WO2004072642A1 - Dispositif d'application de micro-echantillon et reaction d'une puce a adn et procede correspondant - Google Patents

Dispositif d'application de micro-echantillon et reaction d'une puce a adn et procede correspondant Download PDF

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Publication number
WO2004072642A1
WO2004072642A1 PCT/CN2004/000074 CN2004000074W WO2004072642A1 WO 2004072642 A1 WO2004072642 A1 WO 2004072642A1 CN 2004000074 W CN2004000074 W CN 2004000074W WO 2004072642 A1 WO2004072642 A1 WO 2004072642A1
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WO
WIPO (PCT)
Prior art keywords
chip
micro
template
reaction
grooves
Prior art date
Application number
PCT/CN2004/000074
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English (en)
Chinese (zh)
Inventor
Zhanhui Wang
Gang Jin
Original Assignee
Zhanhui Wang
Gang Jin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhanhui Wang, Gang Jin filed Critical Zhanhui Wang
Publication of WO2004072642A1 publication Critical patent/WO2004072642A1/fr

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Classifications

    • 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
    • B01L3/02—Burettes; Pipettes
    • B01L3/0289—Apparatus for withdrawing or distributing predetermined quantities of fluid
    • B01L3/0293—Apparatus for withdrawing or distributing predetermined quantities of fluid for liquids
    • 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
    • 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
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics

