Nitrogen-doped fluorescent carbon dots for trivalent iron detection and their preparation method and application

Abstract

The present invention discloses a nitrogen-doped fluorescent carbon dot for trivalent iron detection and its preparation method and application, which belongs to the field of chemical detection technology. The nitrogen-doped fluorescent carbon dot is prepared by a hydrothermal method using erythromycin and L-isoleucine as raw materials. The present invention prepares red nitrogen-doped fluorescent carbon dots (N-CDs) with a high yield (70%) by hydrothermal treatment of L-isoleucine and erythromycin. The obtained N-CDs have a strong affinity for Fe 3+ With high selectivity and sensitivity, it has broad application prospects in sensors, detection platforms and environmental monitoring systems. Based on the excellent biocompatibility and low toxicity of N-CDs, it can be successfully used for multi-color cell imaging, showing great potential in the biomedical field.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and more particularly relates to nitrogen-doped fluorescent carbon dots for trivalent iron detection, and a preparation method and application thereof. Background Art

[0002] Iron (Fe) is one of the essential trace elements for human body, with a content of about 4-5 grams, mainly in the form of Fe 3+ Iron participates in various life activities in the form of iron. The main function of iron is to participate in the formation of hemoglobin and promote hematopoiesis. Its content in hemoglobin is about 72%. Studies have found that insufficient or excessive iron in the body can lead to serious diseases. Excessive intake can lead to an imbalance in the body's redox system, damage DNA, induce mutations, and increase the risk of cancer. However, insufficient intake can lead to diseases such as anemia, weakened immunity, and developmental disorders. In addition, Fe 3+ It is also one of the main culprits of water pollution. Therefore, the detection of Fe in the external environment and in the body is 3+ It is of great significance to human health.

[0003] In recent years, carbon dots (CDs) have been widely used as bioimaging markers and sensors for metal ions for quantitative detection of metal ions. Most carbon dots have shown great potential in different types of cell bioimaging. CDs as fluorescent probes are characterized by rapidity, simplicity, high specificity and high sensitivity. The detection principle is mainly based on the chelation of functional groups on the surface of CDs with metal ions to produce electron transfer, thereby increasing or decreasing the fluorescence intensity of CDs. Therefore, the sensitivity and selectivity of the sensor depend on the affinity between the active functional groups on the surface of CDs and the target metal ions. It is crucial to improve or construct active functional groups on the surface of CDs by doping heteroatoms to achieve specific recognition of target ions. In addition, doping heteroatoms can also greatly improve the fluorescence quantum yield of CDs. However, the light emitted by current CDs is mainly in the blue or green region, while blue nitrogen-doped fluorescent carbon dots and green nitrogen-doped fluorescent carbon dots have low sensitivity to Fe 3+ The selectivity and sensitivity are poor, and Fe 3+ Therefore, a method for detecting Fe 3+ The nitrogen-doped fluorescent carbon dots are of great significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a nitrogen-doped fluorescent carbon dot for trivalent iron detection and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art and realize Fe 3+ The nitrogen-doped fluorescent carbon dots obtained by the present invention can also be used for multi-color cell imaging, and have great application potential in the fields of sensing and biomedicine.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is to provide a Fe 3+ The nitrogen-doped fluorescent carbon dots are used for detection. The nitrogen-doped fluorescent carbon dots are prepared by a hydrothermal method using erythromycin and L-isoleucine as raw materials.

[0007] The present invention prepares nitrogen-doped fluorescent carbon dots by using erythromycin and L-isoleucine. The nitrogen-doped fluorescent carbon dots only react with Fe after adding different metal ions. 3+ There is a significant response, indicating that it is sensitive to Fe 3+ It has high selectivity and sensitivity, and its fluorescence intensity is completely dependent on Fe 3+ The dose of Fe 3+ , and can also be used for multi-color cell imaging.

[0008] The second technical solution of the present invention is to provide the Fe 3+ The preparation method of nitrogen-doped fluorescent carbon dots for detection comprises the following steps:

[0009] Erythromycin, L-isoleucine and water are mixed and subjected to hydrothermal reaction to obtain the Fe 3+ Nitrogen-doped fluorescent carbon dots were used for detection.

[0010] Preferably, the usage ratio of erythromycin, L-isoleucine and water is 0.1-0.3 g:0.1-0.3 g:10 mL.

