A method for monitoring and imaging the interaction between label-free nanoplastics and single cells

Abstract

The present invention discloses a method for monitoring and imaging the interaction between label-free nanoplastics and single cells, which includes: placing a sensing chip attached to cells on SPRM, and recording the cell SPR images during the interaction process between the cells and nanoplastics on the sensing chip; after comparing the cell SPR images with the cell bright-field images to determine the corresponding regions of single cells in the SPR images, obtaining the deformation data of the cell membranes during the interaction between single cells and nanoplastics according to the SPR signal intensity of the single cell regions, realizing the monitoring of the interaction between nanoplastics and single cells at single-cell resolution, and providing technical support for studying the binding kinetics and cytotoxicity of nanoplastics.

Description

Technical Field

[0001] The present invention relates to the field of cell imaging technology, and particularly relates to a method for monitoring and imaging the interaction between label-free nanoplastics and single cells. Background Art

[0002] As a new type of pollutant, the harm of nanoplastics has attracted much attention. Their extremely small size gives them a very high specific surface area, enabling them to easily penetrate biological membranes and disrupt cell metabolic activities. Therefore, it is crucial to study the interaction between nanoplastics and cells. Many analytical methods have been used in such studies, but they all have certain limitations:

[0003] (1) Fluorescence microscopy and flow cytometry are widely used in related research and have extremely high resolution. For example, CN111850083A discloses a method for detecting the toxicity of nanoplastics, including: after pre-culturing Microcystis aeruginosa in a medium, exposing it to the nanoplastics to be tested, and evaluating the toxic effects of nanoplastics on the growth and / or metabolite of Microcystis aeruginosa by the growth inhibition rate of Microcystis aeruginosa, the content of chlorophyll a, the fluorescence intensity of phycoerythrin, the production and release of microcystin, and / or the changes in the metabolites of Microcystis aeruginosa. By detecting physiological and biochemical indexes, it can be preliminarily judged whether the sample to be tested is contaminated by nanoplastics, providing a new method for the toxicological detection of nanoplastics.

[0004] CN118546282A discloses an aggregation-induced emission fluorescent material and a nanoplastics particle doped with palladium metal for visualizing and quantitatively detecting the distribution law and migration and transformation dynamic process of nanoplastics in organisms.

[0005] However, these methods usually require labeling the nanoplastics with fluorescent dyes. The surface attachment of the fluorescent dyes will change the surface properties of the nanoplastics, thus changing their interaction mode with cells and affecting the reliability of experimental data. In addition, many fluorescence-based analytical methods use end-point measurements to collect signals at discrete time points and cannot provide continuous kinetic data on the binding of nanoplastics to cell membranes.

[0006] (2) Methods such as surface plasmon resonance and quartz crystal microbalance can achieve label-free real-time measurement, but such technologies are limited to the study of the binding process between model lipid structures and nanoplastics and have limited analytical capabilities for real cells. Summary of the Invention

[0007] In view of the limitations of existing methods in the label-free and real-time monitoring of the interaction between nanoplastics and cells, the present invention provides a method for monitoring the interaction between single cells and nanoplastics based on surface plasmon resonance imaging. This method can measure the interaction between nanoplastics and cell membranes in situ at single-cell resolution, and accurately analyze the nanoscale deformation of cell membranes during the binding and dissociation processes of nanoplastics, thereby providing technical support for studying the binding kinetics and cytotoxicity of nanoplastics.

[0008] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A label-free monitoring imaging method for the interaction between nanoplastics and single cells, comprising the steps of:

[0010] Step 1: Incubate cells on a sensing chip to obtain a sensing chip with adhered cells.

[0011] Step 2: Place the sensing chip with adhered cells on the SPRM, and obtain a bright-field image of the cells on the sensing chip through illumination by the bright-field light source of the SPRM.

[0012] Step 3: Adjust the imaging mode of the SPRM (surface plasmon resonance microscope) to SPR, adjust the incident angle of the laser to generate an evanescent field on the sensing chip, and record the cell SPR images during the interaction between the cells and nanoplastics on the sensing chip.

