WO2015025634A1 - Substrate for biochips and method for producing same - Google Patents

Substrate for biochips and method for producing same Download PDF

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WO2015025634A1
WO2015025634A1 PCT/JP2014/068442 JP2014068442W WO2015025634A1 WO 2015025634 A1 WO2015025634 A1 WO 2015025634A1 JP 2014068442 W JP2014068442 W JP 2014068442W WO 2015025634 A1 WO2015025634 A1 WO 2015025634A1
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containing polymer
substrate
amino group
carboxyl group
biochip
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PCT/JP2014/068442
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French (fr)
Japanese (ja)
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亮 森下
竹林　恭志
圭 山口
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日本軽金属株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2610/00—Assays involving self-assembled monolayers [SAMs]

Definitions

  • the present invention relates to a biochip substrate for immobilizing proteins, nucleic acids, peptide derivatives, sugar chains and derivatives thereof, natural products, small molecule compounds and other biological materials as probes.
  • Biochips such as protein chips, peptide chips, and DNA chips are widely used for diagnosis and research of various diseases.
  • a biochip that has been widely used is generally obtained by immobilizing a biological substance such as protein, peptide, or DNA on a glass substrate such as a slide glass.
  • a biochip substrate has been proposed in which a carbon-containing layer having an active group is formed on a metal substrate, and a biological substance is bound to the active group (Patent Document 1).
  • This biochip substrate does not generate autofluorescence, has an excellent property that it is easy to immobilize a biological substance, the substrate is easy to process, and the flatness and surface accuracy are high.
  • a biochip substrate is also proposed in which an amino group or carboxyl group-containing polymer is covalently bonded to a substrate having at least a surface made of carbon (Patent Document 2).
  • the biological substance is covalently bonded to this amino group. Similar to the biochip substrate described in Patent Document 1, this biochip substrate also has excellent properties.
  • the known biochip substrate described above has excellent properties, but if the amount of amino groups that can be immobilized can be further increased, it is possible to immobilize biological materials at a higher density. .
  • An object of the present invention is to provide a novel biochip substrate capable of increasing the amount of amino groups that can be immobilized and a method for producing the same, as compared with known biochip substrates.
  • the inventors of the present application can immobilize an amino group-containing polymer or a carboxyl group-containing polymer directly on a metal substrate or via a NiP layer, rather than a known biochip substrate.
  • the inventors have found that it is possible to increase the amount of amino groups and have completed the present invention.
  • the present invention provides a biochip substrate comprising a metal substrate and an amino group-containing polymer layer or a carboxyl group-containing polymer layer immobilized on the metal substrate directly or via a NiP layer.
  • the present invention also provides use of the substrate of the present invention as a biochip substrate.
  • the present invention includes applying an amino group-containing polymer or a carboxyl group-containing polymer directly on a metal substrate or on a NiP layer formed on the metal substrate, and irradiating with ultraviolet rays.
  • a method for producing a biochip substrate is provided.
  • amino groups or carboxyl groups are bonded to the substrate surface in a high density, uniformly and stably, and biologically related substances such as proteins and nucleic acids using the amino groups or carboxyl groups.
  • the probe immobilization rate is high, the immobilization density is uniform, and the non-specific adsorption of the protein is prevented to increase detection sensitivity and reproducibility.
  • the amino group-containing polymer layer or the carboxyl group-containing polymer layer is immobilized on the metal substrate directly or via the NiP layer, like a known metal substrate biochip, Since the carbon-containing layer is not included, the structure is simple, and the cost for forming the carbon-containing layer can be reduced.
  • the biochip substrate of the present invention (hereinafter sometimes simply referred to as “substrate”) is characterized in that an amino group-containing polymer or a carboxyl group-containing polymer is immobilized on the substrate surface.
  • the “amino group” means a primary amino group, that is, —NH 2 .
  • the amino group or carboxyl group-containing polymer is preferably 50% or more, more preferably 90% or more of the structural unit constituting the amino group or carboxyl group-containing polymer in a state before being immobilized on the substrate surface. More preferably, 99% or more each have at least one amino group.
  • the amino group- or carboxyl group-containing polymer is preferably formed by addition polymerization of a vinyl monomer having an amino group, and polyallylamine (PAA) is particularly preferable.
  • PAA polyallylamine
  • the carboxyl group-containing polymer is preferably 50% or more, more preferably 90% or more, more preferably 99% of the structural unit constituting the carboxyl group-containing polymer before being immobilized on the substrate surface. % Or more each have at least one carboxyl group.
  • the carboxyl group-containing polymer is preferably formed by addition polymerization of a vinyl monomer having a carboxyl group, and poly (meth) acrylic acid is particularly preferable (“poly (meth) acrylic acid” A generic name for acrylic acid (PAc) and polymethacrylic acid.
  • poly (meth) acrylic acid A generic name for acrylic acid (PAc) and polymethacrylic acid.
  • the average molecular weight (weight average molecular weight) of the amino group-containing polymer or carboxyl group-containing polymer is preferably 1000 or more, more preferably 2000 or more in terms of PAA or PAc, from the viewpoint of better achieving the effects of the present invention.
  • PAA conversion and “PAc conversion” mean conversion based on the number of amino groups or carboxyl groups in one molecule, respectively (the same applies hereinafter).
  • the upper limit of the molecular weight of the amino group or carboxyl group-containing polymer is not particularly limited as long as there is no problem in handling properties such as solubility and stability of the coating solution, preferably 60,000 or less, 6000 or less is preferable.
  • Non-specific adsorption of protein does not occur even if the probe is immobilized on a substrate on which only an amino group or carboxyl group-containing polymer is immobilized, but it further prevents non-specific adsorption of proteins due to hydrophobic bonds, etc.
  • a hydrophilic polymer can be covalently immobilized on the substrate surface.
  • the hydrophilic polymer a polymer having low reactivity with an amino group or a carboxyl group is used. Examples of the hydrophilic polymer include polyethers, polyacrylamide, agarose and the like.
  • polyethylene glycol (hereinafter sometimes abbreviated as “PEG”) having a controlled molecular weight distribution can be used, is highly stable, and is preferable because it does not inactivate biological materials.
  • the average molecular weight of the hydrophilic polymer is preferably a molecular weight that does not inhibit the reactivity of the amino group or carboxyl group-containing polymer with the linker or probe, and is 200 to 10,000, more preferably 1,000.
  • the amino group- or carboxyl group-containing polymer and the hydrophilic polymer are uniformly mixed in order to ensure a uniform distribution of amino groups. That is, it is preferable to fix a uniform mixture of an amino group or carboxyl group-containing polymer and a hydrophilic polymer on the substrate surface by the method described later.
