WO2022205399A1 - 一种细胞捕获与拉伸一体式的阵列化微流控芯片 - Google Patents

一种细胞捕获与拉伸一体式的阵列化微流控芯片 Download PDF

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WO2022205399A1
WO2022205399A1 PCT/CN2021/085168 CN2021085168W WO2022205399A1 WO 2022205399 A1 WO2022205399 A1 WO 2022205399A1 CN 2021085168 W CN2021085168 W CN 2021085168W WO 2022205399 A1 WO2022205399 A1 WO 2022205399A1
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capture
channel
arrayed
cell
inlet
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PCT/CN2021/085168
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English (en)
French (fr)
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邹文婧
杨馥与
朱铭杰
张星辰
李志伟
郑昕宇
杨浩
孙立宁
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苏州大学
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/38—Caps; Covers; Plugs; Pouring means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
    • C12M37/04—Seals
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0663—Stretching or orienting elongated molecules or particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0668—Trapping microscopic beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0463—Hydrodynamic forces, venturi nozzles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure

Definitions

  • the invention relates to the technical field of microfluidic chips, in particular to an arrayed microfluidic chip integrating cell capture and stretching.
  • Microfluidic chip technology integrates basic operation units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes into a micron-scale chip to automatically complete the entire analysis process. Due to its huge potential in biology, chemistry, medicine and other fields, it has developed into a new research field that intersects with biology, chemistry, medicine, fluids, electronics, materials, machinery and other disciplines.
  • the growth, differentiation, division, apoptosis and other biological processes of cells in life are affected by a variety of environmental factors including mechanical factors.
  • biomechanical factors are closely related to the biological properties of cells and directly affect the shape, structure, growth and function. Therefore, by studying the biomechanical properties of cells, the physiological functions of cells can be characterized to a certain extent. By stretching the cells, the mechanical properties of each cell can be measured by the difference in the amount of stretching deformation.
  • Existing measurement techniques mainly include microtubule sucking, optical tweezers, magnetic particle torsion, and atomic force microscopy, but most of these methods are harmful to cells, difficult to operate, and unable to achieve high throughput.
  • Microtubule sucking technology cannot achieve high throughput and is easy to damage cell samples; optical tweezers are also difficult to achieve high throughput, complicated to operate, and expensive; magnetic particle torsion technology is difficult to operate; atomic force microscopy relies on the adhesion of cells itself, which is more difficult to operate. big.
  • the existing technology has more or less problems such as low degree of automation, difficult operation, low efficiency, inability to achieve high throughput, and high price.
  • the purpose of the present invention is to provide an arrayed microfluidic chip that integrates cell capture and stretching.
  • An arrayed microfluidic chip integrating cell capture and stretching comprising a conductive substrate, a chip body matched with the conductive substrate, the chip body comprising a cover plate and a microchannel opened in the cover plate , the microchannel includes a first inlet, a cell arrayed capture area and a first outlet, the cell arrayed capture area includes a first channel, a capture channel, at least one sub-channel and a second channel that are connected in sequence, the The first channel communicates with the first inlet, at least one side wall of the capture channel is spaced with a plurality of capture ports, and the second channel communicates with the first outlet.
  • the capture port includes a bell mouth and a through hole communicating with the bell mouth.
  • the outer width of the bell mouth is 10-20 ⁇ m
  • the inner width is 4-12 ⁇ m
  • the width of the capture channel is 80-120 ⁇ m.
  • the outer width of the bell mouth is 15 ⁇ m
  • the inner width is 8 ⁇ m
  • the width of the capture channel is 100 ⁇ m.
  • the conductive substrate is tightly bonded with the chip body.
  • the conductive substrate is ITO glass, and the ITO electrode of the ITO glass is aligned with the bell mouth.
  • the edge of the ITO electrode is aligned with the bell mouth.
