A nitrogen-doped carbon dot, a preparation method thereof and application of the nitrogen-doped carbon dot in colorimetric / fluorescent dual-mode visual detection of pH and histamine

Abstract

The application belongs to the technical field of fluorescent nanomaterials, and provides nitrogen-doped carbon dots, a preparation method thereof and application of the nitrogen-doped carbon dots in colorimetric / fluorescent dual-mode visual detection of pH and histamine, to solve the problem that regular colorimetric signals of current carbon dots are difficult to achieve, which limits the application of the carbon dots in biomedicine and photoelectric devices. The nitrogen-doped fluorescent carbon dots are synthesized by one-step hydrothermal reaction of o-phenylenediamine and 2,6-pyridinedicarboxylic acid, water and hydrochloric acid. The preparation method is simple and fast, the synthesized carbon dots have good water solubility and biocompatibility, stable optical properties, high fluorescence quantum yield, can quickly and visually detect intracellular pH and histamine in food spoilage process, have excellent selectivity and anti-interference ability, fast response speed, high sensitivity, and can realize on-site and instant detection by means of a smart phone and a test strip.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fluorescent nanomaterials, and particularly relates to nitrogen-doped carbon dots, a preparation method thereof and application of the nitrogen-doped carbon dots in colorimetric / fluorescent dual-mode visual detection of pH and histamine. BACKGROUND

[0002] Cell pH value is one of important physical and chemical parameters in an aqueous solution system, and plays an important role in chemical reactions, life processes, environmental governance and food safety and many other fields. Among them, intracellular pH value plays an important role in regulating the functions and activities of cells and organisms, mainly including signal transduction, proliferation and apoptosis. At the same time, abnormal intracellular pH value will cause major changes in metabolism, promote the occurrence and metastasis of tumors, and further induce cancer. Therefore, it is very important to detect the fluctuation of intracellular pH value in real time and accurately.

[0003] Histamine is an organic nitrogen-containing compound produced by histidine under the action of decarboxylase, is a key medium in various physiological and pathological processes of the human body, is mostly produced by microorganisms in deteriorated food, and will cause an allergic reaction of the body when excessive intake, leading to a series of symptoms such as low blood pressure, skin irritation, headache, nausea, vomiting and the like, and is considered as an important indicator for monitoring food quality. Therefore, it is crucial to develop a rapid and sensitive method for detecting histamine for protecting food safety.

[0004] Carbon dots have been widely concerned and applied in many fields such as biological imaging and environmental analysis due to their good biocompatibility, simple synthesis, easy surface functionalization and modification and ultra-high optical stability. At present, most of the synthesized carbon dots have blue-green fluorescence, and are seriously interfered by the background environment in fluorescence detection and imaging. In addition, regular colorimetric signals of most carbon dots are also difficult to achieve, which greatly limits their convenient and visual application in biomedicine and optoelectronic devices. Therefore, it is of extremely important significance to design and synthesize carbon dots with colorimetric and fluorescent dual-mode response signals in the construction of pH and histamine biosensing platforms. SUMMARY

[0005] The present application provides a kind of nitrogen-doped carbon dots and its preparation method and application in colorimetric / fluorescent dual-mode visual detection of pH and histamine to solve the problem that current carbon dots regularity colorimetric signal is difficult to achieve, which limits its application in biomedical and optoelectronic devices.The nitrogen-doped carbon dots prepared by simple raw materials and relatively simple synthesis route have colorimetric and fluorescent dual-response signals, which can be used as good colorimetric / fluorescent biosensors for sensitive and visual detection of intracellular pH and histamine in food spoilage process.In addition, the carbon dots also show highly distinguishable daylight / fluorescent color change in the sensing process, which can be used as excellent color sensors for rapid and on-site identification of the change of histamine content in food spoilage process, and has extremely important significance for monitoring food safety and preventing food poisoning.

[0006] The present application is realized by the following technical solutions: a kind of nitrogen-doped carbon dots, with o-phenylenediamine and 2,6-pyridine dicarboxylic acid as raw materials, o-phenylenediamine, 2,6-pyridine dicarboxylic acid and water, hydrochloric acid are mixed, and nitrogen-doped fluorescent carbon dots are synthesized by one-step hydrothermal reaction;Wherein: the mass-volume ratio of o-phenylenediamine, 2,6-pyridine dicarboxylic acid and water is 1:0.5-2:10-20 g / g / mL;After mixing, hydrothermal reaction is carried out at 160-200 DEG C for 1-6h.

