Preparation method and application of dark red fluorescent carbon dots for detecting hypochlorite and methionine

Abstract

The invention aims to provide a preparation method and application of a dark red fluorescent carbon dot for detecting hypochlorite and methionine, and belongs to the technical field of fluorescent carbon dots, and the preparation method of the fluorescent carbon dot comprises the following steps: 1) weighing azurol B and citric acid, dissolving in secondary water, and carrying out ultrasonic treatment to obtain a uniformly mixed solution; 2) transferring the solution into a hydrothermal reaction kettle, reacting for 4-8 hours at 150-200 DEG C, standing and cooling to room temperature after the reaction is stopped, centrifuging to remove insoluble substances, taking supernate, and dialyzing for at least three days in a glass container through a 500-1000Da dialysis bag to obtain a pure carbon dot aqueous solution; 3, the carbon dot aqueous solution is subjected to freeze drying, and then the carbon dots emitting dark red fluorescence are obtained.The prepared dark red fluorescence carbon dots can be used for sequentially detecting pypocholoride and methionine and are good in selectivity and high in sensitivity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent carbon dots, and in particular relates to a preparation method and application of deep red fluorescent carbon dots for detecting hypochlorite and methionine. Background Art

[0002] Hypochlorite anion (ClO - ) is widely used in daily life as a household bleach, a disinfectant in drinking water and cooling water treatment and disinfection. In addition, as one of the most important reactive oxygen species, ClO - It plays a key role in the signal transduction of organisms. Endogenous ClO produced by immune cells - It can protect the body from pathogens. However, excessive ClO - It can damage DNA, proteins, RNA, and cholesterol through oxidative stress, leading to a variety of diseases such as neuronal degeneration, immune deficiency, kidney disease, myocardial damage, and cancer. Therefore, it is imperative to develop an effective strategy to quantitatively monitor ClO in biological systems with high sensitivity and specificity. - .

[0003] Methionine is an organic compound and one of the essential amino acids that make up the human body. It is involved in protein synthesis. Since it cannot be produced in the body itself, it must be obtained from the outside. If methionine is deficient, protein synthesis in the body will be blocked, causing damage to the body. Excessive oxidation of membrane lipids caused by oxygen free radicals in the body is the cause of various damages to the body. Lipid peroxides can damage primary and secondary lysosomal membranes, release acid phosphatases contained in lysosomes as hydrolysis, and cause damage to important organelles such as cell and mitochondrial membranes. Methionine fights these damages in many ways. Excessive methionine in the human body has a dangerous cumulative effect. Therefore, it is crucial to achieve methionine in organisms.

[0004] Carbon dots have good application prospects in many fields such as bioimaging, environmental monitoring and nanomaterials due to their excellent luminescence properties, good chemical stability, biocompatibility and adjustable surface functions. Currently, most of the synthesized carbon dots emit blue-green fluorescence, which limits their application in biomedical and optoelectronic devices. Therefore, it is of great research significance to design and synthesize long-wavelength emission fluorescent carbon dots to construct a biosensing platform for hypochlorite and methionine. Summary of the invention

[0005] The object of the present invention is to provide a preparation method and application of deep red fluorescent carbon dots for detecting hypochlorite and methionine, wherein the carbon dots have specific recognition effects on hypochlorite and methionine.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps: S1, weigh Azure B and citric acid according to proportion, dissolve in secondary water, and obtain a uniform mixed solution by ultrasonication; S2, transferring the mixed solution to a hydrothermal reactor for reaction, and after the reaction stops, cooling to room temperature, centrifuging to remove insoluble matter, taking the supernatant, and dialyzing the supernatant through a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution; S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

[0007] Furthermore, the mass ratio of Azure B, citric acid and secondary water in step S1 is 0.1-0.8:10:200.

[0008] Furthermore, in step S2, the reaction temperature is 150-200° C., and the reaction time is 4-8 h.

[0009] A deep red fluorescent carbon dot was applied to the detection of hypochlorite.

[0010] A deep red fluorescent carbon dots and hypochlorite complex was applied to the detection of methionine.

[0011] A deep red fluorescent carbon dot was applied to the sequential detection of hypochlorite and methionine.

[0012] A deep red fluorescent carbon dot for application in zebrafish imaging.

