JP2007071650A - Chip and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip constituted so as to exert no effect on the surface smoothness of a substrate, capable of being easily formed of a material easy to obtain and capable of preventing the adhesion of a specimen other than the fixing region of a carrier, and its manufacturing method. <P>SOLUTION: The chip T is constituted so as to support the carrier, which has the component reacting with a component serving as a detection target, on the surface of the substrate 1. The region of a coating film 2 of a phosphorylcholine like group-containing polymer and a fixing region 3 which keeps the surface of the substrate 1 exposed are formed to the surface of the substrate 1. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a chip and a method for producing the same, and in particular, in a cell chip and an antibody chip widely used in the fields of chemical engineering, biotechnology, environment, medicine, etc., high-speed drug screening, animal experiment substitution, cell The present invention relates to a chip used for research and the manufacturing method thereof.

  Techniques have been developed that specifically detect and measure specific substances contained in biological components such as blood, wastewater from factories, foods, etc., using the high substance recognition ability of living organisms. In such a technology, protein or DNA, which is a biomolecule, is supported on a substrate, and a specific reaction or interaction between the biomolecule and a measurement target substance is observed by an electrochemical or optical method. Thus, it becomes possible to detect the substance to be measured and measure the concentration.

In general, when performing an assay using a specific reaction between a biomolecule and a substance to be measured using a biomolecule in this way, a chip having a large number of various biomolecules immobilized for the purpose of a large-scale parallel assay is used. There are many opportunities to be used.
The chip is also referred to as an array, and is obtained by regularly arranging and fixing a large number of biomolecules on a substrate.
Depending on the biomolecules arrayed and fixed on the substrate, a variety of chips such as DNA chips with DNA fragments arrayed, antibody chips (protein chips) with antibodies arrayed fixed, cell chips with cells arrayed fixed, etc. Exists.

As a method for producing such a chip, many methods are known, such as a method of applying a photolithography technique used in a semiconductor manufacturing technique.
Moreover, as an improved method of these methods, a technique has been developed that uses a substrate that is hydrophobic and has a surface that becomes hydrophilic by the action of light or heat (for example, Patent Document 1).
In the technique of Patent Document 1, a photomask is disposed between a light source and a substrate, and irradiation of active light, scanning exposure of laser light, application of heat, and the like are performed through this photomask.
As described above, the portion to which energy such as light, laser light, or heat is applied through the photomask becomes hydrophilic, so that the hydrophilic region becomes a cell region that receives the DNA probe.

However, the method using the conventional photolithography technique as described above has a problem that a complicated operation is required and it takes time and cost to produce a chip.
Further, it is necessary to design a corresponding photomask every time the shape of the cell region formed on the chip is changed, and there is a problem that it takes time and effort.
In the technique of Patent Document 1, it is necessary to use a special substance having a surface that is hydrophobic and hydrophilic by the action of light or heat as the substrate of the chip. For this reason, there are problems such as an increase in cost for chip production.
Further, in order to process the substrate itself, energy for processing is also applied to the cell region. For this reason, the surface shape of the cell region is affected, the smoothness of the cell region may be impaired, and biomolecules immobilized on the cell region may not be regularly aligned.

Accordingly, it has long been desired to develop a chip that does not affect the surface of the substrate (particularly the cell region) and can be easily produced with readily available materials.
Further, it is more preferable that the material is prevented from adhering to the area other than the cell region.

JP 2003-028864 A

  The present invention has been made in view of the above circumstances, and its object is not to affect the smoothness of the surface of the substrate, and can be easily produced with easily available materials, and the fixed region of the support. It is another object of the present invention to provide a chip capable of preventing the specimen from adhering to the chip and a method for manufacturing the chip.

That is, according to the chip according to claim 1, the above-described problem is a chip capable of supporting a carrier having a component that reacts with a component to be detected on the substrate surface, and the surface of the substrate includes: This is solved by forming a film region coated with a phosphorylcholine-like group-containing polymer and a fixing region where the substrate surface is exposed.
At this time, the carrier is carried on the fixed region.
The surface of the chip according to the present invention is coated with a polymer having phosphorylcholine-like groups in the molecule. This phosphorylcholine-like group is a functional group similar to the phosphorylcholine group possessed by the phospholipid that is a component of the cell membrane, and the polymer having such a functional group in the molecule is a biological component such as a protein or blood cell like the cell membrane. And has a property of suppressing the adsorption and denaturation of these biological components.
The immobilization region is a region for supporting an antibody or the like, but this portion is not covered with a polymer having a phosphorylcholine-like group in the molecule.
Therefore, the immobilization region can reliably carry antibodies and the like, and the surface other than the immobilization region is coated with a polymer having a phosphorylcholine-like group in the molecule, so that it is possible to attach a specimen such as a cell. Can be prevented.

