A high-throughput single-particle microplastic detection method based on raman flow spectroscopy technology

Abstract

The application discloses a high-throughput single-particle microplastic detection method based on a Raman flow spectrum technology, and belongs to the technical field of environmental pollutant detection. The method comprises the following steps: preparing microplastic microsphere standard solutions with different particle sizes and materials; sending the standard solutions and water solutions of to-be-detected samples into a Raman flow cytometer; keeping the solutions in a flowing state and performing Raman spectrum detection on the microplastic particles one by one; and finally determining the material, particle size and concentration of the microplastics according to characteristic peak signals, peak intensities and the number of spectrum diagrams per unit time. The application can directly detect water solutions without filtering, enriching and complex pretreatment, and realizes multi-dimensional synchronous analysis of microplastics at a high-throughput single-particle level.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollutant detection technology, specifically relating to a high-throughput method for detecting single-particle microplastics based on Raman flow cytometry. Background Technology

[0002] Microplastics refer to plastic particles with a size between 1 μm and 5 mm. Due to the mass production and use of plastic products, coupled with the relatively stable chemical structure of plastics themselves, microplastics are now widely present in various environmental media such as water, soil, and atmosphere. As a new pollutant, microplastic pollution has become a significant global environmental problem, and its potential threats to ecosystems and human health are receiving increasing attention. Therefore, accurate detection of microplastics in the environment has become particularly important.

[0003] Currently, the main methods for detecting microplastics include pyrolysis gas chromatography-mass spectrometry (PCC-MS), Fourier transform infrared spectroscopy (FTIR), and confocal Raman spectroscopy (DRA). PCC-MS can detect the material and mass concentration of a sample, but it cannot obtain information on the size and particle concentration of microplastics. FTIR can detect the material and size of microplastics; however, due to the diffraction limit of infrared spectroscopy, it can only detect microplastic particles larger than 20 μm, making it difficult to effectively identify smaller particles. DRA, compared to FTIR, can detect microplastic particles smaller than 20 μm and has higher spatial resolution; however, DRA has low throughput, slow detection speed, and complex sample pretreatment, making it difficult to meet the high-throughput, rapid detection requirements for large numbers of microplastic particles in environmental samples.

[0004] Therefore, there is an urgent need in this field to develop a method that can achieve high-throughput, single-particle-level detection of microplastics, so as to simultaneously obtain multi-dimensional information such as the material, particle size and concentration of microplastics, and is simple to operate and does not require complicated sample pretreatment. Summary of the Invention

[0005] This invention provides a high-throughput single-particle microplastic detection method based on Raman flow cytometry to solve the technical problems of low detection throughput, inability to simultaneously obtain multi-dimensional information on material, particle size, and concentration, and complex sample pretreatment in existing microplastic detection technologies.

[0006] To solve the above-mentioned technical problems, the technical solution proposed in this application is as follows:

[0007] This invention provides a high-throughput method for detecting single-particle microplastics based on Raman flow cytometry, characterized by comprising the following steps: S1: Standard aqueous solutions for preparing microplastic microspheres of different particle sizes and materials; S2: Prepare standard aqueous solutions of microplastic microspheres at different concentrations; S3: The standard aqueous solutions of microplastic microspheres with different particle sizes and materials prepared in step S1, the standard aqueous solutions of microplastic microspheres with different concentrations prepared in step S2, and the aqueous solution of the sample to be tested are directly fed into a Raman flow cytometer. In the Raman flow cytometer, each aqueous solution is kept in a flowing state. The microplastic particles flowing through each aqueous solution are focused and Raman spectroscopically detected one by one without filtration and enrichment. The Raman spectrum of each sample is collected in a unit time. The injection flow rate of the Raman flow cytometer is 0.1 μL / min to 40 μL / min. S4: Based on the characteristic peak signals, characteristic peak intensities, and number of spectra per unit time in the Raman spectrum collected in step S3, determine the material, particle size, and concentration of microplastics in the sample to be tested.

[0008] Furthermore, in step S1, the particle size of the microplastic microspheres is below 40 μm.

[0009] Further, in step S1, the particle size of the microplastic microspheres is selected from at least one of 1μm, 2μm, 5μm, 10μm, 20μm and 40μm.

[0010] Further, in step S1, the microplastic microspheres of different materials are selected from at least one of polystyrene microspheres, polyvinyl chloride microspheres, polymethyl methacrylate microspheres, polyethylene terephthalate microspheres, and polylactic acid microspheres.