  • a first check valve is installed at the first inlet, and a first micro-flow pump is connected to the first outlet.
  • the microchannel further includes a second inlet and a second outlet, the second inlet communicates with the second channel, and the second outlet communicates with the first channel.
  • a second micro-flow pump is connected to the second inlet, and a second check valve is installed at the second outlet.
  • the microfluidic chip of the present invention can be used to capture multiple single cells through the design of microchannels and electrode structures, realize array capture and stretching of cells, and realize capture and stretching at the level of single cells and release, which facilitates the analysis of its mechanical properties at the single-cell level.
  • the chip can be reused without disassembly, making it easier to release cells and repeat operations after release, improve chip utilization, and also easier Combine with automation technology to improve the degree of automation.
  • the capture area can be emptied through the cell release function to achieve repeated stretching operations, which can provide more effective stretching deformation data, improve the reliability of the data and the accuracy of the results.
  • FIG. 1 is a schematic structural diagram of a substrate of a preferred embodiment of the present invention.
  • FIG. 2 is a schematic structural diagram of a conductive substrate according to a preferred embodiment of the present invention.
  • Fig. 3 is the enlarged schematic diagram of A in Fig. 2;
  • FIG. 4 is a schematic structural diagram of a chip body according to a preferred embodiment of the present invention.
  • Fig. 5 is the enlarged schematic diagram of B in Fig. 4;
  • Fig. 6 is the enlarged schematic diagram of C in Fig. 5;
  • FIG. 7 is a schematic structural diagram of a microfluidic chip according to a preferred embodiment of the present invention.
  • Fig. 8 is the effect diagram of single cell pre-capture and release experiment performed without ITO electrode
  • Fig. 9 is the experimental effect diagram of capture and release after bonding the ITO electrode
  • Figure 10 is a schematic diagram of cell capture, stretch and release
  • Figure 11 is the actual drawing of the cell stretching experiment
  • an embodiment of the present invention provides an arrayed microfluidic chip integrated with cell capture and stretching, including a conductive substrate 10 and a chip body 20 .
  • the chip body 20 includes a cover plate 201 and a microchannel opened in the cover plate 201.
  • the microchannel includes a first inlet 202, a cell arrayed capture area and a first outlet 203 that are connected in sequence.
  • the second channel 207 is communicated with the first outlet 203.
  • the cells are stuck in the capture port 208.
  • the first cell blocks one capture port 208, so that other cells flow to the other capture port 208. After all the capture ports 208 have been captured, the excess cells will flow out from the subsequent sub-channels 206 through the second channel 207 .
  • the capture port 208 includes a bell mouth 209 and a through hole 210 communicating with the bell mouth 209 . It communicates with the through hole 210, and the through hole 210 communicates with the sub-channel 206, so as to facilitate the formation of streamlines and trap the cells.
  • two opposite side walls of the capture channel 205 are spaced apart with a plurality of capture ports 208, and the number of sub-channels 206 is two, and each sub-channel 206 has two.
  • the channels 206 are all communicated with the capture channel 205 and the second channel 207 .
  • the outer width d1 of the bell mouth 209 is preferably 10-20 ⁇ m
  • the inner width d2 is 4-12 ⁇ m
  • the width d3 of the capture channel 205 is 80-120 ⁇ m, so that the cell capture efficiency is high.
  • the outer width d1 of the bell mouth 209 is 15 ⁇ m
  • the inner width d2 is 8 ⁇ m
  • the width d3 of the capture channel 205 is 100 ⁇ m, which is used to capture single cells of 10-13 ⁇ m to achieve the best capture rate.
  • the diameter of the through hole 210 is the same as the inner width d2 of the bell mouth 209 .
  • a first one-way valve (not shown in the figure) is preferably installed at the first inlet 202.
