Preparation method and bioimaging application of multicolor luminescent carbon dots

Abstract

The invention belongs to the technical field of carbon material luminescent materials, and particularly relates to a preparation method of a multicolor luminescent carbon dot and bioimaging application of the multicolor luminescent carbon dot. The invention provides a simple and controllable multicolor luminescent carbon dot and a preparation method thereof. By adjusting the reaction time in the heat treatment process of o-phenylenediamine and thiourea solvents, various luminescent carbon dots, including green light, yellow light and red light carbon dots, are successfully separated. The carbon dot provided by the invention can realize controllable adjustment of fluorescence performance by regulating and controlling the conjugation degree of the carbon core and the content of graphite nitrogen. According to the method, the fluorescence mechanism in the carbon dot forming process is understood more deeply, and guidance is provided for designing the structure and optical performance of the carbon dots and achieving controllable synthesis of the high-performance carbon dots. The multicolor luminescent carbon dot provided by the invention has excellent optical performance, stable morphological structure and low cytotoxicity, and has a good application prospect in biological imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of carbon material luminescent materials, and in particular relates to a preparation method of multicolor luminescent carbon dots and biological imaging application thereof. Background Art

[0002] Multicolor luminescent carbon dots (CDs) are a new type of carbon-based fluorescent material. They have attracted extensive attention due to their excellent optical properties, good biocompatibility, abundant raw material sources, low cost, easy synthesis, and the presence of multiple functional groups on the surface. In recent years, carbon dots have been rapidly developed and applied in the fields of fluorescent bioimaging, therapy, detection, light-emitting diodes, photocatalysis, anti-counterfeiting and antibacterial.

[0003] Currently, most carbon dots show emission related to the excitation wavelength, and their maximum emission light is limited to the blue-green light region. However, efficient red-light emitting carbon dots are more important because red light has deep tissue penetration in biological imaging and is one of the primary colors of white light diodes. Currently, there are few reports on the synthesis methods of carbon dots that emit red light, and the reaction raw materials are expensive. Therefore, it is necessary to develop a preparation method with easily available raw materials to prepare carbon dots that emit red light.

[0004] Although considerable progress has been made in the synthesis, optical properties and potential applications of carbon dots, their formation process and fluorescence mechanism are still unclear. In particular, the relationship between chemical structure and optical properties is not yet fully understood. It is impossible to design the structure and optical properties of carbon dots through theoretical guidance to achieve controllable synthesis of high-performance carbon dots. At present, the trial and error method is still commonly used to prepare carbon dots with specific optical properties and explore their applications, which limits the further development of luminescent carbon dots. Therefore, it is of great significance to synthesize multi-color luminescent carbon dots with adjustable fluorescence under controllable and comparable conditions.

[0005] Due to the lack of a clear understanding of the structure and luminophore, controlling the emission wavelength by adjusting the synthesis conditions is another way to synthesize carbon dots with different optical properties. In current research, effective strategies for preparing carbon dots with adjustable fluorescence mainly include selecting different precursors, adjusting the reaction solvent and temperature, etc. However, there are problems such as cumbersome operation.

[0006] Therefore, developing a simple and controllable preparation method with easily available raw materials to prepare luminescent carbon dots, especially red-light carbon dots, is a problem that needs to be solved in the art. Summary of the invention

[0007] In view of the defects of the prior art, the present invention provides a simple and controllable preparation method, the purpose of which is to prepare multi-color luminescent carbon dots with controllable emission wavelength.

[0008] The present invention provides a luminescent carbon dot, which comprises a conjugated aromatic carbon core, and the surface of the conjugated carbon core contains oxygen- and nitrogen-containing functional groups;

[0009] The absorption band of the luminescent carbon dots in water is located at 270-500 nm, and the emission band is located at 510-600 nm. The error ranges of the absorption band and the emission band are both ±5%.

[0010] Preferably, the luminescent carbon dots are prepared according to the following steps:

[0011] Step 1, mixing carbon source and nitrogen source molecules with sulfur and nitrogen dopants to prepare a precursor reaction solution;

[0012] Step 2, reacting the precursor reaction liquid in a reactor for 2-10 hours to obtain.

[0013] Preferably, the reaction time is 6-10 hours.

