WO2014071642A1 - 一种微流体细胞捕获芯片及其制备方法 - Google Patents

一种微流体细胞捕获芯片及其制备方法 Download PDF

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WO2014071642A1
WO2014071642A1 PCT/CN2012/084603 CN2012084603W WO2014071642A1 WO 2014071642 A1 WO2014071642 A1 WO 2014071642A1 CN 2012084603 W CN2012084603 W CN 2012084603W WO 2014071642 A1 WO2014071642 A1 WO 2014071642A1
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hard material
channel
wedge
chip
cell capture
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PCT/CN2012/084603
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English (en)
French (fr)
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刘侃
汪胜祥
张南刚
周鹏飞
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武汉友芝友生物制药有限公司
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Priority to US14/418,122 priority Critical patent/US20150196913A1/en
Priority to JP2015540988A priority patent/JP6163560B2/ja
Publication of WO2014071642A1 publication Critical patent/WO2014071642A1/zh

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    • 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
    • B01L3/502707—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 manufacture of the container or its components
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • 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
    • B01L3/502753—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 bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • 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
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • 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
    • B01L2300/12—Specific details about materials
    • 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
    • B01L2300/16—Surface properties and coatings
    • 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
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N2015/0288—Sorting the particles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T29/00—Metal working
    • Y10T29/49—Method of mechanical manufacture
    • Y10T29/49826—Assembling or joining
    • Y10T29/49888—Subsequently coating

Definitions

  • the invention relates to a cell capture tool and a preparation method thereof, in particular to a microfluidic cell capture chip for separating, enriching and recognizing circulating tumor cells and a preparation method thereof, and the microfluidic cell capture chip can be used as a tumor diagnosis and adjuvant therapy. And a useful tool for biochemical analysis.
  • tumor diagnosis is usually diagnosed based on a large number of pathological conditions.
  • Such methods such as biopsy or rectal cancer, usually involve invasive analysis with a degree of trauma and are not conducive to the patient.
  • biomarkers in peripheral blood are also used for examination, such as serological parameters (PSA).
  • PSA serological parameters
  • metastases For many cancer patients, the death is mainly due to metastases.
  • the current detection method is difficult to respond to the treatment situation in time, identify the metastasis, and guide the follow-up chemoradiotherapy process, which leads to the patient missing the optimal treatment timing and unable to adjust the invalid treatment and medication plan in time. Therefore, it is not possible to successfully treat all metastases, resulting in the eventual death of the patient. From a clinical perspective, metastases can be seen as conclusive events in the natural development of cancer.
  • Circulating tumor cells refers to living solid tumor cells that are present at very low levels in the blood. With the deepening of research on circulating tumor cells, enrichment and identification of these cells has become a method to assist in the diagnosis of cancer. Regulatory agency (eg FDA Some clinical applications based on circulating tumor cell capture and identification systems have been approved.
  • the traditional porous membrane method has the following disadvantages: (1) the pore size is inconsistent with the actual clinical patient cell size diversity, and there is a missing phenomenon; (2) The reagent consumption is large, and it is difficult to carry out the post-identification work; (3) the clogging phenomenon is easy to occur, which affects the experimental results; (4) The special filter membrane preparation process is complicated and the cost is high.
  • the immunological recognition enrichment method mainly includes two types of magnetic bead enrichment and chip enrichment. Due to the complexity and diversity of organisms, tumor cells in body fluids may have degeneration of cell signature recognition groups, resulting in decreased efficiency of immune recognition, leading to false negative or false positives. Regardless of magnetic bead enrichment and chip enrichment, there is a low probability that the cells are in contact with the immune recognition group, and the binding is not strong, which affects the final detection and diagnosis. At the same time, such chips are costly and not easy to process. To this end, the existing methods for separation and enrichment of circulating tumor cells are to be improved.
  • the technical problem to be solved by the present invention is to propose a microfluidic cell capture chip capable of separating and enriching rare cells from a liquid sample of a human body, thereby solving the problems of intrusion sampling and the like in the prior art.
  • a microfluidic cell capture chip comprising an upper hard material and a lower hard material, a channel formed between the upper hard material and the lower hard material, the channel having an inlet and an outlet, At least one of the upper hard material and the lower hard material is a transparent material, and the height of the channel from the inlet to the outlet gradually transitions from high to low and is wedge-shaped or the channel portion is wedge-shaped, and the channel is at the lowest point. Or less than at least one target cell size .
  • the channel has a width of 0.05 to 200 mm and a length of 1 to 500. Millimeter.
  • a layer of a nanoparticle layer or a nanofiber layer or a micro/nano structure for increasing the frictional resistance between the cell and the contact surface is deposited on the upper and lower surfaces of the channel.
  • the nanoparticle layer or the nanofiber layer is nano TiO 2 , SiO 2 or Fe 2 O 3 .