（式中、Ｒ^１は炭素数２〜１０の２価の炭化水素基を示し、Ｒ^２は炭素数２〜４の２価の炭化水素基を示し、Ｒ^３，Ｒ^４及びＲ^５は同一あるいは異なってもよく、水素原子あるいは炭素数１〜６の炭化水素基を示し、Ｒ^６は水素またはメチル基であり、Ｒ^７は炭素数１〜６の２価の炭化水素基を示し、Ｒ^８，Ｒ^９，Ｒ^１０は同一あるいは異なってもよく、炭素数１〜３のオキシアルキレン基であり、このうち１または２つはメチル基であってもよく、Ｒ^１１は水素またはメチル基であり、ｌとｍは各構成単位の割合を示し、ｌは０．７５から０．９９、ｍは０．０１から０．２５の範囲であり、ゲルパーミエーションクロマトグラフにより標準ポリエチレングリコールを用いて換算した重量平均分子量が１０，０００から１，０００，０００の範囲である。）で示される共重合体であると好適である。
このように構成されているため、基板をホスホリルコリン類似基含有重合体で簡易に被覆することができる。
つまり、ホスホリルコリン類似基含有重合体中の有機ケイ素基は、加水分解性のオキシアルキレン基を分子内に有しているため、水中で加水分解してシラノール基（Ｓｉ−ＯＨ）を形成する。このシラノール基は不安定であるため、例えばガラスのような、表面にシラノール基を有する基材（シラノール基含有基材）のシラノール基と脱水反応を起こし、安定なシロキサン結合（Ｓｉ−Ｏ−Ｓｉ）を形成する。このように、基板をホスホリルコリン類似基含有重合体で簡易に被覆することができる。 In this case, as in claim 3, at least the surface of the substrate is composed of a silanol group-containing base material, and the phosphorylcholine-like group-containing polymer is:
The following general formula (1)

(In the formula, R ¹ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ² represents a divalent hydrocarbon group having 2 to 4 carbon atoms, and R ³ , R ⁴ and R ⁵ are the same. Alternatively, they may be different and each represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R ⁶ represents hydrogen or a methyl group, R ⁷ represents a divalent hydrocarbon group having 1 to 6 carbon atoms, R ⁸ , R ⁹ , and R ¹⁰ may be the same or different, and are an oxyalkylene group having 1 to 3 carbon atoms, of which 1 or 2 may be a methyl group, and R ¹¹ is hydrogen or a methyl group. Yes, l and m represent the proportion of each structural unit, l is in the range of 0.75 to 0.99, m is in the range of 0.01 to 0.25, and standard polyethylene glycol is used by gel permeation chromatography. The converted weight average molecular weight is 10,000 to 1,000. , It is preferable that the copolymer represented by a range of 000.).
Since it is comprised in this way, a board | substrate can be easily coat | covered with a phosphorylcholine analog group containing polymer.
That is, since the organosilicon group in the phosphorylcholine-like group-containing polymer has a hydrolyzable oxyalkylene group in the molecule, it is hydrolyzed in water to form a silanol group (Si—OH). Since this silanol group is unstable, it causes a dehydration reaction with a silanol group of a substrate having a silanol group on the surface (silanol group-containing substrate), such as glass, and a stable siloxane bond (Si-O-Si). ). Thus, the substrate can be easily coated with the phosphorylcholine-like group-containing polymer.

In addition, according to the chip manufacturing method according to claim 4, the above-described problem is a chip manufacturing method capable of supporting a carrier having a component that reacts with a component to be detected on a substrate surface, A first step of coating the surface of the substrate with a phosphorylcholine-like group-containing polymer, and laser ablation by irradiating the substrate surface with a laser beam, whereby the phosphorylcholine-like group-containing weight at the laser light irradiation position is A second step of removing the union and exposing a part of the substrate to form a fixed region; and a third step of injecting the carrier to the fixed region and supporting the carrier to the fixed region. And solving the problem.
As described above, according to the present invention, the fixing region can be easily formed by removing the coating of the phosphorylcholine-like group-containing polymer by laser ablation.
At this time, only the phosphorylcholine-like group-containing polymer in the portion irradiated with the laser beam is removed by the laser beam, and the exposed substrate surface is not processed.
Therefore, the fixed region can be formed without affecting the smoothness of the exposed substrate surface.
In addition, since only the phosphorylcholine-like group-containing polymer in the portion irradiated with the laser light has to be removed, the fixed region can be obtained with less energy than when the substrate is directly cut to form a concave portion (corresponding to the fixed region). Can be formed.

At this time, in the second step, a cover having an opening having a shape substantially the same as the shape of the fixed region is attached to the laser light irradiation port, and the laser light passes through the opening and is applied to the substrate. If it is configured to irradiate, a complicated operation such as a method using a conventional photolithography technique is not required, and time and cost for chip production are reduced.
In addition, no photomask is required.

At this time, in the second step, it is preferable to form a plurality of the fixed regions by moving the laser light irradiation port at a predetermined interval and repeating laser ablation.
Further, it is preferable that the carrier is ejected to the fixed region by an ink jet function because the biomolecule can be reliably ejected to the fixed region by using the ink jet function. Further, since the periphery of the fixed region is coated with a phosphorylcholine-like group-containing polymer, even if the support is sprayed to this portion, it can be easily removed by washing or the like.