  • the hydrophilic polymer is an optional component, it does not need to be immobilized. It is preferable for bonding a large number of amino groups or carboxyl groups on the substrate surface. Moreover, even when immobilizing both an amino group or carboxyl group-containing polymer and a hydrophilic polymer, the amount of amino group or carboxyl group-containing polymer immobilized on the substrate surface is converted to polyallylamine or polyacrylic acid as described above. 4 ⁇ g / cm 2 or more is preferable. On the other hand, the total immobilization amount of the amino group or carboxyl group-containing polymer and the hydrophilic polymer is preferably 40 ⁇ g / cm 2 or less, as in the case of the amino group or carboxyl group-containing polymer alone. The hydrophilicity of the substrate can be adjusted by the amount of the hydrophilic polymer immobilized.
  • the metal substrate is not particularly limited, but a substrate made of a normal metal such as aluminum or stainless steel can be preferably used.
  • the amino group or carboxyl group-containing polymer layer is applied by applying an amino group or carboxyl group-containing polymer solution to the surface of the metal substrate directly or via the NiP layer (that is, coating on the NiP layer formed on the metal substrate). By irradiating with ultraviolet rays, it can be fixed to the metal substrate directly or via a NiP layer.
  • the NiP (nickel-phosphorus) layer can be preferably formed by electroless plating. This method is known per se and is described in Patent Document 1 and Patent Document 2.
  • the immobilization method is a method in which a polymer solution is applied to a substrate surface and then bonded by radicals generated by light irradiation.
  • ultraviolet light having a wavelength of about 150 nm to 260 nm, for example, a wavelength of 184 nm or 254 nm is used. be able to.
  • Light of this wavelength breaks C—C, C—O, and C—H bonds to generate radicals.
  • oxygen molecules and water molecules in the air are also decomposed to generate oxygen radicals and ozone, and oxidative decomposition of the substrate material carbon and the polymer occurs simultaneously, thereby inhibiting the formation of covalent bonds.
  • the inert gas a rare gas element that is difficult to be radicalized even when irradiated with light such as argon or helium is used.
  • the amount of light irradiation may be an amount necessary for covalently bonding the polymer, and the amount of energy is usually about 1 to 6 joules, preferably about 2 to 4 joules per 1 cm 2 of the substrate surface.
  • the polymer coating amount is an amount that achieves the above-described polymer immobilization amount.
  • the concentration of the polymer solution is not particularly limited as long as the required amount of polymer application can be achieved and can be uniformly applied, and is preferably about 0.5 to 3 (w / v)%.
  • a general method can be used to apply the polymer to the substrate surface, and the method is not particularly limited as long as the method can control the amount of the polymer applied.
  • the method can be selected from roll coating, spray coating, spin coating, dip coating, and the like.
  • the substrate of the present invention obtained as described above has amino groups uniformly and densely on the surface.
  • a biochip can be constructed by immobilizing a biological substance by covalent bond.
  • the bio-related substance may be any substance used as a probe in a biochip, and may be any polypeptide (including natural or synthetic proteins and oligopeptides), nucleic acid (DNA and RNA, and artificial nucleic acid). Inclusion), sugars, lipids, complexes thereof (glycoproteins, etc.) and derivatives (modified proteins, nucleic acids, etc.).
  • the biological material can be directly covalently bonded to the amino group or can be bonded via a desired linker. Since the biochip substrate itself having an amino group on its surface is widely commercially available, the covalent bond of the biological substance to the amino group can be easily carried out by a well-known conventional method.
  • a biological substance has a carboxyl group that binds to an amino group, such as a protein, it can be directly bonded. If it does not have such a functional group, or if desired, a linker can be added. It is also possible to couple via.
  • Linkers are also well known. For example, those having a carboxyl group at one end and a maleimide group at the other end are widely used.
  • Example 1 Production of biochip substrate (Part 1) An aluminum alloy (5000 series alloy) rolled plate (75 x 25 mm thickness 1 mm) is ground with a PVA grinding wheel to a thickness of 0.98 mm and the surface roughness Ra is set to 30 nm, and then the entire surface is alkali degreased, desmut nitrate, zinc zincate The treatment was performed, and nickel-phosphorous electroless plating was applied to a thickness of 13 ⁇ m per side. Furthermore, both surfaces were polished 3 ⁇ m per side with an alumina slurry, and the surface roughness Ra was set to 1 nm. A coating solution was prepared by adjusting the concentration of PAA (average molecular weight 3,000) to 1 (w / v)% with ethanol.
  • PAA average molecular weight 3,000
  • the substrate is coated with a baker type applicator in which the coating thickness is set to 3 mil (about 0.076 mm) so that the PAA coating amount is 20 ⁇ g / cm 2.
  • the coating solution was spread over the entire material surface. After the solvent was volatilized, it was further vacuum-dried for 1 hour (vacuum degree -0.098 MPa), and then irradiated with ultraviolet rays (18.5 mW / cm 2 , 254 nm) for 3 minutes under vacuum to immobilize PAA. Further, the unreacted PAA was removed by shaking and washing with ultrapure water for 1 hour, followed by spin drying to obtain the biochip substrate of the present invention.
  • TAMRA fluorescent label
  • TAMRA was applied to a biochip substrate having an aluminum plate / NiP plating layer / carbon layer / PAA layer described in Patent Document 2 in the same manner as described above, and the fluorescence value was measured.
  • the fluorescence value before the formation was about the same as in Example 1 above, but the fluorescence value after the TAMRA was about 3000, and the amount of amino groups was about 4 times higher in the substrate of the present invention.
  • Example 2 Production of biochip substrate (2) A PAA solution containing 2 (w / v)% PAA was applied to a slide glass size SUS plate (plate thickness 2 mm) in the same manner as in Example 1, and PAA was immobilized in the same manner as in Example 1.
  • FIG. FIG. 1 revealed that amino groups were immobilized on the surface of the substrate. This revealed that PAA can be directly immobilized on a stainless steel substrate without using a NiP electroless plating layer.
  • Example 3 Production of biochip substrate (2) A PAc solution containing 2 (w / v)% PAc was applied to a slide glass size SUS plate (plate thickness 2 mm) in the same manner as in Example 1, and PAc was immobilized in the same manner as in Example 1.
  • FIG. FIG. 2 revealed that carboxyl groups were immobilized on the surface of the substrate. This revealed that PAc can be directly immobilized on the stainless steel substrate without using a NiP electroless plating layer.