  • At least one surface of the upper hard material or the lower hard material is immunomodified to enable molecular specific recognition of at least one target cell.
  • a thickness is interposed between the upper hard material and the lower hard material.
  • a 50-200 micron thick steel sheet having a thickness of 1 to 50 between the upper hard material and the lower hard material at the exit of the channel A micron thick steel sheet, the channel being formed between the two steel sheets.
  • the upper hard material and the lower hard material are both glass or acrylic materials.
  • Another technical solution adopted to solve the technical problem of the present invention is to provide a microfluidic cell capture chip.
  • the manufacturing method comprises the following steps:
  • Step 1 overlapping the upper hard material and the lower hard material
  • Step 2 inserting a thick steel sheet at one end of the upper hard material and the lower hard material and pressing it with a clamp, and inserting a thin steel sheet at the other end of the upper hard material and the lower hard material The clamp is pressed to form a wedge-shaped channel between the upper hard material and the lower hard material;
  • Step 3 sealing the side of the upper hard material and the lower hard material with polydimethylsiloxane, and then drying, so that the human liquid sample cannot be from the upper hard material and the lower hard material. Side of the stream;
  • Step 4 Both ends of the wedge-shaped channel are also coated with polydimethylsiloxane, and then dried, and a through hole is formed at the thick steel sheet to form an inlet of the wedge-shaped channel at the thin steel sheet.
  • a through hole is provided to form an outlet of the wedge channel, and a human body liquid sample can flow in from the inlet of the wedge channel and out from the outlet of the wedge channel.
  • the method further includes: Step 5. Inserting a hole needle through which the human body liquid sample can pass, respectively, at the inlet and the outlet of the wedge structure.
  • the thickness of the thick steel sheet is 50 to 200 ⁇ m, and the thickness of the thin steel sheet is 1 to 50. Micron.
  • the channel has a width of 0.05 to 200 mm and a length of 1 to 500 mm.
  • a surface of the upper hard material and the lower hard material is deposited with a layer of nano-particles or nano-fibers or a micro-layer which is beneficial to increase the friction between the cells and the contact surface.
  • Nano structure wherein the nanoparticle layer or the nanofiber layer is nano TiO 2 , SiO 2 or Fe 2 O 3 .
  • At least one surface of the upper hard material or the lower hard material is immunomodified to enable molecular specific recognition of at least one target cell.
  • the step of modifying the at least one surface is: Step 1: Preparing 4% with absolute ethanol a solution of 3-mercaptopropyltrimethoxysilane, which is filled into the chip channel, and reacted at room temperature for 1 hour and then rinsed with absolute ethanol for 5 minutes; Step 2, using dimethyl sulfoxide to crosslink the protein 4
  • Maleimidobutyric acid -N- succinimide ester is formulated into a solution of 1 ⁇ mol/mL, which is then injected into the chip channel, and reacted at room temperature for 45 minutes and then rinsed with absolute ethanol for 5 minutes.
  • Step 3 Prepared with phosphate buffer solution.
  • Step 4 Inject the epithelial cell adhesion factor antibody solution into the chip channel and allow the reaction to stand at room temperature for 1-2 hours, then rinse the channel with PBS for 5 minutes.
  • Both the upper hard material and the lower hard material are glass or acrylic materials.
  • the invention adopts a non-invasive manner to extract a human body liquid sample from a patient, and injects a human body liquid sample from the entrance of the microchannel chip. Since the height of the microchannel is wedge-shaped, the target cell will automatically separate and enrich when passing through the channel. set.
  • the microfluidic cell capture chip of the invention has the advantages of simple structure, convenient manufacture and low cost, and can rapidly and efficiently separate and enrich cells of different sizes and specific molecular expression.
  • Figure 1 is a schematic view showing the structure of an embodiment of the microfluidic cell capture chip of the present invention
  • FIG. 2 is a schematic enlarged longitudinal cross-sectional view of a microfluidic cell capture chip of the present invention
  • Figure 3 is a schematic illustration of cell separation of the microfluidic cell capture chip of the present invention.
  • inventions of the present invention provide a microfluidic cell capture chip 100 for capturing microfluidic cells. 200.
  • the microfluidic cell capture chip 100 includes an upper transparent glass sheet 10 and a lower transparent glass sheet 20, and a channel is formed between the upper transparent glass sheet 10 and the lower transparent glass sheet 20. 30, the channel has an inlet 32 and an outlet 34 from the inlet 32 to the outlet 34 The height gradually transitions from high to low and is wedge-shaped or the channel portion is wedge-shaped, the channel being at the lowest or near at least one target cell size.
  • the microfluidic cells 200 For circulating tumor cells.
