CN114540021A - Fluorescent carbon dots mPD-CDs and preparation method and application thereof - Google Patents
Fluorescent carbon dots mPD-CDs and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses fluorescent carbon dots mPD-CDs and a preparation method and application thereof, wherein the fluorescent carbon dots mPD-CDs are prepared by one-step hydrothermal carbonization of m-phenylenediamine (mPD). The fluorescent carbon dots can be successfully used for wash-free multicolor cell imaging of animal cells, fungi and bacteria, and have high photostability and biocompatibility. Meanwhile, the fluorescent carbon dots can realize rapid fluorescence detection of various ions in the environment or in the organism. In addition, due to the characteristic of multicolor imaging of the fluorescent carbon dots mPD-CD, the fluorescent carbon dots can be used together with other fluorescent dyes with different excitation wavelengths, so that the application range is further widened.
Description
Technical Field
The invention belongs to the field of nano materials and preparation methods and applications thereof, and particularly relates to fluorescent carbon dots mPD-CDs and a preparation method and application thereof.
Background
Ion is vital to life, in catalysis, osmoregulation, informationThe basic processes of signal conduction, biomineralization and the like play a vital role, and some ions have toxicity and pose serious threats to human health. For example, iron (Fe)3+) Zinc (Zn)2+) Copper (Cu)2+) And iodine (I)-) Is a ubiquitous and indispensable element in all life forms and has important physiological and pathological functions. The abnormal concentration of these ions can lead to various diseases such as anemia, wilson/alzheimer/parkinson disease, hepatitis, thyroid diseases, nerve damage, and even cancer. Gadolinium (Gd)3+) Is a lanthanide element widely used in radiosensitization enhancement and Magnetic Resonance Imaging (MRI) contrast agents (biomaterials.2017,121, 109-120.). However, Gd3+May inhibit calcium channels and induce fibrosis of the renal origin in patients with renal insufficiency. Cadmium (Cd)2+) And mercury (Hg)2+) Has strong toxicity, is widely distributed in the environment, and can carry out biological accumulation through a food chain. Cd even at very low concentrations2+It may also cause various diseases such as cancer, lung injury, renal insufficiency, reproductive deficiency and fracture, and Hg2+Various organs and immune systems of the human body may be damaged (Sensors and activators B: chemical.2017,243, 244-253). Therefore, ion detection in vivo is a central topic of bioanalysis and biomedical science, and has important significance for human health and environmental protection.
Based on this, various ion detection methods such as atomic absorption spectroscopy, atomic fluorescence spectroscopy, hyper-rayleigh scattering, and inductively coupled plasma mass spectrometry have been established. These methods have high accuracy and sensitivity, but require complex sample handling and expensive instrumentation, limiting their use in laboratories and industry. Furthermore, these detection methods cannot detect ions in living cells. Therefore, there is an urgent need to develop new efficient and convenient ion detection strategies, particularly in living cells.
Carbon Dots (CDs) have excellent properties including excellent optical characteristics, easy preparation and functionalization, low toxicity, high biocompatibility, and good water solubility, compared to organic dyes and semiconductor quantum dots. Thus, carbon is spotted on biogenesisAnd the application in image and biological sensing is wide. Efforts have been made to develop fluorescent probes based on carbon dots for ion detection, such as metal ion Cd2+、Cu2+、Hg2+、Zn2+、Fe2+、Fe3+And an anion S2-And I-And the like. Generally, only one ion can be detected by one carbon dot. However, the use of one carbon dot for detection of multiple ions would have the advantages of ease of operation, low cost, and time savings. So far, fluorescent carbon dots for detecting two or more ions have been reported.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a multicolor fluorescent carbon dot with high light stability and high biological safety, a preparation method thereof and application of the multicolor fluorescent carbon dot in multicolor cell imaging and biological multi-ion detection.
The technical scheme is as follows: the fluorescent carbon dots mPD-CDs are prepared from m-phenylenediamine serving as a raw material by a one-step hydrothermal method.
The invention also discloses a preparation method of the fluorescent carbon dots mPD-CDs, which comprises the following steps:
(1) dissolving mPD in deionized water to obtain an mPD solution, transferring the mPD solution into a hydrothermal kettle, and reacting for 6-18h at the temperature of 160-200 ℃;
(2) centrifuging the reaction product obtained in step (1) at 15000rpm for 5-20min, collecting the supernatant, and further filtering with 0.22 μm filter membrane to remove large particles to obtain carbon dots;
(3) the resulting carbon spots were dialyzed in ultrapure water using a 1k molecular weight dialysis bag, freeze-dried and stored at 4 ℃.