[0007] The method for preparing the nitrogen-doped carbon dots has the following specific steps:

[0008] (1) raw material mixing: mix o-phenylenediamine and 2,6-pyridine dicarboxylic acid according to the proportion, add water and 10-100 μL of concentrated hydrochloric acid with a mass concentration of 36%-38% in turn, fully stir and ultrasonically dissolve for 10-20 min to prepare a mixed solution;

[0009] (2) hydrothermal reaction: transfer the mixed solution obtained in step (1) to a hydrothermal reaction kettle, and place it in an oven, and heat it from room temperature to 160-200 DEG C at a rate of 4.2 DEG C / min, and react for 2-6h;

[0010] (3) obtaining of target product: after the reaction is completed, the reaction kettle is left to cool naturally, then taken out to obtain a brownish brown solution, the solution is treated by dialysis through a 1000 D filter membrane for 12-36h, then filtered through a 0.22 μm filter membrane to obtain pure brownish yellow carbon dots, then freeze-dried at-42 DEG C under liquid nitrogen for 24h to obtain the brown target product.

[0011] Further, the hydrothermal reaction temperature in step (2) is 160-180 DEG C, and the reaction time is 2-6h.

[0012] The application also provides application of the nitrogen-doped carbon dots in colorimetric / fluorescent dual-mode visual detection of pH and histamine, and application of the nitrogen-doped carbon dots as a colorimetric / fluorescent dual-mode sensor in visual detection of pH and histamine.

[0013] Further, the application provides application of the nitrogen-doped carbon dots as a colorimetric / fluorescent dual-mode sensor in sensitive detection of intracellular pH and histamine.

[0014] The application provides application of the nitrogen-doped carbon dots as a colorimetric / fluorescent dual-mode sensor in detection of histamine in a food spoilage process.

[0015] The application provides application of the nitrogen-doped carbon dots in on-site and real-time detection of changes of histamine in a food spoilage process.

[0016] The colorimetric / fluorescent dual-mode nitrogen-doped carbon dots prepared by the application can specifically recognize pH and histamine. The reason is as follows: the response mechanism of the carbon dots to pH is mainly attributed to the protonation and deprotonation process of the special structure (amino N and pyridine N) on the surface of the CDs. In the process of increasing pH, the potential changes from a positive value to a negative value, indicating that the protonation ability of the carbon dots gradually weakens, the electron-donating ability of the system is enhanced, the electron density is increased, the absorbance of the carbon dots is reduced, and a blue shift occurs, and the fluorescence intensity is enhanced. Therefore, the reaction mechanism of the CDs to pH is the combination of the nitrogen-containing groups on the surface of the CDs and hydrogen ions.

[0017] After a certain amount of histamine is added to the carbon dots in a buffer solution fixed system, the colorimetric / fluorescent intensity of the carbon dots does not change substantially, while the colorimetric / fluorescent intensity of the carbon dots changes in a carbon dot solution without the use of a buffer solution to fix the pH of the system. This is because the pKa value of histamine in an aqueous solution is 9.8, and it is alkaline in an aqueous solution. Therefore, the alkaline histamine enhances the alkalinity of the system when it is added to the carbon dots, the absorbance of the carbon dots is reduced, and a blue shift occurs, and the fluorescence intensity is increased.

[0018] Compared with the prior art, the application has the advantages that: the operation steps of the application are simple, and the carbon dots with colorimetric / fluorescent dual-response signals can be obtained without surface passivation agent treatment or modification. The synthesized carbon dots have good biocompatibility and stable optical properties, and have good solubility in an aqueous solution. The prepared carbon dots have the ability of colorimetric / fluorescent dual-response signals, can specifically recognize and detect pH and histamine, have good selectivity and high sensitivity, and have wide application prospects in the fields of cell imaging and biological sensing. The synthesized carbon dots have highly distinguishable daylight / fluorescent color changes, and can be used as color sensors for real-time and on-site monitoring of changes of the content of histamine in a food spoilage process. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. 1 is a transmission electron microscope image and size distribution diagram of carbon dots of Example 1 of the application.