  • these transparent glass sheet can be replaced by a transparent hard material, such as an acrylic material, and the upper transparent glass sheet and the lower transparent glass sheet can also be replaced by a transparent hard material and an opaque hard material, that is, As long as one of them is a transparent hard material.
  • a transparent hard material such as an acrylic material
  • the upper transparent glass sheet and the lower transparent glass sheet can also be replaced by a transparent hard material and an opaque hard material, that is, As long as one of them is a transparent hard material.
  • the channel 30 has a width of 0.05 to 200 mm and a length of 1 to 500 mm.
  • the At least one surface of the upper transparent glass sheet 10 or the lower transparent glass sheet 20 may be surface-modified.
  • the surface modification step of the at least one surface is: Step 1: Prepare 4% of 3-ethyl acetate ⁇ propyltrimethoxysilane (MPTMS) solution, fill it into the chip channel, react at room temperature for 1 hour, then rinse with absolute ethanol for 5 minutes; Step two, with dimethyl sulfoxide (DMSO)
  • DMSO dimethyl sulfoxide
  • GMBS 4-maleimidobutyric acid-N-succinimidyl ester
  • SA streptavidin
  • Anti-EpCAM epithelial cell adhesion factor antibody
  • a steel sheet 40 having a thickness of 50 to 200 microns is interposed between the upper transparent glass sheet 10 and the lower transparent glass sheet 20, in which the channel 30 is At the outlet 34, between the upper transparent glass sheet 10 and the lower transparent glass sheet 20, a steel sheet 40 having a thickness of 1 to 50 ⁇ m is inserted, and the channel is in the two steel sheets 40. Formed between.
  • the embodiment of the present invention provides a microfluidic cell capture chip capable of separating and enriching cells from a sample of a human organic liquid, wherein the height of the channel 30 in the microfluidic cell capture chip 100 changes according to a certain rule, and the channel 30
  • the upper and lower surfaces are specially treated (ie, a nano-film or nano-fiber such as TiO 2 , SiO 2 or Fe 2 O 3 is deposited on the glass surface of the upper and lower surfaces of the channel or a micro-nano structure which is beneficial to increase the friction between the cells and the contact surface. Its thickness is 5 ⁇ 200 nm) to improve the target cell capture efficiency.
  • the microfluidic cell capture chip of the invention has the advantages of simple structure, convenient manufacture and low cost, and can rapidly and efficiently separate and enrich cells of different sizes and specific molecular expression.
  • Microfluidic cell capture chip 100 of the present invention The cells were isolated and enriched in a non-manual manner. The cells are automatically separated and enriched in a microfluidic cell capture chip based on liquid flow.
  • the microfluidic cell capture chip of the invention is identified by at least one type of tracer marker.
  • the invention adopts a non-invasive manner to extract a human body liquid sample from a patient, and captures a human body liquid sample from the microfluidic cell capture chip.
  • the entrance 32 of 100 is implanted, and since the height of the channel 30 is wedge-shaped, automatic separation and enrichment are achieved when the target cells pass through the channel 30.
  • the invention also provides a method for manufacturing a microfluidic cell capture chip, comprising the following steps:
  • Step 1 overlapping the upper and lower transparent glass sheets 10 and 20;
  • Step 2 Insert a precision steel sheet with a thickness of 50-200 microns thick at one end of the two glass sheets. Pressing with a clamp, inserting a precision steel sheet with a thickness of 1 to 50 microns thick at the other end of the two glass sheets and pressing it with a clamp to form a wedge-shaped channel 30 between the two glass sheets;
  • Step 3 Polydimethylsiloxane (PDMS) on the sides of the two glass sheets Sealing and drying on a heating table so that the human body liquid sample cannot flow out from the side of the glass sheet;
  • PDMS Polydimethylsiloxane
  • Step 4 Both ends of the wedge-shaped channel are also dried and coated with polydimethylsiloxane, and perforated at both ends, so that the human body liquid sample can be obtained from FIG.
  • the inlet 32 of the illustrated wedge channel 30 flows in and out of the outlet 34;
  • Step 5 Inlet 32 and outlet 34 of wedge channel 30 shown in FIG. A hole needle capable of passing a human body liquid sample is inserted, respectively, so that the microfluidic cell capture chip is completed.
  • the present invention adopts a wedge-shaped channel structure design, and the width of the channel formed between the two glass sheets is 0.05 ⁇ a wedge channel of 200 mm with a channel length of 10 to 500 mm;
  • the wedge channel of the present invention has an entrance height of 50 to 200 ⁇ m and an exit height of 1 to 50. Micrometer, when a human body liquid sample flows into the wedge channel through the inlet, the target capturing cells flowing in with the human body liquid sample will be stuck at a specific position due to the limitation of the fluid space;
  • a microfluidic cycle tumor cell capture chip shown in Fig. 1 is produced;
  • tracer substances eg fluorescent dyes such as DAPI or Hoechst) Dye
  • adding an immunological reagent to identify the target cell eg fluorescent dyes such as DAPI or Hoechst
  • the chip is placed under a microscope to observe the captured target cells.
