WO2011150675A1 - 包含多根微通道的生物芯片 - Google Patents

包含多根微通道的生物芯片 Download PDF

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Publication number
WO2011150675A1
WO2011150675A1 PCT/CN2011/000918 CN2011000918W WO2011150675A1 WO 2011150675 A1 WO2011150675 A1 WO 2011150675A1 CN 2011000918 W CN2011000918 W CN 2011000918W WO 2011150675 A1 WO2011150675 A1 WO 2011150675A1
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Prior art keywords
microchannel
array
probe
sample
dimensional
Prior art date
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PCT/CN2011/000918
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English (en)
French (fr)
Inventor
陈宏�
瞿祥猛
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厦门大学
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Publication date
Priority claimed from CN 201010193890 external-priority patent/CN101864360B/zh
Priority claimed from CN 201010193922 external-priority patent/CN101851680B/zh
Priority claimed from CN 201010193906 external-priority patent/CN101886126B/zh
Application filed by 厦门大学 filed Critical 厦门大学
Publication of WO2011150675A1 publication Critical patent/WO2011150675A1/zh
Priority to US13/553,832 priority Critical patent/US20120289429A1/en

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00511—Walls of reactor vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00513—Essentially linear supports
    • B01J2219/0052—Essentially linear supports in the shape of elongated tubes
    • B01J2219/00522—Essentially linear supports in the shape of elongated tubes in a multiple parallel arrangement
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00657—One-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00664—Three-dimensional arrays
    • B01J2219/00666—One-dimensional arrays within three-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00664—Three-dimensional arrays
    • B01J2219/00668—Two-dimensional arrays within three-dimensional arrays
    • 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
    • B01L2300/0636—Integrated biosensor, microarrays
    • 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
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • 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
    • B01L2300/0874—Three dimensional network
    • 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
    • B01L2300/0883—Serpentine channels

Definitions

  • the present invention relates to the field of biochips, and more particularly to a biochip and a method for high throughput hybridization of a biochip.
  • a biochip immobilizes a large number of biomolecules or materials (such as nucleic acid fragments, proteins, drugs or receptors, cells or tissues, etc.) on the surface of a carrier in a pre-designed arrangement, as a probe, and a sample to be tested. Perform specific adsorption or reaction to detect sample information. Thousands of reactions are performed simultaneously on a single chip, with the ability to perform massively parallel analysis. Biochips are generally processed on carrier materials such as glass sheets, silicon wafers, and nylon membranes. The method of making probe arrays is mainly a bit of a method (Schena M, Shalon D, Davis R W. et al. Science., 1995, 20: 467).
  • the spotting method in the manufacturing method of the biochip is to use a spotting device to spot the probe on the surface of the carrier and then perform a fixing reaction to firmly bind the probe to the surface of the carrier, and the in situ synthesis is mainly used for the oligonucleotide.
  • the multi-step reaction attaches the deoxynucleotide monomer to the tail of the probe to achieve extension on the surface of the carrier.
  • These two processing methods require high precision, high processing costs, and slow production speeds, all of which require expensive precision instruments.
  • biochips require a relatively long hybridization reaction time, usually from a few hours to a dozen hours. One chip is used only for one hybridization reaction, and it is difficult to achieve high-throughput and low-cost rapid detection of multiple samples.
  • the hybridization process of conventional biochips is controlled by the diffusion process.
  • the hybridization reaction takes more than ten hours, and the hybridization reaction in a capillary-sized microchannel promotes mixing and diffusion distance due to the flow hybridization process.
  • the utility model patent of CN2483395Y discloses a transparent capillary tube with capillary filaments (strips) as a capillary biochip device, which is used for making capillary lines, corresponding markers, positive and negative control lines in capillary fibers. The wire (strip) is then inserted into the capillary.
  • the sample is sucked into the capillary by the capillary action of the capillary and the capillary filament (strip) is impregnated, and the point on the sample and the capillary filament (strip) in the sample A hybridization reaction occurs between the sample and the label.
  • the spots and markers are made on the capillary filaments (strips) in the capillary rather than on the wall of the capillary.
  • the number of probes is limited and is generally only used for one hybridization reaction, i.e., only for one sample. If multiple samples are to be tested, multiple capillary chips need to be processed, which reduces the efficiency of the analysis.
  • the microfluidic chip uses various micromachining technologies to process microstructures with various functions on chip materials (such as glass, PDMS or PMMA) to realize functions of reaction, separation, detection, etc.
  • chip materials such as glass, PDMS or PMMA
  • the function of the room is integrated into the portable chip. Since the first introduction of Manz and Widmer et al. (Manz A, Graber N, Widemer H M. Sens. Acturators, B, 1990, Bl: 244) in the early 1990s, microfluidic chip technology has developed rapidly. Chemical analysis extends to all directions, including nucleic acid analysis, protein analysis, and cell analysis. Biochip analysis on microfluidic chips can effectively enhance detection signals, improve sensitivity, reduce reagent and sample consumption, and shorten analysis time (Chen H, Wang L, Li PC H.
