Monitoring and imaging method for interaction of label-free nano plastic and single cell

Abstract

The invention discloses a label-free nano plastic and single cell interaction monitoring imaging method which comprises the following steps: placing a sensing chip attached to cells on an SPRM, and recording a cell SPR image in the interaction process of the cells on the sensing chip and nano plastic; after the cell SPR image and the cell bright field image are compared to determine the corresponding area of the single cell in the SPR image, the deformation data of the cell membrane in the interaction process of the single cell and the nano plastic are obtained according to the SPR signal intensity of the area of the single cell, and the monitoring of the interaction of the nano plastic and the single cell under the resolution of the single cell is realized. The technical support is provided for researching the binding kinetics and cytotoxicity of the nano plastic.

Description

Technical Field

[0001] The present invention relates to the field of cell imaging technology, and particularly 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. Its extremely small size endows it with a very high specific surface area, enabling it 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: (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 culture medium, exposing it to the nanoplastics to be tested, and evaluating the toxic effects of nanoplastics on the growth and / or metabolite production 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.

[0003] CN118546282A discloses an aggregation-induced emission fluorescent material and metal palladium-doped nanoplastic particles for visualizing and quantitatively detecting the distribution law and migration and transformation dynamic process of nanoplastics in organisms.

[0004] However, these methods usually require labeling nanoplastics with fluorescent dyes. The surface attachment of fluorescent dyes will change the surface properties of nanoplastics, thereby changing their interaction mode with cells and affecting the reliability of experimental data. In addition, many fluorescence-based analytical methods use endpoint measurements to collect signals at discrete time points and cannot provide continuous kinetic data on the binding of nanoplastics to cell membranes.

[0005] (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

[0006] 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 the 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.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: A label-free monitoring imaging method for the interaction between nanoplastics and single cells, comprising the steps of: Step 1: Incubate cells on a sensing chip to obtain a sensing chip with adhered cells; 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 the illumination of the bright-field light source of the SPRM; 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 process between the cells and nanoplastics on the sensing chip; 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; Step 5: Obtain the deformation data of the cell membrane during the interaction between a single cell and nanoplastics based on the SPR signal intensity in the corresponding region of the single cell in the SPR image, and realize the monitoring of the interaction between nanoplastics and single cells.

[0008] 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 a single cell and nanoplastics is obtained based on the SPR signal intensity in the corresponding region of the single cell in the SPR image. This method can measure the interaction between nanoplastics and cell membranes in situ at the 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.

[0009] Specifically, it can be applied to the real-time monitoring of the binding process between a single cell and nanoplastics: obtain the SPR signal intensity of the single-cell region according to the SPRM imaging result, and convert it into the average distance of the cell from the chip surface according to the above formula. The strength of the interaction between the cell and the nanoparticles is reflected by the changes of the cell during the binding process of the nanoplastics, and then the cytotoxicity of the nanoplastics is inferred.

[0010] Heterogeneity analysis of the interaction between cells and nanoplastics: It is used to analyze the heterogeneous behavior 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.

[0011] Study on the cytotoxicity of nanoplastics with different properties: It is used to monitor the effects of diameter, material, and surface modification on the process of the interaction between nanoplastics and cells, providing a reference for evaluating the cytotoxicity and environmental toxicity of nanoplastics with different properties.

[0012] The process of the interaction 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.

[0013] The concentration of the nanoplastic solution is 0 - 250 μg / mL, and the baseline stage, binding stage, and dissociation stage are repeated three times in ascending order of concentration for testing.

[0014] 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.

[0015] 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, the average image gray value in each single - cell area is calculated by Fiji (defined as the SPR signal intensity I ). I and the reflectivity R are linearly correlated, and the formula is , β is a constant related to the instrument.

[0016] The coverage rate of cells on the sensing chip in step 1 is 20 - 50%. This coverage rate ensures that there are a large number of cells in the image and the cells mostly exist in the form of single cells.

[0017] The particle size of the nanoplastics is 20 - 1000 nm. The method of the present invention can be used for nanoplastics with various particle sizes.

[0018] The nanoplastics include one or more of nano - polyethylene (NPE), nano - polypropylene (NPP), nano - polystyrene (NPS), nano - polyethylene terephthalate (NPET), and nano - nylon 6 (NPA6), or nanoplastics with chemical group modifications on the surface.

[0019] The SPRM includes a microscope based on lens-excited SPR and a microscope based on prism-excited SPR.

[0020] The sensing chip is a glass slide with nano-gold or nano-silver on its surface; The buffer solution is a buffer solution friendly to cell growth.

