A beer-based fluorescent carbon quantum dot, its preparation method, and its application in anthocyanin detection.

Abstract

This invention belongs to the field of fluorescent carbon quantum dot technology, specifically disclosing a beer-based fluorescent carbon quantum dot, its preparation method, and its application in anthocyanin detection. The method is as follows: beer is placed in a reaction vessel and hydrothermally heated for several hours to obtain a deep yellow dispersion of carbon quantum dots. The carbon quantum dot dispersion is centrifuged, and the supernatant is collected. The obtained supernatant is placed in a dialysis bag, and the carbon quantum dots are further purified in ultrapure water to obtain a yellow solution. The carbon quantum dots prepared by this invention are synthesized in one step using a hydrothermal method, possess stable fluorescent properties, and are non-toxic. The highly efficient fluorescent carbon quantum dots of this invention can be used as fluorescent probes for the quantitative measurement of anthocyanins.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent carbon quantum dot technology, and particularly relates to a beer-based fluorescent carbon quantum dot, its preparation method, and its application in anthocyanin detection. Background Technology

[0002] Natural biomass alternatives are being used to synthesize carbon quantum dots. These carbon sources are low-cost, environmentally friendly, and align with sustainable development principles. Beer is one of the most widely consumed alcoholic beverages and is readily available. The protein in beer comes from the grains used in the brewing process, serving as a carbon source. Additionally, nucleic acids and other non-protein components are derived nitrogen sources. Anthocyanins possess a wide range of pharmacological activities, including antioxidant activity, free radical scavenging, maintaining healthy vision, and inhibiting cancer cell growth. Given the potential and widespread applications of anthocyanins, various anthocyanin products have emerged on the market, making the effective and sensitive quantification of anthocyanins in these products crucial.

[0003] Carbon quantum dots possess outstanding properties, including excellent water dispersibility, ease of preparation, tunable photoluminescence, and strong photoluminescence, leading to their widespread application in photocatalysis, imaging, and biosensing. Furthermore, carbon quantum dot-based fluorescent sensors also meet the requirements of sustainable development and green chemistry standards. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention utilizes a one-step hydrothermal method to prepare carbon quantum dots with fluorescent properties and verifies that they can be widely used as fluorescent detectors for detecting anthocyanins.

[0005] This invention is specifically achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a fluorescent carbon quantum dot, wherein the optimal excitation wavelength of the carbon quantum dot is 360 nm, the optimal emission wavelength is 449 nm, and it exhibits blue fluorescence under a 365 nm ultraviolet lamp.

[0007] A second aspect of this invention provides a method for preparing beer-based fluorescent carbon quantum dots, comprising the following steps:

[0008] Step 1: Put the beer into a beaker and place the beaker on a magnetic stirrer and stir for 5 minutes;

[0009] Step 2: Filter the beer through a microporous membrane to obtain the filtrate;

[0010] Step 3: Transfer the above beer filtrate to a Teflon-lined reactor for hydrothermal reaction;

[0011] Step 4: Centrifuge the obtained dark yellow carbon quantum dot dispersion and collect the supernatant;

[0012] Step 5: Place the obtained solution into a dialysis bag, pour in ultrapure water and dialyze to remove impurities, obtaining a yellow solution;

[0013] Step 6: Place the obtained solution in a vacuum freeze dryer to freeze dry, and obtain purified beer-based fluorescent carbon quantum dots.

[0014] In the above-mentioned beer-based fluorescent carbon quantum dot, in step 1, the beer is Tsingtao Beer.

[0015] In the above-mentioned beer-based fluorescent carbon quantum dot, the microporous membrane in step 2 is 0.22 μm.

[0016] In the above-mentioned beer-based fluorescent carbon quantum dot, step 3 involves a hydrothermal reaction at 150°C for 6 hours.

[0017] In step 4 of the aforementioned beer-based fluorescent carbon quantum dot, the centrifugation is performed at 10,000 rpm for 10 minutes.

[0018] In step 5 of the aforementioned beer-based fluorescent carbon quantum dot assay, the dialysis bag used has a specification of 500 Da, and the amount of ultrapure water used is 2 L.

[0019] In the above-mentioned beer-based fluorescent carbon quantum dots, step 6 involves freeze-drying at a temperature of -49°C for 24 hours.

