阅读说明：本技术 一种细胞电阻抗谱测量的微流控芯片 (Micro-fluidic chip for cell electrical impedance spectroscopy measurement ) 是由 姚佳烽 王力 杨璐 刘凯 陈柏 吴洪涛 于 2020-04-20 设计创作，主要内容包括：本发明公开了一种细胞电阻抗谱测量的微流控芯片,由上盖板和下基板组成,上盖板上开通孔构成流体入口和流体出口,下基板的上表面分布有正六边形微孔单元阵列以及微流体通道,每个正六边形微孔单元内部设置有细胞捕获阱,正六边形微孔单元上靠近细胞捕获阱相邻壁面设有一对测量电极,正六边形微孔单元之间通过微流体通道串联成一条液体通道,液体通道与上盖板的流体入口和流体出口相接处设有入口处蓄液池、出口处蓄液池,上盖板与下基板通过键合封接形成密封微流控芯片。本发明将单细胞捕获及细胞阻抗谱测量集成在单块微流控芯片上,通过提供相应的操作方法,实现快速获取并精确分析单细胞阻抗谱。(The invention discloses a micro-fluidic chip for measuring cell electrical impedance spectroscopy, which consists of an upper cover plate and a lower base plate, wherein a through hole is formed in the upper cover plate to form a fluid inlet and a fluid outlet, a regular hexagonal micropore unit array and a micro-fluid channel are distributed on the upper surface of the lower base plate, a cell capture trap is arranged in each regular hexagonal micropore unit, a pair of measuring electrodes are arranged on the regular hexagonal micropore units and close to the adjacent wall surfaces of the cell capture traps, the regular hexagonal micropore units are connected in series to form a liquid channel through the micro-fluid channel, an inlet liquid storage pool and an outlet liquid storage pool are arranged at the joint of the liquid channel and the fluid inlet and the fluid outlet of the upper cover plate, and the upper cover plate and. The invention integrates single cell capture and cell impedance spectrum measurement on a single microfluidic chip, and realizes rapid acquisition and accurate analysis of single cell impedance spectrum by providing a corresponding operation method.)

1. A micro-fluidic chip for cell electrical impedance spectroscopy measurement is characterized in that: consists of an upper cover plate (1) and a lower base plate (2), a fluid inlet (3) and a fluid outlet (4) are formed by through holes on the upper cover plate (1), the upper surface of the lower substrate (2) is distributed with a regular hexagon micropore unit (7) array and a microfluid channel (8), a cell trapping trap (10) is arranged in each regular hexagon micropore unit (7), a pair of measuring electrodes (9) are arranged on the adjacent wall surface of the regular hexagonal micropore unit (7) close to the cell trapping well (10), the regular hexagonal micropore units (7) are connected in series through the microfluidic channel (8) to form a liquid channel, the liquid channel is provided with an inlet liquid storage tank (5) and an outlet liquid storage tank (6) at the joint of the fluid inlet (3) and the fluid outlet (4) of the upper cover plate (1), the upper cover plate (1) and the lower base plate (2) are sealed through bonding to form a sealed microfluidic chip.

2. The microfluidic chip for electrical impedance spectroscopy of claim 1, wherein: the regular hexagonal micropore units (7) are arranged in an array in a staggered mode at equal intervals, two adjacent rows of regular hexagonal micropore units (7) are communicated through a microfluid channel (8) with the same width as the side length of the regular hexagon to form a zigzag channel, and the side length of the regular hexagon ranges from 100 to 200 micrometers.

3. The microfluidic chip for electrical impedance spectroscopy of claim 1, wherein: the cell trapping trap (10) is composed of two bending walls which are horizontally and symmetrically distributed, the distance between the parallel parts of the two bending walls is 1.2-1.5 times of the diameter of a target cell, the distance between the openings of the inward bending parts is 0.5-0.8 times of the diameter of the target cell, and the included angle formed by the bending parts is 60-120 degrees.

