High-selectivity carbon dots for detecting flavonol compounds in colorimetric and fluorescent dual modes as well as preparation method and application of high-selectivity carbon dots

Abstract

The invention belongs to the technical field of fluorescent nano materials and medical examination, and particularly relates to a high-selectivity carbon dot for detecting flavonol compounds in colorimetric and fluorescent dual modes as well as a preparation method and application of the high-selectivity carbon dot. The preparation method of the carbon dots comprises the following steps: preparing a (PEI) solution; the preparation method comprises the following steps: dispersing D-threonine (D-Thr) and a polyethyleneimine (PEI) solution in deionized water, performing ultrasonic treatment, and performing heating reaction; after the reaction, cooling to room temperature, centrifuging, taking supernate, and filtering to obtain a B-CDs solution; the B-CDs solution is subjected to freeze drying, and the yellowish-brown powder B-CDs carbon dots are obtained. The high-selectivity carbon dot based on polyethyleneimine prepared by the invention shows good water solubility, excellent fluorescent property and optical stability, has been successfully applied to quantitative detection of three flavonol compounds including morin, quercetin and kaempferol in complex matrixes such as human serum and urine, and shows a good linear relationship in a range of 0.5-100 [mu] M.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent nanomaterials and medical testing technology, and specifically relates to a highly selective carbon dot for colorimetric and fluorescent dual-mode detection of flavonol compounds, and a preparation method and application thereof. Background Art

[0002] As a bioactive factor closely related to human health, flavonols have multiple physiological functions such as anti-oxidation, anti-inflammatory, antibacterial and anti-cancer. Flavonols play an important role in the prevention and treatment of chronic diseases such as obesity, diabetes, cardiovascular and cerebrovascular diseases, and thus have become an indispensable functional ingredient in human diet. Therefore, the qualitative and quantitative analysis of flavonols in food and metabolites is of great value in the quality control, safety evaluation and effectiveness research of related products.

[0003] Traditional methods for detecting flavonol compounds include: UV spectrophotometry, high performance liquid chromatography, and electrochemical detection. UV spectrophotometry is easily interfered by factors such as the matrix solution and the cleanliness of the absorption cell during the detection of flavonol compounds; compared with UV spectrophotometry, high performance liquid chromatography has the advantages of a wide detection range, low detection limit, and high sensitivity, but it has the disadvantages of high detection equipment cost, long single sample detection time, complex operation steps, high sample pretreatment requirements, and high reagent consumption, which lead to certain limitations in the detection of flavonol compounds. In the electrochemical detection method, the research on the materials, types, and determination methods of the electrodes is relatively narrow, and there are still many limitations. The existing detection methods lack systematicity and sustainability, and are in urgent need of further improvement and perfection. Summary of the invention

[0004] The purpose of the present invention is to provide a highly selective carbon dot for colorimetric and fluorescent dual-mode detection of flavonol compounds and its preparation method and application. Threonine and polyethyleneimine are used as raw materials and a one-pot hydrothermal method is used to synthesize carbon dots with blue fluorescence. The carbon dots have excellent optical properties and stability and can be used to determine flavonol compounds in complex matrices such as human serum and urine.

[0005] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0006] The present invention provides a carbon dot for colorimetric and fluorescence dual-mode detection, and the preparation method of the carbon dot comprises the following steps:

[0007] (1) Prepare (PEI) solution;

[0008] (2) D-threonine (D-Thr) and polyethyleneimine (PEI) solution were dispersed in deionized water, sonicated, and then heated for reaction;

[0009] (3) After the reaction, cool to room temperature, centrifuge, and filter the supernatant to obtain a B-CDs solution;

[0010] (4) The B-CDs solution was freeze-dried to obtain yellow-brown powder B-CDs carbon dots.

[0011] In the above technical solution, further, the concentration of the polyethyleneimine (PEI) solution is 10 to 30 mM·L -1 ; The mass ratio of D-threonine (D-Thr), polyethyleneimine (PEI) and deionized water is (1:1:100) to (3:1:100).

[0012] In the above technical solution, further, in step (2), the ultrasonic time is 5 to 20 minutes, the heating reaction temperature is 150 to 250° C., and the time is 8 to 10 hours.

[0013] In the above technical solution, further, in the step (3), the centrifugation time is 5 minutes, and the filtration is performed using a microporous membrane.

