JP4811355B2 - Biodevice, manufacturing method thereof, and biosensor - Google Patents

Description

  The present invention relates to a biodevice used for detection and analysis of a large number of proteins in a biological sample and a method for producing the same. More specifically, the present invention relates to proteomics and biotechnology used for measurement of gene activity at the intracellular protein level. The present invention relates to a device and a manufacturing method thereof.

  Attempts to decipher the genetic activity and the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more detailed about the biological functions of cells. Provide information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level, rather than at the gene level. It also includes studies of events that are not encoded by genes such as post-translational modifications of proteins and interactions between proteins.

  Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research has become increasingly popular, and as a result, rapid detection of bioactive substances has been accelerated. There is a need for higher efficiency (high throughput). A DNA chip has been developed and put into practical use as a molecular array for this purpose. On the other hand, protein chips have been proposed for the detection of the most complex and highly diverse proteins in biological functions, and research has been promoted in recent years. The protein chip is a general term for a protein or a molecule that captures the protein immobilized on the surface of the chip (micro substrate).

  However, since current protein chips are generally developed on the extended line of DNA chips, studies are being made to immobilize proteins or molecules that capture them on a solid surface such as a glass substrate. (For example, refer to Patent Document 1).

    A protein is a polymer in which amino acids are linked in a straight chain by peptide bonds, requires a certain higher order structure for function expression, and is often composed of a plurality of subunits. When this is immobilized on the solid phase surface, the three-dimensional structure changes due to adhesion to the solid phase surface, which may impair the function of the protein and lose its interaction. Further, depending on the relative positional relationship between the interaction site and the solid surface, the interaction may be sterically hindered. For this reason, there is a need for a biodevice that can immobilize proteins with as little influence on the three-dimensional structure as possible.

In terms of profiling the fluctuations of all proteins (proteomes), microfluidics technology that enables the manipulation of ultra-trace amounts of proteins and ultra-small quantities of solutions such as several nanoliters, and on-chip The concept of “lab-on-a-chip” that targets processing, separation, and detection becomes important. Also in this technique, it is necessary that a physiologically active substance such as a protein as a sample is immobilized in a state where the three-dimensional structure is maintained as much as possible in the flow path.
JP 2001-116750 A

  The present invention relates to a biodevice capable of detecting a physiologically active substance with high sensitivity and high throughput having a field capable of immobilizing a physiologically active substance with as little influence on the three-dimensional structure as possible, and a method for producing the same, and An object of the present invention is to provide a biosensor in which a physiologically active substance is immobilized on the device.

（５）前記一般式［１］において、Ｘがエチレンオキシ基である（４）記載のバイオデバイス、
（６）前記一般式［１］において、Ｒ４がアルキル基である（４）又は（５）記載のバイオデバイス、
（７）前記アルキル基の炭素数が２〜１０であ（６）記載のバイオデバイス。
（８）前記一般式［１］において、Ｙが下記一般式［２］または［３］（式中ｑ、ｒは１〜２０の整数である。）である（４）〜（７）いずれか記載のバイオデバイス。

