A microfluidic chip for measuring tumor cell deformability and a detection method and application thereof

Abstract

The application discloses a microfluidic chip for measuring deformability of tumor cells and a detection method and application thereof. The microfluidic chip comprises an inlet liquid storage tank, a deterministic lateral displacement section and an outlet liquid storage tank. The deterministic lateral displacement section is sequentially connected by an input area, a plurality of groups of longitudinally arranged half-T-shaped micro-column arrays and an output area. According to the flow direction of a sample to be detected, the critical cutoff size Dc of each group of half-T-shaped micro-column arrays is sequentially increased. The input area and the output area are respectively provided with at least two sub-channels, and the inlet liquid storage tank is communicated with the sub-channels. The microfluidic chip is used for detecting the size, deformability and migration capacity of tumor cells, and has high sensitivity and accuracy. Moreover, the detection process only needs 10 muL of tumor cell suspension, and the size measurement can be completed within 2 minutes, and the detection of deformability and migration capacity can be completed within 4 minutes, so that the detection efficiency is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of cell detection technology, and in particular to a microfluidic chip for measuring the deformability of tumor cells, as well as its detection method and application. Background Technology

[0002] Cancer is a prevalent disease, and the spread of cancer cells to distant organs is a leading cause of cancer death. Cellular mechanics is associated with tumor cell transformation, malignancy, and metastasis. Intracellular pathological changes affect the structure and function of the cytoskeleton, making cellular mechanical characteristics a highly sensitive marker of their health. In malignant cells, the cytoskeleton transforms from an ordered, rigid structure to an irregular, flexible state, including a reduction in cytoskeleton polymers and accessory proteins, as well as network reorganization. The cell transforms from a mature state into a replicable, motile, and "immortal" cancerous form. Therefore, malignant cells may be more flexible than mature, fully differentiated cells. Related studies have reported that increased deformability and flexibility of tumor cells lead to highly invasive and metastatic capabilities. To invade distant sites, these aggressive tumor cells must deform to pass through the small spaces within the tumor extracellular matrix (ECM) and endothelial barrier, and circulate through microvessels smaller than the cell itself. Therefore, measuring cellular deformability can provide new evidence for cancer risk assessment and also contribute to guiding personalized medicine and developing novel cancer therapies.

[0003] Currently, the measurement of tumor cell deformability is mainly achieved by quantifying the deformability of individual tumor cells exposed to external shear stress. Related technologies include atomic force microscopy, optical tweezers, and microtubule aspiration. Although these methods can extract the physical properties of tumor cells (such as elastic modulus or viscosity), their detection techniques are demanding, time-consuming, expensive, and difficult to achieve high throughput, thus limiting their application in clinical trials. In recent years, microfluidic chips have attracted much attention as an emerging technology. Due to their characteristics such as single-cell detection, high throughput, large scale, parallelism, low sample consumption, speed, and intuitive data analysis, they have been widely used in the detection of tumor cell deformability. These methods include gradually reducing the gaps between micropillars within the channel to hinder or allow cells with different mechanical properties to pass through the detection device, thereby analyzing the cell deformability; or applying an electric field through a narrow channel to measure the deformability of HeLa cells and their interaction with the electric field. However, these methods are prone to chip clogging in practical applications, reducing the accuracy of cell deformability detection.

[0004] In summary, although there are numerous methods for detecting tumor cell deformability, the detection standards vary, the correlation and consistency between different methods are not ideal, and they suffer from drawbacks such as expensive instruments, time-consuming procedures, low accuracy, and cell damage. Therefore, there is an urgent need to establish simple, non-destructive, accurate, and reliable devices and methods for detecting tumor cell deformability. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a microfluidic chip capable of rapidly and accurately detecting tumor cell deformability.

[0006] The present invention also proposes a method for fabricating a microfluidic chip.

[0007] The present invention also proposes a detection system.

[0008] This invention also proposes the application of a microfluidic chip or detection system in detecting cell deformability, drug screening, analyzing cytoskeleton state, or assessing cell migration ability.

[0009] This invention also proposes a method for detecting the deformability of tumor cells.

[0010] A first aspect of the present invention provides a microfluidic chip comprising an inlet reservoir, a deterministic lateral displacement (DLD) segment, and an outlet reservoir, wherein:

[0011] The deterministic lateral displacement segment is composed of an input region, several sets of longitudinally arranged semi-T-shaped micropillar arrays, and an output region connected in sequence. According to the flow direction of the sample to be tested, the critical cutoff size Dc value of each set of semi-T-shaped micropillar arrays increases sequentially, and the value of Dc ranges from 5 to 20 μm.

[0012] The input area and the output area are each provided with at least two sub-channels, and the inlet liquid storage tank is connected to the sub-channels.

[0013] The microfluidic chip according to embodiments of the present invention has at least the following beneficial effects:

[0014] The microfluidic chip of this invention creatively proposes the use of a semi-T-shaped micropillar array for efficient detection of tumor cell deformability. Using this microfluidic chip, no electric field is required, and the clogging phenomenon during the detection process can be greatly reduced. Efficient sorting can be achieved simply by controlling the critical cutoff size Dc value of the semi-T-shaped micropillar array.

[0015] In addition, gradually increasing the Dc value helps to initially screen larger cells in the front-end micropillar array, effectively reducing the risk of cell accumulation or blockage in subsequent channels, thereby improving fluid flow and detection stability.

