JP2008281381A - High-density microarray and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microarray producible in large quantities extremely inexpensively, capable of measuring a test material or the like in a short time by using always a trace amount of probe and sample. <P>SOLUTION: In the microarray and its manufacturing method, a microchannel forming body provided with a plurality of parallel line-shaped grooves is stuck on a substrate surface having a probe domain immobilized in the shape of a plurality of parallel lines, and a plurality of parallel line-shaped microchannels for the sample are formed in the linear direction and the rectangular direction of the probe domain by the grooves and the substrate surface. A detection method of the test material using the microarray is also provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  According to the present invention, a micro flow path forming body provided with a plurality of parallel line-shaped grooves is attached on a substrate surface having a probe region fixed in a plurality of parallel lines, and the probe is formed by the grooves and the substrate surface. The present invention relates to a microarray in which a plurality of parallel microchannels for a sample are formed in a direction perpendicular to the linear direction of a region, a manufacturing method thereof, a detection method of a test substance using the microarray, and the like.

Conventionally, various types of microarrays have been used for comprehensive analysis methods of many types of test substances. A typical example is a DNA microarray (DNA chip).

This DNA microarray is roughly classified into so-called Affymetrix type (optical lithography type) and Stanford type (spotting type). The optical lithography type is manufactured by synthesizing an oligonucleotide on a substrate using optical lithography and combinatorial chemistry technology (Patent Document 1). On the other hand, the spotting type is a method in which DNA probes prepared in advance are spotted on a substrate at a high density, and an original DNA microarray can be produced according to the researcher's request (Patent Document 2).

However, the optical lithography type is expensive because it takes time and effort to design and manufacture. In addition, since the spotting type drops a small amount of droplets containing DNA probes on the substrate, the dropped droplets diffuse on the substrate surface, and there is a risk of contamination between adjacent spots. Variations in spot density and uniformity are observed, and the accuracy and reproducibility are not sufficient.

Furthermore, a surface plasmon resonance (SPR) optical biosensor (ProteOn XPR36 system) in which an interaction array (6 × 6 = 36 types) is formed on a sensor chip by a multichannel module has been developed (Non-patent Document 1). This has a ligand channel and an analyte channel crossing at right angles. In measurement using this apparatus, several hundred μL of a measurement sample (analyte) is injected into each ligand channel.

On the other hand, solid phase enzyme immunoassay (ELISA) using a microwell or the like is used for measuring various proteins. In this case, several tens of microliters of sample per sample are required. Therefore, it takes several hours as the total reaction time.

In addition, a test substance microarray (Patent Document 3) and an on-chip bioassay kit (Patent Document) using a flow path formed using a resin such as polydimethylsiloxane (hereinafter abbreviated as “PDMS”). 4) etc. have been developed.

Furthermore, Non-Patent Document 2 describes biotin using a flow chamber module (manufactured by PDMS) having six channels (1.5 mm × 22 mm) provided at intervals of 2 mm on an avidin-coated slide glass. The labeled antibody is flowed and fixed, and then a similar flow chamber module is placed on the glass slide so that the immobilized antibody pattern and the flow channel are at right angles, and the sample is flowed into each flow channel (0.3 mL / In addition, a sandwich immunoassay system is described in which after washing, a fluorescently labeled secondary antibody is allowed to flow through each channel (0.75 mL per channel), and fluorescence is measured using a CCD camera.
US Pat. No. 5,744,305 US Pat. No. 5,807,522 JP 2005-30927 A JP-A-2005-46121 Tsafrir Bravman, et al., Anal. Biochem. 2006, 358, 281-288 Chris A. Rowe, et al., Anal. Chem. 1999, 71, 433-439

  The present invention solves the problems in the microarray technology and the like as described above, and can measure a test substance and the like in a short time using a very small amount of probe and sample, and is extremely inexpensive and can be used in large quantities. An object is to provide a microarray that can be produced.

That is, the present invention relates to the following aspects.
[1] A micro-channel forming body provided with a plurality of parallel line-shaped grooves is attached to a substrate surface having a probe region fixed in a plurality of parallel lines, and the probe is formed by the grooves and the substrate surface. A microarray in which a plurality of parallel linear microchannels for a sample are formed in a direction perpendicular to the linear direction of a region.
[2] A method of manufacturing the above microarray,
(1) By introducing a solution containing a probe into a plurality of parallel line-shaped probe microchannels formed on a substrate, the plurality of probes are arranged at positions corresponding to the microchannels on the substrate. (2) a micro-channel forming body provided with a plurality of parallel linear grooves is attached to the substrate, and the grooves and the substrate surface are perpendicular to the linear direction of the probe region. Forming a plurality of parallel micro-channels for samples in a direction.
[3] A method in which a sample solution is introduced into a plurality of parallel linear sample microchannels formed in the microarray, and the test substance is detected based on a reaction between the immobilized probe and the test substance.

