JP4353073B2 - Biochip and manufacturing method thereof - Google Patents

Description

  The present invention relates to a biochip 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 chip 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 the current protein chip is generally developed on the extension line of the DNA chip, studies have been made to immobilize a protein or a molecule for capturing the protein on the glass substrate on the surface of the chip (for example, Patent Document 1).

  After immobilizing a protein-capturing molecule (hereinafter abbreviated as a capturing molecule) on a substrate, it is reacted with another protein (corresponding to an antigen in the case of an antigen-antibody reaction) on the surface, for example, as in the sandwich method. Furthermore, when a labeled protein is reacted and finally detected by a detector or the like, if a protein other than the molecule, that is, an antigen or a labeled protein is immobilized on a portion where the capture molecule is not immobilized, detection is performed. It sometimes becomes noise and causes a decrease in the signal-to-noise ratio (S / N ratio), thereby reducing the detection accuracy (see Non-Patent Document 1, for example).

  For this reason, in the usual sandwich method, after the primary antibody is immobilized, in order to prevent nonspecific adsorption of the antigen and the secondary antibody, coating with an adsorption inhibitor is performed, but these nonspecific adsorption preventing ability is not sufficient. 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 biochip that does not have an adsorption inhibitor coating step after immobilization of the primary antibody and has a small amount of non-specific adsorption of a physiologically active substance.

In terms of the technology to profile fluctuations in 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. In this technology, a physiologically active substance such as protein as a sample reacts specifically with the capture immobilized in the flow path, and suppresses non-specific adsorption to the inner wall of the flow path other than the capture part. Is required.
JP 2001-116750 A “DNA Microarray Practice Manual”, Yoshihide Hayashizaki, Koji Okazaki, Yodosha, 2000, p.57

  The present invention immobilizes an antibody at an arbitrary position on the surface of a substrate without coating an adsorption inhibitor, and suppresses adsorption and binding of an unnecessary physiologically active substance to other parts, with high sensitivity and high throughput. An object of the present invention is to provide a biochip capable of detecting a physiologically active substance and a method for producing the same.

That is, the present invention is as follows.
(1) A biochip in which an antibody is immobilized on the surface of a substrate, the surface being coated with a polymer that is a copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group , A biochip in which an antibody is immobilized on a polymer by adsorption .
(2) The biochip according to (1), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
(3) The biochip according to (1) or (2), wherein the antibody is immobilized in a spot shape on the surface of the substrate.
(4) The biochip as set forth in (3), wherein spots of a plurality of types of antibodies are present in the same section of the substrate surface.
(5) The biochip as set forth in any one of (1) to (4), wherein the substrate has a glass slide shape.
(6) The biochip according to any one of (1) to (5), wherein the substrate has a flow channel, and the antibody is immobilized in the flow channel.
(7) The biochip as set forth in any one of (1) to (6), wherein the base material is plastic.
(8) The biochip according to (7), 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.
(9) The biochip manufacturing method according to any one of (1) to (8),
(1) A step of applying a polymer which is a copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group on the surface of the substrate,
(2) A step of spotting or applying a liquid in which an antibody is dissolved or dispersed in a solvent on the surface of the substrate,
(3) immobilizing the antibody at a temperature of 25 ° C. or higher,
A method for producing a biochip comprising:
(10) The method for producing a biochip as set forth in ( 9 ), wherein in the step (3), the substrate is kept in a dry state with a humidity of 60% or less.
(11) The method for producing a biochip according to (9) or (10), wherein the solvent for dissolving or dispersing the antibody has a pH of 8 to 10.
(12) The method for producing a biochip according to any one of (9) to (11), wherein in the step (3), the antibody immobilization temperature is 37 to 70 ° C.

  According to the biochip of the present invention and the method for producing the same, the antibody is immobilized at an arbitrary position on the surface of the substrate without coating with an adsorption inhibitor, and an unnecessary physiologically active substance is adsorbed and bound to other portions. It is possible to obtain a biochip capable of detecting a physiologically active substance with high sensitivity and high throughput.