  • the wedge-shaped microfluidic cell capture chip used in the experimental device is simple in structure, easy to manufacture, and low in cost, and can rapidly and efficiently separate and enrich cells of different sizes and specific molecular expression.
  • the invention adopts a non-invasive manner to extract a human body liquid sample from a patient, and injects a human body liquid sample from the entrance of the microchannel chip. Since the height of the microchannel is wedge-shaped, the target cell will automatically separate and enrich when passing through the channel. set.
  • the microfluidic cell capture chip of the invention has the advantages of simple structure, convenient manufacture and low cost, and can rapidly and efficiently separate and enrich cells of different sizes and specific molecular expression.

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Abstract

一种微流体细胞（200）捕获芯片（100）及其制备方法，所述芯片（100）包括上部硬质材料（10）和下部硬质材料（20），所述上部硬质材料（10）和下部硬质材料（20）之间形成有一条沟道（30），所述沟道（30）具有入口（32）和出口（34），所述上部硬质材料（10）和下部硬质材料（20）至少一个是透明的材料，所述沟道（30）从入口（32）到出口（34）的高度由高向低逐渐过渡且呈楔形或沟道部分区域呈楔形，所述沟道（30）最低处接近或小于至少一种目标细胞尺寸。所述芯片（100）能够对不同尺寸和具有特异性分子表达的细胞进行快速高效分离、富集。

Description

一种微流体细胞捕获芯片及其制备方法 技术领域
本发明涉及一种细胞捕获工具及其制备方法，尤其涉及一种分离、富集和识别循环肿瘤细胞的微流体细胞捕获芯片及其制备方法，该微流体细胞捕获芯片可用作为肿瘤诊断、辅助治疗、及生化分析研究的有利工具。
背景技术
目前，肿瘤诊断通常根据大量病理状况进行诊断。这类方法，例如活检或直肠癌指检通常采取侵入性分析，具有一定程度的创伤性，不利于病患。
另外也有采用外周血中生物分子标志物进行检查，例如血清学参数（ PSA 剂量）检测，由于其敏感性和特异性都不是很理想，使得癌症诊断和治疗较难取得良好效果。
对许多癌症患者而言，其死亡主要是由于转移瘤引起的。当病人手术切除主要肿瘤后，目前检测手段很难及时反应治疗情况，鉴定转移瘤，指导后续的放化疗过程，从而导致病人错过最佳治疗时机和无法及时有效调整无效治疗和用药方案。因此不能够成功治疗所有的转移瘤，导致病人最终死亡。从临床角度看来，转移瘤可以看作是癌症自然发展过程中的结论性事件。
人们急待开发非创伤性过程从患者提取肿瘤细胞样品的工具。
循环肿瘤细胞（ CTCs ）是指在血液中以极低水平存在的活实体瘤细胞。随着对循环肿瘤细胞研究的不断深入，富集和鉴定这些细胞已经成为辅助癌症诊断的一种方法。监管机构（例如 FDA ）已经批准一些基于循环肿瘤细胞捕获、鉴定系统的临床应用。
为了分离和富集体液中循环肿瘤细胞和扩散肿瘤细胞，人们利用这类细胞尺寸、表面分子表达等特点开发出一些相应的捕获、富集方法（例如，多孔滤膜法、免疫磁珠富集法）。
