Fluorescent carbon quantum dot based on beer, preparation method of fluorescent carbon quantum dot and application of fluorescent carbon quantum dot in anthocyanin detection

Abstract

The invention belongs to the technical field of fluorescent carbon quantum dots, and particularly discloses a beer-based fluorescent carbon quantum dot as well as a preparation method and application thereof in anthocyanin detection. The method comprises the following steps: putting beer into a reaction kettle, and carrying out hydrothermal treatment for several hours to obtain a deep yellow dispersion of carbon quantum dots; centrifuging the carbon quantum dot dispersion, taking supernate, putting the obtained supernate into a dialysis bag, and further purifying the carbon quantum dots in ultrapure water to obtain a yellow solution. The carbon quantum dot prepared by the invention is synthesized by a hydrothermal method in one step, has stable fluorescent property and is non-toxic. The efficient fluorescent carbon quantum dot can be used as a fluorescent probe for quantitative measurement of anthocyanin.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent carbon quantum dots, and in particular relates to fluorescent carbon quantum dots based on beer, a preparation method thereof, and application of the same in anthocyanin detection. Background Art

[0002] Natural biomass substitutes are used to synthesize carbon quantum dots. These carbon sources are low-cost, green and environmentally friendly, and in line with the concept of sustainable development. Beer is one of the most widely consumed alcoholic beverages and is easily accessible. The protein contained in beer comes from the grains used in the brewing process as a carbon source. In addition, nucleic acids and other non-proteins are derived nitrogen sources. Anthocyanins have a wide range of pharmacological activities, including anti-oxidation, scavenging free radicals in the human body, maintaining healthy vision, inhibiting the growth of cancer cells, etc. In view of the potential and wide application of anthocyanins, various types of anthocyanin products have also appeared on the market, which is of great significance for the effective and sensitive quantification of anthocyanins in products.

[0003] Carbon quantum dots have outstanding properties, including excellent water dispersibility, easy preparation, tunable photoluminescence and strong photoluminescence, and are widely used in photocatalysis, imaging, biosensing, etc. In addition, fluorescent sensors based on carbon quantum dots also meet the requirements of sustainable development and green chemistry standards. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention uses a one-step hydrothermal method to prepare carbon quantum dots with fluorescent properties, and verifies that they can be widely used as fluorescent probes for detecting anthocyanins.

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

[0006] A 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 the carbon quantum dot exhibits blue fluorescence under a 365 nm ultraviolet lamp.

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

[0008] Step 1: Place beer in a beaker and place the beaker on a magnetic stirrer for 5 minutes;

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

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

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

[0012] Step 5: The obtained solution is placed in a dialysis bag, and ultrapure water is poured into the dialysis bag to remove impurities, thereby obtaining a yellow solution;

[0013] Step 6: The obtained solution is placed in a vacuum freeze dryer for freeze drying to obtain purified beer-based fluorescent carbon quantum dots.

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

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

[0016] In the above-mentioned beer-based fluorescent carbon quantum dots, in step 3, the hydrothermal reaction is carried out at 150° C. for 6 hours.

[0017] In the above-mentioned beer-based fluorescent carbon quantum dots, in step 4, the centrifugation is performed at 10,000 rpm for 10 minutes.

[0018] In the above-mentioned beer-based fluorescent carbon quantum dots, in step 5, the specification of the dialysis bag used is 500Da, and the amount of ultrapure water used is 2L.

[0019] In the above-mentioned beer-based fluorescent carbon quantum dots, in step 6, the freeze-drying temperature is -49°C and the time is 24 hours.

[0020] The third aspect of the present invention provides the use of the above-mentioned biomass fluorescent carbon quantum dots as fluorescent probes in detecting anthocyanins.

[0021] The above application includes the following steps:

[0022] Step 1: Take 0.2 mL of anthocyanin solution with different concentrations in the range of 0 to 60 μM, add 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots and 0.6 mL of ultrapure water, mix well, and react for 2 minutes;

[0023] Step 2: Place the mixed solution obtained in step 1 into a fluorescence spectrometer, select 360 nm as the excitation light wavelength, set the spectral range to 360-600 nm, and after obtaining a 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 0.2 mg / mL of the above fluorescent carbon quantum dots and 0.6 mL of ultrapure water. After mixing evenly, react for 2 minutes.

[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 a complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm;

[0026] The linear regression equations for anthocyanin concentrations in the range of 0-30 μM and 35-60 μM are:

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

[0028] According to the method of step 1 to step 4, an anthocyanin solution of unknown concentration is taken to obtain the fluorescence intensity values ​​I and I0, and the concentration of anthocyanin is calculated according to the linear equation.