[0014] Preferably, in step 1, the carbon source and nitrogen source molecules are selected from o-phenylenediamine, p-phenylenediamine, and m-phenylenediamine, and the sulfur-nitrogen dopant is selected from thiourea and 3-mercaptopropionic acid;

[0015] And / or, the feed ratio of the carbon source and nitrogen source molecules to the sulfur and nitrogen dopant is 2-6:1 by mass;

[0016] And / or, the carbon source and nitrogen source molecules and the sulfur-nitrogen dopants may also be replaced by sulfur-nitrogen doped carbon dots.

[0017] Preferably, in step 1, the mixture is dissolved in a mixed solvent of N,N-dimethylformamide and water and / or ethanol; the ratio of the carbon source and nitrogen source molecules to the mixed solvent is 2g:25-50mL;

[0018] And / or, in step 2, the reaction temperature is 180-220°C.

[0019] Preferably, the volume ratio of N,N-dimethylformamide to water and ethanol is 3:1:1.

[0020] Preferably, after the reaction, separation and purification are performed; the separation and purification methods include centrifugation, filtration, column chromatography, and dialysis.

[0021] Preferably, the filtration method is to use a 0.22-0.45 μm filter membrane;

[0022] And / or, the column chromatography method uses silica gel column chromatography or gel column chromatography for separation and purification, and the eluent is at least one of the following combinations: dichloromethane and methanol, tetrahydrofuran and n-hexane, n-hexane and chloroform;

[0023] And / or, the dialysis method is performed in water using a dialysis bag with a molecular weight cutoff of 100-3000Da.

[0024] Preferably, the column chromatography method uses silica gel column chromatography for separation and purification, and the specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first using dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing the proportion of methanol to control the ratio of dichloromethane to methanol from 80-200:1 to 10-30:1; during the separation and purification process, using a 320-400nm ultraviolet lamp to monitor the fluorescence color to collect the products respectively.

[0025] The present invention provides a method for preparing the luminescent carbon dots described in any one of the above, comprising:

[0026] Step 1, mixing carbon source and nitrogen source molecules with sulfur and nitrogen dopants to prepare a precursor reaction solution;

[0027] Step 2, reacting the precursor reaction liquid in a reactor for 2-10 hours to obtain.

[0028] The present invention provides the use of any of the above-mentioned luminescent carbon dots in biological imaging, cell labeling and tracing, and preparation of LED devices.

[0029] The present invention provides a simple and controllable multicolor luminescent carbon dot and its preparation method and application. By adjusting the reaction time during the thermal treatment of o-phenylenediamine and thiourea solvent, the multicolor luminescent carbon dots were successfully separated. The carbon dots provided by the present invention can realize controllable regulation of fluorescence performance by regulating the degree of conjugation of the carbon core and the content of graphitic nitrogen. The present invention enables us to have a deeper understanding of the fluorescence mechanism of the carbon dot formation process, and provides guidance for us to design the structure and optical properties of carbon dots and realize the controllable synthesis of high-performance carbon dots. The multicolor luminescent carbon dots provided by the present invention have excellent optical properties, stable morphological structure, and low cytotoxicity, and have good application prospects in biological imaging.

[0030] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0031] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 a is a schematic diagram of the synthesis of multicolor luminescent carbon dots of the present invention; Figure 1 bf are the ultraviolet absorption spectra of the carbon dots prepared in Examples 1 to 5, respectively. Figure 1 gk are the fluorescence emission spectra of the carbon dots prepared in Examples 1 to 5, respectively;

[0033] Figure 2 Figure 1 is the absorption spectrum and emission spectrum of the multicolor luminescent carbon dots prepared in Examples 1 to 5; Figure a is the absorption spectrum of green light carbon dots, Figure b is the absorption spectrum of yellow light carbon dots, Figure c is the absorption spectrum of red light carbon dots, Figure d is the emission spectrum of green light carbon dots, Figure e is the emission spectrum of yellow light carbon dots, and Figure f is the emission spectrum of red light carbon dots.