[0013] Step 4: Compare the cell SPR images obtained in Step 3 with the bright-field images of the cells in Step 2 to determine the corresponding regions of individual cells in the SPR images.

[0014] Step 5: Obtain the deformation data of the cell membrane during the interaction between individual cells and nanoplastics based on the SPR signal intensity of the corresponding regions of individual cells in the SPR images, and realize the monitoring of the interaction between nanoplastics and single cells.

[0015] In the present invention, a surface plasmon resonance microscope is used to record images during the binding process of nanoplastics and cells. After determining the corresponding regions of individual cells in the SPR images in Step 4, the deformation data of the cell membrane during the interaction between individual cells and nanoplastics is obtained based on the SPR signal intensity of the corresponding regions of individual cells in the SPR images. This method can measure the interaction between nanoplastics and cell membranes in situ at single-cell resolution, and accurately analyze the nanoscale deformation of cell membranes during the binding and dissociation processes of nanoplastics, thereby providing technical support for studying the binding kinetics and cytotoxicity of nanoplastics.

[0016] Specifically applicable to the real-time monitoring of the binding process between single cells and nanoplastics: Obtain the SPR signal intensity of the single-cell region according to the SPRM imaging results, and convert it into the average distance of the cell from the chip surface according to the above formula. Reflect the strength of the interaction between cells and nanoparticles through the changes of cells during the binding process of nanoplastics, and then infer the cytotoxicity of nanoplastics to cells.

[0017] Heterogeneity analysis of the interaction between cells and nanoplastics: Used to analyze the heterogeneous behaviors of different cells during the binding process with nanoparticles under the same conditions, facilitating subsequent research on the mechanism of the interaction between nanoplastics and cells.

[0018] Research on the cytotoxicity of nanoplastics with different properties: Used to monitor the effects of diameter, material, and surface modification on the interaction process between nanoplastics and cells, providing a reference for evaluating the cytotoxicity and environmental toxicity of nanoplastics with different properties.

[0019] The interaction process between cells and nanoplastics in step 3 includes a baseline stage, a binding stage, and a dissociation stage; in the baseline stage, buffer solution is flowed into the sensing chip, in the binding stage, nanoplastic solution is flowed into the sensing chip, and in the dissociation stage, buffer solution is flowed into the sensing chip.

[0020] The concentration of the nanoplastic solution is 0 - 250 μg / mL, and the three stages of the baseline stage, binding stage, and dissociation stage are repeated for testing in ascending order of concentration.

[0021] The relationship between the SPR signal intensity and the deformation of the cell membrane in step 5 is: , is the reflectivity of the SPR image, is the average distance of the cell from the sensing chip, is a constant, reflecting the inherent relationship between distance and reflectivity in the area to be measured.

[0022] Reflectivity change is calculated and extracted from the SPR image. The calculation method is: after determining the corresponding area of a single cell in the SPR image in step 4, calculate the average image gray value (defined as the SPR signal intensity I ) in each single-cell area through Fiji. I and the reflectivity R are linearly correlated, and the formula is , β is a constant related to the instrument.

[0023] In step 1, the coverage rate of cells on the sensing chip is 20 - 50%. This coverage rate ensures that there are a large number of cells in the image and that the cells mostly exist in the form of single cells.

[0024] The particle size of the nanoplastic is 20 to 1000 nm. The method of the present invention can be used for nanoplastics of various particle sizes.

[0025] The nanoplastic includes one or more of nano polyethylene (NPE), nano polypropylene (NPP), nano polystyrene (NPS), nano polyethylene terephthalate (NPET), and nano nylon 6 (NPA6), or a nanoplastic with chemically modified surface groups.

[0026] The SPRM includes a microscope based on lens-excited SPR and a prism-excited SPR microscope.

[0027] The sensing chip is a glass sheet with nano gold or silver on its surface;

[0028] The buffer solution is a buffer solution friendly to cell growth.

