Method for detecting microplastics in water environment based on fluorescence spectrum

Abstract

The application discloses a method for detecting polystyrene microplastics in water environment based on fluorescence spectrum, and belongs to the technical field of environmental monitoring. The application adopts a laser-spectrometer to build a 90-degree lateral fluorescence collection light path, and utilizes the interaction between fluorescent dyes and polystyrene microplastics to generate fluorescence signal changes; by additionally adding a centrifugal-supernatant spectrum comparison true-false discrimination step, the light intensity false appearance caused by microplastic particle Mie scattering and the real molecular interaction between dyes-microplastics are distinguished; the application can be compatible with two types of fluorescent dyes, i.e., fluorescence quenching type and fluorescence enhancement type, and realizes quantitative detection of polystyrene microplastics through a standard curve. The application simultaneously discloses a corresponding detection system. The application solves the technical problems that the existing fluorescence detection of microplastics cannot distinguish scattering false signals, the background noise of the collinear light path is high, and there is a lack of true-false identification means, and does not need a fluorescence microscope, has high detection flux, and can be used for rapid screening of polystyrene microplastics in water bodies in a laboratory.

Description

Technical Field

[0001] This invention relates to the field of environmental pollutant monitoring technology, and in particular to a method for detecting polystyrene microplastics in aquatic environments based on fluorescence spectroscopy. Background Technology

[0002] Microplastics are a typical emerging pollutant in the aquatic environment. Polystyrene microplastics are widely used, have a high environmental detection rate, and pose a potential threat to aquatic ecosystems. Traditional detection methods for microplastics include Fourier transform infrared spectroscopy and Raman spectroscopy. These technologies require filtering and picking out individual particles, which is cumbersome and has low detection throughput.

[0003] Fluorescent staining is an important method for rapid screening of microplastics. Mainstream methods rely on fluorescence microscopy to observe individual stained particles, enabling qualitative observation, but this requires large sample volumes and is unsuitable for rapid testing of large batches of suspension samples. A bulk fluorescence testing system built using a laser and a miniature spectrometer can directly acquire spectra of microplastic suspensions without the need for a fluorescence microscope, possessing the potential for high-throughput rapid detection.

[0004] Current bulk fluorescence detection techniques for microplastics have significant drawbacks: First, solid microplastic particles in suspension undergo Mie scattering, which alters the detected fluorescence intensity. Increases or decreases in the spectral signal may originate from genuine interactions between the dye and microplastic molecules, or simply from light loss due to particle scattering; existing techniques lack simple and effective methods for identifying genuine and false results, easily leading to false positives and false negatives. Second, many self-made optical detection devices employ a 0-degree collinear transmission acquisition mode. Filters can only remove the original excitation wavelength laser, failing to eliminate background noise from water molecule Raman scattering, easily drowning out the weak dye response signal. Third, most current research focuses only on fluorescence-enhancing dyes, neglecting the application potential of fluorescence-quenching dyes; a complete baseline control scheme is lacking, and solvent and dye background interference cannot be effectively subtracted. Fourth, some dye stock solutions are prepared with acetone, which can swell or even destroy polystyrene microplastic particles, altering the true state of the sample.

[0005] In summary, there is an urgent need to develop a bulk fluorescence spectroscopy method for detecting polystyrene microplastics that can distinguish scattering pseudo-signals, has a high signal-to-noise ratio, and is compatible with multiple dyes. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting polystyrene microplastics in the aquatic environment based on bulk fluorescence spectroscopy. This method solves the problems of bulk fluorescence detection being unable to distinguish between Mie scattering pseudo-signals and the real interaction between dyes and microplastics, as well as the high background noise in collinear optical paths. It is compatible with both fluorescence quenching and fluorescence enhancement dyes, enabling high-throughput screening and quantification of polystyrene microplastics in aquatic environments. This invention provides a method for detecting polystyrene microplastics in an aquatic environment based on bulk fluorescence spectroscopy, comprising the following steps: S1. Construct a 90-degree lateral fluorescence acquisition optical path; the optical path includes an excitation laser, a quartz cuvette, a long-pass filter, and a spectrometer; the laser beam is incident on the quartz cuvette, the spectrometer acquisition probe is positioned 90 degrees to the side of the laser beam, and the long-pass filter is placed on the light-incident side of the spectrometer probe; three sets of baseline spectra are acquired: solvent blank spectrum, free dye control spectrum, and intrinsic spectrum of bare polystyrene microplastics; S2. Prepare the test mixture; add the fluorescent dye to the aqueous solution containing polystyrene microplastics; the dye stock solution is prepared with anhydrous ethanol, and the final volume fraction of anhydrous ethanol in the test system is ≤1%; after vortex mixing, quickly acquire the spectrum. S3. Sample Spectrum Acquisition and Baseline Correction: Acquire the fluorescence spectrum of the mixture to be tested, and subtract it from the baseline spectrum obtained in S1 to obtain the corrected sample fluorescence spectrum. S4. Authenticity and authenticity determination steps: distinguish between Mie scattering spurious signals and the real interaction between dye and polystyrene microplastics; centrifuge the test mixture system after spectral acquisition at high speed to allow the polystyrene microplastics to settle completely, and collect the supernatant without microplastic particles; collect the fluorescence spectrum of the supernatant and compare it with the free dye control spectrum; If the fluorescence intensity of the supernatant is significantly lower than that of the free dye control spectrum, it is determined that the dye has undergone real molecular interaction with the polystyrene microplastics, and the change in the spectral signal is an effective detection signal. If the fluorescence intensity of the supernatant is basically consistent with the free dye control spectrum, then the change in spectral intensity is determined to be due to the loss of Mie scattering light from the polystyrene microplastic particles, and this set of signals is an invalid pseudo signal. S5. Quantitative detection of microplastics: Prepare multiple sets of polystyrene microplastic standard solutions with different concentrations, repeat steps S2-S4, screen out effective detection signals, and establish a standard curve of fluorescence signal change versus polystyrene microplastic concentration; substitute the effective signal of the sample after authenticity verification into the standard curve, and output the polystyrene microplastic concentration in the sample.