[0014] In the above technical solution, further, the molar ratio of D-Thr to PEI is 168:1, the reaction temperature is heated to 200° C., and the reaction is carried out for 10 hours.

[0015] The present invention also provides the use of the aforementioned colorimetric and fluorescence dual-mode detection carbon dots in the quantitative detection of flavonol compounds.

[0016] The present invention also provides a method for colorimetric and fluorescent dual-mode detection of flavonol compounds, wherein the method uses the aforementioned carbon dots for detection.

[0017] In the above technical solution, further, the detection method includes a fluorescence determination method and a colorimetric determination method;

[0018] The fluorescence determination method comprises the following steps: adding B-CDs carbon dots to standard solutions of morin, quercetin and kaempferol of different concentrations, diluting with PBS buffer solution, fully mixing the test substance and B-CDs, incubating the reaction, and performing fluorescence detection at an excitation wavelength of 373 nm to detect flavonol compounds through changes in fluorescence signals;

[0019] The colorimetric determination method comprises the following steps: adding B-CDs to standard solutions of morin, quercetin and kaempferol of different concentrations, diluting with a PBS buffer solution, fully mixing the test substance and B-CDs, incubating the reaction, detecting the absorbance at 342 nm by an ultraviolet detector, and detecting the flavonol compounds by the color change;

[0020] In the above technical solution, further, the pH of the PBS buffer solution is 7.45, the incubation reaction temperature is 10° C., and the reaction time is 1 min.

[0021] In the above technical solution, further, the fluorescence determination method is to fit a linear curve based on the relationship between the ratio of fluorescence intensity F / F0 and the concentration of the standard solution, calculate the F / F0 of the analyte, and obtain the concentration of the analyte through a linear equation;

[0022] The colorimetric determination method fits a linear curve based on the relationship between the ratio F / F0 of ultraviolet absorbance and the concentration of the standard solution, calculates the F / F0 of the object to be measured, and obtains the concentration of the object to be measured through a linear equation.

[0023] In the above technical solution, further, the test object is human serum or urine; the serum and urine samples are pre-treated, centrifuged at 13000 rpm for 10 minutes to obtain a supernatant, and the supernatant is diluted 50 times with PBS buffer solution for determination.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1) The present invention prepares a blue fluorescent carbon dot suitable for colorimetric and fluorescent dual-mode detection. The prepared highly selective carbon dots based on polyethyleneimine exhibit good water solubility, excellent fluorescence properties and optical stability, and have been successfully applied to the quantitative detection of three flavonol compounds, namely, morin, quercetin and kaempferol, in complex matrices such as human serum and urine, showing a good linear relationship in the range of 0.5-100 μM.

[0026] 2) Compared with other detection methods, this method has specific specificity for flavonol compounds.

[0027] 3) The carbon dots prepared by the present invention can realize colorimetric and fluorescence detection modes, and the dual-function sensor simplifies the detection process while reducing the experimental time and consumables cost. At the same time, the collected data can be cross-validated, thereby improving the accuracy and reliability of the results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the preparation of B-CDs and dual-mode colorimetric fluorescence detection of flavonols;

[0029] Figure 2 Characterization diagram of B-CDs; TEM image of AB-CDs; HRTEM image of BB-CDs; XRD curve of CB-CDs; FT-IR spectrum of DB-CDs;

[0030] Figure 3 XPS spectrum of B-CDs; XPS full scan spectrum of AB-CDs; high-resolution XPS spectrum of BC 1s; high-resolution XPS spectrum of CN 1s; high-resolution XPS spectrum of DO 1s;

[0031] Figure 4 A. Optical properties of B-CDs; UV absorption spectrum (blue line), fluorescence excitation spectrum (orange line) and emission spectrum (green line) of AB-CDs (the inset is the image of B-CDs solution under natural light (left) and UV light (right)); B. Emission spectrum of B-CDs at different excitation wavelengths;

[0032] Figure 5 The stability evaluation results of B-CD; the time stability of AB-CDs; the salt concentration stability of BB-CDs; the stability of CB-CDs under different pH environments; the antioxidant activity of DB-CDs;

[0033] Figure 6 The results of optimization of B-CDs synthesis conditions are shown in Figure 2. A. Fluorescence intensity of B-CDs prepared at different temperatures; B. Fluorescence intensity of B-CDs prepared at different reaction times; Fluorescence intensity of B-CDs prepared at different molar ratios of CD-Thr to PEI;