Abstract

Provided are: a novel substrate for biochips, which is capable of immobilizing more amino groups than publicly known substrates for biochips; and a method for producing this novel substrate for biochips. This substrate for biochips comprises: a metal substrate; and an amino group-containing polymer layer or a carboxyl group-containing polymer layer, which is immobilized on the metal substrate directly or with an NiP layer being interposed therebetween. This method for producing a substrate for biochips comprises a step wherein an amino group-containing polymer or a carboxyl group-containing polymer is applied directly to a metal substrate or to an NiP layer that is formed on the metal substrate, and then is irradiated with ultraviolet light.

Description

バイオチップ用基板及びその製造方法Biochip substrate and method for manufacturing the same
　本発明は、タンパク質、核酸、ペプチド誘導体、糖鎖とその誘導体、天然物、小分子化合物等の生体関連物質をプローブとして固定化するためのバイオチップ用基板に関する。 The present invention relates to a biochip substrate for immobilizing proteins, nucleic acids, peptide derivatives, sugar chains and derivatives thereof, natural products, small molecule compounds and other biological materials as probes.
　タンパク質チップ、ペプチドチップ、DNAチップ等のバイオチップは、各種疾患の診断や研究用として広く用いられている。従来より広く用いられているバイオチップは、通常、スライドガラス等のガラス基板上にタンパク質、ペプチド又はDNAのような生体関連物質を固定化したものである。 Biochips such as protein chips, peptide chips, and DNA chips are widely used for diagnosis and research of various diseases. Conventionally, a biochip that has been widely used is generally obtained by immobilizing a biological substance such as protein, peptide, or DNA on a glass substrate such as a slide glass.
　しかしながら、ガラス基板を用いた従来のバイオチップでは、非特異吸着が発生しやすく、測定の正確性に問題があった。また、ガラス基板は自家蛍光を発生するので、近年多く用いられるようになった蛍光標識を利用した測定においては正確性に問題があった。 However, conventional biochips using glass substrates tend to cause non-specific adsorption and have a problem in measurement accuracy. In addition, since the glass substrate generates autofluorescence, there is a problem in accuracy in measurement using a fluorescent label that has been widely used in recent years.
　これらの問題を解決すべく、金属製の基板上に活性基を有する炭素含有層を形成し、この活性基に生体関連物質を結合するバイオチップ用基板が提案されている（特許文献１）。このバイオチップ用基板は、自家蛍光を発生せず、生体関連物質の固定化が容易であり、基板の加工が容易で平坦性と表面精度が高いという優れた性質を有する。さらに、少なくとも表面が炭素から成る基板上に、アミノ基又はカルボキシル基含有ポリマーを共有結合で結合して成るバイオチップ用基板も提案されている（特許文献２）。生体関連物質は、このアミノ基に共有結合される。このバイオチップ用基板も、特許文献１記載のバイオチップ用基板と同様、優れた性質を有する。 In order to solve these problems, a biochip substrate has been proposed in which a carbon-containing layer having an active group is formed on a metal substrate, and a biological substance is bound to the active group (Patent Document 1). This biochip substrate does not generate autofluorescence, has an excellent property that it is easy to immobilize a biological substance, the substrate is easy to process, and the flatness and surface accuracy are high. Furthermore, a biochip substrate is also proposed in which an amino group or carboxyl group-containing polymer is covalently bonded to a substrate having at least a surface made of carbon (Patent Document 2). The biological substance is covalently bonded to this amino group. Similar to the biochip substrate described in Patent Document 1, this biochip substrate also has excellent properties.
特開2006-329686号公報JP 2006-329686 A 特開2010-008378号公報JP 2010-008378
　上記した公知のバイオチップ用基板は優れた性質を有しているが、固定化できるアミノ基の量をさらに増大させることができれば、生体関連物質をさらに高密度で固定化することが可能になる。 The known biochip substrate described above has excellent properties, but if the amount of amino groups that can be immobilized can be further increased, it is possible to immobilize biological materials at a higher density. .
　本発明の目的は、公知のバイオチップ用基板よりも、固定化できるアミノ基の量を増大させることが可能な新規なバイオチップ用基板及びその製造方法を提供することである。 An object of the present invention is to provide a novel biochip substrate capable of increasing the amount of amino groups that can be immobilized and a method for producing the same, as compared with known biochip substrates.
　本願発明者らは、鋭意研究の結果、金属基板上に直接又はNiP層を介してアミノ基含有ポリマー又はカルボキシル基含有ポリマーを固定化することにより、公知のバイオチップ用基板よりも、固定化できるアミノ基の量を増大させることが可能であることを見出し、本発明を完成した。 As a result of intensive studies, the inventors of the present application can immobilize an amino group-containing polymer or a carboxyl group-containing polymer directly on a metal substrate or via a NiP layer, rather than a known biochip substrate. The inventors have found that it is possible to increase the amount of amino groups and have completed the present invention.
　すなわち、本発明は、金属基板と、該金属基板上に直接又はNiP層を介して固定化されたアミノ基含有ポリマー層又はカルボキシル基含有ポリマー層とを含むバイオチップ用基板を提供する。また、本発明は、上記本発明の基板の、バイオチップの基板としての使用を提供する。更に、本発明は、金属基板上に直接、又は該金属基板上に形成されたNiP層上に、アミノ基含有ポリマー又はカルボキシル基含有ポリマーを塗布し、紫外線照射することを含む、上記本発明のバイオチップ用基板の製造方法を提供する。 That is, the present invention provides a biochip substrate comprising a metal substrate and an amino group-containing polymer layer or a carboxyl group-containing polymer layer immobilized on the metal substrate directly or via a NiP layer. The present invention also provides use of the substrate of the present invention as a biochip substrate. Furthermore, the present invention includes applying an amino group-containing polymer or a carboxyl group-containing polymer directly on a metal substrate or on a NiP layer formed on the metal substrate, and irradiating with ultraviolet rays. A method for producing a biochip substrate is provided.