[0020] Figure 2 Infrared spectrum of the carbon dots of Example 1 of the present application;

[0021] Figure 3 X-ray photoelectron spectroscopy full spectrum of the carbon dots of Example 1 of the present application;

[0022] Figure 4 C 1s spectrum of the X-ray photoelectron spectroscopy of the carbon dots of Example 1 of the present application;

[0023] Figure 5 N 1s spectrum of the X-ray photoelectron spectroscopy of the carbon dots of Example 1 of the present application;

[0024] Figure 6 O 1s spectrum of the X-ray photoelectron spectroscopy of the carbon dots of Example 1 of the present application;

[0025] Figure 7 Effect of the carbon dots of Example 1 of the present application on the activity of HeLa cells at different concentrations;

[0026] Figure 8 Fluorescence intensity change of the carbon dots of Example 1 of the present application under different ionic strengths;

[0027] Figure 9 Fluorescence intensity change of the carbon dots of Example 1 of the present application before and after storage for 3 months;

[0028] Figure 10 Calculated oil-water partition coefficient spectrum of the carbon dots of Example 1 of the present application;

[0029] Figure 11 Ultraviolet absorption spectrum and fluorescence excitation-emission spectrum of the carbon dots of Example 1;

[0030] Figure 12 Spectrum of the fluorescence emission curve of the carbon dots prepared in Example 1 as a function of excitation wavelength;

[0031] Figure 13 Ultraviolet-visible absorption spectrum of the carbon dots solution in the presence of different pH values;

[0032] Figure 14 Linear relationship diagram of the ultraviolet-visible absorption spectrum of the carbon dots solution in the presence of different pH values (between 3.0-5.6);

[0033] Figure 15 Fluorescence emission spectrum of the carbon dots solution in the presence of different pH values;

[0034] Figure 16 Linear relationship diagram of the fluorescence spectrum of the carbon dots solution in the presence of different pH values (between 3.0-5.6);

[0035] Figure 17 Color change of carbon dots solution under sunlight lamp in the presence of different pH;

[0036] Figure 18 Color change of carbon dots solution under UV lamp (365 nm) in the presence of different pH;

[0037] Figure 19 UV-Vis absorption spectrum of carbon dots solution in the presence of different concentration of histamine;

[0038] Figure 20 Linear relationship of UV-Vis absorption spectrum of carbon dots solution in the presence of different concentration of histamine;

[0039] Figure 21 Fluorescence emission spectrum of carbon dots solution in the presence of different concentration of histamine;

[0040] Figure 22 Linear relationship of fluorescence spectrum of carbon dots solution in the presence of different concentration of histamine;

[0041] Figure 23 Color change of carbon dots solution under sunlight lamp in the presence of different concentration of histamine;

[0042] Figure 24 Color change of carbon dots solution under UV lamp (365 nm) in the presence of different concentration of histamine;

[0043] Figure 25 Laser confocal image of carbon dots prepared in Example 1 of the present application at different pH, wherein the cells are HeLa cells;

[0044] Figure 26 Laser confocal image of carbon dots prepared in Example 1 of the present application in the presence of histamine solution, wherein the cells are HeLa cells;

[0045] Figure 27 Selective test of carbon dots prepared in Example 1 of the present application on related interfering substances under pH 3.0 and pH 7.0 conditions;

[0046] Figure 28 Selective test of carbon dots prepared in Example 1 of the present application on substances related to histamine;

[0047] Figure 29 Color change of carbon dots test strip loaded with carbon dots prepared in Example 1 of the present application under sunlight lamp and under UV lamp (365 nm) before and after being placed for 10 days and before and after adding 1000 µM histamine;

[0048] Figure 30The carbon dots prepared in the embodiment 1 of the present application can be used as a colorimetric color change sensor for monitoring the change of histamine in the spoilage process of raw fish samples.

[0049] Figure 31 The carbon dots prepared in the embodiment 1 of the present application can be used as a fluorescent color change sensor for monitoring the change of histamine in the spoilage process of raw fish samples.

[0050] Figure 32 The transmission electron microscopy (TEM) image of the carbon dots of the present application as a function of pH;

[0051] Figure 33 The zeta potential of the carbon dots of the present application as a function of pH;

[0052] Figure 34 The fluorescence spectra of the carbon dots of the present application before and after adding histamine. DETAILED DESCRIPTION

[0053] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials cited and referred to herein will be incorporated by reference to the extent allowed by law.

[0055] Any equivalent techniques of the described specific embodiments known to those skilled in the art through routine experiments are included in the present application.

