Preparation method and application of green fluorescent carbon dots for detecting captopril

Abstract

The invention aims to provide a preparation method and application of a green fluorescent carbon dot for detecting captopril, and belongs to the technical field of fluorescent carbon dots, diphenylamino-4-benzaldehyde and o-phenylenediamine are weighed and dissolved in ethanol, and ultrasonic treatment is performed to obtain a uniformly mixed solution; transferring the solution into a polytetrafluoroethylene reaction kettle, reacting for 2-8 hours at the temperature of 150-220 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 dialysis bag of 500-1000Da to obtain a pure carbon dot aqueous solution; and freeze-drying the carbon dot aqueous solution to obtain a carbon dot solid. The prepared green fluorescent carbon dots can be used for detecting captopril, and are good in selectivity and high in sensitivity. Compared with other carbon dot-based fluorescence sensing methods, the method provided by the invention can be used for directly detecting captopril.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent carbon dots, and in particular relates to a preparation method of green fluorescent carbon dots for detecting captopril and an application thereof. Background Art

[0002] Captopril (CAP) is a first-generation oral angiotensin-converting enzyme inhibitor (ACEI), which was originally derived from a bradykinin in the venom of the Brazilian snake. The sulfhydryl group on captopril binds to the Zn present in the active site of the carboxypeptidase A protein. 2+ The combination forms a strong metal chelate, which can effectively treat various types of hypertension and congestive heart failure, and also has a good effect on diabetes and kidney disease. Captopril drug treatment has the advantages of fast effect and stable blood pressure reduction. However, excessive use of captopril drugs may cause problems with the skin, kidneys, immune system, etc., often manifested as skin rash, chest pain, palpitations, and taste loss. Therefore, it is of great significance to develop a highly selective and sensitive technology for the analysis of captopril content and closely monitor the content of captopril in drugs or human biological samples.

[0003] Fluorescence detection as a traditional analytical method has been widely used in many fields such as analytical chemistry, environmental biology, biochemistry and medicine because of its high sensitivity, low equipment price, instantaneous response and real-time detection. In recent years, carbon dots as a nanomaterial have attracted widespread attention due to their good photostability, water solubility, biocompatibility and other advantages. Therefore, it is urgent to establish a detection method for captopril based on carbon dots. Summary of the invention

[0004] The object of the present invention is to provide a preparation method of green fluorescent carbon dots for detecting captopril and its application, so as to construct a fluorescent sensing platform for captopril.

[0005] The present invention adopts the following technical solution: A method for preparing green fluorescent carbon dots for detecting captopril comprises the following steps: The first step is to weigh diphenylamino-4-benzaldehyde and o-phenylenediamine in proportion and dissolve them in ethanol, and ultrasonicate to obtain a uniform mixed solution; In the second step, the mixed solution obtained in the first step is transferred to a polytetrafluoroethylene reactor, and reacted at 150-220°C for 2-8 h. After the reaction stops, it is cooled to room temperature, centrifuged to remove insoluble matter, and the supernatant is taken and dialyzed in a glass container for at least three days through a 500-1000 Da dialysis bag to obtain a pure carbon dot solution; The third step is to freeze-dry the carbon dot solution to obtain carbon dot solid.

[0006] Furthermore, in the first step, the mass ratio of diphenylamino-4-benzaldehyde, o-phenylenediamine and ethanol is 6-20:10-50:7893.

[0007] A green fluorescent carbon dot was used for the detection of captopril.

[0008] A green fluorescent carbon dot was used for the detection of intracellular captopril.

[0009] The green fluorescent carbon dots prepared by the present invention have a specific recognition effect on captopril. The reasons are as follows: like Fig. 9 As shown in Figure 2, after adding captopril, the transmission electron microscopy image of carbon dots showed that the carbon dots were partially aggregated. Fig.10 As shown in the figure, after adding captopril, the Zeta potential of the carbon dots changes from 0.815 eV to -6.66 eV. This is because after adding captopril, the carbon dots aggregate and form a rigid conjugated structure, which restricts the rotation of the helical structure within the molecule and reduces the non-radiative transition, thereby enhancing the photoluminescence intensity of the system and greatly improving the fluorescence intensity.

[0010] The beneficial effects of the present invention are as follows: 1. The present invention has simple operation steps and can obtain carbon dots emitting green fluorescence without surface passivation agent treatment or modification.

[0011] 2. The carbon dots prepared by the present invention have good solubility and dispersibility.

