Microplastic fluorescence detection method based on carbon quantum dots
By mixing activated sludge-based carbon quantum dots with microplastics in an aqueous buffer solution, the efficiency and stability issues of fluorescent labeling of microplastics in existing technologies have been solved, achieving efficient and stable labeling of different microplastics, which is suitable for detection in complex environmental matrices.
Patent Information
- Application Number
- CN202511986909.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-02-27
AI Technical Summary
Existing microplastic fluorescent labeling technologies suffer from problems such as the use of organic solvents, low labeling efficiency, poor stability, and limited practical applications. In particular, the labeling efficiency varies greatly for different polymer types, and its applicability in complex environmental matrices needs to be verified.
Activated sludge-based carbon quantum dots were mixed with the water sample in an aqueous buffer solution. Fluorescent labeling was performed by adjusting the pH and temperature. Qualitative and quantitative analysis was then conducted using a fluorescence microscope or spectrometer. This method avoids the use of organic solvents and achieves efficient and stable labeling of different microplastics.
A green, rapid, and stable fluorescent labeling method for microplastics has been developed, which is applicable to a variety of microplastics and real-world environments. It exhibits good labeling effects and tolerance, and is suitable for the direct detection of environmental samples.
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Figure CN121577599A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of environmental detection and material science, and relates to a microplastic fluorescence detection method based on carbon quantum dots, in particular to a microplastic fluorescence labeling and detection method, and particularly relates to a method for efficiently and stably labeling common microplastics such as polypropylene (PP), polyethylene (PE) and polystyrene (PS) by using activated sludge-based carbon quantum dots and application of the method in environmental water sample detection. BACKGROUND
[0002] As a new type of environmental pollutant, microplastics (particle size <5 mm) have been widely distributed in water, soil and atmosphere, and pose potential threats to the ecological system and human health. Therefore, it is crucial to develop rapid, accurate and convenient microplastic detection technologies. At present, the detection methods for microplastics mainly include spectroscopic methods (such as Fourier transform infrared spectroscopy, Raman spectroscopy), thermal analysis methods and optical microscopic observation methods. Among them, the fluorescence labeling method based on optical microscopic observation method is concerned due to its high sensitivity, simple operation and strong visualization ability. Nile Red is a commonly used microplastic fluorescence labeling dye, but its staining process usually requires the use of organic solvents such as acetone, which is not environmentally friendly and has poor biocompatibility. In recent years, as a new type of fluorescent nanomaterial, carbon quantum dots (CQDs) have shown great potential in sensing and labeling fields due to their excellent optical stability, low toxicity, good biocompatibility and adjustable optical properties.
[0003] However, there are still challenges in applying carbon quantum dots to the fluorescence labeling of microplastics: first, the existing labeling methods have large differences in labeling efficiency for different polymer types (such as PP, PE and PS), and lack a universal and efficient labeling system; second, the stability of the labeling process is easily affected by environmental conditions (such as pH, temperature and salinity); third, the applicability in complex actual environmental matrices (such as river water and tap water) needs to be verified.
[0004] Therefore, it is of great practical significance to develop a green, rapid, stable and applicable fluorescence labeling method for various microplastics and actual environments. SUMMARY
[0005] In order to overcome the defects of the existing microplastic fluorescence labeling technology such as the use of organic solvents, low labeling efficiency, poor stability and limited actual application, the present application provides a microplastic fluorescence labeling method which is efficient, stable, environmentally friendly and applicable to actual water sample detection.
[0006] To achieve the above purpose, the technical scheme adopted by the present application is as follows:
[0007] A microplastic fluorescence detection method based on carbon quantum dots, the method comprises the following steps:
[0008] Firstly, the water sample to be measured is pretreated;
[0009] The collected water sample to be measured is filtered through a filter membrane to remove large particle impurities;
[0010] Secondly, the filtered water sample to be measured is adjusted to pH 6.5-8.5 using a buffer solution as a labeling medium;
[0011] Thirdly, the water sample to be measured after the second step is labeled with fluorescence;
[0012] The water sample to be measured and the carbon quantum dot solution are mixed in a buffer system and reacted at 25-45°C and pH 5.0-9.0 for 0.25-24 hours to complete the fluorescence labeling; wherein the carbon quantum dots are activated sludge-based carbon quantum dots.
