Application of quenching type fluorescent carbon dots in detection of perfluorobutanesulfonic acid in cells
Quenching fluorescent carbon dots prepared by hydrothermal reaction are used to detect perfluorobutane sulfonic acid in cells, solving the problems of difficulty in detection and expensive equipment in the prior art, and achieving high selectivity and sensitivity detection effects.
Patent Information
- Application Number
- CN202510411420.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to detect perfluorobutane sulfonic acid (PFBS) in cells quickly, conveniently and accurately, and traditional methods require expensive large-scale instruments and complex sample pretreatment.
Quenching fluorescent carbon dots are prepared by hydrothermal reaction. The carbon dots prepared using specific carbon sources such as ortho-phenylenediamine, m-phenylenediamine, etc. have good solubility and selectivity, and can react with perfluorobutane sulfonic acid to cause fluorescence quenching, thereby being used to detect PFBS.
High selectivity and sensitivity detection of intracellular PFBS is achieved, with a detection limit of 1.01 μM, and the method is simple and low cost, so it can achieve accurate quantitative detection in biological samples.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting perfluorobutane sulfonic acid based on quenching type fluorescent carbon dots, and belongs to the technical field of fluorescent carbon nanomaterial preparation. Background Art
[0002] Perfluorobutane sulfonate (PFBS) refers to a new type of persistent environmental pollutant in which the hydrogen attached to the carbon atom in the alkane compound molecule is replaced by a fluorine atom. It has a hydrophobic carbon-fluorine chain and a hydrophilic sulfonic acid functional group. PFBS is widely used in industrial and civilian fields such as adhesives, food packaging, synthetic detergents, cooking utensils, textiles, and flame retardants due to its good thermal stability, chemical stability, low surface tension, and high surface activity. However, with the increasing use, PFBS inevitably penetrates into the natural environment and exists stably for a long time. At present, PFBS has been detected in food, feed, atmosphere, water, soil, and organisms worldwide. Studies have shown that PFBS can gradually accumulate through the food chain and increase its concentration in organisms. Once it enters the body, PFBS has a long half-life and mainly accumulates in the blood and liver. Long-term exposure can cause toxic hazards to the liver, immunity, embryos, reproduction, nerves, etc., further increasing the risk of cancer. Therefore, accurate and effective detection of PFBS is of great significance in many aspects such as disease diagnosis and environmental monitoring.
[0003] At present, the main method for detecting PFBS is the traditional analysis method based on chromatography-mass spectrometry, including gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Although chromatography-mass spectrometry can accurately and sensitively detect PFBS, the above analysis technology requires expensive large instruments, complex sample pretreatment, and is difficult to detect in living cells. Therefore, it is urgent to develop a fast, convenient and accurate imaging and detection analysis technology for intracellular PFBS.
[0004] As a new type of metal-free nanomaterial, carbon dots have been widely applied in the fields of catalysis, biomedicine, energy storage, light-emitting devices, etc. due to their excellent photoluminescence properties, abundant active sites, good biocompatibility, easy modification, anti-photobleaching and other characteristics. In recent years, the potential of carbon dots in the detection of environmental pollutants has also attracted much attention. Some studies have successfully applied them to the highly sensitive detection of perfluorooctane sulfonyl compounds (PFOS) and ammonium perfluorooctanoate (PFOA) in water environment. However, the detection method of PFBS based on carbon dots has not been reported yet. Due to the differences in the structure, physical and chemical properties between PFBS and PFOS / PFOA, the development of a highly selective and sensitive carbon dot sensing strategy for PFBS still faces challenges. Summary of the Invention
[0005] To solve the above problems, the present invention provides a method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots. The carbon dots prepared by the present invention have good selectivity and sensitivity to perfluorobutane sulfonic acid and are successfully used for in-situ imaging and detection of intracellular perfluorobutane sulfonic acid.