[0012] 3. The green fluorescent carbon dots prepared by the present invention have a specific recognition effect on captopril and are used for the detection of captopril with good selectivity and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 A is a transmission electron microscope spectrum of the fluorescent carbon dots prepared in Example 1 of the present invention, Figure 1 B is the particle size distribution diagram corresponding to the spectrum.

[0014] Figure 2 This is the infrared spectrum of the fluorescent carbon dots prepared in Example 1 of the present invention.

[0015] Figure 3 The ultraviolet absorption spectrum and fluorescence excitation-emission spectrum of the fluorescent carbon dots prepared in Example 1 of the present invention.

[0016] Figure 4 This is a spectrum diagram showing the fluorescence emission curve of the fluorescent carbon dots prepared in Example 1 of the present invention changing with the excitation wavelength.

[0017] Figure 5 Figure 2 shows the selectivity and anti-interference properties of carbon dots for captopril detection.

[0018] Figure 6 The graph shows the changes in the fluorescence emission spectra of carbon dots in the presence of different concentrations of captopril.

[0019] Figure 7 Linear fitting diagram of carbon dots detecting captopril concentration; in the figure: A shows that the carbon dots have a good linear relationship when the captopril concentration is 0.25-100 μM; B shows that the carbon dots have a good linear relationship when the captopril concentration is 100-300 μM.

[0020] Figure 8 This is a laser confocal image of the fluorescent carbon dots prepared in Example 1 of the present invention being quenched by captopril, and the cells are Hela cells.

[0021] Fig. 9 Transmission electron micrographs of carbon dots before and after the addition of captopril, wherein A is before the addition and B is after the addition.

[0022] Fig.10 This is the zeta potential diagram of carbon dots before and after the addition of captopril. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1 A method for preparing green fluorescent carbon dots for detecting captopril comprises the following steps: 1) Weighing diphenylamino-4-benzaldehyde and o-phenylenediamine and dissolving them in ethanol, and ultrasonicating to obtain a uniform mixed solution; the mass ratio of diphenylamino-4-benzaldehyde, o-phenylenediamine and ethanol is 13:30:7893; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 200°C for 5 h. After the reaction stopped, it 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 solution. 3) The carbon dot solution is freeze-dried to obtain a carbon dot solid. Taking rhodamine 6G as a reference, its relative quantum yield is 9.52%.

[0025] Example 2 A method for preparing green fluorescent carbon dots for detecting captopril comprises the following steps: 1) Weighing diphenylamino-4-benzaldehyde and o-phenylenediamine and dissolving them in ethanol, and ultrasonicating to obtain a uniform mixed solution; the mass ratio of diphenylamino-4-benzaldehyde, o-phenylenediamine and ethanol is 10:42:7893; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 180°C for 6 h. After the reaction stopped, it 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 solution. 3) The carbon dot solution is freeze-dried to obtain a carbon dot solid. Taking rhodamine 6G as a reference, its relative quantum yield is 6.34%.

[0026] Example 3 A method for preparing green fluorescent carbon dots for detecting captopril comprises the following steps: 1) Weighing diphenylamino-4-benzaldehyde and o-phenylenediamine and dissolving them in ethanol, and ultrasonicating to obtain a uniform mixed solution; the mass ratio of diphenylamino-4-benzaldehyde, o-phenylenediamine and ethanol is 20:29:7893; 2) The mixed solution was transferred to a hydrothermal reactor and reacted at 210°C for 4 h. After the reaction stopped, it 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 solution. 3) The carbon dot solution is freeze-dried to obtain a carbon dot solid. Taking rhodamine 6G as a reference, its relative quantum yield is 4.33%.

[0027] The morphology of the green fluorescent carbon dots prepared in Example 1 of the present invention is characterized as follows: Figure 1 shown. Figure 1 A transmission electron microscopy image shows that the carbon dots are monodispersed. The inset in the upper right corner is a high-resolution transmission electron microscopy image. The lattice spacing of 0.21 nm corresponds to the (100) plane of graphite. Figure 1 B The statistical analysis of the particle size of carbon dots clearly shows that their particle size distribution is between 2 and 5 nm, and the average particle size is about 3.23 nm.

[0028] The structural characterization of the green fluorescent carbon dots prepared in Example 1 of the present invention is as follows Figure 2 As shown. Infrared spectrum shows that the characteristic absorption band of carbon dots appears at 3348 cm -1 This is the result of NH stretching vibration. CO stretching vibration is located at 2922 cm -1 At 1491 cm -1 and 1587 cm -1The peak at 1275 cm indicates the presence of unsaturated C=C bonds. The peak at 1275 cm indicates the presence of unsaturated C=C bonds. -1 and 1319 cm -1 In addition, at 826 cm -1 The broad peak at 40° is caused by CN stretching vibration. The above analysis confirms that the surface of carbon dots is composed of aromatic structures of carboxyl and hydroxyl groups. By comparing with the raw materials, it can be seen that m-phenylenediamine has modified the surface of carbon dots with nitrogen elements, and G-CDs retains the main functional groups of diphenylamino-4-benzaldehyde.

