Single micro / nano plastic quality detection method based on Raman answer sheet imaging

Abstract

The invention belongs to the technical field of environmental pollutant detection, and particularly relates to a single micro / nano plastic quality detection method based on Raman answer sheet imaging. According to the method, the ordered SERS substrate and the laser confocal Raman spectrometer system are combined for use, so that the quantitative detection of the quality of the micro / nano plastic is realized; the method specifically comprises the following steps: transferring to-be-detected micro / nano plastic to an SERS substrate, and when the quality of the micro / nano plastic fluctuates, correspondingly changing the Raman peak signal intensity; the specific Raman peak signal intensity is collected through a laser confocal Raman spectrometer, so that the quality of the micrometer / nanometer plastic is judged. According to the invention, a point acquisition function in a Raman spectrometer system and characteristics of an ordered SERS substrate are combined, a Raman'answer sheet 'imaging technology concept is provided, and verification is carried out through standard PS and PMMA microspheres; furthermore, actual food samples are verified, and the method has the advantages of being visual, accurate, rapid and the like, can be used for quality detection of the single-particle micro / nano plastic and has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental pollutant detection, and in particular relates to a method for detecting the quality of a single micro / nano plastic. Background Art

[0002] The persistence and ubiquity of micro- and nanoplastics have become a critical environmental issue worldwide, attracting significant attention from both the scientific and regulatory communities. Nanoplastics are able to penetrate biological barriers and interact with cells at the molecular level, thus posing serious risks to ecosystems and human health. Research has increasingly focused on the effects of the specific size and mass of nanoplastics on cellular and immune responses. As particle size decreases (<50-500nm), nanoplastics have been shown to induce oxidative stress, inflammatory responses, and even cell death in aquatic organisms (e.g., zebrafish), mammals, and humans. However, due to the small size and complex environmental behavior of these particles, it remains challenging to effectively identify and quantify them. This poses a significant obstacle to accurately assessing the risks of nanoplastics to ecosystems and human health.

[0003] Current detection methods, such as thermogravimetric analysis and mass spectrometry, have limitations that hinder comprehensive analysis. Thermogravimetric analysis can accurately measure the mass of nanoplastics by monitoring weight changes during heating and distinguishing chemical components based on thermal degradation temperatures. But it usually requires large amounts of sample and does not provide data on particle morphology or size. Meanwhile, techniques such as matrix-assisted laser desorption / ionization time-of-flight mass spectrometry and inductively coupled plasma mass spectrometry can identify chemical components and estimate molecular weight. However, these methods require high purity and specific sample preparation, often requiring dissolution or fragmentation, which irreversibly changes the original particle structure. A linear relationship between the mass concentration of polystyrene nanoplastics (>10 mg / mL) using an Ag / NbC substrate and the intensity of the surface-enhanced Raman substrate was reported. However, since the surface plasmon coupling effect between disordered surface-enhanced Raman substrate metal nanoparticles varies greatly due to their aggregation state, it is complicated to determine whether a single nanoplastic is enhanced and leads to difficulties in the reproducibility of the surface-enhanced Raman scattering (SERS) signal. Therefore, the potential for single-particle mass quantification using this method remains hindered. So far, there are no reports documenting the quantitative mass analysis of individual nanoplastic particles by Raman methods. However, comprehensive analysis of individual nanoplastic particles is essential, which poses challenges in accurately locating the detection position of Raman spectroscopy. Addressing these challenges is essential to deepen the understanding of the behavior and risks of nanoplastics in the environmental context.

[0004] By exploring the performance of ordered surface enhanced Raman scattering (SERS), we believe it is possible to overcome the bottleneck of single-particle nanoplastic localization and mass quantification. The present invention establishes an accurate logical correlation between the mass of polystyrene and polymethyl methacrylate standard microspheres and their corresponding Raman peak areas. At the same time, the introduction of a novel Raman "answer card" imaging method can intuitively distinguish the location and spectral quality of micro-nanoplastics. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned deficiencies in the prior art and to provide an intuitive, fast and accurate method for quantitatively detecting the mass of a single micro / nano plastic based on Raman "answer sheet" imaging, so as to achieve mass measurement of micro / nano plastics.

