WO2022057797A1 - 一种基于数字微流控技术的全自动单细胞捕获芯片及其应用 - Google Patents

一种基于数字微流控技术的全自动单细胞捕获芯片及其应用 Download PDF

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WO2022057797A1
WO2022057797A1 PCT/CN2021/118338 CN2021118338W WO2022057797A1 WO 2022057797 A1 WO2022057797 A1 WO 2022057797A1 CN 2021118338 W CN2021118338 W CN 2021118338W WO 2022057797 A1 WO2022057797 A1 WO 2022057797A1
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electrode
digital microfluidic
capture
cell capture
cell
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French (fr)
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杨朝勇
阮庆宇
杨健
邹芬香
张倩倩
许醒
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厦门大学
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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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • 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
    • 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
    • B01L3/502761—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
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/01—Drops
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • 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
    • 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
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/143—Quality control, feedback systems
    • 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

Definitions

  • the invention belongs to the technical field of digital microfluidics, in particular to a fully automatic single particle/single cell capture chip based on the digital microfluidic technology and its application.
  • Cells are the basic unit of biological structure and function, and cell analysis can provide important theoretical basis for physiological processes such as metabolism and signal transduction. Most of the traditional cell analysis is based on the population of cells, which analyzes the result of the average of a large number of cells, which is an average data of the population state. At present, more and more studies have shown that there is heterogeneity between individual cells. A group of cells that appear to be the same may have essential differences within them, such as genome sequence, enzyme activity, protein expression, etc. difference. These differences are reflected in the heterogeneity of cell function at the cellular level, which cannot be ignored in basic and clinical medical research.
  • cancer tissue also has very large heterogeneity, and this heterogeneity often becomes one of the main obstacles to clinical cancer treatment.
  • epidermal growth factor receptor (EGFR) mutation-aided diagnosis is believed to have a crucial role in tyrosine kinase inhibitor-targeted cancer therapy.
  • Effective identification of a small number of EGFR-mutant cells is an urgent need for current clinical treatments.
  • these mutant cell signals are often masked by the noise signals of most normal cells, preventing the effective identification of mutant cells. Therefore, in recent years, scientists have gradually developed a great interest in the study of single cells, hoping to better understand the nature of organisms through in-depth research on these basic units involved in the reactions of organisms.
  • Microfluidic chips use microchannels with different structures and various forms of external force fields to manipulate, process and control trace fluids or samples on a microscopic scale, thus realizing part or all of the functions of traditional laboratories in a microchip integration on .
  • it has the advantages of low cost, fast analysis speed, and low reagent consumption, which represents the development direction of miniaturization of biological and chemical analysis in the future. Therefore, as one of the most important research methods for single-cell isolation and analysis, microfluidic chips are widely used in neuroscience, stem cell biology, developmental biology, cancer diagnosis, and personalized drug screening.
  • the limitations of conventional microfluidic chips are also very obvious.
  • the chip can capture single cells fully automatically, and the capture efficiency of single cells is high.
  • the purpose of the present invention is to provide a fully automatic single cell capture chip based on digital microfluidic technology, which can fully automatically and efficiently capture single cells.
  • a fully automatic selective single-cell capture device based on digital microfluidic technology comprising a digital microfluidic chip, an imaging system located above the digital microfluidic chip, and a connection between the digital microfluidic chip and the digital microfluidic chip, respectively.
  • the control circuit of the imaging system consists of three parts.
  • the digital microfluidic chip includes two parts, an upper plate and a lower plate.
  • the lower plate includes a substrate, an electrode layer, a medium layer and a partially hydrophilized hydrophobic layer.
  • the upper plate is a hydrophobized ground electrode, and the upper and lower plates Opposed in parallel and separated by a gap layer;
  • the electrode layer includes an electrode access port, a reservoir electrode unit, a droplet generation channel electrode array, a single particle/single cell capture electrode array, and a reaction electrode area.
  • the electrode units of the liquid pool are connected one by one, and are arranged symmetrically on both sides with the single particle/single cell capture electrode array and the reaction electrode area as the center;
  • the partially hydrophilized hydrophobic layer is a hydrophobic layer with partially hydrophilized sites, which can be aligned to form a hydrophilized capture site in the center of a designated electrode to capture single cells through the adhesion of the hydrophilic structure;
  • the imaging system is located just above the digital microfluidic chip, and is used for acquiring image information at the hydrophilization capture site in the imaging area. After the droplet reaches the detection point of the hydrophilization capture position, after two consecutive image recognitions in the bright field and fluorescence field, the output image information enters the control circuit and is compared with the set image threshold. According to the comparison result, the control circuit will output the corresponding The control command controls the electrode drive circuit to output the voltage program with the corresponding changing law, so as to realize the judgment of whether the single cell capture is successful or not;
  • the control circuit is respectively connected to the digital microfluidic chip and the imaging system, and is used for outputting the digital microfluidic chip control program and the imaging system operating program respectively.