[0006] The first object of the present invention is to provide a method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots. The quenched fluorescent carbon dots are prepared by dispersing a carbon source in water and then performing a hydrothermal reaction; the carbon source is selected from one or more of o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, and 5-amino-1,10-phenanthroline.
[0007] Thus, the present invention prepares fluorescent carbon dots by hydrothermal synthesis using a specific carbon source. The aromatic structure of the carbon source can not only provide a conjugated backbone, but also the amino groups on the benzene ring can provide nitrogen doping. The prepared carbon dots have good solubility. When reacting with perfluorobutane sulfonic acid, the collision probability between the carbon dots and perfluorobutane sulfonic acid increases, resulting in fluorescence quenching, so that it can be used to detect perfluorobutane sulfonic acid.
[0008] In one embodiment of the present invention, the operating parameters of the hydrothermal reaction are: hydrothermal reaction at 130-200 °C for 4-10 h.
[0009] Thus, by controlling the temperature and time of the hydrothermal reaction, the types and contents of the functional groups on the surface of the prepared fluorescent carbon dots can be adjusted, which helps to increase the quenching reaction probability between the fluorescent carbon dots and perfluorobutane sulfonic acid and improve the sensitivity of detecting perfluorobutane sulfonic acid.
[0010] In one embodiment of the present invention, the preparation method of the quenched fluorescent carbon dots includes the following steps: (1) Disperse the carbon source in water and mix well to obtain a mixed solution; (2) React the obtained mixed solution at 130-200 °C for 4-10 h, cool, purify, and dry to obtain the quenched fluorescent carbon dots.
[0011] In one embodiment of the present invention, the concentration of the carbon source in the mixed solution is 0.01 - 0.05 g / mL.
[0012] In one embodiment of the present invention, the purification is carried out by any one or two of the following methods: microfiltration membrane filtration or dialysis.
[0013] In one embodiment of the present invention, the pore diameter of the microfiltration membrane filtration is 0.22 μm - 0.45 μm.
[0014] In one embodiment of the present invention, the dialysis is carried out at 500 - 3500 Da for 24 - 72 h.
[0015] In one embodiment of the present invention, the drying is vacuum freeze-drying.
[0016] The second object of the present invention is to provide a method for biological detection, which uses the above method to detect perfluorooctanesulfonic acid in cells.
[0017] The present invention has the following beneficial technical effects: 1) The fluorescent carbon dots of the present invention are synthesized by hydrothermal synthesis. o-Phenylenediamine, m-phenylenediamine, p-phenylenediamine, 5-amino-1,10-phenanthroline, etc. are important precursors for the synthesis of quenching-type carbon dots. Their aromatic structures can not only provide a conjugated backbone, but also the amino groups on the benzene ring can provide nitrogen doping. Therefore, the prepared carbon dots have good solubility. When reacting with perfluorooctanesulfonic acid, the collision probability between the carbon dots and perfluorooctanesulfonic acid increases, resulting in fluorescence quenching. The method of the present invention is simple, and the fluorescent carbon dots used can be prepared in large quantities at low cost.
