Microplastic analysis method, analysis device for same, microplastic detection device, and microplastic detection method

Abstract

In the present invention, microplastics in a sample W are stained with a fluorescent material, the sample is irradiated with fluorescence-purpose excitation light and fluorescence emitted from the fluorescent material is detected, the position of the microplastics in the sample is identified by the fluorescence, the identified position is irradiated with Raman-purpose excitation light and Raman scattered light is detected, and the microplastics at the position are analyzed on the basis of the Raman scattered light.

Description

Microplastic analysis method, analysis device, microplastic detection device, and microplastic detection method

[0001] The present invention relates to a method for analyzing microplastics using Raman scattered light.

[0002] As described in Non-Patent Document 1, Raman spectroscopy can be used for the qualitative analysis of microplastics. Measurements using Raman spectroscopy have extremely high spatial resolution, so if, for example, beach sand containing microplastics is used as a sample and subjected to Raman analysis using a Raman microscope or the like, even minute microplastics of 1 μm or less can be detected and their types identified.

[0003] However, these tiny microplastics are extremely difficult to identify with the naked eye or with conventional optical images, and their location cannot be pinpointed. Therefore, the entire sample must be mapped and measured using Raman. Depending on the area of ​​the sample, this can take a long time, from several hours to several days. Furthermore, prolonged laser irradiation can cause heat accumulation, which can result in the microplastics burning or melting.

[0004] Feature Article "HORIBA Group Activities in Japan Regarding Microplastics" Authors: Tomoko Numata, Susumu Yamauchi https: / / www.horiba.com / uploads / media / R54J_12_061_03.pdf

[0005] The present invention has been made to solve the above-mentioned problems, and its main intended objective is to enable the analysis of microplastics contained in a sample having a certain area in a short period of time using Raman spectroscopy.

[0006] In other words, the method for analyzing microplastics according to the present invention is characterized in that it involves staining microplastics in a sample with a fluorescent material, irradiating the sample with fluorescence excitation light to detect the fluorescence emitted from the fluorescent material, identifying the position of the microplastics in the sample using the fluorescence, irradiating the identified position with Raman excitation light to detect Raman scattered light, and analyzing the microplastics at that position based on the Raman scattered light.

[0007] This method uses fluorescence to detect microplastics on the order of 500 nm, which cannot be detected with conventional optical images or the naked eye. Therefore, the location of the microplastics in the sample is first identified, and then Raman excitation light is irradiated only at that identified location, allowing Raman analysis of the composition and type of microplastics present there.

[0008] Therefore, there is no need to perform Raman mapping measurements over the entire sample, and Raman analysis can be performed only on the necessary areas, making it possible to quickly analyze the composition and type of microplastics contained in the sample.

[0009] A specific method for staining microplastics involves immersing the sample in a staining solution in which the fluorescent material is dissolved in a specified solvent, and then washing the sample with a cleaning solution that can remove the fluorescent material attached to components other than microplastics, thereby fluorescently staining the microplastics in the sample.

[0010] To generate high-intensity fluorescence in a short time, it is preferable that the phosphor be Nile Red and the solvent be toluene. In this case, it is preferable to use ethanol, methanol, or water as the washing solution to wash and remove the phosphor from substances other than microplastics in the sample.

[0011] To reliably detect only the fluorescence, an optical filter that transmits the fluorescence and blocks the fluorescence excitation light can be provided. If the wavelength of the Raman excitation light is set to a wavelength that cannot excite the fluorophore, the influence of fluorescence on Raman analysis can be eliminated.

[0012] The present invention may also be an analytical device for microplastics stained with a fluorescent material, which irradiates a sample containing the microplastics with fluorescence excitation light, detects the fluorescence emitted from the fluorescent material, identifies the position of the microplastics in the sample using the fluorescence, irradiates the identified position with Raman excitation light, detects Raman scattered light, and analyzes the microplastics at that position based on the Raman scattered light.

