JP2010126700A - Polymeric compound for bioassay, and substrate for bioassay using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a polymeric compound for bioassays which is capable of controlling nonspecific adsorption or bonding of proteins without coating an adsorption inhibitor and a substrate for bioassays using the same. <P>SOLUTION: The polymeric compound for bioassays is a polymeric compound at least having a unit capable of controlling nonspecific adsorption of proteins and a unit with a hydrophobic group, and a block copolymer. Preferably, the main chain of the polymeric compound has a (meth)acryl skeleton. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a polymer compound for bioassay used for detection and analysis of a target substance in a biological sample, and a bioassay substrate using the same.

  In recent years, bioassay substrates used in biochips or the like that detect specific substances in the presence of biomolecules such as proteins and lipids have been widely applied in disease diagnosis applications and the like. However, in many cases, non-specific adsorption of the coexisting proteins to the substrate surface occurs simultaneously with the specific reaction for detecting a specific substance, which causes noise and hinders high sensitivity. From such a background, a material that suppresses nonspecific adsorption of proteins is demanded.

  For example, in the sandwich method used in ELISA and the like, an anti-adsorption agent is coated to prevent non-specific adsorption of antigen and secondary antibody after immobilizing the primary antibody. Noh is not enough. In addition, since the adsorption inhibitor is coated after immobilizing the primary antibody, it may be coated on the immobilized protein, and there is a problem that it cannot react with the secondary antibody. Therefore, there is a need for a bioassay substrate that does not have an adsorption inhibitor coating step after immobilization of the primary antibody and has a small amount of nonspecific adsorption of a physiologically active substance.

One approach to reduce the amount of nonspecific adsorption of proteins is to support the active component, the crosslinking component, and the matrix-forming component containing functional groups, spacer groups, and binding groups by coating on a support and curing. It is disclosed that a functional surface firmly bonded on a body can be formed (for example, Patent Document 1). However, in this disclosed method, since curing of low molecular components proceeds on the support, there is a risk of warping or deformation when the support is a plastic substrate. In addition, since a matrix entangled with a mesh is formed, the reaction of the functional group for immobilizing the physiologically active substance is suppressed, and the reproducibility of the functional expression of the immobilized physiologically active substance is poor. There was a problem. In addition, there is a problem that nonspecific adsorption cannot be sufficiently suppressed because the protein that has entered the matrix cannot be removed even after washing.
JP-T-2004-53390

  It is an object of the present invention to provide a bioassay polymer compound capable of suppressing non-specific adsorption / binding of proteins without coating with an adsorption inhibitor and a bioassay substrate using the same. The term “substrate” as used herein includes all materials that can be used in bioassays, such as various chips, plates, particles, and containers.

（式中Ｒ₁、Ｒ₂は水素原子またはメチル基を、Ｒ₃は疎水性基を示す。Ｘは炭素数１〜１０のアルキレンオキシ基を示し、ｐは１〜２０の整数を示す。ｍ、ｎは自然数である。）
（４）前記一般式［１］において、Ｘがエチレンオキシ基である（３）記載のバイオアッセイ用高分子化合物、
（５）前記一般式［１］において、Ｒ_３がアルキル基、シクロアルキル基、芳香族類のいずれかである（３）又は（４）記載のバイオアッセイ用高分子化合物、
（６）前記アルキル基またはシクロアルキル基の炭素数が２〜１０である（５）記載のバイオアッセイ用高分子化合物、
（７） （１）〜（６）いずれか記載のバイオアッセイ用高分子化合物を含む表面コーティング材料、
（８） （７）記載の表面コーティング材料を含む層を表面に形成したバイオアッセイ用基材、
（９）前記基材の形状が、スライド形状基板、プレート、容器、マイクロフルイディスク基板のいずれかである（８）記載のバイオアッセイ用基材、
（１０）前記基材の材質がプラスチック製である（８）または（９）記載のバイオアッセイ用基材、
（１１）前記プラスチックが飽和環状ポリオレフィン、ポリオレフィンまたはポリスチレンを含むものである（１１）記載のバイオアッセイ用基材、
（１２）（１）〜（６）いずれか記載のバイオアッセイ用高分子化合物の製造方法であって、タンパク質の非特異吸着を抑制できるユニットを含むモノマー又は疎水性基を有するユニットを含むモノマーのいずれか一方である第一の成分とリビング合成用触媒との溶液中に開始剤を加えて重合を行い、更に他方のモノマーである第二の成分を追加して重合を継続することによりブロックポリマーを得ることを特徴とするバイオアッセイ用高分子化合物の製造方法、
である。 The present invention
(1) A polymer compound for bioassay comprising at least a unit capable of suppressing nonspecific adsorption of a protein and a unit having a hydrophobic group, which is a block copolymer,
(2) The polymer compound for bioassay according to (1), wherein the main chain of the polymer compound is a (meth) acryl skeleton,
(3) The polymer compound for bioassay according to (1) or (2), wherein the polymer compound is represented by the following general formula [1]:

(Wherein R ₁ and R ₂ represent a hydrogen atom or a methyl group, R ₃ represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20 m. , N is a natural number.)
(4) The polymer compound for bioassay according to (3), wherein, in the general formula [1], X is an ethyleneoxy group,
(5) The polymer compound for bioassay according to (3) or (4), wherein, in the general formula [1], R ₃ is any one of an alkyl group, a cycloalkyl group, and an aromatic group,
(6) The polymer compound for bioassay according to (5), wherein the alkyl group or cycloalkyl group has 2 to 10 carbon atoms,
(7) A surface coating material comprising the polymer compound for bioassay according to any one of (1) to (6),
(8) A substrate for bioassay having a layer containing the surface coating material according to (7) formed on the surface,
(9) The bioassay substrate according to (8), wherein the shape of the substrate is any one of a slide-shaped substrate, a plate, a container, and a microfluidic substrate,
(10) The bioassay substrate according to (8) or (9), wherein the material of the substrate is made of plastic,
(11) The bioassay substrate according to (11), wherein the plastic contains saturated cyclic polyolefin, polyolefin or polystyrene,
(12) A method for producing a polymer compound for bioassay according to any one of (1) to (6), wherein a monomer containing a unit capable of suppressing nonspecific adsorption of a protein or a monomer containing a unit having a hydrophobic group A block polymer is obtained by adding an initiator to the solution of either one of the first component and the living synthesis catalyst and polymerizing, and further adding the second component, which is the other monomer, to continue the polymerization. A method for producing a polymer compound for bioassay,
It is.

    When the polymer compound of the present invention is applied to a bioassay substrate, nonspecific adsorption of proteins can be suppressed without coating with an adsorption inhibitor.

    The polymer compound of the present invention is a polymer compound having at least a unit capable of suppressing nonspecific adsorption of a protein and a unit having a hydrophobic group, and is a block copolymer. This polymer compound has at least the following characteristics because each component is blocked. Hydrophobic interactions are easily expressed and are easily adsorbed to a hydrophobic substrate via a unit having a hydrophobic group. Moreover, since the solubility with respect to water falls compared with the random copolymer of the same composition, when this high molecular compound is apply | coated to a base material, drop-off | omission of the polymer in an assay is suppressed. Furthermore, since the unit capable of suppressing nonspecific adsorption of protein is hydrophilic, when the base material coated with the polymer compound is immersed in an aqueous solution, the hydrophilic unit is unevenly distributed at the solution interface. That is, reduction of non-specific adsorption in the assay can be expected.

  The polymer compound of the present invention may be a block copolymer having a unit capable of suppressing nonspecific adsorption of protein and a unit having a hydrophobic group. However, considering the ease of synthesis, diblock copolymers or triblock copolymers are preferred. On the other hand, from the viewpoint of suppressing nonspecific adsorption, a diblock copolymer and a triblock copolymer of hydrophilic block-hydrophobic block-hydrophilic block are preferable.