[0007] Furthermore, the cutoff wavelength of the long-pass filter in S1 is 20 nm or more greater than the laser emission wavelength, and is used to filter out Rayleigh scattering light.

[0008] Furthermore, the centrifugation conditions in S4 are: rotation speed 8000-12000 r / min, centrifugation time 8-15 min.

[0009] Furthermore, when the fluorescent dye is a fluorescence quenching dye, the decrease in fluorescence intensity is used as the quantitative basis; when the fluorescent dye is a fluorescence enhancing dye, the increase in fluorescence intensity is used as the quantitative basis.

[0010] Furthermore, acetone solvent is prohibited in the dye mother liquor of S2 to avoid swelling and damage of polystyrene microplastic particles by acetone.

[0011] Furthermore, during the S2-S3 spectral acquisition process, when particle sedimentation occurs in the sample, it is vortexed and mixed again before detection, which shortens the spectral integration acquisition time and reduces signal deviation caused by dye photobleaching.

[0012] This invention also provides a detection system for detecting polystyrene microplastics in aquatic environments based on bulk fluorescence spectroscopy, used to perform the above-described detection method, comprising: Optical acquisition unit: including excitation laser, quartz cuvette, long-pass filter, and spectrometer, used to build a 90° lateral fluorescence acquisition optical path to acquire baseline spectrum and sample fluorescence spectrum; Pretreatment unit: includes a high-speed centrifuge for settling polystyrene microplastics in the authenticity identification step to obtain a particle-free supernatant; Data processing unit: Built-in baseline correction module, authenticity discrimination module, and quantitative fitting module; the baseline correction module is used for baseline spectral subtraction; the authenticity discrimination module compares the output signal of the supernatant spectrum with that of the free dye control spectrum to determine the validity of the result; the quantitative fitting module stores the standard curve and outputs the microplastic concentration of the sample to be tested; The interactive display unit is used to show the original spectrum, the corrected spectrum, the authenticity judgment results, and the output value of polystyrene microplastic concentration.

[0013] Furthermore, the data processing unit is deployed on computer equipment or embedded edge computing devices that have undergone quantization and compression.

[0014] In summary, the method for detecting microplastics in the aquatic environment based on fluorescence spectroscopy of the present invention has the following advantages compared with traditional technologies: 1. This invention employs a 90° lateral fluorescence acquisition optical path, which avoids direct laser light from entering the detector from the source of the optical path, significantly reducing the background noise of water molecule Raman scattering and improving the signal-to-noise ratio of weak fluorescence signals; 2. An additional step for authenticity determination by comparing the spectra of centrifugation and supernatant can effectively distinguish between the light intensity artifacts caused by Mie scattering of particles and the real molecular interactions of dyes and microplastics, thus avoiding false positives and false negatives. 3. It is compatible with both fluorescence-quenching dyes and fluorescence-enhancing dyes, thus broadening the dye screening range; it does not require a fluorescence microscope and can directly perform bulk phase spectroscopy tests on suspensions, resulting in high detection throughput; 4. Limit the solvent of the dye mother liquor to avoid the problem of acetone swelling damaging polystyrene microplastics and ensure the authenticity of the samples; 5. The entire device has simple hardware and low setup cost. After establishing a standard curve, it can perform quantitative screening of polystyrene microplastics in water.