Further, in the step (1), mPD is dissolved in deionized water, and the concentration is 1-10 mg/mL; the high-pressure kettle is a high-temperature high-pressure hydrothermal kettle with a polytetrafluoroethylene lining, and the volume of the reaction kettle is 25-100 mL; the reaction temperature is 180 ℃, and the reaction time is 12 h.
The fluorescent carbon dots mPD-CDs are applied to multicolor cell imaging and in vivo multi-ion fluorescence detection.
Further, multicolor imaging that can be used for cells includes red, green, and blue.
Further, the cells include animal cells, bacteria and fungi.
Further, the polyion includes: cd [ Cd ]2+、Cu2+、Hg2+、Gd3+、Fe2+、Zn2+、Fe3+And I-。
Further, the application scenario of fluorescence detection includes: laboratory model animals, animal cells, bacteria and fungi.
Further, the laboratory model animals include zebrafish, mice, fruit flies.
Further, the detection instrument comprises a fluorescence spectrometer, a fluorescence confocal microscope or a flow cytometer.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the fluorescent carbon dots mPD-CDs can be used for washing-free multicolor cell imaging and detection of various ions, and are the first to detect Gd3+And has high stability and high biological activity. The fluorescent carbon dot can be used for ion detection in cells and/or living bodies, and an effective method is provided for detection of various metal ions in the environment or in living bodies. In addition, due to the characteristic of multicolor imaging of the fluorescent carbon dots mPD-CD, the fluorescent carbon dots can be used together with other fluorescent dyes with different excitation wavelengths, so that the application range is further widened.
Drawings
FIG. 1 is a transmission electron microscope image of fluorescent carbon dots mPD-CDs;
FIG. 2 is a particle size distribution diagram of fluorescent carbon dots mPD-CDs;
FIG. 3 is a graph showing the detection result of Zeta potential (Zeta potential) of fluorescent carbon dots mPD-CDs;
FIG. 4 is an evaluation of multicolor cytographic ability of fluorescent carbon dots mPD-CDs;
FIG. 5 is an evaluation of the ion detectability of fluorescent carbon dots mPD-CDs;
FIG. 6 shows the fluorescence intensity of fluorescent carbon dots with Zn2+Fluorescence spectrum and linear range of concentration change of (a);
FIG. 7 shows fluorescence intensity of fluorescent carbon dots with I-Fluorescence spectrum and linear range of concentration change of (a);
FIG. 8 is an evaluation of the intracellular multi-ion detectability of fluorescent carbon spots;
FIG. 9 is an evaluation of the multi-ion detectability of fluorescent carbon spots in zebrafish;
FIG. 10 is a stability evaluation of fluorescent carbon dots;
FIG. 11 is an evaluation of cytotoxicity of fluorescent carbon spots on animal cells.
Detailed Description
The technical scheme of the invention is further explained by combining the attached drawings.
EXAMPLE 1 preparation of fluorescent carbon dots mPD-CDs
M-phenylenediamine was dissolved in deionized water at 3 mg/mL. The solution was transferred to a 25mL hydrothermal reactor and reacted at 180 ℃ for 12 h. After cooling to room temperature, centrifuging to take the supernatant, dialyzing for 1d by ultrapure water and permeation molecular weight of 1kDa, and filtering through a 0.22 mu m filter membrane to obtain the fluorescent carbon dots mPD-CDs.
Example 2 scanning electron microscopy of fluorescent carbon spots mPD-CDs:
the filtered fluorescent carbon spots mPD-CDs were diluted with deionized water, 10. mu.L were dropped on a clean copper mesh and observed by transmission electron microscopy (JEM-2100, JEOL Ltd, Japan) as shown in FIG. 1. The observation result of the transmission electron microscope shows that the carbon dots are approximately circular and uniformly distributed.
Example 3 particle size distribution detection of fluorescent carbon dots mPD-CDs
The diameters of the carbon dots mPD-CDs observed by the scanning electron microscope of FIG. 1 were measured on a scale bar, and the particle size distribution thereof was counted, as shown in FIG. 2, showing that the average particle size of the carbon dots was about 8.5nm.
Example 4 potential detection of fluorescent carbon dots mPD-CDs
The obtained fluorescent carbon dots mPD-CDs were placed in a Zeta potential (Zeta potential) detection cell, and the Zeta potential thereof was measured using a nanosize and Zeta potential analyzer (Malvern Instruments, Zetasizer Nano ZS, UK), and the detection results are shown in FIG. 3. Potential detection results show that the fluorescent carbon dots mPD-CDs are negatively charged (-5.7 mV);
example 5 evaluation of multicolor staining ability of fluorescent carbon dot mPD-CD cells
Raw 264.7, Hep G2 and 4T1 cells at 37 ℃ in 5% CO2Complete DMEM containing 10% fetal bovine serum under conditions). Cells were plated at 5X 10 per well3The density of individual cells was seeded in 96-well plates. After overnight growth, the medium in each well was changed to 50. mu.g/mL mPD-CD and incubated at 37 ℃ for 30 min.