（９）前記多糖類が、キチン及びキトサン、ペクチン、グアーガム、キサンタンガム、アラビアガム、ジェランガム、ファーセレラン、カルボキシメチルセルロース、ヘパリン、ヒアルロン酸、プルラン、デキストラン又はその誘導体、カラギーナン、セファロース、及びアガロースから成る群より選ばれる少なくとも１種である、（１）記載のバイオデバイス、
（１０）前記固相基体の材質がプラスチックである（１）〜（９）いずれか記載のバイオデバイス、
（１１）前記プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、及びそれらの共重合体よりなる群より選択された少なくとも１種である（１０）記載のバイオデバイス、
（１２）前記固相基体の材質がガラスである（１）〜（９）いずれか記載のバイオデバイス、
（１３）デバイスの形状がスライド形状基板、チューブ、ビーズ、９６穴ないし３８４穴プレート、マイクロフルイディクス基板及びそれらの複合体よりなる群より選択された少なくとも１種である（１）〜（１２）いずれか記載のバイオデバイス、
（１４）（１）〜（１３）いずれか記載のバイオデバイスの製造方法であって、
（１）ホスホリルコリン基を有するユニット、疎水性基を有するユニット及び一級アミノ基を有するユニットを含む高分子化合物を基体表面に塗布する工程、
（２）上記塗布基体の表面にさらに親水性化合物を化学結合させる工程、
を含むことを特徴とするバイオデバイスの製造方法、
（１５）前記（２）の工程において、親水性化合物を基体表面に塗布する工程を有する（１４）記載のバイオデバイスの製造方法、
（１６）（１）〜（１３）いずれか記載のバイオデバイスに生理活性物質を固定化させたバイオセンサー。
（１７）生理活性物質がＤＮＡ、ペプチド、蛋白質及びそれらの混合物よりなる群より選択された少なくとも１種であるである（１６）記載のバイオセンサー、
である。
That is, the present invention
(1) having a polymer compound containing a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group on the surface of a solid phase substrate, and a hydrophilic compound is bonded to the surface; A biodevice, wherein the active compound is a polysaccharide ,
(2) The biodevice according to (1), wherein the primary amino group is an oxylamino group and / or a hydrazide group,
(3) The biodevice according to (1) or (2), wherein the main chain of the polymer compound is a (meth) acrylic skeleton,
(4) The polymer compound is represented by the following general formula [1] (wherein R1, R2, and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, and X represents an alkyleneoxy group having 1 to 10 carbon atoms). , P represents an integer of 1 to 20. When p is an integer of 2 to 20, repeated Xs may be the same or different, and Y is a spacer containing an alkylene glycol residue. Z is an oxygen atom or NH. L, m, and n are natural numbers.) The biodevice according to any one of (1) to (3),

(5) The biodevice according to (4), wherein, in the general formula [1], X is an ethyleneoxy group,
(6) The biodevice according to (4) or (5), wherein, in the general formula [1], R4 is an alkyl group,
(7) The biodevice according to (6), wherein the alkyl group has 2 to 10 carbon atoms.
(8) In the general formula [1], any one of (4) to (7), wherein Y is the following general formula [2] or [3] (where q and r are integers of 1 to 20). The biodevice described.

( 9 ) The polysaccharide is selected from the group consisting of chitin and chitosan, pectin, guar gum, xanthan gum, gum arabic, gellan gum, farseleran, carboxymethylcellulose, heparin, hyaluronic acid, pullulan, dextran or derivatives thereof, carrageenan, sepharose, and agarose. The biodevice according to ( 1 ), which is at least one selected
( 10 ) The biodevice according to any one of (1) to ( 9 ), wherein the solid phase substrate is made of plastic.
(11) wherein the plastic is polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefins, polypentene, polyamides, and at least one selected from the group consisting of copolymers thereof (10), wherein the bio-devices,
( 12 ) The biodevice according to any one of (1) to ( 9 ), wherein the solid phase substrate is made of glass.
( 13 ) The shape of the device is at least one selected from the group consisting of a slide-shaped substrate, a tube, a bead, a 96-hole to 384-well plate, a microfluidic substrate, and a complex thereof (1) to ( 12 ) Any one of the biodevices,
( 14 ) The method for producing a biodevice according to any one of (1) to ( 13 ),
(1) A step of applying a polymer compound containing a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group to the surface of the substrate,
(2) a step of further chemically bonding a hydrophilic compound to the surface of the coated substrate;
A method for producing a biodevice, comprising:
( 15 ) The method for producing a biodevice according to ( 14 ), wherein in the step (2), the method comprises a step of applying a hydrophilic compound to the surface of the substrate.
( 16 ) A biosensor in which a bioactive substance is immobilized on the biodevice according to any one of (1) to ( 13 ).
( 17 ) The biosensor according to ( 16 ), wherein the physiologically active substance is at least one selected from the group consisting of DNA, peptides, proteins and mixtures thereof,
It is.

  According to the biodevice of the present invention and the method for producing the same, and the biosensor in which the physiologically active substance is immobilized on the device, the physiologically active substance can be immobilized while minimizing the influence on the three-dimensional structure. A biosensor capable of detecting a physiologically active substance with high sensitivity and sensitivity can be obtained.

The biodevice of the present invention has a polymer compound containing a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group on the surface of the substrate, and a hydrophilic compound is bonded to the surface thereof. It is characterized by.
This polymer compound has a property of suppressing physical adsorption (nonspecific adsorption) of the detection target substance to the base material. Furthermore, it also has the property of immobilizing hydrophilic compounds. The phosphorylcholine group plays a role in suppressing nonspecific adsorption, and the primary amino group plays a role in immobilizing the hydrophilic compound.