传统的多孔滤膜法存在缺点有：（ 1 ）孔径单一与实际临床病人细胞尺寸多样性不符，存在遗漏现象；（ 2 ）试剂消耗量大，难以进行后期鉴定工作；（ 3 ）容易出现堵塞现象，影响实验结果；（ 4 ）专用滤膜制备工艺复杂，成本较高。
免疫识别富集法，主要有磁珠富集和芯片富集两种。由于生物的复杂性与多样性，体液中肿瘤细胞可能存在细胞特征识别基团退化，从而导致免疫识别效率下降，导致假阴性或假阳性出现。无论磁珠富集和芯片富集，都存在细胞与免疫识别基团接触几率不高、结合不牢，影响最后检测和诊断情况。同时此类芯片成本较高，不易于加工。为此要对现有的循环肿瘤细胞分离、富集方法进行改进。
 技术问题
本发明要解决的技术问题在于提出一种能够通过从人体液体样本中分离、富集罕见细胞的微流体细胞捕获芯片，以解决现有技术中侵入采样等问题。
技术解决方案
解决本发明的技术问题所采用的技术方案是：提供 一种微流体细胞捕获芯片，其包括上部硬质材料和下部硬质材料，所述上部硬质材料和下部硬质材料之间形成有一条沟道，所述沟道具有入口和出口，所述上部硬质材料和下部硬质材料至少一个是透明的材料，所述沟道从入口到出口的高度由高向低逐渐过渡且呈楔形或者沟道部分区域呈楔形，所述沟道最低处接近或小于至少一种目标细胞尺寸 。
作为本发明的进一步改进， 所述 沟道的宽度为 0.05 ～ 200 毫米，长度为 1 ～ 500 毫米。
作为本发明的进一步改进， 所述 沟道的上下底面沉积一层纳米颗粒层或纳米纤维层或用于增加细胞与接触面间摩擦阻力的微纳结构。其中， 所述 纳米颗粒层或纳米纤维层为纳米 TiO2 、 SiO2 或者 Fe2O3 。
作为本发明的进一步改进， 对所述 上部硬质材料或者下部硬质材料的至少一个表面进行免疫修饰，能够至少对一种目标细胞进行分子特异性识别。
作为本发明的进一步改进， 在所述沟道的入口处，在所述 上部硬质材料和下部硬质材料之间塞有厚度为 50~200 微米厚的钢片， 在所述沟道的出口处，在所述 上部硬质材料和下部硬质材料之间塞有厚度为 1~50 微米厚的钢片，所述沟道在所述两块钢片之间形成。
作为本发明的进一步改进， 所述 上部硬质材料和下部硬质材料均为玻璃或者亚克力材料。
解决本发明的技术问题所采用的另一技术方案是：提供 一种微流体细胞捕获芯片 的 制作方法，其包括如下步骤：
步骤一、将上部硬质材料和下部硬质材料重叠在一起；
步骤二、在所述上部硬质材料和下部硬质材料重叠的一端塞入厚钢片并用夹具压紧，在所述上部硬质材料和下部硬质材料重叠的另一端塞入薄钢片并用夹具压紧，从而在所述上部硬质材料和下部硬质材料中间形成楔形沟道；
步骤三、在所述上部硬质材料和下部硬质材料的侧边用聚二甲基硅氧烷封涂，然后烘干，使人体液体样本不能从所述上部硬质材料和下部硬质材料的侧边流出；
步骤四、所述楔形沟道的两端也用聚二甲基硅氧烷封涂，然后烘干，在厚钢片处设置通孔以形成所述楔形沟道的入口，在薄钢片处设置通孔以形成所述楔形沟道的出口，人体液体样本能从所述楔形沟道的入口流入，并从所述楔形沟道的出口流出。
作为本发明的制作方法的进一步改进， 进一步包括： 步骤五、在所述楔形结构的入口和出口分别插上能使人体液体样本通过的孔针。
作为本发明的制作方法的进一步改进，厚钢片的厚度为 50~200 微米，薄钢片的厚度为 1~50 微米。
作为本发明的制作方法的进一步改进， 所述 沟道的宽度为 0.05 ～ 200 毫米，长度为 1 ～ 500 毫米。
作为本发明的制作方法的进一步改进， 在步骤一中，所述 上部硬质材料和下部硬质材料的表面沉积一层纳米颗粒层或纳米纤维层或有利于增加细胞与接触面摩擦力的微纳结构。其中， 所述 纳米颗粒层或纳米纤维层为纳米 TiO2 、 SiO2 或者 Fe2O3 。
作为本发明的制作方法的进一步改进， 对所述 上部硬质材料或者下部硬质材料的至少一个表面进行免疫修饰，能够至少对一种目标细胞进行分子特异性识别。
作为本发明的制作方法的进一步改进，对所述至少一个表面进行修饰步骤为：步骤一、用无水乙醇配制 4% 的 3- 巯丙基三甲氧基硅烷溶液，将其注满芯片沟道，在常温下反应 1 小时后用无水乙醇冲洗 5 分钟；步骤二、用二甲亚砜将蛋白质交联剂 4- 马来酰亚胺基丁酸 -N- 琥珀酰亚胺酯 配制成 1 μmol/mL 的溶液，再将其注入芯片沟道，在常温下反应 45min 后用无水乙醇冲洗 5 分钟；步骤三、用磷酸盐缓冲液配制 50μg/mL 的链霉亲和素溶液，再将其注入芯片沟道，然后置入 4℃ 冰箱中过夜反应后用 pH=7.2-7.4 的磷酸盐缓冲液清洗 5 分钟；步骤四、将上皮细胞黏附因子抗体溶液注入芯片沟道，并在常温下静置反应 1-2 小时后用 PBS 将沟道冲洗 5 分钟。
