Red / blue double-color carbon dot-based ratiometric fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a ratio fluorescent probe based on red / blue double-color carbon dots as well as a preparation method and application of the ratio fluorescent probe. The ratio fluorescent probe based on the red-blue double-color carbon dots is obtained by mixing a blue fluorescent carbon dot solution and a red fluorescent carbon dot solution according to the volume ratio of 6: 1, and the concentrations of the blue fluorescent carbon dot solution and the red fluorescent carbon dot solution are both 0.5 mg / mL; the blue fluorescent carbon dot solution is prepared by the following steps: adding deionized water into a mixed solution of citric acid and ethidene diamine, carrying out ultrasonic treatment, and then carrying out reaction centrifugation, filtration and dialysis to obtain blue fluorescent carbon dots; the red fluorescent carbon dot solution is prepared by adding deionized water and concentrated sulfuric acid into o-phenylenediamine, L-cysteine and boric acid, carrying out ultrasonic treatment, reacting, centrifuging, filtering, dialyzing and freeze-drying. The ratio fluorescent probe based on the red / blue double-color carbon dots can accurately detect the concentration of quercetin in the concentration range of 0-30.0 [mu] mol / L.

Description

Technical Field

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

[0002] Quercetin is a natural polyphenolic flavonoid compound. As a secondary metabolite of plants, it is widely present in various parts of plants and is an essential component of the human diet. It is found in a variety of fruits and vegetables, such as onions, tomatoes, apples, and tea. When quercetin-rich foods are consumed, quercetin metabolites accumulate in human tissues and exert a certain antioxidant effect. Quercetin's antioxidant effects, which have certain medicinal value, also make it useful in the prevention of diseases such as cancer. Quercetin has also attracted widespread attention due to its antiviral, anti-inflammatory, and anti-allergic activities. Various methods have been developed for the quantitative detection of quercetin, including chromatography, surface-enhanced Raman spectroscopy, electrochemical methods, and fluorescence. Fluorescence has attracted attention due to its simplicity, speed, and high sensitivity.

[0003] Ratio fluorescence method can effectively avoid the interference of external environmental factors and instrument light source, and has its own advantages. Synchronous fluorescence spectroscopy is a method of measuring the fluorescence intensity at a certain wavelength interval (Δλ=λ em -λ ex ) scanning spectrum, characterized by rapid response, high sensitivity, simple spectra, robustness to disturbances, and a narrow spectral bandwidth. This experiment, using a ratiometric fluorescence sensor under synchronous fluorescence spectroscopy, investigated the quenching of carbon dot fluorescence in the presence of quercetin. This combined advantage of synchronous and ratiometric fluorescence methods enabled excellent experimental results under optimized experimental conditions. Summary of the Invention

[0004] In this experiment, a high-quantum-yield blue fluorescent carbon dot was prepared and mixed with red fluorescent carbon dots in a certain ratio to construct a dual-emission ratio fluorescence system. The quercetin content in the solution was determined by synchronous fluorescence. In the synchronous fluorescence spectrum, two fluorescence emission peaks appeared at 390nm and 560nm. The optimal wavelength difference was selected as Δλ = 70nm. By optimizing the experimental conditions, the quercetin concentration and the ratio of the two fluorescence peak intensities (F 390 / F 560 ) was used to establish a standard curve for quercetin detection. This method has good selectivity, sensitivity, and precision, and can be used to detect quercetin in real human serum and urine samples with satisfactory experimental results. This experimental method has certain guiding significance for the detection of quercetin in the medical field.

[0005] The technical solution of the present invention is as follows: a ratiometric fluorescent probe based on red and blue dual-color carbon dots is obtained by mixing a blue fluorescent carbon dot solution and a red fluorescent carbon dot solution in a volume ratio of 6:1;

[0006] The blue fluorescent carbon dot solution was prepared as follows: deionized water was added to a mixture of citric acid and ethylenediamine, followed by sonication for reaction, followed by cooling and centrifugation, and the supernatant was filtered and dialyzed to obtain blue fluorescent carbon dots, thereby preparing a blue fluorescent carbon dot solution with a concentration of 0.5 mg / mL.

