JP2013148483A - Manufacturing method of biochip, and biochip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a biochip used for detection and analysis of a number of protein in a biological sample, and capable of performing highly sensitive, high-throughput detection of a physiologically active substance by immobilizing protein at an arbitrary position on the surface of a substrate, suppressing unnecessary adsorption and connection of a physiologically active substance to the other part without coating an adsorption preventing agent, and maintaining an excellent spot shape.SOLUTION: Provided is a manufacturing method of a biochip in which protein is immobilized on the surface of a substrate, the method comprising the steps of: (1) applying a polymer having a phosphorylcholine group on the surface of a substrate; (2) performing spot application of or applying a liquid in which protein is dissolved or dispersed in a solvent on the surface of the substrate; (3) and immobilizing the protein. Also provided is a biochip manufactured by the manufacturing method.

Description

The present invention relates to a technique for producing a biochip used for detection and analysis of a large number of proteins in a biological sample, and more specifically, immunoassay using antigen-antibody reaction, proteomics, and intracellular protein of gene activity The present invention relates to a method of manufacturing a biochip used for level measurement.

Since current protein chips are generally developed on the extension line of a DNA chip, studies have been made to immobilize a protein or a molecule that captures it on a glass substrate on the surface of the chip (for example, patent documents). 1).
After immobilizing a protein or a molecule that captures it on the substrate, another protein on the surface (for example, in the case of an antigen-antibody reaction, the antibody for a protein, or the molecule for capturing a protein Protein), or when it is detected with a detector, it captures the protein or it

If a protein other than the molecule is immobilized on the portion where the molecule is not immobilized, noise will be generated during detection, causing a reduction in the signal-to-noise ratio (S / N ratio) and reducing the detection accuracy (for example, Non-Patent Document 1). See).

For this reason, normally, after immobilizing a protein or a molecule that captures it, a coating with an adsorption inhibitor is used to prevent nonspecific adsorption of other proteins at the part where these molecules are not immobilized. However, their ability to prevent non-specific adsorption is not sufficient. In addition, since the adsorption inhibitor is coated after the protein or the molecule that captures the protein is immobilized, the adsorption protein may be coated on the immobilized protein or the molecule that captures it. In the reaction, that is, in the reaction with other proteins (for example, in the antigen-antibody reaction, the antibody against the protein, and the protein against the molecule that captures the protein), there is a problem that the reactivity is lowered. 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 at a portion where a protein or a molecule that captures the protein is not immobilized.

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. In this technology, a physiologically active substance such as a 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 section. Is required.

For example, in the inventions of Patent Documents 2 and 3, an attempt was made to achieve this object by combining an amino group-containing polymer for immobilizing a physiologically active substance and a hydrophilic polymer. Non-specific adsorption can be suppressed by increasing the hydrophilicity of the substrate.

However, increasing the hydrophilicity also reduces the advancing contact angle when water is dropped on the substrate surface. As a result, when an aqueous solution containing a physiologically active substance is spotted on the substrate surface, the spot area increases. Furthermore, the spot outer peripheral portion bleeds and does not become a perfect spot.

JP 2001-116750 A Japanese Patent Laying-Open No. 2005-091245 Special table 2010-008378

“DNA Microarray Practice Manual”, Hayashizaki Yoshihide, Okazaki Koji edited, Yodosha, 2000, p. 57

The present invention fixes a protein at an arbitrary position on the surface of a substrate without coating an adsorption inhibitor, suppresses adsorption and binding of unnecessary physiologically active substances to other portions, and has a good spot shape. It is an object of the present invention to provide a biochip manufacturing method capable of detecting a physiologically active substance with high sensitivity and high throughput by maintaining the above.

