Nitrogen-doped fluorescent carbon dots for ferric iron detection as well as preparation method and application of nitrogen-doped fluorescent carbon dots

Abstract

The invention discloses nitrogen-doped fluorescent carbon dots for ferric iron detection and a preparation method and application thereof, and belongs to the technical field of chemical detection. The nitrogen-doped fluorescent carbon dots are prepared by taking erythromycin and L-isoleucine as raw materials through a hydrothermal method. The red nitrogen-doped fluorescent carbon dots (N-CDs) with high yield (70%) are prepared through hydrothermal treatment of L-isoleucine and erythromycin. The obtained N-CDs have high selectivity and sensitivity to Fe < 3 + >, and have wide application prospects in sensors, detection platforms and environment monitoring systems. Based on excellent biocompatibility and low toxicity of N-CDs, the N-CDs can be successfully used for multicolor cell imaging, and has great potential in the field of biomedicine.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection, and more specifically 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 the human body, with a content of about 4 to 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 cause 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, it is necessary to detect Fe in the external environment of the human body and in the body. 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, and most of them have shown great potential in different types of cell bioimaging. CDs as fluorescent probes are fast, simple, highly specific, and highly sensitive. The detection principle is mainly based on the chelation of CDs surface functional groups 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 CDs surface and the target metal ions. It is crucial to improve or construct the active functional groups on the surface of CDs by doping with heteroatoms to achieve specific recognition of the target ions. In addition, doping with 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, it is necessary to develop 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 a preparation method and application thereof, 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 multicolor 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 depends entirely on Fe 3+ The dose of Fe 3+ , and can also be used for multi-color cell imaging.

[0008] Technical solution 2 of the present invention: providing 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, the temperature, time and reactant concentration of the hydrothermal reaction have an important influence on the size and dispersibility of carbon dots. Appropriate reaction conditions can control the size and fluorescence properties of carbon dots, making them monodisperse in the nanoscale range. When the hydrothermal reaction conditions are too intense or the reactant concentration is too high, the carbon dots will grow too fast, become too large and easily aggregate, thus affecting their fluorescence properties and application effects; when the hydrothermal reaction conditions are too mild or the reactant concentration is too low, the carbon dots will not form, which will also affect their fluorescence properties and application effects.

[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 cutoff 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 for detection are in solid or liquid state.

[0016] Furthermore, the Fe prepared by the preparation method of the present invention 3+ The nitrogen-doped fluorescent carbon dots for detection are in liquid state, and 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] Technical solution 4 of the present invention: providing the Fe 3+ Detection of Nitrogen-doped Fluorescent Carbon Dots on Fe 3+ Application in detection.

[0020] Technical solution 5 of the present invention: providing a method for increasing Fe 3+ The detection selectivity and sensitivity of the method are determined by the above 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 high yield (70%) of red nitrogen-doped fluorescent carbon dots. 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, and the obtained 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 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 The surface functional group characterization diagram of N-CDs prepared in Example 1, wherein a is an infrared spectrum diagram, b is an XRD diagram, c is an XPS spectrum diagram, and d is a C1s spectrum diagram;

[0028] Figure 6 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 The fluorescence intensity diagram of N-CDs obtained in Examples 1 to 12;

[0030] Figure 8 Graphs showing the test results of the optical stability 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] Fig. 9 The N-CDs obtained in Example 1 have a 3+ Selectivity test results of the graph, where a is the 3+ The fluorescence intensity difference of N-CDs solution in the presence of 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] Fig.10 The figure is the result of measuring the cytotoxicity of N-CDs obtained in Example 1 by MTT method;

[0033] Fig.11 These are the cell imaging effect diagrams 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. 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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] Fluorescence sensors have attracted much attention due to their simplicity, rapidity, high selectivity and sensitivity, and high spatial resolution. In addition, metal ions in cells and organisms can be intuitively monitored by fluorescence sensor-based imaging techniques. Currently, the most commonly used fluorescence sensors include fluorescent metal nanoclusters, organic dyes, semiconductor quantum dots, fluorescent metal-organic frameworks, etc. However, in practical applications, the application of fluorescence sensors is challenged by their photostability, complex equipment and processing technology, environmental pollution, and cytotoxicity.

[0041] Carbon dots (CDs) are a new type of luminescent carbon nanomaterials. Due to their unique physical and chemical properties, they show wide application potential and research value in many fields. CDs are zero-dimensional nanoparticles with a diameter of less than 10 nm, which have the advantages of 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 disadvantages of low water solubility, difficulty in functionalization, and high toxicity. At present, CDs have been widely used in 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] Thiazole blue (MTT), green streptomycin mixture, trypsin-EDTA digestion solution, and formalin (PFA) were purchased from Beijing Solebow 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 by the chlorides of various metal cations (Tianjin Third Chemical Reagent Factory).

[0050] Other raw materials and test reagents are commercially available unless otherwise specified.

[0051] The dialysis bag used in the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a specification of 500-1000Da (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] Take 0.2g L-isoleucine and 0.2g erythromycin in a 20mL beaker, add 10mL deionized water to dissolve, transfer the mixed solution to a 50mL hydrothermal kettle, and place it in an electric blast drying oven at 200℃ for 3h. After the reaction is completed, cool it to room temperature, take out the reaction solution, and centrifuge it at 8000r / min for 8min to remove large solid particles. Then take the supernatant and dialyze it through a dialysis bag for 48h to obtain a pure N-CDs aqueous solution. If you need to obtain N-CDs powder, you can get it by freeze-drying the N-CDs aqueous solution at -60℃ for 48h, and the N-CDs yield can reach 70%.