  • the first one-way valve is set to only enter and not enter, and the first
  • the outlet 203 is connected with a first microfluidic pump (not shown in the figure), and a 20ml first syringe (not shown in the figure) is installed on the first microfluidic pump, which is not filled with a solution, and the cells are injected by applying negative pressure. The solution is sucked into the chip body 20 .
  • the microchannel also includes a second inlet 213 and a second outlet 214.
  • the second inlet 213 is communicated with the second channel 207, and the second outlet 214 is communicated with the first channel 204.
  • the second The inlet 213 passes the buffer solution into the chip body 20, and the buffer solution flows through the cell array capture area in reverse, and drives the cells to flow out to the second outlet 214 under the action of fluid force to complete the cell release, thereby facilitating the reception of another batch of cells.
  • the second inlet 213 is connected to a second microfluidic pump (not shown in the figure), a second check valve (not shown in the figure) is installed at the second outlet 214, and the second microfluidic pump is installed There is a 20ml second syringe (not shown in the figure), which is filled with DEP buffer.
  • the DEP buffer is passed into the chip body 20 along the second inlet 213, and the second one-way valve is set to only output. not enter, so that the cells are quickly driven to flow out to the second outlet 214 under the action of the fluid force to complete the rapid release of the cells.
  • a first transition channel 215 is communicated between the first inlet 202 and the first channel 204, and the first outlet 203 A second transition channel 216 is communicated with the second channel 207 .
  • a third transition channel 217 is communicated between the second inlet 213 and the second channel 207, and the second outlet A fourth transition channel 218 is communicated between 214 and the first channel 204 .
  • DEP in the DEP buffer is dielectrophoresis, also known as two-dimensional electrophoresis, which is a phenomenon in which an object with a low dielectric constant is subjected to force in a non-uniform electric field.
  • the magnitude of the dielectric force has nothing to do with whether the object is charged or not, but is related to the size, electrical properties of the object, the electrical properties of the surrounding medium, and the field strength, rate of change of field strength, and frequency of the applied electric field.
  • the main components of the DEP buffer in this example are as follows: 100 ml of deionized water, 8.5 g of sucrose, 0.3 g of glucose, and 0.2 mg of calcium chloride. This solution has the following functions, the cell survival time is long (isotonic, more than 4 hours), the role of calcium chloride is to adjust the conductivity, 0.2mg just makes the solution conductivity 50 ⁇ s/cm, the amount of this component can be adjusted.
  • the material of the chip body 20 is polydimethylsiloxane (PDMS), which is a kind of organic silicon. But it is not limited to polydimethylsiloxane (PDMS), and can also be polymethyl methacrylate (PMMA).
  • PDMS polydimethylsiloxane
  • PMMA polymethyl methacrylate
  • the microchannel is fabricated by a soft lithography process.
  • the conductive substrate 10 is ITO glass
  • the ITO glass is a layer of indium tin oxide ( Commonly known as ITO film, that is, a silicon boron-based substrate glass is coated with an indium tin oxide film, that is, an ITO electrode 102 is formed on the substrate 101, and the ITO electrode 102 is aligned with the bell mouth 208.
  • ITO film that is, a silicon boron-based substrate glass is coated with an indium tin oxide film, that is, an ITO electrode 102 is formed on the substrate 101, and the ITO electrode 102 is aligned with the bell mouth 208.
  • the substrate 101 is not limited to boron-silicon-based substrate glass, and can also be a soda-lime-based substrate glass.
  • the edge 1021 of the ITO electrode is aligned with the bell mouth 208 .
  • the ITO electrode 102 in this embodiment is an interdigitated electrode, which is fabricated by using a photolithography process and a wet etching technology.
  • the electrode gap distance d4 of the interdigital electrode is set to 20 ⁇ m, which can make the cells better achieve cell stretching after the electrodes are captured.
  • the conductive substrate 10 is placed on the bottom and the chip body 20 is placed on the top, and the alignment platform is used for alignment, so that the conductive substrate 10 and the chip body 20 are tightly bonded to avoid liquid leakage.
