Micro-fluidic chip integrating single cell capture and elastic modulus measurement and method

Abstract

The invention discloses a micro-fluidic chip integrating single cell capture and elastic modulus measurement and a method, and relates to the technical field of microfluidics, the chip comprises a lower glass layer and an upper PDMS layer; wherein the PDMS layer comprises an integral chip runner which comprises a chip inlet, a gradient shunt channel, a micro-fluidic main channel and a chip outlet which are arranged from left to right; the gradient shunting channel can enable liquid to flow into the micro-fluidic main channel in a shunting manner; a serpentine channel capable of accommodating single cells to orderly enter the micro-fluidic main channel is arranged at a near-inlet section of the micro-fluidic main channel; a capture microstructure capable of capturing single cells is arranged in the microfluidic main channel. According to the technical scheme, the elasticity modulus of the tumor cells can be measured while the tumor single cells are captured in a high-throughput mode, and high application value is achieved.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a microfluidic chip and method integrating single cell capture and elastic modulus measurement. Background Art

[0002] As the basic unit of life, the growth, division, differentiation, apoptosis and other processes of cells in life are affected by a variety of environmental factors including mechanical factors. More and more studies have shown that biomechanical factors are closely related to the biological properties of cells, which directly affect the morphology, structure and function of cells. By studying the biomechanical properties of cells, the physiological functions of cells can be characterized to a certain extent. In recent years, more and more studies on the biomechanical properties of tumor cells have emerged one after another. Studies have shown that with the increase of tumor malignancy, the mechanical properties of tumor cells and tumor tissues will also change. Among them, the most significant is the difference in cell elastic modulus, which is specifically manifested in the different contractility, stretchability and rigidity of tumor cells with different malignant degrees, which further leads to different metastatic abilities. Effectively capturing single tumor cells is a prerequisite for measuring the mechanical properties of single tumor cells. The emergence of single cell analysis technology provides new methods and tools for studying the genetic composition, expression pattern and function of single cells. It is of great significance in life sciences and can help researchers to understand the essence of cell life more deeply and promote the development of biomedical research and treatment. Since capturing and obtaining single cells from a heterogeneous cell population is the most difficult step in single-cell analysis, single-cell capture has always been the focus and difficulty of single-cell analysis. Therefore, the development of a simple and efficient integrated single-cell capture and elastic modulus measurement method is imminent.

[0003] Most of the existing methods for integrating single-cell capture and elastic modulus measurement have defects in some aspects. For example, the micropipette measurement method, which uses a glass capillary to aspirate spherical cells and obtains the elastic modulus of the cells based on the cell deformation data, has a large error in the measurement results of this method; the atomic force microscope measurement method, which uses a precise cantilever free end to press the cell to cause local deformation of the cell surface, and obtains the elastic modulus of the cell based on the deformation and the required force. The equipment required for this method is relatively expensive, the operation is complex, and the measurement efficiency is relatively low. Therefore, based on this problem, the present application proposes a microfluidic chip and method for integrating single-cell capture and elastic modulus measurement. Summary of the invention

[0004] Technical Purpose In order to solve the above problems, the purpose of the present invention is to provide a microfluidic chip and method integrating single-cell capture and elastic modulus measurement, which can not only realize high-throughput capture of single tumor cells, but also realize the measurement of the elastic modulus of tumor cells. The chip and method have a wide range of application scenarios, low device complexity and simple operation.

[0005] Technical Solution In order to achieve the above-mentioned objectives, the present invention provides a microfluidic chip and method integrating single-cell capture and elastic modulus measurement, which mainly performs high-throughput capture of single tumor cells through an inverted triangular capture structure, increases the width of the capture channel by gradient to analyze the influence of different capture microstructures on the elastic modulus of cells, sets different flow resistances for the main channel and the capture channel to improve the success rate of cell capture and force the cells to deform, and calculates the elastic modulus of the cells based on the pressure drop acting on the cells and the steady-state elongation length of the cells after being captured, thereby achieving high-throughput capture of single tumor cells and measurement of the elastic modulus of tumor cells at the same time.