  • the electrode layer includes 96 electrode access ports, eight reservoir electrode units, eight droplet generation channel electrode arrays, 10 single particle/single cell capture electrode arrays and reaction electrode areas .
  • the droplet generation channel electrode arrays are respectively connected to the liquid reservoir electrode units one by one, and are symmetrically arranged on both sides of the single particle/single cell capture electrode array and the reaction electrode area as the center .
  • the shape of the hydrophilic capture site in the hydrophobic layer includes circle, square, half-moon, etc., and the size can be determined by the size of the electrode.
  • hydrophilic sites for capture may be circular in diameter of 50-500 microns, such as 100 microns, 150 microns, 200 microns, 250 microns, 300 microns, 350 microns or 400 microns.
  • the diameter of the particles may be 5-200 microns, such as 5 microns, 10 microns, 15 microns, 20 microns, 30 microns, 70 microns, 80 microns or 90 microns.
  • the diameter of the cells may be 5-100 microns, such as 5 microns, 10 microns, 15 microns, 20 microns, 30 microns, 70 microns, 80 microns or 90 microns.
  • the material of the hydrophilic and hydrophobic layer used in the digital microfluidic chip of the present invention is Teflon or polytetrafluoroethylene PTFE aqueous dispersion, which is constructed on the dielectric layer by local peeling technology to form a localized hydrophilic capture site.
  • the droplets are coated with an oil shell or the droplets are soaked in the oil phase.
  • the reservoir electrode unit can achieve a droplet volume change from 1 nano to 10 microliters.
  • surfactants are added to the droplets to prevent bioadhesion.
  • the basic workflow of the digital microfluidic chip is:
  • Step A Place the liquid with and without fluorescent cells in the reservoir of the digital microfluidic chip, add an appropriate amount of filler oil to wrap each droplet, use the integrated circuit to pre-energize the electrode, and go to the step B;
  • Step B Use the control circuit of the integrated circuit to control the electrode drive circuit, and control the on-off of the electrodes in a certain order, so as to generate a cell-containing droplet of a suitable volume from the electrode unit of the reservoir, and pass the channel electrode array connected to it.
  • the droplet moves to the single-cell capture electrode, and the process is repeated so that each capture electrode is loaded with cell-containing droplets, and the process proceeds to step C;
  • Step C The control circuit of the integrated circuit controls the electrode driving circuit, so that the droplets pass through the single-cell capture electrode array area.
  • the electrode in the single-cell capture electrode array area is powered off, and the liquid After the droplet is left standing for 1 minute, the cells are allowed to settle on the chip surface under the action of gravity, and then the droplet is moved to the next electrode under the driving of the electrode, and then go to step D;
  • Step D use the bright field and the fluorescence field to shoot and output image information in turn, and then go to step E;
  • Step E After two consecutive image recognitions in the bright field and the fluorescent field, the output image information enters the control circuit and is compared with the set image threshold. According to the comparison result, the control circuit will output the corresponding control command, and the control command controls the electrode drive circuit. Output the voltage program with the corresponding change law, so as to realize the judgment of the success of single cell capture. If it is not a single particle/cell or not a target particle/cell, the control command repeats step C. If it is a target single particle/cell, select Sexual capture of single cells is complete, go to step F.
  • the present invention has the following advantages:
  • the digital microfluidic chip of the present invention includes a digital microfluidic chip with a multi-layer pattern and an integrated circuit.
  • the digital microfluidic chip with multi-layer pattern is composed of upper and lower electrode plates.
  • the lower electrode plate includes a substrate, an electrode layer, a medium layer and a partially hydrophilic hydrophobic layer. Ground electrode;
  • the integrated circuit is a digital microfluidic circuit control system. It has the advantages of fully automatic, easy integration and addressability, small volume, less reagent consumption and cost saving;
  • the single cell capture device can capture single cells 100% selectively
  • the present invention can realize a more direct and greater degree of spatiotemporal control of the generated discrete micro-droplets, and has advantages in fully automatic processing and complete integration.
  • FIG. 1 is a schematic diagram of the overall structure of the digital microfluidic chip of the present invention.
  • FIG. 2 is a schematic diagram of the flow chart of the selective capture of single cells according to the present invention.
  • FIG. 3 is a schematic diagram of the effect of selective capture of single cells according to the present invention.
  • FIG. 4 is a capture efficiency diagram of the selective capture of single cells under different mixed cell ratios (blue fluorescence: green fluorescence) according to the present invention.
  • the single-cell capture device includes a digital microfluidic chip 1, an imaging system located above the digital microfluidic chip, and three parts of a control circuit respectively connecting the digital microfluidic chip and the imaging system .