[0013] Fourthly, the water sample to be measured after the third step is subjected to solid-liquid separation, the separated solid material is dispersed in water again, and the fluorescence microscope or fluorescence spectrometer is used for observation and fluorescence intensity determination to realize the qualitative and quantitative analysis of microplastics in the environmental water sample.
[0014] Further, the microplastics detected in the water sample to be measured include at least one of polypropylene PP, polyethylene PE or polystyrene PS, that is, when the water sample to be measured contains at least one of the above three kinds of microplastics, the fluorescence intensity can be displayed.
[0015] Further, the buffer solution in the second step is a sodium citrate-hydrochloric acid buffer, a phosphate buffer or a Tris-HCl buffer.
[0016] Further, the buffer solution in the third step is a sodium citrate-hydrochloric acid buffer, a phosphate buffer or a Tris-HCl buffer.
[0017] Further, the carbon quantum dots in the third step are prepared by hydrothermal method from activated sludge. The specific preparation method is as follows: dry activated sludge is mixed with water, and hydrothermal reaction is carried out at 180-220°C for 6-12 hours, the reaction product is centrifuged and filtered to obtain a clear solution, and the solution is diluted 10 times to obtain the carbon quantum dot solution used for labeling. The preparation method has the advantages of easy raw material and green environmental protection.
[0018] Further, in the third step, 0.1 milliliter of carbon quantum dot solution is added to every 4.9 milliliters of water sample to be measured.
[0019] Compared with the prior art, the beneficial effects of the present application are as follows:
[0020] (1) The carbon quantum dots used in this invention are prepared from activated sludge, realizing the resource utilization of waste; the labeling process is carried out in an aqueous buffer (sodium citrate-hydrochloric acid buffer, phosphate buffer or Tris-HCl buffer), avoiding the use of organic solvents in methods such as Nile Red, which is more in line with the concept of green chemistry.
[0021] (2) The labeling system established by the present invention can realize fluorescent labeling of microplastics with different properties and has good labeling effect, which solves the problem of large difference in labeling efficiency of single dyes on different plastics.
[0022] (3) Experiments of the present invention have confirmed that the method can maintain good labeling performance in actual water samples, has strong tolerance to complex environmental matrices, and has the potential to be directly applied to the detection of microplastic fluorescent labeling in environmental samples. Attached Figure Description
[0023] Figure 1 This is a fluorescence microscope image of microplastics in river water labeled with carbon quantum dots in Example 1 of the present invention.
[0024] Figure 2 This is a fluorescence microscope image of tap water labeled with carbon quantum dots in Example 2 of the present invention.
[0025] Figure 3 The images show transmission electron microscopy (TEM) images and particle size distribution diagrams of the carbon quantum dots prepared in Example 2 of this invention. Figure 1 Image A in the image is a transmission electron microscope (TEM) image of carbon quantum dots. Figure 1 B in the diagram represents the particle size distribution of carbon quantum dots.
[0026] Figure 4 This is a fluorescence microscope image of microplastics labeled with carbon quantum dots in pure water in Example 3 of the present invention.
[0027] Figure 5 The images show the effects of PP, PE, and PS microplastic labeling under different pH conditions in Example 4 of this invention.
[0028] Figure 6 The images show the marking effects of PP, PE, and PS microplastics at different reaction times in Example 5 of this invention. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions will be clearly and completely described below in conjunction with embodiments of this invention. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.
[0030] Example 1
[0031] A method for detecting fluorescence in microplastics based on carbon quantum dots, the method comprising the following steps:
[0032] The first step is to filter the collected river water through a filter membrane to remove large particulate impurities;
[0033] The second step is to adjust the pH of the filtered environmental water sample to 6.5 using a buffer solution as a labeling medium; in this embodiment, the buffer solution is sodium citrate-hydrochloric acid buffer.
[0034] The third step is to apply fluorescent labeling to the water sample after the second step.
[0035] The water sample to be tested was mixed with carbon quantum dot solution in a buffer system and reacted at 25°C and pH 5.0 for 0.25 hours to complete the fluorescent labeling; wherein, the carbon quantum dots are activated sludge-based carbon quantum dots. The buffer system in this embodiment is sodium citrate-hydrochloric acid buffer solution, and 0.1 ml of carbon quantum dot solution was added to 4.9 ml of the water sample to be tested.