[0013] When analyzing environmental samples such as beach sediment, transporting them from the collection site to the lab and then examining them in more detail for the presence or absence of microplastics and their physical properties is time-consuming. In particular, when surveying multiple locations, it is necessary to transport many samples.

[0014] To solve this problem, it would be desirable to have a simple kit that could immediately determine the presence or absence of microplastics on the spot after sample collection. With such a simple kit, for example, if the only investigation required was the presence or absence of microplastics, the results could be obtained on-site, and even if further investigation of the physical properties of microplastics using Raman spectroscopy or other methods was desired, only samples that were confirmed to contain microplastics could be brought back, eliminating the need for unnecessary transportation.

[0015] In order to solve such problems, the present invention may be a microplastic detection device comprising a sample mounting section for mounting a sample containing microplastics stained with a fluorescent substance, a first light source for irradiating the sample mounted on the sample mounting section with fluorescence excitation light, a camera holding section for holding a portable camera in a position where the sample irradiated with the fluorescence excitation light can be imaged, and an optical filter positioned in front of the portable camera held in the camera holding section that transmits the fluorescence but blocks the fluorescence excitation light.

[0016] With such a device, the device itself can be made portable, and if you want to record the detection results as an image, you can simply attach a personal camera-equipped mobile phone to the camera holder, making it easy to detect microplastics in samples at the collection site.

[0017] It is also more preferable if the portable camera is equipped with a calculation unit that calculates the content of microplastics in the sample from the area occupied by microplastics in the image, etc. Image data and data including the content may be transmitted to other devices.

[0018] The microplastic detection method of the present invention is characterized by comprising a sample mounting step of mounting a sample containing fluorescently stained microplastics on a predetermined sample mounting section, a light irradiation step of irradiating the sample mounted on the sample mounting section with fluorescence excitation light, a camera holding step of holding a portable camera in a position where the sample irradiated with the fluorescence excitation light can be imaged, an optical filter placement step of placing an optical filter in front of the portable camera that transmits the fluorescence but blocks the fluorescence excitation light, and an imaging step of imaging the fluorescence emitted from the microplastics in the sample with the camera. Such a detection method can achieve the same effects as the microplastic detection device of the present invention described above.

[0019] Furthermore, it is preferable that the microplastic detection method further includes a staining step in which the microplastics in the sample are fluorescently stained by immersing the sample in a staining solution in which the fluorescent material is dissolved in a specified solvent, and then washing the sample with a cleaning solution capable of removing the fluorescent material attached to components other than microplastics.

[0020] According to the present invention, it is possible to analyze microplastics contained in a sample having a certain area in a short period of time using Raman spectroscopy.

[0021] FIG. 1 is an overall schematic diagram of a Raman spectroscopic analysis device in one embodiment of the present invention. FIG. 2 is a flowchart illustrating a microplastic staining process in the same embodiment. FIG. 3 is a flowchart illustrating a Raman analysis process of microplastics in a sample in the same embodiment. FIG. 4 is a graph showing the emission characteristics of Nile Red in response to irradiation with excitation light of each wavelength in the same embodiment. FIG. 5 is a schematic cross-sectional view of a microplastic detection device in another embodiment of the present invention. FIG. 6 is a schematic oblique view of a microplastic detection device in another embodiment of the present invention.

[0022] 100: Raman spectroscopic analyzer (analysis device) W: Sample 6: Long-pass filter 42: Short-pass filter

[0023] A Raman spectroscopic analyzer according to one embodiment of the present invention will be described below with reference to the drawings.

[0024] FIG. 1 is a schematic block diagram showing the overall configuration of a Raman spectroscopic analyzer 100 according to this embodiment. In the figure, the symbol W indicates a sample. This sample is, for example, sand containing microplastics collected from a beach. This sample W is placed in a thin circular or rectangular cell, and the surface of the cell, which is the region to be analyzed, has a certain area.