[0011] Preferably, the temperature of the hydrothermal reaction is 160-230° C., and the time of the hydrothermal reaction is 2-6 hours.

[0012] During the preparation process, hydrothermal reaction conditions such as temperature, time, and reactant concentration have a significant impact on the size and dispersibility of carbon dots. Appropriate reaction conditions can control the size and fluorescence properties of carbon dots, achieving good monodispersity within the nanoscale. Excessively intense hydrothermal reaction conditions or excessively high reactant concentrations can cause carbon dots to grow too quickly, become oversized, and easily aggregate, thus affecting their fluorescence properties and application effectiveness. Excessively mild hydrothermal reaction conditions or excessively low reactant concentrations can prevent carbon dots from forming, similarly impacting their fluorescence properties and application effectiveness.

[0013] Preferably, after the hydrothermal reaction is completed, a post-treatment step is further included; the post-treatment includes: centrifuging and dialysis the product obtained from the hydrothermal reaction in sequence.

[0014] Preferably, the centrifugal speed is 5000-12000 r / min, and the centrifugal time is 5-10 min; the molecular weight cut-off of the dialysis bag used for dialysis is 500-1000 Da.

[0015] Preferably, the Fe 3+ The nitrogen-doped fluorescent carbon dots used for detection are red nitrogen-doped fluorescent carbon dots; the Fe 3+ The nitrogen-doped fluorescent carbon dots used for detection are in solid or liquid state.

[0016] Furthermore, the Fe 3+ Nitrogen-doped fluorescent carbon dots for detection are in liquid state, which can be freeze-dried to obtain solid Fe 3+ Nitrogen-doped fluorescent carbon dots were used for detection.

[0017] Preferably, the freeze-drying temperature is -80 to -50°C, and the time is 24 to 52 hours.

[0018] The third technical solution of the present invention is to provide the Fe 3+ Detection of Nitrogen-doped Fluorescent Carbon Dots for Cell Imaging.

[0019] The fourth technical solution of the present invention is to provide the Fe 3+ Detection of Nitrogen-doped Fluorescent Carbon Dots on Fe 3+ Application in detection.

[0020] The fifth technical solution of the present invention is to provide a method for increasing Fe 3+ The method of detecting selectivity and sensitivity is carried out by the above-mentioned Fe 3+ Nitrogen-doped fluorescent carbon dots were used for detection.

[0021] The present invention discloses the following technical effects:

[0022] The present invention prepares high yield (70%) red nitrogen-doped fluorescent carbon dots (N-CDs) by hydrothermal treatment of L-isoleucine and erythromycin. The obtained N-CDs have a strong affinity for Fe 3+ With high selectivity and sensitivity, they have broad application prospects in sensors, detection platforms, and environmental monitoring systems. Based on the excellent biocompatibility and low toxicity of N-CDs, they can be successfully used for multicolor cell imaging, and the resulting N-CDs have great potential in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM image of N-CDs prepared in Example 1;

[0024] Figure 2 HRTEM image of N-CDs prepared in Example 1;

[0025] Figure 3 This is the particle size distribution diagram of N-CDs prepared in Example 1;

[0026] Figure 4The fluorescence spectrum and UV-visible absorption spectrum of N-CDs prepared in Example 1, wherein a is the fluorescence spectrum and b is the UV-visible absorption spectrum;

[0027] Figure 5 Surface functional group characterization diagram of N-CDs prepared in Example 1, where a is an infrared spectrum, b is an XRD pattern, c is an XPS spectrum, and d is a C1s spectrum;

[0028] Figure 6 Figure 2 is the relationship between the absorbance and fluorescence integrated intensity of Rhodamine B and N-CDs at an excitation wavelength of 495 nm, where a is Rhodamine B and b is N-CDs;

[0029] Figure 7 Graphs showing the fluorescence intensity of N-CDs obtained in Examples 1 to 12;

[0030] Figure 8 Graphs showing the optical stability test results of N-CDs obtained in Example 1, wherein a is a graph showing the relationship between pH value and fluorescence intensity, b is a graph showing the relationship between potassium chloride solution concentration and fluorescence intensity, c is a graph showing the relationship between exposure time and fluorescence intensity, and d is a graph showing the relationship between storage time and fluorescence intensity.

