JP4882408B2 - Bioassay substrate - Google Patents

Description

The present invention relates to a bioassay base material used for parallel detection and analysis of a large number of proteins, nucleic acids, etc. in a biological sample. More particularly, the invention relates to proteomics, as well as bioassay substrates used for measuring gene activity at the intracellular protein level.

The technique of obtaining information on a sample using a microarray is becoming an indispensable technique in biology and medicine. For example, a DNA microarray has made it possible to study the expression pattern of the entire genome even in a complicated biological system, which has led to an explosive increase in the amount of genetic information.
In the signal detection of the microarray, the S/N ratio means the amount of signal (signal) obtained from the labeled sample specimen, the amount of signal generated from a site other than the signal substance obtained from the labeled sample specimen ( It is a value divided by (noise), and it is generally said that the detection sensitivity increases when the S/N ratio is high.

The material used for the microarray substrate is often made of glass or plastic, but the surface of these materials is usually chemically inactive, so surface modification is performed to immobilize physiologically active substances. There is a need. Since it is difficult to directly introduce various functional groups onto an inert surface such as glass or plastic, it is common to first introduce an amino group and then introduce the functional group via the amino group. is there.
Examples of the method for introducing an amino group onto the substrate surface include treatment with aminoalkylsilane, plasma treatment under a nitrogen atmosphere, and coating with a polymeric substance containing an amino group. However, the treatment is simple, uniform, and reproducible. From the viewpoint, the treatment with aminoalkylsilane is frequently used. Examples of commonly used aminoalkylsilanes include aminoalkylsilanes having a primary amino group such as aminopropyltrimethoxysilane, aminopropyltriethoxysilane, and aminopropylmethyldimethoxysilane.
As a method for introducing a functional group via an amino group, for example, it is known to introduce an aldehyde group into a substrate by treating it with glutaraldehyde having a bifunctional aldehyde (Patent Document 1 and Patent Document 2). , Patent Document 3). When a maleimide group is introduced, it can be treated with N-(6-maleimidocaproyloxy)succinimide, which is a cross-linking agent having a maleimide group at one end and an active ester at one end (Patent Document 4). Similarly, when introducing an N-hydroxysuccinimide active ester, Ethyleneglycol-O,O-bis (succinimidylsuccinate) having active ester groups at both ends is used.
However, in the surface treatment as described above on the solid-phase substrate, the signal surface is low and the S/N ratio is lowered because the functional group density for immobilizing the physiologically active substance is low because the substrate surface is smooth. It was. There is also a problem that causes a decrease in the S/N ratio. Therefore, there has been a demand for a bioassay substrate having a high signal amount.

"DNA Microarray Practical Manual", edited by Yoshihide Hayashizaki, Koji Okazaki, Yodosha, 2000, p.57. JP, 2002-176991, A JP 2002-181817 A Japanese Patent Publication No. 2002-532699 JP-A-11-187900

It is an object of the present invention to provide a bioassay base material having a higher signal intensity than conventional base materials.

The present invention is
(1) A bioassay substrate for immobilizing a physiologically active substance on a solid phase surface of a substrate, wherein p-nitrophenyloxycarbonyl-polyethylene is used as a physiologically active substance binding component on the solid phase surface of the substrate. Glycol methacrylate, 3 -methacryloxypropyltriethoxysilane as a cross-linking component, and a polymer compound having 2-methacryloyloxyethylphosphorylcholine as a matrix component as constituent units, and the particles are intercalated with the base material surface and the polymer compound. A substrate for bioassay, which is bound and held by a chemical covalent bond.
(2) The bioassay substrate according to (1), wherein the particles are coated with the polymer compound.
(3) The bioassay substrate according to (1) or (2), wherein the particles are made of an inorganic compound, an organic compound, or an organic-inorganic composite compound.
(4) The bioassay substrate according to (1) or (2), wherein the particles are titanium oxide particles or silica particles.
(5) The bioassay substrate according to (1) or (2), wherein the particles are polystyrene resin particles, polyimide resin particles, or poly(meth)acrylic resin particles.
(6) The bioassay substrate according to any one of (1) to (5), wherein the particles have an average diameter of 1 to 1000 nm.
(7) The bioassay substrate according to any one of (1) to (6), wherein the substrate is a slide-shaped substrate, a 96-well plate, a container, or a microfluidic disc substrate.
(8) (1) to (7) microarray immobilizing a physiologically active substance on the bioassay substrate according to any one.
(9) The method for producing a bioassay substrate according to any one of (1) to ( 7 ), which comprises dipping or applying a solution containing the polymer compound, the particles and a solvent into the substrate. A method for producing a bioassay base material comprising:
Is.