[0021] The cells are adherent cells, which are live cells and / or cells fixed with paraformaldehyde solution.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 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 interaction process. The present invention does not need to introduce fluorescent dyes for labeling, and the surface properties of nanoplastics 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 height change of cells, providing a powerful tool for accurately monitoring the interaction process between cells and nanoparticles.

[0023] 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 nanoparticle drug delivery and research on the binding mechanism between nanoparticles and cell surfaces. Description of the Drawings

[0024] Figure 1 In [Figure], A is a schematic diagram of the deformation of cells caused by the binding of nanoplastics and cells; Figure 1 In [Figure], B is a schematic diagram of the process of the deformation of the cell membrane during the binding process of nanoplastics 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 reflectivity 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 image of the reflectivity of the cell region at 70.5° as a function of .

[0025] Figure 2 In [Figure], A is a schematic diagram of the surface plasmon resonance microscope device used in the embodiment; Figure 2 In [Figure], B is the bright-field image of the field of view collected by the camera and the corresponding SPR image. Each single-cell region in the bright-field image corresponds to each bright region in the SPR image, and the scale bar is 100 μm; Figure 2Figure C is a schematic diagram of the change in cell deformation signals at different stages during the single-cell deformation measurement process.

[0026] Figure 3 Figure A is a representative SPR image during the measurement of the interaction between fixed cells and 200 nm PSNPs with a concentration of 25 μg / mL in Example 1. The blue frame line is the single-cell region, the red frame line is the background region, and the scale bar is 100 μm. Figure 3 Figure B is the change in the SPR signal of the entire camera field of view during the measurement of the interaction between fixed cells and 200 nm PSNPs with a concentration of 25 μg / mL in Example 1. Figure 3 Figure C is the change in the SPR signal of the background region without cells during the measurement process. Figure 3 Figure D is the change in the SPR signal value of a single-cell region during the measurement process.

[0027] Figure 4 Figure A is the change in the cell deformation size during the measurement of the interaction between the single-cell regions of 10 fixed cells and 200 nm PSNPs with different concentrations in Example 1. The inset is an enlargement of the framed area. Figure 4 Figure B is from Figure 4 Figure A is a statistical chart of the cell deformation sizes of 10 fixed cells caused by 200 nm PSNPs with different concentrations.

[0028] Figure 5 Figure A is the cell deformation caused by the interaction between PSNPs with different particle sizes and fixed cells at different concentrations in Example 2. Figure 5 Figure B is the cell deformation caused by the interaction between PSNPs with different particle sizes and live cells at different concentrations in Example 2. Figure 5 Figure C is based on Figure 5 Figure A and Figure B are comparison charts of the deformation sizes of fixed cells and live cells caused by PSNPs with different particle sizes at a concentration of 125 μg / mL.

[0029] Figure 6 Figure A is the confocal imaging results under a 20-fold microscope before (left) and after (right) incubation of 200 nm PSNPs with a concentration of 25 μg / mL and fixed cells. 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 Figure B is Figure 6 Figure C is the confocal and stimulated emission depletion (STED) imaging results of the framed part in Figure A under a 60-fold microscope. Figure 6 Figure C is for Figure 6 Figure D is the z-axis scanning result of the STED imaging along the orange line of the cell shown in Figure B. The scale bars on the x-axis and z-axis are both 5 μm.

[0030] Figure 7 In Figure A, confocal imaging at 20x magnification of 200 nm PSNPs at 25 μg / mL before (left) and after (right) incubation with live cells is shown. Figure 7 In Figure B, Figure 7 In Figure A, the confocal and STED imaging results at 60x magnification of the framed part are shown; Figure 7 In Figure C, Figure 7 In Figure B, the z-axis scanning result of STED imaging of the cells along the orange line is shown. The scale bars on the x-axis and z-axis are both 5 μm.

[0031] Figure 8 In Figure A, cell deformation caused by the interaction of 200 nm PSNPs with different surface modifications at different concentrations with fixed cells in Example 3 is shown. Figure 8 In Figure B, cell deformation caused by the interaction of 200 nm PSNPs with different surface modifications at different concentrations with live cells in Example 3 is shown. Figure 8 In Figure C, according to Figure 8 Figure A and Figure 8 Figure 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.

[0032] Figure 9 In Figure A, cell deformation caused by the interaction of 200 nm nanoplastics with different materials at different concentrations with fixed cells in Example 4 is shown. Figure 9 In Figure B, 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 Figure C, according to Figure 9 Figure A and Figure 9 Figure 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

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to 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 replacements on the basis of understanding the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall all be covered within the protection scope of the present invention.

