JP2004177378A - Fixing method of biopolymer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for fixing a biopolymer onto a solid phase surface with high density. <P>SOLUTION: The biopolymer having a functional group (A) is allowed to react with a multifunctional compound having at least three functional groups (B) having reaction activity to the functional group (A) in one molecule for bonding the biopolymer and the multifunctional compound; at least one unreacted functional group (B) is allowed to survive in one molecule of the multifunctional compound; and the unreacted functional group (B) surviving in the multifunctional compound is allowed to react to the solid phase surface having a functional group (C) having the reaction activity to the functional group (B), thus fixing the biopolymer onto the solid phase surface. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for immobilizing a biopolymer on a solid phase surface. The method for immobilizing a biological material of the present invention can be used for, for example, immunoassays for immunodiagnosis, DNA microarrays, and so-called protein chips.
[0002]
[Prior art]
For example, a method for immobilizing a biopolymer on a solid phase surface such as a substrate for use in applications such as immunoassays for immunodiagnosis and DNA microarrays includes, for example, contacting the biopolymer with a solid phase on a hydrophobic surface. Immobilization by non-specifically adsorbing on the surface, immobilizing electrostatically on the positively charged surface if it is a negatively charged substance, binding such as antigen-antibody reaction A method for immobilization using high specificity, for example, a method for immobilization using a covalent bond formed by a reaction between a formyl group and an amino group, and the like are known.
[0003]
Regardless of the method of immobilization on any solid phase surface, in order to increase the amount of biopolymer to be immobilized, increase the amount of functional groups serving as anchors to be introduced to the solid phase surface, or make the surface porous. A method of increasing the surface area of a solid phase to which a biopolymer binds has been adopted, such as a method of denaturation and a method of binding to a bead surface.
[0004]
However, because the reaction is between the solid phase surface and the solution, even if the amount of the functional group that serves as the anchor on the solid phase surface is increased, the reaction efficiency is lower than the reaction in which the same reaction is performed in a solution, and the effect is low. It is hardly reflected on the fixed amount of biopolymer and its density. Therefore, there is a limit to the increase in the fixed density.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for immobilizing a biopolymer at a high density on a solid phase surface.
[0006]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to solve the above problems, and as a result, after binding a plurality of biopolymers to a polyfunctional compound, immobilize the polyfunctional compound on a solid phase surface, The present inventors have found that a polymer can be immobilized on a solid phase surface at high density, and have completed the present invention.
[0007]
That is, the present invention relates to a method for immobilizing a biopolymer on a surface of a solid phase, wherein the biopolymer having a functional group (A) has a reaction activity for the functional group (A) in one molecule. Reacting a polyfunctional compound having at least three functional groups (B) to bind the biopolymer and the polyfunctional compound, and to convert an unreacted functional group (B) to one molecule of the polyfunctional compound; At least one unreacted functional group (B) remaining in the polyfunctional compound on the solid-phase surface having a functional group (C) having a reaction activity with respect to the functional group (B). Provided is a method for immobilizing a biopolymer, which comprises reacting and binding.
[0008]
BEST MODE FOR CARRYING OUT THE INVENTION
The biopolymer has a functional group (A) involved in immobilization of the molecule, and the polyfunctional compound has three or more functional groups (B) reactive to the functional group (A) in one molecule. The solid surface has a functional group (C) having a reaction activity with respect to the functional group (B). As used herein, “reactive” means reacting and covalently bonding. The functional group (A) and the functional group (C) may be the same functional group.
[0009]
As these functional groups and combinations thereof, known and commonly used methods can be used. However, since the reaction involves treatment of a biopolymer, minus 20 ° C. to 100 ° C., preferably 0 ° C. to 70 ° C., and furthermore Preferably, a functional group that efficiently reacts in a temperature range of 0 ° C to 50 ° C and a combination thereof are desirable. Further, a functional group whose reaction proceeds only in a solvent in which the function of the biopolymer is inactivated and a combination thereof are not preferable. Considering that the function of the biopolymer is not inactivated, it is preferable to use a functional group having activity even in water and a combination thereof. As such a combination, a combination of an amino group and a functional group that reacts with the amino group is preferable.