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

[0030] The fluorescent carbon quantum dots prepared by one-step hydrothermal method have stable fluorescent properties and are non-toxic. The fluorescent sensor based on carbon quantum dots is the first to construct a unique platform for tracking anthocyanins, which has broad application prospects in the detection of anthocyanins. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The Fourier infrared spectrum of the fluorescent carbon quantum dots prepared by the present invention;

[0032] Figure 2 The fluorescence spectra of the fluorescent carbon quantum dots prepared by the present invention at different excitation wavelengths;

[0033] Figure 3 The best excitation, emission and ultraviolet spectra of the fluorescent carbon quantum dots prepared by the present invention;

[0034] Figure 4 The stability fluorescence intensity diagram of the fluorescent carbon quantum dots prepared by the present invention is used to test the stability of the carbon quantum dots at different times (a), different salt concentrations (b) and different pH values ​​(c);

[0035] Figure 5 This is a graph showing the fluorescence intensity changes of the fluorescent carbon quantum dots prepared by the present invention under different interfering substances;

[0036] Figure 6 This is a graph showing the change in relative fluorescence intensity of the fluorescent carbon quantum dots prepared by the present invention after anthocyanin is added in the presence of other interfering substances;

[0037] Figure 7The fluorescence intensity change of the fluorescent carbon quantum dots P-CDs prepared by the present invention under different anthocyanin dosages;

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

[0039] Fig. 9 Fluorescent carbon quantum dots and flow chart for detecting anthocyanins.

[0040] Specific implementation method

[0041] The present invention is further described below by way of specific examples. The present invention is not limited to the embodiments, and slight variations are possible without departing from the scope described.

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

[0043] The preparation method is as follows:

[0044] Step 1: Put 50 mL of Tsingtao beer in a beaker and place the beaker on a magnetic stirrer for 5 minutes;

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

[0046] Step 3: Transfer the beer filtrate to a 70 ml Teflon-lined reactor and perform hydrothermal reaction at 150 °C for 6 h;

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

[0048] Step 5: The obtained solution was placed in a 500Da dialysis bag, and 2L of ultrapure water was added for dialysis to remove impurities, thereby obtaining a yellow solution;

[0049] Step 6: The obtained solution was placed in a vacuum freeze dryer and freeze-dried 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 infrared (such as Figure 1 ) were used to characterize the relevant functional groups of the prepared beer-based fluorescent carbon quantum dots (P-CDs). 3432cm -1 The peak at 1633 cm corresponds to the stretching vibration of OH / NH. -1 The peak at 1401 cm is attributed to the stretching vibration of the amide C=N. -1 The peak at 1032 cm comes from the stretching vibration of CN.-1 The peak at 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 ). The fluorescence emission spectra of P-CDs at different excitation wavelengths from 300 to 380 nm (measured every 10 nm), such as Figure 2 As shown in the figure, the fluorescence emission intensity of P-CDs is red-shifted (moving to a longer wavelength). This may be due to the complex environment provided by the biomass material, which makes it difficult to ensure that the generated P-CDs have a uniform size, causing the energy defects on the surface of the carbon quantum dots to lead to the emission wavelength related to excitation. When the excitation wavelength is 360nm, the optimal emission wavelength of P-CDs is 449nm, and it shows bright blue fluorescence under 365nm ultraviolet light; the absorption of the ultraviolet absorption spectrum of P-CDs centered at 275nm is attributed to n-π* transition (such as Figure 3 ).

[0054] (3) The stability of P-CDs was tested by testing the fluorescence intensity of carbon quantum dots at different times, different salt concentrations, and different pH values. The results showed that P-CDs had good stability (e.g. Figure 4 ). Figure 4 (a) shows the effect of storage time on the fluorescence intensity of P-CDs under 365nm UV light. The results show that the fluorescence intensity of P-CDs is almost constant within 1h, indicating that the stability of the sensor based on P-CDs is very good and can be introduced under complex conditions. Figure 4 As can be seen in (b), with the increase of NaCl (0~1M) concentration, the fluorescence intensity of P-CDs remains basically unchanged, indicating that P-CDs have good salt tolerance, and the stability of nanoparticles is one of the main contributions to improving the accuracy of chemical detection or biosensors. Figure 4 (c) shows that the fluorescence intensity of P-CDs does not change significantly when the solution is in the pH range of 1 to 10, but the fluorescence intensity decreases under strong alkaline conditions. The results show that P-CDs have good tolerance to pH values ​​and can be used in a wide pH range.

[0055] Example 3 Detection of anthocyanins based on fluorescent carbon quantum dots in beer as fluorescent probes

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

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

[0058] Step 1: 0.2 mL of the interfering substance or anthocyanin test solution was measured respectively, and 0.2 mL of 0.2 mg / mL fluorescent carbon quantum dots prepared in Example 1 and 0.6 mL of ultrapure water were added thereto, and after mixing evenly, the mixture was reacted for 2 min;

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

[0060] Step 3: 0.2 mL of ultrapure water was measured and added to 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, reacted 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 a complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm.