[0034] Schematic diagrams of cell deformation caused by the binding process of cells and nanoplastics are shown in Figure 1 Figure A and Figure 1As shown in B, during the binding process of nanoplastics and cells, the cell membrane deforms, and the adhesion to the sensing chip decreases. By calculating the average image gray value within each single-cell region I , it is converted into SPR reflectivity R , and then further 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 changes during the binding process of the cell to the nanoplastics, and then the cytotoxicity of the nanoplastics is inferred.

[0035] As Figure 1 shown in C, the WinSpall software is used to simulate the SPR signals at different cell heights to obtain the average distances between different cells and the chip surface ; the reflectivity-versus-SPR-angle images are obtained for the cell regions at 70.5° ; plotting the change of the reflectivity of the cell region at 70.5° Figure 1 as shown in D, it can be seen that there is a linear relationship between the average distance between the cell and the chip surface and the reflectivity of the cell region in the SPR image.

[0036] The raw materials used in the following specific embodiments are all purchased from the market. The surface plasmon resonance microscope used is the SPRm 200 (Biosensing Instrument); The change in the SPR signal during the interaction between the cell and the nanoplastics is measured by the device shown in A Figure 2 . Among them, 1 is the bright-field signal camera, 2 is the SPR signal camera, 3 is the laser, 4 is the lens, 5 is the prism, 6 is the gold nanolayer, 7 is the cell, 8 is the nanoplastics, and 9 is the LED light source. The specific experimental process of the test is as follows: Step 1, after assembling the cell culture tank and 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 containing 5% CO 2 so that the cells are completely attached to the sensing chip, and the coverage rate of the cells on the sensing chip is about 30%. The test for fixed cells is the cells fixed with 4% paraformaldehyde solution for 30 minutes.

[0037] Dilute the stock solutions of different nanoplastics materials and different nanoplastics particle sizes with phosphate buffer to prepare nanoplastics solutions with concentrations ranging from low to high, with a concentration range of 0 - 250 μg / mL, for standby.

[0038] Step 2, place the sensing chip with attached cells on the SPRM, illuminate it with the bright-field light source of the SPRM, and obtain the bright-field image of the cells on the sensing chip by the bright-field signal camera 1, as Figure 2 shown in B 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 cells and nanoplastics on the sensing chip with the SPR signal camera 2, as shown in Figure 2 shown in B of In step 3, the interaction process between cells and nanoplastics includes a baseline stage, a binding stage, and a dissociation stage; in the baseline stage, a buffer solution is flowed into the sensing chip, in the binding stage, a nanoplastic solution is flowed into the sensing chip, and in the dissociation stage, a buffer solution is flowed into the sensing chip.

[0039] 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: Calculate the average image gray value (defined as the SPR signal intensity I ) in each single-cell region through Fiji, plot it against time, and obtain the I-t graph of a 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 monitor the interaction between nanoplastics and single cells. Among them, . .

[0040] 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.

[0041] As shown in Figure 2 C, in the baseline stage, when PBS buffer is introduced into the cell culture medium, no nanoplastics bind to the cells, the cell SPR signal hardly changes, and the cell morphology remains basically unchanged; in the binding stage, a certain concentration of nanoplastics is introduced into the above cell culture tank, the nanoplastics bind to the cells, causing changes in cell morphology and the average distance of the cell chassis from the chip surface to change, resulting in a change in the SPR signal in the cell region; in the dissociation stage, pure PBS buffer solution is passed through the above cell culture tank again to wash the cells in the binding stage, the nanoplastics detach from the cell surface or are endocytosed into the cell, the interaction pattern between the nanoplastics and the cells changes, and the trend of the cell SPR signal changes.

[0042] Example 1 Using a surface plasmon resonance microscope as shown in Figure 2 A, the interaction process between 200 nm PSNPs with 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 respectively, and the cells used were HEK293 fixed cells.

[0043] As shown in Figure 3 A, during the measurement of the interaction between fixed cells and 200 nm PSNPs with a concentration of 25 μg / mL, the representative SPR image, the blue frame line is the single-cell region, the red frame line is the background region, and the scale bar is 100 μm.

[0044] As shown in Figure 3 B - Figure 3 D in Example 1 shows the changes in SPR signal values in different regions of the sensing chip during the measurement of the interaction between fixed cells and 200 nm PSNPs with 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 tank, and the orange arrow indicates the time when pure buffer solution began to be introduced into the cell culture tank.