    The unit having a phosphorylcholine group contained in the polymer compound of the present invention is not particularly limited in structure, but as shown in the structural unit on the left side of the structural unit of the following general formula [1], a (meth) acrylic residue And a phosphorylcholine group are preferably bonded via a chain of an alkyleneoxy group X having 1 to 10 carbon atoms. Of these, X is most preferably an ethyleneoxy group. The repeating number of the alkyleneoxy group X in the formula is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon number of the repeated alkyleneoxy group may be the same or different. Although l is naturally a natural number, it may be expressed as a composition ratio of each component.

  The composition ratio of the unit having a phosphorylcholine group contained in the polymer compound of the present invention (ratio of l to the sum of l, m, and n) is not particularly limited, but is 5 for all units of the polymer compound. -98 mol% is preferable, More preferably, it is 10-90 mol%, Most preferably, it is 10-80 mol%. When the composition ratio falls below the lower limit, nonspecific adsorption increases. On the other hand, when the upper limit is exceeded, water solubility increases, and the polymer compound may be eluted during the assay. However, this is not the case when a functional group capable of covalently bonding to the substrate is introduced into any part of the polymer compound and chemically bonded to the substrate. For example, a method of introducing a silane coupling agent into the polymer compound is simple and preferable.

    The unit having a hydrophobic group contained in the polymer compound of the present invention is not particularly limited in structure. However, as represented by the structural unit in the center of the structural unit of the general formula [1], (meth) acrylic A structure in which a hydrophobic group is bonded to a group residue is preferable. Although a hydrophobic group is not specifically limited, An alkyl group and aromatics are mentioned. More preferably, the alkyl group is an alkyl group having 2 to 20 carbon atoms. The structure of the alkyl group is not particularly limited, and may be linear, branched, or cyclic. By including a unit having a hydrophobic group in the polymer compound, the wettability is improved even on a hydrophobic substrate such as a plastic, and coating can be performed without unevenness. In addition, since the hydrophobicity increases, it is possible to prevent the polymer compound from eluting during the assay. In the formula, m is naturally a natural number, but may be expressed as a composition ratio of each component.

  The composition ratio of the unit having a hydrophobic group contained in the polymer compound of the present invention (the ratio of m to the sum of l, m, and n) is not particularly limited, but is based on all units of the polymer compound. 1 to 90 mol% is preferable, more preferably 10 to 80 mol%, and most preferably 20 to 80 mol%. If the upper limit is exceeded, nonspecific adsorption may increase.

  The unit having a primary amino group contained in the polymer compound of the present invention is not particularly limited in structure, but as represented by the structural unit on the right side of the structural unit of the general formula [1], It is preferable that the (meth) acryl group residue and the oxylamino group or hydrazide group have a structure via a spacer Y containing an alkylene glycol residue. In the case of an oxylamino group, Z represents an oxygen atom, and in the case of a hydrazide group, Z represents NH. n is originally a natural number, but may be expressed as a composition ratio of each component. The structure of the spacer Y containing an alkylene glycol residue is not particularly limited, but may be the following general formula [2] or [3] (wherein q and r are integers of 1 to 20). preferable.

    The oxylamino group or hydrazide group can immobilize sugars, sugar chains, glycopeptides, antibodies, and / or hydrophilic compounds having these. Further, since the amino group is separated from the main chain portion due to the presence of the spacer, the hydrophilic compound is easily immobilized. Furthermore, since an alkylene glycol residue is contained in the spacer, non-specific adsorptivity can be expressed. The number of carbon atoms of the alkylene glycol residue is not limited, but an ethylene glycol residue having 2 carbon atoms is most preferable.

  The composition ratio of the unit having a primary amino group contained in the polymer compound of the present invention (ratio of n to the sum of l, m, and n) is not particularly limited, but for all units of the polymer compound 1-94 mol%, more preferably 2-90 mol%, most preferably 5-80 mol%. When the composition ratio is lower than the lower limit, the sugar, sugar chain and / or hydrophilic compound having these cannot be sufficiently immobilized. On the other hand, if the upper limit is exceeded, non-specific adsorption may increase.