According to the bioassay substrate of the present invention, the signal intensity of the substance to be detected is improved, and a microarray with high detection accuracy can be obtained.

The bioassay substrate of the present invention is characterized by having a polymer compound on the solid phase surface of the substrate and retaining particles. By holding the particles on the surface of the base material, the surface area of the base material can be increased, and accordingly, the density of the physiologically active substance-binding group can be increased and the signal intensity can be increased.

The particles used in the present invention are not particularly limited, but particles made of an inorganic compound, an organic compound, or an organic-inorganic composite compound can be preferably used. The average particle diameter is 100 μm or less, preferably 1 to 5000 nm, more preferably 1 to 1000 nm.

There are various particles made of an inorganic compound, but they can be arbitrarily used, for example, carbon, glass, silica, metal, metal oxide, metal hydroxide, metal salt (carbonate, sulfite, sulfate, metal halide, etc.), Examples thereof include metal sulfides, metal nitrides and metal carbides. Specific examples of these include gold, gold hydroxide, magnesium oxide, calcium oxide, aluminum oxide, zirconium oxide, iron oxide (ferrite, etc.), titanium oxide, tin oxide, zinc oxide, copper oxide, nickel oxide, Cobalt oxide, vanadium oxide, tungsten oxide, magnesium hydroxide, calcium hydroxide, aluminum hydroxide, magnesium carbonate, calcium carbonate, calcium sulfite, aluminum sulfate, cuprous chloride, silicon carbide, silicon nitride, sodium silicate, aluminum nitride Etc. are illustrated.

As the carbon beads, carbon particles obtained by carbonizing and firing a raw material containing a large amount of carbon such as coal, pitch, graphite, coke, carbon black, wood (sugar), and resin can also be used.
As a silica particle, sicastar (registered trademark) (micromod company) is known as a commercial product.

The particles made of an organic compound are preferably particles made of a resin. As the resin, phenol resin, urea resin, melamine resin, benzoguanamine resin, alkyd resin, unsaturated polyester resin, vinyl ester resin, diallyl phthalate resin, epoxy resin, urethane resin, furan resin, ketone resin, xylene resin, polyimide resin, poly Carbodiimide resins, styryl pyridine resins, triazine resins, styrene resins, acrylic resins, methacrylic resins, carbonate resins, vinyl alcohol resins, polypropylene resins, cyclopolyolefin resins, etc. are known. As commercially available products, Sumilite Resin ACS series (Sumitomo Bakelite Co., Ltd.), Bell Pearl (Kanebo Co., Ltd.) and Univex (Unitika Co., Ltd.), micromer (registered trademark) (micromod Co.), which are fine phenol resin particles, are known. There is.

The particles in the present invention are preferably chemically bonded to the surface of the base material via a polymer compound. Therefore, if necessary, the particles can be activated by sulfuric acid, hydrochloric acid, plasma treatment or the like to easily bond the polymer compound and the particles.

The polymer compound used in the present invention is preferably one having a physiologically active substance-binding component, a crosslinking component, and a matrix component as constituent units. This polymer compound can be obtained, for example, by copolymerizing a monomer having a physiologically active substance binding component, a monomer having a crosslinking component, and a monomer having a matrix component.

The structure of the monomer having a physiologically active substance-binding component used in the present invention is not particularly limited, but is represented by the general formula [1] (wherein R ₂ represents a hydrogen atom or a methyl group, and Y represents 1 to 10 carbon atoms). Of the formula (1) represents an alkyleneoxy group or an alkyl group of W. W represents a physiologically active substance-bonding group), and W is a chain of an alkyleneoxy group having 1 to 10 carbon atoms or a chain of an alkyl group. It is preferable that the compound is a compound bound by. The repeating number q of the alkyleneoxy group Y is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon numbers of the repeated alkyleneoxy groups may be the same or different.

Examples of the physiologically active substance-binding group W include p-nitrophenyl ester group, N-hydroxysuccinimide ester group, succinimide ester group, phthalic acid imide ester group, 5-norbornene-2,3-dicarboximide ester group, and aldehyde. Examples thereof include a group, an amino group, an epoxy group, and the like, and a p-nitrophenyl ester group or an N-hydroxysuccinimide ester group is preferable because a physiologically active substance can be immobilized at a lower pH.