[0018] 2) The obtained fluorescent carbon dots of the present invention have high selectivity for perfluorooctanesulfonic acid. The fluorescence intensity shows a good linear relationship with the concentration of perfluorooctanesulfonic acid within the range of 5 μM - 100 μM, and the detection limit is 1.01 μM. Therefore, the probe can sensitively and quantitatively detect perfluorooctanesulfonic acid. In addition, the cytotoxicity of the fluorescent carbon dots is small. Therefore, the signal of fluorescence quenching can be used to detect perfluorooctanesulfonic acid in cells. Description of the Drawings
[0019] Figure 1 It is the transmission electron microscope (TEM) photograph of the carbon dots of Example 1; Figure 2 It is the ultraviolet-visible absorption spectrum diagram of the carbon dots of Example 1; Figure 3 It is the fluorescence spectrum diagram of the carbon dots of Example 1; Figure 4 It is the Fourier transform infrared spectrum diagram of the carbon dots of Example 1; Figure 5 Response diagram of carbon dots in Example 1 to different perfluorinated compounds; Figure 6 Fluorescence spectra of the carbon dots in Example 1 after adding different concentrations of perfluorobutanesulfonic acid, where the arrow "↓" indicates the concentration of perfluorobutanesulfonic acid increasing from small to large; Figure 7 Fluorescence linear relationship diagram of the carbon dots in Example 1 after adding different concentrations of perfluorobutanesulfonic acid; Figure 8 Survival rate diagram of cells cultured with the carbon dots in Example 1; Figure 9 Cell imaging diagram of the carbon dots in Example 1; Figure 10 Fluorescence spectra of the carbon dots prepared in Proportion 1; Figure 11 Fluorescence spectra of the carbon dots prepared in Comparative Example 2. Detailed implementation manners
[0020] The present invention will be further described below in conjunction with examples, but the implementation manners of the present invention are not limited thereto.
[0021] Example 1 Weigh 0.2 g of o-phenylenediamine and place it in a 25 mL beaker. Add 15 mL of ultrapure water, ultrasonicate for 5 - 20 min, transfer it to a 20 mL autoclave, and raise the oven temperature to 180 °C for reaction for 6 h. After cooling to room temperature, filter the crude product through a 0.22 μm microporous membrane to remove over-carbonized substances, and dialyze the filtrate through 1000 Da for 24 h. Finally, obtain the fluorescent carbon dot powder through freeze-drying.
[0022] It can be seen from the transmission electron microscope image of the carbon dots that the carbon dot particles are evenly dispersed, and the average particle size is 1.97 nm ( Figure 1 ). Perform ultraviolet-visible absorption test on the obtained carbon dots. As Figure 2 shown, there is an obvious absorption peak at about 200 - 230 nm. This absorption peak may be caused by the intrinsic absorption of the carbon dots. Usually, carbon quantum dots will show absorption peaks near this wavelength, which is related to the electronic structure of the carbon dots and corresponds to π - π* transitions. The absorption peak at 290 nm is attributed to the n-π* transitions of some functional groups or impurities on the surface of the carbon dots. For example, functional groups such as carboxyl and hydroxyl groups on the surface may have characteristic absorption in this wavelength range. The broad absorption peak at about 420 nm is attributed to the molecular state absorption of the carbon dots.
[0023] Characterize the luminescence performance of the carbon dots using a fluorescence spectrometer. As Figure 3As shown, with the change of the excitation wavelength, the luminescence center of the emission wavelength remains unchanged, indicating that the carbon dots have the property of excitation independence. Its maximum excitation wavelength is 420 nm, and the emission center is located at 565 nm.
[0024] The functional groups on the surface of the carbon dots were characterized by Fourier transform infrared spectroscopy, such as Figure 4 shown, the prepared carbon dots have obvious absorption peaks at 3414 cm -1 , 1621 cm -1 , 1500 cm -1 , 1376 cm -1 , and 1088 cm -1 , which are attributed to the stretching vibrations of O‒H / N‒H, C=O, C=C, COO - and C‒O, respectively. This indicates that the surface of the carbon dots has abundant oxygen-containing and nitrogen-containing functional groups.
