaches 64.20%, and the G-CDs has good application potential.
FIG. 6 is an X-ray photoelectron spectrum of G-CDs prepared in example 1, which was found to be composed of three elements, namely, carbon element, oxygen element and nitrogen element, in proportions of 86.42%,6.24% and 7.34%, respectively.
Cytotoxicity of the G-CDs prepared in example 1 was evaluated by WST-1 cell proliferation and cytotoxicity assay kit, and the results are shown in FIG. 7. When G-CDs are added at a concentration of 0 to 500. Mu.g/mL, the cell viability is 95% or more. The toxicity of the G-CDs is lower, and the biocompatibility is good.
FIG. 8 is a confocal image of the imaging of HeLa cells by G-CDs prepared in example 1, where the co-incubation of HeLa cells with DAPI and G-CDs (100. Mu.g/mL), respectively, shows that G-CDs have relatively high targeting to HeLa cell nuclei and that Merged images show mostly complete coincidence of G-CDs fluorescence imaging and DAPI fluorescence imaging. And as can be seen in the bright field plot, heLa cells are now at the end of mitosis, so G-CDs possess the potential to monitor the cell division cycle.
Claims (8)
1. The preparation method of the ultra-bright green carbon quantum dot fluorescent probe is characterized by comprising the following steps of:
(1) Preparation of carbon dots: adding 5% diluted hydrochloric acid into m-phenylenediamine aqueous solution according to the volume ratio of water to diluted hydrochloric acid of 8-11:4, then placing the mixed solution into a reaction kettle, performing hydrothermal reaction at 160-200 ℃, and cooling to room temperature after the reaction is finished to obtain a carbon-point-containing green solution;
(2) Purification of carbon dots: purifying the green solution containing carbon dots by adopting a column chromatography, sequentially carrying out gradient elution by using DCM/methanol mixed eluents with the volume ratio of DCM to methanol of 10:1, 8:1 and 5:1, collecting the eluted solution, and drying to obtain the ultra-bright green carbon quantum dot fluorescent probe.
2. The process of claim 1, wherein in step (1), the volume ratio of water to dilute hydrochloric acid is 8:4.
3. The method according to claim 1, wherein in the step (1), the ratio of m-phenylenediamine, water and dilute hydrochloric acid is 216:18 to 22:8 to 12, mg: mL.
4. The process of claim 1, wherein in step (1), the hydrothermal reaction temperature is 180 ℃.
5. The process of claim 1, wherein the hydrothermal reaction time in step (1) is 2 hours.
6. The method according to claim 1, wherein in the step (2), the elution time of the DCM/methanol mixed eluent having a volume ratio of DCM to methanol of 10:1 is 15 minutes, the elution time of the DCM/methanol mixed eluent having a volume ratio of DCM to methanol of 8:1 is 10 minutes, and the elution time of the DCM/methanol mixed eluent having a volume ratio of DCM to methanol of 5:1 is 10 minutes.
7. The ultra-bright green carbon quantum dot fluorescent probe prepared by the preparation method according to any one of claims 1 to 6.
8. The use of the ultra-bright green carbon quantum dot fluorescent probe according to claim 7 in DNA imaging for non-disease diagnosis.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108516533A (en) * | 2018-04-04 | 2018-09-11 | 桂林理工大学 | A kind of preparation method of hair peak green fluorescent carbon point |
| CN109021971A (en) * | 2018-08-29 | 2018-12-18 | 郑州大学 | A kind of nuclear targeting fluorescent carbon point and its application and method in nuclei images |
| CN109321240A (en) * | 2018-11-23 | 2019-02-12 | 江南大学 | A kind of fluorescent orange carbon dots and preparation method thereof |
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| CN115125000A (en) * | 2022-07-06 | 2022-09-30 | 重庆国科医创科技发展有限公司 | Preparation method of red light emission carbon quantum dot and application of carbon quantum dot |
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| CN108516533A (en) * | 2018-04-04 | 2018-09-11 | 桂林理工大学 | A kind of preparation method of hair peak green fluorescent carbon point |
| CN109021971A (en) * | 2018-08-29 | 2018-12-18 | 郑州大学 | A kind of nuclear targeting fluorescent carbon point and its application and method in nuclei images |
| CN109321240A (en) * | 2018-11-23 | 2019-02-12 | 江南大学 | A kind of fluorescent orange carbon dots and preparation method thereof |
| CN114479844A (en) * | 2022-01-07 | 2022-05-13 | 中国科学院苏州生物医学工程技术研究所 | Fluorescent carbon quantum dot composite material, and preparation method and application thereof |
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| CN115125000A (en) * | 2022-07-06 | 2022-09-30 | 重庆国科医创科技发展有限公司 | Preparation method of red light emission carbon quantum dot and application of carbon quantum dot |
| CN115074122A (en) * | 2022-07-13 | 2022-09-20 | 郑州中科生物医学工程技术研究院 | Red fluorescent carbon dot and preparation method and application thereof |
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