[0011] Furthermore, in step S2, the concentration range of the different concentrations of the microplastic microsphere standard aqueous solution is below 100,000 particles / mL.

[0012] Further, in step S2, the concentration of the different concentrations of the microplastic microsphere standard aqueous solution is selected from at least one of 0 particles / mL, 5000 particles / mL, 10000 particles / mL, 50000 particles / mL and 100000 particles / mL.

[0013] Further, in step S3, the detection parameters of the Raman flow cytometer are set as follows: a 532nm Raman laser, a 5x to 100x Raman acquisition lens, a Raman exposure time of 0.01s to 1s, and an acquisition wavenumber range of 100cm. - ¹Up to 3500cm - ¹, The sample collection time is 10 min.

[0014] Furthermore, in step S3, the Raman acquisition lens of the Raman flow cytometer is a 50x lens, the Raman exposure time is 0.1s, and the injection flow rate is 1μL / min.

[0015] Further, step S4 includes the following sub-steps: S41: Based on the Raman spectrum collected in step S3, extract the signal intensity at the Raman shift of the characteristic peak corresponding to each microplastic material; S42: Compare the intensity of the characteristic peak signal extracted in step S41 with the preset signal-to-noise ratio threshold to determine whether it belongs to the microplastic particles of the target material. S43: Based on the peak intensity at the characteristic peak of each particle of the target material microplastic particles determined in step S42, and combined with the pre-established particle size-peak intensity standard curve, determine the particle size of each microplastic particle. S44: Count the number of spectra of microplastic particles identified as the target material in step S42 within a unit time, and calculate the concentration of microplastics in the sample to be tested by combining the transmission efficiency determined by the pre-established concentration-detection quantity standard curve.

[0016] Further, in step S41, the characteristic peak is that of polystyrene microspheres at 1003 cm⁻¹. - ¹Characteristic peak at Raman shift; In step S42, the preset signal-to-noise ratio threshold is the standard deviation of the signal strength being higher than the background signal strength by 3 times.

[0017] Further, in step S43, the particle size-peak intensity standard curve is pre-established in the following way: Raman flow cytometry is performed on standard aqueous solutions of microplastic microspheres of the same material with different particle sizes under the same detection conditions to obtain the peak intensity of each particle size microplastic at the characteristic peak, and the correlation curve between particle size and characteristic peak intensity is obtained by fitting with particle size as the abscissa and characteristic peak intensity as the ordinate.

[0018] Further, in step S44, the concentration-detection quantity standard curve is pre-established in the following way: Raman flow cytometry is used to detect standard aqueous solutions of microplastic microspheres of the same particle size at different concentrations under the same detection conditions. The number of target material microplastic particles detected per unit time for each concentration sample is counted. The actual injection concentration is used as the abscissa and the number of detected particles is used as the ordinate to fit the curve and obtain the correlation curve between concentration and detection quantity. The slope of the correlation curve is the transmission efficiency.

[0019] Compared with the prior art, the present invention achieves the following beneficial technical effects: This application directly feeds the sample aqueous solution into a Raman flow cytometer, detecting microplastic particles one by one in a flowing state. This eliminates cumbersome operations such as filtration enrichment and membrane transfer, simplifying the pretreatment process and avoiding particle aggregation and loss. Simultaneously, a single detection can obtain multi-dimensional information such as the material, particle size distribution, and particle concentration of microplastics, achieving true high-throughput single-particle analysis. The detection efficiency is significantly superior to traditional microscopic Raman point-by-point scanning methods. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a Raman spectral information diagram of polystyrene microspheres in a high-throughput single-particle microplastic detection method based on Raman flow cytometry technology according to the present invention. Figure 2 In this invention, a high-throughput single-particle microplastic detection method based on Raman flow cytometry is proposed, in which the particle size of polystyrene microspheres is 1003 cm⁻¹. - ¹Chart showing the relationship between characteristic peak intensities; Figure 3 This is a standard curve of polystyrene microspheres of different concentrations in a high-throughput single-particle microplastic detection method based on Raman flow cytometry technology according to the present invention.