According to the present invention, the surface of the substrate is coated with a polymer having a phosphorylcholine-like group in the molecule, but the fixing region, which is a region for supporting an antibody or the like, is a heavy chain having a phosphorylcholine-like group in the molecule. Coalescence has been removed.
Therefore, it is possible to reliably carry an antibody or the like in the fixed region, and to prevent a substance such as a specimen from adhering to the region other than the fixed region.
For this reason, it is difficult for unreacted specimens and the like to adhere to the chip, so that unreacted specimens can be easily recovered by a cleaning operation or the like.

According to the present invention, the fixed region is created by laser ablation. This treatment is merely a treatment for removing the coated phosphorylcholine-like group-containing polymer, and it does not treat the substrate itself. Therefore, since the smoothness of the substrate surface exposed to the fixed region is not affected, it is possible to reliably carry a carrier such as an antibody.
In addition, since it is a process that only removes the coated phosphorylcholine-like group-containing polymer, it can be processed with less energy compared to the conventional technique in which the substrate is directly shaved to form a recess (corresponding to a fixed region). It is possible to reduce the manufacturing cost of the chip.
Furthermore, the chip according to the present invention can be made of an inexpensive and easily available material such as glass (a silanol group-containing substrate). Therefore, it can be produced at low cost without using a special material.

Below, the chip | tip concerning this invention and its manufacturing method are demonstrated.
It should be noted that the materials, instruments, conditions, and the like described below are not intended to limit the present invention, and various modifications can be made in accordance with the spirit of the present invention.

As shown in FIG. 1, the chip T according to the present embodiment is a substantially rectangular chip, and mainly includes a substrate 1, a coating 2, and a plurality of fixed regions 3.
In the chip T, a plurality of fixed regions 3 are loaded with biomolecules that specifically bind to the specimen to be detected, the specimen is applied to the chip T, and the carried biomolecules and the specimen are specific. It is used to detect the mechanical binding.
The chip T is generally called an array because a plurality of fixed regions 3 are regularly arranged.

The substrate 1 according to this embodiment is a substantially rectangular silanol group-containing base material. In the present embodiment, a general slide glass is used.
The film 2 according to the present embodiment is a film that is provided on the surface of the slide glass.
The coating 2 is a polymer having a phosphorylcholine-like group.
Since this polymer contains a phosphorylcholine-like group having a structure similar to that of a phospholipid polar group (phosphorylcholine group), which is one of the components of a biological membrane, it interacts with biomolecules such as proteins and DNA. Very weak. For this reason, these organic substances are very difficult to adhere to the portion covered with the coating 2 of the substrate 1.
The structure of the polymer having the phosphorylcholine-like group will be described later.

The fixed region 3 according to the present embodiment is a substantially rectangular recess.
The fixed region 3 is a concave portion having a depth corresponding to the thickness of the coating 2 and is formed by digging the coating 2 into a substantially rectangular shape.
A plurality of fixed regions 3 are formed on the chip T, and in the present embodiment, the fixed regions 3 are formed in a substantially square shape having a side of about 50 μm. Furthermore, the distance between the adjacent fixed regions 3 and 3 is about 50 μm.
For ease of understanding, FIG. 1 illustrates the fixed region 3 in an enlarged manner.
Further, the surface of the substrate 1 is exposed at the bottom surface of the fixed region 3.
For this reason, biomolecules can be immobilized on the bottom surface of the fixed region 3, but other than the fixed region 3, various organic substances including biomolecules are difficult to adsorb.
Therefore, after the sample is applied, the sample that has not reacted with the biomolecule fixed in the fixing region 3 is difficult to be adsorbed on the substrate 1.
For this reason, it is possible to easily collect the specimen that has not reacted with the biomolecule only by washing the chip T.
In the present embodiment, biomolecules such as antibodies, DNA, RNA, proteins, and the like are used as the support. However, the present invention is not limited to this, and a component that can specifically bind to the target component. Any substance may be used as long as it has the following.

（式中、Ｒ^３，Ｒ^４及びＲ^５は同一あるいは異なってもよく、水素原子あるいは炭素数１〜６の炭化水素基を示し、ｎは２以上の整数を示す。）
具体的には、被膜２は、下記一般式（１）で示される共重合体Ａである。

（式中、Ｒ^１は炭素数２〜１０の２価の炭化水素基を示し、Ｒ^２は炭素数２〜４の２価の炭化水素基を示し、Ｒ^３，Ｒ^４及びＲ^５は同一あるいは異なってもよく、水素原子あるいは炭素数１〜６の炭化水素基を示し、Ｒ^６は水素またはメチル基であり、Ｒ^７は炭素数１〜６の２価の炭化水素基を示し、Ｒ^８，Ｒ^９，Ｒ^１０は同一あるいは異なってもよく、炭素数１〜３のオキシアルキレン基であり、このうち１または２つはメチル基であってもよく、Ｒ^１１は水素またはメチル基であり、ｌとｍは各構成単位の割合を示し、ｌは０．７５から０．９９、ｍは０．０１から０．２５の範囲であり、ゲルパーミエーションクロマトグラフにより標準ポリエチレングリコールを用いて換算した重量平均分子量が１０，０００から１，０００，０００の範囲である。） Next, the structure of the polymer having a phosphorylcholine-like group will be described.
The coating 2 used in this embodiment is made of a phosphorylcholine-like group-containing polymer.
A phosphorylcholine-like group-containing polymer means a functional group having the same or similar structure as a phospholipid polar group (phosphorylcholine group) that is one of the constituent components of a biological membrane. Specifically, the functional group represented by the following general formula (2) is desirable.