[0025] Reference numeral 1 denotes a Raman light source that irradiates Raman excitation light onto the surface of the sample W. Here, a laser light source that emits monochromatic laser light (having a wavelength of approximately 785 nm) is used as the Raman light source 1, but the present invention is not limited to this.

[0026] Reference numeral 2 denotes a spectroscope that separates the Raman scattered light generated by irradiation with the Raman excitation light. Here, a grating is used as the spectroscope, but the present invention is not limited to this.

[0027] Reference numeral 3 denotes a photodetector that detects light of each wavelength dispersed by the spectrometer and outputs a Raman detection signal having a value corresponding to the light intensity of each detected wavelength. This photodetector 3 includes a photosensor such as a CCD or a photomultiplier tube, and a signal converter that performs, for example, impedance conversion processing or digitization processing on the output signal of the photosensor to generate the Raman detection signal.

[0028] Reference numeral 4 denotes a fluorescence light source that irradiates the entire analysis target area on the surface of the sample W with broad fluorescence excitation light having an upper wavelength limit of approximately 550 nm. Here, this fluorescence light source 4 is composed of a white LED 41 and an optical filter (short-pass filter 42) that is provided on the optical path of the white LED 41 and transmits light with wavelengths of approximately 550 nm or less. The upper wavelength limit of this fluorescence light source 4 is not limited to 550 nm, but may be in the range of 400 nm to 600 nm. Furthermore, the fluorescence excitation light may not only be broad light, but also single-wavelength light emitted from an LED or the like. In this case, an optical filter is not required.

[0029] Reference numeral 5 denotes a two-dimensional area sensor (here, a CCD camera 5) that captures an image of an area to be analyzed on the sample W in one shot and outputs the image data. An optical filter (long-pass filter 6) that transmits light with a wavelength of approximately 625 nm or more is installed in front of the CCD camera 5. Note that the long-pass filter 6 may be any filter that transmits light with a wavelength of 600 nm to 700 nm or more.

[0030] Reference numeral 7 denotes an XYZ stage 7, which is a moving mechanism for relatively moving the irradiation position of the Raman excitation light with respect to the sample W. This XYZ stage 7 can be moved in the horizontal X and Y directions by an actuator 8 such as a motor, and a cell containing the sample W is placed on the XYZ stage 7.

[0031] Reference numeral 9 denotes an information processing device that receives the Raman detection signal output from the photodetector 3 and processes it to perform sample analysis, etc. This information processing device 9 is a so-called computer having a CPU, memory, I / O ports, etc., and performs functions such as a pre-processing unit 91, a sample analysis unit 92, and an output unit 93 by the CPU and peripheral devices working together in accordance with a program stored in advance in the memory.

[0032] The preprocessing unit 91 receives Raman detection signals for each wavelength from the photodetector 3, interpolates the values, i.e., the light intensity for each wavelength, and performs baseline correction to generate Raman spectrum data that can be used for analysis. The sample analysis unit 92 analyzes and identifies the physical properties of the sample W based on the Raman spectrum data. The output unit 93 outputs the analysis results from the sample analysis unit 92 in a predetermined format. Furthermore, in this embodiment, the information processing device 9 also functions as a position identification unit 94 and an actuator control unit 95.

[0033] The position identification unit 94 receives image data of the sample W from the CCD camera 5, processes the image data, specifically by performing binarization processing, for example, to identify the position where fluorescence is being generated.

[0034] The actuator control unit 95 sends a command signal to the actuator 8 to move the XYZ stage 7 so that the Raman excitation light is irradiated onto the position identified by the position identifying unit 94 .

[0035] Next, a procedure for analyzing microplastics in a sample W using the Raman spectroscopic analysis device 100 configured as described above will be described.

[0036] <Staining process> As shown in Figure 2, first, Nile Red (C20H18N2O2), a dye for lipid bilayer membranes, is dissolved in toluene, a solvent, to produce a Nile Red solution (Step S11). Then, the sample W is immersed in this Nile Red solution (Step S12). At this time, the toluene slightly melts the surface of the microplastics, allowing the Nile Red to penetrate into the solution, while other particles (glass, stone, etc.) are thought to be barely affected by the toluene, with only the Nile Red adhering to their surfaces.