[0006] In the present invention, the Raman "answer sheet" imaging technology is a SERS-based nanoplastic detection and quantification method, similar to the way of marking answers on standardized test paper; since the SERS substrate is an array grid structure, the "answer sheet" imaging technology detects the array grid on the SERS substrate and uses each detection point as a potential nanoplastic particle positioning position to achieve rapid scanning and precise positioning of the particles; using the correlation between the characteristic Raman spectrum peak intensity generated by the particles and their mass, the signal is mapped to the array grid to generate an intuitive particle spatial distribution map. In addition, a standard curve established using calibrated PS and PMMA microspheres is used, and the logical relationship between mass and Raman peak area is used as the core. By matching the signal of an unknown particle with a reference standard, its mass can be accurately determined, thereby achieving high-sensitivity detection and quantitative analysis of nanoplastics. The single micro / nano plastic mass quantitative detection method provided by the present invention is based on surface enhanced Raman spectroscopy technology, specifically by combining an ordered SERS substrate with a laser confocal Raman spectrometer to achieve quantitative detection of the mass of micro / nano plastics; the specific steps are:

[0007] The object to be detected (such as a pretreated food container sample) is transferred to the ordered SERS substrate and detected under a Raman spectrometer. The obtained spectrum is preprocessed by subtracting the baseline and cosmic rays through software to obtain the Raman signal of the micro / nano plastic. The Raman spectrometer is then used to collect the specific Raman peak signal intensity to determine the quality of the micro / nano plastic.

[0008] In the present invention, the detection under the Raman spectrometer is specifically to manually select a 5×5 grid each time for detection, the size of the detection area is 20μm×20μm, and then the Raman spectrometer performs automatic detection, and a total of 25 spectra are collected for each grid.

[0009] In the present invention, the specific Raman peak signal refers to the Raman characteristic peak of micro / nano plastics; specifically, the Raman characteristic peak of polystyrene (PS) micro / nano plastics is located at 1601 cm -1 The Raman characteristic peak of polymethyl methacrylate (PMMA) micro / nano plastic is located at 1452cm -1 Place.

[0010] In the present invention, the specific Raman peak signal is the peak area of ​​the characteristic peak of the Raman spectrum.

[0011] The present invention also includes using the same method to establish a logistic function relationship between the Raman peak area and mass of micro / nano plastics.

[0012] In the present invention, the laser confocal Raman spectrometer specifically includes an optical microscope, a laser light source, a sample device, a filter, a monochromator (or an interferometer), a detector, and the like.

[0013] Working principle of the method of the present invention: The logistic model can effectively capture the nonlinear relationship observed in the experimental data, reflecting the inherent saturation behavior in Raman scattering. For smaller microspheres, the peak area initially increases rapidly with increasing mass, but as the particle size approaches or exceeds the diffraction-limited spot size, the peak area begins to stabilize. This saturation effect is driven by two key factors: (1) saturation of the scattering cross section, and (2) size-dependent enhancement on the SERS substrate. Initially, the scattering cross section increases with increasing particle size due to enhanced interaction with the incident light. However, as the particle size approaches or exceeds the diffraction-limited spot size, the scattering effect gradually saturates, resulting in a stable Raman peak area. In addition, smaller particles are more effectively localized in these high-intensity regions on the SERS substrate, resulting in stronger Raman signals. This size-dependent enhancement further promotes the rapid increase in peak area for smaller particles, thereby reinforcing the logical trend seen in the experimental data.

[0014] The Raman "answer card" imaging technology is used to measure the mass of a single micro / nano plastic. The specific process is as follows:

[0015] (1) transferring the pretreated sample to be detected to the SERS substrate and placing it on the sample device stage of the laser confocal Raman spectrometer;

[0016] (2) Determine the position and size of the sample to be tested on the substrate through an optical microscope and accurately adjust the focal length;

[0017] (3) Turn on the laser light source and collect the Raman spectrum of the sample on the SERS substrate;

[0018] (4) Establishing a standard curve by comparing the known mass of micro / nano plastics with the corresponding Raman signal intensity;