4. The microfluidic chip for electrical impedance spectroscopy of claim 3, wherein: the distance between the center of the narrow opening of the cell trapping trap (10) and the nearest vertex angle of the regular hexagon micropore unit 7 is 0.2-0.5 times of the side length of the regular hexagon.

5. The microfluidic chip for electrical impedance spectroscopy of claim 3, wherein: the height of the wall surface of the cell trapping trap (10) is 0.8-1.2 times of the diameter of a target cell.

6. The microfluidic chip for electrical impedance spectroscopy of claim 1, wherein: the measuring electrodes (9) are symmetrically arranged in pairs on the nearest adjacent wall of the cell trapping well (10).

7. The microfluidic chip for electrical impedance spectroscopy of claim 6, wherein: the length of the measuring electrode (9) is 0.5 times of the side length of the regular hexagon, and the width of the measuring electrode is the same as the depth of the regular hexagon micropore unit (7).

8. The microfluidic chip for electrical impedance spectroscopy of any one of claims 1 to 7, wherein: and the fluid inlet (3) and the fluid outlet (4) are connected with a syringe pump for driving the liquid to flow.

9. The microfluidic chip for electrical impedance spectroscopy of any one of claims 1 to 7, wherein: the measuring electrode (9) is connected with an external impedance analyzer through a metal wire deposited on the surface of the substrate.

10. The microfluidic chip for electrical impedance spectroscopy of any one of claims 1 to 7, wherein: the micro-fluidic chip is manufactured and molded by combining Polydimethylsiloxane (PDMS) with a soft lithography method.

The technical field is as follows:

the invention relates to a micro-fluidic chip for measuring cell electrical impedance spectroscopy.

Background art:

the impedance spectrum measurement is to apply a small amplitude alternating voltage to two ends of a sample to be measured, and then obtain the variation relation of the impedance of the particle group in the sample along with the frequency through a signal analyzer. Impedance spectroscopy measurement is an important method for researching the electrical characteristics of cells or other micro-nano particles, and can realize the characterization of micro-nano particle populations. Electrical impedance testing and analysis are widely applied in the fields of drug screening, disease diagnosis and treatment, food inspection, environmental monitoring and the like. Impedance spectroscopy for biological samples is called Bio-impedance technology (Bio-impedance technology), which has the characteristics of being fast, label-free and easy to operate, and gradually becomes an effective analysis tool in biomedical research.

The current impedance spectroscopy measurement technology is directed to a cell cluster, the capture and measurement research on single cells is not complete, and the measurement on a specific single cell in the cell cluster cannot be realized. In many cases, it is necessary and important to study the impedance spectrum of a single cell. For example, peripheral blood of a patient with early cancer contains Circulating Tumor Cells (CTC) which are separated from a focus, the CTC is widely regarded as a marker product of early cancer in the medical field, and the Circulating Tumor cells are distinguished from a large number of blood cells through differences among impedance spectrum parameters, so that the method is beneficial to timely diagnosis of cancer and improvement of cure rate.

Therefore, on the basis of inheriting the basic principle of impedance spectrum measurement, how to realize impedance measurement on a single cell in a cell population is a key problem for solving the limitation.

The invention content is as follows:

the invention provides a micro-fluidic chip for cell electrical impedance spectroscopy measurement, which aims to solve the problems in the prior art, can quickly extract single cells from a cell suspension, integrates single cell capture, flushing and impedance spectroscopy measurement on a single micro-fluidic chip, and realizes the accurate measurement of single cell scale impedance spectroscopy through simple operation.

The technical scheme adopted by the invention is as follows: a micro-fluidic chip for measuring cell electrical impedance spectroscopy comprises an upper cover plate and a lower cover plate, wherein a through hole is formed in the upper cover plate to form a fluid inlet and a fluid outlet, regular hexagonal micropore unit arrays and micro-fluid channels are distributed on the upper surface of the lower cover plate, a cell capture trap is arranged in each regular hexagonal micropore unit, a pair of measuring electrodes are arranged on the regular hexagonal micropore units and close to the adjacent wall surfaces of the cell capture traps, the regular hexagonal micropore units are connected in series through the micro-fluid channels to form a liquid channel, an inlet liquid storage pool and an outlet liquid storage pool are arranged at the joint of the liquid channel and the fluid inlet and the fluid outlet of the upper cover plate, and the upper cover plate and the lower cover plate are sealed and connected through bonding.