[0031] Figure 9 The N-CDs Fe obtained in Example 1 3+ Selectivity test results diagram, where a is the 3+ The fluorescence intensity difference of N-CDs solution in the presence of Fe and other metal ions (F-F0), b is the ... 3+ The effect on the fluorescence intensity of N-CDs, c is the fluorescence intensity difference between N-CDs solution (F-F0) and Fe 3+ concentration, d is the fluorescence intensity difference (F-F0) of N-CDs solution and Fe 3+ Linear relationship graph of concentration;

[0032] Figure 10 The figure shows the cytotoxicity of N-CDs obtained in Example 1 measured by MTT assay;

[0033] Figure 11 These are the cell imaging effect diagrams of N-CDs obtained in Example 1, where a is the cell image when excited by a 488 nm light source, b is the cell image when excited by a 543 nm light source, c is the cell image under bright field, d is the cell image under the combined field of a and c, and e is the cell image under the combined field of b and c. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The present invention first describes the background of fluorescence sensors and carbon dots (CDs):

[0040] Fluorescent sensors have attracted considerable attention due to their simplicity, rapidity, high selectivity, sensitivity, and high spatial resolution. Furthermore, fluorescence-based imaging techniques can be used to visually monitor metal ions in cells and organisms. Currently, the most commonly used fluorescent sensors include fluorescent metal nanoclusters, organic dyes, semiconductor quantum dots, and fluorescent metal-organic frameworks. However, in practical applications, the application of fluorescent sensors is challenged by their photostability, complex equipment and processing techniques, environmental contamination, and cytotoxicity.

[0041] Carbon dots (CDs) are a new type of luminescent carbon nanomaterial. Due to their unique physicochemical properties, they exhibit broad application potential and research value in multiple fields. CDs are zero-dimensional nanoparticles with a diameter of less than 10 nm. They possess advantages such as simple synthesis, excellent optical sensitivity and stability, low toxicity, and high physiological clearance. Compared with traditional semiconductor quantum dots and organic dyes, CDs overcome the shortcomings of low water solubility, difficulty in functionalization, and high toxicity. Currently, CDs have been widely used in fields such as ion sensing, catalysis, optoelectronic devices, anti-counterfeiting, bioimaging, and drug delivery.

[0042] The L-isoleucine (C6H 13 NO2) and erythromycin (C 37 H 67 NO 13 ) was purchased from Tianjin Dengfeng Chemical Co., Ltd.

[0043] The reagents used in the test examples of the present invention and their manufacturers are as follows:

[0044] Disodium hydrogen phosphate and potassium dihydrogen phosphate were purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.;

[0045] Rhodamine B was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0046] High glucose medium (DMEM) was purchased from Shanghai Biono Biotechnology Co., Ltd.;

[0047] Fetal bovine serum (FBS) was purchased from Hangzhou Siqi Bioengineering Co., Ltd.;

[0048] Thiazolyl blue (MTT), green streptomycin mixture, trypsin-EDTA digestion solution, and formalin (PFA) were purchased from Beijing Solaibao Technology Co., Ltd.;

[0049] Various metal cations (Cr 3+ , Pb 2+ , Ca 2+ 、Fe 3+ 、Bi 3+ , K + 、Na + 、Cu 2+ 、Ba 2+ 、Mn 2+ 、Ni 2+ Mg 2+ 、Cd 2+ 、Zn 2+ 、Al 3+ 、Co 2+ ), prepared from the chlorides of various metal cations (Tianjin Third Chemical Reagent Factory).

[0050] Unless otherwise specified, other raw materials and test reagents are commercially available products.

[0051] The dialysis bag used in the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a specification of 500-1000 Da (molecular weight cut-off).

[0052] The DMEM culture medium used in the present invention is prepared by mixing a double-antibody (penicillin-streptomycin) solution, fetal bovine serum, and DMEM high-glucose culture medium in a volume ratio of 1:10:90, wherein the final concentration of penicillin is 100 U / mL and the final concentration of streptomycin is 100 μg / mL.