[0034] Figure 3 The absorption spectrum, emission spectrum and excitation spectrum of the multicolor luminescent carbon dots prepared in Examples 5 and 6; G-CDs, Y-CDs, O-CDs are the multicolor luminescent carbon dots prepared in Example 5, and g-CDs, y-CDs, o-CDs are the multicolor luminescent carbon dots prepared in Example 6; ac is an ultraviolet absorption spectrum, de is a fluorescence emission spectrum at different excitation wavelengths, and gf is an excitation spectrum;

[0035] Figure 4 The superposition diagrams are the absorption spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5 and the emission spectra at different excitation wavelengths; Figure a is the superposition diagram of D-CDs, Figure b is the superposition diagram of G-CDs, Figure c is the superposition diagram of Y-CDs, and Figure d is the superposition diagram of O-CDs;

[0036] Figure 5 The figures are superimposed graphs of the excitation spectra and the absorption spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is a superimposed graph of D-CDs, Figure b is a superimposed graph of G-CDs, Figure c is a superimposed graph of Y-CDs, and Figure d is a superimposed graph of O-CDs;

[0037] Figure 6 The photoluminescence attenuation spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is a photoluminescence attenuation spectra of D-CDs, Figure b is a photoluminescence attenuation spectra of G-CDs, Figure c is a photoluminescence attenuation spectra of Y-CDs, and Figure d is a photoluminescence attenuation spectra of O-CDs;

[0038] Figure 7 The absolute quantum yield spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is the absolute quantum yield spectrum of D-CDs, Figure b is the absolute quantum yield spectrum of G-CDs, Figure c is the absolute quantum yield spectrum of Y-CDs, and Figure d is the absolute quantum yield spectrum of O-CDs;

[0039] Figure 8TEM images of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is a TEM image of D-CDs, Figure b is a TEM image of G-CDs, Figure c is a TEM image of Y-CDs, and Figure d is a TEM image of O-CDs;

[0040] Fig. 9 Figure 1 is a particle size distribution diagram of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is a particle size distribution diagram of D-CDs, Figure b is a particle size distribution diagram of G-CDs, Figure c is a particle size distribution diagram of Y-CDs, and Figure d is a particle size distribution diagram of O-CDs;

[0041] Fig.10 Figure 1 is a structural characterization diagram of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is an XRD diagram, Figure b is a Raman spectrum diagram, Figure c is a Fourier infrared spectrum diagram, and Figure d is an XPS full spectrum diagram;

[0042] Fig.11 High-resolution XPS spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; the first row is a high-resolution XPS spectrum of D-CDs, the second row is a high-resolution XPS spectrum of G-CDs, the third row is a high-resolution XPS spectrum of Y-CDs, and the fourth row is a high-resolution XPS spectrum of O-CDs;

[0043] Fig.12 The results of the nuclear magnetic resonance test of the multi-color luminescent carbon dots prepared in Example 1 and Example 5; Figure a is 13 CNMR spectrum, Figure b is 1 H NMR spectra;

[0044] Fig.13 These are cell experiment pictures of the red light carbon dots prepared in Example 5; Figure a is a picture of the cytotoxicity experiment results, Figure b is a bright field microscope picture of MG63 cells, and Figure c is a fluorescence microscope image of MG63 cells. DETAILED DESCRIPTION

[0045] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0046] Example 1 A multi-color luminescent carbon dot and its preparation method

[0047] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0048] 1. Prepare precursor reaction solution

[0049] 2 g of o-phenylenediamine and 0.5 g of thiourea were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0050] 2. Preparation of carbon dots

[0051] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 2 h. After the reaction was completed, the reactor was naturally cooled to room temperature.

[0052] 3. Obtain carbon dots

[0053] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0054] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with low polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate the bright green fluorescent component by adjusting the polarity of the eluent.

[0055] (3) The obtained fluorescent component was placed in a dialysis bag with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain a solid powder, which is the green light carbon dot D-CDs.

[0056] Example 2 A multi-color luminescent carbon dot and its preparation method

[0057] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0058] 1. Prepare precursor reaction solution

[0059] 2 g of o-phenylenediamine and 0.5 g of thiourea were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0060] 2. Preparation of carbon dots

[0061] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 4 hours. After the reaction was completed, the reactor was cooled naturally to room temperature.

[0062] 3. Obtain carbon dots

[0063] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0064] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected separately according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted at the same time to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with less polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate two green and one yellow fluorescent components by adjusting the polarity of the eluent.

[0065] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain solid powders, namely green carbon dots D-CDs, green carbon dots G-CDs and yellow carbon dots Y-CDs.

[0066] Example 3 A multi-color luminescent carbon dot and its preparation method

[0067] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0068] 1. Prepare precursor reaction solution

[0069] 2 g of o-phenylenediamine and 0.5 g of thiourea were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0070] 2. Preparation of carbon dots

[0071] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 6 hours. After the reaction was completed, the reactor was cooled naturally to room temperature.