  • the microfluidic chip cannot perform high temperature bonding, which brings great difficulties to chip fabrication, experimental operation, system integration and development of new applications; on the other hand, manual alignment of microchannels and For probe arrays, a large number of repetitive probes must be present in the probe array to ensure that the desired probes are contained within the microchannels. The number and density of probes is greatly limited and the surface of the carrier cannot be effectively utilized.
  • the invention includes the following steps -
  • the sample molecule After the sample solution enters the microchannel, the sample molecule will be specifically bound to the specific probe due to sequence complementation or affinity, and will be retained in the region where the specific probe is located, and the corresponding sample molecule will be obtained according to the information of the probe.
  • the sample molecule has been labeled with at least one of a bar code label, a fluorescent label, a quantum dot label, a photonic crystal label, a Raman label label, an infrared spectrum label, an electrochemical label, etc., to determine whether the sample molecule is retained in the probe region And information such as where to keep it;
  • the sample molecules bound to the probe are eluted from the probe by a denaturation technique and cleaned to recover the probe array;
  • step 1) the solution is introduced into the microchannel, and the inlet of the microchannel can be inserted into a reservoir or a small tube to introduce the solution into the microchannel through the inlet;
  • the microchannel can be a capillary or a micro
  • the flow control chip or the like the capillary tube may be a glass capillary tube, a quartz capillary tube or a polymer capillary tube, etc., and the diameter of the capillary tube may be Inn!
  • the capillary is provided with at least one, a plurality of capillary tubes may be connected in parallel or in series;
  • the microfluidic chip may be a glass microfluidic chip, a quartz microfluidic chip, a silicon microfluidic chip or a high polymer micro a flow control chip or the like;
  • the channel size of the microfluidic chip may be 1 nm to 5 cm;
  • the microchannel may be a different shape, such as a straight channel, or a coiled channel, or a rectangular channel It may also be a spiral channel;
  • the channel may be a single channel or a plurality of parallel channels.
  • steps 1), 2), 3), 4) after the solution enters the microchannel, the sample solution flows in the microchannel, and the driving force of the flow may be connected by electric field, capillary action, surface tension or by channel
  • the outlet of the syringe pump, gravity, etc. is provided.
  • step 5 the information of whether the sample molecules are retained in the probe region and the position where they are retained may be detected by a corresponding method to determine whether the sample molecules are retained in the probe region and remain in the position. And other information.
  • the denaturation includes thermal denaturation, extreme pH denaturation, ionic strength denaturation, denaturant Denaturation and so on.
  • the probe and sample may be biological, organic or inorganic substances capable of producing an affinity such as a nucleic acid, a small molecule compound, a polypeptide, a protein, an antigen, a polysaccharide, a ligand, a drug, a receptor, a cell or a tissue.
  • the invention has the advantages that: in the case of low reagent consumption, the probe array can be processed simply and efficiently in the closed microchannel to produce probe arrays of various densities; the nucleic acid hybridization reaction is carried out in the closed microchannel, Rapid analysis of samples; then high-throughput analysis of multiple samples on a capillary or on the same chip using denaturation technology; reduced reliance on expensive instruments, reduced processing and analysis costs, faster fabrication and analysis, and multi-sample detection , to achieve low-cost, high-throughput biochip analysis.
  • Figure 1 is a schematic illustration of the processing of a probe array on the inner wall of a capillary tube of the present invention.
  • Figure 2 is a schematic illustration of the simultaneous processing of a plurality of capillary processing probe arrays in accordance with the present invention.
  • Fig. 3 is a schematic view showing the use of a syringe as a driving force in the present invention.
  • FIG. 4 is a schematic illustration of the processing of a probe array on a microfluidic chip of the present invention.
  • Figure 5 is a schematic illustration of the simultaneous processing of a probe array for a plurality of parallel channels of the present invention.
  • Figure 6 is a schematic illustration of a processing probe array on a microfluidic chip having a spiral channel of the present invention.
  • Figure 7 is a schematic illustration of a process probe array on a microfluidic chip employing a capillary inlet of the present invention.
  • Fig. 8 is a schematic view showing the processing of a probe array using a syringe as a driving force of the present invention.
  • Figure 9 is a bar graph showing the fluorescence signal intensity of a fixed probe array of the present invention.
  • the abscissa is the probe concentration ( ⁇ ⁇ ) and the ordinate is the fluorescence signal intensity.
  • Figure 10 is a schematic illustration of a hybridization reaction with a capillary to which a probe array is immobilized in accordance with the present invention.
  • Figure 11 is a schematic illustration of the simultaneous hybridization reaction with a plurality of parallel capillaries in accordance with the present invention.
  • Figure 12 is a schematic illustration of a hybridization reaction using a syringe as a driving force in accordance with the present invention.
  • Figure 13 is a bar graph of fluorescence signal intensities for hybridization of two different samples to a probe array in accordance with the present invention.
  • the abscissa is the sample solution concentration ( ⁇ ) and the ordinate is the fluorescence signal intensity; where a is CLN1 ', b is CLN5,.