[0053] Example 1

[0054] This embodiment provides a method for preparing red nitrogen-doped fluorescent carbon dots (N-CDs), specifically:

[0055] Dissolve 0.2g of L-isoleucine and 0.2g of erythromycin in a 20mL beaker and add 10mL of deionized water. Transfer the mixture to a 50mL hydrothermal autoclave and allow to react in a 200°C electric blower drying oven for 3 hours. After the reaction is complete, cool to room temperature, remove the reaction mixture, and centrifuge at 8000 rpm for 8 minutes to remove large solid particles. The supernatant is then dialyzed through a dialysis bag for 48 hours to obtain a pure N-CDs aqueous solution. To obtain N-CDs powder, freeze-dry the aqueous solution at -60°C for 48 hours, achieving a yield of up to 70%.

[0056] The N-CDs obtained by the invention are easily soluble in water and emit bright red fluorescence under ultraviolet light.

[0057] The obtained N-CDs were characterized by the following instruments:

[0058] Transmission electron microscopy (TEM) images were obtained using a FEI Tecnai G2 F20 instrument; fluorescence spectra were measured using an F-280 fluorescence spectrometer (Tianjin Gangdong Technology Development Co., Ltd.); UV-visible absorption spectra were obtained using a Cary 50Bio-UV-vis spectrometer (Varian, USA); infrared spectra were obtained using an FTIR-8400S (Shimadzu, Japan) spectrometer; X-ray diffraction spectra were obtained using a Rigaku Miniflex 600; and X-ray photoelectron spectroscopy (XPS) was obtained using a ThermoScientific K-Alpha electron spectrometer. The results are shown in Figure 2. Figures 1 to 5 shown.

[0059] Figure 1 TEM image of N-CDs prepared in Example 1; Figure 2 HRTEM image of N-CDs prepared in Example 1. Figure 1 and Figure 2 It can be seen that the present invention successfully prepared N-CDs using erythromycin and L-isoleucine; the obtained N-CDs were spherical and well dispersed; and the N-CDs had a crystalline structure, indicating that the N-CDs had a graphite-like structure.

[0060] Figure 3 The particle size distribution diagram of N-CDs prepared in Example 1. Figure 3 It can be seen that the average particle size of the obtained N-CDs is 4.6 nm.

[0061] Figure 4 The fluorescence spectrum and UV-visible absorption spectrum of N-CDs prepared in Example 1 are shown in Figures a and b, respectively, where a is the fluorescence spectrum and b is the UV-visible absorption spectrum. Figure 4 It can be seen from the fluorescence spectrum ( Figure 4 a) It can be seen that as the excitation wavelength gradually increases, the fluorescence intensity of N-CDs first increases and then decreases, and the emission wavelength red-shifts. This characteristic of fluorescence being highly dependent on the excitation wavelength is crucial for its application in the biomedical field. UV-visible absorption spectrum of N-CDs ( Figure 4 b) shows an absorption peak centered at 510 nm, attributed to the n-π transition on the molecular surface. The optimal emission wavelength of N-CDs is 608 nm, and the optimal excitation wavelength is 540 nm.

[0062] Figure 5 The surface functional group characterization diagram of N-CDs prepared in Example 1, wherein a is an infrared spectrum, b is an XRD pattern, c is an XPS spectrum, and d is a C1s spectrum. Figure 5 It can be seen that the infrared spectrum shows that the surface of N-CDs mainly contains OH / NH (3421cm -1 )、CH(2965cm -1 )、C=O(1593cm -1 ) and C=C(1503cm -1 ) and other functional groups. The XPS spectrum shows three peaks at 285.1, 401.1 and 531.1 eV, which are C1s, N1s and O1s ( Figure 5 c). High-resolution spectrum of C1s ( Figure 5 d) shows the presence of three different types of carbon, with signals at 284.6, 285.7, and 287.9 ​​eV corresponding to C=C / CC, CO / CN, and C=O, respectively. The XPS spectra are consistent with the FTIR spectra, indicating that the nitrogen element was successfully doped into the CDs.

[0063] The fluorescence quantum yield of the obtained N-CDs was measured using rhodamine B as a reference solution. To prevent the reabsorption effect, a series of N-CDs solutions and reference solutions (using methanol as the solvent) with different concentrations were prepared. By adjusting the concentration, the absorbance value at 495nm was between 0 and 0.1. The fluorescence quantum yield of N-CDs (Φ S ) According to the formula: Φ S =Φ R (Grad S / Grad R )·(η 2 S / η 2 R ) is determined, where Φ R is the fluorescence quantum yield of the reference solution, Grad S is the gradient (slope) of the N-CDs fluorescence integral intensity and absorbance, Grad R is the gradient (slope) of the integrated fluorescence intensity and absorbance of the reference solution, η S is the refractive index of N-CDs solvent, η R is the refractive index of the reference solution solvent. Figure 6 and as shown in Table 1.