[0034] Figure 7 The results of the optimization of the incubation reaction conditions of B-CDs; the pH value optimization of mulberry yellow in the detection of AB-CDs; the time optimization of mulberry yellow in the detection of BB-CDs; the temperature optimization of mulberry yellow in the detection of CB-CDs; the temperature optimization of quercetin in the detection of DB-CDs; the pH optimization of quercetin in the detection of EB-CDs; the time optimization of quercetin in the detection of FB-CDs; the pH optimization of kaempferol in the detection of GB-CDs; the time optimization of kaempferol in the detection of HB-CDs; the temperature optimization of kaempferol in the detection of IB-CDs;

[0035] Figure 8 B-CDs detect the fluorescence spectra of flavonol compounds and the linear relationship between their concentrations and F / F0; AB-CDs fluorescence spectra at different mulberry yellow tin concentrations (inset: images of mulberry yellow tin at different concentrations under ultraviolet light); B. exponential function and linear relationship between mulberry yellow tin concentration and F / F0; CB-CDs fluorescence spectra at different quercetin concentrations (inset: images of quercetin at different concentrations under ultraviolet light); D. exponential function and linear relationship between quercetin concentration and F / F0; EB-CDs fluorescence spectra at different kaempferol concentrations (inset: images of kaempferol at different concentrations under ultraviolet light); F. exponential function and linear relationship between kaempferol concentration and F / F0;

[0036] Fig. 9UV-visible spectra of flavonol compounds detected by B-CDs and the linear relationship between their concentrations and F / F0; UV-visible spectra of AB-CDs at different mulberry yellow tin concentrations (inset: images of mulberry yellow tin at different concentrations under sunlight); B. exponential function and linear relationship between mulberry yellow tin concentration and F / F0; UV-visible spectra of CB-CDs at different quercetin concentrations (inset: images of quercetin at different concentrations under sunlight); D. exponential function and linear relationship between quercetin concentration and F / F0; UV-visible spectra of EB-CDs at different kaempferol concentrations (inset: images of kaempferol at different concentrations under sunlight); F. exponential function and linear relationship between kaempferol concentration and F / F0;

[0037] Fig.10 The IFE quenching mechanism of B-CDs; (A) The fluorescence lifetime of B-CDs in the presence or absence of flavonols; (B) The UV-visible absorption spectrum of mulberry yellow and the excitation and emission fluorescence spectra of B-CDs; (C) The UV-visible absorption spectrum of quercetin and the excitation and emission fluorescence spectra of B-CDs; (D) The UV-visible absorption spectrum of kaempferol and the excitation and emission fluorescence spectra of B-CDs;

[0038] Fig.11 The static fluorescence quenching mechanism of B-CDs; A. UV absorption spectrum of the mixed system before and after adding mulberry yellow; (B) UV absorption spectrum of the mixed system before and after adding quercetin; (C) UV absorption spectrum of the mixed system before and after adding kaempferol;

[0039] Fig.12 A. The interference and selectivity results of B-CDs fluorescence detection; A. The effect of anions and interfering substances in the body on B-CDs in fluorescence detection; B. The effect of flavonoids and amino acids on B-CDs in fluorescence detection; C. The effect of anions and interfering substances in the body on B-CDs in colorimetric detection; D. The effect of anions and interfering substances in the body on B-CDs in colorimetric detection. DETAILED DESCRIPTION

[0040] The following is a further detailed description of the claims of the present invention in conjunction with specific implementation methods, but does not constitute any limitation to the present invention. Any limited modifications made within the scope of protection of the claims of the present invention are still within the scope of protection of the claims of the present invention.

[0041] Example 1 Preparation of B-CDs

[0042] like Figure 1As shown, first, 4g of polyethyleneimine (PEI) was dissolved in 20mL of deionized water to prepare a 20mM PEI solution. 0.2g of D-threonine (D-Thr) and 500μL of PEI solution were dispersed in 10mL of deionized water, ultrasonically treated for 10min, and heated at 200℃ for 10h. After cooling naturally to room temperature, centrifuged for 5min, and the supernatant was filtered through a microporous filter membrane (0.22μm) to obtain a B-CDs solution. After a large amount of synthesis, the solution was freeze-dried at -80℃ for 48h to obtain a yellow-brown powder B-CDs for subsequent experiments.