[0036] The fourth step involves solid-liquid separation of the environmental water sample solution processed in the third step. The separated solids are then redispersed in water, and observed and their fluorescence intensity measured using a fluorescence microscope or fluorescence spectrometer to achieve qualitative and quantitative analysis of microplastics in the environmental water sample. The results are as follows: Figure 1 The fluorescence microscope showed obvious green fluorescent spots (circled in red), indicating that the river water contained at least one of polypropylene (PP), polyethylene (PE), or polystyrene (PS).
[0037] In this embodiment, the carbon quantum dots in the third step are prepared from activated sludge via a hydrothermal method. Specifically, the preparation method involves mixing dried activated sludge with water and subjecting the mixture to a hydrothermal reaction at 180°C for 6 hours. After centrifugation and filtration, a clear solution is obtained. This solution is then diluted 10 times and used as the carbon quantum dot solution for labeling. This preparation method utilizes readily available raw materials and is environmentally friendly.
[0038] Example 2
[0039] A method for detecting fluorescence in microplastics based on carbon quantum dots, the method comprising the following steps:
[0040] The first step is to filter the collected tap water through a filter membrane to remove large particulate impurities;
[0041] The second step is to adjust the pH of the filtered environmental water sample to 7.5 using a buffer solution as a labeling medium; in this embodiment, the buffer solution is a phosphate buffer solution.
[0042] The third step is to apply fluorescent labeling to the water sample after the second step.
[0043] The water sample to be tested was mixed with carbon quantum dot solution in a buffer system and reacted at 35°C and pH 7.0 for 10 hours to complete the fluorescent labeling; wherein, the carbon quantum dots were activated sludge-based carbon quantum dots. In this embodiment, the buffer system was phosphate buffer solution, and 0.1 g of carbon quantum dot solution was added to 4.9 mL of the water sample to be tested.
[0044] The fourth step involves solid-liquid separation of the environmental water sample solution processed in the third step. The separated solids are then redispersed in water, and observed and their fluorescence intensity measured using a fluorescence microscope or fluorescence spectrometer to achieve qualitative and quantitative analysis of microplastics in the environmental water sample. The results are as follows: Figure 2 The results show that obvious green fluorescent spots (circled in red) can be seen under a fluorescence microscope, indicating that the tap water contains at least one of polypropylene (PP), polyethylene (PE), or polystyrene (PS).
[0045] In this embodiment, the carbon quantum dots in the third step are prepared from activated sludge via a hydrothermal method. The specific preparation method is as follows: dried activated sludge is mixed with water and subjected to a hydrothermal reaction at 200°C for 10 hours. The reaction product is centrifuged and filtered to obtain a clear solution. This solution is then diluted 10 times and used as the carbon quantum dot solution for labeling. This preparation method uses readily available raw materials and is environmentally friendly. Characterization showed that the aqueous solution of these carbon quantum dots exhibits the strongest fluorescence emission peak at 410 nm under 340 nm excitation light. Figure 3 As shown in Figure A, the carbon quantum dots prepared in this invention are spherical particles. Their particle size is statistically analyzed, as follows... Figure 3 B shows that its average particle size is 5 nm, which is consistent with the size of classic carbon quantum dots.
[0046] Example 3
[0047] A method for detecting fluorescence in microplastics based on carbon quantum dots, the method comprising the following steps:
[0048] The first step is to filter the purified water through a filter membrane to remove large particulate impurities;
[0049] The second step is to adjust the pH of the filtered environmental water sample to 8.5 using a buffer solution as a labeling medium; in this embodiment, the buffer solution is Tris-HCl buffer solution.
[0050] The third step is to apply fluorescent labeling to the water sample after the second step.
[0051] The water sample to be tested was mixed with carbon quantum dot solution in a buffer system and reacted at 45°C and pH 9.0 for 24 hours to complete the fluorescent labeling; wherein, the carbon quantum dots were activated sludge-based carbon quantum dots. The buffer system in this embodiment was Tris-HCl buffer solution, and 0.1 ml of carbon quantum dot solution was added to 4.9 ml of the water sample to be tested.
[0052] The fourth step involves solid-liquid separation of the environmental water sample solution processed in the third step. The separated solids are then redispersed in water, and observed and their fluorescence intensity measured using a fluorescence microscope or fluorescence spectrometer to achieve qualitative and quantitative analysis of microplastics in the environmental water sample. The results are as follows: Figure 4 The results show that obvious green fluorescent spots (circled in red) can be seen under a fluorescence microscope, indicating that the purified water contains at least one of polypropylene (PP), polyethylene (PE), or polystyrene (PS), but in less quantity than the river water sample in Example 1 and the tap water sample in Example 2.