Definitions

  • the invention relates to a method and a device for preparing a biomolecule chip, and in particular, to a device and method for directly performing micro-sample addition on a biomolecule chip, and directly performing a biomolecule fixing and detection reaction on the biochip. Background technique
  • Biomolecule chip is an integrated parallel biological detection technology developed in recent years. It can integrate a variety of ligands on a small geometric scale, so that it can detect multiple indicators of a small amount of samples at the same time. Due to the high price of biomolecule samples and the minimum amount required, it is required to miniaturize the biomolecule reagents and samples used in biomolecule chips, which also requires chip loading and miniaturization of the reaction device. At present, the main application of the biochip is the spotting instrument.
  • one is contact type, first use the spotting needle to dip the ligand to be used, and then touch the chip surface to place the ligand on the chip;
  • one type is spray printing, first Use a hollow spotting needle to suck a small amount of ligand to be spotted, and then add the ligand to the surface of the chip in a manner similar to an inkjet printer.
  • the common disadvantages of these two methods are the uneven spotting amount and the uneven distribution of the areal density of the ligand molecules in a single spot. This will seriously affect the quality of the test results and is difficult to quantify.
  • most of the biochip reactions use the overall reaction method, that is, the entire chip is immersed in the sample solution to be tested. This method requires a large amount of sample solution, a long reaction time, and low sensitivity.
  • microchannel technology Another chip loading and reaction technology is the use of microchannel technology.
  • chips commonly used in microchannel technology are integrated, that is, the chip and microchannel are made on the same material, such as reference 1 : Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays.
  • reference 1 Dielectrophoretic cell separation and gene expression profiling on microelectronic chip arrays.
  • the microfluidic channel used in this method is a one-time use.
  • the microfluidic channel is complicated to make and the cost is high, which limits the application. Summary of the invention
  • the purpose of the present invention is to overcome the shortcomings of the prior art described above, to greatly reduce the manufacturing cost of biochips and to simplify the process of biomolecule immobilization and detection reactions, so as to provide a set of biochip fabrication and Integrated device and method for directly carrying out biomolecule immobilization and detection reactions on a chip.
  • the object of the present invention is achieved as follows:
  • the device for micro-sample addition and reaction of a biomolecule chip provided by the present invention includes:
  • At least one microchannel template 3 has grooves 2 arranged in an array on the surface, and two openings 10 are formed at both ends of each groove 2 as liquid inlets 7 and 8 respectively; the microchannel template 3 has grooves The surface of 2 is buckled on the chip 1, and a cavity 9 is formed between the groove 2 and the chip 1.
  • the liquid inlet and outlet and the cavity 9 form a microchannel 5 in and out (as shown in FIG. 2a).
  • the liquid inlet 7 and outlet 8 of the device can also be installed with a microtube 12, and the microtube 12 is in communication with the micro pump.
  • a micro-channel template 3 ' is further provided, and a through-hole is also formed on the micro-channel template 3'.
  • the position and number of the through-holes are set as required.
  • the surface of the microchannel template 3 'with the groove 2 is buckled on the microchannel template 3, and its through holes communicate with the liquid inlet 7 and the liquid outlet 8 on the microchannel template 3, respectively.
  • the groove 2 communicates the remaining liquid inlet 7 and the liquid outlet 8 on the micro-channel template 3 to form a micro-channel 5 in and out (as shown in FIG. 2b).
  • a micro-tube 12 and a micro-tube 12 can be respectively connected to the liquid inlet 7 and the outlet 8 of the micro-channel template 3 '.
  • the through hole 15 communicates with the through hole 10 of the micro channel template 3, and the hole size is the same ( (As shown in Fig. 3a);
  • Another piece of rigid material 4 ' is installed under the chip 1, and is clamped together with a common clamp as a whole, or at least 2 holes are opened in each piece of rigid material 4, and a magnet is placed in the hole.
  • the magnets embedded on two rigid material blocks are of opposite polarity. Under the action of magnetic force, the groove 2 on the microchannel template 3 and the surface of the smooth chip 17 are sandwiched to form a cavity. 9, forming a microchannel 5 in and out.
  • a micro tube 12 can also be installed in the through hole 15 of the rigid material block, and the micro tube 12 is in communication with the micro pump (as shown in Fig. 4a).
  • the above device may further include a third rigid material block 4 ", which has an inlet 19 and an outlet 20 thereon.
  • the position and number of the inlet and outlet are set as required.
  • it is up that is, two through holes are opened in the rigid material block 4 ", one through hole corresponds to the liquid inlet 7 on the microchannel template 3, and the other through hole corresponds to the liquid outlet 8 of the last groove 2;
  • the second The grooved surface of the microchannel template 3 ' covers the first rigid material block 4, and the liquid inlet 7 on the microchannel template 3 is connected to the liquid outlet 8 on the microchannel template 3, and the third A rigid material block 4 "covers the second microfluidic template 3 '.
  • a through hole 10 in the microfluidic template 3 communicates with the inlet 19 on the rigid material block; the other outlet 20 communicates with the microfluidic template 3
  • the liquid outlet 8 of the last groove is connected; the inlet 20 and the outlet 21 of the rigid material block 4 "are installed with microtubes 12 for sample addition to form a flow channel to realize the communication of all the flow channels (as shown in Figure 4b) As shown).
  • the micro pump sends the solution to be tested into the first groove through the through hole 7, and then the solution to be tested will sequentially flow through all the grooves connected in series (ie, the microchannel 5). Outflow.
  • liquid inlet 7 and the liquid outlet 8 are provided at the positions corresponding to the through holes of the first and third grooves of the rigid material block 4 ".
  • a microtube 12 is installed to form three grooved serial flow channels.
  • the method for manufacturing the integrated biochip provided by the present invention and the method for directly and immediately carrying out the biomolecule fixation and detection reaction on the manufactured biochip include: using the device of the present invention (as shown in FIG. 4)
  • test solutions can be delivered to different grooves and fixed by pumps.