　本発明のバイオチップ用基板では、基板表面上にアミノ基又はカルボキシル基が高密度に、均一に、かつ安定に結合され、このアミノ基又はカルボキシル基を利用してタンパク質や核酸等の生体関連物質から成るプローブを固定化することにより、プローブ固定化率が高く、且つ固定化密度が均一であり、さらに、タンパク質の非特異吸着を防止することにより検出感度が高く且つ再現性が高くなる。また、本発明のバイオチップ用基板では、アミノ基含有ポリマー層又はカルボキシル基含有ポリマー層が金属基板上に直接又はNiP層を介して固定化されており、公知の金属基板バイオチップのように、炭素含有層を含まないので、構造が単純であり、炭素含有層を形成するコストを削減することができる。 In the biochip substrate of the present invention, amino groups or carboxyl groups are bonded to the substrate surface in a high density, uniformly and stably, and biologically related substances such as proteins and nucleic acids using the amino groups or carboxyl groups. The probe immobilization rate is high, the immobilization density is uniform, and the non-specific adsorption of the protein is prevented to increase detection sensitivity and reproducibility. Further, in the biochip substrate of the present invention, the amino group-containing polymer layer or the carboxyl group-containing polymer layer is immobilized on the metal substrate directly or via the NiP layer, like a known metal substrate biochip, Since the carbon-containing layer is not included, the structure is simple, and the cost for forming the carbon-containing layer can be reduced.
下記実施例で作製した、ステンレス鋼基板上に直接ポリアリルアミン層を固定化したバイオチップ用基板の表面を全面TAMRA化し、蛍光を測定した結果を示す図である。It is a figure which shows the result of having carried out the whole surface TAMRA of the surface of the board | substrate for biochips which fixed the polyallylamine layer directly on the stainless steel board | substrate produced in the following Example, and measured the fluorescence. 下記実施例で作製した、ステンレス鋼基板上に直接ポリアクリル酸層を固定化したバイオチップ用基板の表面を全面TAMRA化し、蛍光を測定した結果を示す図である。It is a figure which shows the result of having made whole surface TAMRA the surface of the board | substrate for biochip which fixed the polyacrylic acid layer directly on the stainless steel board | substrate produced in the following Example, and measured the fluorescence.
　本発明のバイオチップ用基板（以下、単に「基板」と略すことがある）では、基板表面にアミノ基含有ポリマー又はカルボキシル基含有ポリマーが固定化されている点に特徴がある。ここで、「アミノ基」は、一級アミノ基、すなわち、-NH2を意味する。アミノ基又はカルボキシル基含有ポリマーとしては、好ましくは、基板表面に固定化される前の状態で、前記アミノ基又はカルボキシル基含有ポリマーを構成する構成単位の５０％以上、さらに好ましくは９０％以上、さらに好ましくは９９％以上が、それぞれアミノ基を少なくとも１個有するものである。このようなアミノ基又はカルボキシル基含有ポリマーは、１分子中に多数のアミノ基を有するので、基板表面上にアミノ基が均一かつ高密度に結合される。アミノ基又はカルボキシル基含有ポリマーは、アミノ基を有するビニル系モノマーが付加重合することにより形成されたものであることが好ましく、特にポリアリルアミン(PAA)が好ましい。また、カルボキシル基含有ポリマーとしては、好ましくは、基板表面に固定化される前の状態で、前記カルボキシル基含有ポリマーを構成する構成単位の５０％以上、さらに好ましくは９０％以上、さらに好ましくは９９％以上が、それぞれカルボキシル基を少なくとも１個有するものである。このようなカルボキシル基含有ポリマーは、１分子中に多数のカルボキシル基を有するので、基板表面上にカルボキシル基が均一かつ高密度に結合される。カルボキシル基含有ポリマーは、カルボキシル基を有するビニル系モノマーが付加重合することにより形成されたものであることが好ましく、特にポリ（メタ）アクリル酸が好ましい（「ポリ（メタ）アクリル酸」は、ポリアクリル酸(PAc)とポリメタクリル酸の総称）。なお、アミノ基含有ポリマー又はカルボキシル基含有ポリマーが基板に「固定化」されている場合には、水中で１時間振とう洗浄してもポリマーが離脱しない。 The biochip substrate of the present invention (hereinafter sometimes simply referred to as “substrate”) is characterized in that an amino group-containing polymer or a carboxyl group-containing polymer is immobilized on the substrate surface. Here, the “amino group” means a primary amino group, that is, —NH 2 . The amino group or carboxyl group-containing polymer is preferably 50% or more, more preferably 90% or more of the structural unit constituting the amino group or carboxyl group-containing polymer in a state before being immobilized on the substrate surface. More preferably, 99% or more each have at least one amino group. Since such an amino group or carboxyl group-containing polymer has a large number of amino groups in one molecule, the amino groups are uniformly and densely bound on the substrate surface. The amino group- or carboxyl group-containing polymer is preferably formed by addition polymerization of a vinyl monomer having an amino group, and polyallylamine (PAA) is particularly preferable. The carboxyl group-containing polymer is preferably 50% or more, more preferably 90% or more, more preferably 99% of the structural unit constituting the carboxyl group-containing polymer before being immobilized on the substrate surface. % Or more each have at least one carboxyl group. Since such a carboxyl group-containing polymer has a large number of carboxyl groups in one molecule, the carboxyl groups are uniformly and densely bound on the substrate surface. The carboxyl group-containing polymer is preferably formed by addition polymerization of a vinyl monomer having a carboxyl group, and poly (meth) acrylic acid is particularly preferable (“poly (meth) acrylic acid” A generic name for acrylic acid (PAc) and polymethacrylic acid. In the case where the amino group-containing polymer or the carboxyl group-containing polymer is “immobilized” on the substrate, the polymer is not detached even if it is washed with shaking for 1 hour in water.
　アミノ基含有ポリマー又はカルボキシル基含有ポリマーの平均分子量（重量平均分子量）は、本発明の効果をより良く達成する観点からPAA換算又はPAc換算で１０００以上が好ましく、さらに好ましくは２０００以上である。ここで、「PAA換算」及び「PAc換算」は、それぞれ、１分子中のアミノ基又はカルボキシル基の数を基準として換算することを意味する（以下同じ）。一方、アミノ基又はカルボキシル基含有ポリマーの分子量の上限は、特に限定されず、溶解性及び塗布液の安定性等の取扱い性に問題がない範囲であればよく、６万以下が好ましく、さらには６０００以下が好ましい。 The average molecular weight (weight average molecular weight) of the amino group-containing polymer or carboxyl group-containing polymer is preferably 1000 or more, more preferably 2000 or more in terms of PAA or PAc, from the viewpoint of better achieving the effects of the present invention. Here, “PAA conversion” and “PAc conversion” mean conversion based on the number of amino groups or carboxyl groups in one molecule, respectively (the same applies hereinafter). On the other hand, the upper limit of the molecular weight of the amino group or carboxyl group-containing polymer is not particularly limited as long as there is no problem in handling properties such as solubility and stability of the coating solution, preferably 60,000 or less, 6000 or less is preferable.