[0072] 3. Obtain carbon dots

[0073] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0074] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected separately according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with low polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate the bright green, yellow, and orange fluorescent components by adjusting the polarity of the eluent.

[0075] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain solid powders, namely green light carbon dots G-CDs, yellow light carbon dots Y-CDs, and red light carbon dots O-CDs.

[0076] Example 4 A multi-color luminescent carbon dot and its preparation method

[0077] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0078] 1. Prepare precursor reaction solution

[0079] 2 g of o-phenylenediamine and 0.5 g of thiourea were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0080] 2. Preparation of carbon dots

[0081] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 8 hours. After the reaction was completed, the reactor was cooled naturally to room temperature.

[0082] 3. Obtain carbon dots

[0083] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0084] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected separately according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with low polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate the bright green, yellow, and orange fluorescent components by adjusting the polarity of the eluent.

[0085] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain solid powders, namely green light carbon dots G-CDs, yellow light carbon dots Y-CDs, and red light carbon dots O-CDs.

[0086] Example 5 A multi-color luminescent carbon dot and its preparation method

[0087] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0088] 1. Prepare precursor reaction solution

[0089] 2 g of o-phenylenediamine and 0.5 g of thiourea were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0090] 2. Preparation of carbon dots

[0091] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 10 h. After the reaction was completed, the reactor was naturally cooled to room temperature.

[0092] 3. Obtain carbon dots

[0093] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0094] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected separately according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with low polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate the bright green, yellow, and orange fluorescent components by adjusting the polarity of the eluent.

[0095] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain solid powders, namely green light carbon dots G-CDs, yellow light carbon dots Y-CDs, and red light carbon dots O-CDs.

[0096] Example 6 A multi-color luminescent carbon dot and its preparation method

[0097] The method for preparing multicolor luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, it includes the following steps:

[0098] 1. Prepare precursor reaction solution

[0099] 2 g of the carbon dots prepared in Example 1 were placed in a beaker, and 30 mL of N,N-dimethylformamide (DMF), 10 mL of anhydrous ethanol and 10 mL of deionized water were added thereto, and the mixture was completely dissolved by magnetic stirring to obtain a precursor reaction solution.

[0100] 2. Preparation of carbon dots

[0101] The precursor reaction solution was transferred to a 100 mL reactor, and then heated to 200°C for reaction for 8 hours. After the reaction was completed, the reactor was cooled naturally to room temperature.

[0102] 3. Obtain carbon dots

[0103] (1) The reaction solution in the reactor was first filtered with a 0.22 μm filter membrane to remove large particles of reaction products.

[0104] (2) The reaction solution obtained by filtration is separated by a silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp is used for observation, and the fluorescent components are collected separately according to the fluorescent color of the product, and the ratio of dichloromethane and methanol in the eluent is adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first use pure dichloromethane as the eluent to let the product with low polarity flow out first, and then gradually increase methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, so as to better separate the bright green, yellow, and orange fluorescent components by adjusting the polarity of the eluent.

[0105] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000Da and dialyzed in deionized water for 72 hours to remove the unreacted organic solution. The initial product in the dialysis bag was collected and placed in a freeze dryer for 24 hours to obtain solid powders, namely green light carbon dots G-CDs, yellow light carbon dots Y-CDs, and red light carbon dots O-CDs.

[0106] The technical solution of the present invention is further described below through experiments. The samples D-CDs, G-CDs, Y-CDs and O-CDs tested in the following experimental examples are prepared by the methods of Examples 1-6, respectively.

[0107] Experimental Example 1 Optical Performance

[0108] 1. Experimental Methods

[0109] The multicolor luminescent carbon dots prepared in Examples 1 to 6 were subjected to UV absorption spectrum and fluorescence emission spectrum tests. The multicolor fluorescent carbon dots prepared in Examples 1 and 5 were subjected to emission (Em) spectrum test (excitation wavelength), excitation (Ex) spectrum test, time-resolved light scattering (TRPL) decay curve test, and quantum yield test.

[0110] Emission (Em) spectrum test:

[0111] The multicolor fluorescent carbon dots prepared in Example 1 and Example 5 were tested for emission (Em) spectra. For D-CDs, the excitation wavelengths were set to 380nm, 400nm, 420nm, 440nm, and 460nm, respectively; for G-CDs, the excitation wavelengths were set to 360nm, 370nm, 380nm, 390nm, and 400nm, respectively; for Y-CDs, the excitation wavelengths were set to 420nm, 440nm, 460nm, 480nm, and 500nm, respectively; for O-CDs, the excitation wavelengths were set to 440nm, 460nm, 480nm, 500nm, and 520nm, respectively.