[0037] Next, the sample W is dried and washed with ethanol (step S13). As a result, the other particles in the sample W are thought to only have Nile red attached to their surfaces, so the Nile red is washed away, leaving only the microplastics stained with Nile red. Note that a washing liquid that barely dissolves microplastics is preferred, and methanol or water can also be used.

[0038] <Position Identification Step> As shown in FIG. 3, the sample W in which the microplastics have been dyed by the dyeing step described above is placed in a cell and set in the Raman spectroscopic analysis device 100 (step S21).

[0039] The fluorescence light source 4 is then turned on, and the entire analysis area on the surface of the sample W is irradiated with fluorescence excitation light (step S22). Nile red is maximally excited by light of 553 nm, but can also emit fluorescence of sufficient intensity with light of 400 nm to 600 nm. Therefore, as in this embodiment, it also emits fluorescence with fluorescence excitation light that has passed through a short-pass filter 42 that transmits light with wavelengths of 550 nm or shorter. The fluorescence wavelength spectrum of Nile red in relation to excitation light is shown in FIG. 5, and its maximum fluorescence wavelength is generally said to be approximately 637 nm.

[0040] In this state, the CCD camera 5 captures an image of the entire analysis region on the surface of the sample W (step S23). As described above, the long-pass filter 6, which transmits light with wavelengths of 625 nm or more, is provided in front of the CCD camera 5. Since the fluorescence has sufficient intensity even at wavelengths of 625 nm or more, it passes through the long-pass filter 6. However, since the fluorescence excitation light has a wavelength of less than 550 nm, the light scattered and reflected (Rayleigh scattered) by the sample is blocked by the long-pass filter 6. Therefore, the CCD camera 5 captures and images only this fluorescence, and the image data of the sample W will be such that only the fluorescent regions glow red and the other regions are black (in a black-and-white image, the fluorescent regions will be white and the other regions will be black).

[0041] Next, the position specifying unit 94 specifies the areas in the sample that are emitting fluorescence (hereinafter also referred to as fluorescent areas) by, for example, binarizing the image data, and stores the position data in memory (step S24). At this time, the number, size, shape, etc. of the fluorescent areas may also be stored. Then, the fluorescence light source 4 is turned off.

[0042] 4, the actuator control unit 95 sends a command signal to the actuator 8 to move the XYZ stage 7 so that the fluorescent site is irradiated with Raman excitation light (step S31). Once the sample position has been set in this manner, the Raman light source 1 is turned on, and the fluorescent site is irradiated with Raman excitation light (step S32).

[0043] The Raman scattered light emitted from the fluorescent site is separated by the spectroscope 2 and output by the photodetector 3 as a Raman detection signal indicating the light intensity for each wavelength (step S33).

[0044] The preprocessing unit 91 then receives the Raman detection signal and generates Raman spectral data (step S34). Based on this Raman spectral data, the sample analysis unit 92 analyzes the physical properties of the sample at the fluorescent site. This Raman spectral data and analytical data are linked to positional data indicating the fluorescent site and stored in memory (step S35). This Raman analysis process is repeated until the entire range or a desired range of the fluorescent site has been analyzed (step S36). As a result, Raman spectral data or analytical data for each fluorescent site is obtained.

[0045] In this embodiment, the output unit 93 outputs the Raman spectrum data or analysis data with position data attached to the image data obtained by, for example, the CCD camera 5. By performing arithmetic processing on this data, it becomes possible to, for example, click on a fluorescent portion of the sample image displayed on the screen to display the composition of microplastics and Raman spectrum data at that portion.