  • the digital microfluidic chip includes an upper electrode plate and a lower electrode plate, and the upper electrode plate sequentially includes a hydrophobic layer, a grounded conductive layer and an upper electrode plate substrate from bottom to top;
  • the lower electrode plate includes a glass substrate 1 (in other embodiments, it can also be other material substrates), an electrode layer, a dielectric layer and a partially hydrophilized hydrophobic layer, and the electrode layer is made of glass by wet etching technology.
  • the surface of the substrate in other embodiments, it can also be a method such as circuit board printing technology and printing technology
  • the dielectric layer is uniformly coated on the upper surface of the electrode layer
  • the partially hydrophilized hydrophobic layer is used for single particle/single cell capture
  • a specific hydrophilic pattern is formed on the surface of the dielectric layer by techniques such as partial peeling, and a hydrophobic pattern is formed on the rest of the dielectric layer;
  • the electrode layer is a chromium electrode array 2 with a specific pattern (in other embodiments, it can also be other conductive metals), including six reservoir electrode units 4, six droplet generation channel electrode arrays 5, ten single particles /Single cell capture electrode array and reaction electrode area 6 (in Figure 1, 3 is a hydrophilic site, single particle/single cell capture electrode array and reaction electrode area 6 are located below hydrophilic site 3), droplet generation channel electrode
  • the arrays 5 are respectively connected with the electrode units 4 in the reservoir, and are arranged symmetrically on both sides of the single particle/single cell capture electrode array and the reaction electrode area (in other embodiments, other numbers, electrode arrays of other shapes);
  • a partially hydrophilized hydrophobic layer is a hydrophobic layer formed by a partially exfoliated technique with locally hydrophilized sites aligned to form a circular hydrophilized capture site at the center of a given electrode (in other implementations). For example, other shapes of hydrophilized capture sites are also possible).
  • the imaging system is located just above the digital microfluidic chip, and is used for acquiring image information at the hydrophilization capture site in the imaging area. After the droplet reaches the detection point of the hydrophilization capture position, after two consecutive image recognitions in the bright field and fluorescence field, the output image information enters the control circuit and is compared with the set image threshold. According to the comparison result, the control circuit will output the corresponding The control command controls the electrode drive circuit to output the voltage program with the corresponding changing law, so as to realize the judgment of whether the single cell capture is successful or not;
  • the control circuit is respectively connected to the digital microfluidic chip and the imaging system, and is used for outputting the digital microfluidic chip control program and the imaging system operating program respectively.
  • the digital microfluidic control process of the present invention is as follows:
  • a 300-nanometer-thick chromium layer is vapor-deposited on the glass substrate of the lower pole plate using a magnetron sputtering method, and a chromium electrode array with a specific structure is formed by wet etching;
  • the dielectric layer material is an insulating material with high dielectric constant, uniform thickness, and strong breakdown resistance.
  • photoresist is used to form by spin coating;
  • the material of the hydrophobic layer is generally Teflon.
  • PTFE aqueous dispersion is used to partially peel off the hydrophobic layer through local peeling and overetching technology, exposing the relatively hydrophilic surface below.
  • the center forms the site of local hydrophilization.
  • localized hydrophilization sites may also be formed by other existing means.
  • the upper plate base is any insulating transparent material, such as glass;
  • the material of the grounding conductive layer is selected from materials with high light transmittance and good visibility, such as ITO, etc., which are formed by deposition;
  • the hydrophobic material of the upper electrode plate is generally the same as the hydrophobic material of the lower electrode plate, which is generally Teflon.
  • a polytetrafluoroethylene PTFE aqueous dispersion is used to form through a spin coating annealing process.
  • the upper and lower plates are separated by a gap layer with a certain thickness, forming a sandwich structure with the droplets in them. Introduce. After the injection is completed, the digital microfluidic chip is oil-sealed to isolate pollution and promote the movement of droplets.
  • the selected cells are H1975 cells expressing EGFR, and the cell diameter is 10-20 microns.
  • the specific working process of the present invention is:
  • Step A Load each reagent into the electrode unit of the reservoir of the digital microfluidic chip, add an appropriate amount of filling oil to wrap each droplet, use an integrated circuit to pre-energize the electrode, and go to Step B;
  • Step B use the control circuit of the integrated circuit to control the electrode drive circuit, and control the on-off of the electrodes in a certain order, so that the droplets are broken under the traction of the electrodes, thereby generating a suitable volume of cell-containing droplets from the electrode unit of the reservoir , move the droplet to the single-cell capture electrode through the channel electrode array connected to it, repeat this process to make each capture electrode load the cell-containing droplet, and transfer to step C;
  • Step C The control circuit of the integrated circuit controls the electrode driving circuit, so that the droplets pass through the single-cell capture electrode array area.
  • the electrode in the single-cell capture electrode array area is powered off, and the liquid After the droplet sits for 1 minute, the particles/cells are allowed to settle on the chip surface under the action of gravity, and then the droplet is moved to the next electrode under the driving of the electrode.
