Preparation method of multi-color luminescent carbon dots and biological imaging application thereof

Abstract

The application belongs to the technical field of carbon material luminescent material, and particularly relates to a preparation method of multi-color luminescent carbon dots and biological imaging application thereof. The application provides a simple and controllable multi-color luminescent carbon dot and a preparation method thereof. By adjusting the reaction time in a solvothermal treatment process of o-phenylenediamine and thiourea, a plurality of luminescent carbon dots, including green light, yellow light and red light carbon dots, are successfully separated. The carbon dots provided by the application can realize controllable adjustment of fluorescent performance by adjusting the conjugation degree of carbon cores and the content of graphitic nitrogen. The application makes us have a deeper understanding of the fluorescent mechanism of the carbon dot formation process, provides guidance for designing the structure and optical performance of the carbon dots and realizing controllable synthesis of high-performance carbon dots. The multi-color luminescent carbon dots provided by the application have excellent optical performance, stable morphological structure and low cytotoxicity, and have a good application prospect in biological imaging.

Description

Technical Field

[0001] The present 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 applications thereof. Background Art

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

[0003] Currently, most carbon dots exhibit excitation wavelength-dependent emission, with maximum emission limited to the blue-green region. However, efficient red-light-emitting carbon dots are more important because red light has deep tissue penetration in bioimaging and is one of the primary colors of white light diodes. Currently, there are few reports on the synthesis of red-emitting carbon dots, and the raw materials required for the reaction are expensive. Therefore, there is a need to develop a method for preparing red-emitting carbon dots with readily available raw materials.

[0004] While significant progress has been made in the synthesis, optical properties, and potential applications of carbon dots, their formation process and fluorescence mechanism remain unclear. In particular, the relationship between chemical structure and optical properties is not fully understood. This has hindered the controllable synthesis of high-performance carbon dots through theoretical guidance to tailor their structure and optical properties. Currently, a trial-and-error approach is commonly used to prepare carbon dots with specific optical properties and explore their applications, limiting the further development of luminescent carbon dots. Therefore, the synthesis of multicolor luminescent carbon dots with tunable fluorescence under controllable and comparable conditions is of great significance.

[0005] Due to a lack of clear understanding of the structure and luminophore, another approach to synthesizing carbon dots with varying optical properties is to manipulate the emission wavelength by adjusting the synthesis conditions. Currently, effective strategies for preparing carbon dots with tunable fluorescence primarily include selecting different precursors, adjusting the reaction solvent, and adjusting the temperature. However, these strategies are often laborious and require complex procedures.

[0006] Therefore, developing a simple and controllable preparation method with easily available raw materials to prepare luminescent carbon dots, especially red-emitting carbon dots, is a problem that needs to be solved in this field. 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, wherein 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: react 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 dopant can 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 is to use 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, and n-hexane and chloroform;

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

[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: react the precursor reaction liquid in a reactor for 2-10 hours to obtain.

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

[0029] The present invention provides a simple and controllable multi-color 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 multi-color luminescent carbon dots were successfully isolated. The carbon dots provided by the present invention can realize controllable regulation of fluorescence properties 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 to achieve controllable synthesis of high-performance carbon dots. The multi-color 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, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0031] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 a is a schematic diagram of the synthesis of multi-color 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, Figure 1 gk are the fluorescence emission spectra of the carbon dots prepared in Examples 1 to 5;

[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 carbon dots, Figure b is the absorption spectrum of yellow carbon dots, Figure c is the absorption spectrum of red carbon dots, Figure d is the emission spectrum of green carbon dots, Figure e is the emission spectrum of yellow carbon dots, and Figure f is the emission spectrum of red carbon dots.

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

[0035] Figure 4 Figure 1 is an overlay of 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 an overlay of D-CDs, Figure b is an overlay of G-CDs, Figure c is an overlay of Y-CDs, and Figure d is an overlay of O-CDs.