In the microarray of the present invention, since the area on which the probe is immobilized is extremely small, only a very small amount of probe solution is required. For this reason, the immobilization density of the probe becomes uniform, and the measurement accuracy and reproducibility become very excellent. Furthermore, in order to react with the immobilized probe by flowing the sample solution through the microchannel, only a very small amount of sample solution is required. As a result, a very small amount of test substance can be obtained in a very short time. It is possible to measure. Furthermore, since the unit area of the detection (reaction) spot to be formed is very small, the entire probe region can be formed with high density.
If it becomes possible to produce a microarray in a micro area in this way, it becomes possible to fit the entire microarray area in which the reactivity to a plurality of test substances can be detected in one field of view of the microscope. As a result, when detecting binding by a fluorescent signal, etc., a mechanical driving unit such as scanning required for a conventional microarray fluorescence detection device is unnecessary, contributing to high sensitivity and simplification of the detection device. Can do.
Furthermore, quality control when manufacturing the microarray is easy, and mass production can be performed at a very low cost.

[Microarray]
In the microarray of the present invention, the number, width, and length of each line in the probe region fixed in a plurality of parallel lines, and the interval between adjacent lines are determined by those skilled in the art depending on the number and type of probes required. However, in order to efficiently perform comprehensive detection, it is preferable that the width and interval of each line on which the probe is immobilized be as small as possible in consideration of detection sensitivity and density. For example, by producing a microarray by the manufacturing method of the present invention, the width of each line of the probe region is 0.1 to 100 μm and the length is 1 to 100 mm, and the interval between the probes can be 10 to 100 μm. .

The number, width, and length of the sample microchannels formed on the substrate and the interval between the microchannels can be appropriately selected by those skilled in the art according to the number of samples to be measured at the same time. In order to efficiently perform detection, it is preferable that the width and interval of each microchannel be as small as possible in consideration of detection sensitivity and density. For example, by producing a microarray by the manufacturing method of the present invention, the microchannel for a sample has a width of 0.1 to 100 μm, a height (depth) of 1 to 100 μm, and a length of 1 to 100 mm. The interval between the flow paths can be 10 to 100 μm.

  Therefore, the groove provided on one side of the microchannel forming body to be used has a width of 0.1 to 100 μm, a depth of 1 to 100 μm, and a length of 1 to 100 mm, and the interval between the grooves is 10 to 100 μm. Are preferred.

As long as it is possible to provide the microchannel as described above by micrometer and nanometer processing, the material of the microchannel forming body is not particularly limited, and any material known to those skilled in the art can be appropriately selected. Is possible. Typical examples are UV and visible light sensitive polymers that can be processed by photolithography, transparent silicone rubbers such as PDMS (polydimethylsiloxane: PDMS) that can be cast, and PMMA (polymethyl methacrylate) that can be injection molded. And the like, and thermoplastic resins such as COP (cycloolefin polymer) and photo-curing resins that can be applied to nanoimprints that transfer nanometer-sized structures. Furthermore, materials such as various plastics, glass, and quartz can also be used.

PDMS is a kind of silicon elastomer. It has extremely high transparency and excellent optical characteristics, and has very little absorption in a wide wavelength region, particularly in the visible light region, and is suitable for fluorescence detection. Further, by using the template method, microfabrication of a microstructure having an arbitrary microstructure of nano to micron order is easy. Furthermore, PDMS itself has good adsorptivity and releasability to plastics such as glass and acrylic resin, and by attaching a substrate made of these materials to a finely processed PDMS member, Paths and chambers can be easily formed. Furthermore, PDMS is excellent in elasticity and is also a biocompatible material.
COP (cycloolefin polymer) is a resin that has been developed and commercialized as a resin that has transparency, low birefringence, heat resistance, and low hygroscopicity for optical purposes. It is used as a material for camera lenses and optical pickup lenses. In particular, in recent years, it has become possible to construct an arbitrary fine structure of nanometer and micrometer size in a COP resin by thermal transfer nanoimprint technology. Furthermore, since autofluorescence, which is a problem in fluorescence observation, is similar to quartz, it is also suitable for fluorescence observation under a microscope.