^(Wherein, R 3, ^{R 4} and ^{R 5} may be the same or different, represent a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, n represents an integer of 2 or more.)
Specifically, the film 2 is a copolymer A represented by the following general formula (1).

(In the formula, R ¹ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ² represents a divalent hydrocarbon group having 2 to 4 carbon atoms, and R ³ , R ⁴ and R ⁵ are the same. Alternatively, they may be different and each represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R ⁶ represents hydrogen or a methyl group, R ⁷ represents a divalent hydrocarbon group having 1 to 6 carbon atoms, R ⁸ , R ⁹ , and R ¹⁰ may be the same or different, and are an oxyalkylene group having 1 to 3 carbon atoms, of which 1 or 2 may be a methyl group, and R ¹¹ is hydrogen or a methyl group. Yes, l and m represent the proportion of each structural unit, l is in the range of 0.75 to 0.99, m is in the range of 0.01 to 0.25, and standard polyethylene glycol is used by gel permeation chromatography. The converted weight average molecular weight is 10,000 to 1,000. It is in the range of 000.)

  The copolymer A used in the present invention has an organosilicon group in the molecule. This organosilicon group forms a siloxane bond with the silanol group. By this siloxane bond, the copolymer A can be bonded to the surface of the silanol group-containing substrate having a silanol group.

  Further, the copolymer A has a phosphorylcholine-like group in the molecule. This phosphorylcholine-like group has a structure similar to a phospholipid polar group (phosphorylcholine group), which is one of the components of biological membranes, and therefore has very weak interaction with biomolecules such as proteins and DNA. The organic matter is suppressed from adsorbing to the silanol group-containing substrate.

  Further, the copolymer A is composed of a phosphorylcholine-like group-containing monomer (a1) having a phosphorylcholine-like group in the molecule and an organosilicon group-containing monomer (a2) having an organosilicon group in the molecule. It is preferable that it is a copolymer.

（式中、Ｒ^１は炭素数２〜１０の２価の炭化水素基を示し、Ｒ^２は炭素数２〜４の２価の炭化水素基を示し、Ｒ^３，Ｒ^４及びＲ^５は同一あるいは異なってもよく、水素原子あるいは炭素数１〜６の炭化水素基を示し、Ｒ^６は水素またはメチル基である。） The phosphorylcholine-like group-containing monomer (a1), which is one of the constituent units of the copolymer A, has a phosphorylcholine-like group having a property similar to that of the phosphorylcholine group of the phospholipid that is a constituent of the cell membrane as a side chain. And a monomer having a polymerizable (meth) acryloyl group in the molecule.
Specifically, a compound represented by the following general formula (3) is preferably used.

(In the formula, R ¹ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ² represents a divalent hydrocarbon group having 2 to 4 carbon atoms, and R ³ , R ⁴ and R ⁵ are the same. Or may be different and represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, and R ⁶ is hydrogen or a methyl group.)

Examples of such compounds include 2- (meth) acryloyloxyethyl-2- (trimethylammonio) ethyl phosphate, 3- (meth) acryloyloxypropyl-2- (triethylammonio) ethyl phosphate, 4- ( (Meth) acryloyloxybutyl-2- (triethylammonio) ethyl phosphate, 5- (meth) acryloyloxypentyl-2- (triethylammonio) ethyl phosphate, 6- (meth) acryloyloxyhexyl-2- (triethylammonio) ) Ethyl phosphate, 2- (meth) acryloyloxyethyl-2- (triethylammonio) ethyl phosphate, 2- (meth) acryloyloxyethyl-2- (tripropylammonio) ethyl phosphate, 2- (meth) acryloyl Oxyethyl-2- (tributylammonio) ethyl phosphate, 2- (meth) acryloyloxypropyl-2- (trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxybutyl-2- (trimethylammonio) ethyl phosphate, Examples include 2- (meth) acryloyloxypentyl-2- (trimethylammonio) ethyl phosphate, 2- (meth) acryloyloxyhexyl-2- (trimethylammonio) ethyl phosphate, and the like. Here, “(meth) acryloyl” means methacryl and / or acryl, and so on.
In addition, the said compound can be used individually or as a mixture in use.
Among these, 2- (methacryloyloxy) ethyl-2-trimethylammonio) ethyl phosphate {= 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as MPC)} is preferable because it is particularly easy to obtain.

  The copolymer A further includes an organosilicon group-containing monomer (a2) as a structural unit. A silane coupling agent is mentioned as a monomer which has an organosilicon-containing group reactive functional group in such a side chain.

The silane coupling agent has a general formula: Y— (CH ₂ ) nSiX ₃ (n = 1 to 6, X is a hydrolyzable substituent such as an alkoxy group or halogen, Y is a vinyl group, an acryloxy group, a methacryloxy group, Reactive substituents such as epoxy groups and amino groups). When such a silane coupling agent is selected as a copolymerization partner of the phosphorylcholine-like group-containing monomer (a1), the reactive substituent of the silane coupling agent (ie, Y in the above general formula) and the phosphorylcholine-like group The reactive substituent of the containing monomer reacts to form a copolymer, and the resulting copolymer has a hydrolyzable substituent (that is, (CH ₂ ) nSiX _{3 in the} above general formula) in the side chain. ).