　アミノ基又はカルボキシル基含有ポリマーのみを固定化した基板にプローブを固定化したものであってもタンパク質の非特異吸着はあまり起きないが、疎水結合等によるタンパク質等の非特異吸着をさらに防止することが望まれる場合には、アミノ基又はカルボキシル基含有ポリマーに加え、親水性ポリマーを基板表面に共有結合で固定化することができる。親水性ポリマーとしては、アミノ基又はカルボキシル基との反応性の低いものを使用する。親水性ポリマーの例として、ポリエーテル類、ポリアクリルアミド、アガロース等を挙げることができる。特にポリエーテル類の中でもポリエチレングリコール（以下、「ＰＥＧ」と略すことがある）は分子量分布を制御したものが使用可能であり、安定性も高く、生体物質を不活性化しないため好ましい。親水性ポリマーの平均分子量はアミノ基又はカルボキシル基含有ポリマーのリンカーまたはプローブとの反応性を阻害しない分子量が好ましく、２００～１０，０００であり、より好ましくは１，０００である。 Non-specific adsorption of protein does not occur even if the probe is immobilized on a substrate on which only an amino group or carboxyl group-containing polymer is immobilized, but it further prevents non-specific adsorption of proteins due to hydrophobic bonds, etc. In addition to the amino group- or carboxyl group-containing polymer, a hydrophilic polymer can be covalently immobilized on the substrate surface. As the hydrophilic polymer, a polymer having low reactivity with an amino group or a carboxyl group is used. Examples of the hydrophilic polymer include polyethers, polyacrylamide, agarose and the like. In particular, among the polyethers, polyethylene glycol (hereinafter sometimes abbreviated as “PEG”) having a controlled molecular weight distribution can be used, is highly stable, and is preferable because it does not inactivate biological materials. The average molecular weight of the hydrophilic polymer is preferably a molecular weight that does not inhibit the reactivity of the amino group or carboxyl group-containing polymer with the linker or probe, and is 200 to 10,000, more preferably 1,000.
　親水性ポリマーを固定化する場合には、アミノ基の均一な分布を確保するために、アミノ基又はカルボキシル基含有ポリマーと親水性ポリマーは均一に混合されていることが好ましい。すなわち、アミノ基又はカルボキシル基含有ポリマーと親水性ポリマーの均一な混合物を後述する方法で基板表面上に固定化することが好ましい。 When immobilizing the hydrophilic polymer, it is preferable that the amino group- or carboxyl group-containing polymer and the hydrophilic polymer are uniformly mixed in order to ensure a uniform distribution of amino groups. That is, it is preferable to fix a uniform mixture of an amino group or carboxyl group-containing polymer and a hydrophilic polymer on the substrate surface by the method described later.
　親水性ポリマーは、任意成分であるので、固定化しなくてもよいが、固定化する場合には、その固定化量は、アミノ基又はカルボキシル基含有ポリマーの固定化量以下にすることが、十分な数のアミノ基又はカルボキシル基を基板表面上に結合する上で好ましい。また、アミノ基又はカルボキシル基含有ポリマーと親水性ポリマーの両方を固定化する場合でも、アミノ基又はカルボキシル基含有ポリマーの基板表面への固定化量は、上記の通りポリアリルアミン換算又はポリアクリル酸換算で４μｇ／ｃｍ２以上であることが好ましい。一方、アミノ基又はカルボキシル基含有ポリマーと親水性ポリマーの合計固定化量は、アミノ基又はカルボキシル基含有ポリマー単独の場合と同様、４０μｇ／ｃｍ２以下であることが好ましい。親水性ポリマーの固定化量により、基板の親水性を調節することができる。 Since the hydrophilic polymer is an optional component, it does not need to be immobilized. It is preferable for bonding a large number of amino groups or carboxyl groups on the substrate surface. Moreover, even when immobilizing both an amino group or carboxyl group-containing polymer and a hydrophilic polymer, the amount of amino group or carboxyl group-containing polymer immobilized on the substrate surface is converted to polyallylamine or polyacrylic acid as described above. 4 μg / cm 2 or more is preferable. On the other hand, the total immobilization amount of the amino group or carboxyl group-containing polymer and the hydrophilic polymer is preferably 40 μg / cm 2 or less, as in the case of the amino group or carboxyl group-containing polymer alone. The hydrophilicity of the substrate can be adjusted by the amount of the hydrophilic polymer immobilized.
　金属基板としては、特に限定されないが、アルミニウム、ステンレス鋼等の通常の金属から成る基板を好ましく用いることができる。 The metal substrate is not particularly limited, but a substrate made of a normal metal such as aluminum or stainless steel can be preferably used.
　アミノ基又はカルボキシル基含有ポリマー層は、アミノ基又はカルボキシル基含有ポリマー溶液を、金属基板表面に直接又はNiP層を介して（すなわち、金属基板上に形成されたNiP層上に塗布する）塗布し、紫外線照射することにより、金属基板に直接又はNiP層を介して固定化することができる。なお、NiP（ニッケル－リン）層は、好ましくは、無電解めっきにより形成することができ、この方法自体は公知であり、特許文献１及び特許文献２にも記載されている。 The amino group or carboxyl group-containing polymer layer is applied by applying an amino group or carboxyl group-containing polymer solution to the surface of the metal substrate directly or via the NiP layer (that is, coating on the NiP layer formed on the metal substrate). By irradiating with ultraviolet rays, it can be fixed to the metal substrate directly or via a NiP layer. The NiP (nickel-phosphorus) layer can be preferably formed by electroless plating. This method is known per se and is described in Patent Document 1 and Patent Document 2.