作为本发明的制作方法的进一步改进， 所述 上部硬质材料和下部硬质材料均为玻璃或者亚克力材料。
 有益效果
本发明采用非侵入式方式从患者提取人体液体样本，将人体液体样本从微沟道芯片的入口注入，由于微沟道的高度呈楔形分布，目标细胞在沟道中通过时将实现自动分离和富集。本发明的微流体细胞捕获芯片结构简单、便于制作、成本低廉，能够对不同尺寸和具有特异性分子表达的细胞进行快速高效分离、富集。
附图说明
下面将结合附图及实施例对本发明作进一步说明，附图中：
图 1 为以本发明微流体细胞捕获芯片的实施例的结构示意图；
图 2 为本发明微流体细胞捕获芯片的纵截面放大示意图；
图 3 为本发明微流体细胞捕获芯片的细胞分离的示意图。
 本发明的实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
如图 1 至图 3 所示，本发明的实施例提供了一种微流体细胞捕获芯片 100 ，用于捕获微流体细胞 200 。该微流体细胞捕获芯片 100 包括上部透明玻璃片 10 和下部透明玻璃片 20 ，该上部透明玻璃片 10 和下部透明玻璃片 20 之间形成有一条沟道 30 ，该沟道具有入口 32 和出口 34 ，该沟道 30 从入口 32 到出口 34 的高度由高向低逐渐过渡且呈楔形或者沟道部分区域呈楔形，该沟道最低处接近或小于至少一种目标细胞尺寸 。在本实施例中，微流体细胞 200 为循环肿瘤细胞。在本实施例中，不限于本发明的这些透明玻璃片，这些 透明玻璃片可以用透明的硬质材料替代，，例如亚克力材料，并且，上部透明玻璃片和下部透明玻璃片也可以用一个透明的硬质材料和一个不透明的硬质材料替代，也就是说，两者之中只要其中一个是透明的硬质材料即可。
该 沟道 30 的宽度为 0.05 ～ 200 毫米，长度为 1 ～ 500 毫米。 该 上部透明玻璃片 10 或者下部透明玻璃片 20 的至少一个表面可进行表面修饰。对该至少一个表面进行表面修饰步骤为：步骤一、用无水乙醇配制 4% 的 3- 巯丙基三甲氧基硅烷（ MPTMS ）溶液，将其注满芯片沟道，在常温下反应 1 小时后用无水乙醇冲洗 5 分钟； 步骤二、用二甲亚砜（ DMSO ）将蛋白质交联剂 4- 马来酰亚胺基丁酸 -N- 琥珀酰亚胺酯 （ GMBS ）配制成 1 μmol/mL 的溶液，再将其注入芯片沟道，在常温下反应 45min 后用无水乙醇冲洗 5 分钟；步骤三、用磷酸盐缓冲液（ PBS ）配制 50μg/mL 的链霉亲和素（ SA ）溶液，再将其注入芯片沟道，然后置入 4℃ 冰箱中过夜反应后用 PBS （磷酸盐缓冲液， pH=7.2-7.4 ）溶液清洗 5 分钟；步骤四、将上皮细胞黏附因子抗体（ Anti-EpCAM ）溶液注入芯片沟道，并在常温下静置反应 1-2 小时后用 PBS 将沟道冲洗 5 分钟。在经过表面修饰以后可对至少一种目标细胞进行分子识别的特异性抗体。 在该沟道 30 的入口 32 处，在该 上部透明玻璃片 10 和下部透明玻璃片 20 之间塞有厚度为 50~200 微米厚的钢片 40 ， 在该沟道 30 的出口 34 处，在该 上部透明玻璃片 10 和下部透明玻璃片 20 之间塞有厚度为 1~50 微米厚的钢片 40 ，该沟道在该两块钢片 40 之间形成。
本发明实施例提供的一种能够通过从人体有机液体样本中分离、富集细胞的微流体细胞捕获芯片，该微流体细胞捕获芯片 100 内的沟道 30 高度按一定规律变化，且沟道 30 上下底面通过特殊处理（即在该沟道上下底面的玻璃表面沉积一层 TiO2 、 SiO2 或者 Fe2O3 等纳米薄膜或者纳米纤维或有利于增加细胞与接触面摩擦力的微纳结构，其厚度为 5~200 纳米）提高目标细胞捕获效率。本发明的微流体细胞捕获芯片结构简单、便于制作、成本低廉，能够对不同尺寸和具有特异性分子表达的细胞进行快速高效分离、富集。
