Preparation method and application of long-wave carbon dots sensitive to pH

Abstract

The application discloses a preparation method of pH-sensitive long-wave carbon dots and application of the carbon dots in fluorescent ink, and belongs to the technical field of carbon nanomaterials. The application is characterized in that neutral red and L-cysteine are ultrasonically dissolved in deionized water at room temperature, then a heating reaction is carried out, the solution is naturally cooled to room temperature and filtered, and then the carbon dots aqueous solution is obtained by dialysis through a dialysis membrane, and L-CDs are obtained by freeze-drying. The L-CDs prepared by the application have high sensitivity in non-labeled detection of pH and hypochlorite, low cost, a simple detection process, small harm to biological samples, and are more safe and reliable. The L-CDs can also be used for intracellular imaging.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of carbon nanomaterials, and particularly relates to a preparation method of pH-sensitive long-wave carbon dots and application thereof in fluorescent ink. BACKGROUND

[0002] The stability of intracellular pH plays an important role in signal transduction, cell proliferation, cell apoptosis and other metabolic physiological processes. Abnormal intracellular pH can lead to the occurrence of various pathological symptoms of cancer, chronic kidney disease, arthritis and the like. The normal pH value of healthy cells is 7.2-7.4, and the imbalance and fluctuation of pH often occur in the pathological tissues of many secondary diseases. So far, various technologies have been used for the detection of intracellular pH, such as microelectrode, nuclear magnetic resonance and absorption spectrum. However, the traditional methods have the disadvantages of high cost, complex sampling process and complicated operation, which are difficult to overcome. The fluorescent detection has the advantages of high sensitivity, low cost and simple detection process, and the fluorescent detection strategy based on CDs has attracted widespread attention due to its low toxicity, low cost, high biocompatibility and rapid detection. Therefore, it is of great significance to develop a preparation method of pH-sensitive CDs for monitoring the change of intracellular pH value.

[0003] In recent years, counterfeiting has become a serious social problem, so it is essential to use high-tech solutions to prevent and stop counterfeiting. Among various existing anti-counterfeiting technologies, fluorescent anti-counterfeiting is widely used due to its strong anti-counterfeiting ability, fast identification and simple operation. The commonly used fluorescent materials for preventing counterfeiting mainly include inorganic phosphors, quantum dots and organic fluorescent dyes. However, the synthesis of inorganic phosphors is complex and the raw materials are expensive, quantum dots contain heavy metals which have adverse effects on human beings and the environment, and the thermal stability, light stability and chemical stability of organic dyes are poor and the light bleaching resistance is poor. Therefore, it is of great significance to develop fluorescent anti-counterfeiting materials with good light stability, low toxicity and good light bleaching resistance for the development of fluorescent anti-counterfeiting technology. Carbon quantum dots can be used as high-quality new fluorescent ink in data encryption and storage due to their good biocompatibility, environmental friendliness, low cost and excellent light bleaching resistance. The luminescent ink prepared by carbon quantum dots can be packaged in inkjet printers or injected into fountain pens, neutral pens or dipped with a brush, and then printed or written on papers, textiles, leathers, glasses and the like without fluorescent agents, and double encryption can also be realized by combining two-dimensional codes and bar codes. In addition, the water solubility of carbon quantum dots is excellent, and the anti-counterfeiting ink prepared by carbon quantum dots has smooth ink flow and does not cause blockage, so it is of great significance to develop carbon quantum dots with excellent performance for the development of fluorescent anti-counterfeiting technology. SUMMARY

[0004] In view of the above problems, the application provides a preparation method and application of pH-sensitive long-wave carbon dots.

[0005] To achieve the above object, the present application adopts the following technical solutions:

[0006] A preparation method of a pH-sensitive long-wave carbon dot, comprising the following steps: ultrasonic dissolution of neutral red and L-cysteine in deionized water at room temperature; then heating reaction, natural cooling to room temperature, filtering, and then dialysis through a dialysis membrane to obtain a carbon dot aqueous solution, and freeze-drying to obtain L-CDs.

[0007] Further, the mass ratio of neutral red and L-cysteine is 5.9:11.8.

[0008] Further, the ultrasonic dissolution time is 3min-5min.

[0009] Further, the heating temperature of the heating reaction is 160℃-220℃, and the heating time is 4h-8h.

[0010] Further, the amount of deionized water added is 20mL.

[0011] Further, the filtering is filtering through a 0.22μm filter membrane.