　上記固定化方法は、ポリマー溶液を基板表面に塗布後、光照射によって生成するラジカルにより結合させるもので、照射する光としては、波長１５０ｎｍ～２６０ｎｍ程度、例えば波長１８４ｎｍや２５４ｎｍ等の紫外線を使用することができる。この波長の光によりＣ－Ｃ、Ｃ－Ｏ、Ｃ－Ｈ結合が切断され、ラジカルが発生する。このとき空気中の酸素分子、水分子も分解されて酸素ラジカル、オゾンが発生し基板材料カーボンとポリマーの酸化分解も同時に起こり共有結合生成阻害の原因となる。これを防止するため本製造方法は減圧下または、不活性ガス雰囲気下で光照射を実施することが好ましい。減圧は大気圧を基準（０ＭＰａ）として－０．０５ＭＰａ以下、より好ましくは－０．０８ＭＰａ以下の真空度で実施する。不活性ガスはアルゴン、ヘリウム等の光照射を受けてもラジカル化しにくい希ガス元素を使用する。光の照射量は、ポリマーを共有結合するのに必要な量であればよく、エネルギー量として、基板表面１ｃｍ２当たり通常、約１～６ジュール、好ましくは約２～４ジュール程度である。例えば、基板表面１ｃｍ２当たり１８．５ｍＷの紫外線を通常１～５分、好ましくは２～４分照射する。また、ポリマーの塗布量は、上記したポリマーの固定化量を達成する量である。ポリマー溶液の濃度は、必要なポリマー塗布量を達成でき、均一に塗布できる濃度であればよく、0.5～３(w/v)％程度が好ましい。 The immobilization method is a method in which a polymer solution is applied to a substrate surface and then bonded by radicals generated by light irradiation. As light to be irradiated, ultraviolet light having a wavelength of about 150 nm to 260 nm, for example, a wavelength of 184 nm or 254 nm is used. be able to. Light of this wavelength breaks C—C, C—O, and C—H bonds to generate radicals. At this time, oxygen molecules and water molecules in the air are also decomposed to generate oxygen radicals and ozone, and oxidative decomposition of the substrate material carbon and the polymer occurs simultaneously, thereby inhibiting the formation of covalent bonds. In order to prevent this, it is preferable to carry out light irradiation in this production method under reduced pressure or in an inert gas atmosphere. Depressurization is performed at a vacuum degree of −0.05 MPa or less, more preferably −0.08 MPa or less with reference to atmospheric pressure (0 MPa). As the inert gas, a rare gas element that is difficult to be radicalized even when irradiated with light such as argon or helium is used. The amount of light irradiation may be an amount necessary for covalently bonding the polymer, and the amount of energy is usually about 1 to 6 joules, preferably about 2 to 4 joules per 1 cm 2 of the substrate surface. For example, 18.5 mW of ultraviolet light per 1 cm 2 of the substrate surface is usually irradiated for 1 to 5 minutes, preferably 2 to 4 minutes. The polymer coating amount is an amount that achieves the above-described polymer immobilization amount. The concentration of the polymer solution is not particularly limited as long as the required amount of polymer application can be achieved and can be uniformly applied, and is preferably about 0.5 to 3 (w / v)%.
　ポリマーの基板表面への塗布は、一般的な手法を用いることができ、ポリマーの塗布量を制御できる手法であれば、特にその方法を限定するものではない。例えば、ロールコート、スプレーコート、スピンコート、ディップコート等から選択し実施可能である。 A general method can be used to apply the polymer to the substrate surface, and the method is not particularly limited as long as the method can control the amount of the polymer applied. For example, it can be selected from roll coating, spray coating, spin coating, dip coating, and the like.
　上記のようにして得られる本発明の基板は、表面に均一かつ高密度にアミノ基を有する。このアミノ基を利用して、生体関連物質を共有結合により固定化することによりバイオチップを構成することができる。生体関連物質としては、バイオチップにおいてプローブとして用いられているいずれの物質であってもよく、任意のポリペプチド（天然又は合成のタンパク質、オリゴペプチドを包含）、核酸（DNA及びRNA並びに人工核酸を包含）、糖、脂質、これらの複合体（糖タンパク質等）並びに誘導体（修飾タンパク質や核酸等）が挙げられる。 The substrate of the present invention obtained as described above has amino groups uniformly and densely on the surface. By using this amino group, a biochip can be constructed by immobilizing a biological substance by covalent bond. The bio-related substance may be any substance used as a probe in a biochip, and may be any polypeptide (including natural or synthetic proteins and oligopeptides), nucleic acid (DNA and RNA, and artificial nucleic acid). Inclusion), sugars, lipids, complexes thereof (glycoproteins, etc.) and derivatives (modified proteins, nucleic acids, etc.).
　生体関連物質は、アミノ基に直接共有結合することもできるし、所望のリンカーを介して結合することもできる。表面にアミノ基を有するバイオチップ用基板自体は広く市販されているので、生体関連物質のアミノ基への共有結合は、周知の常法により容易に行なうことができる。生体関連物質がタンパク質のように、アミノ基と結合するカルボキシル基等を有している場合には直接結合することができ、このような官能基を有していない場合、あるいは所望により、リンカーを介して結合することも可能である。リンカーも周知であり、例えば、一端にカルボキシル基、他端に例えばマレイミド基等を有するものが広く用いられている。 The biological material can be directly covalently bonded to the amino group or can be bonded via a desired linker. Since the biochip substrate itself having an amino group on its surface is widely commercially available, the covalent bond of the biological substance to the amino group can be easily carried out by a well-known conventional method. When a biological substance has a carboxyl group that binds to an amino group, such as a protein, it can be directly bonded. If it does not have such a functional group, or if desired, a linker can be added. It is also possible to couple via. Linkers are also well known. For example, those having a carboxyl group at one end and a maleimide group at the other end are widely used.
　以下、本発明を実施例及び比較例に基づきより具体的に説明する。もっとも、本発明は下記実施例に限定されるものではない。 Hereinafter, the present invention will be described more specifically based on examples and comparative examples. However, the present invention is not limited to the following examples.
実施例１
１．　バイオチップ用基板の作製（その１）
　アルミニウム合金（5000系合金）の圧延板（７５×２５ｍｍ厚さ１ｍｍ）をＰＶＡ砥石で厚さ０．９８ｍｍまで研削し、表面粗さをＲａ３０ｎｍとした後、全面をアルカリ脱脂、硝酸デスマット、亜鉛ジンケート処理を行い、ニッケル－リン無電解めっきを片面あたり厚さ１３μｍつけた。さらに、両面をアルミナスラリーにより片面あたり３μｍ研磨し表面粗さＲａを１ｎｍとした。　ＰＡＡ（平均分子量3,000）をエタノールで1(w/v)％となるように濃度調整したものをコーティング液とした。ＰＡＡコーティング液を適量ピペットで分取し、基板材料表面に滴下した後、ＰＡＡ塗布量が２０μｇ／ｃｍ２になるように塗工厚さを３ｍｉｌ(約0.076mm)に設定したベーカー式アプリケーターにより基板材料表面全体にコーティング液を塗り広げた。溶媒が揮発してからさらに1時間真空乾燥した（真空度-0.098ＭＰａ）のち、そのまま真空下で3分間紫外線を照射（18.5ｍＷ／ｃｍ２，254ｎｍ）しＰＡＡを固定化した。さらに超純水で1時間振とう洗浄して未反応のＰＡＡを除去してからスピン乾燥させ本発明のバイオチップ用基板を得た。 Example 1
1. Production of biochip substrate (Part 1)
An aluminum alloy (5000 series alloy) rolled plate (75 x 25 mm thickness 1 mm) is ground with a PVA grinding wheel to a thickness of 0.98 mm and the surface roughness Ra is set to 30 nm, and then the entire surface is alkali degreased, desmut nitrate, zinc zincate The treatment was performed, and nickel-phosphorous electroless plating was applied to a thickness of 13 μm per side. Furthermore, both surfaces were polished 3 μm per side with an alumina slurry, and the surface roughness Ra was set to 1 nm. A coating solution was prepared by adjusting the concentration of PAA (average molecular weight 3,000) to 1 (w / v)% with ethanol. After a suitable amount of PAA coating solution is dispensed with a pipette and dropped onto the substrate material surface, the substrate is coated with a baker type applicator in which the coating thickness is set to 3 mil (about 0.076 mm) so that the PAA coating amount is 20 μg / cm 2. The coating solution was spread over the entire material surface. After the solvent was volatilized, it was further vacuum-dried for 1 hour (vacuum degree -0.098 MPa), and then irradiated with ultraviolet rays (18.5 mW / cm 2 , 254 nm) for 3 minutes under vacuum to immobilize PAA. Further, the unreacted PAA was removed by shaking and washing with ultrapure water for 1 hour, followed by spin drying to obtain the biochip substrate of the present invention.