[0062] In the absence of anthocyanins, comparing I and I0, all potential interfering ions and interfering substances have no significant effect on the fluorescence intensity of carbon quantum dots, and the fluorescence intensity of P-CDs containing anthocyanins is much lower than that of blank samples. The above proves that only anthocyanins can effectively quench the fluorescence of P-CDs, indicating that P-CDs has excellent selectivity for anthocyanins (such as Figure 5 ).

[0063] Considering the complexity of actual samples, it is necessary to further investigate the effects of interfering substances on the P-CDs and anthocyanin system through interference tests.

[0064] Step 1: 0.2 mL of the interfering substance test solution was measured respectively, and 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 were added thereto, and after mixing evenly, the mixture was reacted for 2 min;

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

[0066] Step 3: 0.2 mL of anthocyanin solution was measured and added to 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, reacted 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 a complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm.

[0068] In the presence of anthocyanins, by comparing I and I0, when other interfering ions and interfering substances coexist with anthocyanins, all interfering ions and interfering substances will not react with anthocyanins, and will not cause obvious interference to the P-CDs and anthocyanin sensing systems. Anthocyanins can still effectively quench the P-CDs solution, indicating that P-CDs has excellent anti-interference ability in complex and changing environments (such as Figure 6 ).

[0069] Different concentrations (0-60 μM) of anthocyanins were added to the carbon quantum dots. Figure 7 The fluorescence intensity changes of P-CDs under different anthocyanin dosages are shown. In the range of 0-60 μM, the fluorescence quenching shows a good linear relationship with the increase of anthocyanin concentration. The linear regression equations of anthocyanin concentrations in the range of 0-30 μM and 35-60 μM are: y=0.00791x+0.02405, R 2 =0.992; y=0.01411x-0.19468, R 2 =0.993, which can quantitatively detect anthocyanins (such as Figure 8 ).

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

Claims

1. A beer-based fluorescent carbon quantum dot, characterized in that: The preparation method comprises the following steps: Step 1: Put beer in a beaker and place the beaker on a magnetic stirrer to stir; Step 2: filtering the beer through a microporous membrane to obtain a filtrate; Step 3: transferring the beer filtrate to a Teflon-lined reactor for hydrothermal reaction; Step 4: Centrifuge the obtained dark yellow carbon quantum dot dispersion and take the supernatant; Step 5: The obtained solution is placed in a dialysis bag, and ultrapure water is poured into the dialysis bag to remove impurities, thereby obtaining a yellow solution; Step 6: The obtained solution is placed in a vacuum freeze dryer for freeze drying to obtain purified beer-based fluorescent carbon quantum dots.

2. A beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 1, the beer is Tsingtao Beer.

3. The beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 2, the microporous membrane is 0.22 μm.

4. The beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 3, the hydrothermal reaction is carried out at 150° C. for 6 hours.

5. The beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 4, the centrifugation is performed at 10000 rpm for 10 min.

6. The beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 5, the specification of the dialysis bag used is 500Da, and the amount of ultrapure water used is 2L.

7. The beer-based fluorescent carbon quantum dots according to claim 1, characterized in that: In step 6, the freeze-drying temperature is -49°C and the time is 24 hours.

8. Use of the beer-based fluorescent carbon quantum dots according to any one of claims 1 to 7 as fluorescent probes in detecting anthocyanins.

9. The use according to claim 8, characterized in that: The steps include: Step 1: Take 0.2 mL of anthocyanin solution of different concentrations in the range of 0 to 60 μM, add 0.2 mL of 0.2 mg / mL of the beer-based fluorescent carbon quantum dots according to any one of claims 1 to 7 and 0.6 mL of ultrapure water, mix well, and react for 2 minutes; Step 2: Place the mixed solution obtained in step 1 into a fluorescence spectrometer, select 360 nm as the excitation light wavelength, set the spectral range to 360-600 nm, and after obtaining a 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 0.2 mg / mL of the fluorescent carbon quantum dots according to claim 7 and 0.6 mL of ultrapure water, and after mixing evenly, react for 2 minutes; 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 a complete fluorescence spectrum, read the fluorescence intensity value I0 at 449 nm; The linear regression equations for anthocyanin concentrations in the range of 0-30 μM and 35-60 μM are: y=0.00791x+0.02405,R 2 =0.992;y=0.01411x-0.19468,R 2 =0.993 According to the method of step 1 to step 4, an anthocyanin solution of unknown concentration is taken to obtain the fluorescence intensity values ​​I and I0, and the concentration of anthocyanin is calculated according to the linear equation.

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