[0043] B-CDs were characterized and the results are as follows:

[0044] TEM Figure 2 As shown in (A), B-CDs carbon dots are spherical and well dispersed in aqueous solution. Figure 2 As shown in (B), B-CDs have an obvious lattice structure with a lattice spacing of 0.27nm, similar to the (100) plane of graphite carbon. Figure 2 As shown in (C), there is an obvious diffraction peak at 2θ = 23.5°, which is associated with the (002) plane of the graphite lattice spacing. The characterization results of HRTEM and XRD both confirm that N-CDs are highly crystalline materials with graphite structure. The FT-IR spectrum is shown in Figure 2 (D) as shown, 3446cm -1 The broad and blunt characteristic peak at 1634 cm is derived from the stretching vibration of OH or NH. -1 The significant absorption peaks at 1413 and 1327 cm-1 can be attributed to carbonyl and CN. -1 The characteristic peaks at are all from the bending vibration of CH.

[0045] The chemical structure and elemental composition of B-CDs were further analyzed by XPS. Figure 3 As shown in (A), the full scan spectrum shows three different binding energy states of B-CDs: C1s, N 1s and O1s. The three characteristic peaks are located at 285.75, 399.73 and 531.46 eV, and the percentage contents of the three elements are 71.27%, 16.56% and 12.16%, respectively. The high-resolution spectra of C, N and O are shown in Figure 3 As shown in (B, C, D), the fitting results show that the characteristic peaks of C 1s at 285.8 and 288.32 eV can be attributed to CN and C=O; the N 1s high-resolution spectrum can be interpreted as two fitting peaks at 399.78 and 401.6 eV, which are attributed to C-NH2 and NH respectively; the fitting peaks of O1s high-resolution spectrum at 531.18 and 532.2 eV can be attributed to C=O and CO.

[0046] Example 2B-Study on the optical properties of CDs

[0047] The optical properties of B-CDs were studied based on UV-visible absorption and fluorescence spectra. Figure 4 As shown in (A), the shoulder peaks and absorption peaks near 270nm and 368nm in the UV spectrum are attributed to the π-π* and n-π* transitions of B-CDs, and B-CDs exhibit maximum excitation and emission at 373nm and 469nm. Figure 4 As shown in the inset (A), the B-CDs aqueous solution appears light yellow and blue under natural light and UV light, respectively. Figure 4 As shown in (B), the fluorescence emission wavelength of B-CDs was studied to see whether it changes with the excitation wavelength, and its fluorescence properties under the excitation wavelength range of 300-400nm were tested. The results of the study on the fluorescence spectrum of B-CDs showed that when the excitation wavelength gradually increased from 300nm to 400nm, the emission peak red-shifted from 455nm to 478nm, indicating that B-CDs showed obvious excitation dependence. In addition, the Abs-QY of B-CDs at a wavelength of 373nm was measured to be 4.43%.

[0048] Example 3B-Stability Study of CDs

[0049] The stability of B-CDs was investigated by measuring the fluorescence intensity and antioxidant capacity of B-CDs at different times, different ion concentrations and different pH values. Figure 5 As shown in (A), the fluorescence intensity of B-CDs was detected within one month with a cycle of 5 days, and no obvious change was found in the fluorescence value, which proved the time stability. Figure 5(B) and (C) respectively studied the tolerance of B-CDs in high salt environment and different acid-base environments. The results of the bar graphs are clearly visible. The change in fluorescence intensity of B-CDs in high salt concentration (0-2M) is negligible. At the same time, in a pH environment of 3-11, B-CDs can exist stably and is not affected by the acid-base environment. In addition, the anti-bleaching whitening property of B-CDs was studied. After 90 minutes of irradiation of the B-CDs solution under ultraviolet light, the fluorescence emission intensity remained above 95% of the initial intensity, confirming the excellent photostability of B-CDs. An ABTS free radical scavenging experiment was performed to evaluate the antioxidant capacity of B-CDs. First, 7.4mM ABTS and 2.6mM potassium persulfate were mixed in equal volumes to prepare an ABTS free radical stock solution. The reaction was carried out at room temperature in the dark for 16 hours, and then diluted with water to an absorbance of 0.70±0.02 at 734nm. Then, 1 mL of B-CDs solution with different concentration gradients was prepared, and 1 mL of ABTS solution was added to mix well. The mixture was reacted in the dark for 20 min, and the absorbance was measured. Ascorbic acid (AA) was used as a positive control, and each sample was measured in triplicate. Figure 5 As shown in (D), the scavenging effect of ABTS free radicals showed a significant upward trend with the increase of sample concentration, and the color of the solution gradually faded from blue-purple to colorless. When the concentration of B-CDs was 200μg / mL, the scavenging rate of ABTS free radicals reached 99.1%, and the solution also turned colorless, proving that B-CDs had significant activity in scavenging ABTS free radicals, that is, excellent antioxidant properties.