２．　評価
　実施例１で得られたバイオチップ用基板の表面を３つの領域に分け、各領域の全面に市販の蛍光標識であるTAMRA（テトラメチルローダミン）を結合した。この全面TAMRA化は、具体的に次のようにして行った。0mM N,N,N',N'-テトラメチル-(5又は6）-カルボキシローダミン（ＴＡＭＲＡ）、10ｍＭ ＨＢＴＵ（２－１H［ベンゾトリアゾルー１－イル］―１，１，３，３，テトラメチルウロニウムーヘキサフルオロホスフェート）、20ｍＭ ＤＩＥＡ（ジイソプロピルエチルアミン）／ＤＭＦ（ジメチルホルムアミド）溶液に基板を浸漬し室温で1時間振とうさせてアミノ基にＴＡＭＲＡを結合させた。続いてメタノール洗浄で過剰なＴＡＭＲＡを洗い流した後、基板を乾燥した。この方法から明らかなように、基板に結合されたTAMRAの量は、基板表面上のアミノ基の量が多くなるほど多くなる。従って、基板に結合されたTAMRAの量を測定することにより、基板表面上のアミノ基の相対量を測定することができる。 2. Evaluation The surface of the biochip substrate obtained in Example 1 was divided into three regions, and commercially available fluorescent label TAMRA (tetramethylrhodamine) was bound to the entire surface of each region. This full-scale TAMRA conversion was specifically performed as follows. 0 mM N, N, N ′, N′-tetramethyl- (5 or 6) -carboxyrhodamine (TAMRA), 10 mM HBTU (2-1H [benzotriazol-1-yl] -1,1,3,3 The substrate was immersed in a tetramethyluronium-hexafluorophosphate), 20 mM DIEA (diisopropylethylamine) / DMF (dimethylformamide) solution and shaken at room temperature for 1 hour to bind TAMRA to the amino group. Subsequently, excess TAMRA was washed away by methanol washing, and then the substrate was dried. As is apparent from this method, the amount of TAMRA bound to the substrate increases as the amount of amino groups on the substrate surface increases. Therefore, by measuring the amount of TAMRA bound to the substrate, the relative amount of amino groups on the substrate surface can be measured.
　水洗後、蛍光スキャナーを用い以下の条件で測定した。 
励起波長：532 nm（フィルター570nm）、635nm(フィルター670nm)、 
レーザー出力：固定 、PMT：50 ％、解像度：10 mm
各励起波長で測定された蛍光値を下記表１に示す。 After washing with water, measurement was performed under the following conditions using a fluorescent scanner.
Excitation wavelength: 532 nm (filter 570 nm), 635 nm (filter 670 nm),
Laser output: Fixed, PMT: 50%, Resolution: 10 mm
The fluorescence values measured at each excitation wavelength are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１に示す結果から、基板上にアミノ基が固定化されていることが確認された。 From the results shown in Table 1, it was confirmed that the amino group was immobilized on the substrate.
　比較のため、特許文献２に記載されている、アルミニウム板／NiPめっき層／カーボン層／PAA層を持つバイオチップ用基板に上記と同様に全面TAMRA化を行い、蛍光値を測定したところ、TAMRA化前の蛍光値では上記実施例１と同程度であったが、TAMRA化後の蛍光値は約3000程度であり、本発明の基板の方がアミノ基の量が約４倍多かった。 For comparison, TAMRA was applied to a biochip substrate having an aluminum plate / NiP plating layer / carbon layer / PAA layer described in Patent Document 2 in the same manner as described above, and the fluorescence value was measured. The fluorescence value before the formation was about the same as in Example 1 above, but the fluorescence value after the TAMRA was about 3000, and the amount of amino groups was about 4 times higher in the substrate of the present invention.
実施例２
１．　バイオチップ用基板の作製（その２）
　スライドガラスサイズのSUS板（板厚2mm)に、2(w/v)%のPAAを含むPAA溶液を実施例１と同様に塗布し、実施例１と同様にしてPAAを固定化した。 Example 2
1. Production of biochip substrate (2)
A PAA solution containing 2 (w / v)% PAA was applied to a slide glass size SUS plate (plate thickness 2 mm) in the same manner as in Example 1, and PAA was immobilized in the same manner as in Example 1.
２．　評価
　実施例１と同様にして全面TAMRA化を行い、水洗後、FLA-8000で蛍光強度を測定した。測定条件は以下の通りであった。
　PMT 50%，励起波長532nm，蛍光フィルター570nm，解像度10μm 2. Evaluation In the same manner as in Example 1, TAMRA was formed on the entire surface, and after washing with water, the fluorescence intensity was measured with FLA-8000. The measurement conditions were as follows.
PMT 50%, excitation wavelength 532nm, fluorescence filter 570nm, resolution 10μm
　結果を図１に示す。図１から、基板の表面にアミノ基が固定化されていることが明らかになった。このことから、NiP無電解メッキ層を介することなく、ステンレス鋼基板上に直接PAAを固定化することが可能であることが明らかになった。 The results are shown in FIG. FIG. 1 revealed that amino groups were immobilized on the surface of the substrate. This revealed that PAA can be directly immobilized on a stainless steel substrate without using a NiP electroless plating layer.