[0010]
As the functional group that reacts with the amino group, an epoxy group, a haloformyl group, an isocyanato group, a carboxylic acid group, an acid anhydride, a maleimide group, and a formyl group are preferable. Among these, the reaction of the functional group that reacts with the amino group is preferred. Considering properties and stability, a formyl group and an epoxy group are preferred. If the biopolymer is stably present in the range of 37 ° C to 100 ° C, it is preferable to use an epoxy group.
[0011]
Preferred combinations satisfying the above conditions include, for example, (1) a formyl group as the functional group (A), an amino group as the functional group (B), an epoxy group as the functional group (C), and (2) a functional group (A). An amino group, a carboxyl group as the functional group (B), an amino group as the functional group (C), (3) a formyl group as the functional group (A), an amino group as the functional group (B), and an acid as the functional group (C). Combinations such as anhydrides are exemplified.
[0012]
The biopolymer having the functional group (A) is optional, and is, for example, DNA, RNA, peptide, protein, glycoprotein and the like. The method for introducing the functional group (A) into the biopolymer is also arbitrary, and a known and commonly used method can be used. For example, when the biopolymer is an oligonucleotide, various functional groups can be converted into functional groups (through an amino group in a base or a terminal amino group in an oligonucleotide having an aminated 5 ′ or 3 ′ end). A) can be introduced as Further, for example, when the biopolymer is a peptide, it is possible to introduce various functional groups as the functional group (A) via the amino terminal and the amino group in lysine. Further, the peptide can be modified by a photochemical reaction such as an azide compound containing the functional group (A).
[0013]
The polyfunctional compound having three or more functional groups (B) in the molecule is arbitrary, and examples thereof include polyallylamine, poly-L-lysine, polyglutal, and polycarboxylic acid. The polyfunctional compound is preferably water-soluble.
[0014]
The polyfunctional compound needs to have three or more functional groups (B) in one molecule. If the polyfunctional compound has only two functional groups (B), only one molecule of the polyfunctional compound can immobilize one biomolecule on a solid phase. There is no difference from the method of modifying and immobilizing polymers, and it is not possible to immobilize biopolymers at high density. When the polyfunctional compound has only one functional group (B), the functional group (B) reacts with the functional group (A) of the biopolymer to form a functional group ( Cannot react with C).
[0015]
The number of functional groups (B) in one molecule of the polyfunctional compound is preferably 5 or more, more preferably 10 or more, and most preferably 30 or more. Since the number of functional groups is large and there is no inconvenience due to itself, it is not necessary to set an upper limit.However, if the number is excessive, the molecular weight of the polyfunctional compound tends to be excessively high, and a portion of the chain polymer described later The disadvantages described below occur.
[0016]
The polyfunctional compound is preferably a chain polymer. By using a chain polymer, the polyfunctional compound can have many functional groups (B) in the molecule, and the flexible main chain serves as a spacer between the solid phase and the biopolymer. Functions such as affinity of the biopolymer are enhanced. Here, the chain polymer refers to a polymer that is not a crosslinked polymer, and may be a linear polymer, a branched polymer, a star polymer, or the like.
[0017]
The average molecular weight of the chain polymer is arbitrary, but is preferably 1,000,000 or less, more preferably 100,000 or less. If the molecular weight of the chain polymer is excessively high, problems such as easy gelation, a decrease in solubility, and a biopolymer being off-cladded by the polyfunctional compound and not appearing on the surface arise.
[0018]
The lower limit of the average molecular weight of the chain polymer is only required to have three or more functional groups (B) in one molecule as described above. For example, the molecular weight of an oligomer region such as about 200 or about 500 may be used. good. In order for the chain polymer to exhibit a function as a flexible spacer, the average molecular weight is preferably 1,000 or more.
[0019]
The method for synthesizing the polyfunctional compound is also arbitrary, and may be polymerization or copolymerization of a monomer having a functional group (B), or introduction of the functional group (B) into a chain natural polymer or synthetic polymer.