It is possible to measure a large number of types of test substances at the same time by using different types of probes as the probes immobilized in a linear form in the probe region. The probe is a substance that can specifically recognize and bind to a test substance to be detected, and can be appropriately selected by those skilled in the art depending on the test substance. Examples thereof include various nucleic acid molecules such as DNA and fragments thereof such as oligonucleotides, proteins and peptides such as antibodies, lipids and saccharides, or low molecular compounds such as drugs. These may be those prepared from body fluids such as blood and cells / tissues by an appropriate extraction or purification method, those obtained from nature and the environment, and various synthetic substances. Therefore, the microarray of the present invention can be used for various applications such as a DNA chip, a protein chip, an antibody chip, and a sugar chain chip depending on the type of substance immobilized as a probe.

The material used as the substrate can be appropriately selected by a person skilled in the art from known substances according to the measurement means / device used in the detection method using the microarray of the present invention. For example, glass, quartz, plastic, or Examples include synthetic resins represented by transparent silicone rubbers such as PDMS. The size of the substrate depends on the size of the microchannel forming body that forms the microchannel for the sample, the area of the probe region fixed in a plurality of parallel lines, the measurement means / device used for detection, etc. Although those skilled in the art can select as appropriate, the length is usually 15 to 30 mm, the width is 15 to 50 mm, and the thickness is about 1.0 to 3.0 mm.

In addition, through holes as solution inlets and outlets are provided at positions corresponding to the upstream and downstream portions of the plurality of parallel linear grooves provided in the microchannel forming body adhered to the substrate surface. Through which the solution can flow into the microchannel. The plurality of parallel linear grooves in the microchannel forming body communicate with through holes provided at positions corresponding to the upstream portion and the downstream portion, respectively, and these through holes function as solution inlets and outlets. Also, the plurality of grooves can be joined at the downstream portion to form one groove, which can communicate with one common through hole (discharge port). Furthermore, in order to make the operation at the time of injecting the solution easier, the plurality of parallel linear grooves spread out in a fan shape in the upstream portion thereof, and each injection port is, for example, about 2.0 to 5.0 mm. It is preferable to have an interval. In addition, by connecting a thin tube such as a microtube having an appropriate length to the through-holes serving as the injection port and the discharge port, the solution can be easily injected and discharged.

[Microarray manufacturing method]
The microarray of the present invention can be manufactured, for example, by the following steps.
(1) By introducing a solution containing a probe into a plurality of parallel line-shaped probe microchannels formed on a substrate, the plurality of probes are arranged at positions corresponding to the microchannels on the substrate. (2) a micro-channel forming body provided with a plurality of parallel linear grooves is attached to the substrate, and the grooves and the substrate surface are perpendicular to the linear direction of the probe region. Forming a plurality of parallel micro-channels for samples in the direction.

In step (1), a microchannel forming body similar to the microchannel forming body used in step (2) is attached to the substrate to form a plurality of parallel microchannels for probes. Is preferred.

The microchannel forming body used in the present invention can be easily manufactured and processed by any method known to those skilled in the art depending on the material and the like. As a method for forming a groove on the substrate surface, for example, a method of removing a groove portion by a mechanical cutting method using a micro processing drill, broad, etc., ionizing radiation lithography such as photolithography, electron beam lithography, or AFM lithography, The groove can be directly formed on the microchannel forming body by using a method of removing the groove portion by physical or chemical etching or a combination of these methods. Furthermore, it is also possible to form a groove indirectly by transferring the spatial structure using a mold having a convex structure that reverses the desired groove structure produced by such a method. The injection molding used or a press mold at a high temperature can be used. In particular, when PDMS is used as a material, PDMS has a high followability with respect to a mold, and therefore a method of forming using a mold is particularly preferable. Once the mold is made, the manufacturing cost can be reduced, and a large number of microarrays can be produced simply and inexpensively.

In addition, when the microchannel forming body is attached to the substrate surface, the substrate surface is treated with oxygen using a plasma etching apparatus or the like, whereby stronger adhesion can be achieved. In addition, when PDMS is used as the micro-channel forming body, it is possible to attach without using an adhesive by removing organic substances and dust from a substrate such as glass using an appropriate detergent.

In step (1), different types of probes can be immobilized in parallel lines by flowing solutions containing different types of probes in each microchannel. The immobilization depends on the type of probe, such as physical adsorption method, chemical immobilization method such as covalent bond, electrostatic bond and ionic bond, carrier binding method such as biological specific bond, cross-linking method, It can be carried out by any method known to those skilled in the art, such as a wrapping method enveloping with a molecular substance. Furthermore, when immobilizing the probe, treatments such as ultraviolet irradiation, cross-linking with a chemical crosslinking agent, blocking of the substrate surface, and washing can be performed as necessary.