Furthermore, the regular hexagonal micropore unit arrays are arranged in a staggered mode at equal intervals, two adjacent rows of regular hexagonal micropore units are communicated through a microfluid channel with the same width as the side length of the regular hexagon to form a zigzag channel, and the side length of the regular hexagon is 100-200 mu m.

Furthermore, the cell trapping trap is composed of two bending walls which are horizontally and symmetrically distributed, the distance between the parallel parts of the two bending walls is 1.2-1.5 times of the diameter of a target cell, the distance between the openings of the inward bending parts is 0.5-0.8 times of the diameter of the target cell, and the included angle formed by the bending parts is 60-120 degrees.

Further, the distance between the center of the narrow opening of the cell trapping trap and the nearest vertex angle of the regular hexagon micropore unit 7 is 0.2-0.5 times of the side length of the regular hexagon.

Furthermore, the height of the wall surface of the cell trapping trap is 0.8-1.2 times of the diameter of the target cell.

Further, the measuring electrodes are symmetrically arranged in pairs on the nearest adjacent wall surface of the cell trapping well.

Further, the length of the measuring electrode is 0.5 times of the side length of the regular hexagon, and the width of the measuring electrode is the same as the depth of the regular hexagon micropore unit.

Further, the fluid inlet and the fluid outlet are connected with a syringe pump for driving the liquid to flow.

Further, the measuring electrode is connected with an external impedance analyzer through a metal wire deposited on the surface of the substrate.

Further, the microfluidic chip is molded by combining Polydimethylsiloxane (PDMS) with a soft lithography method.

The invention has the following beneficial effects:

1. the invention realizes the capture and the impedance spectrum measurement of single cells on one microfluidic chip, and brings several remarkable advantages to the accurate analysis of the single cells in the molecular biology field and the medical diagnosis field related by the invention: firstly, the accuracy of analysis is greatly improved, so that whether abnormal cells exist in a large number of normal cell populations can be detected; and secondly, compared with the conventional single cell analysis method, the adopted impedance measurement method applies tiny alternating current voltage signals which do not influence the physiological state of the cells, the integrity of the cell structure is kept, repeated measurement can be realized for many times, the flow channel is prevented from being polluted by introducing chemical reagents, and the accuracy of subsequent molecular biological analysis is improved.

2. The cell trapping well structure provided by the invention is positioned in the hexagonal micropore unit, and by utilizing the fluid mechanics principle and the inertial focusing characteristic of particles, once cells are trapped, the surrounding low-speed flow field hardly influences the position, so that the accuracy and reliability of impedance spectrum measurement are ensured.

3. Reliable capture of single cells and accurate measurement of impedance spectra are achieved.

4. The cell capture structure is stable and reliable.

5. The label-free analysis method does not affect the activity of the cells and can continuously repeat the measurement.

6. The operation is simple and the cleaning is convenient.

Description of the drawings:

fig. 1 is an overall view of a microfluidic chip according to an embodiment of the present invention.

Fig. 2 is a top view of a lower substrate of a microfluidic chip according to an embodiment of the present invention.

Fig. 3 is a longitudinal sectional view of a lower substrate of the microfluidic chip according to the embodiment of the present invention.

Fig. 4 is a partially enlarged view of a in fig. 3.

Fig. 5 is an enlarged view of a hexagonal microporous unit in a microfluidic chip according to an embodiment of the present invention.

Fig. 6 is a perspective view of a bonded microfluidic chip according to an embodiment of the present invention.

Fig. 7 is a partially enlarged view of B in fig. 6.

The specific implementation mode is as follows:

the embodiments of the present invention will be described in detail below. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.