The unit capable of suppressing non-specific adsorption contained in the polymer compound of the present invention is not particularly limited in structure, but as shown by the structural unit on the left side of the structural unit of the following general formula [1], (meth) acrylic A structure in which a residue and a phosphorylcholine group are bonded via a chain of an alkyleneoxy group X having 1 to 10 carbon atoms is preferable. Of these, X is most preferably an ethyleneoxy group. The repeating number p of the alkyleneoxy group X in the formula is an integer of 1-20. Although m is naturally a natural number, it may be expressed as a composition ratio of each component. R ₁ and R ₂ represent a hydrogen atom or a methyl group.

  The composition ratio of the unit capable of suppressing nonspecific adsorption contained in the polymer compound of the present invention (m is the ratio of m to the sum of m and n in the case of general formula [1]) is not particularly limited. The amount is preferably 10 to 90 mol%, more preferably 20 to 80 mol%, and most preferably 30 to 70 mol% with respect to all units of the polymer compound. When the composition ratio falls below the lower limit, nonspecific adsorption increases. On the other hand, when the value exceeds the upper limit, water solubility increases, and even if it is a block copolymer, 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. In this case, it is more preferable to introduce the unit having a hydrophobic group. For example, a method of introducing a unit having a silane coupling agent 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, but as represented by the structural unit on the right side 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, a cycloalkyl group, and aromatics are mentioned. More preferably, the alkyl group is an alkyl group having 2 to 6 carbon atoms, a cycloalkyl group, or an aromatic group. The alkyl group is not particularly limited in structure, and may be linear or branched. In the formula, n is originally 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 (m in the case of General Formula [1] as an example, the ratio of n to the sum of n) is not particularly limited, 10-90 mol% is preferable with respect to all the units of the polymer compound, more preferably 20-80 mol%, and most preferably 30-70 mol%.

  In addition to the unit capable of suppressing nonspecific adsorption and the unit having a hydrophobic group, a unit having an arbitrary function may be introduced into the polymer compound of the present invention. As an example other than the functional groups that can be covalently bonded to the substrate, a functional group that immobilizes a physiologically active substance can be introduced. The polymer compound thus obtained can be used for a biochip that can capture a specific protein or the like via a physiologically active substance.

    Examples of the functional group for immobilizing the physiologically active substance include, but are not limited to, a chemically active group, a receptor group, and a ligand group. Specific examples include aldehyde groups, active esters, epoxy groups, vinyl sulfone groups, biotin, thiol groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, acrylate groups, maleimide groups, hydrazide groups, azide groups, Examples include, but are not limited to, amide groups, sulfonate groups, streptavidin, and metal chelates. Among these, an aldehyde group, an active ester, an epoxy group, and a vinyl sulfone group are preferable from the viewpoint of reactivity with an amino group contained in a large amount in the physiologically active substance, and biotin having a high binding constant with the physiologically active substance is preferable. Of these, an active ester group is most preferable from the viewpoint of storage stability of the monomer.

    The functional group includes various physiologically active substances such as various proteins, antigens, antibodies, DNA, RNA, sugars, sugar chains, glycolipids, glycopeptides, and / or physiologically active substances having these depending on the type. Can be immobilized. As a method for immobilizing these physiologically active substances on the substrate, a method of spotting a solution in which the physiologically active substance is dissolved using a spotter, or dispensing a solution in which the physiologically active substance is dissolved into a container or the like. There is a way to fix

    The method for synthesizing the polymer compound of the present invention is not particularly limited, but various living polymerizations are preferable because of easy structure control. Among them, living radical polymerization is simple and preferable. For living radical polymerization, various synthetic methods such as living radical polymerization via nitroxide, polymerization using organic tellurium compounds, iodine transfer polymerization, reversible addition-cleavage chain transfer type radical polymerization have been developed. Among them, the atom transfer radical polymerization method is preferable because of its versatility. Examples of transition metal complexes used in the atom transfer radical polymerization method include copper, ruthenium, iron, nickel and the like.