[0036] Figure 5 Figure 1 is an overlay of the excitation and absorption spectra of the multicolor luminescent carbon dots prepared in Example 1 and Example 5; Figure a is an overlay of D-CDs, Figure b is an overlay of G-CDs, Figure c is an overlay of Y-CDs, and Figure d is an overlay of O-CDs.

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

[0038] Figure 7 Absolute quantum yield spectra of the multicolor luminescent carbon dots prepared in Examples 1 and 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 8Transmission electron micrographs of the multicolor luminescent carbon dots prepared in Examples 1 and 5; Figure a is a transmission electron micrograph of D-CDs, Figure b is a transmission electron micrograph of G-CDs, Figure c is a transmission electron micrograph of Y-CDs, and Figure d is a transmission electron micrograph of O-CDs;

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

[0041] Figure 10 Figure 1 is a structural characterization diagram of the multi-color 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 transform infrared spectrum diagram, and Figure d is an XPS full spectrum diagram;

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

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

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

[0045] In the following examples and experimental examples, reagents and materials not otherwise specified are commercially available.

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

[0047] The method for preparing multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate the bright green fluorescent component.

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

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

[0057] The method for preparing multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 4 h. After the reaction was completed, the reactor was naturally cooled 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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate two green and one yellow fluorescent components.

[0065] (3) The obtained fluorescent components were placed in dialysis bags with a cutoff of 3000 Da and dialyzed in deionized water for 72 h to remove the unreacted organic solution. The initial products in the dialysis bags were collected and placed in a freeze dryer for 24 h 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 multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 h. After the reaction was completed, the reactor was naturally cooled 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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate the bright green, yellow, and orange fluorescent components.

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

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

[0077] The method for preparing multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 h. After the reaction was completed, the reactor was naturally cooled 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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate the bright green, yellow, and orange fluorescent components.

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

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

[0087] The method for preparing multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate the bright green, yellow, and orange fluorescent components.

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

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

[0097] The method for preparing multi-color luminescent carbon dots provided in this embodiment is as follows: Figure 1 As shown in a, the following steps are included:

[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 h. After the reaction was completed, the reactor was naturally cooled 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 was separated by silica gel chromatography column; during the separation process, a 5W 365nm ultraviolet lamp was used for observation, and the fluorescent components were collected according to the fluorescent color of the product. At the same time, the ratio of dichloromethane and methanol in the eluent was adjusted to better separate the fluorescent components. The specific operation of adjusting the eluent was as follows: using dichloromethane and methanol as eluents, first using pure dichloromethane as the eluent to allow the product with less polarity to flow out first, and then gradually increasing methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:1, thereby adjusting the polarity of the eluent to better separate the bright green, yellow, and orange fluorescent components.

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

[0106] The technical solution of the present invention is further illustrated by experiments below. The samples D-CDs, G-CDs, Y-CDs and O-CDs tested in the following experimental examples were 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 1-6 were subjected to UV absorption and fluorescence emission spectroscopy tests. The multicolor fluorescent carbon dots prepared in Examples 1 and 5 were subjected to emission (Em) spectroscopy (excitation wavelength), excitation (Ex) spectroscopy, time-resolved photoluminescence (TRPL) decay curve tests, and quantum yield tests.

[0110] Emission (Em) spectrum test:

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

[0112] 3. Excitation (Ex) spectrum test

[0113] The multicolor fluorescent carbon dots prepared in Example 1 and Example 5 were subjected to emission (Em) spectrum tests 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 FIG. Figure 2 shown.

[0117] The emission peaks of the green carbon dots D-CDs and G-CDs prepared in Examples 1-5 were both around 510 nm. However, the absorption peaks of the green carbon dots D-CDs prepared in Examples 1 and 2 differed from those of the green carbon dots G-CDs prepared in Examples 2-5, specifically in the low-energy region of the UV absorption curve. This indicates that D-CDs and G-CDs are two different types of 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, by regulating the reaction time during the solvent thermal treatment process, 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.