[0016] In some embodiments of the present invention, the semi-T-shaped micropillar array comprises several rows of parallel semi-T-shaped micropillars.

[0017] In some preferred embodiments of the present invention, one end of the two sides of the semi-T-shaped micropillar is vertically connected, and the other end is connected through a third side, which is arc-shaped.

[0018] In some embodiments of the present invention, the lengths of the two sides of the semi-T-shaped micropillar are 25±2μm.

[0019] In some embodiments of the present invention, the number of groups of the semi-T-shaped micropillar array in the DLD segment is 10 to 30, and the increment of the Dc value corresponding to two adjacent groups of the semi-T-shaped micropillar array is 0.3 μm to 0.8 μm.

[0020] In some embodiments of the present invention, the critical cutoff dimension Dc value of each group of the semi-T-shaped micropillar arrays is given by formula D. c = 1.4 × G × (tanθ) 0.48 The decision includes:

[0021] G is the interval between two adjacent semi-T-shaped micropillars, ranging from 15 to 33 μm, and θ is the offset angle of the semi-T-shaped micropillars in each group of the semi-T-shaped micropillar array.

[0022] When testing the deformability of tumor cells, cells smaller than Dc moved in a zigzag pattern, while cells larger than Dc moved in a lateral displacement pattern during collisions.

[0023] In some preferred embodiments of the present invention, the number of groups of the semi-T-shaped micropillar array is 18 to 25, and the number of adjacent groups of L... -1 The corresponding Dc value increment for the micropillar array is 0.4 μm to 0.6 μm.

[0024] In some embodiments of the present invention, the Dc value ranges from 6μm to 16μm, and the adjacent two groups of L -1 The corresponding Dc value increment for the micropillar array is 0.5 μm.

[0025] In some embodiments of the present invention, the value of θ ranges from 1.13 to 23.11°.

[0026] In some embodiments of the present invention, the length of each group of the semi-T-shaped micropillar arrays decreases according to the flow direction of the sample to be tested toward the outlet storage tank, and the length of the semi-T-shaped micropillar arrays ranges from 200 to 1800 μm.

[0027] In some embodiments of the present invention, the input region is provided with at least 10 sub-channels for the sample to be tested to enter the semi-T-shaped micropillar array for separation.

[0028] In some preferred embodiments of the present invention, the input area is provided with 25 to 35 sub-channels.

[0029] In some embodiments of the present invention, the output region is provided with at least 10 sub-channels for the separated test samples to flow out of the semi-T-shaped micropillar array.

[0030] In some preferred embodiments of the present invention, the output area is provided with 25 to 35 sub-channels.

[0031] In some embodiments of the present invention, the diameter of the outlet storage tank is about 2.0 to 4.0 mm, preferably 3.5 ± 0.2 mm.

[0032] A second aspect of the present invention provides a method for fabricating a microfluidic chip, comprising: fabricating a PDMS microchannel based on the microfluidic chip described in any one of the first aspects, punching holes at corresponding positions of the inlet reservoir and the outlet reservoir, and then assembling it with a glass slide to obtain the chip.

[0033] The microfluidic chip according to the embodiments of the present invention has at least the following beneficial effects: the microfluidic chip preparation method of the present invention is simple, low in cost, and suitable for industrial production.

[0034] In some embodiments of the present invention, the method for preparing PDMS microchannels includes: silanizing a silicon template with the microfluidic chip microchannels, then pouring the prepared PDMS adhesive onto the silicon template, removing bubbles, and after curing, peeling the formed PDMS microchannels off the silicon template to obtain the microchannels.

[0035] In some embodiments of the present invention, the silanization process includes: taking a clean silicon template, purging it with nitrogen gas, and then dripping trimethylchlorosilane onto the silicon template for treatment.

[0036] Silanization helps to subsequently peel the cured PDMS microchannels from the silicon template.

[0037] In some embodiments of the present invention, the diameter of the hole in the inlet storage tank is about 0.5 to 2.5 mm, preferably 1.0 ± 0.2 mm.

[0038] A third aspect of the present invention provides a detection system comprising: the microfluidic chip, fluid driving device, and imaging device described in the first aspect.

[0039] In some embodiments of the present invention, the fluid driving device includes an injection pump for injecting a sample of tumor cells to be tested into a microfluidic chip at a certain flow rate.

[0040] In some embodiments of the present invention, the imaging device includes a charge-coupled device (CCD) camera and a microscope. It is used to record the size distribution of tumor cells in a microfluidic chip.

[0041] In a fourth aspect, the present invention provides the application of the microfluidic chip described in the first aspect or the detection system described in the third aspect in detecting cell deformability, drug screening, analyzing cytoskeleton state, or assessing cell migration ability.

[0042] In some embodiments of the present invention, the detection of cell deformability includes the detection of tumor cell deformability.

[0043] A fifth aspect of the present invention provides a method for detecting the deformability of tumor cells, comprising:

[0044] The average size distribution of tumor cells under test is detected using the microfluidic chip described in the first aspect or the detection system described in the third aspect at different flow rates, and the deformability of the tumor cells under test is determined based on the difference in the average size distribution of the tumor cells.

[0045] The detection method according to embodiments of the present invention has at least the following beneficial effects:

[0046] The tumor cell deformability detection method of the present invention is simple, has good repeatability and stability, and is highly sensitive to changes in tumor cell deformability (such as changes in cell deformability induced by MeβCD and Chol). It can be used not only for screening drugs related to tumor treatment, but also for evaluating the basic state of the cytoskeleton and cell migration ability, and has broad application prospects.

