A method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots

Abstract

This invention discloses a method for measuring methylene blue based on the inner filter effect of chlorophyll carbon quantum dots (CQDs). This method uses CQDs as fluorescent probes and exploits the inner filter effect of CQD fluorescence quenching to detect the CQD content. The method is simple and convenient to operate, highly sensitive, and can rapidly complete sample determination. The CQDs are produced from biomass, resulting in low cost and broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of methylene blue detection, and more specifically, to a method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots. Background Art

[0002] Azobenzene dyes are commonly used in the printing and dyeing industry. Dyeing wastewater often contains residual azobenzene dyes. Improper treatment of these wastewaters can pollute the environment and has become a major source of pollution in the printing and dyeing industry. Methylene blue is a typical example of this type of organic dye. Because methylene blue is readily soluble in water, it is easily absorbed by organisms after contaminating water, causing growth distortions and even carcinogenicity. Therefore, detecting methylene blue in water is of great significance for both environmental protection and human health.

[0003] Current methods for determining methylene blue include high-performance liquid chromatography, capillary electrophoresis, resonance Rayleigh scattering spectroscopy, and chemiluminescence. While these methods each have their own advantages and can be applied to a variety of sample types, they still fall short of meeting the analytical requirements of modern samples. Therefore, the development of simple, rapid, and highly sensitive analytical methods remains crucial.

[0004] Carbon quantum dots (CDs), also known as carbon dots, are a type of carbon-based nanomaterial that can be engineered to exhibit a variety of excellent properties. Fluorescence analysis methods based on CDs have been widely used in various fields due to their numerous advantages. Summary of the Invention

[0005] An object of the present invention is to address at least the above-mentioned disadvantages and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a method for determining methylene blue based on the inner filter effect of chlorophyll carbon quantum dots. This method is simple and convenient to operate, highly sensitive, and can rapidly complete sample determination. Because the carbon dots are produced from biomass, they are inexpensive and have broad application prospects.

[0007] In order to achieve these purposes and other advantages according to the present invention, the present invention provides a method for determining methylene blue based on the inner filter effect of chlorophyll carbon quantum dots fluorescence, which uses chlorophyll carbon dots as fluorescent probes and utilizes the inner filter effect of methylene blue quenching the chlorophyll carbon dots fluorescence to realize the detection of methylene blue content.

[0008] Preferably, the method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots comprises the following steps:

[0009] Step 1: prepare a series of methylene blue standard solutions with different concentrations;

[0010] Step 2: Each portion of the methylene blue standard solution is mixed with the chlorophyll carbon dot solution to obtain a series of standard solutions to be tested;

[0011] Step 3: Measure the fluorescence spectrum of each standard solution to be tested at an excitation wavelength of 405 nm, record the fluorescence intensity at a wavelength of 620-760 nm, and establish a linear relationship between the fluorescence intensity and the methylene blue concentration;

[0012] Step 4: Mix the sample to be tested with the chlorophyll carbon dot solution, measure the fluorescence intensity at a wavelength of 620-760 nm under an excitation wavelength of 405 nm, and obtain the methylene blue content in the sample to be tested based on the linear relationship between the fluorescence intensity and the methylene blue concentration.

[0013] Preferably, in the method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots, the preparation process of the methylene blue standard solution in step 1 is specifically as follows:

[0014] Prepare a 20 mg / mL methylene blue standard stock solution using methylene blue standard and ultrapure water;

[0015] Different volumes of methylene blue standard stock solution were measured and diluted to the same volume with 100 mmol / L PBS buffer to obtain a series of methylene blue standard solutions with different concentrations.

[0016] Preferably, in the method for determining methylene blue based on the fluorescent inner filter effect of chlorophyll carbon quantum dots, a series of methylene blue standard solutions of different concentrations include 9 methylene standard solutions, and the concentrations are 0 μM, 0.1 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM, respectively.