[0013] The deep red fluorescent carbon dots prepared by the present invention have specific recognition effects on hypochlorite and methionine. The reasons are as follows: like Figure 8 As shown in Figure 2, after adding hypochlorite, the UV-visible absorption spectrum of carbon dots changed and a new absorption peak appeared. Fig. 9 As shown in Figure 3, the fluorescence lifetime of carbon dots did not change before and after the addition of hypochlorite. Therefore, it is speculated that the quenching mechanism is static quenching.

[0014] like Fig.10 As shown in the figure, after adding methionine to the Cdot@hypochlorite complex, the zeta potentials of Cdots, Cdot@hypochlorite, and Cdot@hypochlorite@methionine are -0.84 mV, -11.8 mV, and -0.89 mV, respectively. After adding methionine, the fluorescence and zeta potential of Cdots quenched by hypochlorite are restored. Therefore, it is speculated that the mechanism is that after adding methionine, due to competition, methionine reacts with hypochlorite to form a complex, thereby restoring the fluorescence of Cdots.

[0015] The beneficial effects of the present invention are as follows: 1. The present invention has simple operation steps and can obtain carbon dots with deep red fluorescence emission without surface passivation agent treatment or modification.

[0016] 2. The carbon dots prepared in the present invention have good solubility and dispersibility in aqueous solution.

[0017] 3. The deep red fluorescent carbon dots prepared by the present invention have specific recognition effects on hypochlorite and methionine, and are used for sequential detection of hypochlorite and methionine with good selectivity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transmission electron microscopy image of the deep red fluorescent carbon dots prepared in Example 1 of the present invention.

[0019] Figure 2 This is an infrared spectrum of the deep red fluorescent carbon dots prepared in Example 1 of the present invention, where the abscissa is the detection wavelength and the ordinate is the transmittance.

[0020] Figure 3 The ultraviolet absorption spectrum and fluorescence excitation-emission spectrum of the deep red fluorescent carbon dots prepared in Example 1 of the present invention.

[0021] Figure 4 This is a spectrum diagram showing the fluorescence emission curve of the deep red fluorescent carbon dots prepared in Example 1 as a function of the excitation wavelength.

[0022] Figure 5 The fluorescence changes of carbon dots in the presence of different concentrations of hypochlorite.

[0023] Figure 6 The fluorescence change diagram of the carbon dots@hypochlorite complex in the presence of different concentrations of methionine.

[0024] Figure 7 This is a laser confocal image of the carbon dots prepared in Example 1 of the present invention, which were quenched by hypochlorite and then recovered by methionine. The animal is a zebrafish.

[0025] Figure 8 UV-visible absorption spectra of carbon dots, carbon dots@hypochlorite and hypochlorite.

[0026] Fig. 9 The fluorescence lifetime diagram of carbon dots before and after adding hypochlorite.

[0027] Fig.10 The zeta potential diagram of carbon dots before and after the addition of hypochlorite and methionine. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The embodiments provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0029] Example 1 A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps: 1) Weigh a certain amount of Azure B and citric acid and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution; the mass ratio of Azure B, citric acid and secondary water is 0.152:10:200; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 180°C for 6 h. After the reaction stopped, the mixture was allowed to stand and cool to room temperature. The insoluble matter was removed by centrifugation and the supernatant was taken. The supernatant was dialyzed in a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution. 3) The carbon dot aqueous solution was freeze-dried to obtain carbon dots emitting deep red fluorescence, with a relative quantum yield of 9.8% using cresyl violet as a reference.

[0030] Example 2 A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps: 1) Weigh a certain amount of Azure B and citric acid, dissolve them in secondary water, and obtain a uniform mixed solution by ultrasonic treatment; the mass ratio of Azure B, citric acid and secondary water is 0.302:10:200; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 150°C for 8 h. After the reaction stopped, the mixture was allowed to stand and cool to room temperature. The insoluble matter was removed by centrifugation and the supernatant was taken. The supernatant was dialyzed in a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution. 3) The carbon dot aqueous solution is freeze-dried to obtain carbon dots emitting deep red fluorescence, with a relative quantum yield of 6.5% using cresyl violet as a reference.

[0031] Example 3 A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps: 1) Weigh a certain amount of Azure B and citric acid and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution; the mass ratio of Azure B, citric acid and secondary water is 0.652:10:200; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 200°C for 4 h. After the reaction stopped, the mixture was allowed to stand and cool to room temperature. The insoluble matter was removed by centrifugation and the supernatant was taken. The supernatant was dialyzed in a glass container for at least three days through a 500-1000 Da dialysis bag to obtain a pure carbon dot aqueous solution; 3) The carbon dot aqueous solution was freeze-dried to obtain carbon dots emitting deep red fluorescence, with a relative quantum yield of 3.3% using cresyl violet as a reference.