[0047] In some embodiments of the present invention, the microfluidic chip is pretreated with a nonionic surfactant before detection.

[0048] In some embodiments of the present invention, the detection method specifically includes: storing a suspension of tumor cells to be tested in the inlet reservoir, controlling the tumor cells to be tested to enter the deterministic lateral displacement segment at a specific flow rate, and then recording and statistically analyzing the average cell size distribution (i.e., D) of the tumor cells to be tested in the semi-T-shaped micropillar array. app value).

[0049] In some embodiments of the present invention, the dilution buffer includes PBS buffer.

[0050] In some embodiments of the present invention, the flow rate range is 2 μL / min to 30 μL / min.

[0051] In some embodiments of the present invention, the flow rates are 2-3 μL / min and 20-30 μL / min, respectively.

[0052] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0054] Figure 1 This is a schematic diagram of the microfluidic chip structure used in this invention to measure the deformability of tumor cells;

[0055] Figure 2 This is a flowchart of the feasibility verification experiment for the tumor cell deformability detection method of the present invention;

[0056] Figure 3 This is the result of the feasibility verification of the tumor cell deformability detection method of the present invention;

[0057] Figure 4 The average Di of various tumor cells in the DLD microfluidic chip under different fluid flow rates is shown in this invention. app Fitting a linear graph;

[0058] Figure 5 The results show the stability test of the tumor cell deformability detection method based on the microfluidic chip of this invention.

[0059] Figure 6 The results represent the sensitivity evaluation of the tumor cell deformability detection method based on the microfluidic chip of this invention.

[0060] Figure 7 The following are the detection results related to the application of the microfluidic chip of this invention in analyzing the basic state of tumor cell cytoskeleton, where A represents the results of different treatment groups B16-F10 at a flow rate of 25 μL / min. app Output frequency ridge map, B is the corresponding average D app Bar chart (n=6), C represents the final calculated ΔD app ;

[0061] Figure 8 The results are related to the application of the microfluidic chip of this invention in evaluating the migration ability of tumor cells, where A represents the ΔD of four types of tumor cells. appB shows Transwell images of four types of tumor cells after MeβCD treatment (scale bar: 100 μm); C shows a statistical graph of the number of migrating cells (n=3); D shows the Transwell experiment and ΔD. app Correlation analysis; E shows scratch migration images of four types of tumor cells after MeβCD treatment; F shows migration rate statistics (n=3); G shows the relationship between scratch experiment and ΔD. app Correlation analysis;

[0062] Figure 9 The following are the relevant detection results of the application of the microfluidic chip of this invention in evaluating the deformability of solid tumor cells in vivo, where A is a flowchart of the detection of solid tumor cell degeneration in mice; B is the result of D in mice at a fluid flow rate of 25 μL / min for tumor cells in each group of solid tumors. app Output frequency ridge plot; C represents D for each group at a fluid flow rate of 25 μL / min. app Bar chart; D represents the results of tumor cell deformability detection within solid tumors. Detailed Implementation

[0063] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0064] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0065] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0066] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0067] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0068] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0069] Inventive concept

[0070] In a first aspect, the present invention provides a microfluidic chip for measuring the deformability of tumor cells. This microfluidic chip is a deterministic lateral displacement (DLD) microfluidic chip based on semi-T-shaped micropillars, and its structure is as follows: Figure 1 As shown, it includes: an inlet storage tank, a DLD section, and an outlet storage tank. The DLD section is formed by sequentially connecting an input area, at least two sets of semi-T-shaped micro-pillar arrays (i.e., DLD sub-segments), and an output area. The input area and the output area are each provided with at least two sub-channels, and the inlet storage tank is connected to the sub-channels.

[0071] In the process of determining the deformability of tumor cells, the suspension of tumor cells to be tested is added to the inlet reservoir. Then, the back-pushing function of the syringe pump is used to pump the tumor cells to be tested from the reservoir into the DLD section through the pipeline. After being sorted by the semi-T-shaped microcolumn array, the cells flow out from the sub-channel of the output area and enter the outlet reservoir.

[0072] In some specific implementations, each group of semi-T-shaped micropillar arrays has a specific DLD critical cut-off size (Dc), the value of which is determined by the following empirical formula:

[0073] Dc = 1.4 × G × (tanθ) 0.48 ;

[0074] In the formula, G represents the spacing between two adjacent micropillars, in μm; θ represents the L of each half-T-shaped micropillar array. -1 The offset angle of the micro-column.

[0075] In some specific embodiments, the input area is provided with a number of sub-channels, the diameter of which can be designed according to the size of the tumor cells; in some preferred embodiments, the input area is provided with at least 10 sub-channels; more preferably, the input area is provided with 25, 26, 27, 28, 29 or 30 sub-channels to facilitate the rapid passage of tumor cells.

[0076] In some specific embodiments, the semi-T-shaped micropillar array can be set in 10 to 30 groups, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 groups, more preferably 18, 19, 20, 21, 22, 23 or 24 groups. By adjusting the number of semi-T-shaped micropillar arrays and input region sub-channels, the size of different tumor cells can be characterized.