[0006] First, a microfluidic chip integrating single cell capture and elastic modulus measurement is provided. It includes a glass layer at the bottom and a PDMS (polydimethylsiloxane) layer at the top. The PDMS layer includes the entire chip flow channel, including the chip inlet, gradient diversion channel, microfluidic main channel and chip outlet arranged from left to right; The gradient shunt channel can allow liquid to be shunted into the microfluidic main channel; The near-entrance section of the microfluidic main channel is provided with a serpentine channel capable of accommodating single cells to enter the microfluidic main channel in an orderly manner; A capture microstructure capable of capturing single cells is arranged in the microfluidic main channel.

[0007] Furthermore, the thickness of the glass layer is 0.2-5 mm, preferably 0.5-3 mm, more preferably 1-2 mm, and most preferably 1 mm.

[0008] Furthermore, the thickness of the PDMS layer is 1-10 mm, preferably 2-8 mm, more preferably 3-5 mm, and most preferably 5 mm.

[0009] Furthermore, the microfluidic main channels are symmetrically distributed and the number is at least 2, preferably 2-10, more preferably 4-8, and most preferably 6.

[0010] Furthermore, the capture microstructure has a triangular capture structure above and a capture channel below. The triangular capture structure is 25 μm in height, and the upper opening is 38 μm in width and 25 μm in height. The capture channels are 25 μm in length and 6 μm in height, and the spacing between two adjacent capture microstructures is 50 μm.

[0011] Furthermore, each of the microfluidic main channels is provided with 90 capture microstructures, and the capture channel widths of the first 30, middle 30, and last 30 capture microstructures increase in sequence. The capture channel width gradient increases, and cells with different elastic moduli are separated, which is used to analyze the effects of different capture microstructures on the elastic modulus of cells, and can provide a deeper understanding of the physical properties of tumor cells.

[0012] Furthermore, in the capture microstructure of each microfluidic main channel, the first thirty capture channels have a width of 8 μm, the middle thirty capture channels have a width of 9 μm, and the last thirty capture channels have a width of 10 μm. By precisely controlling the shape and size of the capture microstructure, the variability in the experiment is reduced and the repeatability of the experimental results is improved.

[0013] Second, a microfluidic system integrating single cell capture and elastic modulus measurement is provided, including: A control module, used for controlling the injection of a cell suspension into the microfluidic chip described in the first aspect; Microfluidic chip for single cell capture and elastic modulus measurement; An observation module is used to observe and record the cell suspension in the microfluidic chip in real time; The recovery module is used to recover the cell suspension flowing out of the microfluidic chip.

[0014] Furthermore, the control module includes a programmable injection pump and a syringe; the programmable injection pump generates a required driving signal, and the syringe injects the cell suspension into the microfluidic chip. Furthermore, the microfluidic chip physically captures cells by optimizing the flow resistance of the main channel and the flow resistance of the capture channel; when the cells are captured, the pressure difference between the main channel and the capture channel forces the cells to deform, thereby capturing a large number of single cells at the same time through an effective capture mechanism.

[0015] Furthermore, the flow resistance ratio of the main channel and the capture channel is higher than 2. By adjusting the flow resistance, the cells can be more effectively guided to the capture area, thereby improving the success rate of single cell capture; by precisely controlling the flow resistance, the interference of non-target cells or impurities is reduced, thereby improving the purity of captured target cells.

[0016] Furthermore, based on the semi-infinite space theory and assuming that the cell is a homogeneous elastic body, the elastic modulus formula of the cell is calculated as:

[0017] in, represents the elastic modulus of the cell; represents the pressure drop acting on the cell; represents a constant that depends on the trapping channel geometry; represents the equivalent radius of the capture channel; represents the steady-state elongation length of cells after arrest.

[0018] It can accurately measure the elastic modulus of cells by quantitatively analyzing the pressure drop and cell elongation length; it helps to distinguish cells of different types or states with different elastic moduli, which is of great significance for disease diagnosis and research.

[0019] Furthermore, in the calculation formula of the elastic modulus of the cell, the equivalent radius of the capture channel is The calculation formula is:

[0020] in, Indicates the width of the capture channel; Indicates the height of the capture channel.

