CN114956049B - Long wavelength ratio fluorescent carbon dot and preparation method and application thereof - Google Patents
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Abstract
The invention provides a long wavelength ratio fluorescent carbon dot, a preparation method and application thereof. The carbon dot preparation step comprises the following steps: the neutral red, aloe and secondary water are put into a hydrothermal reaction kettle together for hydrothermal reaction; centrifuging and filtering the obtained product to remove insoluble substances, dialyzing with a dialysis bag to remove impurities, and freeze-drying to obtain the ratio fluorescent carbon dots. The preparation method is simple and low in cost, and the prepared ratio fluorescent carbon dots can be used as ratio fluorescent sensors for continuously detecting Hg in aqueous solution, living cells and zebra fish with high selectivity and high sensitivity 2+ And GSH, also can be used as an intensity sensor to detect pH in aqueous solutions, living cells, and in zebra fish.
Description
Technical Field
The invention relates to preparation and application of fluorescent nano materials, in particular to a ratio fluorescent carbon dot and a preparation method and application thereof.
Background
The carbon dots being quasi-spherical particles of a size less than 10nm, generally in sp 2 The conjugated carbon of (2) is a carbon core, and a large number of oxygen-containing functional groups such as carboxyl, hydroxyl or aldehyde groups are enriched on the surface of the carbon core. In 2004, xu et al have found carbon dots by accident during the electrophoretic separation of arc-discharged soot (X.Y.Xu, R.Ray, Y.L.Gu, et al, electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments, j.am. Chem. Soc.,2004,126,12736-12737.). As a novel fluorescent material, the carbon dot has the advantages of simple synthesis, low raw material cost, good biocompatibility and the like, and is widely applied to fluorescent sensing and livingThe carbon dots are also a powerful competitor to those currently used for heavy metal semiconductor quantum dots in the fields of imaging, nanomedicine, photoelectrocatalysis, energy conversion and storage, and the like.
The preparation method of the carbon nano-dots is various and can be roughly classified into a laser graphite stripping method, an electrochemical oxidation method, an arc discharge method, a microwave synthesis method, a chemical oxidation method, a pyrolysis method and the like, and main carbon sources include graphene, carbon nano-tubes, activated carbon, small molecular organic compounds and the like.
Currently, most carbon dots are single fluorescent. From the perspective of sensing applications, the development of well-resolved bifluorescent carbon dots remains highly appreciated because such carbon dots can be designed as ratiometric fluorescent probes using the ratio of the intensities of two well-resolved fluorescent peaks. Compared with a single fluorescence intensity probe, the ratio fluorescence probe measures the fluorescence intensity of the probe material at two different wavelengths, and takes the ratio as a signal parameter to determine the target. The fluorescence ratio signal is not influenced by the fluctuation of the light source intensity and the sensitivity of the instrument, so that the ratio fluorescence probe can improve the accuracy, the sensitivity and the dynamic response range of an analysis method.
Currently, carbon spot sensors for ratiometric fluorescence have been reported. Literature (Y.F.Gao, Y.Jiao, H.L.Zang, et al, one-step synthesis of a dual-emitting carbon dot-based ratiometric fluorescent probe for the visual assay of Pb) 2+ and PPi and development of a paper sensor, J.Mater.chem.B,2019,7,5502-5509.) for detecting Pb 2+ And PPi; document (Z.Han, D.Y.Nan, H.Yang, et al carbon quantum dots based ratiometric fluorescence probe for sensitive and selective detection of Cu 2+ and glutethione, sens. Operators bchem 2019,298,126842.) Cu was synthesized for ratio detection using o-phenylenediamine and citric acid as raw materials 2+ Is a ratio of fluorescent carbon dots; the ratio fluorescent carbon dots synthesized in literature (W.Song, W.X.Duan, Y.H.Liu, et al, ratio Detection ofIntracellular Lysine and pH with One-Pot Synthesized Dual Emissive Carbon dots, animal, chem.) were used for lysine and pH detection. Biological fluorescence imaging collarDomain, long wavelength ratio fluorescent carbon dots are more advantageous. To our knowledge, hg was detected based on the ratio fluorescent carbon dots of both red and yellow colors 2+ And GSH sensors have not been reported. Thus, search for detection of Hg 2+ And the study of the long wavelength ratio fluorescent carbon dots of GSH. pH homeostasis is critical in biological systems, but short wavelength probes have great limitations in pH sensing in biological systems due to the presence of autofluorescence. Therefore, it is significant to develop a pH probe of long wavelength and apply to biological imaging.
