Red carbon dot / rhodamine B-based ratiometric fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent carbon dots, in particular to a red carbon dot / rhodamine B-based ratiometric fluorescent probe as well as a preparation method and application thereof. The preparation method comprises the following steps: adding deionized water and concentrated sulfuric acid into o-phenylenediamine, L-cysteine and boric acid, carrying out ultrasonic treatment, transferring into a high-pressure reaction kettle for reaction, cooling, centrifuging, and filtering, dialyzing and freeze-drying supernate to obtain the red carbon dots. The ratio (F582 / F622) of the concentration of malachite green to the fluorescence intensity shows a good linear relationship, and the method has good detection limit and sensitivity, and can effectively realize the detection of malachite green in a water sample.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent carbon dots, and in particular to a ratiometric fluorescent probe based on red carbon dots / rhodamine B, and a preparation method and application thereof. Background Art

[0002] Malachite green (MG) is a green crystalline substance with a metallic luster. Due to its broad antimicrobial activity and low price, it has been widely used as a fungicide and insecticide for many years. However, as an industrially synthesized triphenylmethane organic compound, MG is highly toxic. While MG is an effective treatment for saprolegniasis in fish, some fish farmers use it illegally, potentially causing water pollution. Scientific research has shown that ingestion of MG can adversely affect organs such as the liver and kidneys, and may even cause cancer. Therefore, the detection of MG has attracted widespread attention from various research groups. Currently, high-performance liquid chromatography (HPLC), surface-enhanced Raman spectroscopy (SERS), liquid chromatography-mass spectrometry (LC-MS), electrochemical detection, and fluorescence spectroscopy are all commonly used analytical methods for MG. Fluorescence spectroscopy, compared to other methods, has attracted widespread attention due to its ease of use, rapid response, and sensitivity.

[0003] Carbon dots are fluorescent nanomaterials with unique advantages. Their size is generally less than 10 nm, and they exhibit excellent photostability, tunable emission, low cytotoxicity, and good biocompatibility. Currently, there are reports of detecting malachite green using a single-emission fluorescence signal. However, such detection methods are easily affected by interference from factors such as the instrument and the experimental environment. Using ratiometric fluorescence detection, due to the presence of two distinct fluorescence emission signals in the system, the two fluorescence signals of the ratiometric sensor will vary when detecting the target (MG). Common types of changes include changes in both fluorescence signals, or changes in one fluorescence signal while the other remains unchanged. During the test process, the presence of two fluorescence signals, compared to a single fluorescence signal, can effectively eliminate environmental interference, reduce test errors, and thus effectively improve detection sensitivity. Summary of the Invention

[0004] The present invention constructs a method for detecting malachite green in water using a ratiometric fluorescence sensor based on red carbon dots and rhodamine B. Rhodamine B is a common organic dye with good optical stability, excellent photophysical properties and high quantum yield. Using it as a reference element can effectively avoid interference from environmental factors during the detection process. Therefore, the present method adds rhodamine B to a red carbon dot solution, and the mixed solution exhibits dual fluorescence emissions of 582nm and 622nm at an excitation wavelength of 560nm. When a certain amount of malachite green is added, the fluorescence intensity of the carbon dots decreases significantly, while the fluorescence intensity of rhodamine B remains almost unchanged. Therefore, the fluorescence intensity of rhodamine B is used as a reference signal. Under optimized experimental conditions, within a certain concentration range, the ratio of the concentration of malachite green to the fluorescence intensity (F 582 / F 622 ) showed a good linear relationship. This method has good detection limit and sensitivity and can effectively realize the detection of malachite green in water samples.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: a ratiometric fluorescent probe based on red carbon dots Rhodamine B, the preparation method of which is as follows: deionized water and concentrated sulfuric acid are added to o-phenylenediamine, L-cysteine, and boric acid, ultrasonicated, transferred to a high-pressure reactor for reaction, cooled and centrifuged, the supernatant filtered, dialyzed, and freeze-dried to obtain red carbon dots.

[0006] The above-mentioned ratiometric fluorescent probe based on red carbon dot rhodamine B has a mass ratio of o-phenylenediamine, L-cysteine, and boric acid of 2:1:0.2-0.23.

[0007] The above-mentioned ratiometric fluorescent probe based on red carbon dot Rhodamine B is reacted at 160° C. for 12 hours.