[0077] In some specific embodiments, the Dc value corresponding to each group of semi-T-shaped micropillar arrays exhibits an increasing trend, and the Dc value ranges from 5 to 20 μm, preferably from 6.0 to 16.0 μm. In some more preferred embodiments, the increment of the Dc value corresponding to each group of semi-T-shaped micropillar arrays is 0.3 to 0.8 μm, preferably 0.5 μm.

[0078] In some specific embodiments, the output region is provided with a number of sub-channels, the diameter of which can be adjusted according to the size of the tumor cells; in some preferred embodiments, the output region is provided with at least 10 sub-channels; more preferably, the output region is provided with 25, 26, 27, 28, 29 or 30 sub-channels, preferably 26, 27, 28, 29 or 30.

[0079] The number of subchannels in the output region is usually consistent with that in the input region, and the diameters of the subchannels in both the input and output regions can be adjusted according to the separation target. For example, when separating tumor cells, the diameter of the subchannels should be at least one-third larger than the normal size of the tumor cells.

[0080] A second aspect of the present invention provides a method for preparing a microfluidic chip for measuring the deformability of tumor cells, specifically comprising the following steps:

[0081] In a vacuum chamber, a silicon template with microchannels is silanized. Then, the prepared PDMS adhesive is poured onto the silicon template, bubbles are removed, and after curing, the formed PDMS microchannels are peeled off from the silicon template. Holes are drilled at the inlet and outlet reservoirs, followed by plasma treatment. Finally, the obtained PDMS microchannels are irreversibly bonded to a glass slide, sealed, and dried to obtain the final product.

[0082] In some specific embodiments, the silanization process includes: taking a clean silicon template, purging it with nitrogen, placing the silicon template in a petri dish, and then dripping trimethylchlorosilane around it for treatment. The silanization process facilitates the subsequent peeling of the cured PDMS from the silicon template.

[0083] In some specific embodiments, the perforation diameter of the inlet reservoir is approximately 0.5–2.5 mm, preferably 1.0 ± 0.2 mm; the perforation diameter of the outlet reservoir is approximately 2.0–4.0 mm, preferably 3.5 ± 0.2 mm. The perforation design of the inlet reservoir is primarily to prevent clogging and backflow. A larger orifice diameter in the outlet reservoir reduces the risk of particle or cell accumulation or clogging at the outlet, thereby maintaining fluid flow.

[0084] In some preferred embodiments, after drilling, the holes are cleaned with ethanol and ultrapure water in sequence, and then dried with nitrogen.

[0085] In some specific embodiments, plasma treatment includes plasma treatment using an air plasma generator.

[0086] A third aspect of the present invention provides a detection system comprising: a microfluidic chip, a fluid drive device, and an imaging device for measuring the deformability of tumor cells, as described in the first aspect.

[0087] In some specific embodiments, the fluid drive device can be an injection pump, and the imaging device can be a charge-coupled device (CCD) camera and a microscope.

[0088] In some specific implementations, all visual data on cell movement in the input region of the DLD segment of the microfluidic chip, the arrays of semi-T-shaped micropillars, and the output region are obtained using a microscope and a CCD camera.

[0089] In some specific implementations, the actual projection path of tumor cells distributed within the capture area of ​​the semi-T-shaped micropillar array is obtained using a microscope and a CCD camera, and then tumor cell size data is acquired.

[0090] Specifically, images can be collected in the input and output regions separately and saved as uncompressed ".avi" format. Then, MATLAB software can be used to count and analyze the tumor cells in the input and output regions. All data can be exported to an Excel spreadsheet for statistical analysis of the size distribution of tumor cells.

[0091] A fourth aspect of the present invention provides a method for detecting the deformability of tumor cells, specifically comprising the following steps:

[0092] The tumor cell suspension to be tested was injected into the inlet reservoir, and then the sample was pumped into the DLD segment of the DLD microfluidic chip through a polyethylene tube using the back-pull function of the syringe pump. The average D of tumor cells at different cell flow rates was statistically analyzed. app Distribution, and based on the average D app The difference (ΔD) app Assessing tumor cell deformability, ΔD app The smaller the value, the lower the deformability.

[0093] In some specific embodiments, to reduce the adhesion of tumor cells to the column and channel walls during the workflow, a nonionic surfactant pretreatment can be used before injecting the tumor cell sample to be tested. Preferably, the DLD segment of the microfluidic chip is modified with poloxamer 188 (1% m / v), and then the remaining poloxamer 188 inside is rinsed with PBS solution.

[0094] In some specific embodiments, the different cell flow rates are set to 2–3 μL / min and 20–30 μL / min, respectively. The average D obtained at the above flow rate range is selected. app The difference indicates that the deformability of tumor cells is highly feasible and does not easily damage the microfluidic chip.

[0095] Based on the average D of tumor cells app The flow rate is highly correlated with fluid velocity and exhibits a linear relationship on a logarithmic scale. Considering that excessively high fluid velocities can easily damage the DLD microfluidic chip and tumor cell structures (cell membrane, cytoskeleton, cell nucleus, and other structures that constitute the mechanical properties of cells), affecting the lifespan of the microfluidic chip and the accuracy of the detection results, while excessively low flow rates can easily cause particles to clog the chip, the detection data at flow rates of 2–3 μL / min and 20–30 μL / min were selected after optimization.

[0096] In some preferred embodiments, the different cell flow rates are set to 2.5 μL / min and 25 μL / min, respectively.