[0022] Figure 4 This is a schematic diagram of a high-throughput single-particle microplastic detection method based on Raman flow cytometry technology according to the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] This invention provides a high-throughput method for detecting single-particle microplastics based on Raman flow cytometry. Unlike traditional micro-Raman detection methods that require pre-concentrating microplastic particles on a filter membrane or slide before static point-by-point scanning, this invention directly feeds the sample aqueous solution into the Raman flow cytometer, allowing the microplastic particles to pass sequentially through the detection zone in a flowing state, achieving high-throughput continuous detection of single particles without the need for filtration and concentrating. The method includes three main steps: standard preparation, sample loading and detection, and data processing. The specific embodiments of this invention are described in detail below with reference to the accompanying drawings.

[0025] In one embodiment of this application, see Figure 4 The method includes the following steps: S1: Preparation of standard aqueous solutions of microplastic microspheres with different particle sizes and materials. Specifically, microplastic microsphere standards of different particle sizes are selected, dispersed in deionized water or ultrapure water, and then ultrasonically dispersed to obtain uniformly dispersed standard aqueous solutions of microplastic microspheres. The ultrasonic dispersion parameters can be selected from 100W to 200W ultrasonic power and 10min to 15min ultrasonic time to ensure that the microplastic microspheres are fully dispersed in the solution and to avoid particle agglomeration affecting the accuracy of subsequent detection.

[0026] S2: Preparation of standard aqueous solutions of microplastic microspheres at different concentrations. Specifically, a standard of microplastic microspheres with a fixed particle size is selected and dispersed in deionized water or ultrapure water to prepare a series of standard aqueous solutions with different particle concentrations. After preparation, the solutions are ultrasonically dispersed to ensure uniform dispersion of the microplastic microspheres in the solution. Blank control samples are used for background signal subtraction and signal-to-noise ratio evaluation in subsequent detection processes.

[0027] S3: The standard aqueous solutions of microplastic microspheres with different particle sizes and materials prepared in step S1, the standard aqueous solutions of microplastic microspheres with different concentrations prepared in step S2, and the aqueous solution of the sample to be tested are directly fed into a Raman flow cytometer. In the Raman flow cytometer, each aqueous solution is kept in a flowing state. The microplastic particles flowing through each aqueous solution are focused and Raman spectroscopically detected one by one without filtration and enrichment. The Raman spectrum of each sample is collected within a unit time. The injection flow rate of the Raman flow cytometer is 0.1 μL / min to 40 μL / min.

[0028] Raman flow cytometry is an instrument that combines the fluid focusing technology of flow cytometry with Raman spectroscopy detection technology. Its working process is as follows: the sample aqueous solution forms a single-track flowing micro-fluid under the sheath fluid, and microplastic particles in the fluid sequentially pass through the laser focusing area. When a single particle passes the laser focus, it is excited to generate a Raman scattering signal, which is collected by a high numerical aperture objective lens and transmitted to the spectrometer for dispersion and detection. This "single-track flow, individual detection" working mode allows for direct detection of single particles without pre-concentrating them on a filter membrane, fundamentally avoiding problems such as particle aggregation, clogging, and transfer loss that may occur with filter membrane concentrating. The sample flow rate is a key parameter affecting detection throughput and signal quality: the higher the flow rate, the more particles pass through the detection zone per unit time, and the greater the detection throughput; however, if the flow rate is too high, the time for particles to pass through the detection zone is shortened, the number of photons collected during the exposure time decreases, and the signal quality may deteriorate. This application sets the injection flow rate to 0.1 μL / min to 40 μL / min, which can meet the dual requirements of detection throughput and signal quality within this flow rate range.

[0029] In this step, the detection parameters of the Raman flow cytometer are further set as follows: A 532nm laser is used as the excitation source for the Raman laser. This wavelength is in the visible light band and has the advantages of a large Raman scattering cross section and high excitation efficiency. The Raman acquisition lens uses a 5x to 100x lens. High-magnification objectives (such as 50x or 100x) have a larger numerical aperture, enabling the collection of more Raman scattered light and improving detection sensitivity; low-magnification objectives (such as 5x or 10x) have a larger field of view, suitable for rapid pre-scanning. The Raman exposure time is set to 0.01s to 1s. Shorter exposure times are beneficial for increasing detection throughput, while longer exposure times increase signal intensity but reduce the number of particles detected per unit time. The acquisition wavenumber range is 100cm. - ¹Up to 3500cm - ¹, This range covers the main characteristic peak ranges of most plastic materials, including the CC skeletal vibration region (800 cm⁻¹). - ¹Up to 1200cm - ¹), CH2 bending vibration zone (1400cm) - ¹ to 1500cm - ¹) and the CH2 stretching vibration zone (2800cm) - ¹Up to 3000cm - ¹) Key fingerprint regions. The collection time for each sample was 10 minutes, which was sufficient to obtain statistically significant particle count data.