[0112] 3. Excitation (Ex) spectrum test

[0113] The multicolor fluorescent carbon dots prepared in Example 1 and Example 5 were tested for emission (Em) spectra respectively.

[0114] 2. Experimental Results

[0115] (1) Effect of reaction time of solvent thermal treatment on carbon dots

[0116] The results of the absorption peaks of the carbon dots prepared in Examples 1-5 are as follows: Figure 1 bf shows the results of the emission peak. Figure 1 The comparison results of G-CDs, Y-CDs, and O-CDs in each embodiment are shown in Figure 2 shown.

[0117] The emission peaks of the green carbon dots D-CDs and G-CDs prepared in Examples 1-5 are both around 510 nm, but the absorption peaks of the green carbon dots D-CDs prepared in Examples 1 and 2 and the green carbon dots G-CDs prepared in Examples 2-5 are different, specifically, the absorption peaks in the low energy section of the ultraviolet absorption curve are different, indicating that D-CDs and G-CDs are two different green carbon dots.

[0118] The emission peaks of the yellow carbon dots Y-CDs prepared in Examples 2-5 are all near 560 nm and have similar ultraviolet absorption curves, indicating that the yellow carbon dots Y-CDs prepared in Examples 2-5 are the same type of yellow carbon dots.

[0119] The emission peaks of the red light carbon dots Y-CDs prepared in Examples 2-5 are all around 600 nm and have similar ultraviolet absorption curves, indicating that the red light carbon dots O-CDs prepared in Examples 2-5 are the same type of red light carbon dots.

[0120] The results show that in Examples 1-5, two green light carbon dots (D-CDs, G-CDs), one yellow light carbon dot (Y-CDs), and one red light carbon dot (O-CDs) were prepared respectively by regulating the reaction time during the solvent thermal treatment process.

[0121] (2) Conversion of carbon dots

[0122] The results of the ultraviolet absorption spectra and fluorescence emission spectra of the carbon dots prepared in Example 5 and Example 6 are as follows: Figure 3 shown.

[0123] The green, yellow and red carbon dots of Example 5 have similar emission spectra to those of Example 6. The UV-visible absorption spectra also show similar absorption bands. The excitation spectra have almost the same optimal excitation wavelengths. Therefore, the green carbon dots D-CDs can be further converted into green carbon dots G-CDs, yellow carbon dots Y-CDs and red carbon dots O-CDs using the method of the present invention.

[0124] (3) UV-visible absorption properties

[0125] The results of Example 1 and Example 5 are as follows Figure 4 As shown. In the UV region, two absorption peaks were observed at 273nm and 280nm for D-CDs and G-CDs. A new absorption peak was also observed at 310nm for Y-CDs. For O-CDs, the two peaks at 273nm and 280nm were completely replaced by the absorption peak at 310nm, indicating that a carbon core with a larger conjugated structure was formed. In the low-energy UV absorption region, the multicolor luminescent carbon dots showed obvious absorption bands at wavelengths of 420nm, 380nm, 460nm and 480nm, respectively. Another absorption band was observed at a wavelength of 360nm for Y-CD and O-CD, indicating that the four typical carbon dots have different surface states.

[0126] (4) Emission (Em) spectrum test results

[0127] The test results of Example 1 and Example 5 are as follows Figure 4 As shown. The maximum emission wavelength of D-CDs and G-CDs is 510nm, and the optimal excitation wavelength is 420nm and 380nm, respectively. For Y-CDs and O-CDs, the maximum emission wavelength is 560nm and 600nm, and the optimal excitation wavelength is 460nm and 480nm, respectively. They all show emission characteristics that are independent of excitation, that is, when the excitation wavelength changes, their emission peaks will not move.

[0128] (5) Excitation (Ex) spectrum test results

[0129] The test results of Example 1 and Example 5 are as follows Figure 5 As shown. The optimal excitation wavelength of carbon dots corresponds to the absorption band in the low energy region. The excitation (Ex) spectra of four typical carbon dots at the maximum emission wavelength correspond to the absorption band in the low energy region, indicating that the emission of carbon dots mainly comes from the absorption band in the low energy region.