The present invention is as follows (1) to (14).
(1) A biochip manufacturing method in which a protein is immobilized on a substrate surface, (1) a step of applying a polymer having a phosphorylcholine group to the substrate surface, (2) a protein dissolved or dispersed in a solvent on the substrate surface A method for producing a biochip, comprising: a step of spotting or applying the applied liquid; and (3) a step of immobilizing a protein.
(2) The biochip manufacturing method according to (1), wherein in the step (3), the substrate is kept in a dry state with a humidity of 50% or less.
(3) The biochip manufacturing method according to (1) or (2), wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group.
(4) The polymer is a copolymer of a monomer having a phosphorylcholine group, a monomer having a butyl methacrylate group, and a monomer having a functional group that immobilizes a protein (1) to (3) A method for producing a biochip according to claim 1.
(5) The method for producing a biochip according to any one of (1) to (4), wherein the protein in the immobilization in the step (3) is immobilized by a chemical bond.
(6) The method for producing a biochip according to (5), wherein the functional group for immobilizing the protein is an active ester group.
(7) The method for producing a biochip according to (5) or (6), wherein the active ester group is p-nitrophenyl ester or N-hydroxysuccinimide ester.
(8) The method for producing a biochip according to any one of (1) to (7), wherein the protein is immobilized in a spot shape on the surface of the substrate.
(9) The method for producing a biochip as set forth in (8), wherein a plurality of kinds of protein spots are present in the same section of the substrate surface.
(10) The biochip manufacturing method according to any one of (1) to (9), wherein the substrate has a glass slide shape.
(11) The biochip manufacturing method according to any one of (1) to (10), wherein the base material is plastic.
(12) The biochip manufacturing method according to (11), 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.
(13) The biochip production method according to any one of (1) to (12), wherein the protein is an antibody (14) produced by the production method according to any one of (1) to (13) A biochip characterized by

According to the present invention, a protein is immobilized at an arbitrary position on the surface of a substrate without coating with an adsorption inhibitor, and adsorption and binding of unnecessary physiologically active substances to other portions are suppressed, and a good spot is obtained. It has become possible to provide a method for producing a biochip capable of detecting a physiologically active substance with high sensitivity and high throughput while maintaining its shape.

The figure which shows the measurement result in the presence of the antigen of Example 1. The figure which shows the measurement result without an antigen of Example 1. The figure which shows the measurement result in the presence of the antigen of the comparative example 1. The figure which shows the measurement result without the antigen of the comparative example 1.

The method for producing a biochip of the present invention is characterized in that it includes a step of coating a substrate surface with a polymer having a phosphorylcholine group.

(Polymer composition)
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, I shi
hara K, Tsuji T, Kurosaki T, Nakabayashi N, Journalof Biomedical Materials Research, 2 8 (2), pp. 2 2 5-2 3 2, (1 9 9 4) 4, etc.

Examples of the phosphorylcholine group used in the present invention include 2-methacryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethoxyethylphosphorylcholine, 6-methacryloyloxyhexylphosphorylcholine, 10-methacryloyloxyethoxynonylphosphorylcholine, allylphosphorylcholine, butenylphosphorylcholine, hexenylphosphorylcholine, octyl Examples thereof include tenenyl phosphorylcholine and decenyl phosphorylcholine, and 2-methacryloyloxyethyl phosphorylcholine is preferable.

Furthermore, the polymer used in the present invention preferably has a functional group for immobilizing a physiologically active substance. Examples of the functional group for immobilizing the physiologically active substance used in the present invention include, but are not limited to, a chemically active group, a receptor group, and a ligand group. Specific examples include aldehyde groups, active ester groups, epoxy groups, vinyl sulfone groups, thiol groups, amino groups, isocyanate groups, isothiocyanate groups, hydroxyl groups, acrylate groups, maleimide groups, hydrazide groups, azide groups, amide groups. , But not limited to, sulfonate groups. Among these, an aldehyde group, an active ester group, an epoxy group, and a vinyl sulfone group are preferable from the viewpoint of reactivity with amino groups contained in a large amount of physiologically active substances. In particular, an active ester group is most preferred because it is widely used in bonding with physiologically active substances and has a proven record.

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.
Further, the polymer used in the present invention may contain other groups in addition to the phosphorylcholine group, a monomer having a phosphorylcholine group, a monomer having an active ester group, and a single group having a butyl methacrylate group which is a hydrophobic group. A terpolymer with a monomer is preferred.