[0029] The cells are adherent cells, including live cells and / or cells fixed with paraformaldehyde solution.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] Based on the surface plasmon resonance imaging technology, the present invention can monitor the binding process of cells and nanoplastics in real time with single-cell resolution without labeling, with high sensitivity and high throughput, and obtain kinetic parameters during the action process. The present invention does not need to introduce fluorescent dyes for labeling, and the surface properties of the nanoplastic will not change, ensuring the accuracy of the results; there are multiple cells in one camera field of view, and multiple cell signals can be obtained simultaneously, improving the detection efficiency; due to the existence of the evanescent field, the present invention is very sensitive to the change of cell height, providing a powerful tool for accurately monitoring the interaction process between cells and nanoparticles.

[0032] This method can be applied to environmental fields such as pollution monitoring and toxicity research of nanoplastics, and can also be applied to biological and medical fields such as delivery of nano drugs and research on the binding mechanism between nanoparticles and cell surfaces. Description of the Drawings

[0033] Figure 1 In [Figure], A is a schematic diagram of the deformation of cells caused by the binding of nanoplastic and cells; Figure 1 In [Figure], B is the process schematic diagram of the deformation of the cell membrane during the binding process of nanoplastic and cells, resulting in the change of the average distance ( ) between the cell chassis and the chip surface; Figure 1 In [Figure], C is the reflectance of the cell region obtained by simulation with WinSpall software at different R as a function of the SPR angle; Figure 1 In [Figure], D is the reflectance of the cell region at 70.5° as a function of The changed image.

[0034] Figure 2 In A, it is a schematic diagram of the surface plasmon resonance microscope device used in the embodiment; Figure 2 In B, it is the bright-field image in the field of view collected by the camera and the corresponding area's SPR image. Each single-cell area in the bright-field image corresponds to each bright area in the SPR image, and the scale bar is 100 μm; Figure 2 In C, it is a schematic diagram of the change of the cell deformation signal at different stages during the single-cell deformation measurement.

[0035] Figure 3 In A, it is a representative SPR image during the measurement of the interaction between the fixed cells and 200 nm PSNPs with a concentration of 25 μg / mL in Example 1. The blue frame line is the single-cell area, the red frame line is the background area, and the scale bar is 100 μm. Figure 3 In B, it is the change of the SPR signal of the entire camera field of view during the measurement of the interaction between the fixed cells and 200 nm PSNPs with a concentration of 25 μg / mL in Example 1, Figure 3 In C, it is the change of the SPR signal in the background area where there are no cells during the measurement process, Figure 3 In D, it is the change of the SPR signal value in a single-cell area during the measurement process.

[0036] Figure 4 In A, it is the change of the cell deformation size during the measurement of the interaction between the single-cell areas of 10 fixed cells and 200 nm PSNPs with different concentrations in Example 1. The inset is the magnification of the framed area. Figure 4 In B, it is from Figure 4 In A, it is a statistical chart of the cell deformation sizes of 10 fixed cells caused by 200 nm PSNPs with different concentrations.

[0037] Figure 5 In A, it is the measurement of the cell deformation caused by the interaction between PSNPs with different particle sizes and fixed cells at different concentrations in Example 2. Figure 5 In B, it is the measurement of the cell deformation caused by the interaction between PSNPs with different particle sizes and live cells at different concentrations in Example 2. Figure 5 In C, it is according to Figure 5 In A and B, it is a comparison chart of the deformation sizes of fixed cells and live cells caused by PSNPs with different particle sizes at a concentration of 125 μg / mL.

[0038] Figure 6In A, the confocal imaging results at 20× magnification before (left) and after (right) incubation of 200 nm PSNPs at 25 μg / mL with fixed cells are shown. The red color represents the cell membrane, stained with CellMask (red), and the cyan color represents Nile red-labeled 200 nm PSNPs. The scale bar is 50 μm. Figure 6 In B, Figure 6 In A, the confocal and stimulated emission depletion (STED) imaging results at 60× magnification of the framed part are shown. Figure 6 In C, Figure 6 In B, the z-axis scanning results of STED imaging along the orange line of the cells shown are presented. The scale bars on both the x-axis and z-axis are 5 μm.