[0020]
The method of the present invention comprises the step of reacting the biopolymer with the polyfunctional compound to bind the biopolymer to the polyfunctional compound and to allow the unreacted functional group (B) to react with the polyfunctional compound. Leave at least one in the molecule. The upper limit of the remaining number is a value obtained by subtracting 2 from the number of functional groups (B) of the polyfunctional compound. If the remaining amount is excessive, the fixed amount of the biopolymer decreases and the conventional method is not changed, and the probability that the biopolymer is embedded by the polyfunctional compound increases. When the remaining amount is less than 1 or zero, the biopolymer is not fixed to the solid phase. The number of functional groups (B) remaining in the polyfunctional compound can be measured by a known and commonly used method, for example, NMR. The optimum value can be determined by a simple experiment in the reaction system used.
[0021]
Any method may be used for reacting the biopolymer with the polyfunctional compound to bond the same, and leaving at least one unreacted functional group (B) on average in one molecule of the polyfunctional compound. A method in which the reaction is carried out under conditions in which the functional group (B) is excessive with respect to the functional group (A), or when the functional group (B) is charged in an amount equal to or less than an equimolar amount relative to the functional group (A). Even in this case, a method of interrupting the reaction when the reaction is not completed can be adopted. Among these, the latter is superior to the reaction speed, but the former is preferable because of good reproducibility.
[0022]
The shape of the solid phase for immobilizing the biopolymer in the present invention is arbitrary, and may be, for example, a plate, a sheet, an inner surface of a microwell, an inner surface of an Eppendorf tube, a bead, or a porous body. The shape of the solid phase surface is also arbitrary, and may be a smooth surface, an uneven surface, or a porous shape.
[0023]
The method for introducing the functional group (C) into the solid phase of the present invention is also arbitrary, and a known and commonly used method can be used. Examples include surface treatment with a silane coupling agent having a functional group (C), polymerization or copolymerization of a monomer having a functional group (C), introduction of an azide compound having a functional group (C) by a photoreaction, and the like. Can be.
[0024]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to the scope of the following examples. In the following examples and comparative examples, “parts” is “parts by mass”.
[0025]
[Energy beam irradiation device]
A UE031 type exposure apparatus manufactured by Eye Graphics Co., Ltd. was used as an energy ray irradiation apparatus, and a MOS-L31 type lamp was used as a light source. The ultraviolet intensity is 50 mw / cm ² unless otherwise stated.
[0026]
[Fluorescence intensity measurement method]
The measurement was performed using a confocal laser microscope TCS-NT manufactured by Leica Corporation. The measurement conditions are a PMT sensitivity of 449 V and a pinhole of 0.65.
[0027]
[Example 1]
[Preparation of energy ray-curable composition (i)]
80 parts of a trifunctional urethane acrylate oligomer having an average molecular weight of about 2000 (“Unidick V-4263” manufactured by Dainippon Ink and Chemicals, Inc.) as an active energy ray crosslinkable polymerizable compound, and 1,6-hexanediol diacrylate 20 parts of “New Frontier HDDA” manufactured by Daiichi Kogyo Seiyaku Co., Ltd. and 5 parts of 1-hydroxycyclohexylphenyl ketone (“Irgacure 184” manufactured by Ciba Geigy) as a photopolymerization initiator are uniformly mixed and composed. The product (i) was prepared.
[0028]
[Preparation of energy ray-curable composition (ii)]
As the active energy ray crosslinkable polymerizable compound, 60 parts of a trifunctional urethane acrylate oligomer having an average molecular weight of about 2000 (“Unidick V-4263” manufactured by Dainippon Ink and Chemicals, Inc.), and 1,6-hexanediol diacrylate 30 parts of "New Frontier HDDA" manufactured by Daiichi Kogyo Seiyaku Co., Ltd., 10 parts of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.), and 1-hydroxycyclohexyl phenyl ketone (Ciba Geigy) as a photopolymerization initiator. (“Irgacure 184”) manufactured by Nissan Co., Ltd. was uniformly mixed to prepare composition (ii).