（式中、Ｒ^７は炭素数１〜６の２価の炭化水素基を示し、Ｒ^８，Ｒ^９，Ｒ^１０は同一あるいは異なってもよく炭素数１〜３のオキシアルキレン基であり、このうち１または２つはメチル基であってもよく、Ｒ^１１は水素またはメチル基である。） As such a silane coupling agent, the compound shown by following General formula (4) is mentioned.

(In the formula, R ⁷ represents a divalent hydrocarbon group having 1 to 6 carbon atoms, and R ⁸ , R ⁹ and R ¹⁰ may be the same or different and are oxyalkylene groups having 1 to 3 carbon atoms, 1 or 2 of them may be a methyl group, and R ¹¹ is hydrogen or a methyl group.)

Examples of such compounds include (meth) acryloxymethyltrimethoxysilane, (meth) acryloxymethylmethyldimethoxysilane, (meth) acryloxymethyldimethylmethoxysilane, (meth) acryloxymethyltriethoxysilane, ( (Meth) acryloxymethylmethyldiethoxysilane, (meth) acryloxymethyldiethoxysilane, (meth) acryloxymethyltripropoxysilane, (meth) acryloxymethylmethyldipropoxysilane, (meth) acryloxymethyldipropoxysilane 2- (meth) acryloxyethyltrimethoxysilane, 2- (meth) acryloxyethylmethyldimethoxysilane, 2- (meth) acryloxyethyldimethylmethoxysilane, 2- (meth) acryloxyethyltriethoxy Run, 2- (meth) acryloxyethylmethyldiethoxysilane, 2- (meth) acryloxyethyldiethoxysilane, 2- (meth) acryloxyethyltripropoxysilane, 2- (meth) acryloxyethylmethyldipropoxy Silane, 2- (meth) acryloxyethyldipropoxysilane, 3- (meth) acryloxypropyltrimethoxysilane, 3- (meth) acryloxypropylmethyldimethoxysilane, (meth) acryloxypropyldimethylmethoxysilane, 3- (Meth) acryloxypropyltriethoxysilane, 3- (meth) acryloxypropylmethyldiethoxysilane, 3- (meth) acryloxypropyldiethoxysilane, 3- (meth) acryloxypropyltripropoxysilane, 3- ( (Meta) acryloxyp Pills methyl dipropoxy silane, 3- (meth) acryloxy propyl dipropoxy silane, and the like.
In addition, the said compound can be used individually or as a mixture in use.
Among these, 3-methacryloxypropyltrimethoxysilane (hereinafter referred to as 3-MPMS)} is obtained because it is easily available and has excellent polymerizability with the phosphorylcholine-like group-containing monomer (a1). preferable.

  The copolymer A has a phosphorylcholine-like group derived from the phosphorylcholine-like group-containing monomer (a1) represented by the general formula (3) in the molecule. As described above, this phosphorylcholine-like group has a structure similar to a phospholipid polar group (phosphorylcholine group), which is one of the components of a biological membrane, and thus exhibits excellent biocompatibility. The interaction of is very small.

  Moreover, the copolymer A of this invention has the organosilicon group derived from the organosilicon group containing monomer (a2) shown by the said General formula (4) in a molecule | numerator. Since this organosilicon group has a hydrolyzable oxyalkylene group in the molecule, it is hydrolyzed in water to form a silanol group (Si—OH). Since this silanol group is unstable, it causes a dehydration reaction with a silanol group of a substrate having a silanol group on a surface such as glass (hereinafter referred to as a silanol group-containing substrate), and a stable siloxane bond (Si—O—Si). ). In addition, the silanol group of the copolymer A is partially condensed with the silanol group of another polymer and other silanol groups in the molecule with the passage of time, and is stably crosslinked between polymer molecules or within the polymer molecule. Form a structure.

Therefore, the copolymer is firmly bonded to the surface of the silanol group-containing substrate due to the siloxane bond with the silanol group present on the surface of the silanol group-containing substrate, and is difficult to peel off from the film surface.
Further, the copolymer A forms a stable crosslinked structure within the polymer molecule and between the molecules as described above. Such a cross-linked structure forms a surface layer in which phosphorylcholine-like groups are densely assembled on the surface of the silanol group-containing substrate, and therefore organic substances such as proteins in the solvent are less likely to adhere to the surface of the silanol group-containing substrate. Furthermore, the solubility in the solvent is small and it is difficult to elute into the solvent, and the surface of the silanol group-containing substrate is coated stably over a long period of time.
Therefore, the silanol group-containing substrate surface-treated with such a copolymer can efficiently prevent nonspecific adsorption of biomolecules contained in the sample to the surface of the silanol group-containing substrate. It becomes.

  Each of the monomers (a1) and (a2) described above has a (meth) acryloyl group in the molecule. Therefore, it is easy to form a polymer with other (meth) acryloyl groups, and a polymer having a desired weight molecular weight can be relatively easily adjusted by adjusting conditions such as the concentration of the polymerization initiator and the reaction time. Can be synthesized.