[0015] The technical method of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the 90° lateral fluorescence acquisition optical path structure of the present invention; Figure 2 This is a comparison of the measured fluorescence spectra of R640 dye in Example 1 of the present invention.

Claims

1. A method for detecting polystyrene microplastics in an aquatic environment based on bulk fluorescence spectroscopy, characterized in that, Includes the following steps: S1. Construct a 90-degree lateral fluorescence acquisition optical path; the optical path includes an excitation laser, a quartz cuvette, a long-pass filter, and a spectrometer; the laser beam is incident on the quartz cuvette, the spectrometer acquisition probe is positioned 90 degrees to the side of the laser beam, and the long-pass filter is placed on the light-incident side of the spectrometer probe; three sets of baseline spectra are acquired: solvent blank spectrum, free dye control spectrum, and intrinsic spectrum of bare polystyrene microplastics; S2. Prepare the test mixture; add the fluorescent dye to the aqueous solution containing polystyrene microplastics; the dye stock solution is prepared with anhydrous ethanol, and the final volume fraction of anhydrous ethanol in the test system is ≤1%; after vortex mixing, quickly acquire the spectrum. S3. Sample Spectrum Acquisition and Baseline Correction: Acquire the fluorescence spectrum of the mixture to be tested, and subtract it from the baseline spectrum obtained in S1 to obtain the corrected sample fluorescence spectrum. S4. Authenticity and authenticity determination steps: distinguish between Mie scattering spurious signals and the real interaction between dye-polystyrene microplastics. The test mixture system, after completing the spectral acquisition, was centrifuged at high speed to allow the polystyrene microplastics to settle completely. The supernatant containing no microplastic particles was then collected. The fluorescence spectrum of the supernatant was collected and compared with the free dye control spectrum. If the fluorescence intensity of the supernatant is significantly lower than that of the free dye control spectrum, it is determined that the dye has undergone real molecular interaction with the polystyrene microplastics, and the change in the spectral signal is an effective detection signal. If the fluorescence intensity of the supernatant is basically consistent with the free dye control spectrum, then the change in spectral intensity is determined to be due to the loss of Mie scattering light from the polystyrene microplastic particles, and this set of signals is an invalid pseudo signal. S5. Quantitative detection of microplastics: Prepare multiple sets of polystyrene microplastic standard solutions with different concentrations, repeat steps S2-S4, screen out effective detection signals, and establish a standard curve of fluorescence signal change versus polystyrene microplastic concentration; substitute the effective signal of the sample after authenticity verification into the standard curve, and output the polystyrene microplastic concentration in the sample.

2. The method according to claim 1, characterized in that, The cutoff wavelength of the long-pass filter in S1 is 20 nm or more greater than the emission wavelength of the laser, and is used to filter out Rayleigh scattering light.

3. The method according to claim 1, characterized in that, The centrifugation conditions in S4 are: rotation speed 8000-12000 r / min, centrifugation time 8-15 min.

4. The method according to claim 1, characterized in that, When the fluorescent dye is a fluorescence quenching dye, the decrease in fluorescence intensity is used as the quantitative basis; when the fluorescent dye is a fluorescence enhancing dye, the increase in fluorescence intensity is used as the quantitative basis.

5. The method according to claim 1, characterized in that, Acetone solvent is prohibited in the dye mother liquor of S2 to avoid swelling and damage of polystyrene microplastic particles by acetone.

6. The method according to claim 1, characterized in that, During the S2-S3 spectral acquisition process, if particle sedimentation occurs in the sample, it is vortexed and mixed again before detection to shorten the spectral integration acquisition time and reduce signal deviation caused by dye photobleaching.

7. A detection system for polystyrene microplastics in an aquatic environment based on fluorescence detection, characterized in that, For performing the detection method according to any one of claims 1-6, comprising: Optical acquisition system: including excitation laser, quartz cuvette, long-pass filter, and spectrometer, used to build a 90-degree lateral fluorescence optical path to acquire baseline spectrum and sample fluorescence spectrum; Pretreatment unit: includes a high-speed centrifuge for settling polystyrene microplastics in the authenticity identification step to obtain a particle-free supernatant; Data processing unit: Built-in baseline correction module, authenticity discrimination module, and quantitative fitting module; the baseline correction module is used for baseline spectral subtraction; the authenticity discrimination module compares the output signal of the supernatant spectrum with that of the free dye control spectrum to determine the validity of the result; the quantitative fitting module stores the standard curve and outputs the microplastic concentration of the sample to be tested; The interactive display unit is used to show the original spectrum, the corrected spectrum, the authenticity judgment results, and the output value of polystyrene microplastic concentration.

8. The detection system according to claim 7, characterized in that, The data processing unit is deployed on computer equipment or embedded edge computing devices that have undergone quantization and compression.

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