[0017] Preferably, in the method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots, in step 2, the chlorophyll carbon dot solution in step 2 comprises chlorophyll carbon dots, PBS buffer and ultrapure water.

[0018] Preferably, the method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots comprises the following steps:

[0019] Step a, weighing washed lettuce leaves, chopping them and placing them in a beaker, adding a mixture of anhydrous ethanol and acetone in a volume ratio of 1:1 to the beaker, soaking the lettuce leaves for extraction for 30 minutes, and filtering to obtain a chlorophyll extract;

[0020] Step b, placing the chlorophyll extract in a polytetrafluoroethylene autoclave, reacting with magnetic stirring at 180° C., cooling to room temperature after 6 hours, filtering, adjusting the pH to neutral, and then transferring to a dialysis bag, dialyzing with ultrapure water in a dark environment for 24 hours to obtain carbon dots;

[0021] Step c: vacuum-dry the carbon dots and then prepare a 0.5 mg / mL carbon dot stock solution with ultrapure water.

[0022] Preferably, in the method for determining methylene blue based on the fluorescent inner filter effect of chlorophyll carbon quantum dots, in step 2, in each standard solution to be tested, the volume of methylene blue standard solution is 100 μL, the volume of chlorophyll carbon dots is 100 μL, the volume of PBS buffer is 700 μL, and the volume of ultrapure water is 100 μL.

[0023] Preferably, in the method for determining methylene blue based on the fluorescent inner filter effect of chlorophyll carbon quantum dots, the concentration of the PBS buffer is 100 mmol / L and the pH is 7.

[0024] Preferably, in the method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots, the excitation slit and emission slit of fluorescence detection in step three and step four are both 10 nm.

[0025] The present invention has at least the following beneficial effects: the present invention is simple and convenient to operate, has high sensitivity, and can quickly complete sample determination. The present invention uses biomass to prepare carbon dots, which is low-cost and has broad application prospects.

[0026] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a fluorescence spectrum obtained when the chlorophyll carbon quantum dot fluorescent probe is used to detect standard solutions of different concentrations at an excitation wavelength of 405 nm in Example 3 of the present invention;

[0028] Figure 2 is the linear relationship between fluorescence intensity and methylene blue concentration established in Example 3 of the present invention;

[0029] Figure 3 TEM scan image of carbon dots in Example 2 of the present invention;

[0030] Figure 4 This is a graph showing the stability of carbon dots under 365nm UV light in Example 2 of the present invention;

[0031] Figure 5 The ultraviolet absorption spectra of methylene blue and chlorophyll carbon dots in Example 2 of the present invention are shown;

[0032] Figure 6 This is a fluorescence lifetime diagram of methylene blue and chlorophyll carbon dots in Example 2 of the present invention. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0034] It should be noted that the experimental methods described in the following examples, unless otherwise specified, are conventional methods, and the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0035] 1. Materials

[0036] Lettuce leaves were purchased from the Golden Triangle Vegetable Market in Youjiang District, Baise City. Ultrapure water was used in the experiment.

[0037] PBS buffer was purchased from Solebol;

[0038] Methylene blue, ethanol, and acetone were purchased from Sinopharm Reagent Group Co., Ltd.; other chemical reagents were of analytical grade.

[0039] 2 Experimental instruments

[0040] UV2600 ultraviolet spectrophotometer (Shimadzu, Japan); fluorescence spectrophotometer (Agilent Technologies, Inc.); SZCL-2 intelligent digital display constant temperature magnetic stirrer (Zhengzhou Kehua Instrument Equipment Co., Ltd.); PHS-3C pH meter (Shanghai Leici); vacuum drying oven (Shanghai Jinghong).

[0041] Example 1

[0042] Preparation of methylene blue standard solution

[0043] Prepare a 20 mg / mL methylene blue standard stock solution using methylene blue standard and ultrapure water: Accurately weigh 5.0 mg of methylene blue standard and dissolve it in a 250 mL brown volumetric flask with ultrapure water to obtain a 20 mg / mL methylene blue standard stock solution. Store in a refrigerator at 4°C until use.