  • the biomolecules are reacted, and then washed with a buffer solution after the reaction;
  • Another device for micromolecular sampling and reaction of a biomolecule chip of the present invention includes:
  • a rigid material block 4 a microchannel template 3 and a microchannel template 33.
  • the micro-channel template 33 (shown in FIG. 6) is provided with grooves 14, and the pitch of the grooves 14 is equal to the length of the groove 2 on the micro-channel template 3.
  • One end of each of the channels 14 corresponds to a recess.
  • the other hole 6 of the through hole 10 of the groove 2 is extended to the edge; a horizontal groove 11 is also connected between the two grooves 14 corresponding to each groove 2; and the microchannel templates 3 and 33 are bonded together
  • the grooves and grooves face outward, and the holes communicate; the rigid material block 4 is covered on the grooves of the micro-channel template 33, and the four sides are sealed and bonded together, and the chip 1 is covered on the grooves of the micro-channel template 3
  • a closed microfluidic channel is formed between the microfluidic template and the rigid material block on the surface of the groove 2 (as shown in FIG. 5). In this way, each groove on the microchannel template 3 is connected to two closed microchannels.
  • Each microchannel has a tactile switch 16 and
  • the method for manufacturing the integrated biochip provided by the present invention and directly performing the biomolecule immobilization and detection reaction on the produced biochip, using the device of the present invention includes:
  • step (3) Rinse the biomolecule-immobilized chip substrate prepared in step (2) with a buffer solution, and transfer the buffer solution to different areas on the chip surface through a microfluidic channel; wash away the components not fixed on the chip surface Base molecule
  • the micro-channel template includes: made of silicone, rubber or other elastic plastic materials.
  • the rigid material block is made of plastic, metal, plexiglass and other materials, and has a thickness of 1 mm to 10 dishes.
  • the strip-shaped groove is a groove, which groove strip area from 0.01 2 bandit bandit 2-1; bandit depth from ⁇ - 1; the number of which at least two strip-shaped grooves, for example, from 1 -500.
  • the inner diameter of the through hole 10 can be from 10 ⁇ m to 1 leg.
  • the inner diameter of the microchannel can be from 10 ⁇ m to 1 ⁇ m.
  • the inner diameter of the groove can be from 10 ⁇ m to 1 band.
  • the chip base material includes: silicon, glass, metal, plastic, coated silicon wafer and other materials or the above-mentioned composite materials, preferably silicon.
  • the advantage of the present invention is that the method provided by the present invention is to use a plurality of tiny grooves arranged in an array on a sheet of elastic material (such as silicone), fasten these grooves to the chip, and select a solution input port and The output port, and a microtube is installed on the solution input port and the output port to form a plurality of closed cavities.
  • Each cavity has one microchannel and one microchannel, and the biomolecule solution enters the cavity through the microchannel.
  • Contact with the chip surface it is fixed on the chip surface.
  • the proteins fixed in each groove can be individually reacted with the test solution through the microfluidic channel, or all the grooves can be connected in series or grouped in series with one or more of the to-be-measured through the microfluidic plug or switch.
  • the solution was reacted. Makes the use of the chip very flexible and convenient. It is more worth mentioning that the device made by this method can be reused after being washed clean.
  • the method of the present invention can simultaneously and independently perform multi-point sampling on a chip; the amount of sampling and the size of the points can be controlled.
  • the area on the chip where the ligand biomolecules are fixed is strictly defined by the groove.
  • the surface after fixing and reaction is washed with buffer solution, so that the size of the dots on the chip and the density of the biomolecules within the points are consistent, Effectively improve detection quality.
  • the chip reaction is limited to a small area, and in a flowing state, the mass transfer rate of biomolecules is accelerated, the reaction time is effectively shortened, and the sensitivity is improved.
  • Arbitrary multiple points can be connected in series, making the use of the chip more flexible.
  • trace The sample can react with all points, saving the amount of solution to be tested.
  • the method of the invention enables the preparation, reaction, and detection of the biochip to be implemented in the same device, and can also be reused, which effectively reduces the preparation cost of the chip.
  • FIG. La is a schematic plan view of a microchannel template structure with microchannels in the device of the present invention
  • Figure lb is a cross-sectional view taken along AA 'of Figure la
  • Figure 2a is a schematic cross-sectional view of the first composition of the device of the present invention
  • Figure 2b is a schematic cross-sectional view of the second composition of the device of the present invention.
  • Figure 3a is a schematic plan view of the rigid material block structure of the present invention
  • Figure 3b is a cross-sectional view taken along AA 'of Figure 3a
  • Figure 4a is a schematic cross-sectional view of the third composition in the device of the present invention (representing the structure of the device when the sample is fixed or the detection reaction is performed at each point separately)
  • 4b is a schematic cross-sectional view of a fourth composition in the device of the present invention (representing a structural diagram when directly preparing a biomolecule chip, the through holes at the two ends of the groove of the microchannel template are staggered with the through holes of the rigid material block to form a micro Flow channel cooperation diagram)
  • FIG. 5 is a schematic structural diagram of another microfluidic device according to the present invention.
  • Figure 6 is a top view of the microchannel template shown in Figure 5
  • FIGS. 1a and 1b Take a piece of silicone sheet as the micro-channel template 3, which has twelve grooves 2 formed on the silicone sheet, and the groove area is lrnrn 2 5mm ⁇ , a depth of 0. 1mm; the inner diameter of the through hole 10 at both ends of the groove 2 is 0. 5mm.
  • the rigid material block 4 is plexiglass, which is opened with a through hole 15 having a pore diameter of 0.5mra, which The position of the through hole 15 corresponds to the through hole 10 of the microchannel template 3; and a hole with an inner diameter of 5 mm is opened on each side of the rigid material block 4, and a magnetic block 13 is placed in the hole.
  • the microtube 12 is a polytetrafluoroethylene tube.
  • the material of the chip 1 is silicon.