[0097] The following section provides a detailed description of a microfluidic chip for measuring tumor cell deformability, its detection method, and its application, using specific examples.

[0098] Example 1: Microfluidic chip for measuring tumor cell deformability

[0099] 1. Microfluidic chip:

[0100] This embodiment provides a microfluidic chip (hereinafter referred to as a semi-T-shaped column array chip) for measuring the deformability of tumor cells, and its structural schematic diagram is shown below. Figure 1As shown, the chip includes: an inlet reservoir, a DLD section, and an outlet reservoir.

[0101] The DLD segment is formed by sequentially connecting the input region, 21 DLD sub-segments (i.e., the semi-T-shaped micropillar array, as shown in Table 1), and the output region. Both the input and output regions are equipped with 28 sub-channels for tumor cells to enter or flow out of the semi-T-shaped micropillar array. The inlet reservoir is connected to the input region through a pipe, and the outlet reservoir is connected to the output region.

[0102] Each DLD segment of this chip has a specific Dc value, which is determined by the following empirical formula:

[0103] Dc = 1.4G(tanθ) 0.48 ;

[0104] In the formula, G refers to the interval between two adjacent micropillars, with the unit being μm; θ represents the micropillar offset angle of each group of semi-T-shaped micropillar arrays.

[0105] The relevant parameter settings for the microfluidic chip in this embodiment are shown in Table 1.

[0106] Table 1: Segmentation parameters of the microfluidic chip DLD in this embodiment

[0107]

[0108]

[0109] 2. Microfluidic chip fabrication method:

[0110] This embodiment also provides a method for preparing the above-mentioned microfluidic chip for measuring tumor cell deformability, including the following:

[0111] In a vacuum chamber, a silicon wafer with microchannels is placed in a vacuum chamber filled with trimethylchlorosilane (TMCS) and allowed to stand at room temperature for 30 minutes to complete the silanization process. The silicon template is then rinsed three times with isopropanol, followed by three rinses with deionized water, and finally dried with nitrogen. Next, PDMS prepolymer and curing agent are mixed at a 10:1 ratio and thoroughly stirred. This mixture is poured onto the silicon template, bubbles are removed, and after curing, the formed PDMS microchannels are peeled off the template. Holes are drilled at the inlet and outlet reservoirs; all inlet reservoirs have 1.0 mm holes, and the outlet reservoir has a 3.5 mm hole. These holes can be connected to a microfluidic injection pump via a pump tube. After drilling, an air plasma treatment is performed. Finally, the resulting PDMS microchannels are irreversibly bonded to a glass slide, sealed, and dried.

[0112] Example 2: Detection system for determining tumor cell deformability

[0113] This embodiment provides a detection system for determining the deformability of tumor cells. The detection system mainly consists of an injection pump, the microfluidic chip described in Example 1, a charge-coupled device camera (CCD camera, Leica DFC 360FX), and a microscope (Leica DM IL LED).

[0114] In this process, all visual data regarding the input and output regions and cell movement in the microfluidic chip were acquired using a microscope and a CCD camera. During the detection process, images were collected at 10 frames per second in both the input and output regions, and the data was saved in uncompressed ".avi" format. Then, MATLAB software was used to count and analyze the tumor cells in the input and output regions. All data could be exported to an Excel spreadsheet for statistical analysis of tumor cell size distribution.

[0115] Example 3: Method for determining tumor cell deformability

[0116] This embodiment provides a method for rapid and non-destructive determination of tumor cell deformability using the aforementioned microfluidic chip. The specific experimental procedure is as follows: Figure 2 As shown, it includes the following steps:

[0117] (1) Pretreatment before sample loading:

[0118] To reduce the adhesion of tumor cells to the column and channel walls during the workflow, the microfluidic chip of Example 1 was first modified with poloxamer 188 (1% m / v), and then the remaining poloxamer 188 inside was rinsed with PBS solution for later use.

[0119] (2) Sample loading, testing, and data acquisition:

[0120] 10 μL of tumor cell suspension was added to the sample reservoir, and the sample was pumped into the microfluidic chip through a polyethylene tube using the back-pump function. The flow rate of the tumor cell suspension was controlled at 2.5 μL / min. Visual data of the input and output regions and cell movement were acquired using a microscope (Leica DM IL LED) and a CCD camera (Leica DFC 360FX). 50 and 200 images were collected at 10 frames / second in the input and output regions, respectively. The data were saved as uncompressed ".avi" files. MATLAB software was then used to count and analyze the tumor cells in the input and output regions. All data were exported to an Excel spreadsheet, and the average Do of the tumor cells at a flow rate of 2.5 μL / min was calculated. app .

[0121] Repeat the same steps as above to further detect and statistically analyze the average D of the tumor cell suspension at a flow rate of 25 μL / min. app Then, the average D under different flow rates was statistically analyzed. app The difference (ΔD) app ), and determine the deformability of tumor cells, ΔD app The smaller the value, the lower the deformability.