  • the microfluidic chip is placed under the microscope camera to observe the capture of cells on a connected computer. First, put the hose of the first inlet 202 in the DEP solution, add Jurkat cells to the DEP solution, turn on the first microfluidic pump, and the flow rate set by the first microfluidic pump is 2 ⁇ l/min. The negative pressure sucks the cell solution in the first inlet 202 into the microfluidic chip, and enters the capture channel 205 through the first transition channel 215 and the first channel 204 in turn.
  • the cells Under the action of dielectrophoresis, the cells will flow to the edge 1021 of the ITO electrode , so as to be captured in the bell mouth 209, and the capture situation of the cells is observed in real time under the microscope.
  • the capture ports 208 complete the capture, the first microfluidic pump is turned off, and the expected single cell physical capture is achieved at this time.
  • FIG. 9 is the experimental effect diagram of capture and release after bonding the ITO electrode 102
  • FIG. 9( a ) is the experimental effect diagram of cell capture after bonding the ITO electrode 102
  • FIG. 9( b ) is the cell release experiment renderings.
  • Figure 10 is a schematic diagram of cell capture, stretching and release, in which Figure 10(a) is the physical capture of cells using the principle of hydrodynamics when cells are flowing in the forward direction, and Figure 10(b) is under the action of dielectrophoresis.
  • Figure 10(a) is the physical capture of cells using the principle of hydrodynamics when cells are flowing in the forward direction
  • Figure 10(b) is under the action of dielectrophoresis.
  • Fig. 10(c) shows the cell stretching completed by increasing the voltage under the action of dielectrophoresis
  • Fig. 10(d) shows that after the stretching is completed, by Reverse the process of flushing to release cells.