[0025] To detect the selectivity of the fluorescent carbon dots to PFBS, different types of perfluorinated compounds (20 μM) were added to the carbon dot solution (0.3 mg / mL, 2 mL), including: perfluorobutanesulfonic acid (PFBS), 1H,1H,2H,2H-perfluorooctanesulfonic acid (6:2FTSA), 1H,1H,2H,2H-perfluorooctanol (6:2FTOH), 2-perfluorooctylethanol (8:2FTOH), perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid (HFPO-TA), perfluoroheptanoic acid (PFH P A), perfluorobutyric acid (PFBA). The excitation wavelength was 420 nm, and the fluorescence intensity corresponding to the maximum emission wavelength of 560 nm was measured. Among them, F0 refers to the fluorescence intensity of the carbon dots at the maximum emission center of 560 nm under the excitation wavelength of 420 nm, and F refers to the fluorescence intensity corresponding to the addition of perfluorinated compounds under the same test conditions. The results are as Figure 5 shown, the common perfluorinated compounds have little effect on the fluorescence intensity of the carbon dots. The addition of PFBS will significantly weaken the fluorescence intensity of the fluorescent carbon dots (where the fluorescence intensity F of the fluorescent carbon dots after the addition of PFBS is less than the original intensity F0 before the addition of PFBS), indicating that the prepared carbon dots have a specific response to PFBS.
[0026] The response results of the prepared carbon dots to different concentrations of PFBS are as Figure 6 shown. Figure 6 The results show that with the continuous increase of the PFBS concentration, the fluorescence intensity of the fluorescent carbon dots at 560 nm gradually weakens. Therefore, this carbon dot is called a quenching-type carbon dot, and there is a good linear relationship within 5‒100 μM ( Figure 7 ), and the detection limit is 1.01 μM.
[0027] The cytotoxicity of the quenched carbon dots was tested using a CCK-8 kit. The HepG2 cell line was selected and cultured with carbon dots at different concentrations (1.0, 2.5, 5.0, 7.5, 10, 15, 25, and 30 μg / mL). After co-incubation for 24 h, the results were as Figure 8 shown. It can be seen that the cell survival rate was still close to 100%, indicating that the carbon dots had little cytotoxicity and could be used for the detection of PFBS in cells.
[0028] The change in the fluorescence signal of PFBS in cells by the carbon dots was observed using a laser confocal microscope. The results were as Figure 9 shown. When there was no PFBS in the cells, there was a bright yellow fluorescence signal of the carbon dots in the fluorescence channel. When there was 50 μM of PFBS in the cells, the fluorescence signal in the yellow channel was significantly weakened, indicating that the prepared carbon dots could be used for the detection of PFBS in cells.
[0029] Example 2 Weigh 0.25 g of m-phenylenediamine and place it in a beaker. Add 15 mL of ultrapure water, stir well, and then transfer it to a reaction kettle. Raise the oven temperature to 150 °C and react for 6 h. Then, after cooling to room temperature, filter through a 0.22 μm microporous membrane, take the filtrate, and then dialyze through a 500 Da membrane for 24 h. Finally, obtain the carbon dot powder through freeze-drying.
[0030] Using the method of Example 1 to detect the fluorescent carbon dots prepared in this example, it was found that the prepared fluorescent carbon dot particles were evenly dispersed, the maximum excitation wavelength was 420 nm, the emission peak was located at 560 nm, had high selectivity for PFBS, strong anti-interference ability, little cytotoxicity, had a good linear relationship within the concentration range of 5 - 100 μM for PFBS, and could sensitively detect PFBS in cells.
[0031] Example 3 Weigh 0.3 g of p-phenylenediamine and place it in a beaker. Add 15 mL of ultrapure water, stir well, and then transfer it to a reaction kettle. Raise the oven temperature to 200 °C and react for 7 h. Then, after cooling to room temperature, filter through a 0.22 μm microporous membrane, take the filtrate, and then dialyze through a 1000 Da membrane for 48 h, and obtain the carbon dot powder through freeze-drying.
[0032] Using the method of Example 1 to detect the fluorescent carbon dots prepared in this example, it was found that the prepared fluorescent carbon dot particles were evenly dispersed, the maximum excitation wavelength was 420 nm, the emission peak was located at 560 nm, had high selectivity for PFBS, strong anti-interference ability, little cytotoxicity, had a good linear relationship within the concentration range of 5 - 100 μM for PFBS, and could sensitively detect PFBS in cells.