[0064] Table 1 Absorbance and fluorescence integrated intensity data under 360nm excitation

[0065]

[0066] Figure 6 The graph shows the relationship between the absorbance and fluorescence integrated intensity of Rhodamine B and N-CDs at an excitation wavelength of 495 nm, where a is Rhodamine B and b is N-CDs. Figure 6 As shown in Table 1, the fluorescence quantum yield of N-CDs was 9.2% when Rhodamine B (QY=89%) was used as a reference solution.

[0067] Example 2

[0068] The difference from Example 1 is that the hydrothermal treatment temperature is adjusted to 160° C., and the rest is the same as Example 1.

[0069] Example 3

[0070] The difference from Example 1 is that the hydrothermal treatment temperature is adjusted to 180° C., and the rest is the same as Example 1.

[0071] Example 4

[0072] The difference from Example 1 is that the hydrothermal treatment temperature is adjusted to 220° C., and the rest is the same as Example 1.

[0073] Example 5

[0074] The difference from Example 1 is that the hydrothermal treatment time is adjusted to 2 hours, and the rest is the same as Example 1.

[0075] Example 6

[0076] The difference from Example 1 is that the hydrothermal treatment time is adjusted to 4 hours, and the rest is the same as Example 1.

[0077] Example 7

[0078] The difference from Example 2 is that the hydrothermal treatment time is adjusted to 2 hours, and the rest is the same as Example 2.

[0079] Example 8

[0080] The difference from Example 2 is that the hydrothermal treatment time is adjusted to 4 hours, and the rest is the same as Example 2.

[0081] Example 9

[0082] The difference from Example 3 is that the hydrothermal treatment time is adjusted to 2 hours, and the rest is the same as Example 3.

[0083] Example 10

[0084] The difference from Example 3 is that the hydrothermal treatment time is adjusted to 4 hours, and the rest is the same as Example 3.

[0085] Example 11

[0086] The difference from Example 4 is that the hydrothermal treatment time is adjusted to 2 hours, and the rest is the same as Example 4.

[0087] Example 12

[0088] The difference from Example 4 is that the hydrothermal treatment time is adjusted to 4 hours, and the rest is the same as Example 4.

[0089] Figure 7 The fluorescence intensity diagram of N-CDs obtained in Examples 1 to 12 is shown in FIG. Figure 7 It can be seen that the hydrothermal treatment temperature and time have a significant effect on the fluorescence intensity of N-CDs, and the optimal hydrothermal treatment conditions for preparing N-CDs are 200℃ and 3h.

[0090] Test Example 1

[0091] The optical stability of the N-CDs obtained in Example 1 was tested:

[0092] Test method: Adjust the pH value of the environment in which N-CDs are located within the range of 2 to 12, and measure the relationship between pH value and fluorescence intensity; place N-CDs in potassium chloride solutions of different concentrations, and measure the relationship between potassium chloride solution concentration and fluorescence intensity; expose N-CDs to light at room temperature (365nm, 16W) for 4 hours, and measure the relationship between exposure time and fluorescence intensity; store N-CDs in a refrigerator (5°C) for 3 months, and measure the relationship between storage time and fluorescence intensity. Figure 8 shown.

[0093] Figure 8 The test results of the optical stability of N-CDs obtained in Example 1 are shown in Figure a, where a is the relationship between pH value and fluorescence intensity, b is the relationship between potassium chloride solution concentration and fluorescence intensity, c is the relationship between exposure time and fluorescence intensity, and d is the relationship between storage time and fluorescence intensity. Figure 8 It can be seen that the fluorescence intensity of N-CDs remains basically unchanged in the pH range of 2 to 12. Similarly, different concentrations of potassium chloride solution will not affect the fluorescence intensity of N-CDs. Figure 8 c and Figure 8 d shows that the fluorescence intensity of N-CDs is largely unaffected even after continuous exposure to light (365 nm, 16 W) for 4 h at room temperature and storage for 3 months. These results demonstrate that N-CDs have excellent stability and resistance to photobleaching, which is of great significance for their application in the biological field.