(Polymer coating)
The biochip of the present invention can easily impart the property of suppressing nonspecific adsorption of a physiologically active substance and the property of immobilizing a specific physiologically active substance by coating the surface of the substrate with the polymer. is there.
For coating the polymer on the substrate, for example, a polymer solution in which the polymer is dissolved in an organic solvent so as to have a concentration of 0.05 to 10% by weight is prepared and applied to the substrate surface by a known method such as dipping or spraying. It is carried out by drying at room temperature or under heating.

As the organic solvent to be used, a single solvent such as 2-butanone, ethanol, methanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl ethyl ketone, or a mixed solvent thereof is used. Of these, ethanol and methanol are preferable because they do not denature the plastic substrate and are easy to dry. Moreover, when hydrolyzing a polymer in a solution, it is preferable because it is mixed with water at an arbitrary ratio.

After applying the solution in which the polymer of the present invention is dissolved to the substrate surface, it is preferable to remove the solution, wash it with an organic solvent, and then centrifuge dry.

(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 at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide and copolymers thereof, for heat resistance, chemical resistance, low fluorescence, and moldability. It is more preferable to use a particularly excellent saturated 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 slide glass substrate, a multiwell plate, 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 protein)
In the present invention, when the protein is immobilized on the substrate, a method of spotting a liquid in which the protein is dissolved or dispersed with a solvent is preferable.
The pH of the solvent for dissolving or dispersing the protein is preferably 8 to 10, more preferably pH 9.0 to 9.9. As for the environment in the protein immobilization step, the temperature needs to be 20 ° C. or higher, preferably 25 to 70 ° C.

In this case, it is important to keep the humidity at 0 to 50% in the immobilization process. In particular, when the humidity is 35 to 45%, spot shape abnormality due to spot bleeding or blurring is not observed, and the signal intensity is stable, which is more preferable. After immobilization, it is preferable to wash with pure water or a buffer solution in order to remove proteins that have not been immobilized.

When the protein is immobilized on the surface of the substrate in the form of spots, under the conditions of the present invention, the occurrence of spot shape abnormalities due to spot bleeding or blurring can be suppressed, so that a plurality of types of protein spots can be It can exist in the same compartment. This makes it possible to simultaneously measure a plurality of types of proteins in the same specimen.

Next, specific examples of the present invention will be described.

1. Preparation of Polymer (1) Polymer Synthesis 2-Methacryloyloxyethyl phosphorylcholine (hereinafter referred to as “MPC”), n-butyl methacrylate (hereinafter referred to as “BMA”), p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate ( Hereinafter, described as “MEONP”) was dissolved in dehydrated ethanol.
Further, 2,2-azobisisobutyronitrile (hereinafter referred to as “AIBN”, manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto, and the mixture was stirred until uniform to prepare a monomer mixed solution.
In addition, each molar ratio in a monomer mixed solution is 70: 27: 3 in order of MPC, BMA, and MEONP.
Then, after making it react at 60 degreeC under argon gas atmosphere for 6 hours, the reaction solution was dripped in diethyl ether, and the 1st polymer was obtained by collecting precipitation.
The above-mentioned MEONP was synthesized as shown in (2) below.

(2) Synthesis of p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP) 0.01 mol of polyethylene glycol monomethacrylate (manufactured by NOF Corporation, “Blenmer PE-200”) was dissolved in 20 mL of chloroform, and then −30 Cooled to ° C.
While maintaining the temperature at −30 ° C., 0.01 mol of p-nitrophenyl chloroformate (manufactured by Aldrich), 0.01 mol of triethylamine (manufactured by Wako Pure Chemical Industries) and 20 mL of chloroform were added to this solution. The homogeneous solution was slowly added dropwise.
After reacting at −30 ° C. for 1 hour, the solution was further stirred at room temperature for 2 hours. Thereafter, the salt was removed from the reaction solution by filtration, and the solvent was distilled off to obtain MEONP.

2. Detection of antigen [Example 1] (when RAT ALBUMIN antibody is immobilized in a dry state with a humidity of 50% or less)

(1) First, the following members and raw materials were prepared for detection of RAT ALBUMIN.
A polyethylene resin substrate provided with a well having a diameter of 7 mm and a depth of 1 mm was prepared as a substrate. A 0.5 wt% ethanol solution of the polymer was prepared on the substrate, spotted on the well of the molded product, dried, then washed with ethanol, and the polymer was introduced into the bottom of the well.