[0029]
(Preparation of solid phase for fixing biopolymer)
A composition (i) was applied to a plate made of polyacrylate ("Acrylite L 000" manufactured by Mitsubishi Rayon Co., Ltd.) as a substrate (1) using a spin coater at 2,000 rpm for 50 seconds. The coating film (2) was semi-cured by irradiating 50 mw / cm ² of ultraviolet light in a nitrogen atmosphere for 3 seconds.
[0030]
The composition (ii) was applied on the semi-cured coating film (2) using a spin coater at 2000 rpm for 50 seconds, and irradiated with ultraviolet rays of 50 mw / cm ^{2 in} a nitrogen atmosphere for 60 seconds. Then, the coating film (2) was cured to obtain a solid phase (A) for immobilizing a biopolymer.
[0031]
(Preparation of biopolymer)
1 μl of a gene chain A (5′-GTTCGACGGTCATCGTCTCGATCTCCG-3 ′) modified with an amino group at the 5 ′ end and an FITC (fluorescein isothiocyanate) at the 3 ′ end, and a gene chain B complementary to the gene sequence of the gene chain A ( A PCR reaction tube containing 1 μl of 5′-CGAGATCGAGCGATGACGTCGAAC-3 ′), 1 μl of 10 × buffer for PCR (10 × Ex Taq Buffer manufactured by Takara Shuzo Co., Ltd.), and 7 μl of sterilized water was added to the PCR reaction cycler using a thermal cycler for PCR reaction. By giving a temperature change of 95 ° C for 2 minutes, 80 ° C for 2 minutes, 70 ° C for 2 minutes, 60 ° C for 2 minutes, 50 ° C for 2 minutes, 40 ° C for 2 minutes, 30 ° C for 2 minutes and 4 ° C for 2 minutes, the gene chain A And B were hybridized to prepare a biopolymer solution (iii).
[0032]
(Preparation of biopolymer for immobilization)
The prepared biopolymer solution (iii) was prepared by diluting a 25% glutaraldehyde solution (a 25% glutaraldehyde solution manufactured by Wako Pure Chemical Industries) with a phosphate buffer solution (manufactured by Wako Pure Chemical Industries) 10-fold. 2 μl of a 2.5% glutaraldehyde solution was added, and the mixture was reacted in a water bath at 37 ° C. for 2 hours. Then, 100% ethanol was added thereto to precipitate the gene chain. Got. A 10% polyallylamine solution (PAA-10C manufactured by Nitto Bo) was added to a phosphate buffer (a solution prepared by dissolving “phosphate buffer powder” manufactured by Wako Pure Chemical Industries in 1 liter of sterilized water). 4 μl of a 5% polyallylamine solution prepared by double dilution was added and reacted in a water bath at 37 ° C. for 2 hours to prepare an immobilized biopolymer (α) in which polyallylamine was bonded to an aldehyde-modified gene chain.
[0033]
[Immobilization of biopolymer]
The prepared biopolymer for immobilization and 4 μl of microspotting solution (manufactured by Alliatt) were spotted on the solid phase for biopolymer immobilization (A) in an amount of about 0.5 μl, and then the humidity was 100% and the temperature was 50 ° C. For 12 hours.
[0034]
[Washing of immobilized biopolymer]
After washing the solid phase A held for 12 hours with a 0.2 × SSC / 0.1% SDS (sodium lauryl sulfate) solution at room temperature for 5 minutes, washing with a 0.2 × SSC solution at room temperature for 5 minutes three times. This was repeatedly used as a measurement sample.
[0035]
[Comparative Example 1]
(Preparation of solid phase for fixing biopolymer)
A composition (i) was applied to a plate made of polyacrylate ("Acrylite L 000" manufactured by Mitsubishi Rayon Co., Ltd.) as a substrate (1) using a spin coater at 2,000 rpm for 50 seconds. The coating film (2) was semi-cured by irradiating 50 mw / cm ² of ultraviolet light in a nitrogen atmosphere for 3 seconds.