  • Figure 11 shows the actual cell stretching experiment. After the capture is completed, the ITO electrode 102 near the capture area is used to achieve electrical capture and stretching under the action of dielectrophoresis.
  • Figure 11(a) shows the cells before stretching.

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Abstract

本发明公开了一种细胞捕获与拉伸一体式的阵列化微流控芯片，包括导电基底、与导电基底相配合的芯片本体，芯片本体包括盖板、开设在盖板内的微通道，微通道包括依次相连通的第一入口、细胞阵列化捕获区和第一出口，细胞阵列化捕获区包括依次相连通的第一通道、捕获通道、至少一个分通道和第二通道，第一通道与所述第一入口相连通，捕获通道的至少一个侧壁间隔开设有多个捕获口，第二通道与所述第一出口相连通。本发明可以对多个单细胞进行捕获，并实现单细胞水平上的捕获、拉伸与释放，有利于在单细胞水平上分析其力学特性；使芯片在无拆卸下重复利用，提升芯片利用率，提高自动化程度；可以提供更多有效的拉伸形变量数据。

Description

一种细胞捕获与拉伸一体式的阵列化微流控芯片 技术领域
本发明涉及微流控芯片技术领域，尤其涉及一种细胞捕获与拉伸一体式的阵列化微流控芯片。
 背景技术
微流控芯片技术是把生物、化学、医学分析过程的样品制备、反应、分离、检测等基本操作单元集成到一块微米尺度的芯片上，自动完成分析全过程。由于它在生物、化学、医学等领域的巨大潜力，已经发展成为一个生物、化学、医学、流体、电子、材料、机械等学科交叉的崭新研究领域。
生命体内细胞的生长、分化、分裂、凋亡等生物学过程会受到包括力学因素在内的多种环境因素的影响，其中，生物力学因素与细胞的生物性能密切相关，直接影响细胞的形态、结构、生长及功能。因而通过研究细胞的生物力学特性可以在一定程度上表征细胞的生理功能。对细胞进行拉伸操作就可以通过拉伸形变量的不同，测量出各个细胞的力学特性。现有的测量技术主要包括微管吸吮、光镊、磁微粒扭转、原子力显微镜，但是这些方法大多都对细胞损伤大、操作困难且无法实现高通量。微管吸吮技术无法实现高通量，容易损害细胞样品；光镊也难以实现高通量，操作复杂，价格昂贵；磁微粒扭转技术操作困难；原子力显微镜依赖细胞自身的粘附性，操作难度较大。总之，现有的技术或多或少存在着自动化程度低、操作困难、效率较低、无法实现高通量、价格昂贵等问题。
 技术解决方案
针对现有技术不足，本发明的目的在于提供一种细胞捕获与拉伸一体式的阵列化微流控芯片。
为了实现上述目的，本发明一实施例提供的技术方案如下：
一种细胞捕获与拉伸一体式的阵列化微流控芯片，包括导电基底、与所述导电基底相配合的芯片本体，所述芯片本体包括盖板、开设在所述盖板内的微通道，所述微通道包括第一入口、细胞阵列化捕获区和第一出口，所述细胞阵列化捕获区包括依次相连通的第一通道、捕获通道、至少一个分通道和第二通道，所述第一通道与所述第一入口相连通，所述捕获通道的至少一个侧壁间隔开设有多个捕获口，所述第二通道与所述第一出口相连通。
作为本发明的进一步改进，所述捕获口包括喇叭口、与所述喇叭口相连通的通孔。
作为本发明的进一步改进，所述喇叭口的外部宽度为10-20μm、内部宽度为4-12µm，所述捕获通道的宽度为80-120μm。
作为本发明的进一步改进，所述喇叭口的外部宽度为15μm、内部宽度为8µm，所述捕获通道的宽度为100μm。
作为本发明的进一步改进，所述导电基底与所述芯片本体紧密键合。
作为本发明的进一步改进，所述导电基底为ITO玻璃，所述ITO玻璃的ITO电极对准所述喇叭口。
作为本发明的进一步改进，所述ITO电极的边缘对准所述喇叭口。
作为本发明的进一步改进，所述第一入口处安装有第一单向阀，所述第一出口连接有第一微流泵。
作为本发明的进一步改进，所述微通道还包括第二入口和第二出口，所述第二入口与所述第二通道相连通，所述第二出口与所述第一通道相连通。
作为本发明的进一步改进，所述第二入口连接有第二微流泵，所述第二出口处安装有第二单向阀。
 有益效果