[0056] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment unless otherwise specified. The experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0057] Embodiment 1: A nitrogen-doped carbon dot, which is prepared by using o-phenylenediamine and 2,6-pyridinedicarboxylic acid as raw materials, mixing o-phenylenediamine, 2,6-pyridinedicarboxylic acid, water and hydrochloric acid, and then synthesizing brown-yellow nitrogen-doped fluorescent carbon dots by one-step hydrothermal reaction; wherein the mass ratio of o-phenylenediamine to 2,6-pyridinedicarboxylic acid is 1:0.5; and the mixture is subjected to hydrothermal reaction at 180℃ for 3h.

[0058] The specific preparation method is as follows:

[0059] 1) Place o-phenylenediamine and 2,6-pyridinedicarboxylic acid in a mass ratio of 1:0.5 in a 100 mL beaker. Add 20 mL of water and 50 µL of hydrochloric acid. Stir thoroughly and ultrasonically dissolve for 10-20 minutes to prepare a mixed solution.

[0060] 2) The mixed solution was transferred to a hydrothermal reactor and placed in an oven at 180°C for 3 hours. After the reaction ceased, the reactor was allowed to cool naturally before removal, yielding a brown solution. This solution was dialyzed through a 1000D filter membrane for 24 hours and then filtered through a 0.22 μm filter membrane to obtain a pure carbon dot solution.

[0061] 3) The carbon dot aqueous solution was freeze-dried to obtain a brown carbon dot solid powder with a fluorescence quantum yield of 26.13%.

[0062] The prepared nitrogen-doped carbon dots were characterized by transmission electron microscopy, infrared spectroscopy, and X-ray photoelectron spectroscopy to determine the particle morphology, chemical structure, and element distribution of the carbon dots. Figure 1 、 2 , 3, among which, the transmission electron microscopy image ( Figure 1 ) The results showed that the obtained nitrogen-doped carbon dots were monodispersed quasi-spherical particles with an average particle size of 1.88±0.31 nm. In the infrared spectrum ( Figure 2 ), the stretching vibration of OH and NH makes the nitrogen-doped carbon dots at 3407 cm −1 and 3336 cm −1 There is an absorption peak at 1551 cm −1 and 1627 cm −1 The sharp peak at 1360 cm is the amide bond (–CONH-), which strongly proves that o-phenylenediamine and 2,6-pyridinedicarboxylic acid undergo dehydration condensation during the formation of carbon dots. -1 The peak at 1500 cm is caused by the stretching vibration of CN. -1 Peak. 2605 cm -1 The absorption peak at 2903 cm is attributed to the C=O bond. The stretching vibration of CH makes the carbon dots -1 The characteristic absorption peak appears at . The X-ray photoelectron spectroscopy further proves the successful synthesis of nitrogen-doped carbon quantum dots. The full XPS spectrum of nitrogen-doped carbon dots ( Figure 3 ) has three obvious peaks, located at 281.6 eV (C1s), 396.8 eV (N1s) and 529 eV (O1s). The high-resolution C1s peak ( Figure 4) can be further divided into C-C / C=C (284.8 eV), C-N / C=N (285.8 eV) and -COOH (288.8 eV). For high resolution N1s spectrum (Fig. 6b), Figure 5 ), the main peaks are the characteristic peaks of pyridine nitrogen and pyrrole nitrogen with protonation ability, which are located at 399.4 eV and 400.6 eV, respectively. The high resolution O1s spectrum (Fig. 6c) Figure 6 ) is mainly the C=O peak at 532.6 eV and the C-OH / C-O-C peak at 531.4 eV. The above results show that the nitrogen-doped carbon dots are quasi-spherical nanoparticles containing C, N and O elements, and are rich in various functional groups.

[0063] The biocompatibility, optical stability and solubility in water of the prepared nitrogen-doped carbon dots were verified. Figure 7 The cytotoxicity of nitrogen-doped carbon dots at different concentrations in cells was determined. When the concentration of nitrogen-doped carbon dots reached 0.40 mg / mL, the cell survival rate was still higher than 80%, which indicated that the cell had good biocompatibility. Figure 8 and Figure 9 The optical stability of nitrogen-doped carbon dots under ion strength and long-term storage was determined, respectively. Figure 8 It was shown that when the concentration of NaCl was 0.7 mM, the fluorescence intensity of nitrogen-doped carbon dots did not change significantly. Figure 9 After placing the nitrogen-doped carbon quantum dots for 3 months, the fluorescence intensity only slightly decreased, which proved that the optical properties of the nitrogen-doped carbon quantum dots were stable. The solubility of carbon dots in water was calculated from the oil-water partition coefficient (Log P) of carbon dots (according to Chem Draw20.0 software, Figure 10 ), Figure 10 which showed that the Log P values of raw materials o-phenylenediamine and 2,6-pyridine dicarboxylic acid were 0.43 and 0.66, respectively, and the Log P value of the nitrogen-doped carbon dots formed by the two was 1.12. Further according to the solubility calculation formula (good water solubility requires: Log S>-2): Log S=1.17-1.38Log P, the Log S values of o-phenylenediamine, 2,6-pyridine dicarboxylic acid and the predicted carbon quantum dot structure were 0.5766, 0.2592 and -0.3756, respectively. The Log S values of the three were all less than -2, which further indicated that the nitrogen-doped carbon quantum dots had good water solubility.