[0039] Figure 7 In A, the confocal imaging at 20× magnification before (left) and after (right) incubation of 200 nm PSNPs at 25 μg / mL with live cells is shown. Figure 7 In B, Figure 7 In A, the confocal and STED imaging results at 60× magnification of the framed part are shown; Figure 7 In C, Figure 7 In B, the z-axis scanning results of STED imaging along the orange line of the cells shown are presented. The scale bars on both the x-axis and z-axis are 5 μm.

[0040] Figure 8 In A, the cell deformation caused by the interaction of 200 nm PSNPs with different surface modifications at different concentrations with fixed cells in Example 3 is measured. Figure 8 In B, the cell deformation caused by the interaction of 200 nm PSNPs with different surface modifications at different concentrations with live cells in Example 3 is measured. Figure 8 In C, Figure 8 In A and Figure 8 In B, a comparison chart of the deformation sizes of fixed cells and live cells caused by 200 nm PSNPs with different surface modifications at a concentration of 250 μg / mL is obtained.

[0041] Figure 9 In A, the cell deformation caused by the interaction of 200 nm nanoplastics with different materials at different concentrations with fixed cells in Example 4 is measured. Figure 9 In B, the cell deformation caused by the interaction of 200 nm nanoplastics with different materials at different concentrations with live cells in Example 4 is measured. Figure 9 In C, Figure 9 In A and Figure 9 In B, a comparison chart of the deformation sizes of fixed cells and live cells caused by 200 nm nanoplastics with different materials at a concentration of 250 μg / mL is obtained. Detailed implementation manners

[0042] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Those skilled in the art who make modifications or equivalent substitutions based on the understanding of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention.

[0043] A schematic diagram of the cell deformation caused by the binding process of cells and nanoplastics is as shown in Figure 1 A and Figure 1 B in the figure. During the binding process of nanoplastics and cells, the cell membrane will deform, and the adhesion to the sensing chip will decrease. By calculating the average image gray value in each single-cell region I , it is converted into SPR reflectivity R , and then converted into the change in the average distance between the cell and the chip surface . The strength of the interaction between the cell and the nanoparticle is reflected by the change of the cell during the binding process of the nanoplastics, and then the cytotoxicity of the nanoplastics is inferred.

[0044] As shown in Figure 1 C in the figure, the WinSpall software is used to simulate the SPR signals of different cell heights to obtain the reflectivity image of the average distance between different cells and the chip surface changing with the SPR angle; the reflectivity of the cell region at 70.5° is plotted against the change of to obtain as shown in Figure 1 D in the figure. It can be seen that the average distance between the cell and the chip surface has a linear relationship with the reflectivity of the cell region in the SPR image.

[0045] The raw materials used in the following specific embodiments are all purchased from the market. The surface plasmon resonance microscope used is SPRm 200 (Biosensing Instrument);

[0046] The change of the SPR signal during the interaction process between the cell and the nanoplastics is measured by the device shown in Figure 2 A in the figure. Among them, 1 is a bright-field signal camera, 2 is an SPR signal camera, 3 is a laser, 4 is a lens, 5 is a prism, 6 is a gold nanolayer, 7 is a cell, 8 is a nanoplastics, and 9 is an LED light source. The specific experimental process of the test is as follows:

[0047] Step 1: After assembling the cell culture chamber with the clean sensing chip, inject an appropriate amount of cell suspension solution into it, and culture it overnight in a humidified sterile incubator at 37°C with 5% CO2 to allow the cells to fully adhere to the sensing chip. The cell coverage rate on the sensing chip is approximately 30%. The cells for the fixed cell test are the cells fixed with 4% paraformaldehyde solution for 30 minutes.

[0048] Dilute the stock solutions of different nano-plastic materials and different nano-plastic particle sizes with phosphate buffer to prepare nano-plastic solutions with concentrations ranging from low to high, with a concentration range of 0 - 250 μg / mL, and set aside for use.

