JP2004125462A - Biochip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a biochip for measuring SPR for immobilizing a biomolecular or a biological molecule aggregate to a combined site, while maintaining activity almost without non-specific adsorption at a background section. <P>SOLUTION: The biochip is used to immobilize the biomolecular or the biological molecule aggregate to the surface. A substance, that becomes the starting point for immoblilization or a substance having a functional group, is immobilized to a section (immobilization site) for immobilizing the biomolecular or the biological molecule aggregate. A hydrophilic macromolecule is immobilized to a background section, other than the immobilization site. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a biochip capable of evaluating an interaction between substances.
[0002]
[Prior art]
In recent years, attempts have been made to examine the functions of biomolecules such as DNA and proteins, or to evaluate the interaction between biomolecules as a means for clarifying expressed genes and proteins. As one of the methods, a method of measuring a molecule by immobilizing the molecule on a solid surface is known. As a method of immobilizing a biomolecule on a solid phase, there is a method of immobilizing a biomolecule by a covalent bond or an ionic bond, starting from a functional group introduced on the surface. Alternatively, a method is also possible in which a molecule having a highly specific binding partner such as biotin or streptavidin is immobilized on the surface, and a biomolecule into which a paired molecule is introduced is immobilized on the surface.
[0003]
However, in most of the conventional techniques, a functional group or a binding molecule is introduced into the entire chip, and there is no distinction between an immobilized site and a background portion in a state where the biomolecule is not immobilized. Therefore, non-specific adsorption of a sample to a functional group or a binding protein present in the background may not be ignored. In addition, a technique of spotting a biomolecule solution at a specific position in a very small amount is also indispensable, and an expensive spotting device is required.
[0004]
U.S. Patents disclose a means for preparing an array in which a biomolecule is immobilized on an immobilization site and a hydrophilic polymer having no functional group or binding molecule as a starting point in a background portion is immobilized (for example, Patent Document 1). According to the present invention, when a biomolecule or a biomolecule aggregate is immobilized on a surface, a reversible hydrophobic protecting group is immobilized on a background portion, and spotting is easy. However, after the biomolecules or biomolecule aggregates are immobilized on the surface, it is necessary to remove the hydrophobic protecting groups using a basic organic solvent, and immobilize the hydrophilic polymer. There is a concern that the basic organic solvent may adversely affect the biomolecules or biomolecule aggregates. In addition, there is a concern that the immobilization of the hydrophilic polymer may adversely affect the immobilized biomolecules or biomolecule aggregates. Specifically, the point is that the hydrophilic polymer binds to the immobilized biomolecule, and the function of the biomolecule is impaired.
[0005]
Therefore, there is a demand for a biochip which has almost no non-specific adsorption in the background portion and can immobilize a biomolecule or biomolecule aggregate at an immobilization site while maintaining its activity.
[0006]
The present invention provides a biomolecule in which a hydrophilic polymer that suppresses nonspecific adsorption is immobilized on a background portion, and a substance or a substance having a functional group serving as a starting point for immobilization is immobilized on the immobilization site. It is possible to get chips.
[0007]
[Patent Document 1]
US Pat. No. 6,127,129 [0008]
[Problems to be solved by the invention]
An object of the present invention is to provide a biochip which has almost no non-specific adsorption on a background portion and can immobilize a biomolecule or a biomolecule aggregate on an immobilization site while maintaining its activity.
[0009]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
[0010]
1. A biochip for immobilizing a biomolecule or a biomolecule assembly on a surface, and a portion (immobilization site) for immobilizing the biomolecule or the biomolecule assembly is a substance or a starting point for immobilization. A biochip, wherein a substance having a functional group is immobilized, and a hydrophilic polymer is immobilized on a background portion other than the immobilization site.
[0011]
2. 2. The biochip according to 1, wherein the size of the immobilization site is 2500 μm 2 or more.
[0012]
3. 3. The biochip according to 1 or 2, wherein a transparent glass or a transparent plastic on which gold is deposited is used as a substrate.
[0013]
4. A biochip for analyzing a reaction to a biomolecule or a biomolecule aggregate immobilized on the chip using a surface plasmon resonance (SPR) phenomenon; A biochip as described in Crab.