[0130] (6) Time-resolved light scattering (TRPL) decay curve test results

[0131] The test results of Example 1 and Example 5 are as follows Figure 6 The test results can be fitted by a double exponential formula, which includes a short-lived component τ1 (about 1ns) and a long-lived component τ2 (about 10ns), which are attributed to the recombination process of the core state and the surface state, respectively. From D-CDs to O-CDs, the average lifetime increases from 2.1ns to 10.2ns

[0132] (7) Quantum yield test results

[0133] The test results of Example 1 and Example 5 are as follows Figure 7 The absolute quantum yield (QY) of carbon dots was measured by integrating sphere at the optimal excitation wavelength, and O-CDs had the highest QY of 23.68%.

[0134] The above results show that the present invention prepares two green carbon dots (D-CDs, G-CDs), one yellow carbon dot (Y-CDs), and one red carbon dot by regulating the reaction time during the solvent thermal treatment process. The green carbon dots D-CDs can be further converted into multicolor luminescent carbon dots by the method of the present invention. These luminescent carbon dots are consistent in optical characteristics, that is, each carbon dot has a similar absorption structure and fluorescence center. The generation of different carbon dots can be attributed to the carbon core and surface states controlled by different reaction times. The optical uniformity of these carbon dots is helpful to further study their photoluminescence mechanism based on the comparison of their composition and structure.

[0135] Experimental Example 2 Structural Performance

[0136] 1. Experimental Methods

[0137] The multicolor fluorescent carbon dots prepared in Example 1 and Example 5 were subjected to transmission electron microscopy test, XRD test, Raman test, Fourier transform infrared spectroscopy (FT-IR) test, XPS test, and NMR test, respectively.

[0138] 2. Experimental Results

[0139] (1) Transmission electron microscopy test results

[0140] Test results such as Figure 8 , 9 As shown. These carbon dots are uniform and well dispersed, with average particle sizes of about 9.8, 5.2, 3.1 and 2.8 nm for D-CDs, G-CDs, Y-CDs and O-CDs, respectively. High-resolution TEM images show that all samples have similar well-resolved lattice fringes with a spacing of 0.21 nm, which is attributed to the (100) lattice spacing of graphitic carbon.

[0141] (2) XRD test results

[0142] Test results such as Fig.10 As shown in a. After continuous polymerization and carbonization, a more ordered structure is gradually formed from D-CDs to O-CDs.

[0143] (3) Raman test results

[0144] Test results such as Fig.10 b. At 1348 and 1587 cm -1There are two peaks at , corresponding to disordered structures or defects (D band) and graphitic carbon domains (G band). The intensity ID / IG of D-, G-, Y-, and O-CD are 1.22, 1.06, 0.97, and 0.86, respectively, indicating that the size of sp2-domains gradually increases.

[0145] (4) FT-IR test results

[0146] Test results such as Fig.10 c. The stretching vibration of OH is at 3454 cm -1 , NH at 3118cm -1 , =CH at 2928cm -1 , -SCN at 2055cm -1 , C=O at 1720cm -1 , C=N at 1625cm -1 , C=C at 1460cm -1 , CN at 1352cm -1 , CO at 1172cm -1 , CN at 1460cm -1 , CN at 1352cm -1 、CO at 1172cm -1 The CH in aromatic benzene is at 742 cm -1 , indicating that conjugated aromatic structures and oxygen / nitrogen-containing surface functional groups are formed in the carbon dots during the reaction. In addition, the stretching vibrations of =CH, C=N, and C=C are significantly enhanced from D-CDs to O-CDs, indicating that the aromatic rings of the carbon dots are gradually carbonized to form a larger conjugated structure. From D-CDs to O-CDs, the stretching vibrations of CN and CO are significantly enhanced, and some new functional groups are formed, indicating that O-CDs have more oxygen / nitrogen-containing groups. From D-CDs to O-CDs, the stretching vibration of -SCN is significantly weakened. Fourier transform infrared spectroscopy results show that all carbon dots have similar surface functional groups and chemical bonds, but the reaction time changes the content of these functional groups and generates some new functional groups.