  • the surface of the twelve grooves 2 formed on the above-mentioned silicone sheet 3 is buckled on a silicon chip 1 to form a microchannel 5 having a liquid inlet 7 and a liquid outlet 8; the liquid inlet 7 and a A PTFE microtube 12 can also be installed in the liquid outlet 8, and the microtube 12 is in communication with a micro pump.
  • a micro-flow channel template 3 ' is further included.
  • the face of the flow channel template 3' with the groove 2 is buckled on the micro-flow channel template 3, and the liquid inlet 7 and the micro-flow of the micro-flow channel template 3 '
  • the channel template 3 communicates with the liquid outlet 8 to form a microfluidic channel 5 in and out;
  • a microtube 12 can also be installed in the through hole, and the microtube 12 is in communication with the micro pump.
  • the rigid material block is a metal material and has a thickness of 2 mm.
  • One of them has a plurality of through holes 15 and is mounted on the micro-channel template 3'.
  • the through-hole 15 communicates with the through-hole 10 of the micro-channel template 3 ', and the hole size is the same;
  • another rigid material block 4' is installed under the chip 1, and is clamped as a whole with a common clamp, or each block
  • the rigid material block 4 is provided with at least two holes, and a magnetic block 13 is placed in the hole; the magnets embedded in the two rigid material blocks have opposite polarities, and the concave of the micro-channel template 3 is under the action of the magnetic force.
  • the slot 2 is sandwiched between the surface of the smooth chip 1 to form a cavity 9, which forms a microchannel 5 in and out; a microtube 12 can also be installed in the through hole, and the microtube 12 is in communication with the micro pump .
  • the microtube 12 is first removed from the microchannel opening, and then a microchannel template 3 'and a rigid material block 4 "are set on the rigid material block 4". Only one liquid input hole 7 and output hole 8 are opened, and then a microchannel template 3 'with a groove 2 is placed on the rigid material block 4, with the groove 2 facing downward, and the microchannel template 3' on the microchannel template 3 '
  • the liquid inlet 7 is installed through the liquid outlet 8 of the micro-channel template 3 and communicates with the inlet 19 and the outlet 20 on the rigid material block 4 "to form a micro-channel 5.
  • each sealed cavity has one inlet and one inlet.
  • the silicon wafer 1 is installed under the groove 2 of the micro-channel template 3;
  • the phosphate buffer solution is delivered to the surface of the chip 1 prepared in the above step (2) through the microchannel, and the biomolecules not fixed on the surface of the silicon wafer are washed away; It is fixed on the surface of the chip. After washing with buffer solution, the DNA not fixed on the chip surface is discharged.
  • the solution to be tested enters the micro-channel from the inlet through the pump, flows through all the grooves, and is fixed on the chip surface.
  • the DNA is subjected to a hybridization reaction, and finally discharged from the outlet, and then washed with a buffer solution. After the reaction, the chip is removed from the device for detection. The device can be used for the next DNA fixation and reaction.
  • the silicon wafer When preparing a gene chip, under the action of external pressure, the silicon wafer is in close contact with the microarray template, so that 1000 independent sealed cavities are formed between the silicon wafer and the groove, and each cavity has one Enter and exit two micro-flow channels;
  • test solution Inject 100 microliters of the test solution into the microchannel with a flow rate of 10 microliters / minute.
  • the test solution flows through the 12 grooves in sequence and flows out through the outlet, and then rinsed with phosphate buffer solution (as shown in Figure 4). .
  • a device with 48 grooves is prepared to prepare a 48-point protein chip to react with two solutions to be tested.
  • the structure of the device is the same as the embodiment 1 of the same. 05mm ⁇ The area of each groove is reduced to 0.3 mm 2 and the depth is 0.05 mm. The inner diameter of the microchannel was reduced to 0.3 bandits. Teflon tubing was replaced by stainless steel tubing. Because protein detection results often need to be compared, the 48 grooves in this embodiment are evenly divided into two groups, and 24 grooves in each group are connected in series. The protein fixation procedure was the same as in Example 1. The two solutions to be tested are injected into two sets of grooves connected in series to react with the fixed protein. The results of the reaction are displayed on the same chip and can be easily compared.
  • a device for making 400 grooves is used for fixing and hybridizing reactions of 400 kinds of DNA fragments: a microchannel template 33 (shown in FIGS. 5 and 6) with microchannels and a block 4 of rigid material are produced.
  • the micro-channel template material in this embodiment is rubber, the rigid material block is plexiglass, and the chip base material is gold-plated glass.
  • the microchannel template 33 is provided with a groove 14.
  • the groove is 0.1 legs wide and 0. lmm deep.
  • One end of the groove 14 has a through hole corresponding to the groove on the microchannel template 3, and the other port 6 of the groove 14 is extended to the edge.
  • There is a groove of the same shape between the two grooves corresponding to each groove. 11 are connected.
  • the microchannel template 3 take a rubber sheet with an area of 20mmx20mm as the microchannel template 3, and the microchannel template 3 with 400 grooves 2 engraved on the surface, each strip groove 2 depth 1mm 2 ⁇ It is 0.01mm, the cross-sectional area is 0.1mm2.
  • the 400 pieces are regularly arranged on the rubber sheet 1 in 20 rows, and 20 grooves 2 are arranged in one row. 1mm, 800 ⁇
  • the microfluidic template 3 has the groove 2 facing up, and each end of each of the strip-shaped grooves 2 has a through hole 10, the inner diameter of the through hole 10 is 0. 1mm, a total of 800.
  • the plexiglass block 4 is covered on the grooves of the microchannel template 33, and the periphery of the two is bonded together.
  • a closed microchannel is formed between the microchannel template and the plexiglass block 4, which can correspond to The holes are connected.
  • the micro-channel template 3 and the micro-channel template 33 are bonded together, and the corresponding holes are communicated.
  • each groove in the microchannel template 3 is connected to two closed microchannels.
  • Each microfluidic channel is provided with a pressure switch 16.
  • a horizontal groove 11 is provided between the two microfluidic channels, and a pressure switch 17 is provided thereon. Both of these switches close the microfluidic channel by pressing the elastic material with external force.
  • the chip 1 Under the action of external pressure, the chip 1 is in close contact with the microchannel template 3, so that 400 independent cavities are formed between the chip and the groove. Each cavity has one microchannel and one microchannel.
  • 400 kinds of DNA solutions enter the cavity through the microfluidic channel and contact the surface of the silicon wafer, so that the DNA