[0064] The ultraviolet absorption spectrum, fluorescence excitation and emission spectrum of the nitrogen-doped carbon dots were scanned, and the results are shown in Figure 11 The carbon dots had characteristic ultraviolet absorption peaks at 275 nm and 415 nm, respectively, corresponding to the π-π* transition of C=C and the n-π* transition of C-O / C-N. Further combined with Figure 12It can be obtained that according to the fluorescence intensity obtained after excitation at different excitation wavelengths, the optimal excitation wavelength of nitrogen-doped carbon quantum is 420nm, and the optimal emission wavelength is 570nm.

[0065] The prepared nitrogen-doped carbon dots were used as colorimetric and fluorescence dual-mode sensors to determine the linear range of pH. A certain concentration of nitrogen-doped carbon dot solution was diluted into 2 mL of PBS buffer at different pH conditions, and the UV-visible absorption spectra ( Figure 13 , Figure 14 ) and fluorescence emission spectra ( Figure 15 , Figure 16 ), the absorbance value at 448nm and the emission intensity value at 570nm were measured. Figure 13 It can be seen that when the pH is 3.0, the optimal absorbance position of the solution is 448nm. When the pH value of the solution increases, the absorbance position blue-shifts to 414nm. When the pH value of the solution is in the range of 3.0-5.6 ( Figure 14 ), with an excellent linear relationship y=−0.08165x+1.04571(R 2 =0.9943), when the pH is 3.0, the optimal emission position of the solution is 530 nm. As the pH value of the solution increases, the fluorescence emission intensity gradually increases and gradually red-shifts to 572 nm. In the pH range of 3.2-6.8 ( Figure 16 )has an excellent linear relationship y=2084.19x-5806.72(R 2 =0.9975).

[0066] Nitrogen-doped carbon quantum dots mixed with solutions of different pH values ​​were placed in a cuvette, and their color changes under fluorescent light were observed. They were photographed using a smartphone (OPPO, Reno 5), and the R, G, and B values ​​of the colors under different pH conditions were read using color recognition software. Figure 17 As shown. In the range of 3.0-7.0, it has good linearity y=-0.02018x+0.52401, R 2 =0.9947. Under the same conditions, the carbon dots at different pH values ​​were placed in a fluorescent cuvette. Under ultraviolet light (excitation light was 365 nm), obvious color changes occurred. Photos were taken with a smartphone (OPPO, Reno 5), and the R, G, and B values ​​of the colors under different pH conditions were read using color recognition software ( Figure 18 ). Its linear range is 3.0-5.8, and the linear equation is: y=0.06947x+0.04648, R 2 =0.9928.

[0067] The prepared nitrogen-doped carbon dots were used as a colorimetric and fluorescent dual-mode sensor for the determination of histamine. A certain concentration of nitrogen-doped carbon dot solution was diluted to 2 mL, and the pH was adjusted to a certain value using hydrochloric acid or sodium hydroxide solution, and then different concentrations (0-1000 µM) of histamine solution were added. Then the absorbance value at 447 nm and the emission intensity value at 570 nm were measured by ultraviolet-visible absorption spectrum ( Figure 19 , Figure 20 ) and fluorescence emission spectrum ( Figure 21 , Figure 22 ), respectively. Further according to the ultraviolet-visible absorption spectrum combined with Figure 19 and Figure 20 , it is known that the nitrogen-doped carbon quantum dots have a good linear relationship for histamine detection y = -1.1244*10 -4 x + 0.57137, R 2 = 0.9932, the linear range is 30-1000 µM, and the detection limit is 10.11 µM. Using fluorescence spectrum combined with Figure 21 and Figure 22 , it is known that the nitrogen-doped carbon quantum dots have a good linear relationship for histamine detection y = 2.4535x + 797.02, R 2 = 0.9981, the linear range is 25-1000 µM, and the detection limit is 23.19 µM.