In addition, the board | substrate produced at the process (1) is divided | segmented, the board | substrate which has the probe area | region fix | immobilized by the same number of parallel lines as the original board | substrate is produced, and each of the obtained several board | substrate is, By performing the step (2), the microarray of the present invention can be produced more rapidly.

[Method of detecting test substance using microarray].
When a sample substance that specifically binds to the immobilized probe is contained in the sample solution by introducing the sample solution into the plurality of parallel linear sample microchannels formed in the microarray. The test substance is fixed to the substrate via the probe, and as a result, the test substance can be detected.

  Here, the sample solution is derived or extracted from nature such as biological cells / tissues / organs, water or soil, or artificially produced, etc., and an appropriate method known to those skilled in the art. -Prepared by appropriate pretreatment by means. In this case, the analyte to be measured is preferably labeled with an appropriate compound such as a fluorescent substance, an enzyme, and biotin by any method known to those skilled in the art.

  In addition, the introduction of the probe solution and the sample solution into the microchannel can be performed using appropriate supply means and recovery means known to those skilled in the art. Examples of such means include, for example, glass syringes, plastic syringes, micropipetters, fluoroethylene resin tubes / silicon tubes, PVC tubes, peristaltic pumps, plunger pumps, microsyringe pumps, or appropriate combinations thereof. I can do it.

  For example, in the microarray of the present invention, when the width of one line of the probes fixed in a plurality of parallel lines is about 10 μm, and the width of the sample microchannel is also about 10 μm, each formed The detection (reaction) spot has a very small area of approximately 10 μm square. Also, if 10 probes are solidified on the substrate and the distance between each probe is about 20 μm, 10 probes will be accommodated in about 280 μm, and detection (reaction) in the reaction (probe) region will occur. The spot density is comparable to conventional Affymetrix microarrays. Furthermore, if the width of the microchannel for the sample is 10 μm and the height (depth) is 20 μm, the volume of the channel is only about 56 pL. Therefore, the amount of each solution introduced into the probe and sample microchannels is only about 1 μL.

The test substance is preferably labeled with an appropriate compound such as a fluorescent substance. Alternatively, a secondary reactant such as a labeled antibody can be introduced into the sample microchannel, and the measurement can be performed after the secondary reactant is further bound to the bound analyte. is there. The detection of the test substance can be performed using any measurement method and apparatus known to those skilled in the art depending on the type of the test substance or its labeling substance. In the detection, as in the step (1), the microchannel can be appropriately washed and blocked by an appropriate method known to those skilled in the art.

  Hereinafter, the present invention will be described with reference to examples. It should be noted that the technical scope of the present invention is not limited to the description of these examples, and modifications and corrections appropriately made by those skilled in the art based on these descriptions are also included in the present invention. FIG. 1 shows an outline of the microarray of the present invention, a production method thereof, and a detection method using the same.

[Immobilization of probe] A slide glass for protein immobilization (Nunc # 231641: epoxy silane) is used as a substrate, on which a width of 25 μm, a depth of 30 μm and a length of 20 mm are provided, and intervals between the grooves are 25 μm (grooves). A PDMS film having a plurality of parallel line-shaped grooves with a center line interval of 50 mm) is pasted, and then 0.5 mg is placed in the probe microchannel formed between the slide glass and the PDMS film. / ml Alexa488-conjugated protein A (Invitrogen # P11047) was introduced. The plate was allowed to stand overnight at 4 ° C., and protein A was immobilized on the substrate surface. Next, the inside of the probe microchannel was washed to remove unbound protein A, and then the PDMS film was peeled off.

[Washing / Blocking] Using 0.05% PBS-Tween 20, the slide glass on which the probe was fixed was washed (10 minutes × 3). Unreacted active groups on the substrate were inactivated by incubation with Quenching buffer (0.5M Ethanolamine, 0.5M NaCl, pH. 8.3) for 1 hour at room temperature. Thereafter, the plate was washed with 0.05% PBS-Tween20 (10 minutes × 3), incubated in 0.5% α-casein (sigma # C3082) /0.05% PBST for 1 hour at room temperature, and then again 0.05% PBS-Tween20. (10 minutes × 3). Next, the same PDMS film used for fixing the probe is attached to a sufficiently dried slide glass, and the plurality of parallel linear sample micro-streams are perpendicular to the fixed multiple parallel linear probe. A microarray with paths was made. In the microarray thus obtained, Alexa488 having a plurality of parallel lines was detected on the substrate over a width of 25 μm and a length of 20 mm (FIG. 2A).