  • Figure 14 is a schematic illustration of a hybridization reaction with a microfluidic chip immobilized with a probe array in accordance with the present invention.
  • Figure 15 is a schematic illustration of simultaneous hybridization reactions in multiple parallel channels in accordance with the present invention.
  • Figure 16 is a schematic illustration of hybridization reactions on a microfluidic chip of a spiral channel in accordance with the present invention.
  • Figure 17 is a schematic illustration of the hybridization reaction carried out on a microfluidic chip employing a capillary as an inlet in accordance with the present invention.
  • Figure 18 is a schematic illustration of a hybridization reaction using a syringe as a driving force in accordance with the present invention.
  • Figure 19 is a bar graph of fluorescence signal intensities for hybridization of two different samples to a probe array in accordance with the present invention.
  • the abscissa is the probe concentration ( ⁇ ⁇ ) and the ordinate is the fluorescence signal intensity; where a is ALN1, b is ALN5.
  • Figure 20 is a schematic illustration of the processing of a probe array by combining a plurality of capillaries into a three dimensional array in accordance with the present invention.
  • Figure 21 is a schematic diagram showing the processing of a probe array by superimposing a plurality of microfluidic chips processed with a plurality of parallel microchannels to form a three-dimensional array according to the present invention.
  • Figure 22 is a diagram of a capillary array erected in accordance with the present invention to form a stereoscopic array for use in immobilization and sample hybridization of probe arrays.
  • Figure 23 is a diagram showing a microfluidic chip processed with a plurality of parallel microchannels stacked together in accordance with the present invention to form a stereo which can be used for probe array immobilization and sample hybridization.
  • the inlet of the capillary 101 is inserted into the liquid storage cuvette A, and the liquid storage cuvettes A are arranged with the mineral oil 102 and the probe solution 103, respectively.
  • the outlet of the capillary 101 is connected to a horizontal reservoir 104, which is driven by gravity to effect the flow of the carrier 105 in the capillary 101 to carry the flow of the droplet sequence 106. After flowing to the predetermined position, the flow is stopped and the immobilization reaction of the probe is performed to complete the processing of the biochip.
  • capillary 201 is a plurality of parallel capillaries, each having an inlet and an outlet, and processing multiple parallel probe arrays for multiple parallel biochip analyses.
  • the components indicated by the respective marks are the same as those of Fig. 1. '
  • the outlet of the capillary 301 is connected to a syringe, and the negative pressure generated by the syringe drives the flow of the liquid.
  • the components indicated by the respective marks are the same as those of Fig. 1.
  • Example 4 Referring to Fig. 4, the microchannel 403 of the chip 401 has a disk shape, and its inlet 402 is processed into a sharp needle shape and inserted into the liquid storage cuvette A.
  • the liquid storage cuvettes A are respectively arranged with mineral oil 405 and probe solution 406.
  • the outlet of the microchannel is connected to a horizontal reservoir 404 which is driven by gravity to effect the flow of mineral oil 407 in the microchannel 403 to carry the flow of probe droplets 408. After flowing to the predetermined position, the flow is stopped and the immobilization reaction of the probe is performed to complete the processing of the probe array.
  • Figure 9 is a bar graph showing the fluorescence signal intensity obtained by immobilizing nucleic acid probes of different concentrations for 30 min.
  • the probe contains 20 bases, 3' is labeled with FITC, 5' is labeled with amino groups, and the free aldehyde is modified with the inner surface of the channel. The chemical reaction of the base is fixed in the channel.
  • microchannel 503 of chip 501 is one or more parallel channels, each having an inlet and an exit, and processing a plurality of parallel probe arrays for multiple parallel biochip analysis.
  • the components indicated by other marks are the same as those of Fig. 4.
  • the microchannel 603 is helical for processing the probe array.
  • the components indicated by other marks are the same as those of Fig. 4.
  • the microchannel inlet is replaced by a small section of quartz capillary.
  • the processing method is as follows: Position the microchannel on the side of the chip, drill a small hole with a drill bit, insert a small piece of quartz capillary and fix it with glue as the channel inlet.
  • the components indicated by other marks are the same as those of Fig. 4.
  • a syringe 804 is connected to the outlet, and the negative pressure generated by the syringe provides a driving force to drive the liquid to flow in the microchannel.
  • the components indicated by other marks are the same as those of Fig. 4.
  • the inlet of the capillary 1001 is inserted into the liquid storage cuvette A, and the liquid storage cuvette A is separately arranged with the sample solution 1002, the washing buffer 1003, and the denaturant 1004.
  • the outlet of the capillary 1001 is connected to a horizontal reservoir 1005, which uses gravity to drive liquid flow.
  • the probe array has been pre-fixed on the inner wall of the capillary 1007, allowing the sample to flow through the entire channel and undergoing a hybridization reaction. After the reaction is completed, the channel 1006 is washed and detected, and then the denaturing agent is passed through the channel to denature the hybridized sample. Remove the probe and clean it, and restore the probe array. This process is repeated to complete high throughput analysis of the sample.