[0053] In this embodiment, the carbon quantum dots in the third step are prepared from activated sludge via a hydrothermal method. Specifically, the preparation method involves mixing dried activated sludge with water and subjecting the mixture to a hydrothermal reaction at 220°C for 12 hours. The reaction product is then centrifuged and filtered to obtain a clear carbon quantum dot solution. This preparation method utilizes readily available raw materials and is environmentally friendly.
[0054] Example 4: Labeling effect under acidic pH conditions
[0055] Accurately weigh the appropriate masses of sodium citrate, disodium hydrogen phosphate, and tris(hydroxymethyl)aminomethane, and dissolve them in deionized water to prepare corresponding solutions. The final concentrations of the three solutions are 0.1 M, 10 mM, and 10 mM, respectively. Adjust the pH of the above solutions with 1 M hydrochloric acid aqueous solution to prepare buffer systems with pH values of 5.5, 6.5, and 6.8.
[0056] Add 0.02 g of PE, PP, or PS microplastics and 0.1 mL of the carbon quantum dot solution prepared in Example 1 (diluted 10 times) to 4.9 mL of each pH buffer solution, and mix thoroughly. Place the mixture in a 35 °C constant temperature water bath and react in the dark for 1 h. After the reaction is complete, collect the solid by filtration and redisperse it in 5 mL of deionized water. Take 100 μL of the suspension on a glass slide, observe and photograph it under a fluorescence microscope, and analyze the fluorescence intensity.
[0057] The results are as follows Figure 2 As shown, fluorescent labeling of microplastics with carbon quantum dots can be achieved under pH conditions ranging from 5.5 to 6.8. Analysis of the fluorescence intensity values reveals no significant difference in labeling efficiency for the three types of microplastics.
[0058] Example 5: Labeling effect under alkaline pH conditions
[0059] Accurately weigh the appropriate masses of sodium citrate, disodium hydrogen phosphate, and tris(hydroxymethyl)aminomethane, and dissolve them in deionized water to prepare corresponding solutions. The final concentrations of the three solutions are 0.1 M, 10 mM, and 10 mM, respectively. Adjust the pH of the above solutions with 1 M sodium hydroxide aqueous solution to prepare buffer systems with pH values of 7.3, 7.8, 8.0, 8.5, and 9.0.
[0060] Add 0.02 g of PE, PP, or PS microplastics and 0.1 mL of the carbon quantum dot solution prepared in Example 1 (diluted 10 times) to 4.9 mL of each pH buffer solution, and mix thoroughly. Place the mixture in a 35 °C constant temperature water bath and react in the dark for 1 h. After the reaction is complete, collect the solid by filtration and redisperse it in 5 mL of deionized water. Take 100 μL of the suspension on a glass slide, observe and photograph it under a fluorescence microscope, and analyze the fluorescence intensity.
[0061] The results are as follows Figure 2 As shown, fluorescent labeling of microplastics with carbon quantum dots can be achieved under pH conditions ranging from 7.3 to 9.0. Analysis of the fluorescence intensity values reveals no significant difference in labeling efficiency for the three types of microplastics.
[0062] Example 6: Marking effect at different reaction temperatures
[0063] Prepare a buffer solution with a pH of 6.5 as described in Example 2. Add 0.02 g of PE, PP, or PS microplastics and 0.1 mL of the carbon quantum dot solution prepared in Example 1 (diluted 10 times) to 4.9 mL of the buffer solution, and mix thoroughly. Place the mixture in a constant temperature water bath at 25, 35, and 45°C and react in the dark for 1 h. After the reaction is complete, collect the solid by filtration and redisperse it in 5 mL of deionized water. Take 100 μL of the suspension on a glass slide, observe and photograph it under a fluorescence microscope, and analyze the fluorescence intensity.
[0064] The results are shown in Table 1 below. Carbon quantum dots can be used to fluorescently label three types of microplastics at temperatures of 25, 35, and 45°C.