[0019] (5) Based on the standard curve model, the PS and PMMA standard microspheres of known sizes were detected by Raman point scanning, and the Raman point scanning results were mapped to the matching degree with the standard spectrum to locate the position of micro / nano plastics and obtain the Raman peak signal of specific micro / nano plastics. The specific Raman peak signal and the mass value of micro / nano plastics in the scanning Raman mapping were substituted into the standard curve to verify whether the proposed Raman "answer card" imaging technology is suitable for the quality detection of single micro / nano plastics;

[0020] (6) Detecting the Raman imaging spectrum of the sample on the SERS substrate by Raman point scanning, mapping the signal to match the standard spectrum, generating a Raman "answer card" imaging result, and obtaining the Raman signal peak intensity of the sample;

[0021] (7) Detect the SEM image of the sample and calculate the mass of the single micro / nano plastic detected in the substrate using ImageJ.

[0022] The mass calculated by ImageJ of the Raman signal peak intensity of the sample was put into the standard curve to verify the accuracy of the established standard curve.

[0023] Beneficial effects of the present invention: By combining surface enhanced Raman technology with Raman "answer card" imaging technology, the mass of a single micro / nano plastic as low as femtogram level can be directly and quantitatively measured. This method can detect nanoplastics with a mass as low as 25.65 femtograms. The intensity change of the nanoplastic spectrum and the "answer card" imaging technology in the laser confocal Raman spectrometer system can intuitively display the position of the micro / nano plastic and its spectral quality. The nanosilver used for the SRES substrate improves the signal intensity of the Raman spectrum by enhancing the group spectral vibration state, thereby ensuring the accuracy of the measured micro / nano plastic mass and realizing the detection of the micro / nano plastic mass.

[0024] This method combines the "answer card" imaging technology in the Raman spectrometer system to measure the sample quality through the change in the Raman peak intensity of micro / nano plastics. It has the advantages of being intuitive, accurate, and fast. It can be used for the quality detection of single micro / nano plastics and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a diagram of the single micro / nano plastic quality detection method of the present invention.

[0026] Figure 2is the Raman detection result of the standard PS ball and the Raman detection result based on the logistic function relationship between the PS Raman peak area and mass. Where a is the Raman detection result of the standard PS ball, and the Raman shift is 200 cm -1 -3200cm -1 , b is the Raman detection result of the standard PS ball, the Raman shift is 1550cm -1 -1650cm -1 , c is the logistic function relationship between the Raman peak area and mass of standard PS spheres with sizes of 360nm, 500nm, 700nm, 1000nm and 2μm, d is the standard curve of the standard PS spectrum results with sizes of 360nm, 500nm and 1000nm, e is a pseudo-color picture of the matching results of the 360nm PS spectrum and the standard spectrum, presented in the form of an "answer sheet", f is a pseudo-color picture of the matching results of the 500nm PS spectrum and the standard spectrum, presented in the form of an "answer sheet", and g is a pseudo-color picture of the matching results of the 1000nm PS spectrum and the standard spectrum, presented in the form of an "answer sheet".

[0027] Figure 3 is the Raman detection result of the standard PMMA ball and the logistic function relationship between the PMMA Raman peak area and mass. Where a is the Raman detection result of the standard PMMA ball, and the Raman shift is 200cm -1 -3200cm -1 , b is the Raman detection result of the standard PS ball, the Raman shift is 1550cm -1 -1650cm -1 , c is the logistic function relationship between the Raman peak area and mass of standard PMMA balls with sizes of 360nm, 500nm, 1000nm, 2μm, and 5μm, d is the standard curve of the matching results of standard PMMA spectra with sizes of 360nm, 500nm, and 1000nm, e is a pseudo-color picture of the matching results of the 360nm PMMA spectrum and the standard spectrum, presented in the form of an "answer sheet", f is a pseudo-color picture of the matching results of the 500nm PMMA spectrum and the standard spectrum, presented in the form of an "answer sheet", and g is a pseudo-color picture of the matching results of the 1000nm PMMA spectrum and the standard spectrum, presented in the form of an "answer sheet".