  • Figure 13 is a bar graph of the signal intensity obtained by two different concentrations of sample solution added to a capillary for hybridization.
  • capillary 1101 is a plurality of parallel capillaries, each of which has an inlet and an outlet for multiple parallel biochip analyses.
  • the components indicated by other marks are the same as those of Fig. 10.
  • a syringe 1205 is connected to the outlet of the capillary 1201 for driving the flow of liquid within the microchannel.
  • the components indicated by other marks are the same as those of Fig. 10.
  • the microchannel 1403 of the chip 1401 has a disk shape, and the probe array 1408 is preliminarily fixed on the inner surface of the microchannel, and the inlet 1402 is processed into a sharp needle shape and inserted into the liquid storage cuvette B.
  • the liquid storage small tube B is arranged with a sample solution 1405, a washing buffer 1406 and a denaturant 1407, respectively.
  • the outlet is connected to a horizontal reservoir 1404.
  • the gravity is used to drive the liquid flow.
  • the sample flows through the entire channel and a hybridization reaction occurs. After the reaction is completed, the channel 1409 is cleaned and detected, and then the denaturing agent flows through the channel to the hybridized sample. Denatured, the sample molecules are detached from the probe and cleaned, and the probe array is restored. This process is repeated to complete a high throughput analysis of the sample.
  • Figure 19 is a bar graph showing the signal intensity obtained by hybridization of two different samples into a microchannel, wherein the probe arrays were each obtained by fixed reaction on the inner surface of the channel using different concentrations of probe solution.
  • the microchannel 1503 of the chip 1501 is one or more parallel channels, each of which is machined with an inlet and an outlet for performing multiple parallel biochip analyses.
  • the components indicated by other marks are the same as those of Fig. 14.
  • Microchannel 1603 is helical for biochip analysis.
  • the components indicated by other marks are the same as those of Fig. 14.
  • the microchannel inlet is replaced by a small section of quartz capillary.
  • a small hole is drilled with a drill bit, a small piece of quartz capillary is inserted and fixed with glue as the channel inlet.
  • the components indicated by other marks are the same as those of Fig. 14.
  • a syringe 1804 is connected to the outlet of the microchannel for driving the flow of liquid in the microchannel.
  • the components indicated by other marks are the same as those of Fig. 14.
  • capillary 2001 is a plurality of parallel capillaries, each having an inlet and an outlet, forming a three-dimensional array that can process multiple parallel probe arrays for multiple parallel biochip analyses.
  • the image on the right is a cross-sectional view along the AA' direction in the left image.
  • the components indicated by the respective marks are the same as those of Fig. 1.
  • the microchannel 2103 of the chip 2101 is one or more parallel channels, each of which is processed with an inlet and an outlet, and a plurality of microfluidic chips are stacked to form a three-dimensional array, which can be used for multiple parallels.
  • the image on the right is a cross-sectional view along the AA' direction in the left image.
  • the other reference numerals are the same as those of Fig. 4.
  • the capillary 2201 is a plurality of parallel capillaries, each of which has an inlet and an outlet, and an array is formed and then erected to form a stereoscopic array, and a plurality of parallel probe arrays can be processed for a plurality of parallel biochips. analysis.
  • the components indicated by the respective marks are the same as those of Fig. 1.
  • the microchannel 2303 of the chip 2301 is one or more parallel channels, each of which is processed with an inlet and an outlet, and a plurality of microfluidic chips are superimposed and erected to form a stereoscopic array, and multiple parallel processing is performed.
  • the array is used for multiple parallel biochip analyses.
  • the components indicated by other marks are the same as those of Fig. 4.