[0030] In this step, the aqueous solution of the sample to be tested is an environmental water microplastic extract, a soil microplastic extract, or an atmospheric microplastic extract. Environmental samples can be extracted using methods known in the art: water samples can be obtained as a microplastic suspension by sieving or filtering followed by ultrasonic elution; soil samples can be obtained as a microplastic extract by density separation combined with filtration; atmospheric samples can be obtained by wet sampling to capture airborne microplastic particles in a liquid medium. The above extracts can be directly used for instrumental analysis without further filtration, enrichment, or transfer to a filter membrane.

[0031] S4: Based on the characteristic peak signals, peak intensities, and number of spectra per unit time in the Raman spectra acquired in step S3, determine the material, particle size, and concentration of microplastics in the sample. Specifically, the acquired raw data consists of a series of Raman spectra, each corresponding to a particle flowing through the detection zone. Through the identification of characteristic peaks, determination of signal-to-noise ratio, quantitative relationship between peak intensity and particle size, and conversion between detection count and concentration, a comprehensive characterization of microplastics in the sample is achieved.

[0032] In one embodiment of this application, the particle size range of the microplastic microspheres described in step S1 is below 40 μm. This particle size range is set based on two considerations: firstly, according to the general definition of microplastics, the upper limit of microplastic size is 5 mm, but the most common and difficult-to-detect particles in the environment are those below 40 μm; secondly, the design of the flow channel and detection zone of Raman flow cytometers is typically designed for micron-sized particles, and a particle size range below 40 μm matches the optimal detection window of the Raman flow cytometer, ensuring that particles pass through the flow channel in a single file without clogging, thus ensuring the continuity and stability of the detection. Furthermore, microplastic particles below 40 μm are precisely the lower limit of particle size that traditional Fourier transform infrared spectroscopy struggles to detect effectively, and the technical solution of this application fills the detection gap in this particle size range.

[0033] In one embodiment of this application, the particle size of the microplastic microspheres described in step S1 is selected from at least one of 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, and 40 μm. These specific particle size values ​​are representative: 1 μm and 2 μm represent submicron to micron-scale particles, which are the most time-consuming and difficult-to-quantify particle size range in traditional Raman detection; 5 μm and 10 μm represent the median particle size of common microplastic particles in environmental samples; and 20 μm and 40 μm correspond to the detection limit of Fourier transform infrared spectroscopy. By establishing a standard curve covering the above particle size range, this application can accurately quantify any particle falling within this particle size range.

[0034] In one embodiment of this application, the microplastic microspheres of different materials mentioned in step S1 are selected from at least one of polystyrene microspheres, polyvinyl chloride microspheres, polymethyl methacrylate microspheres, polyethylene terephthalate microspheres, and polylactic acid microspheres. These materials cover the most common types of plastics in the environment. Polystyrene and polyvinyl chloride are the main components of environmental microplastics; polymethyl methacrylate is widely used in building materials and optical devices; polyethylene terephthalate is a major raw material for beverage bottles and textile fibers; and polylactic acid, as a biodegradable plastic, has seen rapid growth in usage in recent years. Each of the above materials has its own distinctive Raman fingerprint spectrum; for example, the characteristic peak of polystyrene is 1003 cm⁻¹. - ¹、1602cm - ¹ and 3053cm - ¹; The characteristic peak of polyvinyl chloride is 635 cm⁻¹. - ¹、692cm - ¹ and 1428cm - ¹; The characteristic peak of polymethyl methacrylate is 600 cm⁻¹. - ¹、812cm - ¹ and 1728cm - ¹; The characteristic peak of polyethylene terephthalate is 633 cm⁻¹.- ¹、1096cm - ¹ and 1614cm - ¹; The characteristic peak of polylactic acid is 872 cm⁻¹. - ¹、1042cm - ¹ and 1770cm - ¹. By matching characteristic peaks and quantifying peak intensities, it is possible to identify and determine the particle size of microplastics made of different materials.

[0035] In one embodiment of this application, the concentration range of the different concentrations of microplastic microsphere standard aqueous solutions in step S2 is below 100,000 particles / mL. This upper limit of concentration is set based on the detection throughput limitation of the Raman flow cytometer: when the concentration exceeds 100,000 particles / mL, the number of particles passing through the detection zone per unit time is too high, which may exceed the data acquisition and processing capacity of the spectrometer, leading to missed detections or spectral overlap. This concentration range covers the typical order of magnitude of microplastic concentration in environmental water samples, ranging from clean water (close to 0 particles / mL) to severely polluted water.