[0068] The nitrogen-doped carbon quantum dots containing different concentrations of histamine solution were placed in a cuvette, and the color change under daylight lamp was observed, and a smart phone (OPPO, Reno 5) was used to take a picture, and the R, G, B values of the color under different pH conditions were identified and read using the color identifier software, as shown in Figure 23 . There is a good linear relationship in the range of 50-1000 µM y = -5.3385*10 5 x + 0.41708, R 2 = 0.9921, and the detection limit is 10.75 µM. Under the same conditions, the carbon dots in the presence of different concentrations of histamine were placed in a fluorescence cuvette, and under ultraviolet lamp (excitation light is 365 nm), obvious color change (from green to yellow) occurred, and a smart phone (OPPO, Reno 5) was used to take a picture, and the R, G, B values of the color under different concentrations of histamine were identified and read using the color identifier software ( Figure 24 ). The linear range is 50-1000 µM, the linear equation is y = 3.91542*10 -5 x + 0.33499, R 2 = 0.9945, and the detection limit is 18.87 µM.

[0069] In a 5% CO2 incubator, HeLa cells in the exponential phase were cultured at a rate of 1×10 6 The cells were seeded at an initial density of 100 cells / mL in a 15 mm glass dish in DMEM containing 10% FBS and cultured at 37°C. + The fluorescence response of carbon dots to different intracellular pH values ​​was studied in buffer. The obtained nitrogen-doped carbon dots (0.2 mg / mL) were incubated with HeLa cells for 10 minutes and then gently washed three times with PBS (pH 7.40). Then, 10 μg / mL nigericin was used to detect the fluorescence of carbon dots at different pH values ​​(3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.5, 7.0, 8.0). + The cells were incubated in a buffer solution (120.0 mM KCl, 30.0 mM NaCl, 0.5 mM MgSO4, 1.0 mM CaCl2, 20.0 mM HEPES, 20.0 mM NaOAc, 1.0 mM NaH2PO4, and 5.0 mM glucose) for 10 min. Finally, fluorescence images of the red channel (Ex = 405 nm, Em = 500 nm-600 nm) were recorded using a laser confocal scanning microscope. Figure 25 It can be seen that the fluorescence signal of carbon dots is weak at pH 3.0, and as the pH value gradually increases, the fluorescence signal inside the cell is significantly enhanced. This result shows that the carbon dots can be used to monitor pH changes in living cells. Figure 26 It was found that after adding 200 μM histamine to HeLa cells pretreated with nitrogen-doped carbon dots, the fluorescence signal in the cells became enhanced. Based on this, the carbon dots can be used for pH imaging of HeLa cells and identification of intracellular histamine. Figure 25 and Figure 26 As can be seen from the figure, from top to bottom they are: dark field cell image, bright field cell image (red), and bright field and dark field overlay image.

[0070] To verify that the fluorescence enhancement of the prepared carbon dots was pH-specific, the effects of possible interfering substances on the fluorescence intensity of the carbon dots were investigated. The fluorescence intensity of the carbon dots in the presence of cations, anions, or amino acids at pH 7.0 and pH 3.0 was further investigated. The results showed that under constant pH conditions, the addition of other interfering substances did not cause significant fluorescence changes ( Figure 27 ), indicating that the carbon dots have high selectivity to pH changes. (Interfering substances: 0, Blank, 1, Na + (150 mM), 2, K + (150 mM), 3, Mg 2+ (10 mM), 4, Ca 2+ (10 mM), 5, Ba2+ (10 mM), 6, Cu 2+ (2 mM), 7, Fe 2+ (2 mM), 8, Fe 3+ (2 mM), 9, Zn 2+ (2 mM), 10, Cd 2+ (2 mM), 11, Cl - (10 mM), 12, SO4 2- (10 mM), 13, SO3 2- (10 mM), 14, NO - (10 mM), 15, Ac - (10 mM), 16,H2O2(0.1 mM), 17, ClO - (0.1 mM), 18, Cys (1 mM), 19, GSH (1 mM), 20, HCy (1mM), 21, Asp(1 mM), 22, Glu(1 mM), 23,AA(1 mM), 24, Lyr(1 mM), 25, Leu(1 mM),26, Tyr(1 mM), 27, Phe(1 mM), 28, DA(1 mM))。

[0071] To verify that the fluorescence enhancement of the prepared carbon dots is caused by the specificity of histamine, the influence of possible interfering substances on the fluorescence intensity of the carbon dots was studied. Ethylenediamine (EDA), five biological amines (spermidine, tryptamine, cadaverine, putrescine, spermine), and three basic amino acids (arginine, lysine, histidine) were chosen as common parts of interference in actual samples. These samples were tested under the same conditions. It can be seen from Figure 28 that the reaction of the carbon dots to histamine shows sufficient specificity, i.e. only when histamine concentration (400 µM) is present, the fluorescence changes significantly, while almost no significant change is observed when other analytes (1 mM) are added at 2.5 times the concentration of histamine, which indicates that the fluorescent carbon dots have excellent selectivity to histamine.