（式中、ｌとｍは各構成単位の割合を示し、ｌは０．７５から０．９９、ｍは０．０１から０．２５の範囲であり、ゲルパーミエーションクロマトグラフにより標準ポリエチレングリコールを用いて換算した重量平均分子量が１０，０００から１，０００，０００の範囲である。）で示される共重合体が好ましい。 Copolymer A was synthesized by combining MPC as the phosphorylcholine-like group-containing monomer (a1) and 3-MPMS as the organosilicon group-containing monomer (a2), for reasons such as the availability of raw materials. Preferred is a copolymer. Specifically, the following general formula (5)

(In the formula, l and m represent the proportion of each structural unit, l is in the range of 0.75 to 0.99, m is in the range of 0.01 to 0.25, and standard polyethylene glycol is obtained by gel permeation chromatography. The weight average molecular weight converted by use is in the range of 10,000 to 1,000,000.).

The content ratio l of the phosphorylcholine-like group-containing monomer (a1) in the copolymer may be 0.75 to 0.99 in terms of molar fraction. In this case, the content ratio m of the organosilicon group-containing monomer (a2) is 0.01 to 0.25.
In particular, the content ratio l in the copolymer of the phosphorylcholine-like group-containing monomer (a1) is preferably in the range of 0.8 to 0.97 in terms of molar fraction. If the molar fraction of the phosphorylcholine-like group-containing monomer (a1) is 0.75 or less, the reaction density of the silanol groups increases and the cross-linked structure due to condensation of the silanol groups becomes dense, which is not preferable because it is insoluble in the solvent.
Moreover, since the content ratio of the phosphorylcholine-like group in the copolymer is low, the efficiency of preventing nonspecific adsorption of biological components on the surface of the silanol group-containing substrate is unfavorable.

  On the other hand, when the content ratio l of the phosphorylcholine-like group-containing monomer (a1) is 0.99 or more in terms of molar fraction, the content ratio of the organosilicon group-containing monomer (a2) in the copolymer is low. Therefore, it is not preferable because a siloxane bond between a silanol group and a hydroxyl group on the surface of the silanol group-containing substrate is hardly formed, or a stable cross-linked structure due to condensation of the silanol group is hardly formed.

  Further, the weight average molecular weight of the copolymer A is not particularly limited. Preferably, the weight average molecular weight converted using standard polyethylene glycol by gel permeation chromatography is 10,000 to 1,000,000. Good. More preferably, it is 100,000-800,000. When the weight average molecular weight of the polymer is less than 10,000, the solubility in a solvent is increased, which is not preferable. On the other hand, if the weight average molecular weight is more than 1,000,000, the solubility in a solvent may be too low, which is not preferable.

Examples of the copolymer of the phosphorylcholine-like group-containing monomer (a1) and the organosilicon group-containing monomer (a2) include known polymers such as random copolymers, block copolymers, and graft copolymers. Any of these may be used.
The polymerization of each monomer is performed using a known method such as solution polymerization, emulsion polymerization, suspension polymerization or the like. At this time, if necessary, the polymerization system is replaced with an inert gas such as nitrogen or argon, or under the inert gas atmosphere, a polymerization temperature of 0 to 100 ° C. and a polymerization time of 10 minutes to 48 hours. It can be prepared by a radical polymerization method or the like.

  In the polymerization, a known radical polymerization initiator can be used. Examples of the radical polymerization initiator include 2,2-azobis (2-amidinopropyl) dihydrochloride, 4,4-azobis (4-cyanovaleric acid), 2,2-azobis (2- (5-methyl- 2-Imidazolin-2-yl) propane) dihydrochloride, 2,2-azobisisobutyramide dihydrate, 2,2-azobisisobutyronitrile, ammonium persulfate, potassium persulfate, benzoyl peroxide, diisopropyl Peroxydicarbonate, t-butylperoxy-2-ethylhexanoate, t-butylperoxypivalate, t-butylperoxydiisobutyrate, lauroyl peroxide, azobisisobutyronitrile, 2,2-azobis (2, 4-dimethylvaleronitrile), t-butylperoxyneodecanoate and the like. In particular, 4,4-azobis (4-cyanovaleric acid) or 2,2-azobisisobutyronitrile is preferable.

These radical polymerization initiators may be used alone or in a mixture. Various redox accelerators may be used as the polymerization initiator. The amount of the polymerization initiator used is preferably 0.05 to 3.0 parts by weight with respect to 100 parts by weight of the monomer composition. The reaction temperature is preferably from 40 to 80 ° C, particularly preferably from 50 to 70 ° C.
By appropriately selecting the type and concentration of the radical polymerization initiator, the reaction time, the reaction temperature, and the like, it is possible to obtain the copolymer A having a desired weight average molecular weight.
The obtained copolymer A can be purified by a general purification method such as a reprecipitation method, a dialysis method, a microfiltration method, or an ultrafiltration method.