Candida albicans (c. albicans) and saccharomyces cerevisiae (s. cerevisiae) were grown in YM medium at a rate of 100rpm at 37 ℃. Coli (e.coli) and staphylococcus aureus (s.aureus) were each grown on LB medium overnight at 200rpm at 37 ℃. Microbial cells were collected by centrifugation and treated with 100. mu.g/mL carbon spots in the corresponding medium. After incubation for 1h, microbial cells were collected by centrifugation at 10000rpm for 3min for confocal microscopy imaging. Then, cell imaging was examined using a confocal laser scanning microscope (CLSM, Leica SP8) with 63 x oil immersion objective. Confocal images were taken using blue, green and red channels within the fluorescence emission ranges of 410-, 480-, 500-, 580-and 581-700nm after excitation at 405, 488 and 552nm, respectively.
As shown in FIG. 4, the fluorescent carbon dots mPD-CD can carry out fluorescence imaging on cells under three channels, and the fluorescent carbon dots are proved to have the capability of multicolor fluorescence staining on the cells.
Example 6 evaluation of Effect of ion-detecting ability of fluorescent carbon dot mPD-CD
Will contain different concentrations of ions (Cu)2+、Hg2+、Gd3+、Fe2+、Fe3+、I-、Cd2+And Zn2+) To 30. mu.g/mL of CD in water, respectively, to achieve different final ion concentrations. The fluorescence intensity of these mixtures was recorded at a wavelength of 340nm using a fluorescence spectrophotometer, the excitation wavelength being 280 nm. Calculation (F)0-F)/F0，F0And F is the fluorescence intensity of the carbon spot without and with the ion to be detected, respectively. As shown in FIG. 5, Cu2+、Hg2+、Gd3+、Fe2+、Fe3+、I-Can enhance the fluorescence of the fluorescent carbon dots mPD-CD, and Cd2+And Zn2+Will weaken the fluorescent carbon dots mPThe fluorescence intensity of D-CD indicates that the fluorescent carbon dot mPD-CD can be used for detecting the ions.
Example 7 detection of Zn by fluorescent carbon dots mPD-CD2+And I-Linear range determination of
CD was added to aqueous solutions containing varying concentrations of the ions to be tested until the final concentration of CD was 30. mu.g/mL. The fluorescence intensity of the solution was recorded at a wavelength of 340nm using a spectrofluorometer, with an excitation wavelength of 280 nm. Working curve is represented by log (F)0-F)/F0And the measured ion concentration. F0And F is the fluorescence intensity of the carbon spot without and with the ion to be detected, respectively. As shown in FIGS. 6 and 7, CD vs. Zn are shown, respectively2+And I-Linear range of detection capability of (1).
The limit of detection (LOD) is calculated as follows:
LOD＝3σ/K
where K is the slope of the linear range and σ is the standard deviation of the blank (n-11).
Example 8 evaluation of Effect of intracellular Multi-ion detectability of fluorescent carbon dot mPD-CD
Similarly to example 5, after culturing the cells, the cells were cultured at 5X 10 per well3Density of individual cells, cells were added to 96-well plates and incubated overnight. The overnight cultured cells were then incubated with different ions (Cd) at a concentration of 100M2+、Cu2+、Hg2+、Gd3+、Fe2 +、Zn2+、Fe3+Or I-) Incubation for 2h, wherein Zn2+The concentration was 200M. The medium was then replaced with 50. mu.g/mL CD in DMEM. After 30min of incubation, cells were imaged by confocal laser scanning microscopy (CLSM, Leica SP8) with 63 × oil immersion objective. Fluorescence emission upon excitation at 405nm was 410nm to 480 nm. Ion detection in microorganisms including Saccharomyces cerevisiae is also performed in a similar manner. Finally, the fluorescence intensity of the cells of different samples was recorded using a flow cytometer. As shown in FIG. 8, Cd is contained in the cells2+And Zn2+When the ion is generated, the fluorescence intensity of the carbon dot mPD-CD is weakened, and Cu is contained in the cell2+、Hg2+、Gd3+、Fe2+、Fe3+Or I-When ions exist, the fluorescence intensity of the carbon dots mPD-CD is enhanced, and the fluorescent carbon dots have the capability of detecting the concentration of ions in cells.