[0014]
5. The biochip according to any one of claims 1 to 4, wherein the biochip is for immobilizing a plurality of biomolecules or biomolecule aggregates on the chip and analyzing a plurality of reactions simultaneously by SPR imaging. Chips.
[0015]
6. 6. The biochip according to any one of 1 to 5, wherein the substance serving as a starting point of the immobilization site is at least one selected from the group consisting of biotin, glutathione, streptavidin, amylose, and antibody.
[0016]
7. The functional group of the substance having a functional group as a starting point of the immobilization site is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, a maleimide group, a succinimide group, and a nitrosotriacetic acid group. 7. The biochip according to any one of 1 to 6, wherein
[0017]
8. 8. The biochip according to any one of 1 to 7, wherein the hydrophilic polymer immobilized on the background is polyethylene glycol.
[0018]
9. 9. The biochip according to 8, wherein the molecular weight of polyethylene glycol is 1,000 or more.
[0019]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail. The biochip immobilizes biomolecules or biomolecule aggregates on the surface, and is effective for interaction analysis and screening of expressed genes and proteins. In the present invention, a portion for immobilizing a biomolecule or a biomolecule assembly (immobilization site) and a background portion other than the immobilization site are clearly distinguished. A substance serving as a starting point for immobilization or a substance having a functional group is immobilized on the immobilization site. The biomolecule or biomolecule aggregate is immobilized on the surface by a hydrogen bond, a covalent bond, an ionic bond or a hydrophobic bond via a starting substance or a functional group. In the background area, there is basically no starting substance or functional group present at a high density at the immobilization site, and a hydrophilic polymer is immobilized to suppress non-specific adsorption of substances existing in the sample. Have been.
[0020]
The binding molecule or functional group to be introduced into the immobilization site is not particularly limited, but is preferably one that can easily bind and immobilize the target biomolecule or biomolecule assembly as a starting point of the binding. Preferred binding molecules are biotin, streptavidin, glutathione, amylose and antibodies. It is known that biotin and streptavidin bind tightly to each other. When glutathione-S-transferase fusion protein is used for glutathione, maltose binding protein is used for amylose, and when an anti-mouse antibody is used as an antibody, a mouse-derived antibody can be used.
[0021]
The functional group serving as the starting point of the immobilization site is preferably a carboxyl group, an amino group, a thiol group, a maleimide group, a succinimide group, or a nitrosotriacetic acid group.
[0022]
The method for introducing a binding molecule or a functional group serving as a starting point of the immobilization site is not particularly limited. In the case of a gold-deposited surface, a thiol-terminated compound is used to directly or indirectly introduce the compound to the surface. be able to. Alternatively, in the case of a glass surface, a substance can be introduced to the surface by using a silane coupling agent. As another method, a substance having a binding molecule or a functional group can be coated on the surface by spin coating.
[0023]
In the present invention, it is easy to bind and immobilize a biomolecule or a biomolecule assembly immobilized on an immobilization site, but the background substance is basically composed of a starting substance or a functional group present in the immobilization site. Biomolecules or biomolecule aggregates cannot be immobilized. By clarifying the immobilization site and the background part, more precise analysis becomes possible. Means for separating the immobilization site from the background portion include a patterning technique by light irradiation, a stamp technique, and the like.
[0024]
As a material of the biochip, glass or transparent plastic is preferable. Glass or transparent plastic on which gold is deposited is particularly preferable because it is easy to introduce a functional group or a bonding molecule through a gold-sulfur bond. Alkanethiols having a binding molecule or a functional group at the end can be bound to the gold surface. Alternatively, an alkanethiol having a base functional group or binding molecule at the end is bound to the gold surface, and a binding molecule or compound having a functional group to be introduced is bound to the binding molecule or functional group present on the surface and immobilized. Or a means for introducing a binding molecule or a functional group using a crosslinking agent or the like in a plurality of steps.
[0025]
In addition, the method of patterning by irradiating light can be applied to the gold surface, and the alkanethiol can be newly bonded to the gold surface by oxidizing and removing the gold-sulfur bond by ultraviolet rays.