[0012] Further, the dialysis through a dialysis membrane is dialysis through a dialysis membrane with a molecular weight of 500-1000Da for 2-3d.

[0013] Further, the dissolved solution is loaded into a Teflon liner, and then sealed into a stainless steel high-pressure hydrothermal reaction kettle for heating reaction.

[0014] A long-wave carbon dot prepared by the above preparation method and application thereof in fluorescent ink.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] 1. The operation steps of the present application are simple, and the carbon dots with long-wave emission can be obtained through a simple one-step hydrothermal method.

[0017] 2. The L-CDs prepared by the present application have high sensitivity, low cost, simple detection process, and small harm to biological samples, and are more safe and reliable in non-labeled detection of pH and hypochlorite. The L-CDs can also be used for intracellular imaging.

[0018] 3. The L-CDs prepared by the present application have excellent light stability and chemical stability, and the fluorescent ink prepared based on the L-CDs can respond to pH changes, and has good application potential in the field of anti-counterfeiting. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1(a) TEM and HRTEM images of L-CDs; (b) size distribution of L-CDs; (c) AFM image of L-CDs;

[0020] Figure 2 FTIR image of L-CDs;

[0021] Figure 3 (a) XPS full spectrum (b) high resolution element spectra of C 1s, (c) N 1s, (d) O 1s and (e) S 2p of L-CDs;

[0022] Figure 4 (a) UV-Vis absorption, excitation and emission spectra of L-CDs; (b) emission spectra of L-CDs under different excitation light;

[0023] Figure 5 (a) Fluorescence intensity of L-CDs solution under different pH; (b) Fluorescence intensity of L-CDs solution under different KCl concentration; (c) Fluorescence intensity of L-CDs after xenon lamp irradiation for different time;

[0024] Figure 6 (a) pH titration curve of L-CDs; (b) Boltzmann nonlinear fitting of fluorescence intensity and pH of L-CDs and linear fitting of pH (c) 4.2-5.8, (d) 5.8-7.4;

[0025] Figure 7 Zeta potential of L-CDs solution under different pH;

[0026] Figure 8 (a) Hypochlorite titration curve of L-CDs; (b) nonlinear fitting of fluorescence quenching degree and hypochlorite concentration of L-CDs and (c) linear fitting; (d) Selective detection of L-CDs;

[0027] Figure 9 (a) Fluorescence lifetime curve of L-CDs solution with and without hypochlorite; (b) UV-Vis absorption spectra of L-CDs solution with and without hypochlorite and hypochlorite solution;

[0028] Figure 10 Relationship between Hela cell survival rate and L-CDs concentration;

[0029] Figure 11 (a) Confocal imaging of L-CDs on Hela cells at pH 4, 7, 9; (b) Confocal imaging of L-CDs and L-CDs / ClO- on Hela cells;

[0030] Figure 12The relevant photos of handwriting "Shanxi University SXU" on filter paper under (top) sunlight, (bottom) 365 nm ultraviolet light, (a) using 0.26 mg / mL L-CDs solution and (b) 0.26 mg / mL L-CDs solution (PH = 10) and (c) 0.26 mg / mL L-CDs solution (PH = 2). DETAILED DESCRIPTION

[0031] Example 1

[0032] A preparation method of long-wave carbon dots for pH and hypochlorite non-labeled detection, comprising the following steps:

[0033] 5.9 mg of NR and 11.8 mg of L-cys were added in 20 mL of deionized water, the solution was ultrasonically dissolved for 3 min, the solution was loaded into a 50 mL Teflon-lined, sealed into a stainless steel high-pressure hydrothermal reactor, and placed in an oven for heating at 180°C for 4 h. After the reaction was completed, the obtained solution was naturally cooled to room temperature, filtered with a 0.22 μm filter membrane, then dialyzed with a 500-1000 Da dialysis membrane for 3 d, and then freeze-dried to obtain L-CDs powder. Rhodamine B was used as a reference, and the relative quantum yield of L-CDs was 2.53%.

[0034] Example 2

[0035] The TEM image, size distribution and AFM image characterization of L-CDs prepared in Example 1 of the present application are shown in Figure 1 The carbon dots show regular spherical morphology characteristics, monodisperse arrangement, uniform dispersion of L-CDs nanoparticles, and interlayer lattice spacing of 0.21 nm, indicating the presence of graphene structure; the average particle size is 2.43 nm; and the AFM image characterizes the height of the L-CDs nanoparticles.