本发明的微流体细胞捕获芯片 100 以非手动方式分离、富集该细胞。该细胞是基于液体流动在微流体细胞捕获芯片里自动分离和富集。本发明的微流体细胞捕获芯片，该细胞至少用一种类型的示踪物标记识别。本发明采用非侵入式方式从患者提取人体液体样本，将人体液体样本从微流体细胞捕获芯片 100 的入口 32 注入，由于沟道 30 的高度呈楔形分布，目标细胞在沟道 30 中通过时将实现自动分离和富集。
本发明还提供了一种微流体细胞捕获芯片的制作方法，其包括如下步骤：
步骤一、将上下两片透明玻璃片 10 、 20 重叠在一起；
步骤二、在两玻璃片重叠的一端塞入厚度为 50~200 微米厚的精密钢片 40 并用夹具压紧，在两玻璃片重叠的另一端塞入厚度为 1~50 微米厚的精密钢片并用夹具压紧，从而在两玻璃片中间形成楔形沟道 30 ；
步骤三、两玻璃片的侧边用聚二甲基硅氧烷（ PDMS ）封涂，然后在加热台上烘干，使人体液体样本不能从玻璃片的侧边流出；
步骤四、该楔形沟道的两端也用聚二甲基硅氧烷封涂烘干，并在两端打孔，使人体液体样本能从图 1 所示的楔形沟道 30 的入口 32 流入，并从出口 34 流出；
步骤五、在图 1 所示的楔形沟道 30 的入口 32 和出口 34 分别插上能使人体液体样本通过的孔针，这样，该微流体细胞捕获芯片就制作完成了。
实验原理：
（ 1 ）如图 1 所示，本发明采用楔形沟道结构设计，在两玻璃片中间形成的沟道的宽度为 0.05 ～ 200 毫米，沟道长度为 10 ～ 500 毫米的楔形沟道；
（ 2 ）本发明的楔形沟道的入口高度为 50~200 微米，出口高度为 1~50 微米，当人体液体样本通过入口流入楔形沟道时，由于流体空间的限制，随人体液体样本流入的目标捕获细胞将在特定的位置卡住；
（ 3 ）此实验的基本思路是将待检测的患者的人体液体样本通过本发明的楔形微流体细胞捕获芯片，最终使不同尺寸的目标细胞在沟道中通过时实现自动分离和富集。
实验步骤：
（ 1 ）依据发明的制作方法，制作出带有图 1 所示的微流楔形循环肿瘤细胞捕获芯片；
（ 2 ）将待检测人体液体样本从上述芯片的入口注入，并在芯片的出口处收集目标细胞分离后的人体液体样本；
（ 3 ）继续通过入口加入磷酸盐缓冲溶液（即 PBS 溶液， pH=7.2-7.4 ， NaCl 137mmol/L ， KCl 2.7 mmol/L ， Na2HPO4 4.3mmol/L ， KH2PO4 1.4mmol/L ）进行清洗；
（ 4 ）继续通过入口选择加入示踪物质（如：染细胞核的荧光染料如 DAPI 或者 Hoechst 染料）或加入免疫试剂，对目标细胞进行识别；
（ 5 ）再次通过入口加入 PBS 进行清洗；
（ 3 ）将芯片放置在显微镜下观察捕获的目标细胞。
实验效果分析
（ 1 ）、如图 2 所示，分别选择区域一、区域二、区域三、区域四中的四个观测点，可以看到目标细胞在上述芯片中的分离和富集。
（ 2 ）、在上述四个区域的四个观测点观测到的目标细胞的分布可以看出，目标细胞的分离和富集是由于目标细胞的尺寸和楔形沟道的尺寸相互作用的结果，大尺寸细胞富集在离出口远处，小尺寸细胞富集在离出口近处。
（ 3 ）、本实验能实现不同尺寸的目标细胞的分离、捕获，操作简单，可重复性强。
（ 4 ）、本实验装置使用的楔形微流体细胞捕获芯片结构简单、便于制作、成本低廉，能够对不同尺寸和具有特异性分子表达的细胞进行快速高效分离、富集。
本发明采用非侵入式方式从患者提取人体液体样本，将人体液体样本从微沟道芯片的入口注入，由于微沟道的高度呈楔形分布，目标细胞在沟道中通过时将实现自动分离和富集。本发明的微流体细胞捕获芯片结构简单、便于制作、成本低廉，能够对不同尺寸和具有特异性分子表达的细胞进行快速高效分离、富集。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (17)

  1. 一种微流体细胞捕获芯片，其包括上部硬质材料和下部硬质材料，所述上部硬质材料和下部硬质材料之间形成有一条沟道，所述沟道具有入口和出口，所述上部硬质材料和下部硬质材料至少一个是透明的材料， 其特征在于， 所述沟道从入口到出口的高度由高向低逐渐过渡且呈楔形或者沟道部分区域呈楔形，所述沟道最低处接近或小于至少一种目标细胞尺寸 。
  2. 如权利要求 1 所述的微流体细胞捕获芯片，其特征在于 ：所述 沟道的宽度为 0.05 ～ 200 毫米，长度为 1 ～ 500 毫米。