    The monomer used in the polymerization is not particularly limited as long as the polymer of the present invention can be obtained. Monomers having a unit capable of suppressing non-specific adsorption include those containing a phosphorylcholine group such as 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxyhexyl phosphorylcholine. Examples thereof include 10- (meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability. Examples of the monomer not containing a phosphorylcholine group include methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, methoxyethyl (meth) acrylate, ethoxyethyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, and 2 -Hydroxypropyl (meth) acrylate and mono-substituted ester thereof, 2-hydroxybutyl (meth) acrylate and mono-substituted ester thereof, glycerol mono (meth) acrylate, (meth) acrylate having polypropylene glycol as a side chain, 2-ethoxyethyl (meth) acrylate, methoxydiethylene glycol (meth) acrylate, ethoxydiethylene glycol (meth) acrylate, ethoxypoly Liethylene glycol (meth) acrylate and the like can be mentioned, but methoxypolyethylene glycol (meth) acrylate, ethoxypolyethylene glycol (meth) acrylate, methoxyethyl acrylate and the like are preferable because of low nonspecific adsorption and availability. In the present invention, (meth) acryl represents acryl and / or methacryl, and (meth) acrylate represents acrylate and / or methacrylate.

    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 (Meth) acrylate, phenyl methacrylate and the like can be mentioned. Among these, n-butyl methacrylate, iso-butyl methacrylate, and t-butyl methacrylate cyclohexyl methacrylate are preferable.

    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 method for synthesizing the block polymer of the present invention is not particularly limited, but the first polymer which is either a monomer containing a unit capable of suppressing nonspecific adsorption of a protein or a monomer containing a unit having a hydrophobic group. A method of obtaining a block polymer by adding an initiator to a solution of the component and the living synthesis catalyst to perform polymerization, and further adding the second component, which is the other monomer, to continue the polymerization is preferred. Moreover, when adding a 2nd component, you may add together a solvent. When synthesizing a triblock polymer, a third component may be added in the same manner.

  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.

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

    The organic solvent is not particularly limited as long as the polymer compound of the present invention is dissolved, but alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, and cyclohexanol. , Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, cyclohexanone, etc. be able to. These solvents are used alone or in combination of two or more. Among these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, or a mixed solvent mainly composed of these are preferable because they do not denature the plastic substrate and are easily dried.

    The substrate used in the present invention is not particularly limited, but a slide-shaped substrate, a 96-hole plate, a 384-hole plate, a container, and a microfluidic substrate are preferable. Examples of the material include plastic, glass, and a substrate having a metal vapor deposition film. Specific examples of the plastic include various polyolefins such as cycloolefin polymer, polypropylene and polyethylene, and various (meth) acrylic resins such as polysalophone, polyimide, polycarbonate and polymethyl methacrylate.

Example 1
(Synthesis example of polymer compound)
2-methacryloyloxyethyl phosphorylcholine (MPC) 6 mmol, CuBr 0.12 mmol, 2,2′-bipyridyl 0.24 mmol, and methanol 6 ml were formulated in a sealed tube in a glove box controlled to 1 ppm or less for both oxygen and water vapor. 0.12 mmol of ethyl bromo-2-methylpropionate was added and sealed. After reacting at 40 ° C. for 4 hours, 1 ml of ethyl acetate and 8.4 mmol of n-butyl methacrylate (BMA) were added in the glove box, and the reaction was further continued for 20 hours. The resulting polymerization solution was purified by reprecipitation to obtain a block polymer.

<< Comparative Example 1 >>
(Comparative synthesis examples of polymer compounds)
MPC 6 mmol, BMA 6 mmol, CuBr 0.12 mmol, 2,2′-bipyridyl 0.24 mmol, methanol 6 ml, and ethyl acetate 1 ml were formulated in a sealed tube in a glove box, and further ethyl 2-bromo-2-methylpropionate 0.12 mmol Was added and sealed. After reacting at 40 ° C. for 24 hours, a random polymer was obtained by reprecipitation purification of the obtained polymerization solution.

  The obtained polymer was measured by 1H-NMR, and the composition ratio of the polymer compound was calculated. Table 1 shows the results. A block polymer and a random polymer having almost the same composition were obtained.