  • image recognition it is judged whether the hydrophilic area is a single cell, and if so, the capture is completed. ; If there are multiple particles/cells or no particles/cells, repeat the above operation to realize the capture of multiplex single cells on the chip, and then transfer to step D.
  • Step D For rare samples, driving the removed droplets back into the electrode of the reservoir can achieve almost non-destructive capture; for ordinary samples, directly driving the removed droplets into the waste pool, and then go to Step E.
  • Step E Control the electrode drive circuit to draw out the cell lysate (0.2% Triton X-100) from another reservoir electrode unit, drive the cell lysate to the single-cell capture structure, and lyse the cells. After cell lysis, it contains The cellular contents of the cells are released into the droplet and proceed to step F.
  • Step F Control the electrode drive circuit to draw out the single-cell whole genome amplification reagent from the electrode unit of the next reservoir, mix the reaction process by means of droplet driving, and take out the droplet for subsequent analysis and research after the reaction is completed.
  • the two cells stained with blue fluorescence and green fluorescence were mixed in a certain proportion and placed in the reservoir electrode unit of the digital microfluidic chip.
  • 10 hydrophilic micropore arrays with a diameter of 200 ⁇ m are arranged on the lower plate of the chip.
  • the cell droplets are passed into the chip and driven to the hydrophilic site area for cell loading.
  • a single cell with specific fluorescent staining can enter the hydrophilic site, while other cells do not enter the area. Pulling the cell away from the hydrophilic site results in the generation of a single-cell-containing hydrophilic droplet through negative generation, enabling cell-specific single-cell separation (Figure 2).
  • the present invention illustrates the detailed features and detailed methods of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed features and detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed characteristics and detailed methods to be able to implement.
  • Those skilled in the art should understand that any improvement to the present invention, equivalent replacement of the selected components of the present invention, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