[0036] Example 3

[0037] The infrared spectrum (FTIR) characterization of L-CDs prepared in Example 1 of the present application is shown in Figure 2 The carbon dots show regular spherical morphology characteristics, monodisperse arrangement, uniform dispersion of L-CDs nanoparticles, and interlayer lattice spacing of 0.21 nm, indicating the presence of graphene structure; the average particle size is 2.43 nm; and the AFM image characterizes the height of the L-CDs nanoparticles. -1 Seven characteristic peaks were observed at 614, 1101, 1405, 1630, 1711, 3210 and 3306 cm

[0038] Example 4

[0039] The X-ray photoelectron spectroscopy (XPS) characterization of L-CDs prepared in Example 1 of the present application is shown in Figure 3 Four obvious binding energy peaks can be observed at 195, 282, 396 and 528 eV, respectively, corresponding to S2p , C 1s, N 1s and O 1s four elements, indicating that the carbon dots are composed of C, N, O, S four elements. Figure 3 b, 3c, 3d and 3e are C 1s, N 1s, O 1s and S 2p High-resolution XPS spectra. The C 1s element spectrum can be divided into three sub-peaks of C-C / C=C at 284.8eV, C=O at 285.3eV and C-O at 287.25eV; in the N 1s element spectrum, 398.25eV, 398.85eV and 400.65eV three sub-peaks correspond to pyridine N, pyrrole N and graphene N respectively; in the O 1s element spectrum, C-O and C=O two sub-peaks are obtained at 398.85eV and 400.65eV respectively; in the S 2p Element spectrum, two sub-peaks (S 2p 1 / 2 at 167.65eV and S 2p 3 / 2 at 168.85eV) can be obtained. The elemental analysis results show that S is successfully doped on L-CDs, and various nitrogen-containing, oxygen-containing and sulfur-containing groups exist on the surface of L-CDs.

[0040] Example 5

[0041] The ultraviolet-visible absorption spectrum, excitation spectrum and emission spectrum of L-CDs prepared in Example 1 of the present application are shown in Figure 4 a, and the emission spectrum of L-CDs under different excitation light is shown in Figure 4 b. Two absorption peaks can be observed at 272nm and 531nm for the L-CDs, respectively attributed to π→π* transition of C=C bond and n→π* transition of C=O / C-N bond, and the maximum excitation and emission wavelengths are 520nm and 587nm respectively. Figure 4 b indicates that the fluorescence of L-CDs shows a slight red shift with the increase of excitation wavelength.

[0042] Example 6

[0043] The fluorescence intensity of L-CDs prepared in Example 1 of the present application in different pH, salt concentration environments and the stability under xenon lamp irradiation are shown in Figure 5 ; the fluorescence intensity of the L-CDs gradually quenches with the increase of pH, indicating that the L-CDs can produce fluorescence response to the change of pH Figure 5 (a). In different concentrations of KCl solution (0-4mol / L), the fluorescence intensity of L-CDs remains relatively stable, indicating that L-CDs has good tolerance to solution salt concentration Figure 5 (b). The results show that the L-CDs have good light stability Figure 5 (c) after continuous irradiation of L-CDs with xenon lamp for 60min.

[0044] Example 7

[0045] The L-CDs prepared in Example 1 of this invention are used for label-free pH detection as follows: Figure 6 As shown in the figure, a series of BR buffer solutions with pH values ​​ranging from 2 to 10 and intervals of 0.2 were first prepared using boric acid, phosphoric acid, and glacial acetic acid. 60 μL of L-CDs solution (0.26 mg / mL) was added to 2 mL of BR buffer solutions at different pH values, and the fluorescence spectra of the solutions were measured to observe the relationship between pH value and the maximum fluorescence intensity of the L-CDs solution. Figure 6 As shown in (a), the fluorescence intensity of L-CDs significantly decreased as pH gradually increased. The Boltzmann equation was used to perform a nonlinear fitting of the relationship between the fluorescence intensity of L-CDs and pH. Figure 6 b) Two linear ranges were obtained, corresponding to 4.2-5.8 and 5.8-7.4 respectively. Figure 6 (c) shows the linear fitting results when pH is between 4.2 and 5.8. Figure 6 (d) shows the linear fit of pH in the range of 5.8-7.4, indicating that L-CDs have the ability to monitor pH changes.

[0046] Example 8

[0047] The response mechanism of the L-CDs prepared in Example 1 of this invention to label-free pH detection is as follows: Figure 7 As shown, the Zeta potential of L-CDs solutions varies between -23.4 mV and 23.6 mV at different pH levels. The Zeta potential of L-CDs decreases as the pH gradually increases, and this change is particularly pronounced at pH 2-5. Based on this, it is speculated that the pH response mechanism of L-CDs is the protonation and deprotonation of nitrogen-containing functional groups on the L-CDs surface in different pH environments.