[0046] Thus, according to this embodiment configured as described above, there is no need for Raman mapping measurements or the like over the entire sample W, and it is sufficient to perform Raman analysis only on the necessary areas, making it possible to quickly analyze the composition and type of microplastics contained in the sample W. Furthermore, the fluorescence detection method of this embodiment can detect even minute microplastics on the order of 500 nm, making it possible to fully utilize the spatial resolution of Raman analysis.

[0047] Furthermore, because toluene is used as the solvent for the fluorescent dye solution (Nile Red solution), staining takes only a few seconds, which also contributes to shortening analysis time. On the other hand, toluene dissolves some types of plastic, so there is a risk that tiny microplastics will melt and be lost from the sample. To avoid this, an organic solvent with low plastic solubility, such as n-hexane (normal hexane), can be used. However, in this case, staining may take several hours.

[0048] Furthermore, a long-pass filter 6 that transmits the fluorescence but blocks the fluorescence excitation light is installed before the CCD camera 5, ensuring that only the fluorescence is detected. In addition, the wavelength of the Raman excitation light is set to approximately 785 nm, a wavelength that cannot excite Nile Red, so the influence of fluorescence on the Raman analysis can also be eliminated.

[0049] The present invention is not limited to the above-described embodiment. For example, a broad wavelength source such as a mercury lamp may be used as the light source for fluorescence excitation light. Conversely, a monochromatic LED that emits light of a narrow wavelength may be used. In this case, the short-pass filter may be omitted depending on the monochromatic LED selected.

[0050] In the above embodiment, Nile Red is used as the phosphor, but this is not limiting. For example, any fluorescent substance may be used as the phosphor as long as it is soluble in an organic solvent such as toluene, and commercially available fluorescent substances such as fluorescent chalk may also be used.

[0051] In the above embodiment, the sample W is placed in a thin circular or rectangular cell, but this is not limiting. In other embodiments, the sample W may be placed on a thin glass plate, acrylic plate, or the like, so that the analysis target region on its surface has a certain area. In this case, an adhesive member such as double-sided tape may be provided on the surface of the glass plate or acrylic plate on which the sample W is placed, to prevent the position of the placed sample W from shifting. Furthermore, a scale may be provided on the surface of the glass plate or adhesive member on which the sample W is placed.

[0052] Furthermore, it may be possible to detect the presence of microplastics in a sample only by fluorescence, without being able to identify the type of microplastics. Specific examples of this are shown in Figures 6 and 7.

[0053] This microplastic detection device 200 has a rectangular parallelepiped housing 201 that is portable in size and weight. On the top surface of the bottom plate of this housing 201, there is provided a sample placement section 202 for placing a sample W containing microplastics stained with a fluorescent material.

[0054] A fluorescence light source 4, for example, including a green LED 41 and a short-pass filter 42, is provided on one side of the lower surface of the upper plate of the housing. The fluorescence excitation light emitted from the fluorescence light source 4 is irradiated onto the surface of the sample W placed on the sample placement portion 202. The green LED may be a white LED, for example. Alternatively, for example, both a green LED and a white LED may be provided, allowing the emitted light to be switched between green and white. Instead of or in addition to the white LED, an openable opening for inserting and removing the sample W may be provided in the wall of the housing 201, and light for observing the sample W may be introduced into the housing 201 by opening the opening. The inner surface of the housing 201 may be coated with a light-absorbing material to prevent diffuse reflection of the fluorescence excitation light emitted from the fluorescence light source 4 within the housing 201.

[0055] Furthermore, in order to reduce unevenness in illumination of the fluorescence excitation light onto the sample W, a light diffusing member such as a diffusion plate or diffusion sheet may be provided in front of the fluorescence light source 4 in the light emission direction and between the sample W and the light source 4. Furthermore, the fluorescence light sources 4 may be provided not only on the top surface of the sample W within the housing 201 but also on the side surfaces thereof, and the fluorescence excitation light may be irradiated onto the sample W from above and from the sides, thereby reducing uneven illumination.