    As long as the chemical structure of the polymer compound of the present invention is a structure including at least a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group, the bonding method is any of random, block, graft, etc. It may be in a form.

    The method for synthesizing the polymer compound of the present invention is not particularly limited, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a primary amino group are previously protected with a protecting group for ease of synthesis. A production method comprising a step of radical copolymerization of the monomer obtained and a step of removing a protecting group from the polymer compound obtained by the step is preferred. Alternatively, a step of radical copolymerizing a monomer having at least a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having a functional group capable of introducing a primary amino group, and the polymer compound obtained by the step having a primary amino group The manufacturing method including the step of introducing is preferable.

    The monomer having a phosphorylcholine group is not particularly limited in structure. For example, 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine, 10 -(Meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine and the like can be mentioned, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability.

    Specific examples of the monomer having a hydrophobic group include n-butyl (meth) acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl ( (Meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate, neopentyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) acrylate, heptyl (meth) acrylate, n- Octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n-decyl (meth) acrylate, iso-decyl ( (Meth) acrylate, n-dodecyl (Meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meta ) Acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, cyclohexyl (meth) acrylate, isobonyl Examples include (meth) acrylate. Of these, n-butyl methacrylate, n-dodecyl methacrylate, and n-octyl methacrylate are most preferable.

The monomer in which the primary amino group is previously protected with a protecting group is not particularly limited in structure, but the following general formula [4] (wherein R ₃ is a hydrogen atom or a methyl group, Y is a spacer containing an alkylene glycol residue, Z represents an oxygen atom or NH, and W represents a protecting group.) (Meth) acrylic group and oxylamino group or hydrazide group have a structure through a spacer Y containing an alkylene glycol residue. Preferably there is.

  The protecting group W is not limited as long as it can protect the amino group, and can be arbitrarily used. Of these, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferably used.

Specific examples of the monomer are those represented by the following formula.

  Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, or anhydrous hydrogen fluoride.

  On the other hand, the method for introducing a primary amino group after polymerizing a polymer compound is not limited at all, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having an alkoxy group are radicalized. A method of producing a hydrazide group by reacting an alkoxy group introduced into the polymer compound with hydrazine after copolymerization is preferred. As the alkoxy group, a methoxy group, an ethoxy group, a propoxy group, a t-butoxy group and the like are preferable.

Specific examples of the monomer having an alkoxy group are those represented by the following formula.

    As the synthetic solvent for the polymer compound of the present invention, any solvent may be used as long as each monomer can be dissolved. For example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol are used. , Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether And ethylene glycol monobutyl ether. These solvents are used alone or in combination of two or more.

    The polymerization initiator may be any ordinary radical initiator, such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.

  The molecular weight of the polymer compound of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer compound and unreacted monomer can be easily separated and purified.

    By coating the substrate surface with the polymer compound of the present invention, it is possible to easily impart the property of suppressing nonspecific adsorption of the hydrophilic compound and the property of immobilizing the hydrophilic compound.

    For coating the substrate surface with a polymer compound, for example, a polymer solution in which the polymer compound is dissolved in an organic solvent so as to have a concentration of 0.05 to 50% by weight is prepared, and then a known method such as dipping or spraying is used. After coating on the surface of the substrate, the drying is performed at room temperature or under heating.

    Examples of organic solvents include ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, Examples thereof include butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, and cyclohexanone. These solvents are used alone or in combination of two or more. Of these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, and cyclohexanol are preferable because they do not denature the plastic substrate and can be easily dried.

    The shape of the substrate used in the present invention is not particularly limited, and examples thereof include a slide-shaped substrate, a tube, a bead, a 96- to 384-well plate, a microfluidic substrate, and a composite thereof.

  The substrate material can be usually glass, metal or the like, but as the substrate material used in the present invention, plastic is used from the viewpoint of ease of surface treatment and mass productivity, and particularly a thermoplastic resin. Preferably there is. As a thermoplastic resin, a thing with little fluorescence generation amount is preferable. For example, it is preferable to use linear polyolefin such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, saturated cyclic polyolefin, fluorine-containing resin, etc., and saturation that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. It is more preferable to use a cyclic polyolefin. Here, the saturated cyclic polyolefin refers to a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.