[0049] Step 2: Place the sensing chip with adhered cells on the SPRM. Through the bright-field light source illumination of the SPRM, obtain the bright-field image of the cells on the sensing chip by the bright-field signal camera 1, as shown in Figure 2 B in

[0050] Step 3: Adjust the imaging mode of the SPRM to SPR, adjust the laser incident angle to 70.5° to generate an evanescent field on the sensing chip, and record the cell SPR images during the interaction between the cells and the nano-plastic on the sensing chip by the SPR signal camera 2, as shown in Figure 2 B in

[0051] The process of the interaction between the cells and the nano-plastic in Step 3 includes a baseline stage, a binding stage, and a dissociation stage; in the baseline stage, buffer solution is flowed into the sensing chip, in the binding stage, nano-plastic solution is flowed into the sensing chip, and in the dissociation stage, buffer solution is flowed into the sensing chip.

[0052] Step 4: Compare the cell SPR images obtained in Step 3 with the cell bright-field images in Step 2 to determine the corresponding regions of individual cells in the SPR images;

[0053] Step 5: Calculate the average image gray value (defined as the SPR signal intensity I ) within each single-cell region by Fiji, and plot it against time to obtain the I-t graph of single-cell single measurement. Then convert I to reflectivity R ( ), and calculate the average distance change of the cells from the sensing chip according to to achieve the monitoring of the interaction between nano-plastic and single cells. Among them, .

[0054] In step 3, the baseline stage, the binding stage, and the dissociation stage can be repeated with nanoplastic solutions of increasing concentrations to obtain a series of image sequences and the interaction patterns between cells and nanoplastics of different concentrations. Alternatively, the interaction patterns between solutions of different nanoplastic particle sizes and cells can be tested, or the interaction patterns between nanoplastics of different materials and cells can be tested, or the interaction patterns between different cell types and nanoplastics can be tested.

[0055] As Figure 2 shown in C, during the baseline stage, when PBS buffer was introduced into the cell culture medium, no nanoplastics bound to the cells, the cell SPR signal hardly changed, and the cell morphology remained basically unchanged. During the binding stage, a certain concentration of nanoplastics was introduced into the above cell culture chamber, and the nanoplastics bound to the cells, causing changes in cell morphology and the average distance of the cell chassis from the chip surface, resulting in changes in the SPR signal in the cell region. During the dissociation stage, pure PBS buffer solution was passed through the above cell culture chamber again to wash the cells in the binding stage. The nanoplastics detached from the cell surface or were endocytosed into the cells, and the interaction pattern between the nanoplastics and the cells changed, as did the trend of the cell SPR signal.

[0056] Example 1

[0057] Using the surface plasmon resonance microscope shown in A Figure 2 , the interaction process between 200 nm PSNPs of different concentrations and fixed cells was monitored in real time. The nanoplastic solutions used were 200 nm PSNPs solutions with concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, 125 μg / mL, and 250 μg / mL, and the cells used were HEK293 fixed cells.

[0058] As Figure 3 shown in A, the representative SPR image during the interaction measurement between fixed cells and 200 nm PSNPs at a concentration of 25 μg / mL. The blue frame line is the single-cell region, the red frame line is the background region, and the scale bar is 100 μm.

[0059] As Figure 3 shown in B- Figure 3 shown in D in Example 1, the changes in the SPR signal values in different regions of the sensing chip during the interaction measurement between fixed cells and 200 nm PSNPs at a concentration of 25 μg / mL. The black arrow indicates the time when 25 μg / mL 200 nm PSNPs began to be introduced into the cell culture chamber, and the orange arrow indicates the time when pure buffer solution began to be introduced into the cell culture chamber.

[0060] The curve of the entire sensing chip (Figure 3 Middle B) represents the result obtained by conventional SPR sensor, which shows the difference with the background area ( Figure 3 (C, where the black line is the original It curve and the red line is the fitted line) show similar kinetic patterns and signal amplitudes, demonstrating that at the cell density required for single-cell imaging, the signal of nanoplastic binding to cells measured by the traditional sensor system is mainly caused by the background signal (PSNP adhered to the sensor surface).