[0026]
The hydrophilic polymer that suppresses nonspecific adsorption is preferably nonionic. This is for suppressing non-specific adsorption due to ionic bonds. Specific examples include synthetic polymers such as polyethylene glycol and polyvinyl alcohol, and saccharides such as dextran, and polyethylene glycol is particularly preferable. Further, those having a molecular weight of 1,000 or more have an effect of suppressing nonspecific adsorption, and are therefore preferable. The method of immobilization may be direct or indirect on the surface. In the case of immobilization directly on the gold surface, a method of immobilizing a hydrophilic polymer at the terminal of a thiol group may be used. When indirectly immobilizing on a gold surface, it is necessary to first immobilize an alkanethiol having a functional group at the terminal on the gold surface, and then immobilize a hydrophilic polymer starting from the functional group of the alkanethiol. it can. Alternatively, a method of coating the surface with a hydrophilic polymer is also possible.
[0027]
A chip in which a thin gold layer is deposited on a transparent substrate can not only be analyzed for interaction by labeling with general fluorescence or radioisotope, but also can be detected by surface plasmon resonance (SPR). SPR is a label-free technique capable of real-time interaction analysis, and monitors reflected light near the SPR resonance angle.
[0028]
In particular, SPR imaging is a method of irradiating the entire chip with light and capturing the reflected light with a CCD camera. Using the chip of the present invention, a plurality of biomolecules or biomolecule aggregates are immobilized, and a plurality of interactions are simultaneously performed. It is possible to analyze.
[0029]
For observation by SPR imaging, the size of the immobilized site is preferably 2500 μm ² or more. If it is less than this, the image is not clear and observation is difficult.
[0030]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to Examples.
[0031]
[Example]
After depositing 1 nm of chromium on a transparent glass substrate made of SF10 having a thickness of 1 mm and 18 mm × 18 mm, gold was deposited to a thickness of 45 nm. The thickness of the evaporation was monitored with a crystal oscillator. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 16 hours to form a self-assembled surface of 8-AOT. Was.
[0032]
Succinimide group-terminated polyethylene glycol having a molecular weight of 2,000 (mPEG-SPA, manufactured by Sheawater Polymers) was dissolved in a phosphate buffer at 10 mg / ml, reacted with 8-AOT on the gold surface for 2 hours, and PEG was immobilized on the surface. . A photomask in which a pattern was drawn with chromium on quartz was placed on the substrate, and irradiation was performed with a 1000 W xenon arc lamp manufactured by Oriel for one hour to perform patterning. The pattern of the photomask is one in which 100 squares of 500 μm × 500 μm are arranged.
[0033]
After irradiation, the plate was washed with Milli-Q water and ethanol, immersed in a 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 16 hours, and the irradiated portion was irradiated with 7-CHT. Formed a self-assembled surface. Next, 0.2 M water-soluble carbodiimide (manufactured by Nacalai Tesque) and 0.05 M N-hydroxysuccinimide (manufactured by Nacalai Tesque) are dissolved in a phosphate buffer and reacted for 15 minutes to activate the carboxyl group of 7-CHT. It was made. Immediately, 1 mM AB-NTA (manufactured by Dojindo Laboratories) was reacted for 30 minutes to introduce a nitrosotriacetic acid (NTA) group into the immobilization site. Unreacted succinimide groups were blocked with a 1 M ethanolamine solution (pH 8.5). The biochip thus obtained has an NTA group at the immobilization site, and the background is immobilized with polyethylene glycol, which is a hydrophilic polymer.
[0034]
This biochip was immersed in a phosphate buffer solution of 2.5 mM nickel (II) chloride for 1 hour to form NTA-Ni chelate, and then His-tagged Myc peptide, His-tagged Flag peptide, and His-tagged Activated tyrosine hydroxylase (TH) was spotted at four locations in the center of the chip in the pattern shown in FIG. 1 and immediately set on an SPR imaging device (SPRImager: manufactured by GWC instruments) 5 minutes later. The observation was performed in a buffer of 10 mM Hepes, 150 mM NaCl, 0.005% Nonidet P-40, pH 7.2. Photographs were taken before and after the addition of 1 μg / ml anti-Myc antibody (manufactured by Santa Cruz) and 15 minutes after the addition, and the difference between the images before and after was shown in FIG. 2. position). Further, the difference between the images before and after the anti-Flag antibody (manufactured by Sigma) is inserted is shown in FIG. 3 (the dotted frame in the figure is the spot position). It was possible to observe that the anti-Myc antibody selectively bound to the Myc peptide and the anti-Flag antibody selectively bound to the Flag antibody.