Referring to fig. 1 to 5, the microfluidic chip for electrical impedance spectroscopy measurement of cells according to the present invention comprises an upper cover plate 1 and a lower base plate 2, wherein a through hole is formed in the upper cover plate 1 to form a fluid inlet 3 and a fluid outlet 4, an array of regular hexagonal microporous cells 7 is distributed on the upper surface of the lower base plate 2, a cell capture well 10 is disposed inside each regular hexagonal microporous cell 7 for capturing a single cell in a cell suspension, and a pair of measurement electrodes 9 is disposed on the regular hexagonal microporous cells 7 and near the adjacent wall surface of the cell capture well 10 for applying a small-amplitude ac voltage to excite the cell impedance spectroscopy measurement; the regular hexagonal micropore units 7 are connected in series through the micro-fluid channel 8 to form a liquid channel; the liquid channel is provided with an inlet liquid storage tank 5 and an outlet liquid storage tank 6 at the joint of the fluid inlet 3 and the fluid outlet 4 of the upper cover plate 1; the upper cover plate 1 and the lower base plate 2 are sealed and connected through bonding to form a sealed microfluidic chip.

In a preferred embodiment, the regular hexagonal micropore unit arrays 7 are arranged in a staggered mode at equal intervals, two adjacent rows of regular hexagonal micropore units 7 are communicated through a microfluid channel 8 with the same width as the side length of the regular hexagon to form a zigzag channel, and the side length of the regular hexagon is 100-200 μm.

In a preferred embodiment, the cell trap 10 is composed of two horizontally symmetrically distributed bent walls, the bent walls are two micro-wall surfaces connected with each other at a certain included angle, the distance between the parallel portions of the two bent walls is 1.2-1.5 times of the diameter of a target cell, the distance between the openings of the inward bent portions is 0.5-0.8 times of the diameter of the target cell, and the included angle formed by the bent portions is 60-120 °.

In a preferred embodiment, the distance between the center of the narrow opening of the cell trap 10 and the nearest top corner of the regular hexagonal microporous unit 7 is 0.2-0.5 times of the side length of the regular hexagon.

In a preferred embodiment, the height of the wall of the cell trap 10 is 0.8 to 1.2 times the diameter of the target cell.

In the preferred embodiment, the inlet 5 and outlet 6 reservoirs are axially coincident with the fluid inlet 3 and fluid outlet 4 of the upper deck 2, and the inlet 5 and outlet 6 reservoirs are larger in diameter than the fluid inlet 3 and fluid outlet 4.

In a preferred embodiment, a syringe pump for driving the flow of liquid is connected to the fluid inlet 3 and the fluid outlet 4.

In a preferred embodiment, the impedance measuring electrodes are connected to an impedance analyzer via wires deposited on the surface of the substrate.

In a preferred embodiment, the microfluidic chip is fabricated by combining Polydimethylsiloxane (PDMS) with soft lithography.

The result and position of cell capture are determined by an optical microscope, so that the upper cover plate 1 is made of transparent material, and the upper cover plate 1 is compatible with the material of the lower substrate 2 to form a sealed microfluidic channel. Polydimethylsiloxane (PDMS) is an optimal material for manufacturing the upper cover plate 1 and the lower base plate 2 of the microfluidic chip because of its non-toxicity, chemical inertness, good light transmittance, biocompatibility, and high structure elasticity.

The arrangement of the fluid inlet 3, the fluid outlet 4, the liquid storage pool 5 at the inlet and the liquid storage pool 6 at the outlet can be freely designed according to the shape of the fluid channel of the lower substrate 2, and the diameter of the liquid storage pool can be properly changed according to the flow requirement of the measuring system.