[0094] Test Example 2

[0095] Test Example 1 obtained N-CDs Fe 3+ Selectivity:

[0096] Test method: First, 400 μL of N-CDs aqueous solution (0.5 mg / mL) was added to 2000 μL of PBS (pH = 5) buffer solution, and its original fluorescence intensity (denoted as F0) was measured. Then, various metal cations (Cr 3+ , Pb 2+ , Ca 2+ 、Fe 3+ 、Bi 3+ , K + 、Na + 、Cu 2+ 、Ba 2+ 、Mn 2+ 、Ni 2+ Mg 2+ 、Cd 2 + 、Zn 2+ 、Al 3+ 、Co 2+) 3 μL each, and the fluorescence intensity (denoted as F) was measured after 10 seconds of reaction. 3+ For the sensitivity test, under the same conditions as above, first measure the original fluorescence intensity (denoted as F0), then add Fe 3+ The solution was prepared and its concentration was gradually increased (0-150 μmol / L). The fluorescence intensity at each concentration was measured and recorded (denoted as F). The above operation was repeated three times. The excitation wavelength was set to 540 nm, and the excitation and emission slit widths were both set to 5 nm. The results are shown in Figure 2. Figure 9 shown.

[0097] Figure 9 The N-CDs Fe 3+ Selectivity test results diagram, where a is the selectivity of Fe 3+ The fluorescence intensity difference of N-CDs solution in the presence of Fe and other metal ions (F-F0), b is the ... 3+ The effect on the fluorescence intensity of N-CDs, c is the fluorescence intensity difference between N-CDs solution (F-F0) and Fe 3+ concentration, d is the fluorescence intensity difference (F-F0) of N-CDs solution and Fe 3+ The linear relationship between the concentration of Figure 9 It can be seen that adding Fe 3+ The fluorescence intensity of N-CDs can be significantly enhanced, while the addition of other metal ions has almost no effect on the fluorescence intensity of N-CDs, indicating that N-CDs have a strong effect on Fe 3+ Further sensitivity experiments showed that the fluorescence intensity of N-CDs depends on Fe 3+ The dose ( Figure 9 b) With Fe 3+ With the increase of Fe 3+ Applicability of fluorescent probes. Figure 9 c shows the fluorescence intensity of N-CDs and Fe 3+ The relationship between the concentrations is linear in the range of 2.1 to 37.5 μM. Figure 9 d) According to the detection limit formula LOD = 3σ / S (σ is the signal-to-noise ratio of the blank sample, S is the linear slope), the N-CDs to Fe 3+ The detection limit was 0.17 μM.

[0098] Test Example 3

[0099] The N-CDs obtained in Example 1 were tested for Fe in actual samples. 3+ The test results:

[0100] Test method: First, centrifuge tap water at 8000 r / min for 8 minutes to remove large particles, then filter through a 0.45 μm pore size filter membrane. Dilute 400 μL of N-CDs aqueous solution (0.5 mg / mL) with 2000 μL of PBS (pH = 5) buffer solution and measure the initial fluorescence intensity (denoted as F0). 3+ The tap water sample of the standard solution was added to the N-CDs solution (Fe 3+ The final concentrations of Fe were: 8.33, 16.67 and 25 μM, and the fluorescence intensity was measured again (denoted as F). 3+ The concentration can be calculated according to the linear equation. The results are shown in Table 2.

[0101] Table 2 Effects of N-CDs on Fe in tap water samples 3+ Detection

[0102]

[0103] As shown in Table 2, the N-CDs obtained in the present invention are suitable for Fe 3+ Quantitative detection of .

[0104] Test Example 4

[0105] Evaluation of the cytotoxicity of N-CDs obtained in Example 1:

[0106] The method is as follows: HeLa cells were seeded into a 96-well culture plate for 18 hours to allow complete attachment. The DMEM medium was then removed and the cells were washed three times with PBS (pH 7.4). The cells were then incubated with fresh DMEM containing varying concentrations of N-CDs for 24 hours. The old DMEM medium was again removed from the culture plate and washed three times with PBS (pH 7.4). 200 μL of fresh DMEM containing 20 μL of MTT (5 mg / mL) was then added, and the cells were incubated at 37.5°C for another 5 hours. Finally, the supernatant was aspirated, and 150 mL of DMSO was added to the cells, allowing them to rest for 10 minutes. The absorbance at 490 nm was recorded using a microplate reader. The results are shown in Figure 10.