[0029] The optical property spectrum of the green fluorescent carbon dots prepared in Example 1 of the present invention is as follows: Figure 3 , 4 The carbon dots show obvious ultraviolet absorption at 288 nm, 356 nm and 456 nm, among which the characteristic peak at 288 nm comes from the core state sp 2 The broad absorption peaks at 356 nm and 456 nm can be attributed to the n-π* transition of surface states, and the fluorescence emission peak of carbon dots is located at 520 nm. Figure 4 It is the emission spectrum of the carbon dots under different excitation wavelengths, indicating that the carbon dots are independent of the excitation wavelength.

[0030] Take an appropriate amount of dry carbon dot powder and put it into a 10 mL volumetric flask, dilute it with PBS buffer and ethanol (volume ratio 1:1) to make a fixed volume and shake it well to prepare a carbon dot mother solution. Captopril was gradually added to the carbon dot mother solution, mixed thoroughly, incubated at room temperature for 3 min, and the change in fluorescence intensity was recorded. In order to evaluate the anti-interference ability of carbon dots for captopril detection, a series of interferences were selected and added to the solution for fluorescence testing. All measurements were repeated three times. Figure 5 It shows that carbon dots have good selectivity and anti-interference for the detection of captopril.

[0031] Take 5 mg of dry carbon dot powder and put it into a 10 mL volumetric flask, dilute it with a mixed solution of PBS buffer and ethanol (ratio 1:1) and shake it to make a carbon dot mother solution (0.18 mg / mL). Add 10 μL of captopril solution of different concentrations to 2.0 mL of carbon dot mother solution, mix thoroughly, incubate at room temperature for 3 min, and record the change in fluorescence intensity. The green fluorescent carbon dots prepared in Example 1 of the present invention are sensitive to captopril. Figure 6 As shown, Figure 7 A shows a linear range of 0.25-100 μM, Figure 7 B shows that the linear range is 100-300 μM. The limit of detection (LOD) is calculated according to the formula 3σ / slope and is as low as 78nM.

[0032] The carbon dot solution was added to the Hela cells and incubated for 15 min. The residual carbon dots in the culture dish were removed by suction and rinsed three times with PBS buffer (pH=7.4). Subsequently, a small amount of PBS buffer was added to maintain the physiological activity of the cells. The culture dish was placed under a laser scanning confocal microscope to observe the cell fluorescence, collect data and record it. Finally, under the same operating conditions as above, 10 μL of captopril solution (10 μM) was added, and the cell images were observed and recorded under a microscope. Figure 8 As shown, the cell morphology is good and the carbon dots have no cytotoxicity and can be used for living cell labeling. Figure 8 The laser confocal images of cells in the presence of captopril in the carbon dots prepared in Example 1 are, from left to right: bright field cell image, dark field cell image (green), bright field and dark field overlay image. After adding captopril, the green fluorescence in the cells is significantly enhanced, indicating that the green fluorescent carbon dots can be used to construct a fluorescent sensing platform for intracellular captopril.

Claims

1. A method for preparing green fluorescent carbon dots for detecting captopril, characterized in that: The steps include: The first step is to weigh diphenylamino-4-benzaldehyde and o-phenylenediamine in proportion and dissolve them in ethanol, and ultrasonicate to obtain a uniform mixed solution; In the second step, the mixed solution obtained in the first step is transferred to a polytetrafluoroethylene reactor, and reacted at 150-220°C for 2-8 h. After the reaction stops, it is cooled to room temperature, centrifuged to remove insoluble matter, and the supernatant is taken and dialyzed in a glass container for at least three days through a 500-1000 Da dialysis bag to obtain a pure carbon dot solution; The third step is to freeze-dry the carbon dot solution to obtain carbon dot solid.

2. The method for preparing green fluorescent carbon dots for detecting captopril according to claim 1, characterized in that: The mass ratio of diphenylamino-4-benzaldehyde, o-phenylenediamine and ethanol in the first step is 6-20:10-50:7893.

3. The green fluorescent carbon dots prepared by the preparation method as claimed in claim 1 are used for the detection of captopril.

4. Green fluorescent carbon dots prepared by the preparation method as claimed in claim 1 are used for detecting intracellular captopril.

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