Disclosure of Invention
The invention aims to provide a long-wavelength ratio fluorescent carbon dot and a preparation method thereof, the method has the advantages of convenient and easily obtained raw materials and low preparation condition requirements, and the prepared ratio fluorescent carbon dot has high stability, good water solubility, low toxicity and good biocompatibility, and can be used as a ratio fluorescent sensor for detecting Hg in aqueous solution, living cells and zebra fish 2+ And GSH, also can be used as an intensity sensor to detect pH in aqueous solutions, living cells, and in zebra fish.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a method for preparing a long wavelength ratio fluorescent carbon dot, comprising the steps of:
(1) Adding neutral red and aloe into secondary water according to the mass ratio of 1:20-180:500-1500 to prepare mixed solution;
(2) Transferring the mixed solution obtained in the step (1) into a hydrothermal reaction kettle for hydrothermal reaction, wherein the temperature of the hydrothermal reaction is 80-180 ℃ and the time is 0.25-4 h;
(3) Centrifuging and filtering the product obtained in the step (2) to remove insoluble substances, and dialyzing for 12-24 hours by using a dialysis bag with the molecular weight cutoff of 500-1000 Da to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the ratio fluorescent carbon dot solution obtained in the step (3) to obtain target ratio fluorescent carbon dots.
In the step (1), the mass ratio of neutral red to aloe to secondary water is 1:20-160:500-1500.
The temperature of the hydrothermal reaction in the step (2) is 100-180 ℃ and the time is 0.5-4 h.
The ratio fluorescent carbon dots prepared by the method can be used as ratio fluorescent sensors for detecting Hg in aqueous solution, living cells and zebra fish 2+ And GSH, also can be used as an intensity sensor to detect pH in aqueous solutions, living cells, and in zebra fish.
Compared with the prior art, the invention has the advantages that:
(1) The ratio fluorescent carbon dots prepared by the invention have good luminous performance and can be used as ratio fluorescent sensors for detecting Hg 2+ And GSH, system error and background error of the instrument caused by external unstable factors are eliminated, so that the detection accuracy is greatly improved.
(2) The ratio fluorescent carbon dots prepared by the invention can be used as an intensity sensor for detecting pH, and can be used for detecting Hg due to the pH and Hg 2+ Different from GSH detection method, the two detection methods are not interfered with each other, thus realizing dual-mode detection.
(3) The ratio fluorescent carbon dots prepared by the invention have high stability, small toxic and side effects, good water solubility and biocompatibility, and the ratio contrast colors are yellow and red with long wavelength, so the ratio fluorescent carbon dots have wide application prospects in the fields of biosensing, cell imaging and the like.
Drawings
FIG. 1 is a transmission electron micrograph and a size distribution diagram of a ratiometric fluorescent carbon spot prepared in example 1
FIG. 2 is an infrared spectrum of the ratiometric fluorescent carbon dots prepared in example 1
FIG. 3 is an X-ray photoelectron spectrum of a ratio fluorescent carbon dot prepared in example 1
FIG. 4 is a graph showing the ultraviolet absorption spectrum of the ratiometric fluorescent carbon dots prepared in example 1
FIG. 5 is a graph showing fluorescence emission spectra of the ratiometric fluorescent carbon dots prepared in example 1 at different excitation wavelengths
FIG. 6 is a graph of Hg in Hg for a ratiometric fluorescent carbon dot solution prepared in example 1 2+ Fluorescence emission spectrum of concentration variation
FIG. 7 is a graph of the ratio of fluorescent carbon dots to Hg prepared in example 1 2+ Fluorescence of the mixed solution of (2) with GSH concentrationEmission spectrum graph
FIG. 8 is a ratio fluorescent carbon dot solution, ratio fluorescent carbon dots and Hg prepared in example 1 2+ Mixed liquor and ratio of fluorescent carbon dots to Hg 2+ Fluorescence pictures of mixed liquid (left, middle and right in sequence) of GSH under excitation of 400nm
FIG. 9 is a semi-quantitative Hg assay for HeLa cells labeled with ratiometric fluorescent carbon dots prepared in example 1 2+ Laser confocal map of GSH
FIG. 10 is a semi-quantitative Hg detection for a ratiometric fluorescent carbon dot labeled zebra fish prepared in example 1 2+ Laser confocal map of GSH
FIG. 11 is a fluorescence emission spectrum of the ratiometric fluorescent carbon dot solution prepared in example 1 at different pH values
FIG. 12 is a laser confocal plot of pH measurements of ratiometric fluorescent carbon dot labeled HeLa cells prepared in example 1
FIG. 13 is a laser confocal plot of the pH detected by the ratiometric fluorescent carbon dot labeled zebra fish prepared in example 1
Detailed Description
The following examples further illustrate the invention, but the invention is not limited to these examples.