(Verification of solubility in water)
A 0.3 wt% ethanol solution of the polymer of Example 1 and Comparative Example 1 was prepared, applied to a slide substrate made of cyclic polyolefin (COC), and air-dried. The substrate was immersed in water, and the film thickness and contact angle before and after that were investigated. The results are shown in FIG.

  The film thickness is decreased by immersion in both the examples and the comparative examples, but the examples tend to maintain the film thickness. As for the contact angle, the substrate coated with the polymer of the example has a low contact angle even after immersion, but the substrate coated with the polymer of the comparative example is affected by the substrate because the film thickness is reduced. The contact angle became high.

  These facts suggest that the polymer of the present invention is prevented from falling off from the substrate during the assay, and the blocks that suppress nonspecific adsorption of proteins are unevenly distributed on the surface.

(Measurement of non-specific adsorption amount of each polymer)
A 0.3 wt% ethanol solution of the polymer of Example 1 and Comparative Example 1 was prepared, dispensed to each well of a cyclic polyolefin (COC) 96-well plate, and allowed to stand for 10 minutes. The solution was then collected and the plate was air dried. Pure water was dispensed, allowed to stand overnight, and then removed. Before the surface was dried, 100 μl of HRP-labeled BSA PBS buffer solution adjusted to 1 μg / ml was dispensed, sealed and allowed to stand for 1 hour. The wells were washed 3 times with PBS buffer containing 0.05 wt% Triton X-100. After this step, the HRP-labeled BSA remaining in the wells was regarded as non-specifically adsorbed, and detection was performed using a SUMILON (registered trademark) peroxidase coloring kit (ML-1120T, substrate: TMBZ). 100 μl of a mixed solution of a color former and a substrate (color developing solution) was dispensed into each well and allowed to react for 15 minutes under light shielding. Thereafter, 100 μl of the stop solution was dispensed, and the absorbance at 450 nm was measured. The results are shown in Table 2.

    It can be seen that the plate coated with the block polymer of the example has less non-specific adsorption of proteins despite the almost the same composition as the plate coated with the random polymer of the comparative example.

Film thickness and contact angle after coating and drying and after immersion in water

Claims (12)

A polymer compound for bioassay, which is a polymer compound having at least a unit capable of suppressing nonspecific adsorption of a protein and a unit having a hydrophobic group, which is a block copolymer. The polymer compound for bioassay according to claim 1, wherein the main chain of the polymer compound is a (meth) acryl skeleton. The polymer compound for bioassay according to claim 1 or 2, wherein the polymer compound is represented by the following general formula [1].

(Wherein R ₁ and R ₂ represent a hydrogen atom or a methyl group, R ₃ represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20 m. , N is a natural number.) The high molecular compound for bioassay according to claim 3, wherein X in the general formula [1] is an ethyleneoxy group. In the general formula [1], R ₃ is an alkyl group, a cycloalkyl group, according to claim 3 or 4 Bioassay for polymer compound according either of aromatics. The polymer compound for bioassay according to claim 5, wherein the alkyl group or cycloalkyl group has 2 to 10 carbon atoms. A surface coating material comprising the polymer compound for bioassay according to claim 1. A bioassay substrate on which a layer containing the surface coating material according to claim 7 is formed. The bioassay substrate according to claim 8, wherein the shape of the substrate is any one of a slide-shaped substrate, a plate, a container, and a microfluidic substrate. The bioassay substrate according to claim 8 or 9, wherein the material of the substrate is made of plastic. The bioassay substrate according to claim 11, wherein the plastic contains saturated cyclic polyolefin, polyolefin or polystyrene. It is a manufacturing method of the high molecular compound for bioassays in any one of Claims 1-6, Comprising: It is either a monomer containing the unit which can suppress the nonspecific adsorption | suction of protein, or a monomer containing the unit which has a hydrophobic group. The polymerization is performed by adding an initiator to the solution of the first component and the living synthesis catalyst, and the block component is obtained by adding the second component, which is the other monomer, and continuing the polymerization. A method for producing a polymer compound for bioassay.

Images