[0122] Example 4: Feasibility Verification of Tumor Cell Deformability Detection

[0123] This embodiment verifies the feasibility of using the microfluidic chip of the present invention to detect tumor cell deformability based on the method of Embodiment 2 above. The specific verification process is as follows:

[0124] The deformability of tumor cells was modulated by controlling cholesterol content on the cell membrane using methyl-β-cyclodextrin (MeβCD) and cholesterol (Chol). Specifically, B16-F10 cancer cells collected in culture medium were washed once with DMEM, centrifuged at 1200 rpm for 5 minutes at room temperature, and then centrifuged at 5 × 10⁻⁶ ppm. 5 Cells were resuspended in DMEM at a concentration of 2 million cells / mL. To deplete cholesterol on the plasma membrane, cells were incubated with DMEM supplemented with MeβCD (5 mM) at 37°C for 30 minutes; to replenish cholesterol on the plasma membrane, cells were incubated with DMEM supplemented with cholesterol (0.5 mM) at 37°C for 30 minutes and washed three times with PBS. All cells were resuspended in PBS at a concentration of 2 million cells / mL and then analyzed using the method described in Example 3.

[0125] Test results as follows Figure 3 As shown, after MeβCD treatment, the average D of tumor cells at different flow rates can be observed. app The difference (ΔD) app The significant reduction indicates decreased deformability, consistent with expectations. Furthermore, after Chol treatment, the average Do of tumor cells at different flow rates... app The difference (ΔD) app The significant increase indicates increased deformability, consistent with the expected results. These results demonstrate that the microfluidic chip and detection method of this invention can accurately characterize the deformability of tumor cells.

[0126] Example 5: Effect of fluid flow rate on cell deformability

[0127] Since tumor cells are deformable biological particles whose morphology and apparent size change under external forces, this embodiment investigates the effect of different fluid flow rates on the deformability of tumor cells using the microfluidic chip described in the above embodiment. The specific method is as follows:

[0128] The microfluidic chip described in Example 1 was used to detect the average Dg of melanoma cells B16-F10, colon cancer cells HCT-116, and breast cancer cells MDA-MB-231 and MCF-7 at different fluid flow rates (set to 2.5 μL / min, 5 μL / min, 10 μL / min, and 25 μL / min, respectively). app Each group was repeated 3 times. For specific experimental methods, please refer to Example 3.

[0129] The average D of B16-F10, HCT-116, MDA-MB-231, and MCF-7 under different fluid flow rates app Fitted linear graph as follows Figure 4 As shown, the average D of the detected tumor cells is... app The average Di is highly correlated with fluid flow rate and exhibits a linear relationship on a logarithmic scale. However, considering that excessively high fluid flow rates can easily damage the chip and cellular structures (cell membranes, cytoskeleton, nuclei, etc., which constitute the mechanical properties of cells), affecting chip lifespan and the accuracy of detection results, while excessively low flow rates can easily cause particle blockage of the chip, it is preferable to select the average Di obtained from fluid flow rates in the ranges of 2.5 μL / min and 25 μL / min. app The difference (ΔD) app The result is represented by the Tumor Cell Deformability (TCD) test result.

[0130] Example 6: Repeatability and Stability Test

[0131] To verify the repeatability and stability of the microfluidic chip of the present invention, this embodiment repeatedly measured the same diluted B16-F10 melanoma cell suspension using six batches of microfluidic chips prepared according to the method of Example 1 (three microfluidic chips were randomly selected from each batch). The specific detection method is as described in Example 3. After the detection was completed, ΔD was analyzed and calculated. app The relative standard deviation (RSD) of intraday measurements.

[0132] Test results as follows Figure 5 As shown, the ΔD measured for the same batch of chips is displayed. app The relative standard deviations were all less than or equal to 7.59%, and the RSD of different batches of chips (n=6) tested on the same day using the same diluted cell suspension samples was 1.66%, with an RSD of 4.34% for 18 tests. This demonstrates that the microfluidic chip and detection method of this invention exhibit good stability in detecting the deformability of tumor cells.

[0133] Example 7: Sensitivity Assessment

[0134] In this embodiment, B16-F10 cells were treated with MeβCD, which is known to alter cell deformability, and then the microfluidic chip prepared in Example 1 was used for sensitivity detection. The specific method is as follows:

[0135] A suspension of melanoma cells (B16-F10) to be tested was obtained, and then treated with different concentrations (0, 2.5, 5, 7.5, 10 mM) of MeβCD. The mean D of tumor cells at flow rates of 2.5 μL / min and 25 μL / min was detected using the detection method described in Example 3. app And based on the average D app The difference (ΔD) app )Analyze the deformability of tumor cells.

[0136] Test results as follows Figure 6 As shown, the reduction in tumor cell deformability induced by MeβCD is significantly dose-dependent, and it also demonstrates that the microfluidic chip of the present invention can sensitively detect the effect of trace amounts of tumor cell sclerosing agent (MeβCD) on the deformability of B16-F10 cells.

[0137] Example 8: Application in analyzing the basic state of the tumor cytoskeleton

[0138] This embodiment analyzes the changes in the basic cytoskeleton state of melanoma cells B16-F10 under cholesterol regulation using the microfluidic chip prepared in Example 1. The specific method is as follows:

[0139] A suspension of melanoma cells (B16-F10) was obtained, and then the B16-F10 cells were treated with MeβCD and Chol. The detection method described in Example 3 was used for analysis. The average Dc of tumor cells in different treatment groups at flow rates of 5 μL / min and 25 μL / min was also statistically analyzed. app Distribution, and average D app The difference (ΔD) app ).