[0021] Furthermore, the observation module includes a fluorescence microscope and a high-speed camera, through which the capture process is observed in real time, and the cell capture image is processed to measure the contour of the cell in the capture channel.

[0022] In a third aspect, the present invention further provides a microfluidic method for integrating single cell capture and elastic modulus measurement, the method being based on the system described in the second aspect, comprising: Control the cell suspension to be injected into the microfluidic chip through a control module; Single cell capture and elastic modulus measurement are completed through microfluidic chips; The observation module observes and records the capture process in real time; The cell suspension flows out of the microfluidic chip and is injected into the recovery module.

[0023] In a fourth aspect, the present invention further provides a computer-readable storage medium, wherein the computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the aforementioned microfluidic method of integrating single-cell capture and elastic modulus measurement is implemented.

[0024] The present invention uses an inverted triangular capture structure to perform high-throughput capture of single tumor cells. The width of the capture channel is increased by gradient to analyze the effect of different capture microstructures on the elastic modulus of cells. Different flow resistances are set for the main channel and the capture channel to increase the success rate of cell capture and force the cells to deform. The elastic modulus of cells is calculated based on the pressure drop acting on the cells and the steady-state elongation length of the cells after being captured. The chip and method have a wide range of application scenarios, and can measure the elastic modulus of tumor cells while capturing single tumor cells at high throughput.

[0025] Beneficial Effects By implementing the microfluidic chip and method for integrating single cell capture and elastic modulus measurement provided by the present invention, the following technical effects are achieved: (1) High-throughput capture of single tumor cells is performed through an inverted triangular capture structure. The width of the capture channel is increased by gradient to analyze the effect of different capture microstructures on the elastic modulus of cells. By precisely controlling the shape and size of the capture microstructure, the variability in the experiment is reduced and the repeatability of the experimental results is improved. The width of the capture channel is increased by gradient to separate cells with different elastic moduli, which can provide a deeper understanding of the physical properties of tumor cells and simultaneously capture a large number of single cells through an effective capture mechanism.

[0026] (2) Different flow resistances are set for the main channel and the capture channel to improve the success rate of cell capture and force the cells to deform. By adjusting the flow resistance, the cells can be more effectively guided to the capture area, thereby improving the success rate of single-cell capture. By precisely controlling the flow resistance, the interference of non-target cells or impurities is reduced, thereby improving the purity of captured target cells.

[0027] (3) The elastic modulus of the cells is calculated based on the pressure drop acting on the cells and the steady-state elongation length of the cells after being captured. By quantitatively analyzing the pressure drop and the cell elongation length, the elastic modulus of the cells can be accurately measured, which helps to distinguish cells of different types or states with different elastic moduli, which is of great significance for the diagnosis and research of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the above-mentioned integrated single-cell capture and elastic modulus measurement microfluidic chip and method of the present invention more obvious and easy to understand, the drawings required for use in the specific embodiments of the present invention are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 Schematic diagram of the microfluidic method integrating single-cell capture and elastic modulus measurement; Figure 2 Schematic diagram of the microfluidic system integrating single-cell capture and elastic modulus measurement; Figure 3 Schematic diagram showing the distribution of captured microstructures; Figure 4 Schematic diagram showing the capture of microstructural details. DETAILED DESCRIPTION

[0030] Embodiment 1: Provided are a microfluidic chip and method for integrating single cell capture and elastic modulus measurement, and a microfluidic method for integrating single cell capture and elastic modulus measurement. Figure 1 As shown in Figure 2, the microfluidic system integrating single cell capture and elastic modulus measurement is shown in Figure 2. Figure 2 As shown, the system includes: a control module, a microfluidic chip, an observation module and a recovery module, as described in detail below.

[0031] A control module is used to control the injection of cell suspension into the microfluidic chip described in the first aspect above, and the control module includes a programmable injection pump and a syringe; the required driving signal is generated by the programmable injection pump, and the cell suspension is injected into the microfluidic chip by the syringe.