[0033] Example 4 Weigh 0.5 g of 5 - aminophenanthroline and place it in a beaker. Add 15 mL of ultrapure water, stir well, then transfer it to a reaction kettle. Raise the oven temperature to 160 °C and react for 10 h. Then, after cooling to room temperature, filter through a 0.45 μm microporous membrane, take the filtrate, and then dialyze it through a 3500 Da membrane for 36 h. Obtain carbon dot powder through freeze - drying.
[0034] Using the method of Example 1 to detect the fluorescent carbon dots prepared in this example, it can be found that the prepared fluorescent carbon dot particles are evenly dispersed, the maximum excitation wavelength is 420 nm, the emission peak is located at 560 nm, with high selectivity for PFBS, strong anti - interference ability, very low cytotoxicity, and a good linear relationship within the range of 5 - 100 μM of PFBS concentration, and can sensitively detect PFBS in cells.
[0035] Comparative Example 1 Referring to Example 1, weigh 0.2 g of β - naphthol and replace 0.2 g of o - phenylenediamine and place them in a beaker to mix well. Keep other reaction conditions unchanged. The obtained carbon dots are called CDs - 1, with a maximum excitation wavelength of 350 nm and the center of the maximum emission wavelength located at 439 nm. As Figure 10 shown, when adding 20 μM PFBS to a 0.3 mg / mL carbon dot solution, its fluorescence signal weakens, and there is a good linear relationship within the range of 5 - 100 μM, and it can sensitively detect PFBS in cells. However, the carbon dots obtained in Comparative Example 1 emit short - wavelength light when detecting PFBS, and the short - wavelength light is likely to cause cell damage and is not suitable for detecting PFBS in cells.
[0036] Comparative Example 2 Referring to Example 1, weigh 0.2 g of salicylic acid and replace 0.2 g of o - phenylenediamine and place them in a beaker to mix well. Keep other reaction conditions unchanged. The obtained carbon dots are called CDs - 2, with a maximum excitation wavelength of 470 nm and the center of the maximum emission wavelength located at 515 nm. As Figure 11 shown, when adding 20 μM PFBS to a 0.3 mg / mL carbon dot solution, its fluorescence signal intensity remains basically unchanged, and the wavelength blue - shifts to 510 nm, and there is no linear relationship within the range of 5 - 100 μM, so it cannot be used for the detection of PFBS in cells.
[0037] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims
1. A method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots, characterized in that: The quenched fluorescent carbon dots are prepared by dispersing a carbon source in water and then subjecting it to a hydrothermal reaction; the carbon source is selected from one or more of o-phenylenediamine, m-phenylenediamine, p-phenylenediamine and 5-amino-o-phenanthroline.
2. A method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 1, characterized in that: The operating parameters of the hydrothermal reaction are: hydrothermal reaction at 130-200° C. for 4-10 h.
3. A method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 1, characterized in that: The preparation method of the quenched fluorescent carbon dots comprises the following steps: (1) dispersing the carbon source in water and mixing to obtain a mixed solution; (2) The resulting mixed solution is reacted at 130-200°C for 4-10 h, cooled, purified and dried to obtain quenched fluorescent carbon dots.
4. A method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 3, characterized in that: The concentration of the carbon source in the mixed solution is 0.01-0.05 g / mL.
5. The method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 3, characterized in that: The purification is performed by any one or both of the following methods: microporous membrane filtration or dialysis.
6. A method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 5, characterized in that: The micropore diameter of the microporous membrane filtration is 0.22 μm-0.45 μm.
7. The method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 5, characterized in that: The dialysis was performed at 500-3500 Da for 24-72 h.
8. The method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots according to claim 3, characterized in that: The drying is vacuum freeze drying.
9. A method for biological detection, characterized in that: The method uses a method for detecting perfluorobutane sulfonic acid based on quenched fluorescent carbon dots as described in any one of claims 1 to 8 to detect perfluorobutane sulfonic acid in cells.