The monomer having a crosslinking component used in the present invention is a monomer having a crosslinkable functional group, and is not particularly limited as long as the reaction of the crosslinkable functional group does not proceed during the synthesis of the polymer compound. Absent.
As the crosslinkable functional group, for example, a functional group that produces a silanol group by hydrolysis or a glycidyl group is used, but a functional group that produces a silanol group by hydrolysis is preferable because it can be crosslinked at a lower temperature.

The monomer having a crosslinking component used in the present invention is a monomer represented by the general formula [2] (wherein R ₃ represents a hydrogen atom or a methyl group, and Z represents an alkyl group having 1 to 20 carbon atoms). However, Z may be omitted. It is preferable that at least one of A ₁ , A ₂ , and A ₃ is a hydrolyzable group and the other is an alkyl group).

The monomer having a functional group capable of generating a silanol group by hydrolysis represented by the general formula [2] is a (meth)acryl group and an alkyl group having a functional group capable of generating a silanol group by hydrolysis has 1 to 20 carbon atoms. It is a compound that is attached through a chain or directly. The functional group that produces a silanol group by hydrolysis is a group that is easily hydrolyzed to form a silanol group when contacted with water, and examples thereof include a halogenated silyl group, an alkoxysilyl group, a phenoxysilyl group, and an acetoxysilyl group. Etc. can be mentioned. Of these, an alkoxysilyl group is preferable because it easily forms a silanol group.

Examples of the monomer containing an alkoxysilyl group include 3-(meth)acryloxypropenyltrimethoxysilane, 3-(meth)acryloxypropylbis(trimethylsiloxy)methylsilane, and 3-(meth)acryloxypropyldimethyl. Methoxysilane, 3-(meth)acryloxypropyldimethylethoxysilane, 3-(meth)acryloxypropylmethyldimethoxysilane, 3-(meth)acryloxypropylmethyldiethoxysilane, 3-(meth)acryloxypropyltrimethoxy Silane, 3-(meth)acryloxypropyltriethoxysilane, 3-(meth)acryloxypropyltris(methoxyethoxy)silane, 8-(meth)acryloxyoctanyltrimethoxysilane, 11-(meth)acryloxyundenyl Examples thereof include (meth)acryloxyalkylsilane compounds such as trimethoxysilane. Among them, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-methacryloxypropyldimethylmethoxysilane and 3-methacryloxypropyldimethylethoxysilane are copolymerizable with a monomer having a phosphorylcholine group. Is preferable and easy to obtain. These alkoxysilyl group-containing monomers are used alone or in combination of two or more.

The monomer having a matrix component used in the present invention is not particularly limited in structure, but is preferably a (meth)acrylic group-bonded compound represented by the general formula [3]. (In the formula, R ₁ represents a hydrogen atom or a methyl group, and X represents a group having a phosphorylcholine group or a group having an alkylene glycol residue).

Examples of the monomer having a phosphorylcholine group include 2-(meth)acryloyloxyethylphosphorylcholine, 2-(meth)acryloyloxyethoxyethylethylphosphorylcholine, 6-(meth)acryloyloxyhexylphosphorylcholine, 10-(meth)acryloyloxyethoxy. Nonylphosphorylcholine, 2-(meth)acryloyloxypropylphosphorylcholine and the like can be mentioned, but 2-methacryloyloxyethylphosphorylcholine is preferable from the viewpoint of availability.

The alkylene glycol residue is, for example, a group composed of repeating alkylene glycol residues T, and the number of repetitions is an integer of 1 to 100, more preferably an integer of 2 to 100, and further preferably 2 to 95. It is an integer, and most preferably an integer of 20 to 90. When the number of repetitions is 2 or more and 100 or less, the number of carbon atoms of the repeated alkylene glycol residue T may be the same or different.
The alkylene glycol residue T has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms, further preferably 2 to 4 carbon atoms, still more preferably 2 to 3 carbon atoms, and most preferably 2.

Examples of the monomer having an alkylene glycol residue include hydroxyl groups such as methoxy polyethylene glycol (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 2-hydroxypropyl (meth)acrylate, and 2-hydroxybutyl (meth)acrylate. Mono-substituted ester (meth)acrylates, glycerol mono(meth)acrylate, (meth)acrylate having polypropylene glycol as a side chain, 2-methoxyethyl (meth)acrylate, 2-ethoxyethyl (meth)acrylate, methoxydiethylene glycol Examples thereof include (meth)acrylate, ethoxydiethylene glycol (meth)acrylate, and ethoxypolyethylene glycol (meth)acrylate, with methoxypolyethylene glycol methacrylate being preferred from the viewpoint of availability.