1、本发明微流控芯片通过微通道以及电极结构的设计，可以用来对多个单细胞进行捕获，实现细胞的阵列化捕获和拉伸，并实现单细胞水平上进行的捕获、拉伸与释放，有利于在单细胞水平上分析其力学特性。
2、使用两个入口、两个出口以及两个单向阀的设计，使芯片在无拆卸的情况下可重复利用，更易释放细胞以及释放后的反复操作，提升芯片利用率，同时也更容易与自动化技术相结合，提高自动化程度。
3、可以通过细胞释放功能清空捕获区进而实现反复拉伸操作，可以提供更多有效的拉伸形变量数据，提高数据的可靠性以及结果的准确性。
 附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明的优选实施例的基底的结构示意图；
图2为本发明的优选实施例的导电基底的结构示意图；
图3为图2中A的放大示意图；
图4为本发明的优选实施例的芯片本体的结构示意图；
图5为图4中B的放大示意图；
图6为图5中C的放大示意图；
图7为本发明的优选实施例的微流控芯片的结构示意图；
图8为在未加ITO电极下进行的单细胞预捕获和释放实验效果图；
图9是键合ITO电极之后，捕获与释放的实验效果图；
图10为细胞捕获、拉伸与释放的原理图；
图11为实际进行的细胞拉伸实验图；
图中：10、导电基底，101、基底，102、ITO电极，1021、ITO电极的边缘，20、芯片本体，201、盖板，202、第一入口，203、第一出口，204、第一通道，205、捕获通道，206、分通道，207、第二通道，208、捕获口，209、喇叭口，210、通孔，213、第二入口，214、第二出口，215、第一过渡通道，216、第二过渡通道，217、第三过渡通道，218、第四过渡通道。
 本发明的实施方式
为了使本技术领域的人员更好地理解本发明中的技术方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本发明保护的范围。
如图1-图7所示，本发明一实施例提供了一种细胞捕获与拉伸一体式的阵列化微流控芯片，包括导电基底10和芯片本体20。
芯片本体20包括盖板201、开设在盖板201内的微通道，微通道包括依次相连通的第一入口202、细胞阵列化捕获区和第一出口203，细胞阵列化捕获区包括依次相连通的第一通道204、捕获通道205、至少一个分通道206和第二通道207，第一通道204与第一入口202相连通，捕获通道205的至少一个侧壁间隔开设有多个捕获口208，第二通道207与第一出口203相连通，利用流体动力学原理，使细胞卡在捕获口208，第一个细胞会堵住一个捕获口208，从而使其他细胞往另外的捕获口208流动，当所有捕获口208都完成捕获之后，多余的细胞会从后面的分通道206经由第二通道207流出。
请参阅图5、图6，在本实施例中，捕获口208包括喇叭口209、与喇叭口209相连通的通孔210，喇叭口209的外部与捕获通道205相连通，喇叭口209的内部与通孔210相连通，通孔210与分通道206相连通，便于形成流线，卡住细胞。
为了提高捕获效率，缩短捕获时间，提升细胞测量数据量，本实施例优选捕获通道205相对的两个侧壁均间隔开设有多个捕获口208，分通道206的数量为两个，每个分通道206均与捕获通道205、第二通道207相连通。
本实施例优选喇叭口209的外部宽度d1为10-20μm、内部宽度d2为4-12µm，捕获通道205的宽度d3为80-120μm，对细胞的捕获效率高。
进一步优选喇叭口209的外部宽度d1为15μm、内部宽度d2为8µm，捕获通道205的宽度d3为100μm，用于捕获10-13μm的单细胞，达到最佳的捕获率。具体地，通孔210的孔径与喇叭口209的内部宽度d2相同。
本实施例优选第一入口202处安装有第一单向阀（图中未示出），当往第一入口202通入细胞溶液时，第一单向阀设置成只进不出，第一出口203连接有第一微流泵（图中未示出），第一微流泵上安装有一个20ml的第一注射器（图中未示出），未装有溶液，通过施加负压将细胞溶液吸入芯片本体20。
微通道还包括第二入口213和第二出口214，第二入口213与第二通道207相连通，第二出口214与第一通道204相连通，当细胞捕获并拉伸完成之后，在第二入口213将缓冲液通入芯片本体20，缓冲液反向流经细胞阵列化捕获区，在流体力的作用下带动细胞向第二出口214流出，完成细胞释放，从而便于接收另一批细胞。