[0050] Example 4 Optimization of experimental conditions

[0051] During the synthesis process, factors such as temperature, time, and molar ratio of raw materials have an impact on the luminescence of B-CDs. Figure 6 As shown in the figure, when the molar ratio of D-Thr to PEI is 168:1, the fluorescence intensity of B-CDs reaches the maximum at 373nm, so this molar ratio is designated as the optimal ratio for synthesizing carbon dots with blue emission. The fluorescence intensity of B-CDs is not significantly enhanced in the range of 120℃-180℃. When the reaction temperature reaches 200℃, its fluorescence intensity has a significant leap, which may be related to the physical properties of B-CDs. Therefore, D-Thr:EI=168:1 and reaction at 200℃ for 10h are selected as the optimal conditions for the synthesis process of B-CDs.

[0052] The experimental system studied the effects of environmental acidity, alkalinity, reaction time, and temperature on the detection of flavonols by B-CDs. 50 μL of B-CDs was added to 100 μL of standard solutions of mulberry yellow, quercetin, and kaempferol at different concentrations, and then diluted to 2 mL with PBS buffer solutions of different pH values. After the analyte and carbon dots were fully mixed, they were incubated and fluorescence detection was performed at an excitation wavelength of 373 nm to explore the optimal pH for the fluorescence quenching reaction. The incubation reaction time and temperature were changed to explore the optimal reaction time and temperature for the fluorescence quenching reaction. All optimization experiments were performed three times. The effect of pH on the system is as follows. Figure 7 As shown in (A, E, G), the fluorescence intensity of B-CDs itself and the system after B-CDs added to the test substance remained stable in the pH range of 3-11, so the PBS buffer solution with pH = 7.45 was selected as the best reaction condition. In addition, after a rapid reaction of 1 minute, the reaction system in a low temperature environment can produce the maximum quenching, so according to the intensity of quenching, pH = 7.45, reaction 1 minute, 10 ° C were selected as the best detection conditions.

[0053] Example 5

[0054] Dual-mode detection of flavonol compounds:

[0055] In this experiment, a fluorescence colorimetric dual-mode detection was established, and the fluorescence experiment was performed under the detection conditions optimized in Example 4. 50 μL of B-CDs was added to 100 μL of standard solutions of Morin, Quercetin, and Kaempferol at different concentrations, and then diluted to 2 mL with PBS buffer solution (pH = 7.45). After the analyte and the carbon dots were fully mixed, the reaction was incubated under the optimal conditions, and the fluorescence detection was performed at an excitation wavelength of 373 nm. The results are shown in FIG. Figure 8 As shown in the figure, as the concentration of flavonols increases, the fluorescence of the system is gradually quenched. The ratio of fluorescence intensity F / F0 is exponentially related to the concentration gradient of flavonoids in the range of 0.5-100 μM, and linearly related in the range of 0.5-50 μM. The linear equation fitted with Morin is F / F0=-0.0098[Morin]+0.9893, R 2 =0.992, the detection limit is 0.28 μM; the linear relationship with quercetin is F / F0=-0.0115[Quercetin]+0.9816, R 2 =0.995, and the detection limit was 0.11 μM; the response of kaempferol was similar to the above, and the fitting equation in the range of 0.5-50 μM was F / F0=-0.0109[Kaempferol]+0.998, R 2 =0.992, and the detection limit is 0.13 μM.