[0147] (5) XPS test results

[0148] Test results such as Fig.10d and 11. All carbon dots are composed of C, O, N and S elements. Compared with D-CDs, G-, Y- and O-CDs have significantly higher C contents, indicating a higher degree of carbonization. In addition, the N content of O-CDs is higher than that of G- and Y-CDs, while the relative contents of O and S elements are similar. The high-resolution C1s XPS spectrum can be fitted into three types of C: CC / C=C (284.8 eV), CN / CO (285.8 eV) and COOH (289.5 eV). The results clearly show that the content of CC / C=C groups increases significantly from D-CDs to Y-CDs, indicating a larger conjugated structure; O-CDs have more CN / CO content, indicating more surface functional groups. For N1s, the high-resolution XPS spectra can be converted into pyridinic N (398.4 eV), amino N (399.5 eV) and graphitic N (401.2 eV), respectively. The relative content of pyridinic N in D- and G-CDs is significantly higher than that in Y- and O-CDs, indicating that pyridinic N is the main reason for the green fluorescence of Cdots. The relative content of graphitized N increases significantly from D- and G-CDs to Y- and O-CDs, indicating that graphitized N has a significant effect on the red shift of the emission wavelength of Cdots. The high-resolution XPS spectrum of O1s contains two components related to CO at 534 eV and C=O at 531.7 eV. The relative content of C=O decreases slightly from D- and G-CDs to Y- and O-CDs (Table S4), indicating an increase in the degree of dehydration and carbonization. The high-resolution XPS spectrum of S2p can be decomposed into SC (162.4 eV), SH (163.2 eV), SS (164.7 eV), and S=O (169.8 eV) peaks. The results show that Cdots have large conjugated sp 2 The carbon core of the domains and the abundant oxygen / nitrogen-containing surface groups indicate that when their maximum emission wavelengths are red-shifted, both the conjugated structure and the graphitic N content increase, while the pyridinic N content decreases.

[0149] (6) NMR test results

[0150] Test results such as Fig.12 All carbon dots 1 H NMR spectra all showed obvious aromatic hydrogen signals in the range of 7.5-8.5 ppm ( Fig.12 b) In addition, 13 C NMR spectroscopy ( Fig.12 In a), G-, Y-, and O-CDs produce obvious signals in the range of 110-140 ppm, and the signals of Y- and O-CDs are stronger than those of G-CDs, which indicates the presence of sp 2 C atoms. In contrast, D-CDs 13 The signals in the 110–140 ppm range of the CNMR spectrum are weak, which indicates that the sp2 C conjugated structure is less.

[0151] The above results show that the four luminescent carbon dots prepared by the present invention have similar surface functional groups and chemical bonds. With the extension of reaction time, their sizes become smaller, their structures become more ordered, the contents of certain functional groups change, and some new functional groups are generated.

[0152] Experimental Example 3: Application of multicolor luminescent carbon dots in biological imaging

[0153] 1. Experimental Methods

[0154] 1. Determination of cytotoxicity

[0155] The CCK-8 method was used to detect the cytotoxicity of the red light carbon dots prepared in Example 5 to MG63 cells. The cells were cultured at a density of 1×10 4 The cells were seeded at a density of 100 μg / well in a 96-well cell culture plate and cultured in an incubator at 37°C and 5% CO2 for 24 h. The DMEM medium was aspirated and 4% CO2 containing different carbon dot concentrations (0, 20, 50, 100, and 200 μg mL -1 ) fresh DMEM medium and culture for another 24h. Subsequently, the medium was aspirated, and the cells were treated with 10% volume fraction of cck-8 (100 μL per well) and cultured for another 1 hour (37°C, 5% CO2). Finally, the absorbance of cck-8 was recorded at 450nm using an ELISA reader. The cell survival rate was calculated based on the OD values ​​of the experimental group and the control group.

[0156] 2. Cell imaging experiment

[0157] MG63 cells (1×10 4 Each well) was inoculated in a 96-well cell culture plate, and the cells were placed in 100 μL of DMEM medium containing 10% fetal bovine serum and 1% antibiotics, and cultured in an incubator at 37°C, 5% CO2 for 24 hours. Then, the DMEM medium was aspirated, and a medium containing carbon dots (20 μg / mL) was added, and the cells were cultured for 1 hour. Subsequently, the medium containing the material was aspirated, and the cells were washed three times with PBS buffer to remove the carbon dots that were not taken up by the cells, and 100 μL of PBS buffer was added to each well. Cell fluorescence imaging was performed using an inverted microscope.

[0158] 2. Experimental Results

[0159] 1. Cytotoxicity results

[0160] Cytotoxicity results such as Fig.13 As shown in a. It can be seen from the figure that when the working concentration of carbon dots is as high as 200 μg / mL, the cell survival rate is still above 80%, indicating that the carbon dots have low cytotoxicity.