[0036]
The composition (ii) was applied on the semi-cured coating film (2) using a spin coater at 2000 rpm for 50 seconds, and irradiated with ultraviolet rays of 50 mw / cm ^{2 in} a nitrogen atmosphere for 60 seconds. Then, the coating film (2) was cured. A 10% polyallylamine solution (Nittobo PAA-10C) was dissolved in a phosphate buffer (a solution of "phosphate buffer powder" manufactured by Wako Pure Chemical Industries, Ltd. in 1 liter of sterilized water). Then, it was immersed in a 5% polyallylamine solution prepared by diluting with 2 times and reacted in a 37 ° C. water bath for 2 hours to prepare a solid phase (B) for immobilizing a biopolymer having polyallylamine bonded to the surface.
[0037]
(Preparation of biopolymer for immobilization)
A 25% glutaraldehyde solution (25% glutaraldehyde solution manufactured by Wako Pure Chemical Industries, Ltd.) was added to the prepared biopolymer solution (iii) by adding a phosphate buffer solution (“phosphate buffer powder” manufactured by Wako Pure Chemical Industries, Ltd.) to 1 part. 2 μl of a 2.5% glutaraldehyde solution prepared by diluting 10-fold with a solution dissolved in 1 liter of sterilized water), and allowed to react in a 37 ° C. water bath for 2 hours. Was precipitated to obtain a gene chain in which the amino group in the gene chain was modified with aldehyde. To this, 4 μl of a phosphate buffer (a solution of “phosphate buffer powder” manufactured by Wako Pure Chemical Industries, Ltd., dissolved in 1 liter of sterilized water) was added, and the mixture was reacted in a 37 ° C. water bath for 2 hours. An immobilized biopolymer (β) to which an aldehyde-modified gene chain was bonded was prepared.
[0038]
[Immobilization of biopolymer]
4 μl of the prepared biopolymer for immobilization (β) and 4 μl of microspotting solution (manufactured by Alliatt) were spotted on the solid phase for biopolymer immobilization (B) in an amount of about 0.5 μl each. The temperature was kept at 50 ° C. for 12 hours.
[0039]
[Washing of immobilized biopolymer]
The solid phase B held for 12 hours is washed with 0.2 × SSC / 0.1% SDS (sodium lauryl sulfate) solution at room temperature for 5 minutes, and then washed with 0.2 × SSC solution at room temperature for 5 minutes three times. This was repeatedly used as a measurement sample.
[0040]
[Comparative Example 2]
(Preparation of solid phase for fixing biopolymer)
A composition (i) was applied to a plate made of polyacrylate ("Acrylite L 000" manufactured by Mitsubishi Rayon Co., Ltd.) as a substrate (1) using a spin coater at 2,000 rpm for 50 seconds. The coating film (2) was semi-cured by irradiating 50 mw / cm ² of ultraviolet light in a nitrogen atmosphere for 3 seconds.
[0041]
The composition (ii) was applied on the semi-cured coating film (2) using a spin coater at 2000 rpm for 50 seconds, and irradiated with ultraviolet rays of 50 mw / cm ^{2 in} a nitrogen atmosphere for 60 seconds. Then, the coating film (2) was cured. The cured solid phase was prepared by dissolving a 10% polyallylamine solution (Nittobo PAA-10C) in a phosphate buffer ("Phosphate buffer powder" manufactured by Wako Pure Chemical Industries, Ltd. dissolved in 1 liter of sterilized water). Immersed in a 5% polyallylamine solution prepared by diluting with 2 times, and reacted in a 37 ° C. water bath for 2 hours. Further, a 25% glutaraldehyde solution (a 25% glutaraldehyde solution manufactured by Wako Pure Chemical Industries) was phosphorylated. It was immersed in a 2.5% glutaraldehyde solution prepared by diluting 10-fold with an acid buffer ("Phosphate buffer powder" manufactured by Wako Pure Chemical Industries, Ltd. in 1 liter of sterilized water), and then immersed in a 37% solution. The reaction was carried out in a water tank for 2 hours to prepare a solid phase (C) for immobilizing a biopolymer having glutaraldehyde bonded to the surface.