[0028] Figure 4The figure is the "answer sheet" test result of PS micro / nano plastics in disposable PS plastic lunch boxes. Among them, a is the Raman test result of PS micro / nano plastics (1-6) in plastic lunch boxes, b is the standard curve matching the spectrum result of PS micro / nano plastics (1-6) in plastic lunch boxes, c is the pseudo-color image of the matching result of PS micro / nano plastics (1-6) in plastic lunch boxes and the standard spectrum, presented in the form of an "answer sheet", and d is the SEM image of PS micro / nano plastics (1-6) in plastic lunch boxes. DETAILED DESCRIPTION

[0029] The present invention is further described below by way of embodiments in conjunction with the accompanying drawings.

[0030] In this embodiment, the specific process of establishing a standard curve, proposing and verifying the "answer card" imaging technology, and verifying the "answer card" imaging technology with actual sample test results is as follows: Figure 1 As shown, the specific steps are as follows:

[0031] (i) Establishing the logical relationship between the mass of micro / nano plastics and the Raman peak area;

[0032] (1) Use a pipette to take 2 μL of 360 nm, 500 nm, 700 nm, 1000 nm, 2 μm PS and 2 μL of 360 nm, 500 nm, 1000 nm, 2 μm, 5 μm PMMA standard sample diluted solutions and drop them on the SERS substrate. Wait for the solution to dry;

[0033] (2) placing the dried SERS substrate on a sample device in a laser confocal Raman spectrometer system, and obtaining a clear image of a single micro / nano plastic standard by moving the stage of the sample device and focusing the objective lens of the optical microscope;

[0034] (3) Turn on the laser, select appropriate laser and acquisition parameters, and collect Raman signals of micro / nano plastics; the laser used in this embodiment is 532 nm, the acquisition time is 10 seconds, and the number of acquisitions is 1;

[0035] (4) Fitting the specific Raman peak signal in the collected Raman spectrum with the mass value of the micro / nano plastic to establish a standard curve, such as Figure 2 c and Figure 3 As shown in c.

[0036] (ii) Verify Raman “answer sheet” imaging technology;

[0037] (1) Use a pipette to pipette 2 μL of 360 nm, 500 nm, 1000 nm PS and 2 μL of 360 nm, 500 nm, 1000 nm PMMA standard sample diluted solutions onto the SERS substrate and wait for the solutions to dry;

[0038] (2) placing the dried SERS substrate on a sample device in a laser confocal Raman spectrometer system, and obtaining a clear image of the sample by moving the stage of the sample device and focusing the objective lens of the optical microscope;

[0039] (3) Turn on the laser, select the appropriate laser and acquisition parameters, manually select a 5×5 grid (20μm×20μm), and collect Raman signals of micro / nano plastics. The laser used is 532nm, the acquisition time is 10 seconds, and the number of acquisitions is 1;

[0040] (4) Mapping the collected Raman spectrum set to match the standard spectrum, using the Raman "answer card" technology, such as Figure 2 For example, Figure 3 e.g.

[0041] (5) Substitute the specific Raman peak signal and the mass value of micro / nano plastics in the scanning Raman mapping into the standard curve to verify the Raman "answer card" technology, such as Figure 2 d and Figure 3 As shown in d.

[0042] (iii) Testing actual samples to verify the Raman “answer sheet” imaging technology;

[0043] The detection steps for detecting actual samples to verify the Raman "answer sheet" imaging technology are similar to the steps proposed and verified in this embodiment for the Raman "answer sheet" imaging technology, and are as follows:

[0044] (1) Transfer the pretreated sample to the SERS substrate and wait for the solution to dry;

[0045] (2) placing the dried SERS substrate on the sample device stage in the laser confocal Raman spectrometer system, and obtaining a clear image of the sample by moving the sample device stage and magnifying and focusing the optical microscope objective lens;

[0046] (3) Turn on the 532 nm laser, select the acquisition time as 10 seconds, the acquisition number as 1, and manually select a 5×5 grid (20 μm×20 μm) to collect the Raman signal of micro / nano plastics.

[0047] (4) Mapping the collected Raman spectrum set to match the standard spectrum, such as Figure 4 As shown in c;

[0048] (5) SEM images of in-situ test samples, such as Figure 4 As shown in d, the sample quality was obtained by ImageJ calculation;

[0049] (6) Substitute the specific Raman peak signal and the mass value of micro / nano plastics in the scanning Raman mapping into the standard curve to verify the Raman "answer card" technology, such as Figure 4As shown in b.