[0036] In one embodiment of this application, the concentrations of the microplastic microsphere standard aqueous solutions of different concentrations mentioned in step S2 are selected from at least one of 0 particles / mL, 5000 particles / mL, 10000 particles / mL, 50000 particles / mL, and 100000 particles / mL. A blank control of 0 particles / mL is used for background subtraction and signal-to-noise ratio evaluation; a medium concentration range of 5000 to 50000 particles / mL is used to establish a correlation curve between concentration and detection quantity; and a high concentration point of 100000 particles / mL is used to verify the detection performance of the system under high concentration conditions.

[0037] In one embodiment of this application, the detection parameters of the Raman flow cytometer in step S3 are set as follows: a 532nm Raman laser, a 5x to 100x Raman acquisition lens, a Raman exposure time of 0.01s to 1s, and an acquisition wavenumber range of 100cm. - ¹Up to 3500cm - ¹, The sample acquisition time is 10 min. The combination of the above parameters is a suitable parameter window obtained by the inventors of this application through extensive experimental optimization. A 532 nm laser as the excitation source has universal applicability in Raman detection and can generate strong Raman signals for most microplastic materials. The adjustable range of exposure time provides flexibility for both rapid and precise detection: when rapid screening of a large number of samples is required, a short exposure time of 0.01 s can be used, with a detection throughput of hundreds of particles per second; when high-quality spectra are required for accurate material identification, a long exposure time of 1 s can be used, significantly improving the signal-to-noise ratio. The lower limit of the acquisition wavenumber is 100 cm⁻¹. -¹ Capable of covering skeletal vibration modes in the low wavenumber region, with an upper limit of 3500 cm. - ¹It can cover stretching vibration modes such as CH and OH, and this range contains all the characteristic fingerprint information of various plastic materials. The 10-minute acquisition time is a choice that strikes a balance between detection throughput and statistical reliability. Within this time, sufficient particle count data can be accumulated, keeping the concentration statistical error within an acceptable range.

[0038] In one embodiment of this application, the Raman acquisition lens of the Raman flow cytometer described in step S3 is a 50x lens, the Raman exposure time is 0.1s, and the sample injection rate is 1μL / min. These preferred parameter values ​​represent a specific implementation of the present invention. The 50x objective lens combines a high numerical aperture with a suitable field of view, ensuring sufficient light collection efficiency while allowing for convenient observation and adjustment of the liquid flow position during detection. The 0.1s exposure time achieves a good balance between signal quality and detection throughput. The 1μL / min sample injection rate matches the detection zone size of the 50x objective lens. At this flow rate, particles pass through the laser focus at an appropriate linear velocity, ensuring that each particle has sufficient residence time in the detection zone to complete exposure while avoiding signal overlap between adjacent particles.

[0039] In one embodiment of this application, step S4 determines the material, particle size, and concentration of microplastics in the sample to be tested based on the characteristic peak signals, characteristic peak intensities, and the number of spectra per unit time in the Raman spectrum acquired in step S3. Specifically, this includes the following sub-steps: S41: Based on the Raman spectra acquired in step S3, extract the signal intensity at the Raman shift of the characteristic peak corresponding to each microplastic material. This step first preprocesses each Raman spectrum, including background subtraction to remove the broad-spectrum background caused by stray light from the optical system and the sample medium, baseline correction to eliminate fluorescence background bulges, and smoothing to reduce random noise. Then, the net signal intensity at the preset characteristic peak wavenumber position is extracted. Different microplastic materials have their own characteristic Raman peaks; for example, polystyrene microspheres have a characteristic Raman peak at 1003 cm⁻¹. - ¹The Raman shift has a characteristic peak, which belongs to the breathing vibration mode of the benzene ring and is one of the most significant and strongest Raman characteristic peaks of polystyrene.

[0040] S42: Compare the intensity of the characteristic peak signal extracted in step S41 with a preset signal-to-noise ratio (SNR) threshold to determine if the microplastic particles belong to the target material. The SNR is calculated as follows: the intensity of the characteristic peak signal minus the average intensity of adjacent background intervals, then divided by the standard deviation of the background interval intensity. When this ratio is greater than the preset threshold, the particle is determined to belong to the target material. This determination criterion is based on the statistical significance test principle to ensure that the false positive rate is within a controllable range. The SNR threshold is set to eliminate false positive signals caused by background noise and ensure the accuracy of the count.