  The biochip of the present invention is characterized in that the substrate surface is coated with a polymer having a phosphorylcholine group. A polymer having a phosphorylcholine group is a polymer having a structure similar to a biological membrane (phospholipid bilayer membrane), and has an effect of suppressing adsorption of a physiologically active substance (for example, Ishihara K, Tsuji T, Kurosaki T Nakabayashi N, Journal of Biomedical Materials Research, 28 (2), pp.225-232, (1994) 4).

  Examples of the phosphorylcholine group include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine, octenylphosphorylcholine, Although senyl phosphorylcholine etc. are mentioned, 2-methacryloyloxyethyl phosphorylcholine is preferable.

  The bond between the substrate surface and the polymer may be any type of bond such as covalent bond, electrostatic interaction, hydrogen bond, bond by hydrophobic effect, etc., but from the viewpoint of simplicity of surface treatment, the substrate surface It is preferable that the polymer and the polymer are bonded by a hydrophobic effect.

  The polymer used in the present invention may contain other groups in addition to the phosphorylcholine group, and is preferably a binary copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group.

(Base material)
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.

(Base shape)
The shape of the substrate used in the present invention is not particularly limited, and examples thereof include a glass slide substrate and a bead-shaped sphere. A fine channel may be provided on the surface of these substrates, and the antibody can be immobilized in the channel.

(Immobilization of antibodies)
In the present invention, when an antibody is immobilized on a substrate, a method of spotting a liquid in which the antibody is dissolved or dispersed in a solvent is preferable.
The pH of the solvent in which the antibody is dissolved or dispersed is preferably 8 to 10, and more preferably pH 9.0 to 9.9. Regarding the environment in the antibody immobilization step, the temperature is preferably 25 to 70 ° C. and the humidity is preferably 0 to 80%. In particular, the spot signal intensity is increased under conditions of a temperature of 37 to 70 ° C. and a dry state (humidity 0 to 60%), which is more preferable. After immobilization, it is preferable to wash with pure water or a buffer solution in order to remove the unimmobilized antibody.

  The method of immobilizing the antibody on the surface of the substrate is not particularly limited, and in addition to chemical bonds such as covalent bonds and ionic bonds, immobilization by adsorption can be used regardless of covalent bonds.

  When the antibody is immobilized on the substrate surface in the form of a spot, a plurality of types of antibody spots can be present in the same compartment on 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)
A saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm) to prepare a solid phase substrate. The solid phase substrate was immersed in a 0.5 wt% ethanol solution of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer to introduce a polymer having a phosphorylcholine group on the substrate surface.
Next, using an automatic spotter, the solution obtained by dissolving the anti-mouse IgG2a antibody, which is the primary antibody, in a carbonate buffer adjusted to pH 9.5 at the dilution rate (concentration) shown in Table 1 is spotted on the substrate. Then, it was allowed to stand for 24 hours in an environment of a temperature of 70 ° C. and a humidity of 50% to be fixed. After immobilization, washing with PBS containing 0.05% Tween 20 was performed. Thereafter, a mixture of mouse IgG2a antibody and serum protein, which are antigens, was labeled with Cy3 (mouse IgG2a antibody concentration: 19 nmol / L), or a mixture of only serum protein (no antigen) was labeled with Cy3 and each spot was reacted. The fluorescence amount was measured for a portion other than the spot portion (background), and the signal ratio with / without antigen and the S / N ratio (Signal / noise ratio) at that time were calculated. The results are shown in Table 1.