Example 1
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.02g neutral red and 1.6g aloe into 20mL secondary water to obtain mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 2 hours at 180 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 18h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
The transmission electron microscope and the size distribution diagram of the prepared ratio fluorescent carbon dots are shown as A and B in FIG. 1.
The prepared infrared spectrogram of the ratio fluorescent carbon dots is shown in figure 2.
The prepared X-ray photoelectron spectrum of the ratio fluorescent carbon point is shown in figure 3.
The ultraviolet absorption spectrum of the prepared ratio fluorescent carbon dots is shown in fig. 4.
The prepared ratio fluorescent carbon dots have fluorescent emission spectra under different excitation wavelengths shown in figure 5, wherein 1-9 are respectively the fluorescent spectra under excitation of excitation wavelengths of 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 420nm, 460nm and 470 nm.
Example 2
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.02g neutral red and 1.6g aloe into 20mL secondary water to obtain mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 1h at 120 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 12h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
Example 3
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.02g neutral red and 3.2g aloe into 20mL secondary water to obtain mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 2.5h at 160 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 18h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
Example 4
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.02g neutral red and 1.6g aloe into 30mL secondary water to obtain mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 3 hours at 180 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 24h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
Example 5
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.02g neutral red and 0.8g aloe into 10mL secondary water to obtain mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 2 hours at 180 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 12h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
Example 6
Preparation of ratio fluorescent carbon dots:
(1) Dispersing 0.04g neutral red and 1.6g aloe into 30mL secondary water to obtain a mixed solution;
(2) Placing the mixed solution obtained in the step (1) into a hydrothermal kettle, and carrying out hydrothermal reaction for 2 hours at 160 ℃;
(3) Centrifuging the product obtained in the step (2) at a rotating speed of 3000r/min for 20min by using a centrifuge to remove insoluble matters to obtain clear yellow solution, and dialyzing for 18h by using a dialysis bag with a molecular weight cutoff of 500-1000 Da to remove impurities to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the solution of the ratio fluorescent carbon dots obtained in the step (3) to obtain the ratio fluorescent carbon dots.
Example 7
Example 1 preparationIs taken as Hg 2+ Sensitivity experiment of the sensor:
with PBS buffer at ph=7.0 and HgCl 2 Respectively preparing Hg 2+ The concentration is 15 mu mol.L -1 、30μmol·L -1 、45μmol·L -1 、60μmol·L -1 、75μmol·L -1 、90μmol·L -1 、105μmol·L -1 And 120. Mu. Mol.L -1 1.05mg of the ratio fluorescent carbon dots prepared in example 1 were dissolved respectively into 1mL of the above solution containing Hg of different concentrations 2+ In the solution (C), the fixed excitation wavelength was 400nm, fluorescence spectrum detection was performed at room temperature, and the ratio of the intensity at 616nm to the intensity at 550nm was calculated (I 616 /I 550 ) Thereby to Hg 2+ And (5) detecting.