[0044] Different volumes of methylene blue standard stock solution were measured respectively, and diluted to the same volume with PBS buffer of the same concentration to obtain a series of methylene blue standard solutions with different concentrations; a series of methylene blue standard solutions with different concentrations included 9 parts of methylene standard solutions, and the concentrations were 0 μM, 0.1 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM, respectively.

[0045] Example 2

[0046] 1 Preparation of chlorophyll carbon dots

[0047] Step a, weighing washed lettuce leaves, chopping them and placing them in a beaker, adding a mixture of anhydrous ethanol and acetone in a volume ratio of 1:1 to the beaker, soaking the lettuce leaves for extraction for 30 minutes, and filtering to obtain a chlorophyll extract;

[0048] Step b, placing the chlorophyll extract in a polytetrafluoroethylene autoclave, reacting with magnetic stirring at 180° C., cooling to room temperature after 6 hours, filtering, adjusting the pH to neutral, and then transferring to a dialysis bag, dialyzing with ultrapure water in a dark environment for 24 hours to obtain carbon dots;

[0049] Step c: After vacuum drying the carbon dots, prepare a 0.5 mg / mL carbon dot stock solution with ultrapure water, and store it in a refrigerator at 4° C. away from light until use, thereby obtaining chlorophyll carbon dots.

[0050] 2 Carbon dot characterization

[0051] The structure and morphology of chlorophyll carbon dots (carbon dots after vacuum drying in step c) were investigated using TEM. Figure 3 As shown, the prepared chlorophyll carbon dots exhibit good dispersion. The particle size distribution of the chlorophyll carbon dots primarily ranges from 2.0 to 6.0 nm, with an average particle size of 4.0 nm. High-resolution transmission electron microscopy (HRTEM) images (inset) reveal that the synthesized chlorophyll carbon dots have a lattice diameter of 0.21 nm, consistent with the sp2 diffraction parameters of graphitic carbon.

[0052] 3. Stability of carbon dots

[0053] In order to investigate the stability of chlorophyll carbon dots, the effect of ultraviolet light irradiation on their fluorescence intensity was investigated. Figure 4 As shown in FIG, the fluorescence intensity of chlorophyll carbon dots did not change much after continuous irradiation with Xe lamp (365 nm) for 60 minutes. These experimental results indicate that chlorophyll carbon dots have good photostability.

[0054] 4 Experimental principle analysis

[0055] In order to understand the mechanism of the determination of methylene blue based on chlorophyll carbon dots, UV-visible absorption spectroscopy and fluorescence lifetime experiments were carried out. Figure 5 As shown in FIG, under the emission spectrum of 405 nm excitation, the absorption spectrum of methylene blue and the absorption spectrum of chlorophyll carbon dots have a large overlap.

[0056] In order to further confirm that the measurement mechanism is caused by photoinduced electron transfer or resonance energy transfer, the fluorescence lifetime of chlorophyll carbon dots was measured before and after the addition of methylene blue. Figure 6 As shown in the figure, the fluorescence lifetime of chlorophyll carbon dots did not change, indicating that there was no electron or energy transfer between chlorophyll carbon dots and methylene blue. All these results show that the recognition mechanism of methylene blue based on chlorophyll carbon dots fluorescence is caused by the inner filter effect.

[0057] Example 3

[0058] The present invention provides a method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots, which is characterized by comprising the following steps:

[0059] Step 1. Prepare a series of methylene blue standard solutions of different concentrations: Use methylene blue standard and ultrapure water to prepare a 20 mg / mL methylene blue standard stock solution: Accurately weigh 5.0 mg of methylene blue standard and dissolve the standard in a 250 mL brown volumetric flask with ultrapure water to obtain a 20 mg / mL methylene blue standard stock solution. Store in a refrigerator at 4°C until use.