本实施例优选第二入口213连接有第二微流泵（图中未示出），第二出口214处安装有第二单向阀（图中未示出），第二微流泵上安装有一个20ml的第二注射器（图中未示出），装有DEP缓冲液，通过施加正压沿着第二入口213将DEP缓冲液通入芯片本体20，第二单向阀设置成只出不进，从而在流体力的作用下快速带动细胞向第二出口214流出，完成细胞的快速释放。
为了便于细胞溶液从第一入口202进入第一通道204，再通过第一出口203排出，本实施例优选第一入口202与第一通道204之间连通有第一过渡通道215，第一出口203与第二通道207之间连通有第二过渡通道216。为了便于DEP缓冲液从第二入口213进入第二通道207，再通过第二出口214排出，本实施例优选第二入口213与第二通道207之间连通有第三过渡通道217，第二出口214与第一通道204之间连通有第四过渡通道218。
其中，DEP缓冲液中的DEP为介电泳，也称双向电泳，是介电常数较低的物体在非匀强电场中受力的现象。介电力大小与物体是否带电无关，与物体的大小、电学性质、周围介质的电学性质以及外加电场的场强、场强变化率、频率有关。本实施例的DEP缓冲液，主要成分如下：100ml去离子水，8.5g蔗糖，0.3g葡萄糖，0.2mg氯化钙。这个溶液有如下作用，细胞存活时间长（等渗，4个小时以上），氯化钙的作用是调节电导率，0.2mg恰好使溶液电导率为50μs/cm，此成分的量可以调节。
优选芯片本体20的材质为聚二甲基硅氧烷（PDMS），是有机硅的一种，成本低廉，使用简单，与硅片之间具有良好的粘附性，而且具有良好的化学惰性，但并不局限于聚二甲基硅氧烷（PDMS），也可以为聚甲基丙烯酸甲酯（PMMA）。优选微通道采用软光刻工艺制作。
本实施例中，导电基底10为ITO玻璃，ITO玻璃是在材质为硅硼基基片玻璃的基底101上利用溅射、蒸发等多种方法在基底101上镀上的一层氧化铟锡（俗称ITO）膜，即硅硼基基片玻璃的上面镀有一层氧化铟锡膜，也就是在基底101上形成ITO电极102，ITO电极102对准喇叭口208。可以理解的是，基底101并不局限于硅硼基基片玻璃，也可以为钠钙基基片玻璃。
本实施例中，将ITO电极的边缘1021对准喇叭口208。本实施例的ITO电极102为叉指电极，利用光刻工艺以及湿法刻蚀技术制作而成。本实施例中，叉指电极的电极间隙距离d4设置为20μm，可以使细胞更好地在电极捕获后实现细胞拉伸。
装配时，将导电基底10放在下面，芯片本体20放在上面，通过对准平台进行对准，使导电基底10与芯片本体20紧密键合，避免液体的渗漏。
微流控芯片的力学特性检测具体步骤如下：
（1）单细胞捕获。将微流控芯片放在显微摄像头下，用于在连接的电脑上观察细胞的捕获情况。首先将第一入口202的软管放在DEP溶液中，DEP溶液中加入Jurkat细胞，开启第一微流泵，此时第一微流泵设置的流速为2μl/min，第一注射器抽取，施加负压将第一入口202中的细胞溶液吸入微流控芯片中，依次经过第一过渡通道215、第一通道204进入捕获通道205，在介电泳作用下，细胞会向ITO电极的边缘1021流动，从而被捕获在喇叭口209，显微镜下实时观察细胞的捕获情况，当所有捕获口208完成捕获后，关闭第一微流泵，此时实现预期的单细胞物理捕获。
（2）细胞电捕获及拉伸。在完成上述单细胞捕获后，开启与ITO电极102的两端连接的信号发生器，设置电压5Vpp、频率12MHz的正弦波信号，实现Jurkat细胞的电捕获，在显微镜下观察电捕获情况，完成电捕获后，再设置电压8Vpp、频率12MHz的正弦波信号，此时细胞会在电场梯度作用下进行拉伸，在显微摄像头连接的电脑上检测其拉伸情况。
（3）细胞释放。在信号发生器增加电压完成拉伸后，关闭信号发生器，开启第二微流泵，此时第二微流泵设置流速为500μl/min，将第二注射器中的DEP缓冲液依次通过第二入口213、第三过渡通道217、第二通道207、分通道206、捕获通道205、第一通道204、第四过渡通道218，使得被捕获的细胞由第二出口214排出，充分释放微流控芯片中一批被拉伸的细胞，在显微摄像头连接的电脑上观察其释放情况，当细胞阵列化捕获区没有细胞时，关闭第二微流泵，此时微流控芯片中没有细胞，可以继续进行下一批的拉伸实验，实现微流控芯片1的重复利用。