[0161] 2. Cell imaging results

[0162] Cell imaging results Fig.13 As shown in b and c, it can be seen from the figure that carbon dots can be effectively taken up by cells and undergo red fluorescence imaging.

[0163] The above results indicate that the multicolor luminescent carbon dots prepared by the present invention have low cytotoxicity and can be used for red fluorescence imaging of cells.

[0164] Through the above embodiments and experimental examples, it can be seen that the present invention provides a simple and controllable multicolor luminescent carbon dot and its preparation method and application. By adjusting the reaction time during the thermal treatment of o-phenylenediamine and thiourea solvent, the multicolor luminescent carbon dots were successfully separated. The carbon dots provided by the present invention can achieve controllable regulation of fluorescence performance by regulating the degree of conjugation of the carbon core and the content of graphitic nitrogen. The present invention enables us to have a deeper understanding of the fluorescence mechanism of the carbon dot formation process, and provides guidance for us to design the structure and optical properties of carbon dots and realize the controllable synthesis of high-performance carbon dots. The multicolor luminescent carbon dots provided by the present invention have excellent optical properties, stable morphological structure, and low cytotoxicity, and have good application prospects in biological imaging.

Claims

1. A luminescent carbon dot, characterized in that: The luminescent carbon dots include a conjugated aromatic carbon core, and the surface of the conjugated carbon core contains oxygen- and nitrogen-containing functional groups; The absorption band of the luminescent carbon dots in water is located at 270-500 nm, and the emission band is located at 510-600 nm. The error ranges of the absorption band and the emission band are both ±5%.

2. The luminescent carbon dots according to embodiment 1, characterized in that: It is prepared according to the following steps: Step 1, mixing carbon source and nitrogen source molecules with sulfur and nitrogen dopants to prepare a precursor reaction solution; Step 2, reacting the precursor reaction liquid in a reactor for 2-10 hours to obtain.

3. The luminescent carbon dots according to embodiment 2, characterized in that: The reaction time is 6-10h.

4. The luminescent carbon dots according to embodiment 2, characterized in that: Step 1, the carbon source and nitrogen source molecules are selected from o-phenylenediamine, p-phenylenediamine, and m-phenylenediamine, and the sulfur-nitrogen dopant is selected from thiourea and 3-mercaptopropionic acid; And / or, the feed ratio of the carbon source and nitrogen source molecules to the sulfur and nitrogen dopant is 2-6:1 by mass; And / or, the carbon source and nitrogen source molecules and the sulfur-nitrogen dopants may also be replaced by sulfur-nitrogen doped carbon dots.

5. The luminescent carbon dots according to embodiment 2, characterized in that: Step 1, the mixture is dissolved in a mixed solvent of N,N-dimethylformamide and water and / or ethanol; the ratio of the carbon source and nitrogen source molecules to the mixed solvent is 2g: 25-50mL; And / or, in step 2, the reaction temperature is 180-220°C.

6. The luminescent carbon dots according to embodiment 2, characterized in that: After the reaction, separation and purification are performed; the separation and purification methods include centrifugation, filtration, column chromatography, and dialysis.

7. The luminescent carbon dots according to embodiment 6, characterized in that: The filtration method is to filter using a 0.22-0.45 μm filter membrane; And / or, the column chromatography method uses silica gel column chromatography or gel column chromatography for separation and purification, and the eluent is at least one of the following combinations: dichloromethane and methanol, tetrahydrofuran and n-hexane, n-hexane and chloroform; And / or, the dialysis method is performed in water using a dialysis bag with a molecular weight cutoff of 100-3000Da.

8. The luminescent carbon dots according to embodiment 7, characterized in that: The column chromatography method uses silica gel column chromatography for separation and purification, and the specific operation of adjusting the eluent is: using dichloromethane and methanol as eluents, first using dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing the proportion of methanol to control the ratio of dichloromethane to methanol from 80-200:1 to 10-30:1; during the separation and purification process, using a 320-400nm ultraviolet lamp to monitor the fluorescence color to collect the products respectively.

9. The method for preparing luminescent carbon dots according to any one of claims 1 to 8, characterized in that: It includes: Step 1, mixing carbon source and nitrogen source molecules with sulfur and nitrogen dopants to prepare a precursor reaction solution; Step 2, reacting the precursor reaction liquid in a reactor for 2-10 hours to obtain.

10. Use of the luminescent carbon dots according to any one of claims 1 to 8 in biological imaging, cell labeling and tracing, and preparation of LED devices.

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