  Examples of the solvent for dissolving the copolymer A include alcohols such as methanol, ethanol and isopropanol. It is also possible to use a mixed solvent in which two or more kinds of solvents are mixed. Examples of the mixed solvent include dimethylformamide, dimethylsulfoxide, acetonitrile, dioxane, acetone, methyl ethyl ketone, ethyl acetate, tetrahydrofuran, N, N-dimethylacetamide, a mixture of N-methyl-2-pyrrolidone and alcohol. Can be mentioned.

  As described above, the copolymer A forms a stable surface layer by forming silanol groups on the surface of the silanol group-containing substrate and siloxane bonds. The material of the silanol group-containing substrate may be any material having a silanol group on the surface, and examples thereof include known materials such as borosilicate glass, quartz glass, and soda lime glass.

Next, a manufacturing method of the chip T according to the present embodiment will be described with reference to FIG.
First, the substrate 1 is coated with the copolymer A constituting the coating film 2.
This coating is performed by adsorbing the copolymer A to the substrate 1.
When the copolymer A is adsorbed to the silanol group-containing base material, it is preferable to remove organic substances or the like in advance by polishing or washing the surface of the silanol group-containing base material.
As a method of polishing the surface of the silanol group-containing substrate, it is possible to polish with a fine sandpaper, or by rubbing the surface of the silanol group-containing substrate by including alumina powder or diamond paste in a nonwoven fabric etc. Done. After washing, wash away the washing solution with deionized water or pure water.

  Coupling of the copolymer A to the silanol group-containing substrate is performed by applying a solution in which the copolymer A is dissolved to the surface of the silanol group-containing substrate or immersing the silanol group-containing substrate in the solution. By the method. Protons are required for the siloxane bond formation reaction, so an acid selected from acetic acid, citric acid, succinic acid, hydrochloric acid, and the like is added to the reaction system. The concentration of the acid to be added is 10 to 200 μM. The contact time between the copolymer A and the silanol group-containing substrate is about 5 to 30 minutes, but for example, a surface layer with high orientation is formed by performing a long time of about 2 to 5 hours. It becomes possible to do.

  The copolymer A in contact with the surface of the silanol group-containing substrate forms a covalent siloxane bond between the silanol group on the surface of the silanol group-containing substrate and the organosilicon group of the copolymer A. A stable surface layer is formed on the surface of the silanol group-containing substrate.

  After the copolymer A is adsorbed to the silanol group-containing substrate, the surface of the silanol group-containing substrate is washed with a solvent such as water to remove the free copolymer A. After washing, it can be stored in a state immersed in a solvent such as water. Moreover, the silanol group containing base-material surface can also be dried and preserve | saved as needed. In this case, it is good to carry out by drying naturally in air | atmosphere or drying in inert gas atmosphere, such as nitrogen gas.

Next, a plurality of fixed regions 3 are formed on the substrate 1 by irradiating with laser light.
As shown in FIG. 2A, this is performed by irradiating the substrate 1 coated with the film 2 with laser light to perform laser ablation and removing the film 2 in the portion irradiated with the laser light.
Laser light irradiation is performed using a known laser device S.
At this time, a cover 11 in which a substantially square hole 11 a having substantially the same area as the opening area of the fixed region 3 is formed is disposed in the laser light irradiation port S <b> 1.
Therefore, the laser light emitted from the irradiation port S1 passes through the hole 11a, becomes a substantially square shape, and is irradiated onto the substrate 1.
The bond of the film 2 is cut by the energy of the laser beam, and this part of the film 2 is scraped off.
For this reason, the surface of the board | substrate 1 will be exposed to the part irradiated with the laser beam.
The wavelength of the laser beam used is preferably about 200 nm to 450 nm.
In this embodiment, laser light having a wavelength of about 245 nm is used.

Next, the irradiation port S1 is moved relative to the substrate 1, and laser ablation is similarly performed. By repeating this operation, a predetermined number of fixed regions 3 are formed on the substrate 1 to produce a chip T (FIG. 2B).
In the present embodiment, since the distance between the adjacent fixed regions 3 and 3 is set to about 50 μm, the irradiation port S1 is moved by about 50 μm.
However, it is not limited to such a form, You may form the fixing | fixed area | region 3 by a plurality using the cover 11 in which the several hole 11a was formed.

As described above, in the present embodiment, the plurality of fixed regions 3 are formed by performing laser ablation by irradiating laser light through the holes 11 a formed in the cover 11. Therefore, a chip T in which a plurality of fixed regions 3 are formed can be easily formed without going through complicated steps such as development work, etching work, and resist removal work as in the case of creating a chip by photolithography technology. can do.
In the present embodiment, only the coating 2 portion is scraped off by the laser beam, and the substrate 1 itself is not processed.
Therefore, the laser output may be small as compared with a technique in which the substrate 1 is directly scraped to form a concave portion (corresponding to a fixed region). Therefore, energy can be saved and the manufacturing cost is reduced.
Further, since the substrate 1 is not directly cut, the smoothness of the fixed region 3 can be maintained as compared with a technique in which the substrate 1 is directly cut and a concave portion (corresponding to the fixed region) is formed. For this reason, the biomolecule fixed to the fixed area | region 3 can be fixed regularly regularly reliably.
Furthermore, in the present embodiment, the cover 11 having the hole 11a and the irradiation port S1 are integrally moved relative to the substrate 1 to form the plurality of fixed regions 3. The arrangement pattern of the fixed region 3 can be easily obtained.