[0020] The third aspect of this invention provides the application of the above-mentioned biomass fluorescent carbon quantum dots as fluorescent probes in the detection of anthocyanins.

[0021] The above application includes the following steps:

[0022] Step 1: Within the range of 0–60 μM, take 0.2 mL of anthocyanin solutions of different concentrations, add 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots and 0.6 mL of ultrapure water, and after mixing evenly, react for 2 min.

[0023] Step 2: Place the mixed solution obtained in Step 1 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I at 449 nm.

[0024] Step 3: Measure 0.2 mL of ultrapure water and add it to 0.2 mL of the above-mentioned fluorescent carbon quantum dots at a concentration of 0.2 mg / mL and 0.6 mL of ultrapure water. After mixing evenly, react for 2 min.

[0025] Step 4: Place the mixed solution obtained in Step 3 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm.

[0026] The linear regression equations for anthocyanin concentrations in the ranges of 0–30 μM and 35–60 μM are as follows:

[0027] y = 0.00791x + 0.02405, R 2 =0.992; y=0.01411x-0.19468, R 2 =0.993

[0028] Following the methods in steps 1-4, an anthocyanin solution of unknown concentration was taken, and fluorescence intensity values ​​I and I0 were obtained. The concentration of anthocyanin was then calculated based on the linear equation.

[0029] Compared with the prior art, the present invention has the following beneficial technical effects:

[0030] This invention utilizes fluorescent carbon quantum dots prepared in a one-step hydrothermal process, which exhibit stable fluorescence properties and are non-toxic. A novel platform for tracking anthocyanins is constructed based on a carbon quantum dot fluorescent sensor, demonstrating broad application prospects in anthocyanin detection. Attached Figure Description

[0031] Figure 1 Fourier transform infrared spectrum of fluorescent carbon quantum dots prepared in this invention;

[0032] Figure 2 The fluorescence spectra of the fluorescent carbon quantum dots prepared in this invention at different excitation wavelengths are shown.

[0033] Figure 3 The optimal excitation, emission, and ultraviolet spectra of the fluorescent carbon quantum dots prepared in this invention are shown.

[0034] Figure 4 The fluorescence intensity diagram shows the stability of the fluorescent carbon quantum dots prepared in this invention. The stability of the carbon quantum dots at different times (a), different salt concentrations (b), and different pH values ​​(c) was tested.

[0035] Figure 5 The graph shows the fluorescence intensity variation of the fluorescent carbon quantum dots prepared in this invention under different interfering substances.

[0036] Figure 6 The graph shows the relative fluorescence intensity change of the fluorescent carbon quantum dots prepared in this invention after the addition of anthocyanins in the presence of other interfering substances.

[0037] Figure 7The fluorescence intensity changes of fluorescent carbon quantum dots P-CDs prepared in this invention under different anthocyanin dosages;

[0038] Figure 8 Linear relationship between anthocyanin concentration and quenching efficiency;

[0039] Figure 9 Fluorescent carbon quantum dots and a flowchart for detecting anthocyanins.

[0040] Specific implementation methods

[0041] The present invention will be further illustrated by specific examples below. The present invention is not limited to the embodiments described, and minor variations may be made without departing from the scope thereof.

[0042] Example 1: Preparation method of fluorescent carbon quantum dots based on beer

[0043] The preparation method is as follows:

[0044] Step 1: Take 50mL of Tsingtao beer into a beaker, place the beaker on a magnetic stirrer and stir for 5 minutes;

[0045] Step 2: Filter the beer through a 0.22μm microporous membrane to obtain the filtrate;

[0046] Step 3: Transfer the above beer filtrate to a 70ml Teflon-lined reactor and hydrothermally react at 150℃ for 6 hours;

[0047] Step 4: Centrifuge the obtained dark yellow carbon quantum dot dispersion at 10,000 rpm for 10 min and collect the supernatant;

[0048] Step 5: Place the obtained solution into a 500Da dialysis bag, pour in 2L of ultrapure water and dialyze to remove impurities, obtaining a yellow solution;

[0049] Step 6: Place the obtained solution in a vacuum freeze dryer at -49°C for 24 hours to obtain purified carbon quantum dot powder.