Description

包含多根微通道的生物芯片
相关申请
本申请主张如下优先权： 中国发明专利申请： 201010193906. 1, 题为 "一种用于 生物芯片分析的毛细管探针阵列的制备方法" ； 201010193890. 4， "一种用于生物芯 片分析的微流控芯片探针阵列的制备方法" ； 201010193922. 0， "生物芯片高通量杂 交的方法"， 都于 2010年 6月 1日提交。 技术领域
本发明涉及生物芯片领域，特别是涉及一种生物芯片以及一种生物芯片高通量杂 交的方法。
背景技术
生物芯片是将大量的生物分子或材料（如核酸片段、 蛋白质、 药物或受体、 细胞 或组织等）按照预先设计的排列方式固定在载体表面， 以此作为探针， 与样品中待测 物进行特异性的吸附或反应， 实现对样品信息的检测。成千上万的反应在一块芯片上 同时进行， 具有大规模并行分析的能力。 生物芯片一般加工在玻璃片、 硅片、 尼龙膜 等载体材料上， 探针阵列的制作方法主要有点样法 （Schena M, Shalon D, Davis R W. et al. Science. , 1995, 20 : 467- 470)和原位合成法（Fodor S P A, Read J L, Pirrung M C, et al. Science, 1991, 251 : 767-773)。 将样品覆盖在探针阵列表面进行杂交 反应， 然后进行检测 （范金坪， 中国医学物理杂志， 2009， 26： 1115-1117 )。 生物芯 片的制作方法中的点样法是用点样仪将探针点在载体表面再进行固定反应从而将探 针牢固结合在载体表面， 原位合成主要用于寡聚核苷酸， .利用多步反应， 依次将脱氧 核苷酸单体连接在探针尾部， 实现在载体表面的延伸。 这两种加工方法技术要求高、 加工成本高、制作速度慢， 都需要比较昂贵的精密仪器。 而且生物芯片需要比较长的 杂交反应时间， 一般要几个小时到十几个小时， 一块芯片只用于一次杂交反应， 很难 实现多样品的高通量低成本的快速检测。
毛细管成本低廉， 用来进行生物芯片分析能够降低加工难度， 有效减少成本。而 且常规生物芯片的杂交过程受扩散过程的控制， 一般杂交反应需要十几个小时， 而在 毛细管类似尺寸的微通道内进行杂交反应由于流动杂交过程促进混合而且扩散距离
确 认 本 短， 能够缩短反应时间、 增强检测信号、 提高检测灵敏度 （Benn J A, Hu J, Hogan B J, et al. Anal. Biochem. , 2006, 348 : 284-293)。 授权公告号为 CN2483395Y的实 用新型专利公开一种内设有毛细纤维丝（条）的透明毛细管作为毛细管生物芯片装置， 该装置将点样线、 相应标记物、 阳性和阴性对照线制作在毛细纤维丝 （条）上， 然后 插在毛细管内，分析时通过毛细管的毛细作用将样品吸入到毛细管内并浸渍毛细纤维 丝 （条）， 样品中的待测物与毛细纤维丝 （条） 上的点样物和标记物发生杂交反应。 这一方法， 点样物和标记物是制作在毛细管内的毛细纤维丝（条）上， 而不是毛细管 的管壁上。探针数目有限，而且一般只是用于一次杂交反应，即只用于检测一个样品。 如要检测多个样品， 需要加工多根毛细管芯片， 降低了分析效率。
微流控芯片是利用各种微加工技术在芯片材料（如玻璃、 PDMS或 PMMA等其他材 料）上加工出具有各种功能的微结构， 实现反应、 分离、 检测等功能， 最大限度地把 实验室的功能集成到便携的芯片上。自 20世纪 90年代初 Manz和 Widmer等（Manz A, Graber N, Widemer H M. Sens. Acturators, B, 1990, Bl : 244 ) 首次提出以来， 微流控芯片技术得到了快速发展， 已经从单纯的化学分析扩展到各个方向， 包括核酸 分析、 蛋白质分析和细胞分析等。在微流控芯片上进行生物芯片分析， 能够有效增强 检测信号、 提高灵敏度、 减少试剂和样品消耗、 缩短分析时间等（Chen H, Wang L, Li P C H. Lab Chip, 2008, 8 : 826-829 )。 已有报道在微流控芯片上进行核酸杂交分析 (Wang L, Li P C H. J. Agric. Food Chera. , 2007, 55 : 10509-10516)， 将杂交反 应时间缩短到 5min以内，但一般还只是用于一次杂交反应， 即只用于检测一个样品。 如要检测多个样品， 需要加工多块芯片， 极大地降低了分析效率。 而且由于探针不耐 高温， 微流控芯片都无法进行高温键合， 给芯片制作、 实验操作、 系统集成和开发新 应用带来了极大的困难； 另一方面需手工对准微通道和探针阵列，探针阵列中必须出 现大量的重复探针， 以确保所需探针被包含在微通道内，探针的数目和密度受到极大 限制， 无法有效利用载体的表面。
发明内容
本发明的目的在于针对上述方法存在的缺点，提供一种生物芯片高通量杂交的方 法。
本发明包括以下步骤-
1 ) 将探针溶液以载流中的间隔液滴的形式引入到微通道中； 2) 在探针液滴序列在微通道中流动到预定位置侯， 停止流动， 液滴中的探针会 自发或者在光、 电、磁外界引发下通过反应或吸附而被固定到微通道内表面， 固定反 应完成后， 排出残余探针液滴和载流， 然后用缓冲液进行清洗以完成整个步骤；