[0032] Microfluidic chip, gradient flow channel, used for single cell capture and elastic modulus measurement. The chip is divided into two layers, the lower layer is 1mm thick glass, and the upper layer is 5mm thick PDMS layer. The PDMS layer contains the entire chip flow channel. The overall structure of the chip is as follows Figure 3 As shown in the figure, the chip is equipped with inlets and outlets on the left and right sides. The left inlet is equipped with a gradient diversion channel until the liquid enters the six main channels. The six main channels are symmetrical up and down. The front sections of the six main channels are equipped with serpentine channels so that cells can pass through the main channels one by one in an orderly manner during the experiment, achieving a better capture effect. Each main channel is equipped with 90 capture microstructures. The microstructure is a triangular capture structure above and a capture channel below. The capture microstructure is as follows Figure 4 As shown, the capture microstructure is a triangular capture structure with a height of 25μm, a width of 38μm at the top opening, and a height of 25μm, which enables the system to have a better single-cell capture effect, and the distance between two adjacent capture microstructures is 50μm. The design of every thirty capture channels is different, with a length of 25μm and a height of 6μm. The width of the first thirty capture channels is 8μm, the width of the middle thirty capture channels is 9μm, and the width of the last thirty capture channels is 10μm. There are 540 capture microstructures in the six channels of the entire chip, and the different capture channel structures can be used to compare and verify the results of the cell elastic modulus.

[0033] The width gradient of the capture channel is increased to analyze the effect of different capture microstructures on the elastic modulus of cells. The cells are physically captured by setting different flow resistances for the main channel and the capture channel through the triangular capture structure; when the cells are captured, the pressure difference between the main channel and the capture channel forces the cells to deform. The flow resistance ratio of the main channel to the capture channel is higher than 2.

[0034] Based on the semi-infinite space theory and assuming that the cell is a homogeneous elastic body, the elastic modulus formula of the cell is calculated as:

[0035] in, represents the elastic modulus of the cell; represents the pressure drop acting on the cell; represents a constant that depends on the trapping channel geometry; represents the equivalent radius of the capture channel; represents the steady-state elongation length of cells after arrest.

[0036] In the calculation formula of the elastic modulus of the cell, the equivalent radius of the capture channel is The calculation formula is:

[0037] in, Indicates the width of the capture channel; Indicates the height of the capture channel.

[0038] The observation module is used to observe and record the cell suspension in the microfluidic chip in real time. During the experiment, an inverted fluorescence microscope and a matching high-speed camera can be combined to achieve real-time single-cell capture observation and deformation recording of tumor cells.

[0039] The recovery module is used to recover the cell suspension flowing out of the microfluidic chip.

[0040] Embodiment 2: On the basis of the above-mentioned embodiments, a microfluidic method integrating single cell capture and elastic modulus measurement is used to achieve high-throughput capture of single tumor cells while measuring the elastic modulus of tumor cells. The microfluidic chip is fixed on a microscope platform, and a tumor cell line with an average diameter of 15 to 17 mm is prepared into a cell suspension with a concentration of 1×10^6 cells per milliliter. The flow rate of the programmable injection pump is set to 100 microliters per minute to ensure that tumor cells are injected into the microfluidic chip in a single and orderly manner, and the cell suspension is injected into the microfluidic device through a syringe. The cell flow is observed through an inverted fluorescence microscope and a matching high-speed camera to ensure that the cells pass through the main channel in a single and orderly manner. The capture of cells in the capture microstructure is observed under a microscope and the cell capture process is recorded by a high-speed camera.

[0041] For cells trapped in the trapping microstructures, their steady-state elongation length and pressure drop within the trapping channel were recorded.

[0042] Assume that the steady-state elongation length of a cell in the capture channel is 10 mm, the pressure drop is 50 Pa, and the width and height of the capture channel are 8 mm and 6 mm respectively:

[0043]

[0044] It will be appreciated by those skilled in the art that embodiments of the present invention may be provided as methods, systems or computer program products. Therefore, the present invention may take the form of a complete hardware embodiment, a complete software embodiment, or an embodiment combining software and hardware. Moreover, the present invention may take the form of a computer program product implemented on one or more computer-usable non-transient storage media containing computer-usable program code.