[0032] The deep red fluorescent carbon dots prepared in Example 1 of the present invention are characterized as follows: Figure 1 , 2 As shown. Transmission electron microscopy images show that the carbon dots are quasi-spherical, uniform and monodisperse, with an average particle size of 3.33 nm. Infrared spectra show that the carbon dots have a benzene ring structure and sulfur-containing groups on the surface, which confirms the successful doping of sulfur.

[0033] The optical property spectrum of the deep red fluorescent carbon dots prepared in Example 1 of the present invention is as follows: Figure 3 , 4 As shown in Figure 2, the UV-Vis absorption spectrum of the carbon dots has three absorption peaks at around 284 nm, 470 nm and 622 nm. Under the excitation of 625 nm, an emission wavelength of 651 nm appears, showing deep red fluorescence. Figure 3 It is the emission spectrum of the carbon dots under different excitation wavelengths, indicating that the carbon dots are independent of the excitation wavelength.

[0034] Sensing performance of the deep red fluorescent carbon dots prepared in Example 1 of the present invention to hypochlorite anions Figure 5 As shown, 10 mg of dry carbon dot powder was placed in a 10 mL volumetric flask, diluted with PBS buffer to a fixed volume and shaken to prepare a carbon dot mother solution (0.20 mg / mL). 10 μL of sodium hypochlorite solution of different concentrations was added to 2.0 mL of carbon dot mother solution, mixed thoroughly, and incubated at room temperature for 3 min. The changes in fluorescence intensity were recorded, as shown in Figure 2. Figure 5 As shown, its linear ranges are 2.5-125 μM and 125-287.5 μM, and the detection limit is 0.46 μM.

[0035] Sensing performance of the deep red fluorescent carbon dots@hypochlorite anion complex prepared in Example 1 of the present invention on methionine Figure 6 As shown in the figure, 10 μL of methionine solution of different concentrations was added to 2 mL of carbon dot@sodium hypochlorite mixed solution, mixed thoroughly and incubated at room temperature for 3 min, and the change of fluorescence intensity was recorded, where the concentration of carbon dots was 0.20 mg / mL and the concentration of sodium hypochlorite was 600 μM. Figure 6As shown, its linear range is 0.25-6.25 μM and 6.25-16.25 μM, and the detection limit is 80nM.

[0036] The cell imaging image of the deep red fluorescent carbon dots prepared in Example 1 of the present invention is as follows Figure 7 Human cervical cancer cells Hela were incubated in a deep red fluorescent carbon dot aqueous solution (pH = 7.4) for 2 hours. The carbon dots were fully dispersed into the cytoplasm and showed bright red fluorescence in the red channel. When ClO was added - After adding methionine, the carbon dots are ClO - The quenched fluorescence gradually recovered, indicating that the orange-red fluorescent carbon dots can be used to construct a fluorescent sensing platform for hypochlorite and methionine in vivo.

Claims

1. A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine, characterized in that: The steps include: S1. Weigh Azure B and citric acid according to proportion, dissolve in secondary water, and perform ultrasonication to obtain a uniform mixed solution; S2, transferring the mixed solution to a hydrothermal reactor for reaction, and after the reaction stops, cooling to room temperature, centrifuging to remove insoluble matter, taking the supernatant, and dialyzing the supernatant through a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution; S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

2. The method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine according to claim 1, characterized in that: The mass ratio of Azure B, citric acid and secondary water in step S1 is 0.1-0.8:10:

200.

3. The method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine according to claim 1, characterized in that: In step S2, the reaction temperature is 150-200° C. and the reaction time is 4-8 h.

4. The deep red fluorescent carbon dots prepared by the preparation method as claimed in claim 1 are used for the detection of hypochlorite.

5. A deep red fluorescent carbon dots prepared by the preparation method according to claim 1 and a complex of deep red fluorescent carbon dots and hypochlorite are used for the detection of methionine.

6. A deep red fluorescent carbon dots prepared by the preparation method as claimed in claim 1 are used for sequential detection of hypochlorite and methionine.

7. Use of the deep red fluorescent carbon dots prepared by the preparation method according to claim 1 in zebrafish imaging.

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