[0061] By fitting with first-order kinetics (red curve to background) Figure 3 The background sensor diagram of B in Figure 2 gives the binding rate constant , dissociation rate constant and affinity As shown below: =(1.70±0.09)×10 8 M -1 s -1 , =(2.89±0.08)×10 -4 s -1 ,and = (1.71 ± 0.09) pM. The extremely low affinity indicates that the binding of nanoplastics to the background is a highly irreversible adsorption process.

[0062] and Figure 3 Compared with the background area in B, the single cell area ( Figure 3 The black curve in Figure 5D shows a decrease in the SPR signal, shifting toward the negative direction and exhibiting a much smaller amplitude. This signal is hidden in the background of conventional biosensors and cannot be detected by them. This highlights a major limitation of conventional biosensors: their inability to separate useful cell-specific signals from background noise, leading to potentially erroneous conclusions. Our present invention overcomes this limitation by providing spatially resolved SPR imaging, enabling us to capture subtle changes in single-cell behavior.

[0063] like Figure 4 Figure A shows the changes in cell deformation during the interaction between 10 fixed cell regions and 200 nm PSNPs at different concentrations. Figure 4 B is Figure 4 The statistical diagram of the deformation of 10 fixed cells caused by different concentrations of 200 nm PSNPs obtained in Figure A. The height of the cell chassis from the chip surface during the binding phase ( )Continuously increasing, the dissociation stage remains basically unchanged, indicating that the degree of cell deformation increases during the binding stage, and the cell morphology remains basically unchanged during the dissociation stage. The amplitude of cell deformation during the dissociation stage also proves that the adsorption of PSNPs on the cell membrane surface is relatively tight and difficult to elute. The size of cell deformation is also related to the particle concentration, and the greater the concentration, the more obvious the change in cell deformation. At the same time, there are also significant differences in the responses of different cells to the same concentration of PSNP solution, showing heterogeneity among cells.

[0064] Example 2

[0065] Using the surface plasmon resonance microscope as shown in Figure 2 Figure A, the interaction processes between fixed cells and live cells and PSNPs with different diameters were monitored in real time. The nano-plastic solutions used were PSNP solutions with different concentrations and diameters of 20 nm, 50 nm, 100 nm, 200 nm, and 1000 nm respectively. The cells used were HEK293 fixed cells or HEK293 live cells.

[0066] Figure 5 Figure A shows the cell deformation caused by the interaction between PSNPs with different particle sizes and fixed cells at different concentrations. The solid line is the average value of multiple single cells, and the shadow is the standard error (the same as in Figure 8 and Figure 9 ). For each particle size of nano-plastic, the change trend is basically the same as the phenomenon in Example 1. For nano-plastics with different particle sizes at the same concentration, the amplitude of cell deformation is significantly different. The response sizes of cells to PSNPs with different particle sizes are 100 nm > 20 nm > 50 nm > 200 nm > 1000 nm in sequence. This non-monotonic sorting may reflect the size-dependent mechanism of membrane deformation and has nothing to do with cell internalization.

[0067] As shown in Figure 6 Figure A are the confocal imaging results of 200 nm PSNPs at 25 μg / mL before (left) and after (right) incubation with fixed cells under a 20-fold microscope. The red color represents the cell membrane, stained with CellMask (red), and the cyan color represents 200 nm PSNPs labeled with Nile red. The scale bar is 50 μm. Figure 6 Figure B in Figure 7 is the imaging result of the framed part in Figure A in Figure 6 under a 60-fold microscope. The white line is the boundary. The upper left part is the confocal imaging diagram, and the lower right part is the STED imaging result. The scale bar is 5 μm. Figure 6 Figure C in Figure 6It is also proved that nanoplastics do not enter fixed cells after incubation for a period of time.