[0007] The red fluorescent carbon dot solution was prepared by adding deionized water and concentrated sulfuric acid to o-phenylenediamine, L-cysteine, and boric acid, performing sonication, transferring the mixture to a high-pressure reactor for reaction, cooling, and centrifuging. The supernatant was filtered, dialyzed, and freeze-dried to obtain red fluorescent carbon dots, thereby preparing a blue fluorescent carbon dot solution with a concentration of 0.5 mg / mL.

[0008] In the above-mentioned method for preparing a ratiometric fluorescent probe based on red and blue dual-color carbon dots and a blue fluorescent carbon dot solution, the reaction is carried out at 200° C. for 5 hours.

[0009] In the above-mentioned ratiometric fluorescent probe based on red and blue dual-color carbon dots, in the preparation method of the blue fluorescent carbon dot solution, the ratio of citric acid to ethylenediamine is 1 g:1 mL.

[0010] In the above-mentioned method for preparing a ratiometric fluorescent probe based on red and blue dual-color carbon dots and a blue fluorescent carbon dot solution, the dialysis membrane used in the dialysis has a MWCO of 300.

[0011] In the above-mentioned ratiometric fluorescent probe based on red and blue dual-color carbon dots, in the method for preparing the red fluorescent carbon dot solution, the mass ratio of o-phenylenediamine, L-cysteine, and boric acid is 2:1:0.2-0.23.

[0012] In the above-mentioned method for preparing a ratiometric fluorescent probe based on red and blue dual-color carbon dots and a red fluorescent carbon dot solution, the reaction is carried out at 160° C. for 12 hours.

[0013] The above-mentioned application of a ratiometric fluorescent probe based on red and blue dual-color carbon dots in the detection of quercetin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 TEM image (A) and particle size distribution histogram (B) of blue fluorescent carbon dots.

[0015] Figure 2 XRD spectrum of blue fluorescent carbon dots.

[0016] Figure 3 Infrared spectrum of blue fluorescent carbon dots.

[0017] Figure 4 UV-visible absorption spectrum of blue fluorescent carbon dots.

[0018] Figure 5 Fluorescence excitation and emission spectra of blue fluorescent carbon dots.

[0019] Figure 6 Fluorescence emission spectra of carbon dots under different excitation wavelengths of blue fluorescence.

[0020] Figure 7 TEM image (A) and particle size distribution histogram (B) of carbon dots.

[0021] Figure 8 XRD patterns of carbon dots.

[0022] Figure 9 FT-IR spectrum of carbon dots.

[0023] Figure 10 Full XPS spectrum of carbon dots (A) and high-resolution XPS spectra of C (B), N (C), O (D), and S (E).

[0024] Figure 11 UV-visible absorption spectrum of carbon dots.

[0025] Figure 12 Fluorescence excitation and emission spectra of carbon dots.

[0026] Figure 13 Fluorescence emission spectra of carbon dots at different excitation wavelengths.

[0027] Figure 14 Synchronous fluorescence spectra of the ratiometric fluorescence system after adding different amounts of quercetin (A) and the curve of fluorescence ratio value changing with quercetin concentration (B). DETAILED DESCRIPTION

[0028] Example 1

[0029] 1. Preparation of blue fluorescent carbon dots

[0030] 1g of citric acid and 1mL of ethylenediamine were weighed, and 25mL of deionized water was added. The mixture was ultrasonicated for 5 minutes and then transferred to a Teflon-lined autoclave. The reaction temperature was 200°C for 5 hours. The product was cooled to room temperature to obtain a dark brown solution. The solution was centrifuged at 10,000 rpm for 15 minutes, and the supernatant was filtered through an aqueous filter membrane (0.22μm). The filtrate was dialyzed through a dialysis membrane (MWCO = 300) for 48 hours and freeze-dried to obtain a brown carbon dot powder. The carbon dot powder was dispersed in deionized water to prepare a blue carbon dot solution (B-CDs) with a concentration of 0.5mg / mL.