[0140] Test results as follows Figure 7 As shown, A represents the D values ​​of different treatment groups B16-F10 at a flow rate of 25 μL / min. app Output frequency ridge map (the right box shows a superimposed image of 200 real-world images of tumor cells in the corresponding output region), B represents the corresponding average D. app Bar chart (n=6), C represents the final calculated ΔD app The results showed that MeβCD induced melanoma cells to drowsiness at a fluid flow rate of 25 μL / min. app Higher, but with significantly reduced deformability; in contrast, B16-F10 cells exposed to Chol had an average D appSignificantly reduced deformability and enhanced deformability. These results indicate that the biophysical parameters detected using the microfluidic chip of this invention can not only reflect changes in the deformability of tumor cells but also, to a certain extent, reflect changes in the basic state of the corresponding tumor cell cytoskeleton.

[0141] Example 9: Application in assessing the migration ability of tumor cells

[0142] This embodiment evaluates the correlation between tumor cell deformability and cell migration ability based on the microfluidic chip of Embodiment 1 above. The specific method is as follows:

[0143] (1) Cell degeneration detection: Cell suspensions of four types of tumor cells (B16-F10, HCT-116, HT-29, MDA-MB-231, and MCF-7) were obtained and set up as a control group and a MeβCD group. The MeβCD group was treated with 5 mM MeβCD, while the control group was not treated. The detection was then performed using the method described in Example 3.

[0144] (2) Transwell assay: The drug-treated cells were resuspended in serum-free culture medium and the concentration was adjusted to 1–5 × 10⁻⁶. 5 Cells / mL. To test invasiveness, 50–100 μL of Matrigel was coated onto the polycarbonate membrane (8 μm pore size) of the Transwell chamber and incubated at 37°C for 1–2 hours to form a gel. Subsequently, medium containing 10% FBS was added to the lower chamber as a chemotactic agent, and 200 μL of cell suspension was added to the upper chamber. The chamber was incubated at 37°C and 5% CO2 for 24–48 hours. After incubation, unmigrated cells were removed from the upper chamber, gently rinsed with PBS, and fixed with 4% paraformaldehyde for 10–20 minutes. Cells were then stained with crystal violet or DAPI, and rinsed again to remove excess dye. Cells that had passed through the membrane pores were observed under a microscope, and the number of migrating cells per field of view was counted to calculate the average migration rate. One-way ANOVA was performed using SPSS or similar software to evaluate statistical significance. MeβCD (5mM) significantly reduced the migration and invasion abilities of tumor cells, and the changes in migration before and after drug treatment were verified by Transwell assay, which correlated with ΔD. app The results were highly correlated.

[0145] (3) Scratch assay: Cells were seeded into culture plates and cultured until 90-100% confluence to form a monolayer. A straight line was vertically drawn in the center of the cell layer using a sterile 200 μL pipette tip to create a scratch. After scratching, the cells were slowly washed three times with PBS to remove detached cells, and the medium was replaced with serum-free medium to prevent cell proliferation from affecting the experimental results. Images of the initial scratch state (0 hours) were then taken, and the culture plates were placed in a 37°C, 5% CO2 incubator for further culture. Depending on the experimental requirements, images of the scratch healing were taken at 12, 24, or 48 hours, and cell migration was observed under a microscope. Changes in the scratch area or width could be analyzed using software such as ImageJ, and the migration rate was calculated (formula: scratch healing rate (%) = (0h scratch area - 24h scratch area) / 0h scratch area × 100%). One-way ANOVA was then performed using SPSS software to assess the significance of differences.

[0146] Test results as follows Figure 8 As shown, A represents the ΔD of four types of tumor cells (B16-F10, HCT-116, MDA-MB-231, and MCF-7). app B shows Transwell migration images of B16-F10, HCT-116, MDA-MB-231, and MCF-7 after MeβCD treatment (scale bar 100 μm); C shows the number of migrating cells (n=3); D shows the Transwell experiment and ΔD. app Correlation analysis; E represents scratch migration images of B16-F10, HCT-116, MDA-MB-231, and MCF-7 after MeβCD treatment; F represents migration rate statistics (n=3); G represents the correlation between scratch experiment and ΔD. app Correlation analysis was performed. The results showed that the number of migrating cells in the MeβCD group was less than that in the control group, and the overall mean difference in the number of migrating cells was statistically significant (P<0.05), indicating that cell migration was inhibited.

[0147] Furthermore, the scratch assay showed similar results; compared to the control group, the lateral migration rate of cells treated with MeβCD decreased to varying degrees. The results of the Transwell assay, scratch assay, and cell deformability assay (ΔD) were compared. app Correlation analysis showed that the results of tumor cell deformability testing were well correlated with those of Transwell assay (r = 0.817, P < 0.01) and scratch assay (r = 0.938, P < 0.05).

[0148] The above results indicate a negative correlation between tumor cell deformability and migration ability; higher tumor cell deformability correlates with stronger migration ability, while lower tumor cell deformability correlates with weaker migration ability. In conclusion, the microfluidic chip of this invention can be used to accurately measure the deformability of tumor cells and, to a certain extent, to assess their migration ability.