Example 9 evaluation of Multi-ion detectability of fluorescent carbon dot mPD-CD on Zebra fish
Zebrafish of 5dpf (days post fertilization) were cultured at room temperature using standard zebrafish E3 medium. Zebra fish larvae and 200 mu M Cd2+、Zn2+、Cu2+、Hg2+、Gd3+、Fe2+、Fe3+Or I-Incubate for 2h, then treat with 100. mu.g/mL mPD-CD for 1.5 h. Confocal images of zebrafish were then obtained on a confocal laser scanning microscope (CLSM, Leica SP8) with a 10 x objective. Fluorescence emission was 410nm to 500nm upon 488nm excitation. As shown in FIG. 9, the fluorescence intensity of zebra fish is Cu2+、Hg2+、Gd3+、Fe2+、Fe3+Or I-In the presence of ions, the fluorescence intensity is enhanced, while in the presence of Cd2+And Zn2+In the presence of ions, the fluorescence intensity decreases. The fluorescent carbon dot can be used for detecting the ion concentration in organisms such as zebra fish and the like.
Example 10 evaluation of stability of fluorescent carbon dot mPD-CD
The fluorescent carbon dots mPD-CD were recorded at 340nm fluorescence intensity at different temperatures (37, 55, 70 and 90 ℃) by fluorescence spectrophotometer at an excitation wavelength of 280 nm. Also, the fluorescence intensity of CD samples at room temperature was measured at different potassium chloride (KCl) concentrations (0, 25, 100, 150, 200, 250 and 300M). As shown in FIG. 10, the fluorescence intensity of CD is not affected at 37-90 ℃ and 0-300M salt concentration, thus proving that the fluorescent carbon dot has good stability.
Example 11 Cytotoxicity (cytoxicity) study of fluorescent carbon dot mPD-CD on animal cells
4T1 cells and L929 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 100IU/mL penicillin at 37 ℃ with 5% CO2. Will be 5X 103Individual cells were transferred to each well of a 96-well plate and grown for 24 h. Then, the cells were incubated with fluorescent carbon dots mPD-CD for 24hThen 10. mu.L of 5mg/mL 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) was added and incubated for an additional 4 h. After removal of the medium, 150. mu.L DMSO was added. Finally, the absorbance at a wavelength of 570nm was measured with a microplate reader (Multiskan FC, Thermo Scientific, USA). As a result, as shown in FIG. 11, the fluorescent carbon dot mPD-CD was almost non-toxic to human cells.
Claims (10)
1. The fluorescent carbon dot mPD-CDs are characterized by being prepared from m-phenylenediamine serving as a raw material by a one-step hydrothermal method.
2. A method for preparing fluorescent carbon dots mPD-CDs as claimed in claim 1, comprising the following steps:
(1) dissolving mPD in deionized water to obtain an mPD solution, transferring the mPD solution into a hydrothermal kettle, and reacting for 6-18h at the temperature of 160-200 ℃;
(2) centrifuging the reaction product obtained in step (1) at 15000rpm for 5-20min, collecting the supernatant, and further filtering with 0.22 μm filter membrane to remove large particles to obtain carbon dots;
(3) the resulting carbon spots were dialyzed in ultrapure water using a 1k molecular weight dialysis bag, freeze-dried and stored at 4 ℃.
3. The method for preparing fluorescent carbon dots mPD-CDs as claimed in claim 2, wherein in the step (1), mPD is dissolved in deionized water at a concentration of 1-10 mg/mL; the high-pressure kettle is a high-temperature high-pressure hydrothermal kettle with a polytetrafluoroethylene lining, and the volume of the reaction kettle is 25-100 mL; the reaction temperature is 180 ℃, and the reaction time is 12 h.
4. Use of the fluorescent carbon dots mPD-CDs as defined in claim 1 for multicolor cellular imaging and in vivo multi-ion fluorescence detection.
5. Use of polychromatic cell imaging according to claim 4, characterized in that it can be used for polychromatic imaging of cells, comprising red, green and blue.
6. Use of polychromatic cell imaging according to claim 4, characterized in that the cells comprise animal cells, bacteria and fungi.
7. The use of in vivo multi-ion fluorescence detection as claimed in claim 4, wherein said multi-ions comprise: cd [ Cd ]2+、Cu2+、Hg2+、Gd3+、Fe2+、Zn2+、Fe3+And I-。
8. The in vivo multi-ion fluorescence detection application of claim 4, wherein the application scenario of fluorescence detection comprises: laboratory model animals, animal cells, bacteria and fungi.
9. The use of the in vivo polyion fluorescence detection of claim 8, wherein the laboratory model animal comprises zebrafish, mouse, fruit fly.
10. The use of in vivo polyion fluorescence detection of claim 4, wherein the detection apparatus comprises a fluorescence spectrometer, a fluorescence confocal microscope or a flow cytometer.
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