The method of synthesizing the polymer compound used in the present invention is not particularly limited, but from the viewpoint of easiness of synthesis, a monomer having at least a physiologically active substance-binding component, a monomer having a crosslinking component and a matrix component. It is preferable to radically polymerize a mixture containing a monomer having OH in a solvent in the presence of a polymerization initiator.

Any solvent can be used as long as it can dissolve each monomer, and examples thereof include methanol, ethanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, and chloroform. These solvents are used alone or in combination of two or more. When the polymer compound is applied to a plastic substrate, ethanol and methanol are preferable because they do not denature the substrate.

The polymerization initiator may be any ordinary radical initiator, for example, 2,
Examples include azo compounds such as 2'-azobisisobutylnitrile (hereinafter referred to as "AIBN") and 1,1'-azobis(cyclohexane-1-carbonitrile), organic peroxides such as benzoyl peroxide and lauryl peroxide. be able to.

The chemical structure of the polymer compound used in the present invention may be random, block, graft or the like as long as the monomers are copolymerized.

As for the molecular weight of the polymer compound used in the present invention, the number average molecular weight is preferably 5,000 or more, and more preferably 10,000 or more, because separation and purification of the polymer compound and unreacted monomer are facilitated.

An example of the method for coating the surface of the base material or the particles with the polymer compound is shown below. For example, a polymer solution is prepared by dissolving a polymer compound in an organic solvent to a concentration of 0.05 to 10% by weight in a first solvent, and then mixed with particles dispersed in a second solvent. At this time, it is preferable that the particles and the polymer compound are chemically covalently bonded by a reaction. The substrate is applied to the surface of the substrate by a known method such as dipping or spraying in the thus obtained mixed solution, and then dried at room temperature or under heating.

The first solvent and the second solvent may be the same or different, but are preferably organic solvents. For example, a single solvent such as ethanol, methanol, t-butyl alcohol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform or a mixed solvent thereof is used. Among them, ethanol or methanol is preferable because it does not denature the plastic substrate and is easy to dry.

The base material used in the present invention is preferably a slide-shaped substrate, a 96-well plate, a container, or a microfluidic disc substrate. Examples thereof include a plastic substrate, a glass substrate, and a substrate having a metal vapor deposition film. Specific examples of the plastic substrate include substrates made of cycloolefin polymer, polypropylene, polyethylene oxide, polysalophone, polyimide, polycarbonate, and polymethyl petacrylate.

By immobilizing a physiologically active substance using the bioassay base material of the present invention, a bioassay with high signal intensity becomes possible.

Examples of synthesizing the polymer compound used in the present invention and examples of the bioassay substrate are shown below.

(Synthesis of p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP))
0.01 mol of polyethylene glycol monomethacrylate (Blenmer PE-200 (n=4) manufactured by NOF CORPORATION) was dissolved in 20 mL of chloroform and then cooled to -30°C. While maintaining at -30°C, 0.01 mol of p-nitrophenyl chloroformate (manufactured by Aldrich), 0.01 mol of triethylamine (manufactured by Wako Pure Chemical Industries, Ltd.), and 20 ml of chloroform prepared in advance were added to the solution. The homogeneous solution was dropped slowly. After reacting at -30°C for 1 hr, the solution was further stirred at room temperature for 2 hr. After that, salt was removed from the reaction solution by filtration and the solvent was distilled off to obtain p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate (MEONP). The obtained monomer was measured by 1H-NMR in a deuterated chloroform solvent, and it was confirmed that 4.5 units of ethylene glycol residues were contained.

(Synthesis example 1 of polymer compound)
2-methacryloyloxyethylphosphorylcholine (MPC), 3-methacryloxypropyltriethoxysilane (MPTES), and MEONP were added to dehydrated ethanol in order of 0.9 mol/L, 0.05 mol/L, and 0.05 mol/L, respectively. It was made to melt|dissolve and the monomer mixed solution was produced. Further, 0.002 mol/L of AIBN was added thereto, and the mixture was stirred until it became uniform. Then, after reacting at 60° C. for 3 hours in an argon gas atmosphere, the reaction solution was dropped into a mixed solvent of diethyl ether and chloroform to collect a precipitate. The obtained polymer compound was measured by 1 H-NMR, and peaks attributed to methylene of BMA appearing at around 1.46 and 1.65 ppm and peaks attributed to trimethyl of MPC appearing near 3.34 ppm, respectively. The composition ratio of this polymer compound was calculated from the integrated value. The results are shown in Table 1.