[0031] 2. Preparation of Red Fluorescent Carbon Dots

[0032] 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 and the reaction was carried out 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.

[0033] 3. Determination of Fluorescence Quantum Yield of Fluorescent Carbon Dots

[0034] The fluorescence quantum yield (QY) of blue carbon dots was determined using quinine sulfate solution as a reference (QY = 54%). Quinine sulfate solution of appropriate concentration (0.1 mol / L H2SO4, absorbance no greater than 0.05) was prepared, and its absorption and fluorescence emission spectra were measured to calculate the QY of the blue carbon dots.

[0035] The fluorescence quantum yield (QY) of the obtained red carbon dots was determined using rhodamine B solution as a reference (QY = 89%). An appropriate concentration of rhodamine B ethanol solution (absorbance less than 0.05) was prepared, and its absorption and fluorescence emission spectra were measured to calculate the QY of the red carbon dots.

[0036] The QY calculation formula is as follows:

[0037]

[0038] Where "R" represents the reference sample, and the quantum yield of quinine sulfate (QY R ) is 0.54, and the quantum yield of rhodamine B (QY R ) is 0.89, “I” is the integrated area of ​​the fluorescence spectrum, “A” is the absorbance, “n” is the refractive index, and n / n R =1, it is calculated that the quantum yield of blue carbon dots is 41.5%, and the quantum yield of red carbon dots is 20.9%.

[0039] Example 2 Characterization of blue fluorescent carbon dots

[0040] The morphology and size distribution of blue fluorescent carbon dots were analyzed using transmission electron microscopy. Figure 1As shown in (A), the carbon dots are approximately spherical in shape, well dispersed, and have a uniform particle size distribution within a certain range. The particle size distribution histogram shows that the carbon dots are mainly distributed in the range of 0.8nm to 3.0nm, with an average particle size of approximately 1.8nm. Figure 2 Figure 3 is the XRD pattern of carbon dots. A broad peak appears at 25.9°, indicating the existence of amorphous carbon structure, which is consistent with the description in the literature.

[0041] The surface groups of the prepared carbon dots were analyzed using FT-IR spectroscopy. Figure 3 As shown, at 3426cm -1 A broad infrared absorption peak appears at 1654 cm, which is attributed to the stretching vibration of NH or OH; -1 The strong absorption peak near 1171cm -1 and 1387cm -1 The strong absorption peaks at 10 and 11 represent the stretching vibrations of C-C and C=C bonds, respectively. In summary, nitrogen atoms have been successfully doped into carbon dots. At the same time, the surface of CDs is rich in hydrophilic groups such as -COOH, -NH2, and -OH, which is beneficial to improve the hydrophilicity of carbon dots and make them well water-soluble.

[0042] The absorption properties of CDs were studied using UV-visible absorption spectroscopy. Figure 4 As shown in the figure, the broad absorption peak at 350 nm is probably due to the n-π* transition of the C=O structure, while the high-energy UV absorption peak at 239 nm is due to the sp 2 The unique UV characteristics of hybridized carbon dots likely originate from the transformation of molecular fluorophores. At wavelengths above 400 nm, CDs exhibit a broad, low-intensity absorption band extending to approximately 500 nm. This absorption is typically attributed to n-π* transitions associated with functional groups or heteroatom doping on the CD surface.

[0043] Excitation and emission spectra of carbon dot solutions, such as Figure 5 As shown in Figure 2, the maximum excitation wavelength of the prepared CDs is 358 nm, while the maximum emission wavelength is 444 nm, corresponding to a Stokes shift of 86 nm. In addition, the fluorescence emission spectrum of the carbon dots was obtained by changing the excitation light wavelength (338 nm to 398 nm). Figure 6As shown, the fluorescence emission peak under excitation at a wavelength of 358 nm has the highest fluorescence intensity. Furthermore, as the excitation wavelength increases from 338 nm to 398 nm, the position of the carbon dots' fluorescence emission peak shifts accordingly, demonstrating their wavelength-dependent excitation characteristics. In the experiment, using quinine sulfate as a reference substance, the quantum yield of the carbon dots was measured to be 41.5%, demonstrating the excellent luminescence properties of the prepared blue carbon dots.