其中，图8（a）为在未加ITO电极102下进行的单细胞预捕获实验图，图8（b）为在正向捕获完成后利用DEP缓冲液的反向流动来完全释放细胞的实验效果图，可以看到基本多数捕获区域都可以实现单细胞的捕获，其释放效率也非常可观。图9是键合ITO电极102之后，捕获与释放的实验效果图，其中，图9（a）为键合ITO电极102之后，细胞捕获的实验效果图，图9（b）为细胞释放的实验效果图。图10为细胞捕获、拉伸与释放的原理图，其中图10（a）是细胞在正向流动时，利用流体动力学原理实现的细胞物理捕获，图10（b）为在介电泳作用下使ITO电极的边缘1021附近的细胞捕获在ITO电极102上的过程，图10（c）为在介电泳作用下增大电压完成的细胞拉伸，图10（d）为完成拉伸之后，通过反向的冲洗来释放细胞的过程。图11为实际进行的细胞拉伸实验，在捕获完成之后，通过在捕获区域附近的ITO电极102在介电泳作用下实现电捕获及拉伸，其中，图11（a）为拉伸前的细胞图像，图11（b）为拉伸后的细胞图像，通过拉伸前后的比对可以得到其杨氏模量等力学特性参数。
具体实验数据：进行了多次单细胞捕获实验，在本实施例的20个捕获口208中可以捕获不少于14个单细胞，捕获效率不低于70%，反向释放细胞的效率基本可以达到100%。细胞拉伸实验，针对Jurkat细胞，能够在电压5Vpp、频率12MHz的交流信号下实现捕获，在电压8Vpp、频率12MHz的交流信号下实现拉伸。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外，应当理解，虽然本说明书按照实施方式加以描述，但并非每个实施方式仅包含一个独立的技术方案，说明书的这种叙述方式仅仅是为清楚起见，本领域技术人员应当将说明书作为一个整体，各实施例中的技术方案也可以经适当组合，形成本领域技术人员可以理解的其他实施方式。

Claims (10)

  1. 一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，包括导电基底、与所述导电基底相配合的芯片本体，所述芯片本体包括盖板、开设在所述盖板内的微通道，所述微通道包括第一入口、细胞阵列化捕获区和第一出口，所述细胞阵列化捕获区包括依次相连通的第一通道、捕获通道、至少一个分通道和第二通道，所述第一通道与所述第一入口相连通，所述捕获通道的至少一个侧壁间隔开设有多个捕获口，所述第二通道与所述第一出口相连通。
  2. 根据权利要求1所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述捕获口包括喇叭口、与所述喇叭口相连通的通孔。
  3. 根据权利要求2所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述喇叭口的外部宽度为10-20μm、内部宽度为4-12µm，所述捕获通道的宽度为80-120μm。
  4. 根据权利要求3所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述喇叭口的外部宽度为15μm、内部宽度为8µm，所述捕获通道的宽度为100μm。
  5. 根据权利要求1所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述导电基底与所述芯片本体紧密键合。
  6. 根据权利要求1或5所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述导电基底为ITO玻璃，所述ITO玻璃的ITO电极对准所述喇叭口。
  7. 根据权利要求6所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述ITO电极的边缘对准所述喇叭口。
  8. 根据权利要求1所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述第一入口处安装有第一单向阀，所述第一出口连接有第一微流泵。
  9. 根据权利要求1或8所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述微通道还包括第二入口和第二出口，所述第二入口与所述第二通道相连通，所述第二出口与所述第一通道相连通。
  10. 根据权利要求9所述的一种细胞捕获与拉伸一体式的阵列化微流控芯片，其特征在于，所述第二入口连接有第二微流泵，所述第二出口处安装有第二单向阀。
PCT/CN2021/085168 2021-03-30 2021-04-02 一种细胞捕获与拉伸一体式的阵列化微流控芯片 WO2022205399A1 (zh)

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