[0048] Example 9

[0049] The label-free detection of hypochlorite by L-CDs prepared in Example 1 of this invention is as follows: Figure 8 As shown. First, different amounts of hypochlorite solution were added to 60 μL of L-CDs solution (0.26 mg / mL), and then diluted with deionized water to 2 mL to obtain a series of test solutions with different hypochlorite concentrations (0-300 μM). Their fluorescence spectra were measured and recorded at λex / λem of 520 / 587 nm. Figure 8 As shown in Figure a, the fluorescence intensity of the L-CDs solution decreased significantly as the hypochlorite concentration gradually increased from 0 μmol / L to 300 μmol / L. Figure 8 b represents the nonlinear fitting result of the Boltzmann equation on the quenching degree and hypochlorite concentration. Figure 8cThe linear fitting equation is shown, the linear range is 10-162.5 μmol / L, and the detection limit is 1.021 μmol / L. The selectivity of L-CDs to different ions is then detected Figure 8 d), 60 μL of L-CDs solution is diluted to 2 mL with deionized water, different ions are added, and the change of fluorescence intensity is detected. The results show that hypochlorite can cause significant fluorescence quenching of L-CDs, and this quenching has high tolerance to the presence of other ions, indicating that L-CDs has good selectivity for the detection of hypochlorite.

[0050] Example 10

[0051] The response mechanism of L-CDs prepared in Example 1 to hypochlorite is shown in Figure 9 , as shown in Figure 9 (a) is the fluorescence lifetime of L-CDs solution and the fluorescence lifetime of L-CDs solution containing ClO - (concentration of 0.5 mmol / L), and no significant change is observed between the two. Figure 9 (b) After adding hypochlorite to the L-CDs solution, the absorption peak at 272 nm shows a certain degree of red shift and the absorbance decreases slightly, and the absorption peak at 531 nm shows a blue shift and the absorbance decreases significantly, which indicates that the interaction between L-CDs and hypochlorite causes changes in the chemical structure of L-CDs surface, thereby causing changes in optical properties. Combined with the detection results of fluorescence lifetime and UV / Vis absorption spectrum, it is inferred that the fluorescence response mechanism of L-CDs to hypochlorite is static quenching.

[0052] Example 11

[0053] The toxicity test of L-CDs prepared in Example 1 to Hela cells is shown in Figure 10 The present application uses standard MTT colorimetric method to detect the cytotoxicity of L-CDs in Hela cells. After inoculating Hela cells in a 96-well plate, they are placed in a incubator with a temperature of 37℃ and a CO2 volume fraction of 5%, after 1d of culture, the culture solution is aspirated, different concentrations of L-CDs medium are added, after 1d of culture, 10 μL of MTT solution with a concentration of 5 mg / mL is added for 4h of culture, after removing the supernatant, 150 μL of dimethyl sulfoxide is added, after 10 min of oscillation, the absorbance at 490 nm is measured using an enzyme-linked immunoassay instrument. It can be seen from Figure 10 that with the increase of the concentration of L-CDs, the survival rate of cells gradually decreases, but the survival rate of cells in each concentration group can be maintained at a high level (more than 80%). It is inferred that the cytotoxicity of L-CDs is low, and it has application potential in biological monitoring and cell imaging.

[0054] Example 12

[0055] Cell imaging of L-CDs prepared in Example 1 of the present application to Hela cells is shown in Figure 11 As shown, first, HeLa cells were placed in a 15mm glass dish and incubated at 37℃ for 24h. After the cell incubation was completed, the cells were washed with PBS (pH = 7.4) three times to remove impurities, incubated with PBS solutions of different pH values (4, 7, 9) for 2h, and then treated with L-CDs (0.26mg / mL) for 2h. The culture medium was carefully discarded, fresh culture medium containing hypochlorite was added and incubated for 30min, and then the treated cells were washed with a PBS solution and observed by confocal imaging. Figure 11 (a) is a cell image after treatment at different pH values, and the results show that the fluorescence intensity of Hela cells can be obviously observed to gradually decrease with the increase of pH value. Figure 11 b shows that the fluorescence of Hela cells is obviously quenched under the action of hypochlorite relative to cells treated only with L-CDs. The above results show that L-CDs have imaging and monitoring capabilities for intracellular pH and hypochlorite concentration, and have certain application potential in the field of biological monitoring.