Ratio fluorescent carbon dot solution with Hg 2+ The fluorescence emission spectrum of the concentration variation is shown in FIG. 6, wherein 1 to 9 are Hg respectively 2+ The concentration is 0 mu mol.L -1 、15μmol·L -1 、30μmol·L -1 、45μmol·L -1 、60μmol·L -1 、75μmol·L -1 、90μmol·L -1 、105μmol·L -1 And 120. Mu. Mol.L -1 A fluorescence emission spectrum of the ratio fluorescent carbon dot solution; from the figure it can be seen that with Hg 2+ The increase in concentration gradually decreased the fluorescence intensity at 616nm and gradually increased the fluorescence intensity at 550 nm.
Example 8
The ratio of fluorescent carbon dots to Hg prepared in example 1 2+ Is a mixed solution (carbon concentration 1.05 g.L) -1 ，Hg 2+ The concentration is 120 mu mol.L -1 ) Sensitivity experiments for GSH sensors:
adding GSH of different masses to dissolved ratio fluorescent carbon dots and Hg 2+ In the mixed solution of (2), GSH concentration was set to 40. Mu. Mol.L -1 、80μmol·L -1 、120μmol·L -1 、160μmol·L -1 、200μmol·L -1 And 240. Mu. Mol.L -1 The excitation wavelength was fixed at 400nm, and fluorescence spectrum detection was performed at room temperature.
Ratio of fluorescent carbon dots to Hg 2+ Along with the mixed solution GThe fluorescence emission spectrum of SH concentration variation is shown in FIG. 7, wherein 1-7 are GSH concentration of 0. Mu. Mol.L -1 、40μmol·L -1 、80μmol·L -1 、120μmol·L -1 、160μmol·L -1 、200μmol·L -1 And 240. Mu. Mol.L -1 Time ratio fluorescent carbon dots and Hg 2+ Fluorescence emission spectrum of the mixed solution of (2); it can be seen from the graph that as the GSH concentration increases, the fluorescence intensity at 550nm gradually decreases, while the fluorescence intensity at 616nm gradually increases.
Example 9
As shown in FIG. 8, the ratio fluorescent carbon dot solution prepared in example 1 was placed in a cuvette, and the ratio fluorescent carbon dot solution was red fluorescent (left) under excitation at 400nm, hg was added 2+ (concentration 120. Mu. Mol. L) -1 ) After that, the fluorescent material turns into yellow-green fluorescence (medium), and GSH (concentration of 240 mu mol.L) is added -1 ) After that, the red fluorescence is recovered again (right).
Example 10
Semi-quantitative detection of Hg in cell imaging by ratiometric fluorescent carbon dots prepared in example 1 2+ And GSH aspect:
the ratio fluorescent carbon dots prepared in example 1 were used to label HeLa cells, see fig. 9. The excitation wavelength is 405nm, and the emission wavelength is set to 400nm to 575nm (channel 1) and 575nm to 760nm (channel 2). FIG. 9A shows a cell plot of the ratiometric fluorescent carbon dot label, showing yellowish fluorescence at 400nm to 575nm, and bright red fluorescence at 575nm to 760 nm. With all of the above settings unchanged, in the case of Hg addition 2+ After that, the yellow fluorescence at 400 nm-575 nm is gradually increased, the red fluorescence at 575 nm-760 nm is obviously weakened (as shown in figure 9B), GSH is continuously added, the yellow fluorescence at 400 nm-575 nm is weakened, and the red fluorescence at 575 nm-760 nm is weakened (as shown in figure 9C).
Example 11
Semi-quantitative detection of Hg in zebra fish imaging by using ratio fluorescent carbon dots prepared in example 1 2+ And GSH aspect:
the ratiometric fluorescent carbon dots prepared in example 1 were used to label zebra fish, see fig. 10. The excitation wavelength was 405nm, and the emission wavelength was set to400nm to 575nm (channel 1) and 575nm to 760nm (channel 2). FIG. 10A shows a zebra fish plot labeled with ratiometric fluorescent carbon dots, showing yellowish fluorescence at 400 nm-575 nm, and bright red fluorescence at 575 nm-760 nm. With all of the above settings unchanged, in the case of Hg addition 2+ After that, the yellow fluorescence at 400nm to 575nm is gradually increased, while the red fluorescence at 575nm to 760nm is obviously weakened (as shown in fig. 10B), and the yellow fluorescence at 400nm to 575nm is weakened while the red fluorescence at 575nm to 760nm is obviously increased (as shown in fig. 10C) by continuously adding GSH.