    The oxylamino group or hydrazide group can immobilize sugars, sugar chains, glycopeptides, antibodies, and / or hydrophilic compounds having these. As a method for immobilizing these hydrophilic compounds on a substrate, a solution in which the hydrophilic compound is dissolved is dispensed into a container or the like, and the substrate is immersed and immobilized, or a solution in which the hydrophilic compound is dissolved is sprayed. There are methods such as spraying, coating and fixing. In addition, other substances not involved in immobilization with the substrate can be mixed in this solution.

    In the immobilization, the hydrophilic compound can be more effectively immobilized on the base material by oxidizing the sugar moiety of the sugar, sugar chain, glycopeptide, or antibody to an aldehyde with an oxidizing agent. Although it does not specifically limit as an oxidizing agent to be used, Periodic acid is used. These concentrations are 0.04 to 0.16M. In addition, a sodium bicarbonate solution (pH 8.1) is usually used as a buffer for the oxidation reaction. Chemical immobilization can be achieved by reacting the aldehyde group of the hydrophilic compound thus oxidized with the primary amino group on the substrate to produce a Schiff base.

    The hydrophilic compound used in the present invention is not limited as long as it can be immobilized on the substrate coated with the polymer compound, but after that various physiologically active substances are maintained and immobilized. In this sense, polysaccharides are preferred. The type of polysaccharide is not particularly limited. In particular, agarose is easy to gel and is suitable as a support for a physiologically active substance in a state where the three-dimensional structure and activity are maintained. Moreover, these polysaccharides can be used individually or in combination of 2 or more types.

    Various buffers are preferably used as the solution for dissolving the hydrophilic compound. The type of the buffer is not particularly limited, but for example, sodium carbonate, sodium bicarbonate, potassium phosphate, dipotassium hydrogen phosphate, Tris-HCl buffer, Tris acetate buffer, PBS buffer, sodium citrate, sodium acetate, HEPES (N-2-hydroxyethylperazine-N′-ethanesulfonic acid) buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer, and the like are used.

    The pH of the solution for dissolving the hydrophilic compound is preferably 2 to 8 when the sugar or sugar chain is dissolved.

    The concentration of the hydrophilic compound in the solution is not particularly limited, but is preferably 0.0001 mg / ml to 10 mg / ml.

    The temperature for immobilizing the hydrophilic compound solution is preferably 0 ° C to 100 ° C.

The bioactive substance can be immobilized on the surface of the substrate on which the hydrophilic compound is immobilized, and can be used as a biosensor for detecting various molecular biological reactions. When immobilizing a physiologically active substance, a method in which a liquid in which a physiologically active substance is dissolved or dispersed in a solvent is spotted on a substrate is preferable.
The pH of the solvent for dissolving or dispersing the physiologically active substance is preferably 8 to 10, and more preferably pH 9.0 to 9.9. Regarding the environment in the step of immobilizing a physiologically active substance, the temperature is preferably 15 to 70 ° C., and the humidity is preferably 0 to 95%.

  The manner of immobilizing the physiologically active substance on the substrate surface is not particularly limited, and immobilization by adsorption can be used in addition to chemical bonds such as covalent bonds and ionic bonds, and not based on covalent bonds.

  When the physiologically active substance is immobilized on the surface of the substrate in the form of spots, a plurality of types of physiologically active substances can be present in the same section of the substrate surface.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), N- [2- [2- [2- (t-butoxycarbonylaminooxyacetylamino) ethoxy] ethoxy] ethyl] -methacrylamide (OA) The solid phase substrate is immersed in a 0.3 wt% ethanol solution of a polymer compound having a composition molar ratio of 26: 66: 8 of the compound represented by the formula [5] and dried, whereby the above polymer is formed on the substrate surface. A layer containing the compound was introduced.
Next, the substrate was immersed in dibasic hydrochloric acid and stirred at a temperature of 37 ° C. for 4 hours to remove the Boc group as a protecting group.
Subsequently, agarose was dissolved in a PBS buffer adjusted to pH 4.0 at a concentration of 0.2 wt%, the substrate was immersed in this, and allowed to stand at a temperature of 90 ° C. for 12 hours. Immobilized on the substrate. After the reaction, agarose that was not immobilized was removed by immersion in pure water.