3) 将样品溶液引入到微通道内；
4) 样品溶液进入微通道后， 样品分子会由于序列互补或亲和作用而与特定的探 针产生特异性的结合而被保留在特定探针所在区域，根据探针的信息获得样品分子的 相应信息；
5) 样品分子已经条形码标记、 荧光标记、 量子点标记、 光子晶体标记、 拉曼标 签标记、 红外光谱标记、 电化学标记等中的至少一种进行标记， 判断样品分子是否被 保留在探针区域以及保留所在位置等信息；
6) 检测完成后， 利用变性技术将已结合在探针上的样品分子从探针上洗脱下来 并清洗干净， 恢复探针阵列；
7) 恢复探针阵列后， 加入另一种样品， 进行杂交反应和检测， 重复这一过程， 实现生物芯片高通量杂交。
在步骤 1 ) 中， 所述将溶液引入到微通道内， 可将微通道的入口插入到储液池或 小试管中将溶液通过入口引入到微通道内； 所述微通道可为毛细管或微流控芯片等， 所述毛细管可为玻璃毛细管、石英毛细管或高聚物毛细管等， 所述毛细管的管径可为 Inn！〜 5cm; 所述毛细管设有至少 1根， 多根毛细管可以并联或串联； 所述微流控芯片 可为玻璃微流控芯片、 石英微流控芯片、硅微流控芯片或高聚物微流控芯片等； 所述 微流控芯片的通道尺寸可为 lnm〜5cm; 所述微通道可以是不同的形状， 如直形通道， 也可以是盘管形通道， 也可'以是矩形通道， 也可以是螺旋形通道； 所述通道可以是单 根通道， 也可以是多根平行通道。
在步骤 1 )、 2 )、 3 )、 4 )中， 所述溶液进入微通道后， 样品溶液在微通道内流动， 所述流动的驱动力可由电场、 毛细作用、 表面张力或者由连接在通道出口的注射泵、 重力等提供。
在步骤 5) 中， 所述判断样品分子是否被保留在探针区域以及保留所在位置等信 息，可利用相应方法对这些标记进行检测， 从而判断样品分子是否被保留在探针区域 以及保留所在位置等信息。
在步骤 6) 中， 所述变性包括热变性、 极端 pH值变性、 离子强度变性、 变性剂 变性等。
所述探针和样品可以是核酸 \小分子化合物、 多肽、 蛋白质、 抗原、 多糖、 配体、 药物、 受体、 细胞或组织等能产生亲和作用的生物、 有机物质或无机物质。
本发明的优点在于： 在试剂消耗少的情况下， 能简单有效地在封闭的微通道加工 探针阵列， 制作各种不同密度的探针阵列； 在封闭的微通道内进行核酸杂交反应， 对 样品进行快速分析； 然后利用变性技术，在毛细管或同一块芯片上进行多个样品的高 通量分析； 降低对昂贵仪器的依赖， 降低加工和分析成本， 加快制作和分析速度， 进 行多样品检测， 实现低成本高通量的生物芯片分析。
附图说明
图 1是本发明的在毛细管内壁上加工探针阵列的加工示意图。
图 2是本发明同时对多根毛细管加工探针阵列的示意图。
图 3是本发明采用注射器作为驱动力时的示意图。
图 4是本发明的在微流控芯片上加工探针阵列的示意图。
图 5是本发明的对多根平行通道同时加工探针阵列的示意图。
图 6是本发明的具有螺旋通道的微流控芯片上加工探针阵列的示意图。
图 7是本发明的采用毛细管入口的微流控芯片上加工探针阵列的示意图。
图 8是本发明的采用注射器作为驱动力的探针阵列加工示意图。
图 9是本发明的己固定的探针阵列荧光信号强度的柱状图， 在图 6中，横坐标为 探针浓度 （μ πι)， 纵坐标为荧光信号强度。
图 10是根据本发明的与固定有探针阵列的毛细管进行杂交反应的示意图。
图 11是根据本发明的与多根平行毛细管同时进行杂交反应的示意图。
图 12是根据本发明的采用注射器作为驱动力的杂交反应的示意图。
图 13是根据本发明的两个不同样品与探针阵列杂交的荧光信号强度的柱状图， 在图 13中， 横坐标为样品溶液浓度（ηΜ)， 纵坐标为荧光信号强度； 其中 a为 CLN1 '， b为 CLN5，。
图 14 是根据本发明的与固定有探针阵列的微流控芯片上进行杂交反应的示意 图。
图 15是根据本发明的在多根平行通道同时进行杂交反应的示意图。
图 16是根据本发明的在螺旋通道的微流控芯片上进行杂交反应的示意图。 图 17是根据本发明的采用毛细管作为入口的微流控芯片上进行杂交反应的示意 图。
图 18是根据本发明的采用注射器作为驱动力的杂交反应的示意图。
图 19是根据本发明的两个不同样品与探针阵列杂交的荧光信号强度的柱状图， 在图 19中， 横坐标为探针浓度 （μ Μ)， 纵坐标为荧光信号强度； 其中 a为 ALN1， b 为 ALN5。
图 20是根据本发明将多根毛细管组成一个三维阵列， 进行探针阵列加工的示意 图。
图 21是根据本发明将多块加工有多根平行微通道的微流控芯片叠加起来组成一 个三维阵列， 进行探针阵列加工的示意图。
图 22是根据本发明将毛细管阵列竖立起来组成一个立体阵列， 可用来进行探针 阵列的固定和样品杂交。
图 23是根据本发明将叠加在一起的加工有多根平行微通道的微流控芯片竖立起 来组成一个立体， 可用来进行探针阵列的固定和样品杂交。
具体实施方式
以下实施例将结合附图对本发明作进一步的说明。
实施例 1