[Reaction with rabbit IgG]
Cy3 and Cy5-labeled 0.5 mg / ml rabbit IgG was introduced into the sample microchannel. Incubation was performed at room temperature for about 30 minutes to 1 hour. Next, the inside of the microchannel was washed to remove unbound IgG, and then the PDMS film was peeled off and washed with 0.05% PBS-Tween20 (10 minutes × 3).

When this was measured using a KEYENCE BioZero (BZ8000) inverted fluorescence microscope, the fluorescence signal by Alexa488 exhibited a linear signal with a width of 25 μm as shown in FIG. 2 (A), and Cy3 (FIG. 2 (B)) and In Cy5 (FIG. 2C), a rectangular fluorescent signal of 25 μm square was obtained. The above results are summarized and schematically shown in FIG. FIG. 3 shows an outline of the microarray of the present invention and a detection method using the microarray.

  By utilizing the microarray of the present invention, it is possible to construct means / methods for simultaneously detecting a large number of types of test substances contained in a very small amount of sample in a short time with high sensitivity.

The outline of the microarray of this invention and the detection method using the same is shown. The detection result of the test substance (rabbit IgG) using the microarray of the present invention is shown. The outline of the detection method of the test substance using the microarray of the present invention is shown.

Claims (19)

A micro-channel forming body provided with a plurality of parallel line-shaped grooves is attached to a substrate surface having a probe area fixed in a plurality of parallel lines, and the line of the probe area is formed by the grooves and the substrate surface. A microarray in which a plurality of parallel microchannels for samples are formed in a direction perpendicular to the shape direction. The microarray according to claim 1, wherein each line of the probe region has a width of 0.1 to 100 µm and a length of 1 to 100 mm, and an interval between adjacent lines is 10 to 100 µm. The microarray according to claim 1 or 2, wherein the sample microchannel has a width of 0.1 to 100 µm, a height of 1 to 100 µm, and a length of 1 to 100 mm, and an interval between the microchannels is 10 to 100 µm. . The microarray according to any one of claims 1 to 3, wherein the microchannel forming body is made of a resin film. The microarray according to claim 4, wherein the film material is a synthetic resin selected from polydimethylsiloxane, polymethylmethacrylate, or cycloolefin polymer. The microarray according to any one of claims 1 to 5, wherein the probes immobilized in a linear form in the probe region are different types of probes. The microarray according to any one of claims 1 to 6, wherein the probe is a nucleic acid molecule. The microarray according to any one of claims 1 to 7, wherein the probe is an antibody molecule. The microarray according to any one of claims 1 to 8, wherein the substrate is made of glass, quartz, plastic, or polymethylsiloxane. A plurality of parallel linear grooves in the micro-channel forming body communicate with through holes provided at positions corresponding to the upstream portion and the downstream portion, respectively, and these through holes function as solution inlets and outlets. The microarray according to claim 1, wherein the microarray is characterized. The microarray according to any one of claims 1 to 10, wherein one common through hole is provided as an outlet of each microchannel. It is a manufacturing method of the microarray as described in any one of Claims 1-11,
(1) By introducing a solution containing a probe into a plurality of parallel line-shaped probe microchannels formed on a substrate, the plurality of probes are arranged at positions corresponding to the microchannels on the substrate. (2) a micro-channel forming body provided with a plurality of parallel linear grooves is attached to the substrate, and the grooves and the substrate surface are perpendicular to the linear direction of the probe region. Forming a plurality of parallel micro-channels for samples in the direction. In the step (1), a micro flow path forming body provided with a plurality of parallel linear grooves is attached to the substrate, and a plurality of parallel linear probe micro flow paths are formed by the grooves and the substrate surface. The method of claim 12. The groove provided on one side of the microchannel forming body used in step (1) and / or step (2) has a width of 0.1 to 100 μm, a depth of 1 to 100 μm, and a length of 1 to 100 mm, The method of Claim 12 or 13 that the space | interval of each groove | channel is 10-100 micrometers. The microarray according to any one of claims 12 to 14, wherein the microchannel forming body is made of a resin film. The method according to any one of claims 12 to 15, wherein in step (1), a solution containing different types of probes is allowed to flow in each microchannel. The substrate produced in the step (1) is divided to produce a substrate having probe regions fixed in the same number of parallel lines as the original substrate, and the step ( The method according to any one of claims 12 to 16, wherein 2) is performed. A sample solution is introduced into a plurality of parallel linear sample microchannels formed in the microarray according to any one of claims 1 to 11, and the reaction is performed based on a reaction between an immobilized probe and a test substance. A method for detecting a test substance. The detection method according to claim 18, wherein fluorescence of a test substance by a fluorescent label is measured.

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