[0068] Figure 5 In B, it is the cell deformation caused by the interaction of PSNPs with different particle sizes at different concentrations with living cells. For living cells of the same particle size, they show different behaviors from fixed cells, which may be due to the fluidity of the living cell membrane. Although their sizes are different from those of fixed cells, the trend of the response of living cells to nanoplastics with different particle sizes is consistent with that of fixed cells: 100 nm > 20 nm > 50 nm > 200 nm > 1000 nm (at a nanoplastic concentration of 125 μg / mL). However, at a lower concentration (such as 25 μg / mL), the sorting order is 20 nm > 50 nm > 100 nm > 200nm > 1000 nm, which is the same as the sorting of particle sizes. This indicates that the interaction mechanism between cells and nanoplastics may be different at different concentrations.

[0069] Figure 5 In C, it is a comparison chart of the deformation sizes of fixed cells and living cells caused by PSNPs with different particle sizes at a concentration of 125 μg / mL obtained according to Figure 4 Figure A and Figure B in it. In the binding stage, the situation of cells is basically the same as that of fixed cells, both showing a downward trend; however, in the dissociation stage, the signal of living cells continues to decrease. This indicates that due to the dynamic nature of the living cell membrane, the interaction between the membrane and PSNP persists, which may involve the active internalization of nanoplastics.

[0070] Figure 7 In A, it is the Confocal imaging at 20 times magnification of 200 nm PSNPs at 25 μg / mL before incubation (left) and after incubation (right) with living cells. Figure 7 In B Figure 7 The imaging result at 60 times magnification of the framed part in Figure A in it. The white line is the boundary. The upper left is the Confocal result, and the lower right is the STED result. The scale bar is 5 μm. Figure 7 In C, it is Figure 7 the z-axis scanning result of the cells shown in Figure B in it using STED imaging along the orange line part. The scale bar on the x-axis and the scale bar on the z-axis are both 5 μm. It is also proved that living cells endocytose some nanoplastics after incubation for a period of time.

[0071] Example 3

[0072] Using as Figure 2The surface plasmon resonance microscope shown in A was used to monitor in real time the interaction processes of fixed cells and live cells with 200 nm PSNPs with different surface modifications. The nanoplastic solutions used were 200 nm PSNP solutions without modification, surface-modified with amino groups, and surface-modified with carboxyl groups. The cells used were fixed HEK293 cells or live HEK293 cells.

[0073] As Figure 8 shown in A, during the interaction process of PSNPs with different surface modifications and fixed cells, the COOH-modified PSNP showed the highest level of interaction, almost twice the signal of the unmodified 200 nm PSNP. In contrast, the NH2-modified PSNP only produced about one-third of the original response. As Figure 8 shown in B, the measurement of the interaction of PSNPs with different surface modifications and live cells. Different from fixed cells, live cells showed the weakest response to COOH-modified PSNP (only 1 / 5 of the response of fixed cells). Figure 8 Shown in C is Figure 8 based on A and Figure 8 B in Figure, a comparison chart of the deformation sizes of fixed cells and live cells caused by 200 nm nanoplastics of different materials with a concentration of 250 μg / mL. This phenomenon once again illustrates the key role of membrane flexibility and fluidity in regulating nanoplastic binding.

[0074] Example 4

[0075] Using the surface plasmon resonance microscope shown in A, the interaction processes of fixed cells and live cells with 200 nm nanoplastics of different materials were monitored in real time, and the specific steps were as shown above. The nanoplastic solutions used were 200 nm PENP, PETNP, and PSNP solutions, and the cells used were fixed HEK293 cells or live HEK293 cells. Figure 2 For the interaction of nanoplastics of different materials with fixed cells as shown in A, PENP and PETNP generally showed weaker responses than PSNP, following the trend of PSNP > PENP > PETNP. This may be due to the inherent properties of the materials, such as surface chemistry and hydrophobicity, which have different effects on the degree of membrane deformation.