[0149] Example 10: Application in assessing the deformability of solid tumor cells in vivo

[0150] To verify the accuracy and sensitivity of the method of the present invention in detecting the deformability of tumor cells in mouse solid tumors, this embodiment analyzed the changes in biophysical parameters of tumor cells after intratumoral injection of MeβCD or Chol in B16-F10 tumor rats based on the microfluidic chip prepared in Example 1. The flowchart of TCD detection in mouse solid tumors is shown below. Figure 9 As shown in A, the specific method is as follows:

[0151] Nine male C57BL / 6J mice were subcutaneously injected with 1×10⁻⁶ ppm on the right side. 6 A tumor model was established using B16F10 cells (100 μL PBS). During the experiment, C57BL / 6J mice had free access to food and water, and were randomly divided into 3 groups (n = 3 / group) on day 10. To determine the deformability of cancer cells in B16F10 tumors, the model group received a single intratumoral injection of physiological saline (100 μL). The MeβCD group received a single intratumoral injection of MeβCD solution (100 μL, 15 mM physiological saline) for 30 min, and the Chol group received a single intratumoral injection of Chol solution (100 μL, 15 mM physiological saline) for 30 min. Afterwards, cells were collected after passing through a 70 μm cell filter and washed with 5% FBS / 1640 medium. Red blood cells were lysed with ACK lysis buffer for 5 min at room temperature. Finally, the cells were washed three times with PBS, resuspended at a concentration of 2 million cells / ml, and then the deformability of tumor cells in solid tumors was detected according to the method in Example 3.

[0152] Test results as follows Figure 9 As shown, A is the flowchart of TCD detection in mouse solid tumors; B is the flow chart of tumor cells in solid tumors of the mouse control group, MeβCD treatment group, and Chol treatment group at a fluid flow rate of 25 μL / min. app Output frequency ridge plot; C represents D for each group at a fluid flow rate of 25 μL / min. app Bar chart; D represents the results of tumor cell deformability detection within solid tumors. This shows the average D values ​​of single-cell suspensions of solid tumors from the control group, MeβCD group, and Chol group at a fluid flow rate of 25 μL / min. app There was a statistically significant difference (P<0.05) in the MeβCD treatment group, with ΔD... appCompared to the control group, the number of tumor cells decreased by 28.42%, while the number of cells treated with Chol increased by 24.16%. These results indicate that the tumor cell deformability detection method of the present invention has good sensitivity and applicability for in vivo solid tumor cell deformability detection, and can provide timely feedback on the biophysical characteristics of tumor cells in a short detection time (about 2 minutes). In addition, the detection method is non-destructive to cells and not only facilitates the collection of cell characteristics from the chip, but also helps in downstream tumor cell research.

[0153] In summary, this invention provides a microfluidic chip for measuring tumor cell deformability, along with its detection method and applications. The microfluidic chip-based tumor cell deformability detection method is simple, has good repeatability and stability, and exhibits high sensitivity to changes in tumor cell deformability induced by cholesterol regulators (MeβCD and Chol). It can be used not only for screening drugs related to tumor treatment but also for evaluating the basic state of the cytoskeleton and cell migration ability, showing broad application prospects.

[0154] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A microfluidic chip, characterized in that, It includes an inlet storage tank, a deterministic lateral displacement section, and an outlet storage tank, wherein: The deterministic lateral displacement segment is composed of an input region, a longitudinally arranged array of semi-T-shaped micropillars, and an output region connected in sequence. The input region has 10 to 35 sub-channels; the output region has 10 to 35 sub-channels. According to the flow direction of the sample to be tested, the critical cutoff size of each group of the semi-T-shaped micropillar arrays is... Dc The values ​​increase sequentially, the Dc The value ranges from 5 to 20 μm; the number of groups in the semi-T-shaped micropillar array is 10 to 30, and the corresponding values ​​of two adjacent groups of the semi-T-shaped micropillar array are... Dc The value increment is 0.3 μm ~ 0.8 μm; the critical cutoff size of each group of the semi-T-shaped micropillar arrays. Dc The value is determined by the formula. D c = 1.4 × G × (tanθ) 0.48 The decision includes: G is the spacing between two adjacent semi-T-shaped micropillars, ranging from 15 to 33 μm. θ The offset angle of the semi-T-shaped micropillars in each group of the semi-T-shaped micropillar arrays ranges from 1.13 to 23.11°; one end of each of the two sides of the semi-T-shaped micropillar is perpendicularly connected, and the other end is connected through a third side, which is arc-shaped; The inlet storage tank is connected to the sub-channel.

2. A method for fabricating a microfluidic chip, characterized in that, include: PDMS microchannels are fabricated based on the microfluidic chip as described in claim 1, and holes are drilled at corresponding positions in the inlet and outlet reservoirs, which are then assembled with a glass slide to obtain the final product.

3. A detection system, characterized in that, Includes the microfluidic chip, fluid drive device, and imaging device as described in claim 1.

4. The application of the microfluidic chip as described in claim 1 or the detection system as described in claim 3 in the preparation of products for detecting cell deformability, analyzing cytoskeleton state, or assessing cell migration ability.

5. A non-disease diagnostic or therapeutic method for detecting tumor cell deformability, characterized in that, include: The average size distribution of tumor cells under test at different flow rates is detected using the microfluidic chip as described in claim 1 or the detection system as described in claim 3, and the deformability of the tumor cells under test is determined based on the difference in the average size distribution of the tumor cells.

6. The method according to claim 5, characterized in that, The process also includes pretreatment of the microfluidic chip with a nonionic surfactant before detection.

7. The method according to claim 5, characterized in that, The flow rate range is 2 µL / min to 30 µL / min.

8. The method according to claim 7, characterized in that, The flow rates were 2~5 µL / min and 20~30 µL / min, respectively.

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