[0072] To determine the potential of the carbon dots to detect histamine in actual samples, the carbon dots were combined with an equal volume of round test paper to prepare a portable analysis device for the detection of histamine vapor. The obtained carbon dot-loaded test paper strip remained stable in its original absorbance and fluorescence color after being stored for 10 days, which indicates that the carbon dots have good processability and stability (Fig. 6). Figure 29). Then the test strip embedded with carbon dots was fixed at the same position, and the smartphone was fixed at a certain height and angle to take pictures and obtain R, G, B values through color identifier. After the addition of histamine, the test strip produced obvious color changes under the daylight lamp ( Figure 30 ) and the ultraviolet lamp ( Figure 31 ). Then the spoilage process of the raw fish sample in the actual sample was further monitored to determine its freshness. The same test strip loaded with carbon dots was placed in a closed petri dish with 1 g of raw fish sample, and the dynamic freshness change of the fish sample was monitored within 120 hours by storing at four different temperatures (-21, 0, 25 and 35℃). As can be seen from Figure 30 , the test strip loaded with carbon dots acts as a colorimetric color and fluorescent color change sensor. The closed petri dish at different temperatures was taken out and placed at a fixed position, and then a smartphone fixed at a certain height and angle was used to take pictures and identify, and was irradiated and photographed under the same light (daylight lamp, colorimetric sensor) and ultraviolet lamp (fluorescent lamp, fluorescent sensor, UV: 365nm) at a fixed position, respectively. Thus, the color change of the test strip under the daylight lamp or the fluorescent lamp was monitored after the production of histamine during the spoilage process of the raw fish sample at different temperatures and different times. The results show that for the raw fish sample stored at 35℃, the color of the test strip changed obviously to light yellow under the daylight lamp after only 12 hours, and the color of the test strip changed obviously under the ultraviolet lamp; for the raw fish sample stored at 25℃, the color of the test strip changed obviously under the daylight lamp after only 24 hours, and the color changed under the ultraviolet lamp; for the sample stored at 0℃, they can change obviously after 72h, i.e. the color of the test strip changes under the daylight lamp, and the fluorescent color changes under the ultraviolet lamp; and for the fish at -21℃, the color of the test strip changes obviously under the daylight lamp and the ultraviolet lamp after 120h. As can be seen from the above, the test strip loaded with carbon dots acts as a colorimetric and fluorescent color sensor, which can sensitively detect the content of histamine in food under different temperature and time conditions, and further monitor the freshness of food.

[0073] In order to verify the sensing mechanism of carbon dots to pH, transmission electron microscopy was performed under different pH conditions. As can be seen from Figure 32 , compared with the transmission electron microscopy under the condition of pH 3.0, the carbon dots aggregated in the transmission electron microscopy under the condition of pH 7.0, and the particle size increased from 1.88 nm to 6.27 nm, so that the carbon dots are prone to aggregation when the pH increases. In order to better understand the aggregation characteristics, Figure 33For the zeta potential of the obtained carbon dots as a function of pH, the zeta potential at pH 3.0 is +3.57 mV, indicating that the carbon dots have a positive charge and have strong protonation ability; with the increase of pH, the protonation ability of the carbon dots is weakened, the electron-donating ability is enhanced, the deprotonation ability is enhanced, and the electron density and hydrogen bond interaction are further enhanced. Therefore, in this process, the carbon dots have an aggregation-induced fluorescence enhancement effect, further making the emission position red-shifted and the fluorescence intensity enhanced; after the deprotonation process and hydrogen bond interaction are enhanced, due to the generation of a more compact structure, the absorbance position is slightly blue-shifted, and the absorbance is reduced. When the pH continues to increase to pH 9.0, i.e. under alkaline conditions, the protonation process of the functional groups such as carboxyl or hydroxyl on the surface of the carbon dots is interrupted, and the zeta potential value gradually increases.