[0008] The above-mentioned ratiometric fluorescent probe based on red carbon dot Rhodamine B uses a dialysis membrane with a MWCO of 500 for dialysis.

[0009] The above-mentioned application of a ratiometric fluorescent probe based on red carbon dot rhodamine B in the quantitative detection of malachite green.

[0010] The above method is to add the Rhodamine B solution to be tested and the buffer solution to the above solution of the ratio fluorescence probe based on red carbon dot Rhodamine B, measure the fluorescence spectrum after standing, record the fluorescence emission intensity of the solution at 582nm and 622nm, and substitute the ratio of the two into the linear equation F 582 / F 622 =0.587+0.0249[malachite green], the concentration of malachite green is obtained.

[0011] The above method, the linear equation F 582 / F622 =0.587+0.0249 [Malachite green] was obtained as follows: 4 μL of rhodamine B solution, 10 μL of malachite green solutions of varying concentrations, and 1.5 mL of HCl-Na2HPO4 buffer solution at pH 2 were added to 3 mL of carbon dot solution. The mixture was allowed to stand at room temperature for 2 minutes before measuring the fluorescence spectrum. The fluorescence emission intensity of the solution at 582 nm and 622 nm was recorded using a 5 nm slit width and 560 nm excitation wavelength. The malachite green concentration was plotted as the horizontal axis, and the fluorescence intensity ratio F was plotted as the horizontal axis. 582 / F 622 As the vertical axis, in the concentration range of 0 to 2.92 mg / L, the linear equation is F 582 / F 622 =0.587+0.0249[malachite green], the correlation coefficient is R 2 =0.994. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 TEM image (A) and particle size distribution histogram (B) of carbon dots.

[0013] Figure 2 XRD patterns of carbon dots.

[0014] Figure 3 FT-IR spectrum of carbon dots.

[0015] Figure 4 Full XPS spectrum of carbon dots (A) and high-resolution XPS spectra of C (B), N (C), O (D), and S (E).

[0016] Figure 5 UV-visible absorption spectrum of carbon dots.

[0017] Figure 6 Fluorescence excitation and emission spectra of carbon dots.

[0018] Figure 7 Fluorescence emission spectra of carbon dots at different excitation wavelengths.

[0019] Figure 8 Fluorescence spectra (A) and ratio values ​​F of the RB / R-CDs system in the presence of different concentrations of malachite green 582 / F 622 Linear relationship curve with malachite green concentration (B). DETAILED DESCRIPTION

[0020] Example 1 Synthesis of red carbon dots

[0021] 1. Preparation of Red Carbon Dots

[0022] 0.5 g of o-phenylenediamine, 0.25 g of L-cysteine, and 0.065 g of boric acid were weighed, and 20 mL of deionized water and 0.5 mL of concentrated sulfuric acid were added. The mixture was ultrasonicated for 5 minutes and then transferred to a polytetrafluoroethylene (PTFE)-lined autoclave. The reaction temperature was controlled at 160°C for 12 hours. The product was naturally cooled at room temperature to obtain a deep red solution. The solution was centrifuged at 10,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm aqueous filter membrane. The filtrate was dialyzed through a dialysis membrane (MWCO = 500) for 48 hours and then freeze-dried to obtain red carbon dot powder. The carbon dot powder was dispersed in deionized water to prepare a red carbon dot solution (R-CDs) with a concentration of 0.5 mg / mL.

[0023] 2. Fluorescence Quantum Yield Determination

[0024] The fluorescence quantum yield (QY) of the resulting red carbon dots was determined using a rhodamine B (RB) solution as a reference (QY = 89%). An appropriate concentration of rhodamine B ethanol solution (absorbance < 0.05) was prepared, and its absorption and fluorescence emission spectra were measured to calculate the QY of the carbon dots. The QY calculation formula is as follows:

[0025]

[0026] Where "R" represents the reference sample, and the quantum yield of rhodamine B (QY R ) is 0.89, “I” is the intensity integrated area of ​​the fluorescence emission spectrum, “A” is the absorbance, and “n” is the refractive index of the solvent used (n / n R =1). After calculation, QY = 20.9%.