(Comparative example)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm). After subjecting the substrate surface to hydrophilization, the substrate was immersed in a 2% by weight aqueous solution of an amino group-containing alkylsilane, and then heat treated to introduce amino groups on the surface. This was immersed in a 1% by weight glutaraldehyde aqueous solution to react the surface amino groups with glutaraldehyde to introduce aldehyde groups.
Next, a solution prepared by dissolving an anti-mouse IgG2a antibody as a primary antibody in a carbonate buffer adjusted to pH 9.5 at the dilution rate shown in Table 1 using an automatic spotter was spotted on the substrate, It was allowed to stand for 24 hours in an environment of 70 ° C. and humidity of 50% for immobilization. After immobilization, the substrate is dipped in a solution obtained by diluting Blocking Ace, a blocking agent for immunological experiments manufactured by Dainippon Pharmaceutical Co., Ltd., 4 times with pure water to prevent nonspecific adsorption, and gently shaken at room temperature for 1 hour. did. Thereafter, washing was performed with PBS containing 0.05% Tween20. Subsequently, a mixture of mouse IgG2a antibody and serum protein, which are antigens, was labeled with Cy3 (mouse IgG2a antibody concentration: 19 nmol / L), or only a mixture of serum proteins (no antigen) was labeled with Cy3, The amount of fluorescence was measured for the spot and the portion other than the spot (background), and the signal ratio with / without antigen and the S / N ratio (Signal / noise ratio) at that time were calculated. The results are shown in Table 1.

A biochip scanner “ScanArray” manufactured by Packard BioChip Technologies 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 550 nm, measurement wavelength 570 nm, and resolution 30 μm.
In Example, the spot signal intensity with antigen was inferior to that of the comparative example, but the spot signal intensity value without antigen was lower than that of the comparative example, and the signal ratio with / without antigen was larger than that of the comparative example. Furthermore, the fluorescence intensity (background) other than the spot portion was much lower than that of the comparative example, and the S / N ratio was much higher in the example. That is, it can be said that a highly sensitive physiologically active substance was detected.

＊ 固定化抗体濃度は、
希釈倍率２倍：３．３μｍｏｌ／Ｌ
希釈倍率８倍：０．８３μｍｏｌ／Ｌ

* Immobilized antibody concentration is
Dilution factor 2 times: 3.3 μmol / L
Dilution factor 8 times: 0.83 μmol / L

  According to the biochip of the present invention and the method for producing the same, it is possible to control the protein without coating the adsorption inhibitor by controlling at least one of the pH of the solution of the immobilized antibody, the temperature at the time of immobilization, and the humidity. , Or a physiologically active substance such as a molecule that captures it, is immobilized at an arbitrary position on the surface of the substrate and suppresses the adsorption and binding of unnecessary physiologically active substances to other parts of the substrate. Since a biochip capable of detecting a substance can be provided, it can be applied to the production of various biochips including microfluidics.

Claims (12)

A biochip in which an antibody is immobilized on the surface of a substrate, the surface of which is coated with a polymer that is a copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group. A biochip characterized in that antibodies are immobilized by adsorption . The biochip according to claim 1, wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group. The biochip according to claim 1 or 2, wherein the antibody is immobilized in a spot shape on the surface of the substrate. The biochip according to claim 3 , wherein spots of a plurality of types of antibodies are present in the same section of the substrate surface. The biochip according to any one of claims 1 to 4 , wherein the substrate has a glass slide shape. The biochip according to any one of claims 1 to 5 , wherein the substrate has a flow path, and the antibody is immobilized in the flow path. The biochip according to any one of claims 1 to 6 , wherein the substrate is made of plastic. The biochip according to claim 7 , 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. It is a manufacturing method of the biochip in any one of Claims 1-8, Comprising:
(1) A step of applying a polymer which is a copolymer of a monomer having a phosphorylcholine group and a monomer having a butyl methacrylate group on the surface of the substrate,
(2) A step of spotting or applying a liquid in which an antibody is dissolved or dispersed in a solvent on the surface of the substrate,
(3) a step of immobilizing an antibody at a temperature of 25 ° C. or higher;
A method for producing a biochip comprising: The biochip manufacturing method according to claim 9 , wherein in the step (3), the substrate is kept in a dry state with a humidity of 60% or less. The method for producing a biochip according to claim 9 or 10 , wherein the solvent for dissolving or dispersing the antibody has a pH of 8 to 10 . The method for producing a biochip according to any one of claims 9 to 11 , wherein in the step (3), the antibody immobilization temperature is 37 to 70 ° C.