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

Abstract

The present invention provides a method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine, and its application. This method belongs to the field of fluorescent carbon dot technology. The preparation of the fluorescent carbon dots involves the following steps: 1) weighing Azure B and citric acid, dissolving them in secondary water, and sonicating to obtain a uniform mixed solution; 2) transferring the solution to a hydrothermal reactor and reacting at 150-200°C for 4-8 hours. After the reaction ceases, the solution is allowed to cool to room temperature, centrifuged to remove insoluble matter, and the supernatant is dialyzed in a glass container for at least three days through a 500-1000 Da dialysis bag to obtain a purified carbon dot aqueous solution; 3) freeze-drying the carbon dot aqueous solution to obtain carbon dots that emit deep red fluorescence. The prepared deep red fluorescent carbon dots can be used for the sequential detection of hypochlorite and methionine with good selectivity and high sensitivity.

Description

Technical Field

[0001] The present 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, an organic compound, is one of the essential amino acids in the human body and is involved in protein synthesis. Since it cannot be produced internally, it must be obtained externally. A methionine deficiency can hinder protein synthesis and cause damage to the body. Excessive oxidation of membrane lipids by oxygen free radicals in the body is responsible for various types of damage. Lipid peroxides damage primary and secondary lysosomal membranes, releasing acid phosphatases within lysosomes that act as hydrolyzers, damaging important organelles such as cell and mitochondrial membranes. Methionine counteracts this damage through various pathways. Excessive methionine in the human body can have a dangerous cumulative effect. Therefore, maintaining methionine levels in the body is crucial.

[0004] Carbon dots (Cdots) have promising applications in a wide range of fields, including bioimaging, environmental monitoring, and nanomaterials, due to their excellent luminescence properties, good chemical stability, biocompatibility, and tunable surface functionality. Currently synthesized Cdots mostly emit blue-green fluorescence, which limits their application in biomedical and optoelectronic devices. Therefore, the design and synthesis of long-wavelength fluorescent Cdots for the construction of biosensing platforms for hypochlorite and methionine is of vital research significance. 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:

[0007] A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps:

[0008] S1. Weigh Azure B and citric acid in proportion and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution;

[0009] S2. The mixed solution was transferred to a hydrothermal reactor for reaction. After the reaction stopped, the mixture was allowed to 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.

[0010] S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

[0011] Furthermore, the mass ratio of Azure B, citric acid and secondary water in step S1 is 0.1-0.8:10:200.

[0012] Furthermore, in step S2, the reaction temperature is 150-200° C., and the reaction time is 4-8 h.

[0013] A deep red fluorescent carbon dot was applied to the detection of hypochlorite.

[0014] A deep red fluorescent carbon dot-hypochlorite complex was applied to the detection of methionine.

[0015] A deep red fluorescent carbon dot was applied to the sequential detection of hypochlorite and methionine.

[0016] A deep red fluorescent carbon dot for zebrafish imaging.

[0017] The deep red fluorescent carbon dots prepared by the present invention have specific recognition effects on hypochlorite and methionine. The reasons are as follows:

[0018] 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. Figure 9 As shown in Figure 3, the fluorescence lifetime of carbon dots did not change before and after the addition of hypochlorite. Therefore, the quenching mechanism is speculated to be static quenching.

[0019] like Figure 10As shown in the figure, after adding methionine to the Cdot@hypochlorite complex, the zeta potentials of the Cdots, Cdot@hypochlorite, and Cdot@hypochlorite@methionine were -0.84 mV, -11.8 mV, and -0.89 mV, respectively. The addition of methionine restored the fluorescence and zeta potential of the Cdots, which had been quenched by hypochlorite. Therefore, the mechanism is speculated to be that after the addition of methionine, due to competition, methionine reacts with hypochlorite to form a complex, thereby restoring the fluorescence of the Cdots.

[0020] The beneficial effects of the present invention are as follows:

[0021] 1. The present invention has simple operation steps and can obtain deep red fluorescent carbon dots without surface passivation treatment or modification.

[0022] 2. The carbon dots prepared in the present invention have good solubility and dispersibility in aqueous solution.

[0023] 3. The deep red fluorescent carbon dots prepared by the present invention have specific recognition effects on hypochlorite and methionine, and are used for the sequential detection of hypochlorite and methionine with good selectivity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a transmission electron micrograph of the deep red fluorescent carbon dots prepared in Example 1 of the present invention.

[0025] 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.

[0026] Figure 3 These are the ultraviolet absorption spectrum and fluorescence excitation-emission spectrum of the deep red fluorescent carbon dots prepared in Example 1 of the present invention.

[0027] 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.

[0028] Figure 5 Figure 2 is the fluorescence change of carbon dots in the presence of different concentrations of hypochlorite.

[0029] Figure 6 Figure 2 shows the fluorescence changes of the carbon dots@hypochlorite complex in the presence of different concentrations of methionine.

[0030] 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.

[0031] Figure 8UV-visible absorption spectra of carbon dots, carbon dots@hypochlorite, and hypochlorite.

[0032] Figure 9 The fluorescence lifetime diagram of carbon dots before and after adding hypochlorite.

[0033] Figure 10 The zeta potential diagram of carbon dots before and after the addition of hypochlorite and methionine. DETAILED DESCRIPTION

[0034] The present invention will be further described below with reference to 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.

[0035] Example 1

[0036] A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps:

[0037] 1) Weigh a certain amount of Azure B and citric acid, dissolve them in secondary water, and sonicate to obtain a uniform mixed solution; the mass ratio of Azure B, citric acid, and secondary water is 0.152:10:200;

[0038] 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 cool to room temperature. The insoluble matter was removed by centrifugation, and the supernatant was dialyzed through a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution.