[0044] Example 3 Characterization of red fluorescent carbon dots

[0045] Example 1: Red carbon dots were synthesized by hydrothermal method in sulfuric acid medium using o-phenylenediamine, L-cysteine ​​and boric acid as raw materials. TEM characterization was performed to study the morphology and size distribution of the red carbon dots. Figure 7 As shown in (A), the carbon dots have a nearly spherical structure and a uniform particle size distribution. Figure 7 (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 8 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.

[0046] The surface groups of the prepared carbon dots were analyzed by FT-IR spectroscopy. Figure 9 As shown, located at 3428cm -1 The 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.

[0047] The carbon dots were characterized by XPS spectra. Figure 10 As shown. Figure 10 (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 10(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 10 (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 10 (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 10 (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.

[0048] The absorption properties of CDs were studied using UV-visible absorption spectroscopy. Figure 11 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.

[0049] Excitation and emission spectra of carbon dot solution. Figure 12 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 13 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.

[0050] Example 4 Detection of Quercetin

[0051] To 1.5 mL of 0.5 mg / mL blue carbon dot solution, add 0.25 mL of 0.5 mg / mL red carbon dot solution, and then add 1.5 mL of Na2HPO4 -Mix citric acid buffer (pH 4.0) and 1.25 mL of deionized water. Add 10 μL of a 2 mmol / L quercetin solution to the solution, mix thoroughly, and allow to stand at room temperature for 5 minutes before measurement. Synchronous fluorescence spectra were recorded at 390 nm and 560 nm at Δλ = 70 nm. Perform the experiment in triplicate.

[0052] Under the optimal experimental conditions, the effect of quercetin concentration on the synchronous fluorescence spectrum of the mixed system (B-CDs / R-CDs) was investigated. Figure 14 As shown in (A), with the increase of quercetin addition, the intensities of the two fluorescence peaks at 390nm and 560nm decreased. The fluorescence peak at 390nm was used as the main signal and the fluorescence peak at 560nm was used as the reference signal. The ratio of the two synchronous fluorescence peak intensities F 390 / F 560 It showed a linear relationship with the concentration of quercetin. With the concentration of quercetin as the horizontal axis, the synchronous fluorescence peak ratio F 390 / F 560 As the vertical axis, linear fitting is performed, such as Figure 14 As shown in (B), in the concentration range of 0-30.0 μmol / L, F 390 / F 560 =0.0986×[quercetin]+5.20, correlation coefficient R 2 =0.999; in the concentration range of 30.0~66.7μmol / L, F 390 / F 560 =0.0479×[quercetin]+6.69(30.0~66.7μmol / L) correlation coefficient R 2 = 0.964. Clearly, a better linear correlation is achieved within the concentration range of 0–30.0 μmol / L. Since the quercetin content in actual samples generally ranges from 0–30.0 μmol / L, subsequent experiments were conducted to investigate the linear relationship within this range. The method's limit of detection (LOD) was calculated to be 0.58 μmol / L, demonstrating good sensitivity.

[0053] Example 5 Actual Sample Analysis

[0054] The feasibility of the proposed method was investigated by performing spiked recovery experiments on human serum and urine samples. Under optimal experimental conditions, 0.25 mL of a 0.5 mg / mL red carbon dot solution, 0.50 mL of the test sample (at concentrations of 0, 6.67, 13.3, and 20.0 μmol / L), and 0.75 mL of deionized water were added to 1.50 mL of a 0.5 mg / mL blue carbon dot solution. The mixture was mixed thoroughly and allowed to stand for 5 minutes before testing. The assay was repeated three times, and the spiked recoveries and relative standard deviations (RSDs) were calculated. The results are listed in Table 1. The spiked recoveries of quercetin in human serum and urine samples were well within the range of 97.0% to 104%, with relative standard deviations (RSDs) within 1.4%. In summary, this method exhibits excellent precision and accuracy, effectively enabling the detection of quercetin in real samples. The experimental results are satisfactory.