The two adjacent rows of the regular hexagonal micropore units 7 are arranged in a staggered manner to form a similar honeycomb structure, the adjacent micropore units between the two adjacent rows are connected in series to form a complete flow channel by the micro flow channel 8, the micro flow channels are arranged in a zigzag manner, the purpose is to guide fluid to pass in the micro flow channel 8 in a zigzag manner, uneven velocity field distribution is formed in the flow channel region, the fluid velocity at the position, far away from the fluid inlet and outlet, in the regular hexagonal micropore units 7 is low, and the fluid velocity is matched with the cell trapping trap 10, so that single cells are trapped more easily.

The cell trapping well 10 is composed of two bent walls which are horizontally and symmetrically distributed, for each regular hexagonal micro-pore unit 7, after entering from the micro-fluid channel 8, under the resultant force action of fluid shearing force and inertial lifting force, cells flow to the cell trapping well structure and are limited at the narrow opening part of the cell trapping well 10, the whole cell trapping well structure is positioned in a low-speed flow field area of the micro-pores far away from a fluid inlet and a fluid outlet, and the trapped cells are not easily influenced by fluid disturbance and escape.

After the cell is captured by the trap, the flow through the cell trap 10 is further reduced due to the obstruction of the cell, causing other cells to bypass. The cell trap structure is sized to the target trap cell diameter, allowing for the accommodation of a single cell.

The measuring electrode 9 is produced by a sputtering process, the process of which is described below: and sputtering a chromium metal layer with the thickness of 0.1 micrometer on the front surface of the lower substrate 2, sputtering a gold layer with the thickness of 0.5 micrometer on the chromium metal layer, and inclining the lower substrate 2 by 45 degrees and rotating during sputtering so as to ensure that the metal covers the side wall of the micropore. And (3) carrying out vacuum auxiliary gluing and photoetching on the front surface of the lower substrate 2 to define the shape of the required electrode, then corroding the gold layer by using a potassium iodide solution, and corroding the chromium layer by using ammonium ceric nitrate, thus obtaining the measuring electrode 9 in the regular hexagonal micropore unit 7. The wires of the measuring electrodes 9 are led out on the upper surface of the lower substrate 2 in the same manner.

The upper cover plate 1 and the lower base plate 2 are used for preparing a substrate by Polydimethylsiloxane (PDMS) molding technology and preparing a micro-channel structure by combining a soft lithography method. The upper cover plate 1 and the lower substrate 2 are bonded together by an oxygen plasma assisted bonding method to form a complete chip.

And (3) micro-fluidic chip system construction: on the basis of the microfluidic chip, a polytetrafluoroethylene tube is used for connecting a microfluidic injection pump and a fluid inlet and a fluid outlet of an upper cover plate, an electrode leading-out end is connected with a multiplexing gating circuit by a lead and then connected to an impedance analyzer, and the microfluidic chip is placed under a fluorescence microscope, so that the whole set of system can be built.

The working process of the microfluidic chip is described as follows: and respectively filling the cell suspension to be detected and the buffer solution for removing the cells into the two syringes, wherein the syringe filled with the cell suspension slowly injects fluid into the microfluidic channel from the fluid inlet of the microfluidic chip under the action of a push rod of the injection pump, and the suspension flows through each regular hexagonal microporous unit in sequence under the guidance of the zigzag microfluidic channel. For each regular hexagonal micropore unit, after the cells enter from the microfluidic channel, the cell capture trap structure moves and is limited in the low-speed field region due to the self inertia effect and the action of the fluid shear force, and single cell capture is completed. After observing that each microfluidic channel obtains a single cell by using a microscope, switching the injector to introduce a buffer solution into the chip to wash off residual redundant cells in the flow channel and provide a stable background solution for impedance spectrum measurement, and sequentially providing excitation voltage for the measurement electrodes in the micropores by the impedance analyzer through a multiplexing gating circuit and completing the measurement of the cell impedance spectrum in the unit.

The applicant declares that the present invention is described by the above embodiments as the detailed features and the detailed methods of the present invention, but the present invention is not limited to the above detailed features and the detailed methods, that is, it is not meant that the present invention must be implemented by relying on the above detailed features and the detailed methods. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes and the like, are within the scope and disclosure of the present invention.