[0056] On the other hand, a window 203 is opened on the other side of the upper plate of the housing, and below the window 203, a long-pass filter 6 that transmits fluorescence but blocks the fluorescence excitation light, as in the above embodiment, is provided in a removable or movable manner by an attachment / detachment mechanism (not shown).

[0057] On the top surface of the upper plate of the housing, there is provided a camera holder 204 for placing and holding a portable camera, such as a smartphone P. As shown in Fig. 6, this camera holder 204 has an L-shaped ridge 207 that serves as a positioning structure. When the smartphone P is placed with two sides abutting against this ridge 207, the camera surface faces the window 203, and it is configured to be able to capture an image of the fluorescence emitted from the microplastics in the sample W.

[0058] With this type of microplastic detection device 200, the device 200 itself is portable, and if you want to keep an image of the detection results, you simply need to place the smartphone P on the camera holding portion 204, so microplastics in the sample can be easily detected at the collection site.

[0059] Furthermore, because the long-pass filter 6 is removable, removing it allows capturing a normal optical image of the sample W without fluorescence. Moreover, the imaging position of the smartphone P can be kept constant simply by aligning it with the protrusion 207 of the camera holding part 204, so that fluorescent images and normal optical images of the sample W before and after attaching and detaching the long-pass filter 6 can be captured in the same field of view. This makes it easy to, for example, superimpose the optical image and fluorescent image during subsequent image processing.

[0060] Furthermore, if the smartphone P is equipped with a calculation unit (application) that calculates the content of microplastics in the sample W from the area occupied by microplastics in the image, etc., more information can be obtained on the spot, which is more preferable. In addition, various modifications and combinations of the embodiments may be made as long as they do not contradict the spirit and scope of the present invention.

[0061] According to the present invention described above, it becomes possible to analyze microplastics contained in a sample having a certain area in a short period of time using Raman spectroscopy.

Claims

1. A sample placement section for placing a sample containing microplastics stained with a fluorescent material; a fluorescence excitation light source that irradiates the sample placed on the sample placement section with fluorescence excitation light; a camera holder for holding a portable camera at a position where the camera can capture an image of the sample irradiated with the fluorescence excitation light; A microplastic detection device characterized by comprising an optical filter arranged in front of a portable camera held in the camera holding part, which transmits the fluorescence while blocking the fluorescence excitation light.

2. A microplastic detection device as described in claim 1, configured to detect fluorescence emitted from the sample, identify the location of microplastics in the sample using the fluorescence, irradiate the identified location with Raman excitation light to detect Raman scattered light, and analyze the microplastics at that location based on the Raman scattered light.

3. A sample placement step of placing a sample containing microplastics stained with a fluorescent material on a predetermined sample placement part; a light irradiation step of irradiating the sample placed on the sample placement portion with excitation light for fluorescence; a camera holding step of holding a portable camera at a position where the sample irradiated with the fluorescence excitation light can be imaged; an optical filter arrangement step of arranging an optical filter in front of the portable camera, the optical filter transmitting the fluorescent light and blocking the fluorescent excitation light; and imaging the fluorescence emitted from the microplastics in the sample with the camera.

4. The microplastic detection method described in claim 3 further includes a staining step of fluorescently staining the microplastics in the sample by immersing the sample in a staining solution in which the fluorescent material is dissolved in a specified solvent, and then washing the sample with a cleaning solution capable of removing the fluorescent material attached to components other than microplastics.

5. A microplastic detection method as described in claim 4, wherein the fluorescent substance is Nile red, the solvent is toluene, and the cleaning solution is ethanol or methanol.

6. Detecting fluorescence emitted from the phosphor; Identifying the location of microplastics in the sample by the fluorescence; Irradiating the specified position with Raman excitation light and detecting Raman scattered light; A microplastic detection method described in any one of claims 3 to 5, wherein microplastics at the location are analyzed based on the Raman scattered light.

7. A microplastic detection method as described in claim 6, wherein the wavelength of the Raman excitation light is set to a wavelength that cannot excite a fluorescent substance.