実施例３
１．　バイオチップ用基板の作製（その２）
　スライドガラスサイズのSUS板（板厚2mm)に、2(w/v)%のPAcを含むPAc溶液を実施例１と同様に塗布し、実施例１と同様にしてPAcを固定化した。 Example 3
1. Production of biochip substrate (2)
A PAc solution containing 2 (w / v)% PAc was applied to a slide glass size SUS plate (plate thickness 2 mm) in the same manner as in Example 1, and PAc was immobilized in the same manner as in Example 1.
２．　評価
　実施例１と同様にして全面TAMRA化を行い、水洗後、実施例２と同様に蛍光強度を測定した。 2. Evaluation In the same manner as in Example 1, TAMRA was formed on the entire surface. After washing with water, the fluorescence intensity was measured in the same manner as in Example 2.
　結果を図２に示す。図２から、基板の表面にカルボキシル基が固定化されていることが明らかになった。このことから、NiP無電解メッキ層を介することなく、ステンレス鋼基板上に直接PAcを固定化することが可能であることが明らかになった。 The results are shown in FIG. FIG. 2 revealed that carboxyl groups were immobilized on the surface of the substrate. This revealed that PAc can be directly immobilized on the stainless steel substrate without using a NiP electroless plating layer.

Claims (12)

  1. 　金属基板と、該金属基板上に直接又はNiP層を介して固定化されたアミノ基含有ポリマー層又はカルボキシル基含有ポリマー層とを含むバイオチップ用基板。 A biochip substrate comprising a metal substrate and an amino group-containing polymer layer or a carboxyl group-containing polymer layer immobilized on the metal substrate directly or via a NiP layer.
  2. 　前記アミノ基含有ポリマーが、ポリアリルアミンである請求項１記載のバイオチップ用基板。 The biochip substrate according to claim 1, wherein the amino group-containing polymer is polyallylamine.
  3. 　前記カルボキシル基含有ポリマーが、ポリ（メタ）アクリル酸である請求項１記載のバイオチップ用基板。 The biochip substrate according to claim 1, wherein the carboxyl group-containing polymer is poly (meth) acrylic acid.
  4. 　前記アミノ基含有ポリマー又はカルボキシル基含有ポリマー層が、前記金属基板又はNiP層上に前記アミノ基含有ポリマー又はカルボキシル基含有ポリマーの溶液を塗布した後、紫外線照射することにより固定化されたものである請求項１～３のいずれか１項に記載のバイオチップ用基板。 The amino group-containing polymer or carboxyl group-containing polymer layer is fixed by applying an ultraviolet ray after applying a solution of the amino group-containing polymer or carboxyl group-containing polymer on the metal substrate or NiP layer. The biochip substrate according to any one of claims 1 to 3.
  5. 　前記金属基板がアルミニウム製であり、前記アミノ基含有ポリマー又はカルボキシル基含有ポリマーが前記NiP層を介して前記金属基板上に固定化されている請求項１～４のいずれか１項に記載のバイオチップ用基板。 The bio of any one of claims 1 to 4, wherein the metal substrate is made of aluminum, and the amino group-containing polymer or carboxyl group-containing polymer is immobilized on the metal substrate via the NiP layer. Chip substrate.
  6. 　前記金属基板がステンレス鋼製であり、前記アミノ基含有ポリマー又はカルボキシル基含有ポリマーが前記金属基板上に直接固定化されている請求項１～４のいずれか１項に記載のバイオチップ用基板。 The biochip substrate according to any one of claims 1 to 4, wherein the metal substrate is made of stainless steel, and the amino group-containing polymer or carboxyl group-containing polymer is directly immobilized on the metal substrate.
  7. 　請求項１～６に記載の基板の、バイオチップの基板としての使用。 Use of the substrate according to claims 1 to 6 as a biochip substrate.
  8. 　金属基板上に直接、又は該金属基板上に形成されたNiP層上に、アミノ基含有ポリマー又はカルボキシル基含有ポリマーを塗布し、紫外線照射することを含む、請求項１記載のバイオチップ用基板の製造方法。 The biochip substrate according to claim 1, comprising applying an amino group-containing polymer or a carboxyl group-containing polymer directly on the metal substrate or on the NiP layer formed on the metal substrate and irradiating with ultraviolet rays. Production method.
  9. 　前記アミノ基含有ポリマーが、ポリアリルアミンである請求項８記載のバイオチップ用基板の製造方法。 The method for producing a biochip substrate according to claim 8, wherein the amino group-containing polymer is polyallylamine.
  10. 　前記カルボキシル基含有ポリマーが、ポリ（メタ）アクリル酸である請求項８記載のバイオチップ用基板の製造方法。 The method for producing a biochip substrate according to claim 8, wherein the carboxyl group-containing polymer is poly (meth) acrylic acid.
  11. 　前記金属基板がアルミニウム製であり、前記アミノ基含有ポリマー又はカルボキシル基含有ポリマーが前記NiP層を介して前記金属基板上に固定化される請求項８～１０のいずれか１項に記載のバイオチップ用基板の製造方法。 The biochip according to any one of claims 8 to 10, wherein the metal substrate is made of aluminum, and the amino group-containing polymer or carboxyl group-containing polymer is immobilized on the metal substrate via the NiP layer. Manufacturing method for industrial use.
  12. 　前記金属基板がステンレス鋼製であり、前記アミノ基含有ポリマー又はカルボキシル基含有ポリマーが前記金属基板上に直接固定化されている請求項８～１０のいずれか１項に記載のバイオチップ用基板の製造方法。 The biochip substrate according to any one of claims 8 to 10, wherein the metal substrate is made of stainless steel, and the amino group-containing polymer or carboxyl group-containing polymer is directly immobilized on the metal substrate. Production method.
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JP2004097173A (en) * 2002-07-17 2004-04-02 Toyo Kohan Co Ltd Solid support having electrostatic layer and its use
JP2009216555A (en) * 2008-03-11 2009-09-24 Dkk Toa Corp Detection surface and its forming method
JP2010008378A (en) * 2008-06-30 2010-01-14 Hipep Laboratories Biochip substratum and method for production thereof
JP2011027552A (en) * 2009-07-24 2011-02-10 Hitachi High-Technologies Corp Method for inhibition of fine particle aggregation, and preservative solution

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2004097173A (en) * 2002-07-17 2004-04-02 Toyo Kohan Co Ltd Solid support having electrostatic layer and its use
JP2009216555A (en) * 2008-03-11 2009-09-24 Dkk Toa Corp Detection surface and its forming method
JP2010008378A (en) * 2008-06-30 2010-01-14 Hipep Laboratories Biochip substratum and method for production thereof
JP2011027552A (en) * 2009-07-24 2011-02-10 Hitachi High-Technologies Corp Method for inhibition of fine particle aggregation, and preservative solution

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