Example 12
The ratio fluorescent carbon dots prepared in example 1 were tested for pH in aqueous solution:
the ratio fluorescent carbon dots prepared in example 1 were used to detect pH values in aqueous solution and recorded under excitation at 400nm, see fig. 11, where 1-8 are the fluorescence spectra of the ratio fluorescent carbon dots in PBS buffer at ph=3, ph=4, ph=5, ph=6, ph=7, ph=8, ph=9 and ph=10, respectively, with the fluorescence intensity of the carbon dot solution increasing as the pH value increases from 3 to 10.
Example 13
Application experiment of the ratiometric fluorescent carbon dots prepared in example 1 in detecting cell pH:
the ratio fluorescent carbon dots prepared in example 1 were used for HeLa cell pH imaging, see fig. 12. The excitation wavelength is 405nm, and the emission wavelength is set to 400nm to 575nm (channel 1) and 575nm to 760nm (channel 2). Fig. 12A-D show cell plots of ratio fluorescent carbon spot incubations in PBS buffer at ph=6, ph=7, ph=8, and ph=9, respectively, with increasing pH, both yellow fluorescence at 400nm to 575nm and red fluorescence at 575nm to 760nm increasing.
Example 14
Application experiment of the ratiometric fluorescent carbon dots prepared in example 1 in zebra fish pH imaging:
the ratio fluorescent carbon dots prepared in example 1 were used for zebra fish pH imaging, see fig. 13. The excitation wavelength is 405nm, and the emission wavelength is set to 400nm to 575nm (channel 1) and 575nm to 760nm (channel 2). Figures 13A-D show zebra fish plots of ratiometric fluorescent carbon spot incubations in PBS buffer at ph=6, ph=7, ph=8, and ph=9, with increasing pH, both yellow fluorescence at 400nm to 575nm and red fluorescence at 575nm to 760 nm.
Claims (8)
1. The long wavelength ratio fluorescent carbon dot is used as ratio fluorescent sensor in detecting Hg in water solution 2+ And the use of GSH; the preparation method of the long wavelength ratio fluorescent carbon dot comprises the following steps:
(1) Adding neutral red and aloe into secondary water according to the mass ratio of 1:20-180:500-1500 to prepare a mixed solution;
(2) Transferring the mixed solution obtained in the step (1) into a hydrothermal reaction kettle for hydrothermal reaction, wherein the temperature of the hydrothermal reaction is 80-180 ℃ and the time is 0.25-4 h;
(3) Centrifuging and filtering the product obtained in the step (2) to remove insoluble substances, and dialyzing for 12-24 hours by using a dialysis bag with the molecular weight cutoff of 500-1000 Da to obtain a ratio fluorescent carbon dot solution;
(4) And (3) freeze-drying the ratio fluorescent carbon dot solution obtained in the step (3) to obtain target ratio fluorescent carbon dots.
2. The use of a long wavelength ratio fluorescent carbon spot according to claim 1 in the preparation of a fluorescent probe for detection of Hg in living cells 2+ And the use of GSH ratio fluorescence sensors.
3. The use of a long wavelength ratio fluorescent carbon spot as claimed in claim 1 in the preparation of a fluorescent probe for detection of Hg in vivo 2+ And the use of GSH ratio fluorescence sensors.
4. The use of a long wavelength ratio fluorescent carbon dot as claimed in claim 1 as an intensity fluorescent sensor for detecting pH in solution.
5. The use of a long wavelength ratio fluorescent carbon dot as claimed in claim 1 in the preparation of an intensity fluorescent sensor for detecting pH in living cells.
6. The use of a long wavelength ratio fluorescent carbon dot as claimed in claim 1 in the preparation of an intensity fluorescent sensor for detecting pH in a living body.
7. The use according to any one of claims 1 to 6, wherein the mass ratio of neutral red, aloe and secondary water in step (1) is 1:20 to 160:500 to 1500.
8. The use according to any one of claims 1 to 6, wherein the hydrothermal reaction in step (2) is carried out at a temperature of 100 to 180 ℃ for a time of 0.5 to 4 hours.
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