[0074] In order to verify the sensing mechanism of the carbon dots for histamine, the fluorescence spectra of the carbon dots before and after adding histamine under the same pH in different systems (PBS buffer system, aqueous solution system) were verified. Figure 34 It is shown that the fluorescence of the carbon dots solution is significantly enhanced after adding histamine because the alkaline effect of histamine in the aqueous solution changes the pH of the solution, so that the fluorescence intensity of the system is enhanced.

[0075] Example 2: A nitrogen-doped carbon dot, using o-phenylenediamine and 2,6-pyridine dicarboxylic acid as raw materials, mixing o-phenylenediamine, 2,6-pyridine dicarboxylic acid, water and hydrochloric acid, one-step hydrothermal reaction to synthesize nitrogen-doped fluorescent carbon dots; wherein: the mass ratio of o-phenylenediamine, 2,6-pyridine dicarboxylic acid and water is 1:1:10 g / g / mL, and 10 μL of hydrochloric acid is added; after mixing, hydrothermal reaction at 160°C for 6h. The solution is dialyzed by a 1000D filter membrane for 12h, and the rest of the method is the same as the method described in Example 1.

[0076] Example 3: A nitrogen-doped carbon dot, using o-phenylenediamine and 2,6-pyridine dicarboxylic acid as raw materials, mixing o-phenylenediamine, 2,6-pyridine dicarboxylic acid, water and hydrochloric acid, one-step hydrothermal reaction to synthesize nitrogen-doped fluorescent carbon dots; wherein: the mass ratio of o-phenylenediamine, 2,6-pyridine dicarboxylic acid and water is 1:2:15 g / g / mL, and 100 μL of hydrochloric acid is added; after mixing, hydrothermal reaction at 200°C for 1h. The solution is dialyzed by a 1000D filter membrane for 36h, and the rest of the method is the same as the method described in Example 1.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Application of nitrogen-doped carbon dots in colorimetric / fluorescent dual-mode visual detection of pH or histamine, characterized by: Application of the nitrogen-doped carbon dots as colorimetric / fluorescent dual-mode sensors in visual detection of pH or histamine; The nitrogen-doped carbon dots are prepared by mixing o-phenylenediamine and 2,6-pyridinedicarboxylic acid with water and hydrochloric acid, and then reacting the mixture in a one-step hydrothermal method to synthesize nitrogen-doped fluorescent carbon dots. The mass volume ratio of o-phenylenediamine, 2,6-pyridinedicarboxylic acid and water is 1:0.5-2:10-20 g / g / mL. After mixing, the mixture is hydrothermally reacted at 160-200°C for 1-6 hours.

2. The use according to claim 1, characterized in that: The preparation method of the nitrogen-doped carbon dots comprises the following specific steps: (1) Mixing raw materials: Mix o-phenylenediamine and 2,6-pyridinedicarboxylic acid in proportion, add water and 10-100 μL of concentrated hydrochloric acid with a mass concentration of 36%-38% in sequence, stir thoroughly, and then ultrasonically dissolve for 10-20 minutes to prepare a mixed solution; (2) Hydrothermal reaction: The mixed solution obtained in step (1) was transferred to a hydrothermal reactor and placed in an oven. The temperature was raised from room temperature to 160-200°C at a rate of 4.2°C / min and the reaction was continued for 2-6 hours. (3) Obtaining the target product: After the reaction is completed, the reactor is allowed to stand and cooled naturally before being taken out to obtain a brown solution. The solution is dialyzed through a 1000 D filter membrane for 12-36 h, and then filtered through a 0.22 μm filter membrane to obtain pure brown-yellow carbon dots. The solution is then freeze-dried at -42 °C liquid nitrogen for 24 h using a freeze-drying apparatus to obtain a brown target product.

3. The use according to claim 2, characterized in that: The hydrothermal reaction temperature in step (2) is 160-180°C, and the reaction time is 2-6 hours.

4. The use according to claim 1, characterized in that: The nitrogen-doped carbon dots are used as colorimetric / fluorescence dual-mode sensors for sensitive detection of intracellular pH or histamine.

5. The use according to claim 1, characterized in that: The nitrogen-doped carbon dots are used as a colorimetric / fluorescence dual-mode sensor for the detection of histamine in the food spoilage process.

6. The use according to claim 5, characterized in that: The nitrogen-doped carbon dots are used for on-site and immediate detection of histamine changes during food spoilage.

7. The use according to claim 6, characterized in that: The food is seafood.

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