[0060] Different volumes of methylene blue standard stock solution were measured and diluted to the same volume with PBS buffer of the same concentration to obtain a series of methylene blue standard solutions of different concentrations; the series of methylene blue standard solutions of different concentrations included 9 methylene blue standard solutions, and the concentrations were 0 μM, 0.1 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM, respectively;

[0061] Step 2: preparing chlorophyll carbon dots, specifically comprising:

[0062] Step a, weighing 10 g of washed lettuce leaves, chopping them and placing them in a beaker, adding 30 mL of a mixture of anhydrous ethanol and acetone in a volume ratio of 1:1 to the beaker, soaking the lettuce leaves for extraction for 30 minutes, and filtering to obtain a chlorophyll extract;

[0063] Step b: 25 mL of the chlorophyll extract was placed in a polytetrafluoroethylene autoclave and reacted with magnetic stirring at 180° C. After 6 h, the mixture was cooled to room temperature and filtered, the pH was adjusted to neutral, and the mixture was transferred to a dialysis bag and dialyzed with ultrapure water in a dark environment for 24 h to obtain carbon dots;

[0064] Step c, vacuum-drying the carbon dots and then preparing a 0.5 mg / mL carbon dot stock solution with ultrapure water, and storing it in a refrigerator at 4°C away from light until use, thereby obtaining chlorophyll carbon dots;

[0065] Step 3: Take 100 μL of each methylene blue standard solution and add it to a centrifuge tube. Then, add 100 μL of chlorophyll carbon dots, 700 μL of PBS buffer (100 mmol / L, pH 7), and 100 μL of ultrapure water to each centrifuge tube. Mix thoroughly by shaking and keep in a 25°C water bath for 5 minutes to obtain a series of standard solutions to be tested. The excitation and emission slits for fluorescence detection are both 10 nm.

[0066] Step 4: Use a fluorescence cuvette to test each standard solution to be tested, and measure the fluorescence spectrum of each standard solution to be tested at an excitation wavelength of 405 nm. Figure 1 As shown, the fluorescence intensity gradually decreases with the increase of methylene blue concentration. In the figure, the concentrations of methylene blue corresponding to the multiple standard solutions to be tested correspond to lines a to I in order from small to large. The excitation slit and emission slit of fluorescence detection are both 10 nm.

[0067] In addition, the fluorescence intensity at a wavelength of 620 to 760 nm was the strongest and showed a good linear relationship with the methylene blue concentration in the concentration range of 0.1 to 12 μM, as shown in Figure 2. Figure 2 As shown, a linear relationship between fluorescence intensity and methylene blue concentration was established. The fluorescence intensity Y was used as the ordinate and the methylene blue concentration X was used as the abscissa to draw a standard curve. The linear regression equation was Y = 20.304X + 29.509, and the correlation coefficient was R 2 =0.9915. The detection limit can reach 5.0nM (signal-to-noise ratio is 3), which is much lower than the detection limit of methylene blue by fluorescent probe in existing technologies (such as Gong Xianghong, Xu Yingjiang, Ren Chuanbo, et al. HPLC determination of malachite green, methylene blue, crystal violet and their metabolites in aquatic products [J]. Food Science, 2012, 33(4):144-147.; Yang Fang, Fan Kewei, Liu Zhengcai, et al. Detection of methylene blue and its metabolites in aquatic products by ultra-high performance liquid chromatography-tandem mass spectrometry [J]. Journal of Analysis and Testing, 2009, 28(1):32-36.).

[0068] Spike recovery experiment

[0069] As shown in Table 1, the spiked recoveries ranged from 92% to 105%, with a relative standard deviation (RSD, n = 3) of <3.6%, demonstrating the high analytical precision of this method. This result further confirms the reliability and feasibility of the chlorophyll carbon dot fluorescence method for the determination of methylene blue in actual water samples.