(Substrate 1)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm×26 mm×1 mm) to prepare a solid phase substrate, and oxygen plasma treatment was performed.

(Particle 1)
As silica nano beads, sicastar (registered trademark) manufactured by micromod: average particle diameter 70 nm (product number 43-00-701) was used.

Solution 1 (Preparation of solution of polymer compound having particles)
The polymer compound obtained in Synthesis Example 1 and particles 1 were dissolved in ethanol to prepare a solution containing 0.3 wt% of the polymer compound and 0.2 wt% of particles.

Solution 2 (Preparation of polymer compound solution)
The polymer compound obtained in Synthesis Example 1 was dissolved in ethanol to prepare a 0.3 wt% polymer solution.

(Example 1)
The substrate 1 was dipped in the solution 1 and pulled up, and heated at 100° C. for 2 hours to obtain a bioassay base material.

(Comparative Example 1)
A bioassay substrate was obtained in the same manner as in Example 1 except that the solution 2 was used.

(Evaluation)
(Preparation of DNA solution)
DNA solution 1: Oligo DNA having a chain length of 24 bp and having an amino group at the 5'end (TAGAAGCATTTTGCGGTGGACGATG (SEQ ID NO: 1) (manufactured by Sigma Genosys) was added with a predetermined buffer solution to a concentration of 0.1 µg/µl. Dissolved.
DNA solution 2; 24 bp long oligo DNA having a Cy3 label at the 5'end (CATCGTCCACCCGCAAATGCTTCA (SEQ ID NO: 2) (manufactured by Sigma Genosys), 3×SSC, 0 at a concentration of 0.002 μg/μl) It was dissolved in a solution of 2% SDS.

(Spot and hybridization)
In Example 1, the DNA solution 1 was dispensed into a 96-well plate and spotted on the substrate using a micropin type microarray spotter. After the completion of spotting, the spot was kept still in an oven at 80°C.
Then, the active ester group was inactivated by immersing it in a 0.1 N sodium hydroxide solution for 5 minutes to perform a blocking treatment. Next, the DNA solution 2 is spread on this substrate, covered with a cover glass, and left in a humid container at 65° C. for 3 hours to allow hybridization of the immobilized oligo DNA with the Cy3 labeled oligo DNA. went. After that, the substrate was washed in 2×SSC, 0.5% SDS, and then washed with pure water to prepare a substrate after DNA hybridization.

Table 2 shows the results of measuring the fluorescence count value and background value after DNA hybridization and quantifying the fluorescence amount of the spot.
A microarray scanner "ScanArray" manufactured by Packard BioChip Technologies was used for the measurement of the amount of fluorescence. The measurement conditions were 90% laser output, 60% PMT sensitivity, excitation wavelength 649 nm, measurement wavelength 670 nm, and resolution 50 μm.
It was found that the example is a bioassay substrate having a higher hybrid signal amount than the comparative example.

Claims (9)

A bioassay substrate for immobilizing a physiologically active substance on a solid phase surface of a substrate, comprising p-nitrophenyloxycarbonyl-polyethylene glycol methacrylate as a physiologically active substance binding component on the solid phase surface of the substrate. A polymer compound having 3 -methacryloxypropyltriethoxysilane as a cross-linking component and 2-methacryloyloxyethylphosphorylcholine as a constituent unit as a matrix component, and the particles are chemically bonded to the surface of the base material and the polymer compound. A bioassay substrate, which is bound and held by a covalent bond. The bioassay substrate according to claim 1, wherein the particles are coated with the polymer compound. The bioassay substrate according to claim 1 or 2, wherein the particles are made of an inorganic compound, an organic compound, or an organic-inorganic composite compound. 3. The bioassay substrate according to claim 1, wherein the particles are titanium oxide particles or silica particles. The bioassay substrate according to claim 1, wherein the particles are polystyrene resin particles, polyimide resin particles, or poly(meth)acrylic resin particles. The bioassay substrate according to any one of claims 1 to 5, wherein the particles have an average diameter of 1 to 1000 nm. Said substrate slides shaped substrate, 96-well plates, containers, or bioassay substrate according to any one of claims 1 to 6 is a micro sieve disc substrate. Microarray immobilizing a physiologically active substance on the bioassay substrate according to any one of claims 1 to 7. A method of manufacturing a bioassay substrate according to any one of claims 1-7, wherein the polymer compound, for bioassay comprising a step of immersing or applying a solution containing the particles and a solvent to the substrate A method for manufacturing a base material.