[0041] S43: Based on the peak intensity at the characteristic peak of each microplastic particle identified in step S42, and combined with the pre-established particle size-peak intensity standard curve, determine the particle size of each microplastic particle. Since the intensity of the Raman scattering signal and the particle volume are positively correlated within a certain range in the same material, the particle size can be estimated by measuring the absolute intensity of the characteristic peak. The particle size-peak intensity standard curve is pre-established as follows: Raman flow cytometry is performed on standard aqueous solutions of microplastic microspheres of the same material with different particle sizes under the same detection conditions to obtain the peak intensity at the characteristic peak of each particle size. The particle size is then fitted with the characteristic peak intensity as the x-axis and the characteristic peak intensity as the y-axis to obtain the correlation curve between the particle size and the characteristic peak intensity. For unknown particles, their peak intensity at the characteristic peak is substituted into this standard curve to deduce their particle size.

[0042] S44: Count the number of microplastic particles identified as the target material in step S42 within a unit time. Combine this with the transmission efficiency determined by the pre-established concentration-detection quantity standard curve to calculate the concentration of microplastics in the sample. Since the detection efficiency of Raman flow cytometry is not 100%, calibration using a standard curve is required. The concentration-detection quantity standard curve is pre-established as follows: Raman flow cytometry is performed on standard aqueous solutions of microplastic microspheres of the same particle size at different concentrations under the same detection conditions. The number of target material microplastic particles detected per unit time for each concentration sample is counted. A correlation curve between concentration and detection quantity is obtained by fitting the actual injection concentration as the x-axis and the number of detected particles as the y-axis. The slope of this correlation curve is the transmission efficiency. When calculating the concentration of the sample, the number of particles detected per unit time is divided by the transmission efficiency to obtain the actual particle concentration of the sample.

[0043] In one embodiment of this application, the characteristic peak mentioned in step S41 is the peak of polystyrene microspheres at 1003 cm⁻¹. -¹The characteristic peak at the Raman shift. This characteristic peak belongs to the ring breathing vibration mode of the benzene ring in the polystyrene molecule and is the strongest characteristic peak in the polystyrene Raman spectrum, exhibiting extremely high detection sensitivity. Using polystyrene as an example material, this application establishes a complete quantitative framework of "characteristic peak determination → particle size-peak intensity standard curve → concentration-detection quantity standard curve". This framework can be extended to the detection of other microplastic materials; simply replace the characteristic peak with the strongest characteristic peak corresponding to that material.

[0044] In one embodiment of this application, the preset signal-to-noise ratio (SNR) threshold in step S42 is the standard deviation of the signal intensity being more than three times the background signal intensity. This threshold (i.e., an SNR greater than 3) is a recognized detection limit standard in analytical chemistry, ensuring the presence of the target signal can be determined under conditions of high confidence. In practice, the background signal intensity and standard deviation are obtained through repeated measurements of blank samples and are pre-stored as system parameters in the data processing software.

[0045] In one embodiment of this application, the particle size-peak intensity standard curve in step S43 is pre-established as follows: Raman flow cytometry is performed on standard aqueous solutions of microplastic microspheres of the same material with different particle sizes under the same detection conditions to obtain the peak intensity at the characteristic peak of each particle size. A correlation curve between particle size and characteristic peak intensity is obtained by fitting the curve with particle size as the abscissa and characteristic peak intensity as the ordinate. This standard curve is used to convert the characteristic peak intensity of unknown particles into particle size values. Taking polystyrene as an example, standard aqueous solutions of polystyrene microspheres with particle sizes of 1μm, 2μm, 5μm, 10μm, and 20μm are prepared respectively, and Raman flow cytometry is performed under the same instrument parameters. Multiple spectra are collected for each particle size. The peak intensity at 1003 cm⁻¹ is used as the reference value. - The peak intensity value at point ¹ is averaged to obtain the average peak intensity corresponding to that particle size. A correlation curve between particle size and characteristic peak intensity is obtained by fitting the particle size as the x-axis and the average peak intensity as the y-axis.