[0055] Table 1 Detection of quercetin in actual samples (n=3)

[0056]

[0057] Note: ND*, not detected.

Claims

1. A ratiometric fluorescent probe based on red and blue dual-color carbon dots, characterized in that: It is obtained by mixing a blue fluorescent carbon dot solution and a red fluorescent carbon dot solution in a volume ratio of 6:1; The blue fluorescent carbon dot solution was prepared by adding deionized water to a mixture of citric acid and ethylenediamine, performing a reaction after sonication, cooling and centrifuging, filtering and dialyzing the supernatant to obtain blue fluorescent carbon dots, and preparing a blue fluorescent carbon dot solution with a concentration of 0.5 mg / mL. The red fluorescent carbon dot solution was prepared by adding deionized water and concentrated sulfuric acid to o-phenylenediamine, L-cysteine, and boric acid, performing sonication, transferring the mixture to a high-pressure reactor for reaction, cooling, and centrifuging. The supernatant was filtered, dialyzed, and freeze-dried to obtain red fluorescent carbon dots, thereby preparing a blue fluorescent carbon dot solution with a concentration of 0.5 mg / mL.

2. A ratiometric fluorescent probe based on red and blue dual-color carbon dots according to claim 1, characterized in that: In the method for preparing the blue fluorescent carbon dot solution, the reaction is carried out at 200° C. for 5 hours.

3. A ratiometric fluorescent probe based on red and blue dual-color carbon dots according to claim 1, characterized in that: In the preparation method of the blue fluorescent carbon dot solution, the ratio of citric acid to ethylenediamine is 1 g:1 mL.

4. A ratiometric fluorescent probe based on red and blue dual-color carbon dots according to claim 1, characterized in that: In the method for preparing the blue fluorescent carbon dot solution, the dialysis membrane used in the dialysis has a MWCO of 300.

5. The ratiometric fluorescent probe based on red and blue dual-color carbon dots according to claim 1, characterized in that: In the preparation method of the red fluorescent carbon dot solution, the mass ratio of o-phenylenediamine, L-cysteine, and boric acid is 2:1:0.2-0.

23.

6. The ratiometric fluorescent probe based on red and blue dual-color carbon dots according to claim 1, characterized in that: In the method for preparing the red fluorescent carbon dot solution, the reaction is carried out at 160° C. for 12 hours.

7. Use of a ratiometric fluorescent probe based on red and blue dual-color carbon dots according to any one of claims 1 to 6 in the quantitative detection of quercetin.

8. The use according to claim 7, characterized in that The method is as follows: add a citric acid buffer solution with a pH of 4.0, deionized water, and a quercetin solution to be tested to any of the ratiometric fluorescent probes based on red and blue dual-color carbon dots according to any one of claims 1 to 6, mix them, let them stand, and record the synchronous fluorescence spectra at 390 nm and 560 nm respectively under the condition of Δλ = 70 nm. The ratio of the maximum fluorescence intensities of the two is calculated as F 390 / F 560 , substitute into the linear equation F 390 / F 560 =0.0986×[quercetin]+5.20(R 2 =0.999) to obtain the concentration of quercetin.

9. The use according to claim 7, characterized in that The method is as follows: 390 / F 560 =0.0986×[quercetin]+5.20(R 2 =0.999) was obtained by adding 0.25 mL of 0.5 mg / mL red carbon dot solution to 1.5 mL of 0.5 mg / mL blue carbon dot solution, and then adding 1.5 mL of Na2HPO4 with pH = 4.

0. - Mix citric acid buffer solution and 1.25 mL deionized water, add 10 μL of quercetin solution of different concentrations, mix, and let it stand at room temperature. Under the condition of Δλ=70 nm, record the synchronous fluorescence spectra at 390 nm and 560 nm respectively. The quercetin concentration is used as the horizontal axis, and the synchronous fluorescence peak ratio F is used as the horizontal axis. 390 / F 560 As the vertical axis, linear fitting was performed and the linear equation F was obtained in the concentration range of 0 to 30.0 μmol / L. 390 / F 560 =0.0986×[quercetin]+5.20, correlation coefficient R 2 =0.999.

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