[0027] 3. Characterization of Carbon Dots

[0028] In this example, o-phenylenediamine, L-cysteine, and boric acid were used as raw materials to synthesize red carbon dots by hydrothermal method in sulfuric acid medium. TEM characterization was performed to study the morphology and size distribution of the red carbon dots. Figure 1 As shown in (A), the carbon dots have a nearly spherical structure and a uniform particle size distribution. Figure 1 (B) It can be found that the carbon dots are mainly distributed in the range of 1.1 nm to 2.8 nm, with an average particle size of about 1.8 nm. Figure 2 Figure 3 is the XRD pattern of carbon dots, with a broad diffraction peak at 26.1°, indicating the amorphous carbon structure of the carbon dots.

[0029] The surface groups of the prepared carbon dots were analyzed by FT-IR spectroscopy. Figure 3 As shown, located at 3428cm -1The broad infrared absorption peak at 2826 cm comes from the stretching vibration of NH / OH. -1 The absorption peak at 1596 cm comes from the bending vibration of the C-H bond of unsaturated carbon. -1 The strong absorption peak at 1351 cm is attributed to the stretching vibration of C=O. -1 The strong absorption peak at 1123 cm is due to the bending vibration of the C-H bond. -1 The broad absorption peak at 663 cm indicates the presence of OCO bending vibration on the carbon dot surface, while the -1 The small absorption peak at is the characteristic peak of the C-S bond. In summary, heteroatoms such as nitrogen and sulfur have been successfully doped into carbon dots. At the same time, the surface of CDs is rich in the aforementioned hydrophilic groups (-OH, -NH2, -COOH), which effectively improves the hydrophilicity of the carbon dots and makes them well water-soluble.

[0030] The carbon dots were characterized by XPS spectra. Figure 4 As shown. Figure 4 (A) It can be seen that the contents of C, N, O, and S in the carbon dots are 65.81%, 10.96%, 17.23%, and 6.01%, respectively. Figure 4 (B) It can be seen that there are three obvious C peaks, located at 284.6eV, 285.6eV, and 287.2eV, representing the structures of CC, CN / CO, and CS. Figure 4 (C) It can be seen that there are three obvious N peaks, located at 398.5eV and 400.0eV, representing the structures of CN and NH. Figure 4 (D) There are two obvious O peaks, located at 531.3eV and 532.6eV, representing the structures of C=O and COC / C-OH. Figure 4 (E) There are three obvious S peaks, and the peak at 168.4 eV represents C-SO x The XPS spectrum shows that the prepared red carbon dots contain abundant hydrophilic groups, and the conclusion is consistent with that shown by infrared spectroscopy.

[0031] The absorption properties of CDs were studied using UV-visible absorption spectroscopy. Figure 5 As shown, the shoulder peak at 300 nm originates from π-π* transitions of C=C and C=N bonds within the conjugated domains of the carbon core, while a strong UV absorption peak at 327 nm and a tail peak at 400 nm are attributed to n-π* transitions associated with surface groups and heteroatom doping. In particular, the low-energy absorption at 400 nm, originating from surface defect state transitions, is the primary reason for the red fluorescence emission of carbon dots under visible light excitation.

[0032] Excitation and emission spectra of carbon dot solution. Figure 6 It can be found that the maximum excitation wavelength (λex) of the prepared CDs is 560nm, while the maximum emission wavelength (λem) is 622nm, corresponding to a Stokes shift of 62nm. In addition, by changing the excitation light wavelength, the fluorescence spectra of the carbon dots were measured in the range of 520nm to 580nm, as shown in Figure 2. Figure 7 As shown in the figure, the carbon dots' emission peak intensity is highest at an excitation wavelength of 560 nm. Furthermore, the position of the carbon dots' fluorescence emission peak remains almost unchanged as the excitation wavelength increases from 520 nm to 580 nm, demonstrating their excitation wavelength independence. This independence of emission wavelength may be related to the uniform size distribution of the carbon dots and the type of functional groups on their surface.

[0033] Example 2 Application of Red Carbon Dots in Detection of Malachite Green

[0034] To 3 mL of carbon dot solution, add 4 μL of rhodamine B solution, 10 μL of malachite green solutions of varying concentrations, and 1.5 mL of HCl-Na₂HPO₄ buffer (pH 2). Mix thoroughly and let the mixture stand at room temperature for 2 minutes before measuring the fluorescence spectrum. Using a 560 nm excitation wavelength and a 5 nm slit width, record the fluorescence emission intensities at 582 nm and 622 nm. Repeat the experiment three times.