[0046] In one embodiment of this application, the concentration-detection quantity standard curve in step S44 is pre-established as follows: Raman flow cytometry is performed on standard aqueous solutions of microplastic microspheres of the same particle size at different concentrations under the same detection conditions. The number of target material microplastic particles detected per unit time for each concentration sample is counted. A correlation curve between concentration and detection quantity is obtained by fitting the actual injection concentration as the abscissa and the number of detected particles as the ordinate. The slope of the correlation curve represents the transmission efficiency. Taking 2μm polystyrene microspheres as an example, standard aqueous solutions with concentrations of 5000 particles / mL, 10000 particles / mL, 50000 particles / mL, and 100000 particles / mL are prepared respectively. Raman flow cytometry is performed under the same instrument parameters. Each concentration sample is collected for 10 minutes, and the number of target material microplastic particles detected per unit time for each concentration sample is counted. A linear fit is performed with the actual concentration as the abscissa and the number of detected particles as the ordinate. The slope of the fitted line is the transmission efficiency. When calculating the concentration of the sample to be tested, the number of particles detected per unit time is divided by the transmission efficiency to obtain the actual particle concentration of the sample.

[0047] The present invention will be further described below with reference to specific embodiments.

[0048] Example 1: Detection of polystyrene microplastics Preparation of aqueous solutions of standard polystyrene microspheres with different particle sizes Take 10 μL of standard polystyrene microspheres of different particle sizes (1 μm, 2 μm, 5 μm, 10 μm and 20 μm) at a 2.5% mass concentration, dilute them in 5 mL of aqueous solution, and sonicate for 10 min for later use.

[0049] Preparation of standard polystyrene aqueous solutions of different concentrations Take 2μm standard polystyrene microspheres, dilute them to particle concentrations of 5000, 10000, 50000 and 100000 particles / mL, sonicate for 10 min, and set aside.

[0050] On-machine testing Raman flow cytometry acquisition parameters were set as follows: 532nm Raman laser, 50x magnification Raman acquisition lens, exposure time of 0.1s, and acquisition wavenumber range of 100 to 3500cm. - ¹, The injection flow rate was 1 μL / min, and data were collected for 10 min for each sample.

[0051] Data processing The data is saved as a txt file and processed using R or Excel.

[0052] (1) 1003cm -¹The Raman shift peak is a characteristic peak of polystyrene microspheres. A spectrum with a signal intensity higher than the background signal intensity and a signal-to-noise ratio greater than 3 (i.e., the signal intensity is more than 3 times the standard deviation of the background signal intensity) is defined as a polystyrene microplastic particle. Figure 1 As shown.

[0053] (2) Based on the definition of polystyrene plastic particles, calculate the microplastic particles of different sizes at 1003 cm⁻¹. - The peak intensity at position ¹ is used to fit a correlation curve with particle size as the x-axis and Raman peak intensity as the y-axis, as shown below. Figure 2 As shown.

[0054] (3) The transport efficiency was obtained by measuring 2μm standard polystyrene microspheres at different concentrations and fitting the correlation between the number of polystyrene plastic particles detected and the actual concentration of microplastics injected. Figure 3 As shown.

[0055] (4) The number of polystyrene plastic particles collected within the 1 min measurement time is divided by the transmission efficiency to obtain the microplastic concentration in the actual sample.

[0056] To verify the applicability of this method to actual environmental samples, groundwater samples were collected, and microplastic extracts were obtained as the aqueous solution for the test samples. Polystyrene standards of different sizes (particle sizes of 1 μm, 2 μm, 5 μm, 10 μm, and 20 μm) were added to the test samples, and Raman flow cytometry was performed according to the above-mentioned detection parameters. After acquiring the Raman spectra, the data were processed using the same method at a resolution of 1003 cm⁻¹. - The characteristic peak at position ¹ was used as the criterion for polystyrene identification. The number of polystyrene microplastic particles of various sizes was counted, and the particle size-peak intensity correlation curve and concentration-detection quantity correlation curve were combined to obtain the particle size distribution and concentration distribution information of polystyrene standards of different sizes added to environmental groundwater samples. The results show that this method can effectively detect microplastic particles of different sizes added to environmental water samples and simultaneously obtain material information, particle size distribution information, and concentration information, achieving high-throughput single-particle level microplastic detection.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-throughput method for detecting single-particle microplastics based on Raman flow cytometry, characterized in that, Includes the following steps: S1: Standard aqueous solutions for preparing microplastic microspheres of different particle sizes and materials; S2: Prepare standard aqueous solutions of microplastic microspheres at different concentrations; S3: The standard aqueous solutions of microplastic microspheres with different particle sizes and materials prepared in step S1, the standard aqueous solutions of microplastic microspheres with different concentrations prepared in step S2, and the aqueous solution of the sample to be tested are directly fed into a Raman flow cytometer. In the Raman flow cytometer, each aqueous solution is kept in a flowing state. The microplastic particles flowing through each aqueous solution are focused and Raman spectroscopically detected one by one without filtration and enrichment. The Raman spectrum of each sample is collected in a unit time. The injection flow rate of the Raman flow cytometer is 0.1 μL / min to 40 μL / min. S4: Based on the characteristic peak signals, characteristic peak intensities, and number of spectra per unit time in the Raman spectrum collected in step S3, determine the material, particle size, and concentration of microplastics in the sample to be tested.