[0070] Table 1 Spike recovery results (n=5)

[0071]

[0072] Step 5. Take a sample from Chengbi Lake Reservoir in Youjiang District, Baise City as the sample to be tested, and measure the mixture of the sample to be tested and the chlorophyll carbon dot solution (specifically: take 100 μL of the sample to be tested and add it to a centrifuge tube, add 100 μL of chlorophyll carbon dots, 700 μL of PBS buffer (concentration of 100 mmol / L, pH = 7) and 100 μL of ultrapure water to the centrifuge tube in sequence, shake and mix evenly and keep in a water bath at 25°C for 5 minutes), measure the fluorescence intensity at a wavelength of 620-760 nm under an excitation wavelength of 405 nm, and obtain the content of methylene blue in the sample to be tested according to the linear relationship between the fluorescence intensity and the methylene blue concentration; wherein, the excitation slit and emission slit of the fluorescence detection are both 10 nm.

[0073] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiment. They can be applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized.

Claims

1. A method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots, characterized in that: The chlorophyll carbon dots are used as fluorescent probes, and the inner filter effect of methylene blue on the fluorescence quenching of chlorophyll carbon dots is utilized to detect the methylene blue content. The method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots comprises the following steps: Step 1: prepare a series of methylene blue standard solutions with different concentrations; Step 2: Each portion of the methylene blue standard solution is mixed with the chlorophyll carbon dot solution to obtain a series of standard solutions to be tested; the chlorophyll carbon dot solution includes chlorophyll carbon dots, PBS buffer and ultrapure water; Step 3: Measure the fluorescence spectrum of each standard solution to be tested at an excitation wavelength of 405 nm, record the fluorescence intensity at a wavelength of 620-760 nm, and establish a linear relationship between the fluorescence intensity and the methylene blue concentration; Step 4: Mix the sample to be tested with the chlorophyll carbon dot solution, measure the fluorescence intensity at a wavelength of 620-720 nm under an excitation wavelength of 405 nm, and obtain the methylene blue content in the sample to be tested based on the linear relationship between the fluorescence intensity and the methylene blue concentration; The preparation process of chlorophyll carbon dots includes: Step a, weighing washed lettuce leaves, chopping them and placing them in a beaker, adding a mixture of anhydrous ethanol and acetone in a volume ratio of 1:1 to the beaker, soaking the lettuce leaves for extraction for 30 minutes, and filtering to obtain a chlorophyll extract; Step b, placing the chlorophyll extract in a polytetrafluoroethylene autoclave, reacting with magnetic stirring at 180° C., cooling to room temperature after 6 hours, filtering, adjusting the pH to neutral, and then transferring to a dialysis bag, dialyzing with ultrapure water in a dark environment for 24 hours to obtain carbon dots; Step c: vacuum-dry the carbon dots and then prepare a 0.5 mg / mL carbon dot stock solution with ultrapure water.

2. The method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots according to claim 1, wherein: The preparation process of methylene blue standard solution in step 1 is specifically as follows: Prepare a 20 mg / mL methylene blue standard stock solution using methylene blue standard and ultrapure water; Different volumes of methylene blue standard stock solution were measured and diluted to the same volume with 100 mmol / L PBS buffer to obtain a series of methylene blue standard solutions with different concentrations.

3. The method for determining methylene blue based on the chlorophyll carbon quantum dot fluorescence inner filter effect according to claim 2, wherein: A series of methylene blue standard solutions with different concentrations includes 9 methylene blue standard solutions, and the concentrations are 0 μM, 0.1 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, and 12 μM, respectively.

4. The method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots according to claim 3, wherein: In step 2, in each standard solution to be tested, the volume of methylene blue standard solution is 100 μL, the volume of chlorophyll carbon dots is 100 μL, the volume of PBS buffer is 700 μL, and the volume of ultrapure water is 100 μL.

5. The method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots according to claim 4, wherein: The concentration of PBS buffer was 100 mmol / L and the pH was 7.

6. The method for determining methylene blue based on the fluorescence inner filter effect of chlorophyll carbon quantum dots according to claim 1, wherein: The excitation slit and emission slit for fluorescence detection in steps 3 and 4 are both 10 nm.

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