[0056] Example 13

[0057] Application of L-CDs prepared in Example 1 of the present application in anti-counterfeiting is shown in Figure 12 As shown, fluorescent ink made of L-CDs is applied to anti-counterfeiting writing. The specific operation is to encapsulate the fluorescent ink prepared from a 0.26mg / ml L-CDs solution into the ink sac of a pen and perform handwriting on paper. As shown in Figure 12 a, "Shanxi University SXU" written on paper with L-CDs ink is almost invisible under daylight and emits orange fluorescence under 365nm ultraviolet light irradiation. Based on the non-labeled detection of pH by L-CDs in Example 7 above, 0.26mg / ml L-CDs solutions of different pH values were prepared and written on paper according to the above steps. As shown in Figure 12 b, when pH = 10, the carbon dot solution written "Shanxi University SXU" is completely invisible under daylight, and the fluorescence color emitted under ultraviolet light irradiation becomes lighter and the intensity becomes weaker; when the pH of the solution is adjusted to 2, as shown in Figure 12 c, orange fluorescence is emitted again under 365nm ultraviolet light irradiation, and the fluorescence intensity is increased compared with before, which also corresponds to the prepared L-CDs whose fluorescence can respond to pH changes. It is shown that the L-CDs as invisible fluorescent ink have good application potential in anti-counterfeiting.

[0058] In summary, the yellow light-emitting L-CDs were successfully synthesized by one-step hydrothermal method using neutral red and L-cysteine. The L-CDs have specific fluorescence response to pH and hypochlorite concentration. When the environmental pH value is between 4.2 and 7.4, the fluorescence intensity of L-CDs shows a good linear relationship with the pH value. Hypochlorite can cause the fluorescence of L-CDs solution to be obviously quenched, the linear range is 10-162.5 μM, the detection limit is 1.021 μM, and according to the results of ultraviolet-visible absorption spectrum and fluorescence lifetime detection, the response mechanism is static quenching. The results of cell toxicity experiment show that L-CDs have low cell toxicity and good biocompatibility, then the L-CDs are used for cell imaging experiment of Hela cells, and the results show that L-CDs can be used for fluorescence imaging in cells, and can realize fluorescence imaging monitoring of pH change and hypochlorite concentration in cells. This means that L-CDs have potential application value in the field of biological monitoring, thereby further enriching the theory of long-wavelength L-CDs and the application of long-wavelength L-CDs in fluorescence monitoring and biological sensing.

[0059] The contents not described in detail in the specification of the present application belong to the prior art known to those skilled in the art. Although the above describes the specific embodiments of the present application in a descriptive manner, so as to facilitate those skilled in the art to understand the present application, it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

Claims

1. A method for preparing pH-sensitive long-wavelength carbon dots, characterized in that: The process includes the following steps: neutral red and L-cysteine ​​are dissolved in deionized water by ultrasonication at room temperature; then a heating reaction is carried out, and the mixture is naturally cooled to room temperature and filtered. The solution is then dialyzed through a dialysis membrane to obtain an aqueous solution of carbon dots, which is then freeze-dried to obtain L-CDs. The mass ratio of neutral red to L-cysteine ​​is 5.9:11.

8. The heating temperature of the heating reaction is 160℃~220℃, and the heating time is 4h~8h.

2. The method for preparing pH-sensitive long-wavelength carbon dots according to claim 1, characterized in that: The ultrasonic dissolution time is 3 to 5 minutes.

3. The method for preparing pH-sensitive long-wavelength carbon dots according to claim 1, characterized in that: The amount of deionized water added is 20 mL.

4. The method for preparing pH-sensitive long-wavelength carbon dots according to claim 1, characterized in that: The filtration is performed using a 0.22 μm filter membrane.

5. The method for preparing pH-sensitive long-wavelength carbon dots according to claim 1, characterized in that: The dialysis membrane used is a dialysis membrane with a pressure of 500-1000 Da for 2-3 days.

6. The method for preparing pH-sensitive long-wavelength carbon dots according to claim 1, characterized in that: The dissolved solution is placed into a Teflon-lined container and then sealed in a stainless steel high-pressure hydrothermal reactor for heating and reaction.

7. A long-wave carbon dot prepared by the preparation method according to any one of claims 1 to 6.

8. The application of a long-wavelength carbon dot prepared by any one of claims 1 to 6 in a fluorescent ink at different pH values.

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