[0050] In order to improve the stability of nanoplastic detection, surface enhanced Raman technology based on nano-Ag modified SERS substrate was used.

[0051] Compared with the latest reported micro / nano plastic quality detection technology, the quantitative detection method of single micro / nano plastic quality based on Raman "answer card" imaging has good accuracy, wide adaptability, simpler detection, and can be used for actual sample detection. Moreover, SEM images can be detected in situ to obtain various information such as the morphology and size of micro / nano plastics.

[0052] Application areas: environment, food, medicine, bioengineering, chemical engineering and scientific research.

[0053] The preferred specific embodiments of the present invention are described in detail above. However, it should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work, such as replacement of spectral instruments, replacement of SERS substrates, and replacement of micro / nano plastic materials. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A single micro / nano plastic quality detection method based on Raman "answer sheet" imaging, characterized in that: The ordered SERS substrate is combined with a laser confocal Raman spectrometer to achieve quantitative detection of the quality of micro / nano plastics; the SERS substrate is an array grid structure, and the "answer sheet" imaging technology detects the point array grid on the SERS substrate, and each detection point is used as a potential nanoplastic particle location to achieve rapid scanning and precise positioning of the particles; the correlation between the characteristic Raman spectrum peak intensity generated by the particles and their mass is used to map the signal to the point array grid to generate an intuitive particle spatial distribution map; the specific steps are: The object to be detected is transferred to the ordered SERS substrate, and detected under a Raman spectrometer, and the baseline and cosmic rays are subtracted from the obtained spectrum to obtain the Raman signal of the micro / nano plastic; A Raman spectrometer is then used to collect the signal intensity of specific Raman peaks to determine the quality of micro / nano plastics.

2. The single micro / nano plastic quality detection method according to claim 1 is characterized in that: The detection under the Raman spectrometer is performed by manually selecting a 5×5 grid each time, and the size of the detection area is 20 μm×20 μm, and then the Raman spectrometer performs automatic detection, and a total of 25 spectra are collected for each grid; The specific Raman peak signal refers to the Raman characteristic peak of micro / nano plastics; specifically, the Raman characteristic peak of polystyrene (PS) micro / nano plastics is located at 1601 cm -1 The Raman characteristic peak of polymethyl methacrylate (PMMA) micro / nano plastic is located at 1452 cm -1 The specific Raman peak signal is the peak area of ​​the characteristic peak of the Raman spectrum.

3. The single micro / nano plastic quality detection method according to claim 2 is characterized in that: It also includes establishing the logistic function relationship between the Raman peak area and mass of micro / nano plastics.

4. The single micro / nano plastic quality detection method according to claim 3 is characterized in that: The laser confocal Raman spectrometer comprises an optical microscope, a laser light source, a sample device, an optical filter, a monochromator or an interferometer, and a detector.

5. The single micro / nano plastic quality detection method according to claim 4 is characterized in that: The specific process is: (1) transferring the pretreated sample to be detected to the SERS substrate and placing it on the sample device stage of the laser confocal Raman spectrometer; (2) Determine the position and size of the sample to be tested on the substrate through an optical microscope and accurately adjust the focal length; (3) Turn on the laser light source and collect the Raman spectrum of the sample on the SERS substrate; (4) Establishing a standard curve by comparing the known mass of micro / nano plastics with the corresponding Raman signal intensity; (5) Based on the standard curve model, the PS and PMMA standard microspheres of known sizes were detected by Raman point scanning, and the Raman point scanning results were mapped to the matching degree with the standard spectrum to locate the position of micro / nano plastics and obtain the Raman peak signal of specific micro / nano plastics. The specific Raman peak signal and the mass value of micro / nano plastics in the scanning Raman mapping were substituted into the standard curve to verify whether the proposed Raman "answer card" imaging technology is suitable for the quality detection of single micro / nano plastics; (6) Detecting the Raman imaging spectrum of the sample on the SERS substrate by Raman point scanning, mapping the signal to match the standard spectrum, generating a Raman "answer card" imaging result, and obtaining the Raman signal peak intensity of the sample; (7) Detect the SEM image of the sample and calculate the mass of the single micro / nano plastic detected in the substrate using ImageJ.

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