[0050] Example 2 Characterization of fluorescent carbon quantum dots based on beer

[0051] The specific characterization results are as follows:

[0052] (1) Using Fourier transform infrared (e.g.) Figure 1 The functional groups of the prepared beer-based fluorescent carbon quantum dots (P-CDs) were characterized. (3432 cm⁻¹) -1 The peak at 1633 cm⁻¹ corresponds to the stretching vibration of OH / NH. -1 The peak at 1401 cm⁻¹ is attributed to the stretching vibration of the amide C=N. -1 The peak at 1032 cm⁻¹ originates from the stretching vibration of CN.-1 The peak at that point corresponds to the stretching vibration of COC.

[0053] (2) To study the optical properties of P-CDs, the fluorescence spectra of P-CDs were recorded (e.g., Figure 2 Fluorescence emission spectra of P-CDs at different excitation wavelengths in the range of 300–380 nm (measured every 10 nm), such as... Figure 2 As shown, the fluorescence emission intensity of P-CDs exhibits a redshift (shifting to longer wavelengths). This may be due to the complex environment provided by biomass materials, making it difficult to ensure that the generated P-CDs have uniform size, leading to energy defects on the carbon quantum dot surface that result in excitation-related emission wavelengths. When the excitation wavelength is 360 nm, the optimal emission wavelength corresponding to P-CDs is 449 nm, exhibiting bright blue fluorescence under 365 nm UV light illumination. The UV absorption spectrum of P-CDs, with absorption centered at 275 nm, is attributed to n-π* transitions (such as...). Figure 3 ).

[0054] (3) Stability tests were conducted on P-CDs. The fluorescence intensity of carbon quantum dots in P-CDs was measured at different times, salt concentrations, and pH values. The results showed that P-CDs have good stability (e.g., Figure 4 ). Figure 4 (a) shows the effect of storage time on the fluorescence intensity of P-CDs under 365 nm UV light irradiation. The results indicate that the fluorescence intensity of P-CDs remained almost constant within 1 hour, demonstrating the excellent stability of the P-CD-based sensor and its suitability for use under complex conditions. Figure 4 As can be seen in (b), the fluorescence intensity of P-CDs remains basically unchanged with the increase of NaCl (0-1M) concentration, indicating that P-CDs have good salt resistance, and the stability of nanoparticles is one of the main contributions to improving the accuracy of chemical detection or biosensors. Figure 4 Figure (c) shows that the fluorescence intensity of P-CDs does not change significantly when the solution is in the pH range of 1–10, but the fluorescence intensity decreases under strongly alkaline conditions. The results indicate that P-CDs have good pH tolerance and can be used over a wide pH range.

[0055] Example 3: Detection of anthocyanins using beer-derived fluorescent carbon quantum dots as fluorescent probes.

[0056] The specific testing method includes the following steps:

[0057] To test whether fluorescent carbon quantum dots have good selectivity, several interfering ions and substances that may exist in anthocyanin products were selected.

[0058] Step 1: Take 0.2 mL of the interferon or anthocyanin test solution, add 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots prepared in Example 1 and 0.6 mL of ultrapure water, and after mixing evenly, react for 2 min.

[0059] Step 2: Place the mixed solution obtained in Step 1 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I at 449 nm.

[0060] Step 3: Measure 0.2 mL of ultrapure water and add it to 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots prepared in Example 1 and 0.6 mL of ultrapure water. After mixing evenly, react for 2 min.

[0061] Step 4: Place the mixed solution obtained in Step 3 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm.

[0062] In the absence of anthocyanins, compared with I and I0, all potential interfering ions and substances had no significant effect on the fluorescence intensity of carbon quantum dots, while the fluorescence intensity of P-CDs containing anthocyanins was much lower than that of the blank sample. This demonstrates that only anthocyanins can effectively quench the fluorescence of P-CDs, indicating that P-CDs have excellent selectivity for anthocyanins (e.g., ...). Figure 5 ).

[0063] Considering the complexity of actual samples, further interference experiments are needed to investigate the effects of interfering substances on P-CDs and the anthocyanin system.

[0064] Step 1: Take 0.2 mL of the interfering test solution, add 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots prepared in Example 1, 0.2 mL of anthocyanin solution and 0.4 mL of ultrapure water to it, and after mixing evenly, react for 2 min.

[0065] Step 2: Place the mixed solution obtained in Step 1 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I at 449 nm.

[0066] Step 3: Measure 0.2 mL of anthocyanin solution and add it to 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots prepared in Example 1 and 0.6 mL of ultrapure water. After mixing evenly, react for 2 min.