[0107] Figure 10 The figure shows the cytotoxicity of N-CDs obtained in Example 1 measured by MTT method. Figure 10 It can be seen that after incubating HeLa cells with different concentrations of N-CDs for 24 hours, the cell survival rate remained above 85% even at a very high concentration (400 μg / mL) of N-CDs, indicating that N-CDs have low toxicity and excellent biocompatibility.

[0108] Test Example 5

[0109] The cell imaging effect of the N-CDs obtained in Test Example 1 was as follows:

[0110] Test method: HeLa cells (density 1×10 5 / mL) were inoculated into a 35mm culture dish containing DMEM medium and incubated in a constant temperature cell culture incubator at 37.5°C overnight. The old DMSO medium was removed and new DMEM medium containing 500μg / mL N-CDs was added to continue culturing HeLa cells for 2h. After the incubation, the medium was removed and washed three times with 1mL of PBS buffer with pH=7.4 to remove free N-CDs particles. Then, a new 1mL of PBS buffer with pH=7.4 was added and the cells were immediately placed under a laser scanning confocal microscope (LSCM; Leica TCS SP5, Germany) for cell imaging and photography. The results are shown in Figure 2. Figure 11 shown.

[0111] Figure 11 The cell imaging effect diagram of N-CDs obtained in Example 1, wherein a is the cell image when excited by a 488nm light source, b is the cell image when excited by a 543nm light source, c is the cell image under bright field, d is the cell image under the combined field of a and c, and e is the cell image under the combined field of b and c. Figure 11 As shown, N-CDs enter the HeLa cell nuclei and are evenly distributed. Due to the excitation-dependent emission characteristics of N-CDs, multicolor fluorescence images can be obtained by selecting different lasers. When excited by 488nm and 543nm light sources, green and red fluorescence cell images can be obtained. These results demonstrate that N-CDs have promising application prospects in cell imaging.

[0112] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0113] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kind of Fe 3+ Nitrogen-doped fluorescent carbon dots for detection are characterized by: The nitrogen-doped fluorescent carbon dots are prepared by a hydrothermal method using erythromycin and L-isoleucine as raw materials.

2. Fe according to claim 1 3+ The preparation method of nitrogen-doped fluorescent carbon dots for detection is characterized in that: The steps include: Erythromycin, L-isoleucine and water are mixed and subjected to hydrothermal reaction to obtain the Fe 3+ Nitrogen-doped fluorescent carbon dots were used for detection.

3. The preparation method according to claim 2, characterized in that The usage ratio of erythromycin, L-isoleucine and water is 0.1-0.3 g:0.1-0.3 g:10 mL.

4. The preparation method according to claim 2, characterized in that The temperature of the hydrothermal reaction is 160-230° C., and the time of the hydrothermal reaction is 2-6 hours.

5. The preparation method according to claim 2, characterized in that After the hydrothermal reaction is completed, a post-treatment step is further included, and the post-treatment includes: centrifuging and dialysis the product obtained from the hydrothermal reaction in sequence.

6. The preparation method according to claim 5, characterized in that The centrifugal speed is 5000-12000 r / min, and the centrifugal time is 5-10 min; and / or the dialysis bag used for dialysis has a molecular weight cut-off of 500-1000 Da.

7. The preparation method according to claim 2, characterized in that The Fe 3+ The nitrogen-doped fluorescent carbon dots used for detection are red nitrogen-doped fluorescent carbon dots; and / or the Fe 3+ The nitrogen-doped fluorescent carbon dots used for detection are in solid or liquid state.

8. Fe according to claim 1 3+ Detection of Nitrogen-doped Fluorescent Carbon Dots for Cell Imaging.

9. Fe according to claim 1 3+ Detection of Nitrogen-doped Fluorescent Carbon Dots on Fe 3+ Application in detection.

10. A method to increase Fe 3+ A method for detecting selectivity and sensitivity, characterized in that The Fe according to claim 1 3+ Nitrogen-doped fluorescent carbon dots were used for detection.

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