[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 exhibit similar emission spectra to those of Example 6. Their UV-visible absorption spectra also display similar absorption bands. Their excitation spectra share nearly identical 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 methods of this 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 ultraviolet 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 the formation of a carbon core with a larger conjugated structure. In the low-energy ultraviolet 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 in Figure 2, the maximum emission wavelength of D-CDs and G-CDs is 510 nm, with optimal excitation wavelengths of 420 nm and 380 nm, respectively. For Y-CDs and O-CDs, the maximum emission wavelengths are 560 nm and 600 nm, respectively, with optimal excitation wavelengths of 460 nm and 480 nm, respectively. All exhibit excitation-independent emission characteristics, meaning their emission peaks do not shift when the excitation wavelength changes.

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

[0129] The test results of Example 1 and Example 5 are as follows Figure 5 As shown in Figure 3, 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 with 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 using an integrating sphere at the optimal excitation wavelength, and O-CDs had the highest QY of 23.68%.

[0134] The above results show that by regulating the reaction time during the solvent thermal treatment process, the present invention can prepare two green-light carbon dots (D-CDs, G-CDs), one yellow-light carbon dot (Y-CDs), and one red-light carbon dot. The green-light carbon dots D-CDs can be further converted into multi-color 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 helps 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, XRD, Raman, Fourier transform infrared spectroscopy (FT-IR), XPS, and NMR tests, respectively.

[0138] 2. Experimental Results

[0139] (1) Transmission electron microscopy test results

[0140] The test results are as follows Figure 8 、 9 As shown in Figure 3, the carbon dots are uniform and well dispersed, with average particle sizes of approximately 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] The test results are as follows Figure 10 As shown in a. After undergoing continuous polymerization and carbonization processes, a more ordered structure is gradually formed from D-CDs to O-CDs.

[0143] (3) Raman test results

[0144] The test results are as follows Figure 10 b. At 1348 and 1587 cm -1There are two peaks at the center, corresponding to disordered structures or defects (D band) and graphitic carbon domains (G band), respectively. The intensities 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 the sp2-domains gradually increases.

[0145] (4) FT-IR test results

[0146] The test results are as follows Figure 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 At 742 cm, CH in aromatic benzene -1 The carbon dots formed conjugated aromatic structures and oxygen- and nitrogen-containing surface functional groups during the reaction. Furthermore, the stretching vibrations of =CH, C=N, and C=C increased significantly from D-CDs to O-CDs, indicating that the aromatic rings of the carbon dots gradually carbonized and formed larger conjugated structures. The stretching vibrations of CN and CO increased significantly from D-CDs to O-CDs, and some new functional groups were formed, indicating that O-CDs possessed more oxygen- and nitrogen-containing groups. The stretching vibration of -SCN decreased significantly from D-CDs to O-CDs. Fourier transform infrared spectroscopy revealed that all carbon dots possessed similar surface functional groups and chemical bonds, but reaction time altered the content of these functional groups and generated some new functional groups.

[0147] (5) XPS test results

[0148] The test results are as follows Figure 10d and 11. All C-dots are composed of C, O, N, and S elements. Compared to D-CDs, G-, Y-, and O-CDs have significantly higher C contents, indicating a higher degree of carbonization. Furthermore, O-CDs have higher N contents than G- and Y-CDs, while the relative contents of O and S elements are similar. High-resolution C1s XPS spectra can be fitted to three C groups: 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 a higher CN / CO content, indicating a greater number of surface functional groups. For N1s, the high-resolution XPS spectra can be converted to 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 primary driver of the green fluorescence of the Cdots. The relative content of graphitized N increases significantly from D- and G-CDs to Y- and O-CDs, indicating that graphitized N significantly influences the red-shift of the Cdot emission wavelength. The high-resolution XPS spectrum of O1s contains two components associated with 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. These results indicate that the Cdots possess a large conjugated sp 2 The carbon core of the domains and the rich oxygen / nitrogen-containing surface groups indicate that when their maximum emission wavelengths red-shift, both the conjugated structure and the graphitic N content increase, while the pyridinic N content decreases.