[0067] Step 4: Place the mixed solution obtained in Step 3 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360–600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm.

[0068] In the presence of anthocyanins, compared to I and I0, when other interfering ions and substances coexist with anthocyanins, none of them interact with the anthocyanins, and therefore do not significantly interfere with P-CDs or the anthocyanin sensing system. Anthocyanins can still effectively quench the P-CDs solution, indicating that P-CDs have excellent anti-interference capabilities in complex and variable environments (e.g., Figure 6 ).

[0069] Different concentrations (0–60 μM) of anthocyanins were added to carbon quantum dots. Figure 7 The fluorescence intensity changes of P-CDs under different anthocyanin concentrations were shown. Within the range of 0–60 μM, fluorescence quenching showed a good linear relationship with increasing anthocyanin concentration. The linear regression equations for anthocyanin concentration ranges of 0–30 μM and 35–60 μM were y = 0.00791x + 0.02405, R0. 2 =0.992; y=0.01411x-0.19468, R 2 =0.993, which can be used to quantitatively detect anthocyanins (e.g. Figure 8 ).

[0070] In summary, the carbon quantum dots prepared by this invention are synthesized in one step using a hydrothermal method, exhibit stable fluorescence properties, and are non-toxic. These highly efficient fluorescent carbon quantum dots can be used as fluorescent probes for the quantitative measurement of anthocyanins.

Claims

1. An application of beer-based fluorescent carbon quantum dots as fluorescent probes in the detection of anthocyanins, characterized in that, The method for preparing beer-based fluorescent carbon quantum dots includes the following steps: Step 1: Take Tsingtao beer into a beaker and place the beaker on a magnetic stirrer to stir; Step 2: Filter Tsingtao beer through a microporous membrane to obtain the filtrate; Step 3: Transfer the above beer filtrate to a Teflon-lined reactor and perform a hydrothermal reaction at 150°C for 6 hours; Step 4: Centrifuge the obtained dark yellow carbon quantum dot dispersion and collect the supernatant; Step 5: Place the obtained solution into a dialysis bag, pour in ultrapure water and dialyze to remove impurities, obtaining a yellow solution; Step 6: Place the obtained solution in a vacuum freeze dryer to freeze dry, and obtain purified beer-based fluorescent carbon quantum dots.

2. The application of beer-based fluorescent carbon quantum dots as a fluorescent probe in the detection of anthocyanins according to claim 1, characterized in that, In step 2, the microporous membrane has a diameter of 0.22 μm.

3. The application of beer-based fluorescent carbon quantum dots as a fluorescent probe in the detection of anthocyanins according to claim 1, characterized in that, In step 4, the centrifugation is performed at 10,000 rpm for 10 minutes.

4. The application of beer-based fluorescent carbon quantum dots as a fluorescent probe in the detection of anthocyanins according to claim 1, characterized in that, In step 5, the dialysis bag used is 500 Da, and the amount of ultrapure water used is 2 L.

5. The application of beer-based fluorescent carbon quantum dots as a fluorescent probe in the detection of anthocyanins according to claim 1, characterized in that, In step 6, the freeze-drying temperature is -49°C and the time is 24 hours.

6. The application according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Within the range of 0~60μM, take 0.2mL of anthocyanin solution of different concentrations, add 0.2mL of the 0.2mg / mL beer-based fluorescent carbon quantum dots and 0.6mL of ultrapure water, and after mixing evenly, react for 2min. Step 2: Place the mixed solution obtained in Step 1 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360~600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I at 449 nm. Step 3: Measure 0.2 mL of ultrapure water and add it to 0.2 mL of the 0.2 mg / mL fluorescent carbon quantum dots and 0.6 mL of ultrapure water. After mixing evenly, react for 2 min. Step 4: Place the mixed solution obtained in Step 3 into a fluorescence spectrometer, select 360 nm as the excitation wavelength, set the spectral range to 360~600 nm, and after obtaining the complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm. The linear regression equations for anthocyanin concentrations in the ranges of 0–30 μM and 35–60 μM are as follows: y = 0.00791x + 0.02405,R 2 =0.992;y = 0.01411x - 0.19468,R 2 =0.993; Following the methods in steps 1-4, an anthocyanin solution of unknown concentration was taken, and fluorescence intensity values ​​I and I0 were obtained. The concentration of anthocyanin was then calculated based on the linear equation.

Images