2. The method according to claim 1, characterized in that, In step S1, the particle size of the microplastic microspheres is below 40 μm.

3. The method according to claim 2, characterized in that, In step S1, the particle size of the microplastic microspheres is selected from at least one of 1μm, 2μm, 5μm, 10μm, 20μm and 40μm.

4. The method according to claim 1, characterized in that, In step S1, the microplastic microspheres of different materials are selected from at least one of polystyrene microspheres, polyvinyl chloride microspheres, polymethyl methacrylate microspheres, polyethylene terephthalate microspheres, and polylactic acid microspheres.

5. The method according to claim 1, characterized in that, In step S2, the concentration range of the standard aqueous solution of microplastic microspheres of different concentrations is below 100,000 per mL.

6. The method according to claim 5, characterized in that, In step S2, the concentration of the standard aqueous solution of microplastic microspheres of different concentrations is selected from at least one of 0 microspheres / mL, 5000 microspheres / mL, 10000 microspheres / mL, 50000 microspheres / mL and 100000 microspheres / mL.

7. The method according to claim 1, characterized in that, In step S3, the detection parameters of the Raman flow cytometer are set as follows: a 532nm Raman laser, a 5x to 100x Raman acquisition lens, a Raman exposure time of 0.01s to 1s, and an acquisition wavenumber range of 100cm. - ¹Up to 3500cm - ¹, The sample collection time is 10 min.

8. The method according to claim 7, characterized in that, In step S3, the Raman acquisition lens of the Raman flow cytometer is a 50x lens, the Raman exposure time is 0.1s, and the injection flow rate is 1μL / min.

9. The method according to claim 1, characterized in that, Step S4 includes the following sub-steps: S41: Based on the Raman spectrum collected in step S3, extract the signal intensity at the Raman shift of the characteristic peak corresponding to each microplastic material; S42: Compare the intensity of the characteristic peak signal extracted in step S41 with the preset signal-to-noise ratio threshold to determine whether it belongs to the microplastic particles of the target material. S43: Based on the peak intensity at the characteristic peak of each particle of the target material microplastic particles determined in step S42, and combined with the pre-established particle size-peak intensity standard curve, determine the particle size of each microplastic particle. S44: Count the number of spectra of microplastic particles identified as the target material in step S42 within a unit time, and calculate the concentration of microplastics in the sample to be tested by combining the transmission efficiency determined by the pre-established concentration-detection quantity standard curve.

10. The method according to claim 9, characterized in that, In step S41, the characteristic peak is that of polystyrene microspheres at 1003 cm⁻¹. - ¹Characteristic peak at Raman shift; In step S42, the preset signal-to-noise ratio threshold is the standard deviation of the signal strength being higher than the background signal strength by 3 times.

11. The method according to claim 9, characterized in that, In step S43, the particle size-peak intensity standard curve is pre-established in the following way: Raman flow cytometry is used to detect standard aqueous solutions of microplastic microspheres of the same material with different particle sizes under the same detection conditions to obtain the peak intensity of each particle size microplastic at the characteristic peak. The particle size is used as the abscissa and the characteristic peak intensity is used as the ordinate to fit the curve and obtain the correlation curve between the particle size and the characteristic peak intensity.

12. The method according to claim 9, characterized in that, In step S44, the concentration-detection quantity standard curve is pre-established in the following way: Raman flow cytometry is used to detect standard aqueous solutions of microplastic microspheres of the same particle size at different concentrations under the same detection conditions. The number of target material microplastic particles detected per unit time for each concentration sample is counted. The actual injection concentration is used as the abscissa and the number of detected particles is used as the ordinate to fit the curve and obtain the correlation curve between concentration and detection quantity. The slope of the correlation curve is the transmission efficiency.

Images