参见图 1 , 毛细管 101的入口插入到储液小试管 A中， 储液小试管 A间隔排列分 别装有矿物油 102和探针溶液 103。 毛细管 101出口与一水平储液池 104相连， 利用 重力驱动液体流动， 在毛细管 101内实现载流 105携带液滴序列 106流动的形式。流 动到预定位置后， 停止流动并进行探针的固定反应以完成生物芯片的加工。
实施例 2
参见图 2， 毛细管 201为多根平行毛细管， 每根通道均有一个入口和出口， 加工 多个平行的探针阵列用于多个平行的生物芯片分析。在图 2中， 各标记所指部件与图 1的相同。 '
实施例 3
参见图 3, 毛细管 301的出口与一注射器相连， 通过注射器产生的负压驱动液体 的流动。 在图 3中， 各标记所指部件与图 1的相同。
实施例 4 参见图 4， 芯片 401的微通道 403呈盘管状， 其入口 402加工成尖针状， 插入到 储液小试管 A中。 储液小试管 A间隔排列分别装有矿物油 405和探针溶液 406。 微通 道的出口与一水平储液池 404相连， 利用重力驱动液体流动， 在微通道 403内实现矿 物油 407携带探针液滴 408流动的形式。流动到预定位置后， 停止流动并进行探针的 固定反应以完成探针阵列的加工。
图 9是不同浓度的核酸探针固定 30min所得的荧光信号强度的柱状图，探针含有 20个碱基， 3' 标记有 FITC, 5' 标记有氨基， 通过与修饰在通道内表面的游离醛基 发生的化学反应而被固定在通道内。
实施例 5
参见图 5， 芯片 501的微通道 503为一根或多根平行通道， 每根通道均加工有一 个入口和出口， 加工多个平行的探针阵列用于多个平行的生物芯片分析。 在图 5中， 其它标记所指部件与图 4的相同。
实施例 6
参见图 6， 微通道 603呈螺旋形， 用于加工探针阵列。 在图 6中， 其它标记所指 部件与图 4的相同。
实施例 7
参见图 7， 其微通道入口由一小段石英毛细管代替， 加工方法为： 在芯片侧面对 准微通道的位置， 利用钻头钻出一小孔，插入一小段石英毛细管后用胶水固定作为通 道入口。 在图 7中， 其它标记所指部件与图 4的相同。
实施例 8
参见图 8， 其出口连接有一根注射器 804， 注射器产生的负压提供驱动力， 驱动 液体在微通道内流动。 在图 8中， 其它标记所指部件与图 4的相同。
实施例 9
参见图 10， 毛细管 1001的入口插入到储液小试管 A中， 储液小试管 A间隔排列 分别装有样品溶液 1002、 清洗缓冲液 1003和变性剂 1004。 毛细管 1001的出口与一 水平储液池 1005相连， 利用重力驱动液体流动。 探针阵列已经预先固定在毛细管内 壁上 1007， 让样品流经整个通道并发生杂交反应， 反应完成后清洗通道 1006并进行 检测，然后再让变性剂流经通道对己杂交样品进行变性， 样品分子脱离探针并被清洗 干净， 恢复探针阵列。 重复这一过程完成样品的高通量分析。 图 13是两种不同浓度的样品溶液加入到毛细管内进行杂交反应得到的信号强度 的柱状图。
实施例 10
参见图 11， 毛细管 1101为多根平行毛细管， 每根毛细管均有一个入口和出口进 行， 多个平行的生物芯片分析。 在图 11中， 其它标记所指部件与图 10的相同。
实施例 11
参见图 12， 毛细管 1201的出口连接有一根注射器 1205， 用于驱动液体在微通道 内的流动。 在图 12中， 其它标记所指部件与图 10的相同。
实施例 12
参见图 14， 芯片 1401的微通道 1403呈盘管状， 探针阵列 1408预先固定在微通 道内表面， 其入口 1402加工呈尖针状， 插入到储液小试管 B中。 储液小试管 B间隔 排列分别装有样品溶液 1405、清洗缓冲液 1406和变性剂 1407。 出口与一水平储液池 1404相连， 利用重力驱动液体流动， 首先让样品流经整个通道并发生杂交反应， 反 应完成后清洗通道 1409并进行检测， 然后再让变性剂流经通道对已杂交样品进行变 性， 样品分子脱离探针并被清洗干净， 恢复探针阵列。重复这一过程完成样品的高通 量分析。
图 19是两种不同样品加入到微通道内进行杂交反应得到的信号强度的柱状图， 其中探针阵列分别是使用不同浓度的探针溶液在通道内表面进行固定反应得到。
实施例 13
参见图 15， 芯片 1501的微通道 1503为一根或多根平行通道， 每根通道均加工 有一个入口和出口， 进行多个平行的生物芯片分析。 在图 15中， 其它标记所指部件 与图 14的相同。
实施例 14
参见图 16， 微通道 1603呈螺旋形， 用于生物芯片分析。 在图 16中， 其它标记 所指部件与图 14的相同。
实施例 15
参见图 17， 其微通道入口由一小段石英毛细管代替， 在芯片侧面对准微通道的 位置， 利用钻头钻一小孔， 插入一小段石英毛细管并用胶水固定作为通道入口。在图 17中， 其它标记所指部件与图 14的相同。 实施例 16
参见图 18， 其微通道出口处连接有一根注射器 1804， 用于驱动液体在微通道内 的流动。 在图 18中， 其它标记所指部件与图 14的相同。
实施例 17