[0042]
(Preparation of biopolymer for immobilization)
100% ethanol was added to the prepared biopolymer solution (iii) to precipitate the gene chain, and a phosphate buffer solution ("phosphate buffer powder" manufactured by Wako Pure Chemical Industries, Ltd.) was added to 1 liter of sterilized water. Was dissolved in a water bath at 37 ° C. for 2 hours to prepare a biopolymer (γ) for immobilization.
[0043]
[Immobilization of biopolymer]
The prepared biopolymer for immobilization (γ) and 4 μl of microspotting solution (manufactured by Araiat) were spotted on the solid phase for biopolymer immobilization (C) in an amount of about 0.5 μl. The temperature was kept at 50 ° C. for 12 hours.
[0044]
[Washing of immobilized biopolymer]
The solid phase C held for 12 hours is washed with 0.2 × SSC / 0.1% SDS (sodium lauryl sulfate) solution at room temperature for 5 minutes, and then washed with 0.2 × SSC solution at room temperature for 5 minutes three times. This was repeatedly used as a measurement sample.
[0045]
As a result, although observation was sufficiently possible in the examples, it was not possible to observe in comparative examples 1 and 2.
[0046]
【The invention's effect】
The method for immobilizing a biopolymer on a solid surface according to the present invention can fix the biopolymer at a higher density than the conventional method of directly immobilizing a target biopolymer on a functional group on a wall surface. it can. Therefore, it is possible to detect a biopolymer with higher sensitivity. In addition, when the sample amount is very small, or when the immobilization efficiency of the sample is low, by using the method of immobilizing at high density, detection is performed with higher sensitivity than the conventional immobilization method. It becomes possible. By using the method of the present invention, it is possible to detect with higher sensitivity in an experiment for immobilizing a biopolymer such as a DNA microarray or an ELISA method using a microtiter plate.
[Brief description of the drawings]
FIG. 1 is a schematic cross-sectional view of a solid phase for fixing a biopolymer (A), (B) and (C) used in Example 1, Comparative Examples 1 and 2.
[Explanation of symbols]
1 Substrate (polyacrylate plate)
2 Coating film, resin layer, energy ray-curable composition (i)
3. Coating film, resin layer, energy ray-curable composition (ii)
4 Polyallylamine layer 5 Glutaraldehyde layer

Claims (9)

A method for immobilizing a biopolymer on a surface of a solid phase, comprising a biopolymer having a functional group (A) and a functional group (B) having a reaction activity with respect to the functional group (A) in one molecule. Reacting at least three polyfunctional compounds to bind the biopolymer and the polyfunctional compound, and leaving at least one unreacted functional group (B) in one molecule of the polyfunctional compound; And then reacting the unreacted functional group (B) remaining in the polyfunctional compound with a solid phase surface having a functional group (C) having a reaction activity with respect to the functional group (B) to bond it. A method for immobilizing a biopolymer. The method for immobilizing a biopolymer according to claim 1, wherein the functional group (A) is an amino group. The functional group (A) is at least one functional group selected from the group consisting of an epoxy group, a haloformyl group, an isocyanato group, a carboxylic acid group, an acid anhydride, a sulfo group, a halosulfonyl group, a maleimide group, and a formyl group. The method for immobilizing a biopolymer according to claim 1. The functional group (B) according to claim 2, wherein the functional group (B) is at least one functional group selected from the group consisting of an epoxy group, a haloformyl group, an isocyanato group, a carboxylic acid group, an acid anhydride, a maleimide group, and a formyl group. How to fix biopolymers. The method according to claim 3, wherein the functional group (B) is an amino group. The method for immobilizing a biopolymer according to claim 4, wherein the functional group (C) is an amino group. The functional group according to claim 5, wherein the functional group (C) is at least one functional group selected from the group consisting of an epoxy group, a haloformyl group, an isocyanato group, a carboxylic acid group, an acid anhydride, a maleimide group, and a formyl group. How to fix biopolymers. The method for fixing a biopolymer according to claim 1, wherein the polyfunctional compound is a chain polymer having a molecular weight of 1,000 to 100,000. The method according to claim 1, wherein the biopolymer is an oligonucleotide.

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