  • the invention discloses a fully automatic selective single cell capture device based on digital microfluidic technology and its application, comprising a digital microfluidic chip, an imaging system located above the digital microfluidic chip, and a digital Three parts of the microfluidic chip and the control circuit of the imaging system.
  • the single-cell capture device can achieve fully automatic single-cell selective capture, with a capture success rate of 100%, and the obtained single cells have been classified and identified by morphology and molecular biology, with high specificity and high sensitivity, suitable for complete Single-cell isolation and subsequent application of circulating tumor cells in blood have industrial practicality.

Abstract

一种基于数字微流控技术的全自动选择性单细胞捕获装置及其应用，包括数字微流控芯片（1）、位于数字微流控芯片（1）上方的成像系统以及分别连接数字微流控芯片（1）和成像系统的控制电路三部分。基于数字微流控技术的全自动选择性单细胞捕获装置能够实现全自动的单细胞选择性捕获，捕获成功率为100％，且获取的单个细胞经过形态学和分子生物学的分类鉴定，具有高特异性和高灵敏度，适用于完成血液中循环肿瘤细胞的单细胞分离及后续应用。

Description

一种基于数字微流控技术的全自动单细胞捕获芯片及其应用 技术领域
本发明属于数字微流控技术领域，具体为一种基于数字微流控技术的全自动单颗粒/单细胞捕获芯片及其应用。
背景技术
细胞是生物结构组成和功能行使的基本单元，细胞分析可以为新陈代谢和信号传导等生理过程提供重要的理论依据。传统的细胞分析大多是基于群体细胞进行的，其分析的是大量细胞平均下的结果，是群体状态的一个平均性数据。目前，越来越多的研究表明，细胞个体之间存在异质性，看似相同的一群细胞，其内部有可能存在本质的差别，其在基因组序列、酶活性、蛋白表达等方面都存在着差异。这些差异反映在细胞水平上就表现为细胞功能的异质性，在基础和临床医学研究中，这种异质性是无法忽视的。并且在癌症发生过程中，由于基因突变等事件具有一定的偶然性，癌症组织也具有非常大的异质性，这种异质性也常常会成为临床上癌症治疗的主要障碍之一。例如：表皮生长因子受体(EGFR)的突变辅助诊断，在酪氨酸激酶抑制剂靶向癌症的治疗中，被认为具有至关重要的作用。有效识别少量的EGFR-突变细胞是当前临床治疗的迫切需求。但是，这些突变细胞信号往往被大部分正常细胞的噪声信号所掩盖，妨碍了突变细胞的有效识别。因此，近年来科学家们逐渐对单细胞产生了极大的研究兴趣，希望可以通过对这些参与生物体内反应的基本单元的深入地研究，从而更好地理解生物的本质。
尽管单细胞分析技术在很多应用领域中是至关重要的，然而单细胞体积小、所测组分含量低、偏差放大的特点使得单细胞的分离和分析面临着巨大的挑战。而且在细胞数目较多时，需要大量的重复操作，极其耗时，导致实验通量无法提升。
微流控芯片利用结构各异的微通道和形式多样的外加力场，对微量流体或样品在微观尺度上进行操纵、处理与控制，从而实现了传统实验室的部分乃至全部功能在一 块微芯片上的集成。相比于传统方法，其具有成本低、分析速度快、试剂消耗少等优势，代表着未来生物、化学分析走向微型化的发展方向。因此，微流控芯片作为单细胞分离和分析的最主要研究手段之一，被广泛应用于神经科学、干细胞生物学、发育生物学、癌症诊断和个性化药物筛选等领域中。然而，常规微流控芯片的局限性也是非常明显的，其需要机械泵、阀配合使用，集成化难度大，难以实现样品的连续多步处理；对多试剂位置和反应时间的精确控制捉襟见肘；并且无法实现对少量稀有细胞的单细胞俘获，在大量单细胞进行高效率高通量分析领域更是无能为力。
为了解决以上问题，我们提出了一种基于数字微流控技术的全自动单细胞捕获芯片。该芯片可以全自动地捕获单细胞，而且单细胞的捕获效率高。
发明内容
本发明的目的在于提供一种基于数字微流控技术的全自动单单细胞捕获芯片，可以全自动高效率地捕获单单细胞。
为了达到上述目的，本发明采用如下技术方案：
一种基于数字微流控技术的全自动选择性单细胞捕获装置，包括数字微流控芯片、位于所述数字微流控芯片上方的成像系统以及分别连接所述数字微流控芯片和所述成像系统的控制电路三部分。