[0149] (6) NMR test results

[0150] The test results are as follows Figure 12 All carbon dots 1 The H NMR spectra all showed obvious aromatic hydrogen signals in the range of 7.5-8.5 ppm ( Figure 12 b) In addition, 13 C NMR spectroscopy ( Figure 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 There are fewer C conjugated structures.

[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. 4 The cells were seeded at a density of 100 μg / well in a 96-well cell culture plate and cultured in a 37°C and 5% CO2 incubator for 24 hours. The DMEM medium was aspirated and 5% CO2 was added to the wells of a 96-well cell culture plate. -1 ) in fresh DMEM medium and culture for an additional 24 hours. Subsequently, the medium was aspirated and the cells were treated with 10% CCK-8 (100 μL per well) and cultured for an additional hour (37°C, 5% CO2). Finally, the absorbance of CCK-8 was recorded at 450 nm using a microplate reader. Cell viability was calculated based on the OD values ​​of the experimental and control groups.

[0156] 2. Cell Imaging Experiment

[0157] MG63 cells (1×10 4 Cells were seeded into 96-well cell culture plates (100 μL per well) and incubated in 100 μL of DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics in a 37°C, 5% CO2 incubator for 24 hours. The DMEM medium was then aspirated, and medium containing carbon dots (20 μg / mL) was added, and the cells were incubated for 1 hour. Subsequently, the medium containing the material was aspirated, and the cells were washed three times with PBS buffer to remove any carbon dots that were not taken up by the cells. 100 μL of PBS buffer was then added to each well. Fluorescence imaging of the cells was performed using an inverted microscope.

[0158] 2. Experimental Results

[0159] 1. Cytotoxicity results

[0160] Cytotoxicity results such as Figure 13 As shown in a. As can be seen from the figure, 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 Figure 13 As shown in b and c, it can be seen from the figures 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] It can be seen from the above embodiments and experimental examples that the present invention provides a simple, controllable multi-color luminescent carbon dot and its preparation method and application. By adjusting the reaction time during the thermal treatment of o-phenylenediamine and thiourea solvent, multi-color luminescent carbon dots were successfully isolated. The carbon dots provided by the present invention can achieve controllable regulation of fluorescence properties by regulating the degree of conjugation of the carbon core and the content of graphitic nitrogen. The present invention has given us 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 to achieve controllable synthesis of high-performance carbon dots. The multi-color 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 method for preparing luminescent carbon dots, characterized in that: It includes: Step 1: dissolving o-phenylenediamine and thiourea in a mixed solvent of N,N-dimethylformamide, water and ethanol to prepare a precursor reaction solution; The feed ratio of o-phenylenediamine to thiourea is 4:1 by mass, the volume ratio of N,N-dimethylformamide to water and ethanol in the mixed solvent is 3:1:1, and the ratio of o-phenylenediamine to the mixed solvent is 2 g:50 mL; Step 2: react the precursor reaction solution in a reactor at 200° C. for 4-10 hours, and perform separation and purification; The separation and purification method includes sequentially using filtration, column chromatography, and dialysis; The filtration method is to filter using a 0.22-0.45 μm filter membrane; The column chromatography method uses silica gel column chromatography for separation and purification. The specific operation of adjusting the eluent is as follows: using dichloromethane and methanol as eluents, first using dichloromethane as the eluent to allow the less polar product to flow out first, and then gradually increasing the proportion of methanol to control the ratio of dichloromethane to methanol from 100:1 to 20:

1. During the separation and purification process using silica gel column chromatography, the fluorescence color is monitored using a 365nm ultraviolet lamp to collect the products respectively; The dialysis method is to use a dialysis bag with a molecular weight cut-off of 3000Da and dialyze in water.

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