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention porte sur un dispositif d'application d'un micro-échantillon et sur une réaction d'une puce à-ADN et sur son procédé d'analyse. Le dispositif comprend un réseau de rainures formé dans un moule à canaux microfluidiques et des trous traversants situés aux deux extrémités de la rainure. Le moule à canaux microfluidiques pourvu de rainures est placé sur une puce et un microtube est inséré dans le trou traversant, formant un canal microfluidique pourvu d'un orifice d'admission et d'un orifice d'évacuation. Le dispositif de l'invention peut être utilisé pour fabriquer des puces à ADN et effectuer des essais et la détection. Le procédé consiste à : faire passer une solution biologique par le canal microfluidique pour qu'elle pénètre dans la cavité, cette solution biologique étant en contact avec la surface de la puce, et immobiliser la molécule biologique sur la surface ; la liaison protéinique dans chaque rainure pouvant réagir individuellement avec la solution détectée dans le canal microfluidique ou pouvant réagir avec une ou plusieurs solution détectées par raccordement des canaux microfluidiques ou des commutations qui réunissent toutes les rainures en série ou un ensemble de rainures. La puce peut donc être utilisée facilement et de manière appropriée.
PCT/CN2004/000074 2003-02-17 2004-01-20 Dispositif d'application de micro-echantillon et reaction d'une puce a adn et procede correspondant WO2004072642A1 (fr)

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CN 03102659 CN1249437C (zh) 2003-02-17 2003-02-17 用于生物分子芯片微量加样和反应的方法及其装置
CN03102659.1 2003-02-17

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CN112899139A (zh) * 2021-01-14 2021-06-04 北京普若博升生物科技有限公司 一种核酸试纸条卡盒及其使用方法和应用

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CN109745934B (zh) * 2019-03-18 2023-11-21 中国人民解放军军事科学院军事医学研究院 一种阵列式合成装置及喷墨合成仪
CN110501491B (zh) * 2019-09-20 2022-07-26 四川微康朴澜医疗科技有限责任公司 可支持芯片倾斜的多通道孵育装置及试样制备设备
CN110501514B (zh) * 2019-09-20 2023-12-22 四川朴澜医疗科技有限公司 自动检测仪及自动检测系统
CN110628887A (zh) * 2019-09-26 2019-12-31 南京溯远基因科技有限公司 生物分子微阵列及其制备方法与应用

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