所述的数字微流控芯片包括上、下极板两部分，下极板包括基底、电极层、介质层和局部亲水化的疏水层，上极板为疏水化的地电极，上下极板平行相对，被间隙层相隔；电极层包括电极接入口、贮液池电极单元、液滴生成通道电极阵列、单颗粒/单细胞捕获电极阵列和反应电极区，液滴生成通道电极阵列分别与贮液池电极单元一一相连，并以单颗粒/单细胞捕获电极阵列和反应电极区为中心对称分列于其两侧；
局部亲水化的疏水层是具有局部亲水化位点的疏水层，其经对准可在指定电极中央形成亲水化捕获位点，通过亲水结构的粘附作用捕获单细胞；
所述成像系统位于数字微流控芯片正上方，用于获取所述成像区域内亲水化捕获位点处的图像信息。在液滴到达亲水化捕获位检测点后，经明场、荧光场连续两次图 像识别后，输出图像信息进入控制电路与所设定的图像阈值进行比较，根据比较结果控制电路会输出对应的控制指令，控制指令控制电极驱动电路输出相应变化规律的电压程序，从而实现对单细胞捕获成功与否的判断；
所述控制电路分别连接所述数字微流控芯片和所述成像系统，用于分别输出数字微流控芯片控制程序和成像系统操作程序。
本发明的一个优选的实施方式中，电极层包括96个电极接入口、八个贮液池电极单元、八个液滴生成通道电极阵列、10个单颗粒/单细胞捕获电极阵列和反应电极区。
本发明的一个优选的实施方式中，液滴生成通道电极阵列分别与贮液池电极单元一一相连，并以单颗粒/单细胞捕获电极阵列和反应电极区为中心对称分列于其两侧。
本发明的一个优选的实施方式中，疏水层中亲水化捕获位点的形状包括圆形、方形、半月形等，大小可由电极大小决定。一般的，用于捕获的亲水位点可以是50-500微米直径的圆形，例如100微米、150微米、200微米、250微米、300微米、350微米或者400微米。
本发明的一个优选的实施方式中，所述的颗粒的直径可以是5-200微米，例如5微米、10微米、15微米、20微米、30微米、70微米、80微米或者90微米。
本发明的一个优选的实施方式中，所述的细胞的直径可以是5-100微米，例如5微米、10微米、15微米、20微米、30微米、70微米、80微米或者90微米。
本发明的数字微流控芯片所用亲疏水层的材料为特氟龙或聚四氟乙烯PTFE水分散液，通过局部剥离技术构建于电介层上，形成局部亲水化的捕获位点。
为了防止液滴的挥发，对液滴包裹油壳或将液滴浸润在油相中。
对于不同的单颗粒/单细胞捕获体系，所述贮液池电极单元可以实现液滴体积从1纳升至10微升的变化。
为了保证单颗粒/单细胞分析体系中液滴的移动，在液滴中加入表面活性剂用以 防止生物黏附。
为了选择性捕获单颗粒/单细胞，结合图像识别和全自动程序选择性捕获。
本发明的一个优选的实施方式中，所述的数字微流控芯片基本工作流程为：
步骤A:将带有和不带有荧光细胞的液体置于数字微流控芯片的贮液池内，加入适量的填充油将各液滴包裹住，使用集成电路对电极进行预通电，转入步骤B；
步骤B：使用集成电路的控制电路控制电极驱动电路，按一定次序进行电极的通断电控制，从而从贮液池电极单元产生一个合适体积的含细胞液滴，通过与其连接的通道电极阵列将液滴移动至单细胞捕获电极处，重复这一过程使各捕获电极均负载含细胞液滴，转入步骤C；
步骤C：集成电路的控制电路控制电极驱动电路，使液滴从单细胞捕获电极阵列区通过，当单个细胞进入拥有亲水结构的捕获电极时，对单细胞捕获电极阵列区电极断电，液滴静置1分钟后，使细胞在重力作用下沉降于芯片表面，然后在电极驱动下使液滴移动到下一个电极，然后转入步骤D；
步骤D：依次使用明场、荧光场进行拍摄并输出图像信息，转入步骤E；
步骤E：经明场、荧光场连续两次图像识别后，输出图像信息进入控制电路与所设定的图像阈值进行比较，根据比较结果控制电路会输出对应的控制指令，控制指令控制电极驱动电路输出相应变化规律的电压程序，从而实现对单细胞捕获成功与否的判断，若不为单颗粒/细胞或不是目标颗粒/细胞则控制指令重复步骤C，若为目标单颗粒/细胞，则选择性捕获单细胞完成，转入步骤F。
本发明与现有技术相比，其优势在于：
(1)本发明数字微流控芯片包括具有多层图案的数字微流控芯片和集成电路两部分。所述的具有多层图案的数字微流控芯片由上、下极板两部分组成，下极板包括 基底、电极层、介质层和局部亲水化的疏水层，上极板包括疏水化的地电极；集成电路为数字微流控电路控制系统。具有全自动、易集成、可寻址的优点，体积小，消耗试剂少，节约成本；
(2)该单细胞捕获装置可100％选择性捕获单细胞；
(3)基本没有细胞损失，在有限细胞数目的条件下可以使得全部捕获位点捕获有单细胞，能够根据芯片捕获位点的数目控制单细胞捕获数目，特别适用于稀有样品的分析，比如循环肿瘤细胞(CTC)、干细胞等稀有细胞的分析；
(4)本发明可以实现对产生的离散微液滴更加直接和更大程度的时空控制，在全自动处理和完全集成化上具有优势。
附图说明
图1为本发明数字微流控芯片整体结构示意图。
图2为本发明进行单细胞选择性捕获流程示意图。
图3为本发明进行单细胞选择性捕获效果示意图。
图4为本发明在不同混合细胞比例(蓝色荧光：绿色荧光)下进行单细胞选择性捕获的捕获效率图。
具体实施方式
下面结合附图以数字微流控芯片上单细胞全基因组扩增为例，对本发明进行进一步细致描述。
参见图1，该单细胞捕获装置，包括数字微流控芯片1、位于所述数字微流控芯片上方的成像系统以及分别连接所述数字微流控芯片和所述成像系统的控制电路三部分。