[0065] Table 1: Fluorescence intensity of three microplastics at different temperatures at pH=6.5
[0066]
[0067] Example 7: Marking effect at different reaction times
[0068] Prepare a buffer solution with a pH of 6.5 as described in Example 2. Add 0.02 g of PE, PP, or PS microplastics and 0.1 mL of the carbon quantum dot solution prepared in Example 1 (diluted 10 times) to 4.9 mL of the buffer solution, and mix thoroughly. Place the mixture in a 35 °C constant temperature water bath and react in the dark for 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. After the reaction is complete, collect the solid by filtration and redisperse it in 5 mL of deionized water. Take 100 μL of the suspension on a glass slide, observe and photograph it under a fluorescence microscope, and analyze the fluorescence intensity.
[0069] The results are as follows Figure 3 As shown, carbon quantum dots can be used to fluorescently label three microplastics at reaction times of 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. The labeling efficiency is optimal within 1 h, indicating that the method established in this invention can rapidly achieve carbon quantum dot fluorescent labeling of three microplastics.
[0070] Example 8: Application in actual environmental water samples
[0071] River water and tap water were collected and filtered through a 0.22 μm filter membrane. Buffer solutions with pH values of 6.5, 7.8, and 8.5 were prepared using the filtered water samples. Spiked recovery experiments were conducted using these buffer solutions as the labeling medium: 0.02 g of PE, PP, or PS microplastics and 0.1 mL of a carbon quantum dot solution prepared in Example 1 (diluted 10 times) were added to 4.9 mL of the above water sample buffer solution, and the mixture was reacted at 35°C in the dark for 1 h. A deionized water system was used as a control. The spiked recovery rate was calculated by comparing the fluorescence intensity of the actual water sample and the control sample.
[0072] The results are shown in Table 2. In river water and tap water substrates, the spiked recoveries of the three microplastics by this method were all between 93.95% and 104.63%, indicating that common impurities in actual water samples have little interference with the labeling method of this invention. This method can be directly used for the qualitative and semi-quantitative detection of microplastics in environmental water samples.
[0073] Table 2: Labeling efficiency of three microplastics in tap water and river water at different pH levels
[0074]
[0075] The above embodiments are merely illustrative of the implementation methods of the present invention, but should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the protection scope of the present invention.
Claims
1. A method for detecting fluorescence in microplastics based on carbon quantum dots, characterized in that, The method includes the following steps: The first step is to pre-treat the water sample to be tested; The second step is to adjust the pH of the water sample to be tested to 6.5-8.5 using a buffer solution as a labeling medium; The third step is to apply fluorescent labeling to the water sample after the second step. The water sample to be tested was mixed with a carbon quantum dot solution in a buffer system and reacted at 25-45°C and pH 5.0-9.0 for 0.25-24 hours to complete the fluorescent labeling; wherein the carbon quantum dots were activated sludge-based carbon quantum dots. The fourth step involves solid-liquid separation of the water sample solution processed in the third step, redispersing the separated solids in the water, and observing and measuring the fluorescence intensity using a fluorescence microscope or fluorescence spectrometer to achieve microplastic analysis in environmental water samples.
2. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 1, characterized in that, In the first step, pretreatment refers to filtering the water sample to be tested through a filter membrane.
3. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 1, characterized in that, In the first step, the detectable microplastics in the water sample to be tested include at least one of polypropylene (PP), polyethylene (PE), or polystyrene (PS). That is, when the water sample to be tested contains at least one of the above three microplastics, it can be displayed by fluorescence intensity.
4. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 1, characterized in that, The buffer solution in the second step is sodium citrate-hydrochloric acid buffer, phosphate buffer, or Tris-HCl buffer.
5. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 1, characterized in that, The buffer solution in the third step is sodium citrate-hydrochloric acid buffer, phosphate buffer, or Tris-HCl buffer.
6. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 1, characterized in that, The carbon quantum dots in the third step are prepared from activated sludge via a hydrothermal method.
7. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 6, characterized in that, The specific method for preparing carbon quantum dots from activated sludge via hydrothermal reaction is as follows: dry activated sludge is mixed with water and subjected to a hydrothermal reaction at 180-220°C for 6-12 hours. After centrifugation and filtration, a clear solution is obtained. The solution is then diluted 10 times and used as a carbon quantum dot solution for labeling.
8. The method for detecting fluorescence in microplastics based on carbon quantum dots according to claim 7, characterized in that, In the third step, 0.1 ml of carbon quantum dot solution is added to every 4.9 ml of water sample to be tested.