  3. 如权利要求 1 所述的微流体细胞捕获芯片，其特征在于 ：所述 沟道的上下底面沉积一层纳米颗粒层或纳米纤维层或用于增加细胞与接触面间摩擦阻力的微纳结构。
  4. 如权利要求 3 所述的微流体细胞捕获芯片，其特征在于 ：所述 纳米颗粒层或纳米纤维层为纳米 TiO2 、 SiO2 或者 Fe2O3 。
  5. 如权利要求 1 或 4 所述的微流体细胞捕获芯片，其特征在于 ：对所述 上部硬质材料或者下部硬质材料的至少一个表面进行免疫修饰，能够至少对一种目标细胞进行分子特异性识别。
  6. 如权利要求 1 所述的微流体细胞捕获芯片，其特征在于 ：在所述沟道的入口处，在所述 上部硬质材料和下部硬质材料之间塞有厚度为 50~200 微米厚的钢片， 在所述沟道的出口处，在所述 上部硬质材料和下部硬质材料之间塞有厚度为 1~50 微米厚的钢片，所述沟道在所述两块钢片之间形成。
  7. 如权利要求 1 所述的微流体细胞捕获芯片，其特征在于 ：所述 上部硬质材料和下部硬质材料均为玻璃或者亚克力材料。
  8. 一种微流体细胞捕获芯片 的 制作方法，其特征在于，其包括如下步骤：
    步骤一、将上部硬质材料和下部硬质材料重叠在一起；
    步骤二、在所述上部硬质材料和下部硬质材料重叠的一端塞入厚钢片并用夹具压紧，在所述上部硬质材料和下部硬质材料重叠的另一端塞入薄钢片并用夹具压紧，从而在所述上部硬质材料和下部硬质材料中间形成楔形沟道；
    步骤三、在所述上部硬质材料和下部硬质材料的侧边用聚二甲基硅氧烷封涂，然后烘干，使人体液体样本不能从所述上部硬质材料和下部硬质材料的侧边流出；
    步骤四、所述楔形沟道的两端也用聚二甲基硅氧烷封涂，然后烘干，在厚钢片处设置通孔以形成所述楔形沟道的入口，在薄钢片处设置通孔以形成所述楔形沟道的出口，人体液体样本能从所述楔形沟道的入口流入，并从所述楔形沟道的出口流出。
  9. 如权利要求 8 所述的制作方法，其特征在于 ：进一步包括： 步骤五、在所述楔形结构的入口和出口分别插上能使人体液体样本通过的孔针。
  10. 如权利要求 8 所述的制作方法，其特征在于 ： 厚钢片的厚度为 50~200 微米，薄钢片的厚度为 1~50 微米。
  11. 如权利要求 8 所述的制作方法，其特征在于 ：所述 沟道的宽度为 0.05 ～ 200 毫米，长度为 1 ～ 500 毫米。
  12. 如权利要求 8 所述的制作方法，其特征在于 ：在步骤一中，所述 上部硬质材料和下部硬质材料的表面沉积一层纳米颗粒层或纳米纤维层或有利于增加细胞与接触面摩擦力的微纳结构。
  13. 如权利要求 12 所述的制作方法，其特征在于 ：所述 纳米颗粒层或纳米纤维层为纳米 TiO2 、 SiO2 或者 Fe2O3 。
  14. 如权利要求 8 所述的制作方法，其特征在于 ：对所述 上部硬质材料或者下部硬质材料的至少一个表面进行免疫修饰，能够至少对一种目标细胞进行分子特异性识别。
  15. 如权利要求 14 所述的制作方法，其特征在于 ： 对所述至少一个表面进行修饰步骤为：步骤一、用无水乙醇配制 4% 的 3- 巯丙基三甲氧基硅烷溶液，将其注满芯片沟道，在常温下反应 1 小时后用无水乙醇冲洗 5 分钟；步骤二、用二甲亚砜将蛋白质交联剂 4- 马来酰亚胺基丁酸 -N- 琥珀酰亚胺酯 配制成 1 μmol/mL 的溶液，再将其注入芯片沟道，在常温下反应 45min 后用无水乙醇冲洗 5 分钟；步骤三、用磷酸盐缓冲液配制 50μg/mL 的链霉亲和素溶液，再将其注入芯片沟道，然后置入 4℃ 冰箱中过夜反应后用 pH=7.2-7.4 的磷酸盐缓冲液清洗 5 分钟；步骤四、将上皮细胞黏附因子抗体溶液注入芯片沟道，并在常温下静置反应 1-2 小时后用 PBS 将沟道冲洗 5 分钟。
  16. 如权利要求 8 所述的制作方法，其特征在于 ：所述 上部硬质材料和下部硬质材料均为玻璃或者亚克力材料。
  17. 一种根据权利要求 8-16 任意一项制作方法制作出来的微流体细胞捕获芯片。
PCT/CN2012/084603 2012-11-08 2012-11-14 一种微流体细胞捕获芯片及其制备方法 WO2014071642A1 (zh)

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