所述数字微流控芯片包括上极板和下极板，所述上极板从下至上依次包括疏水层、接地导电层和上极板基底；
所述的下极板包括玻璃基底1(在其它实施例中，也可以是其它材料基底)、电 极层、介质层和局部亲水化的疏水层，电极层由湿法刻蚀技术制作于玻璃基底表面(在其它实施例中，也可以是电路板印刷技术和打印技术等方法)，介质层均匀涂覆于电极层上表面，局部亲水化的疏水层用于单颗粒/单细胞捕获，通过局部剥离等技术在介质层表面形成特定的亲水图案，在介质层的其余区域形成疏水图案；
电极层为具有特定图案的铬电极阵列2(在其它实施例中，也可以是其它导电金属)，包括六个贮液池电极单元4、六个液滴生成通道电极阵列5、十个单颗粒/单细胞捕获电极阵列和反应电极区6(图1中，3为亲水位点，单颗粒/单细胞捕获电极阵列和反应电极区6位于亲水位点3下面)，液滴生成通道电极阵列5分别与贮液池电极单元4一一相连，并以单颗粒/单细胞捕获电极阵列和反应电极区为中心对称分列于其两侧(在其它实施例中，也可以是其它数目、其它形状的电极阵列)；
局部亲水化的疏水层是由局部剥离技术形成的具有局部亲水化位点的疏水层，其经对准可在指定电极的中心处形成圆形的亲水化捕获位点(在其它实施例中，也可以是其它形状的亲水化捕获位点)。
所述成像系统位于数字微流控芯片正上方，用于获取所述成像区域内亲水化捕获位点处的图像信息。在液滴到达亲水化捕获位检测点后，经明场、荧光场连续两次图像识别后，输出图像信息进入控制电路与所设定的图像阈值进行比较，根据比较结果控制电路会输出对应的控制指令，控制指令控制电极驱动电路输出相应变化规律的电压程序，从而实现对单细胞捕获成功与否的判断；
所述控制电路分别连接所述数字微流控芯片和所述成像系统，用于分别输出数字微流控芯片控制程序和成像系统操作程序。
本发明的数字微流控制作工艺如下：
下极板的制作：
(a)下极板玻璃基底使用磁控溅射方法蒸镀300纳米厚铬层，通过湿法刻蚀形成具有特定结构的铬电极阵列；
(b)介质层材料为具有高介电常数、厚度均匀、抗击穿能力强的绝缘材料，本实施案例中使用光刻胶通过旋涂的方式形成；
(c)疏水层材料一般为特氟龙，本实施案例中使用聚四氟乙烯PTFE水分散液通过局部剥离和套刻技术使疏水层局部剥落，暴露出下方较为亲水的表面，在指定电极中心形成局部亲水化的位点形成。在其它实施例中，也可以通过其它现有方式形成局部亲水化位点。
上极板的制作：
(a)上极板基底为任意绝缘透明材料，如玻璃；
(b)接地导电层材料选择透光率高、可见性好的材料，如ITO等，通过沉积形成；
(c)上极板疏水材料一般与下极板疏水材料一致，一般为Teflon，本实施案例中使用聚四氟乙烯PTFE水分散液通过旋涂退火工艺形成。
上下极板之间使用具有一定厚度的间隙层分隔开来，与其中的液滴形成三明治结构，液滴通过对应下极板贮液池电极上方的上极板位置开孔，通过自动进样引入。进样完成后，将数字微流控芯片进行油封，起到隔绝污染和促进液滴移动的作用。
作为本发明的优选实施方式，所选用的细胞为EGFR表达的H1975细胞，细胞直径为10-20微米。
作为本发明的优选实施方式，本发明的具体工作过程为：
步骤A：将各试剂分别负载于数字微流控芯片的贮液池电极单元内，加入适量的填充油将各液滴包裹住，使用集成电路对电极进行预通电，转入步骤B；
步骤B：使用集成电路的控制电路控制电极驱动电路，按一定次序进行电极的通断电控制，使液滴在电极的牵引下断裂从而从贮液池电极单元产生一个合适体积的含细胞液滴，通过与其连接的通道电极阵列将液滴移动至单细胞捕获电极处，重复这一过程使各捕获电极均负载含细胞液滴，转入步骤C；
步骤C：集成电路的控制电路控制电极驱动电路，使液滴从单细胞捕获电极阵列区通过，当单个细胞进入拥有亲水结构的捕获电极时，对单细胞捕获电极阵列区电极断电，液滴静置1分钟后，使颗粒/细胞在重力作用下沉降于芯片表面，然后在电极驱动下使液滴移动到下一个电极，结合图像识别判断亲水区是否为单个细胞，若是则捕获完成；若为多个颗粒/细胞或没有颗粒/细胞则重复上述操作，即可实现该芯片上多路单细胞的捕获，然后转入步骤D。
步骤D：对于稀有样品，将移走的液滴驱动回贮液池电极中可以实现几乎无损捕获；对于普通样品，直接将移走的液滴驱动至废液池中，转入步骤E。
步骤E：控制电极驱动电路从另一贮液池电极单元中引出细胞裂解液(0.2％Triton X-100)，将细胞裂解液驱动至单细胞捕获结构处，使细胞裂解，细胞裂解后其含有的细胞内含物释放于该液滴中，转入步骤F。
步骤F：控制电极驱动电路从下一贮液池电极单元中引出单细胞全基因组扩增试剂，通过液滴驱动的方式对反应过程进行混合，反应结束后将液滴取出进行后续的分析研究。
实施例1：
将染有蓝色荧光和绿色荧光的两种细胞按一定比例混合，置于数字微流控芯片的贮液池电极单元内。芯片下极板上排列有孔径为200μm的10个亲水微孔阵列。将细胞液滴通入芯片上并驱动至亲水位点区域进行细胞负载，通过流体的动态调控使得特定荧光染色的单个细胞进入亲水位点，而其他细胞不进入该区域。拉动细胞远离该亲水位点，通过负向生成使得生成一个含有单细胞的亲水液滴，实现特定细胞单细胞分离(图2)。
通过上述选择性捕获操作，在10个亲水微孔阵列的第一排全部捕获绿色荧光染色的细胞，在第二排全部捕获蓝色荧光染色的细胞，如图3。