[0056] At the same time, the colorimetric method was also used to detect flavonol compounds. 50 μL of B-CDs was added to 100 μL of different concentrations of mulberry yellow, quercetin, and kaempferol standard solutions, and then diluted to 2 mL with PBS buffer solution (pH = 7.45). After the analyte and carbon dots were fully mixed, the reaction was incubated under the optimal conditions, and the absorbance was detected at 342 nm by a UV detector. The absorption spectra of B-CDs in the presence of different concentrations of the analyte are shown in Figure 2. Fig. 9 As shown. As the concentration of the analyte increases from 0.5 μM to 50 μM, the absorbance increases and the color of the solution gradually turns yellow, showing a good linear curve. The fitted linear curve of Morin is F / F0=0.0096[Morin]+0.0053, R 2 =0.998, the detection limit is 0.45 μM; the linear curve of quercetin is F / F0=0.0117[Quercetin]+0.0119, R 2 =0.994, the detection limit is 0.25 μM; the linear relationship of kaempferol is F / F0=0.0081[Kaempferol]+0.0109, R 2 =0.996, and the detection limit is 0.23μM. Compared with other detection methods, this study demonstrates the advantages of dual-mode detection. Compared with traditional single-function sensors, dual-function sensors can replace multiple single-function sensors in the same experiment. By measuring the fluorescence signal or color change, the presence and concentration of the target substance can be detected, reducing costs while improving the accuracy and reliability of detection.

[0057] Example 6

[0058] Study on the quenching mechanism of flavonols on B-CDs:

[0059] First, the fluorescence lifetime before and after the addition of the analyte was analyzed and calculated, and the fluorescence lifetime was fitted according to the formula, such as Fig.10 As shown in (A), the fluorescence lifetime τ0 = 1.43 ns before the addition of the analyte, and after the addition of mulberry yellow, quercetin, and kaempferol, the fluorescence lifetimes became 1.43 ns, 1.33 ns, and 1.33 ns, respectively, and the fluorescence lifetimes did not change significantly. Fig.10 The shaded part of (B, C, D) is the overlap of the UV absorption spectra of the three analytes and the excitation spectra of B-CDs. When the absorption band of the analyte overlaps with the excitation band of the fluorophore, the fluorescent substance is quenched but the fluorescence lifetime does not change, and the internal filter effect (IFE) will effectively occur, which proves that the fluorescence quenching may be caused by IFE. Fig.11As shown in the figure, after adding the analyte, the UV absorption peak shifts significantly, and there is an interaction between B-CDs and the analyte to form a stable complex. Static quenching refers to the process of forming a non-fluorescent complex between the ground state fluorescent substance and the quencher. The generated complex absorbs light and can immediately return to the ground state without any photon emission. The fluorescence lifetime has not changed, and the generation of new substances further proves static quenching. In summary, IFE and static quenching jointly regulate the fluorescence quenching of B-CDs.

[0060] Example 7

[0061] Interference and selectivity studies:

[0062] The selectivity and interference of this experiment were evaluated by examining the effects of some common ions, flavonoids, and in vivo interfering substances on the fluorescence intensity of B-CDs. Quercetin was selected as a representative research model of flavonols, and common anions and cations (Na + , K + , Ca 2+ Mg 2+ ), flavonoids (isoflavones - puerarin, dihydroflavonoids - naringenin / hesperidin, dihydroflavonols - dihydroquercetin, flavanols - catechins), in vivo interfering substances (uric acid, urea, glutathione, bovine serum albumin), amino acids (glycine, cysteine, alanine, histidine, proline) were selectively tested. At the same time, 2mM reference substances were mixed with 50μM quercetin for anti-interference experiments. The results are shown in Fig.12 As shown in (A, B), the experiment was conducted using quercetin as a representative of flavonols. Among all interfering substances, only quercetin can cause a significant decrease in fluorescence intensity. In the presence of other potential interfering substances, the fluorescence intensity of B-CDs is not affected by the interfering components. Fig.12 (C, D) are the selectivity and interference experiments of colorimetric detection. The absorbance is low when only interfering substances are present, and the absorbance increases significantly after adding quercetin, which proves the excellent selectivity of colorimetric detection. These results all confirm that the prepared B-CDs have high selectivity for the detection of flavonol compounds.

[0063] Example 8

[0064] Determination of actual samples:

[0065] In this experiment, fluorescence and colorimetric analysis methods for flavonol detection were developed. Human serum and urine were selected as the test samples of actual samples. The human serum sample was provided by the First Affiliated Hospital of Jilin University and approved by the Ethics Committee. Both serum and urine samples were pretreated and centrifuged at 13,000 rpm for 10 minutes to obtain the supernatant. The supernatant was diluted 50 times with PBS buffer solution, and different concentrations of flavonol compounds (10-50 μM) were added by the standard addition method. The recovery rate was determined to investigate the applicability of the method. The results of fluorescence detection are shown in Tables 1-1, 1-2, and 1-3. The recovery rates of flavonols at different concentrations were all in the range of 96.8%-113.3%, and the reproducibility (RSD) value was less than 2.65% (n=3), which proved that fluorescence detection has good accuracy and precision. The colorimetric detection results are shown in Tables 2-1, 2-2, and 2-3. The recoveries of the three flavonols were 95.9%-113.3%, and the reproducibility (RSD) value was lower than 1.79% (n=3). The dual-mode recovery results confirmed the feasibility and applicability of the probe in actual sample detection.