[0035] Under the optimal experimental conditions, the effect of malachite green concentration on the fluorescence intensity of the Rhodamine B / red carbon dots (RB / R-CDs) system was studied. Figure 8 As shown in (A), when malachite green (0-2.92 mg / L) is added, the fluorescence intensity of the carbon dots in the system is quenched to a certain extent. As the concentration of malachite green gradually increases, the fluorescence intensity of the carbon dots decreases, and the fluorescence intensity of rhodamine B also changes to a certain extent. A linear fit is performed on the above spectral data, with the malachite green concentration as the horizontal axis and the fluorescence intensity ratio F as the horizontal axis. 582 / F 622 As the vertical coordinate, the linear equation is F 582 / F 622 =0.587+0.0249[malachite green], the correlation coefficient is R 2 =0.994. Figure 8 As shown in (B), when the concentration of malachite green is in the range of 0 to 2.92 mg / L, the ratio of the fluorescence peaks F 582 / F 622 The detection limit of the method was calculated to be 0.12 mg / L according to the formula LOD = 3σ / S, indicating that the ratiometric fluorescence method has high sensitivity.

[0036] Example 3 Actual sample analysis

[0037] The feasibility of the method was verified by spiking actual water samples. The actual water samples used were water from the Xinkai River in Shenyang and tap water from the laboratory. Three malachite green spike concentrations of 0.61, 0.91, and 1.22 mg / L were selected for the experiment. Each sample was measured three times in parallel according to the experimental method described in Example 2. The results are listed in Table 1. The results showed that the recovery of malachite green in both water samples was controlled between 96.7% and 100%, and the relative standard deviation (RSD, n = 3) was less than 1%, indicating that the method has good precision and accuracy. Therefore, the ratiometric fluorescence sensor constructed in this experiment can be effectively applied to the detection of malachite green in actual samples.

[0038] Table 1 Detection of malachite green in actual water samples (n=3)

[0039]

[0040] Note: ND*, not detected.

Claims

1. A ratiometric fluorescent probe based on red carbon dots / rhodamine B, characterized in that: The preparation method is as follows: deionized water and concentrated sulfuric acid are added to o-phenylenediamine, L-cysteine, and boric acid, ultrasonicated, transferred to a high-pressure reactor for reaction, cooled and centrifuged, and the supernatant is filtered, dialyzed, and freeze-dried to obtain red carbon dots.

2. A ratiometric fluorescent probe based on red carbon dots / rhodamine B according to claim 1, characterized in that: By mass ratio, o-phenylenediamine, L-cysteine, and boric acid = 2:1:0.2-0.

23.

3. A ratiometric fluorescent probe based on red carbon dots / rhodamine B according to claim 1, characterized in that: The reaction was carried out at 160° C. for 12 h.

4. The ratiometric fluorescent probe based on red carbon dots / rhodamine B according to claim 1, characterized in that: The dialysis membrane used for dialysis had a MWCO of 500.

5. Use of a ratiometric fluorescent probe based on red carbon dots / rhodamine B according to any one of claims 1 to 4 in the quantitative detection of malachite green.

6. The method according to claim 5, characterized in that The rhodamine B solution to be tested and the buffer solution are added to the solution of the ratiometric fluorescent probe based on red carbon dots / rhodamine B according to any one of claims 1 to 4. The fluorescence spectrum is measured after standing, and the fluorescence emission intensity of the solution at 582 nm and 622 nm is recorded. The ratio of the two is substituted into the linear equation F 582 / F 622 =0.587+0.0249[malachite green], the concentration of malachite green is obtained.

7. The method according to claim 6, characterized in that The linear equation F 582 / F 622 =0.587+0.0249 [Malachite green] was obtained as follows: 4 μL of rhodamine B solution, 10 μL of malachite green solutions of varying concentrations, and 1.5 mL of HCl-Na2HPO4 buffer solution at pH 2 were added to 3 mL of carbon dot solution. The mixture was allowed to stand at room temperature for 2 minutes before measuring the fluorescence spectrum. The fluorescence emission intensity at 582 nm and 622 nm was recorded using a 5 nm slit width and 560 nm excitation wavelength. The malachite green concentration was plotted as the horizontal axis, and the fluorescence intensity ratio F was plotted as the horizontal axis. 582 / F 622 As the vertical axis, in the concentration range of 0 to 2.92 mg / L, the linear equation is F 582 / F 622 =0.587+0.0249[malachite green], the correlation coefficient is R 2 =0.994.

Images