[0039] 3) The carbon dot aqueous solution was freeze-dried to obtain carbon dots that emitted deep red fluorescence. Using cresyl violet as a reference, the relative quantum yield was 9.8%.

[0040] Example 2

[0041] A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps:

[0042] 1) Weigh a certain amount of Azure B and citric acid, dissolve them in secondary water, and sonicate to obtain a uniform mixed solution; the mass ratio of Azure B, citric acid, and secondary water is 0.302:10:200;

[0043] 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 cool to room temperature. The insoluble matter was removed by centrifugation, and the supernatant was dialyzed through a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution.

[0044] 3) The carbon dot aqueous solution was freeze-dried to obtain carbon dots that emitted deep red fluorescence. Using cresyl violet as a reference, the relative quantum yield was 6.5%.

[0045] Example 3

[0046] A method for preparing deep red fluorescent carbon dots for detecting hypochlorite and methionine comprises the following steps:

[0047] 1) Weigh a certain amount of Azure B and citric acid, dissolve them in secondary water, and sonicate to obtain a uniform mixed solution; the mass ratio of Azure B, citric acid, and secondary water is 0.652:10:200;

[0048] 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 cool to room temperature. The insoluble matter was removed by centrifugation, and the supernatant was dialyzed through a 500-1000 Da dialysis bag in a glass container for at least three days to obtain a pure carbon dot aqueous solution.

[0049] 3) The carbon dot aqueous solution was freeze-dried to obtain carbon dots that emitted deep red fluorescence. Using cresyl violet as a reference, the relative quantum yield was 3.3%.

[0050] The deep red fluorescent carbon dots prepared in Example 1 of the present invention are characterized as follows: Figure 1 、 2 Transmission electron microscopy (TEM) images demonstrate that the carbon dots are quasi-spherical, uniform, and monodisperse, with an average particle size of 3.33 nm. Infrared spectroscopy confirms that the carbon dots have a benzene ring structure and sulfur-containing groups on their surfaces, confirming the successful doping of sulfur.

[0051] 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 The UV-Vis absorption spectrum of the carbon dots exhibits three absorption peaks at approximately 284 nm, 470 nm, and 622 nm. Excitation at 625 nm reveals a deep red fluorescence emission wavelength of 651 nm. Figure 3 Figure 3 is the emission spectrum of the carbon dots at different excitation wavelengths, indicating that the carbon dots are independent of the excitation wavelength.

[0052] Sensing performance of the deep red fluorescent carbon dots prepared in Example 1 of the present invention to hypochlorite anions is as follows: Figure 5 As shown in the figure, 10 mg of dry carbon dot powder was placed in a 10 mL volumetric flask, diluted to volume with PBS buffer and shaken to prepare a carbon dot stock solution (0.20 mg / mL). 10 μL of sodium hypochlorite solution of different concentrations was added to 2.0 mL of carbon dot stock solution, mixed thoroughly, and incubated at room temperature for 3 minutes. The changes in fluorescence intensity were recorded, as shown in Figure 2. Figure 5As shown in Figure 3, the linear ranges were 2.5-125 μM and 125-287.5 μM, with a detection limit of 0.46 μM.

[0053] Sensing performance of the deep red fluorescent carbon dots@hypochlorite anion complex to methionine prepared in Example 1 of the present invention 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 changes in fluorescence intensity were recorded. The concentration of carbon dots was 0.20 mg / mL and the concentration of sodium hypochlorite was 600 μM. Figure 6 As shown in Figure 3, the linear ranges are 0.25-6.25 μM and 6.25-16.25 μM, with a detection limit of 80 nM.

[0054] The cell imaging image of the deep red fluorescent carbon dots prepared in Example 1 of the present invention is shown in FIG. 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 stained by 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. An application of deep red fluorescent carbon dots in the detection of hypochlorite, characterized by: The method for preparing the deep red fluorescent carbon dots comprises the following steps: S1. Weigh Azure B and citric acid in proportion and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution; S2. The mixed solution was transferred to a hydrothermal reactor for reaction. After the reaction stopped, the mixture was allowed to 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; the reaction temperature was 150-200 ° C, and the reaction time was 4-8 h; S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

2. Application of a deep red fluorescent carbon dot-hypochlorite complex in the detection of methionine, characterized in that: The method for preparing the deep red fluorescent carbon dots comprises the following steps: S1. Weigh Azure B and citric acid in proportion and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution; S2. The mixed solution was transferred to a hydrothermal reactor for reaction. After the reaction stopped, the mixture was allowed to 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; the reaction temperature was 150-200 ° C, and the reaction time was 4-8 h; S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

3. Application of deep red fluorescent carbon dots in detecting the order of hypochlorite and methionine, characterized in that: The method for preparing the deep red fluorescent carbon dots comprises the following steps: S1. Weigh Azure B and citric acid in proportion and dissolve them in secondary water, and ultrasonicate to obtain a uniform mixed solution; S2. The mixed solution was transferred to a hydrothermal reactor for reaction. After the reaction stopped, the mixture was allowed to 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; the reaction temperature was 150-200 ° C, and the reaction time was 4-8 h; S3. Freeze-drying the carbon dot aqueous solution to obtain carbon dots emitting deep red fluorescence.

4. The use of the deep red fluorescent carbon dots according to any one of claims 1 to 3, characterized in that: The mass ratio of Azure B, citric acid and secondary water in step S1 is 0.1-0.8:10:200.

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