[0066] Table 1-1 Fluorescence determination of Morin in human serum and urine

[0067]

[0068]

[0069] Table 1-2 Fluorescence determination of quercetin in human serum and urine

[0070]

[0071] Table 1-3 Fluorescence determination of kaempferol in human serum and urine

[0072]

[0073] Table 2-1 Colorimetric determination of mulberry yellow in human serum and urine

[0074]

[0075] Table 2-2 Colorimetric determination of quercetin in human serum and urine

[0076]

[0077] Table 2-3 Colorimetric determination of kaempferol in human serum and urine

[0078]

[0079] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A carbon dot with colorimetric and fluorescence dual-mode detection, characterized in that: The method for preparing the carbon dots comprises the following steps: (1) Prepare (PEI) solution; (2) D-threonine (D-Thr) and polyethyleneimine (PEI) solution were dispersed in deionized water, sonicated, and then heated for reaction; (3) After the reaction, cool to room temperature, centrifuge, and filter the supernatant to obtain a B-CDs solution; (4) The B-CDs solution was freeze-dried to obtain yellow-brown powder B-CDs carbon dots.

2. The carbon dots for colorimetric and fluorescence dual-mode detection according to claim 1, characterized in that: The concentration of the polyethyleneimine (PEI) solution is 10 to 30 mM·L -1 ; The mass ratio of D-threonine (D-Thr), polyethyleneimine (PEI) and deionized water is (1:1:100) to (3:1:100).

3. The carbon dots for colorimetric and fluorescence dual-mode detection according to claim 1, characterized in that: The ultrasonic time in step (2) is 5 to 20 minutes, and the heating reaction temperature is 150 to 250° C. for 8 to 10 hours.

4. The carbon dots for colorimetric and fluorescence dual-mode detection according to claim 1, characterized in that: In the step (3), the centrifugation time is 5 minutes, and the filtration is performed using a microporous filter membrane.

5. The carbon dots for colorimetric and fluorescence dual-mode detection according to claim 1, characterized in that: The molar ratio of D-Thr to PEI is 168:1, the reaction temperature is heated to 200° C., and the reaction is carried out for 10 hours.

6. Use of the carbon dots with colorimetric and fluorescence dual-mode detection according to claim 1 in the quantitative detection of flavonol compounds.

7. A method for colorimetric and fluorescence dual-mode detection of flavonol compounds, characterized in that: The method uses the carbon dots described in any one of claims 1 to 4 for detection.

8. The detection method according to claim 7, characterized in that: The detection method includes a fluorescence determination method and a colorimetric determination method; The fluorescence determination method comprises the following steps: adding B-CDs to standard solutions of morin, quercetin and kaempferol of different concentrations, diluting with a PBS buffer solution, fully mixing the test substance and B-CDs, incubating the reaction, and performing fluorescence detection at an excitation wavelength of 373 nm to detect flavonol compounds through changes in fluorescence signals; The colorimetric determination method comprises the following steps: adding B-CDs to standard solutions of morin, quercetin and kaempferol of different concentrations, diluting with a PBS buffer solution, fully mixing the test substance and B-CDs, incubating the reaction, detecting the absorbance at 342 nm by an ultraviolet detector, and detecting the flavonol compounds by the color change; Preferably, the pH of the PBS buffer solution is 7.45, the incubation reaction temperature is 10° C., and the reaction time is 1 min.

9. The detection method according to claim 8, characterized in that: The fluorescence determination method is to fit a linear curve based on the relationship between the fluorescence intensity ratio F / F0 and the concentration of the standard solution, calculate the F / F0 of the analyte, and obtain the concentration of the analyte through a linear equation; The colorimetric determination method fits a linear curve based on the relationship between the ratio F / F0 of ultraviolet absorbance and the concentration of the standard solution, calculates the F / F0 of the object to be measured, and obtains the concentration of the object to be measured through a linear equation.

10. The detection method according to claim 8, characterized in that: The analyte is human serum or urine.

Images