[0045] The curve of the entire sensing chip ( Figure 3 B) represents the result obtained by a traditional SPR sensor, which shows a kinetic pattern and signal amplitude similar to those of the background region ( Figure 3 C, where the black line is the original I-t curve and the red line is the fitted line), proving 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 adhesion on the sensor surface).

[0046] By fitting the background sensing map in Figure 3 B with first-order kinetics (the red curve for the background), we obtained the association rate constant , dissociation rate constant and affinity as follows: =(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 nanoplastic to the background is a highly irreversible adsorption process.

[0047] Compared with Figure 3 the background region of B in Figure 3 the black curve of D in , the single-cell region shows a reduced SPR signal, shifts in the negative direction, and has a much smaller amplitude. This signal is hidden in the background of traditional biosensors and cannot be detected by traditional SPR sensors. This highlights a major limitation of traditional biosensors: they cannot separate useful cell-specific signals from background noise, leading to potentially false conclusions. This limitation is overcome in the present invention by providing spatially resolved measurement SPR imaging, enabling us to capture subtle changes in single-cell behavior.

[0048] As Figure 4 shown in A of , the change in the size of cell deformation during the interaction between 10 fixed cell regions and 200 nm PSNPs at different concentrations was measured. Figure 4 Shown in B of Figure 4 is the statistical chart of the size of cell deformation caused by 200 nm PSNPs at different concentrations obtained from A of . During the binding stage, the height of the cell chassis from the chip surface ( ) continuously increases, and remains basically unchanged during the dissociation stage, 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, different cells also have significant differences in their responses to the same concentration of PSNP solution, showing heterogeneity among cells.

[0049] Example 2 Using the surface plasmon resonance microscope shown in A of Figure 2 , the interaction processes between fixed cells and live cells and PSNPs with different diameters were monitored in real time. The used nanoplastics solutions were PSNPs solutions with different concentrations and diameters of 20 nm, 50 nm, 100 nm, 200 nm, and 1000 nm respectively, and the used cells were HEK293 fixed cells or HEK293 live cells.

[0050] Figure 5 Shown in A of is 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 nanoplastics, The change trend is basically the same as that in Example 1. For different-sized nanoplastics with the same concentration, the deformation amplitude of cells is significantly different. The response of cells to PSNPs of different sizes is in the order of 100 nm > 20 nm > 50 nm > 200 nm > 1000 nm. This non-monotonic ranking may reflect the size-dependent mechanism of membrane deformation and has nothing to do with cell internalization.

[0051] As Figure 6 In [reference], A shows 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 Nile red-labeled 200 nm PSNPs. The scale bar is 50 μm. Figure 6 In [reference], B is Figure 7 In [reference], the imaging result of the framed part A 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 In [reference], C is for Figure 6 In [reference], the z-axis scanning result of the cell along the orange line shown in B using STED imaging. The scale bars on both the x-axis and z-axis are 5 μm. Figure 6 It also proves that nanoplastics do not enter fixed cells after incubation for a period of time.

[0052] Figure 5 In [reference], B measures the cell deformation caused by the interaction of PSNPs of different sizes with live cells at different concentrations. For live cells of the same size, they show different behaviors from fixed cells, which may be due to the fluidity of the live cell membrane. Although the size is different from that of fixed cells, the trend of the response of live cells to nanoplastics of different 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 ranking order is 20 nm > 50 nm > 100 nm > 200 nm > 1000 nm, the same as the ranking of particle sizes. This indicates that the interaction mechanism between cells and nanoplastics may be different at different concentrations.

[0053] Figure 5 In [reference], C is based on Figure 4Figure A and Figure B show the comparison charts of the deformation sizes of fixed cells and live cells caused by PSNPs with different particle sizes at a concentration of 125 μg / mL. During the binding stage, the situation of live cells is basically the same as that of fixed cells, both showing a downward trend; however, during the dissociation stage, the signal of live cells continues to decrease. This indicates that due to the dynamic nature of the live cell membrane, the interaction between the membrane and PSNP persists, possibly involving the active internalization of nanoplastics.

[0054] Figure 7 In Figure A, confocal imaging at 20× magnification before (left) and after (right) incubating 200 nm PSNPs at 25 μg / mL with live cells is shown. Figure 7 In Figure B Figure 7 The imaging result at 60× magnification of the framed part in Figure A. 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 Figure C Figure 7 The z-axis scanning result of STED imaging along the orange line of the cells shown in Figure B. The scale bars on both the x-axis and the z-axis are 5 μm. This also proves that after incubation for a period of time, live cells endocytose some nanoplastics.