Next, as shown in FIG. 2 (C), the biomolecule is fixed to the fixing region 3. Such biomolecules include antibodies, DNA, RNA, proteins and the like.
In the present embodiment, an antibody is used as a biomolecule, and the antibody is supported on the fixed region 3 by a known technique.
In the present embodiment, biomolecules are applied to the fixed region 3 by ink jet technology.
This is done using a known printing inkjet head U. For example, instead of ink, a solution in which a biomolecule is suspended is filled, and the biomolecule is ejected to the fixed region 3 by a piezoelectric element or the like. By repeating this operation, biomolecules are fixed to all the fixing regions 3. When changing the type of biomolecule to be fixed, the inkjet head U may be replaced with a head U filled with another biomolecule.

By repeating such a jetting operation, the target biomolecule is fixed to all the fixing regions 3 to complete the chip T.
As described above, the coating 2 is applied to the surface of the chip T (portion excluding the fixed region 3).
Therefore, biomolecules overflowing from the fixed region 3 are not attached due to the presence of the coating 2.
For this reason, the biomolecule overflowing from the fixed region 3 can be easily removed.

As shown in FIG. 2 (D), a cell (specifically a cell suspension in the present embodiment) is applied to the chip T thus prepared to detect cells that specifically bind to the antibody. .
As described above, since the portion other than the fixed region 3 of the chip T is coated with the coating 2, cells other than the cells specifically bound to the antibody fixed to the fixed region 3 are stably present on the chip T. Can not survive.
Therefore, by washing the chip T with a buffer solution or the like, the unreacted cells are easily separated from the chip T, so that the unreacted cells can be easily recovered.
Therefore, even when expensive cells or cells that are difficult to obtain in large quantities are used as samples, it is preferable because unreacted cells can be easily collected.
Note that after the sample is applied to the chip T, the sample specifically bound to the antibody fixed to the fixing region 3 is detected by a known method such as fluorescence analysis.

As described above, in the present embodiment, the chip T that can prevent the specimen from adhering to an area other than the fixed area 3 can be easily produced.
Thus, the produced chip T can be used in various scenes such as high-speed drug screening, alternative to animal experiments, cell function studies such as receptor analysis and intracellular metabolism analysis.

It is a perspective view of the chip concerning the present invention. It is explanatory drawing which shows the manufacturing process of the chip | tip concerning this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Coating 3 Fixing area 11 Cover 11a Opening (hole)
S Laser device S1 Irradiation port T Chip U Head

Claims (7)

A chip capable of supporting a carrier having a component that reacts with a component to be detected on a substrate surface,
On the surface of the substrate, a coating region coated with a phosphorylcholine-like group-containing polymer,
And a fixed region where the substrate surface is exposed.   The chip according to claim 1, wherein the carrier is carried on the fixed region. The substrate has at least a surface composed of a silanol group-containing base material,
The phosphorylcholine-like group-containing polymer is
The following general formula (1)

(In the formula, R ¹ represents a divalent hydrocarbon group having 2 to 10 carbon atoms, R ² represents a divalent hydrocarbon group having 2 to 4 carbon atoms, and R ³ , R ⁴ and R ⁵ are the same. Alternatively, they may be different and each represents a hydrogen atom or a hydrocarbon group having 1 to 6 carbon atoms, R ⁶ represents hydrogen or a methyl group, R ⁷ represents a divalent hydrocarbon group having 1 to 6 carbon atoms, R ⁸ , R ⁹ , and R ¹⁰ may be the same or different, and are an oxyalkylene group having 1 to 3 carbon atoms, of which 1 or 2 may be a methyl group, and R ¹¹ is hydrogen or a methyl group. Yes, l and m represent the proportion of each structural unit, l is in the range of 0.75 to 0.99, m is in the range of 0.01 to 0.25, and standard polyethylene glycol is used by gel permeation chromatography. The converted weight average molecular weight is 10,000 to 1,000. In the range of 000.) Chip according to claim 1 or claim 2 characterized in that it is a copolymer represented by. A method for manufacturing a chip capable of supporting a carrier having a component that reacts with a component to be detected on a substrate surface,
A first step of coating the surface of the substrate with a phosphorylcholine-like group-containing polymer;
By performing laser ablation by irradiating the substrate surface with laser light, the phosphorylcholine-like group-containing polymer at the laser light irradiation position is removed, and a part of the substrate is exposed to form a fixing region. Two steps;
A third step of injecting the support to the fixed region and supporting the support to the fixed region;
A chip manufacturing method comprising:   In the second step, a cover having an opening having substantially the same shape as the fixed region is attached to the laser light irradiation port, and the laser light is irradiated to the substrate through the opening. The method of manufacturing a chip according to claim 4.   The said 2nd process WHEREIN: The said fixed area | region is formed by moving the irradiation port of the said laser beam by predetermined space | interval, and repeating laser ablation, The said fixed area | region is characterized by the above-mentioned. Chip manufacturing method.   The chip manufacturing method according to claim 4, wherein the carrier is ejected to the fixed region by an inkjet function.

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