[0056] The N-CDs obtained by the invention are very 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 by FEI Tecnai G2 F20 instrument; fluorescence spectra were measured by F-280 fluorescence spectrometer (Tianjin Gangdong Technology Development Co., Ltd.); UV-visible absorption spectra were obtained by Cary 50Bio-UV-vis spectrometer (Varian, USA); infrared spectra were obtained by FTIR-8400S (Shimadzu, Japan) spectrometer; X-ray diffraction spectra were obtained by Rigaku Miniflex 600; X-ray photoelectron spectroscopy (XPS) was obtained by ThermoScientific K-Alpha electron spectrometer; the results are shown in 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 had good dispersibility; 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 Figure 1, 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 increases first and then decreases, and the emission wavelength red-shifts. This characteristic that fluorescence is 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, which is 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 diagram, b is an XRD diagram, c is an XPS spectrum diagram, and d is a C1s spectrum diagram. 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 results of the XPS spectrum and the IR spectrum are consistent, indicating that the N element was successfully doped into CDs.

[0063] The fluorescence quantum yield of the obtained N-CDs was measured using rhodamine B as a reference solution. In order to prevent the reabsorption effect, a series of N-CDs solutions and reference solutions (using methanol as solvent) with different concentrations were prepared. The concentration was adjusted so that 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 integrated intensity and absorbance, Grad R is the gradient (slope) of the fluorescence integrated 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 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. Figure 6 As shown in Table 1, the fluorescence quantum yield of N-CDs measured using rhodamine B (QY=89%) as a reference solution was 9.2%.

[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] Embodiment 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. 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 where 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 room temperature (365nm, 16W) for 4 hours, and measure the relationship between exposure time and fluorescence intensity; store N-CDs in a refrigerator (5℃) for 3 months, and measure the relationship between storage time and fluorescence intensity. The results are as follows: Figure 8 shown.

[0093] Figure 8 The test results of the optical stability of N-CDs obtained in Example 1 are shown in Figure 1, where 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. 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 basically unaffected even after continuous exposure to light (365 nm, 16 W) for 4 h and storage for 3 months at room temperature. The results show 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 N-CDs obtained 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, the original fluorescence intensity (denoted as F0) was measured first, and then a known concentration of 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. Fig. 9 shown.

[0097] Fig. 9 The N-CDs obtained in Example 1 have a 3+ Selectivity test result diagram, where a is the 3+ The fluorescence intensity difference of N-CDs solution in the presence of 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 Fig. 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 little effect on the fluorescence intensity of N-CDs, indicating that N-CDs have a strong affinity for Fe 3+ Further sensitivity experiments showed that the fluorescence intensity of N-CDs depends on Fe 3+ The dose ( Fig. 9 b) With Fe 3+ With the increase of Fe 3+ Applicability of fluorescent probes. Fig. 9 c shows the fluorescence intensity of N-CDs and Fe 3+ The relationship between the concentrations has a good linear relationship in the range of 2.1 to 37.5 μM ( Fig. 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] Test Example 1 N-CDs obtained for Fe in actual samples 3+ The test results:

[0100] Test method: First, centrifuge tap water at 8000 r / min for 8 min to remove large particles, and then filter through a filter membrane with a pore size of 0.45 μm. 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, respectively, 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 Effect 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 inoculated into a 96-well culture plate for 18 hours to allow them to completely adhere to the wall. Then the DMEM medium was removed, and the cells were washed three times with PBS (pH = 7.4) buffer, and then the cells were incubated with new DMEM medium containing different concentrations of N-CDs for 24 hours, and the old DMEM medium in the culture plate was removed again, and washed three times with PBS (pH = 7.4) buffer. Then 200 μL of new DMEM medium containing 20 μL MTT (5 mg / mL) reagent was added, and incubation continued at 37.5°C for 5 hours. Finally, the supernatant was aspirated, 150 mL DMSO was added to the cells and allowed to stand for 10 minutes. The absorbance at 490 nm was recorded using an enzyme reader. The results are shown in 10.

[0107] Fig.10 The figure is the result of the cytotoxicity of N-CDs obtained in Example 1 measured by MTT method. Fig.10 It can be seen that after incubating HeLa cells with different concentrations of N-CDs for 24 h, 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] Test the cell imaging effect of N-CDs obtained in Example 1:

[0110] Test method: HeLa cells (density 1×10 5 / mL) were inoculated in a 35mm culture dish containing DMEM culture medium and incubated in a constant temperature cell culture incubator at 37.5℃ overnight. The old DMSO culture medium was removed, and new DMEM culture medium containing 500μg / mL N-CDs was added to continue culturing HeLa cells for 2h. After the incubation, the culture medium was removed and washed three times with 1mL pH=7.4 PBS buffer to remove free N-CDs particles. Then, a new 1mL pH=7.4 PBS buffer 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 Fig.11 shown.

[0111] Fig.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. Fig.11 It can be seen that N-CDs enter the nucleus of HeLa cells 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 488 and 543nm light sources, green and red fluorescent cell images can be obtained. The results show that N-CDs have good 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 enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kind of Fe 3+ The detection nitrogen-doped fluorescent carbon dots 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 method for preparing 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 dosage 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 by 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 molecular weight cutoff of the dialysis bag used for dialysis is 500-1000 Da.

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