(Comparative example)
Saturated cyclic polyolefin resin (hydrogenated ring-opening polymer of 5-methyl-2-norbornene, MFR (Melt flow rate): 21 g / 10 min, hydrogenation rate: substantially 100%, heat distortion temperature 123 ° C.) A solid-phase substrate was prepared by processing into a slide glass shape (size: 76 mm × 26 mm × 1 mm). MPC, BMA, p-nitrophenyloxycarbonyl-4.5-ethylene glycol methacrylate (MEO4.5NP) in a 0.3 wt% ethanol solution of a polymer compound having a composition molar ratio of 23: 68: 9 By immersing and drying, a layer containing the polymer compound was introduced onto the substrate surface.

  Next, using an automatic spotter, a DsRed protein, which is a monomer red fluorescent protein, was added to a carbonate buffer adjusted to a pH of 9.5 at a concentration of 10 μmol / L on the substrates obtained in Examples and Comparative Examples. The dissolved solution was spotted. The amount of fluorescence was measured for each spot immediately after the spot. Subsequently, the substrate was allowed to stand at room temperature for the time shown in Table 1, and then the fluorescence amount was measured for each spot in the same manner. The results are shown in Table 1.

A biochip scanner “ScanArray” manufactured by PerkinElmer was used to measure the amount of fluorescence in Examples and Comparative Examples. The measurement conditions were laser output 90%, PMT sensitivity 55%, excitation wavelength 558 nm, measurement wavelength 583 nm, and resolution 10 μm.
In the examples, the color of fluorescence indicating that the protein maintained the activity was observed even after the passage of time, but in the comparative example, the color of fluorescence decreased with the passage of time, and finally it became almost invisible. That is, in the biodevice of the present invention, it can be said that the physiologically active substance can be fixed while maintaining the activity.

Claims (17)

The surface of the solid phase substrate has a polymer compound including a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group, and a hydrophilic compound is bonded to the surface, and the hydrophilic compound is A biodevice characterized by being a polysaccharide . The biodevice according to claim 1, wherein the primary amino group is an oxylamino group and / or a hydrazide group. The biodevice according to claim 1, wherein a main chain of the polymer compound is a (meth) acryl skeleton. The polymer compound is represented by the following general formula [1] (wherein R1, R2, and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents Represents an integer of 1 to 20. When p is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, Z is an oxygen atom or NH. L, m, and n are natural numbers.) The biodevice according to any one of claims 1 to 3.

The biodevice according to claim 4, wherein, in the general formula [1], X is an ethyleneoxy group. The biodevice according to claim 4 or 5, wherein, in the general formula [1], R4 is an alkyl group. The biodevice according to claim 6, wherein the alkyl group has 2 to 10 carbon atoms. The biodevice according to any one of claims 4 to 7, wherein, in the general formula [1], Y is the following general formula [2] or [3] (wherein q and r are integers of 1 to 20).

The polysaccharide is at least selected from the group consisting of chitin and chitosan, pectin, guar gum, xanthan gum, gum arabic, gellan gum, far celerane, carboxymethyl cellulose, heparin, hyaluronic acid, pullulan, dextran or derivatives thereof, carrageenan, sepharose, and agarose. it is one, bio device of claim 1, wherein. Claim 1-9 biodevice according to any one material of said solid substrate is a plastic. The biodevice according to claim 10 , wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof. The biodevice according to any one of claims 1 to 9 , wherein a material of the solid phase substrate is glass. Shaped slide-shaped substrate of the device, tubes, beads, 96 to 384-well plates, microfluidics substrate and bio according to claim 1-12, wherein any one is at least one selected from the group consisting of a complex thereof device. Claim 1-13 A method for producing a bio-device according to any one,
(1) A step of applying a polymer compound containing a unit having a phosphorylcholine group, a unit having a hydrophobic group, and a unit having a primary amino group to the surface of the substrate,
(2) a step of further chemically bonding a hydrophilic compound to the surface of the coated substrate;
A method for producing a biodevice, comprising: The method for producing a biodevice according to claim 14, wherein the step (2) includes a step of applying a hydrophilic compound to the surface of the substrate. Claim 1-13 biosensors to immobilize a physiologically active substance on bio device according to any one. The biosensor according to claim 16 , wherein the physiologically active substance is at least one selected from the group consisting of DNA, peptides, proteins and mixtures thereof.