调整两种不同染色细胞的混合比例，连续不断重复上述单细胞选择性捕获操作，计算不同纯度下的靶细胞捕获效率，得图4。实验证明在极低纯度情况下仍能实现高效的靶细胞捕获。
申请人声明，本发明通过上述实施例来说明本发明的详细特征以及详细方法，但本发明并不局限于上述详细特征以及详细方法，即不意味着本发明必须依赖上述详细特征以及详细方法才能实施。所属技术领域的技术人员应该明白，对本发明的任何改进，对本发明选用组分的等效替换、具体方式的选择等，均属于本发明的保护范围和公开范围之内。
工业实用性
本发明公开了一种基于数字微流控技术的全自动选择性单细胞捕获装置及其应用，包括数字微流控芯片、位于所述数字微流控芯片上方的成像系统以及分别连接所述数字微流控芯片和所述成像系统的控制电路三部分。该单细胞捕获装置能够实现全自动的单细胞选择性捕获，捕获成功率为100％，且获取的单个细胞经过形态学和分子生物学的分类鉴定，具有高特异性和高灵敏度，适用于完成血液中循环肿瘤细胞的单细胞分离及后续应用,具有工业实用性。

Claims (9)

  1. 一种基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：包括数字微流控芯片、位于所述数字微流控芯片上方的成像系统以及分别连接所述数字微流控芯片和所述成像系统的控制电路三部分；
    所述的数字微流控芯片包括上、下极板两部分，下极板包括基底、电极层、介质层和局部亲水化的疏水层，上极板为疏水化的地电极，上下极板平行相对，被间隙层相隔；电极层包括电极接入口、贮液池电极单元、液滴生成通道电极阵列、单颗粒/单细胞捕获电极阵列和反应电极区，液滴生成通道电极阵列分别与贮液池电极单元一一相连；
    局部亲水化的疏水层是具有局部亲水化位点的疏水层，其经对准可在指定电极中央形成亲水化捕获位点，通过亲水结构的粘附作用捕获单细胞；
    所述成像系统位于数字微流控芯片正上方，用于获取所述成像区域内亲水化捕获位点处的图像信息；在液滴到达亲水化捕获位检测点后，经明场、荧光场连续两次图像识别后，输出图像信息进入控制电路与所设定的图像阈值进行比较，根据比较结果控制电路输出对应的控制指令，控制指令控制电极驱动电路输出相应变化规律的电压程序，从而实现对单细胞捕获成功与否的判断；
    所述控制电路分别连接所述数字微流控芯片和所述成像系统，用于分别输出数字微流控芯片控制程序和成像系统操作程序。
  2. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：电极层包括96个电极接入口、八个贮液池电极单元、八个液滴生成通道电极阵列、10个单颗粒/单细胞捕获电极阵列和反应电极区。
  3. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：液滴生成通道电极阵列分别与贮液池电极单元一一相连，并以单颗粒/单细胞捕获电极阵列和反应电极区为中心对称分列于其两侧。
  4. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：局部亲水化的疏水层由局部剥离技术形成。
  5. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：所述的亲水化捕获位点直径为50-500微米。
  6. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：对于不同的单颗粒/单细胞捕获体系，所述贮液池电极单元能够实现液滴体积从1nL至10μL的变化。
  7. 根据权利要求1所述的基于数字微流控技术的全自动选择性单细胞捕获装置，其特征在于：所述上极板采用透明状导电膜。
  8. 一种基于数字微流控技术的全自动选择性单细胞捕获方法，采用权利要求1至7任一项所述的装置，包括如下步骤：
    步骤A:将带有和不带有荧光细胞的液体置于数字微流控芯片的贮液池内，加入适量的填充油将各液滴包裹住，使用集成电路对电极进行预通电，转入步骤B；
    步骤B：使用集成电路的控制电路控制电极驱动电路，按一定次序进行电极的通断电控制，从而从贮液池电极单元产生一个合适体积的含细胞液滴，通过与其连接的通道电极阵列将液滴移动至单颗粒/单细胞捕获电极处，重复这一过程使各捕获电极均负载含颗粒/细胞液滴，转入步骤C；
    步骤C：集成电路的控制电路控制电极驱动，使液滴从单细胞捕获电极阵列区通过，当细胞进入拥有亲水结构的捕获电极时，对单颗粒/单细胞捕获电极阵列区电极断电，液滴静置后，使颗粒/细胞在重力作用下沉降于芯片表面，然后在电极驱动下使液滴移动到下一个电极，然后转入步骤D；
    步骤D：依次使用明场、荧光场进行拍摄并输出图像信息，转入步骤E；
    步骤E：经连续两次图像识别后，输出图像信息进入控制电路与所设定的图 像阈值进行比较，根据比较结果控制电路会输出对应的控制指令，控制指令控制电极驱动电路输出相应变化规律的电压程序，从而实现对单细胞捕获成功与否的判断，若不为单颗粒/细胞或不是目标细胞则控制指令继续重复步骤C，若为目标单细胞，则选择性捕获单细胞完成。
  9. 根据权利要求8所述的基于数字微流控技术的全自动选择性单细胞捕获方法，其特征在于：步骤A中，在带有和不带有荧光细胞的液体中加入表面活性剂用以防止生物黏附。促进单颗粒/单细胞分析体系中液滴的移动。
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