参见图 20， 毛细管 2001为多根平行毛细管， 每根通道均有一个入口和出口， 组 成一个三维阵列， 可加工多个平行的探针阵列用于多个平行的生物芯片分析。右图为 左图中沿 AA' 方向的截视图。 在图 20中， 各标记所指部件与图 1的相同。
实施例 18
参见图 21， 芯片 2101的微通道 2103为一根或多根平行通道， 每根通道均加工 有一个入口和出口，将多块微流控芯片叠加起来组成一个三维的阵列， 可用于多个平 行的生物芯片分析。 右图为左图中沿 AA' 方向的截视图。 在图 21中， 其它标记所指 部件与图 4的相同。
实施例 19
参见图 22， 毛细管 2201为多根平行毛细管， 每根通道均有一个入口和出口， 组 成一个阵列后竖立起来形成一个立体阵列，可加工多个平行的探针阵列用于多个平行 的生物芯片分析。 在图 22中， 各标记所指部件与图 1的相同。
实施例 20
参见图 23， 芯片 2301的微通道 2303为一根或多根平行通道， 每根通道均加工 有一个入口和出口， 将多块微流控芯片叠加后竖立起来形成一个立体阵列， 加工多个 平行的阵列用于多个平行的生物芯片分析。 在图 23中， 其它标记所指部件与图 4的 相同。

Claims

权 利 要 求
1. 一种微通道阵列， 包括：
1 ) 至少一根微通道；
2) 多个探针分布在微通道的内壁上组成一维的探针阵列；
3 ) 一维阵列上的探针被设计为会与样品进行反应。
2. 如权利要求 1所述的阵列， 其特征在于多根所述微通道组成至少二维的微通 道阵列。
3. 如权利要求 2所述的阵列， 其特征在于所述至少二维的微通道阵列为三维微 通道阵列， 包括所述多根微通道垂直于一个基底。
4. 如权利要求 3所述的阵列， 其特征在于所述样品在其中至少一个微通道中排 为液滴序列。
5. 如权利要求 4所述的阵列， 其特征在于所述液滴序列包括不同种类的样品， 所述其中至少一个微通道中包括不同的探针，该液滴序列的液滴和不同的探针在微通 道内实现一一对应。
6. 如权利要求 1所述的阵列， 其特征在于微通道是微流控的微通道或者毛细管 的微通道。
7. 如权利要求 1所述的阵列， 其特征在于所述探针和样品是核酸、 寡聚核苷酸、 蛋白质、 多肽、 肽核酸、 多糖、 抗原、 抗体、 配体、 受体、 药物、 细胞、 细胞器、 细 胞片断、 组织、 小分子或嵌合分子等能产生亲和作用或化学反应的生物、有机或无机 物质。
8. 如权利要求 1所述的阵列， 其特征在于微通道是由玻璃、 高聚物或者半导体 材料制得。
9. 一种生物芯片， 包括-
1 ) 多根微通道；
2) 多个探针分布在每根微通道的内壁上分别组成一维的探针阵列；
3) 一维阵列上的探针被设计为会与样品进行反应；
4) 分别分布有一维通道阵列的多根微通道组成二维、 三维或者多维的微通道阵 列。
10. 如权利要求 9所述的生物芯片，其特征在于微通道是微流控芯片的微通道或 者毛细管的微通道。
11. 如权利要求 9所述的生物芯片， 其特征在于所述探针和样品是核酸、寡聚核 苷酸、 蛋白质、 多肽、 肽核酸、 多糖、 抗原、 抗体、 配体、 受体、 药物、 细胞、 细胞 器、 细胞片断、 组织、 小分子或嵌合分子等能产生亲和作用或化学反应的生物、 有机 或无机物质。
12. 如权利要求 9所述的生物芯片， 其特征在于微通道是由玻璃、 高聚物或者半 导体材料制得。
13. 一种生物芯片高通量杂交的方法， 其特征在于包括以下步骤：
(A) 从微通道的入口引入探针液滴的阵列；
(B) 探针固定到微通道的内壁上， 形成一维的探针阵列；
(C) 使样品流经微通道并与探针阵列发生杂交反应。
14. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于探针完成固 定后对微通道进行清洗和干燥。
15. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于样品会与相 应的探针发生杂交反应。
16. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于样品完成检 测后进一步通过变性方法去除已杂交的样品以恢复探针阵列。
17. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于在恢复探针 阵列后进一步将另一种样品加入到微通道中与变性后的探针阵列进行杂交反应。
18. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于通过毛细作 用或者泵引入探针和样品。
19. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于引入的探针 液滴之间由不互溶的溶剂所间隔。
20. 如权利要求 13所述的生物芯片高通量杂交的方法， 其特征在于不互溶的溶 剂是有机溶剂或者离子液。
PCT/CN2011/000918 2010-06-01 2011-05-31 包含多根微通道的生物芯片 WO2011150675A1 (zh)

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