[0076] As Figure 9 shown in A, for the interaction of nanoplastics of different materials with fixed cells, PENP and PETNP generally showed weaker responses than PSNP, following the trend of PSNP > PENP > PETNP. This may be due to the inherent properties of the materials, such as surface chemistry and hydrophobicity, which have different effects on the degree of membrane deformation.

[0077] As Figure 9 shown in B, when measuring the interaction process of live cells with nanoplastics of different materials, although the ranking trend of the interaction between nanoparticles and live cells is the same as that of fixed cells, the signal amplitude is 1 to 3 times higher. Figure 9 Shown in C is Figure 9 based on A and Figure 9Figure showing the comparison of the deformation sizes of fixed cells and live cells caused by 200 nm nanoplastics of different materials with a concentration of 250 μg / mL in B. This may reflect that the live cell membrane has a stronger deformation ability, and compared with the rigid fixed membrane of fixed cells, the live cell membrane can interact more strongly with nanoparticles.

Claims

1. A method for monitoring and imaging the interaction between label-free nanoplastics and single cells, characterized in that Including the steps: Step 1: Incubate cells on the sensing chip to obtain a sensing chip with adherent cells. Step 2: Place the sensing chip with adherent cells on the SPRM, and obtain the bright-field image of the cells on the sensing chip through the bright-field light source illumination of the SPRM. Step 3: Adjust the imaging mode of the SPRM to SPR, adjust the incident angle of the laser to generate an evanescent field on the sensing chip, and record the cell SPR images during the interaction process between the cells and the nanoplastics on the sensing chip. Step 4: Compare the cell SPR images obtained in Step 3 with the cell bright-field images in Step 2 to determine the corresponding regions of individual cells in the SPR images. Step 5: Obtain the deformation data of the cell membrane during the interaction between individual cells and nanoplastics according to the SPR signal intensity of the corresponding regions of individual cells in the SPR images, and realize the monitoring of the interaction between nanoplastics and single cells. The relationship between the SPR signal intensity and the deformation of the cell membrane in step 5 is as follows: , where is the reflectivity of the SPR image, is the average distance of the cell from the sensing chip, .

2. The method for monitoring and imaging the interaction between label-free nanoplastics and single cells according to claim 1, wherein The interaction process between the cells and the nanoplastics in Step 3 includes a baseline stage, a binding stage, and a dissociation stage; in the baseline stage, buffer solution is flowed into the sensing chip, in the binding stage, nanoplastic solution is flowed into the sensing chip, and in the dissociation stage, buffer solution is flowed into the sensing chip.

3. The method for monitoring and imaging the interaction between label-free nanoplastics and single cells according to claim 2, characterized in that, The concentration of the nanoplastic solution is 0 - 250 μg / mL, and the baseline stage, binding stage, and dissociation stage are repeated for testing in order of increasing concentration.

4. The monitoring imaging method for the interaction between label-free nanoplastics and single cells according to claim 1, wherein In Step 1, the coverage rate of the cells on the sensing chip is 20% - 50%.

5. The monitoring imaging method for the interaction between label-free nanoplastics and single cells according to claim 1, characterized in that, The particle size of the nanoplastics is 20 - 1000 nm.

6. The monitoring imaging method for the interaction between label-free nanoplastics and single cells according to claim 1, characterized in that The nanoplastics are nanopolymer or nanopolymer with chemical group modification on the surface; the nanopolymer includes one or more of nano-polyethylene, nano-polypropylene, nano-polystyrene, nano-polyethylene terephthalate, and nano-nylon 6.

7. The method for monitoring and imaging the interaction between label-free nanoplastics and single cells according to claim 1, characterized in that The SPRM includes a microscope based on lens-excited SPR and a microscope based on prism-excited SPR.

8. The method for monitoring and imaging the interaction between label-free nanoplastics and single cells according to claim 2, wherein The buffer solution is a buffer solution friendly to cell growth.

9. The method for monitoring and imaging the interaction between label-free nanoplastics and single cells according to claim 1, characterized in that, The cells are adherent cells, which are live cells and / or cells fixed with paraformaldehyde solution.

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