[0045] The present invention can provide computer program instructions to a processor of a general-purpose computer, a special-purpose computer, an embedded processor or other programmable data processing device to produce a machine, so that the instructions executed by the processor of the computer or other programmable data processing device produce means for implementing the system.

[0046] These computer program instructions may also be stored in a computer-readable memory that can direct a computer or other programmable data processing device to work in a specific manner, so that the instructions stored in the computer-readable memory produce a product including an instruction device that implements the functions of the system.

[0047] These computer program instructions may also be loaded onto a computer or other programmable data processing device so that a series of operational steps are executed on the computer or other programmable device to produce a computer-implemented process, whereby the instructions executed on the computer or other programmable device provide steps for implementing the functions of the system.

Claims

1. A microfluidic chip integrating single cell capture and elastic modulus measurement, characterized in that: include: It includes a glass layer at the bottom and a PDMS layer at the top. The PDMS layer includes the entire chip flow channel, including the chip inlet, gradient diversion channel, microfluidic main channel and chip outlet arranged from left to right; The gradient shunt channel can allow liquid to be shunted into the microfluidic main channel; The near-entrance section of the microfluidic main channel is provided with a serpentine channel capable of accommodating single cells to enter the microfluidic main channel in an orderly manner; A capture microstructure capable of capturing single cells is arranged in the microfluidic main channel.

2. The microfluidic chip integrating single cell capture and elastic modulus measurement according to claim 1, characterized in that: The thickness of the glass layer is 0.2-5 mm; and / or The thickness of the PDMS layer is 1-10 mm.

3. The microfluidic chip integrating single cell capture and elastic modulus measurement according to claim 1, characterized in that: The microfluidic main channels are symmetrically distributed and the number is at least 2.

4. The microfluidic chip integrating single cell capture and elastic modulus measurement according to claim 1, characterized in that: The capture microstructure has a triangular capture structure above and a capture channel below. The triangular capture structure is 25 μm high, and the upper opening is 38 μm wide and 25 μm high. The capture channels are 25 μm long and 6 μm high, and the distance between two adjacent capture microstructures is 50 μm.

5. The microfluidic chip integrating single cell capture and elastic modulus measurement according to claim 1, characterized in that: Each of the microfluidic main channels is provided with 90 capture microstructures, and the capture channel widths of the first 30, middle 30, and last 30 capture microstructures increase in sequence.

6. The microfluidic chip integrating single cell capture and elastic modulus measurement according to claim 5, characterized in that: In the capture microstructure of each microfluidic main channel, the width of the first thirty capture channels is 8 μm, the width of the middle thirty capture channels is 9 μm, and the width of the last thirty capture channels is 10 μm.

7. A microfluidic system integrating single cell capture and elastic modulus measurement, characterized by include: A control module, used for controlling the injection of a cell suspension into the microfluidic chip according to any one of claims 1 to 6; Microfluidic chip for single cell capture and elastic modulus measurement; An observation module is used to observe and record the cell suspension in the microfluidic chip in real time; The recovery module is used to recover the cell suspension flowing out of the microfluidic chip.

8. The microfluidic system integrating single cell capture and elastic modulus measurement according to claim 7, characterized in that: The control module comprises a programmable injection pump and a syringe; the required driving signal is generated by the programmable injection pump, and the cell suspension is injected into the microfluidic chip by the syringe.

9. A microfluidic method integrating single cell capture and elastic modulus measurement, characterized in that: The method is based on the system according to claim 7 or 8, comprising: Control the cell suspension to be injected into the microfluidic chip through a control module; Single cell capture and elastic modulus measurement are completed through microfluidic chips; The observation module observes and records the capture process in real time; The cell suspension flows out of the microfluidic chip and is injected into the recovery module.

10. A computer-readable storage medium, wherein a computer program is stored in the computer-readable storage medium, characterized in that: When the computer program is executed, the microfluidic method for integrating single cell capture and elastic modulus measurement as claimed in claim 9 is implemented.

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