(2) Next, a 5 μg / mL phosphate buffer solution of a primary antibody (RABBIT IGG FRACTION TO RAT ALBUMIN (manufactured by Cappel)) was prepared, and after spotting this in the hole, a dry state of 45% or less, Allowed to stand overnight at room temperature. Washing was performed 3 times using a washing solution (0.05% triton X100 / PBS).

(3) Next, 70 μg / m of Albumin antigen (Albumin, Rat (−) (Cappel))
An LPBS solution (specimen) was prepared, spotted in a hole, and then allowed to stand in an environment at 37 ° C. for 1.5 hours to carry out an antigen-antibody reaction. Washing was performed 3 times using the washing solution.

(4) Next, a 1 ug / mL PBS solution (0.05% triton X100) of a labeled secondary antibody (Otin-labeled SHEEP IGG FRACTION TO RAT ALBUMIN (Cappel)) was prepared and spotted in the hole. After that, it was allowed to stand in an environment of 37 ° C. for 1 hour to carry out an antigen-antibody reaction. Washing was performed 3 times using the washing solution.

(5) Next, a solution of Cy3-labeled streptavidin in PBS (0.05% triton X100) was prepared, spotted in the hole, and allowed to stand in an environment of 37 ° C. for 0.5 hour. Washing was performed 3 times using the washing solution.

(6) For the measurement, fluorescence was measured for each well (excitation wavelength: 550 nm, fluorescence wavelength: 570 nm).
The results are shown in Figure 1. According to FIG. 1, a fluorescence signal was obtained in an antigen-dependent manner. Also, the shape of each spot was a good shape circle.

[Comparative Example] (When RAT ALBUMIN antibody is immobilized under high humidity of 50% or higher) The same procedure as in Example 1 is carried out under the conditions of 60% humidity in the immobilization of the primary antibody in (2). did.
As a result, an antigen-dependent fluorescence signal was not obtained.

It can be seen that Example 1 and Comparative Example 1 clearly differ in the intensity and shape of the fluorescent signal obtained, and that good results were obtained in Example 1.

INDUSTRIAL APPLICABILITY The present invention can be used for a biochip for detecting and analyzing bioactive substances with high sensitivity and high throughput by suppressing adsorption and binding of unnecessary bioactive substances.

Claims (14)

A method for producing a biochip comprising a protein immobilized on a substrate surface,
(1) A step of applying a polymer having a phosphorylcholine group to the surface of the substrate,
(2) A step of spotting or applying a liquid in which a protein is dissolved or dispersed in a solvent on the surface of the substrate,
(3) a step of immobilizing the protein,
A method for producing a biochip comprising: The biochip manufacturing method according to claim 1, wherein in the step (3), the substrate is kept in a dry state with a humidity of 50% or less. The method for producing a biochip according to claim 1 or 2, wherein the phosphorylcholine group is a 2-methacryloyloxyethyl phosphorylcholine group. 4. The polymer according to claim 1, wherein the polymer is a copolymer of a monomer having a phosphorylcholine group, a monomer having a butyl methacrylate group, and a monomer having a functional group for immobilizing a protein. Biochip manufacturing method. The method for producing a biochip according to any one of claims 1 to 4, wherein the protein is immobilized by chemical bonding in the immobilization in the step (3). 6. The method for producing a biochip according to claim 5, wherein the functional group for immobilizing the protein is an active ester group. The method for producing a biochip according to claim 5 or 6, wherein the active ester group is p-nitrophenyl ester or N-hydroxysuccinimide ester. The method for producing a biochip according to any one of claims 1 to 7, wherein the protein is immobilized in a spot shape on the surface of the substrate. 9. The method for producing a biochip according to claim 8, wherein a plurality of kinds of protein spots are present in the same section of the substrate surface. The biochip manufacturing method according to any one of claims 1 to 9, wherein the substrate has a glass slide shape. The biochip manufacturing method according to claim 1, wherein the substrate is made of plastic. The method for producing a biochip according to claim 11, 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 method for producing a biochip according to any one of claims 1 to 12, wherein the protein is an antibody. A biochip manufactured by the manufacturing method according to claim 1.