[0055] Example 3 Using the surface plasmon resonance microscope as shown in Figure 2 Figure A, the interaction processes between fixed cells and live cells and 200 nm PSNPs with different surface modifications were monitored in real time. 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 HEK293 fixed cells or HEK293 live cells.

[0056] As shown in Figure 8 Figure A, during the interaction process between PSNPs with different surface modifications and fixed cells, the PSNP modified with COOH showed the highest level of interaction, almost twice the signal of the unmodified 200 nm PSNP. In contrast, the PSNP modified with NH 2 produced only about one-third of the original reaction. As shown in Figure 8 Figure B is the measurement of the interaction between PSNPs with different surface modifications and live cells. Different from fixed cells, live cells showed the weakest response to the PSNP modified with COOH (only 1 / 5 of the response of fixed cells). Figure 8 In Figure C Figure 8 Figure A and Figure 8 Figure B show the comparison charts of the deformation sizes of fixed cells and live cells caused by 200 nm nanoplastics of different materials at a concentration of 250 μg / mL. This phenomenon once again illustrates the key role of the flexibility and fluidity of the membrane in regulating nanoplastic binding.

[0057] Example 4 Using a surface plasmon resonance microscope as shown in Figure 2 A, the interaction processes of fixed cells and live cells with 200 nm nanoplastics of different materials were monitored in real time. The specific steps were as shown above. The nanoplastic solutions used were PENP, PETNP, and PSNP solutions with a size of 200 nm, and the cells used were HEK293 fixed cells or HEK293 live cells.

[0058] As Figure 9 shown in A, for the interaction of different material nanoplastics with fixed cells, PENP and PETNP generally showed weaker responses than PSNP, following the trend of PSNP > PENP > PETNP. This may stem from the inherent properties of the materials, such as surface chemistry and hydrophobicity, which have different effects on the degree of membrane deformation.

[0059] As Figure 9 shown in B, when measuring the interaction process of live cells with nanoplastics of different materials, although the ranking trend of nanoparticle interaction with live cells was the same as that of fixed cells, the signal amplitude was 1 - 3 times higher. Figure 9 Shown in C is 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 obtained from Figure 9 A and Figure 9 B. This may reflect that the live cell membrane has a stronger deformation ability. Compared with the rigid fixed membrane of fixed cells, the live cell membrane can interact more strongly with nanoparticles.

Claims

1. A label-free monitoring imaging method for the interaction between nanoplastics and single cells, characterized in that: Includes steps: Step 1, incubating cells on the sensor chip to obtain a sensor chip adhered to the cells; Step 2, placing the cell-attached sensor chip on the SPRM, and obtaining a bright field image of the cells on the sensor chip through bright field light illumination of the SPRM; Step 3, adjusting the imaging mode of SPRM to SPR, adjusting the laser incident angle to generate an evanescent field on the sensor chip, and recording the SPR image of the cells during the interaction between the cells and the nanoplastics on the sensor chip; Step 4, comparing the cell SPR image obtained in step 3 with the cell bright field image obtained in step 2 to determine the corresponding area of ​​the single cell in the SPR image; Step 5, obtaining the deformation data of the cell membrane during the interaction between the single cell and the nanoplastic according to the SPR signal intensity of the corresponding area of ​​the single cell in the SPR image, thereby realizing the monitoring of the interaction between the nanoplastic and the single cell.

2. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 1, characterized in that: The process of cell-nanoplastic interaction in step 3 includes a baseline phase, a binding phase and a dissociation phase; the baseline phase is when a buffer solution flows into the sensor chip, the binding phase is when a nanoplastic solution flows into the sensor chip, and the dissociation phase is when a buffer solution flows into the sensor chip.

3. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 2, characterized in that: The concentration of the nanoplastic solution is 0-250 μg / mL, and the test is repeated in three stages: baseline stage, binding stage and dissociation stage from low to high concentration.

4. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 1, characterized in that: The relationship between the SPR signal intensity and the deformation of the cell membrane in step 5 is: ,in, is the reflectivity of the SPR image, is the average distance between the cell and the sensor chip, .

5. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 1, characterized in that: In step 1, the coverage of cells on the sensor chip is between 20% and 50%.

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

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

8. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 1, characterized in that: The SPRM includes lens-based excitation SPR and prism-based excitation SPR microscopy.

9. The label-free monitoring imaging method for the interaction between nanoplastics and single cells according to claim 1, characterized in that: A glass sheet with nano-gold or silver on the surface of the sensor chip; The buffer is a buffer that is friendly to cell growth.

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

Images