[0035]
[Comparative Example 1]
The same gold-deposited substrate as in the example was immersed in a 1 mM ethanol solution of 7-CHT for 16 hours to form a self-assembled surface of 7-CHT on the entire surface. Next, 0.2 M water-soluble carbodiimide and 0.05 M N-hydroxysuccinimide were dissolved in a phosphate buffer and reacted for 15 minutes to activate the carboxyl group of 7-CHT. Immediately, 1 mM AB-NTA was reacted for 30 minutes to introduce an NTA group into the immobilization site. Unreacted succinimide groups were blocked with a 1 M ethanolamine solution (pH 8.5). The biochip thus obtained has NTA groups on the entire surface of the substrate.
[0036]
This biochip was immersed in a phosphate buffer solution of 2.5 mM nickel (II) chloride for 1 hour to form an NTA-Ni chelate, and then a histidine-tagged protein was spotted. However, the spot shape is not constant and is difficult to observe. In addition, since the NTA-Ni chelate is also formed in the portion where the protein is not bound, the background portion does not function as a blank, making it difficult to collect data.
[0037]
[Comparative Example 2]
The same gold-deposited substrate as in the example was immersed in a 1 mM ethanol solution of 8-AOT for 16 hours to form a self-assembled surface of 8-AOT. 1 mg of Fmoc-OSu (manufactured by Nova Biochem) was dissolved in 300 μl of DMSO, 200 μl of 100 mM TEA buffer at pH 7 was added, and the reaction was performed on a substrate, and the surface was covered with Fmoc. Irradiation was performed for one hour using a photomask to perform patterning in the same manner as in the example. After irradiation, the substrate was washed with Milli-Q water and ethanol, and then immersed in a 1 mM ethanol solution of 8-AOT for 16 hours to form a self-assembled surface of 8-AOT on the irradiated portion. Next, Sulfo-SMCC (manufactured by Pierce) was dissolved in a phosphate buffer, spotted, and reacted for 15 minutes to make the surface of the spot portion a maleimide group.
[0038]
Next, the DNA was immobilized by reacting the 5 ′ thiol-terminal DNA for 16 hours. A basic organic solvent obtained by mixing 0.9 ml of Tris (2-aminoethyl) Amine and 5.1 ml of dimethylformamide was applied to the substrate, and Fmoc in the background was removed. Finally, succinimide group-terminated polyethylene glycol having a molecular weight of 2,000 (mPEG-SPA, manufactured by Sheawater Polymers) is dissolved at 10 mg / ml in a phosphate buffer, and reacted with 8-AOT on the gold surface for 2 hours to immobilize PEG on the surface. did. This method is not only very laborious, but also burdens the immobilized material. The effect of the organic solvent on the immobilized substance and the reactivity of PEG to the immobilized substance when removing Fmoc are problematic. In particular, it is difficult to adapt to the case where proteins are immobilized on the surface.
[0039]
【The invention's effect】
The biochip for SPR of the present invention has almost no non-specific adsorption in the background portion, and can immobilize a biomolecule or a biomolecule aggregate on an immobilization site while maintaining its activity.
[Brief description of the drawings]
FIG. 1 is a diagram showing Myc, Flag, and TH spotting patterns of a biochip for SPR of an example. FIG. 2 is a diagram showing a difference between images before and after an anti-Myc antibody is inserted. FIG. Figure showing the difference between the images before and after the Flag antibody was added

Claims (9)

A biochip for immobilizing a biomolecule or a biomolecule assembly on a surface, and a portion (immobilization site) for immobilizing the biomolecule or the biomolecule assembly is a substance or a starting point for immobilization A biochip, wherein a substance having a functional group is immobilized, and a hydrophilic polymer is immobilized on a background portion other than the immobilization site. The biochip according to claim 1, wherein the size of the immobilization site is 2500 µm ² or more. 3. The biochip according to claim 1, wherein a transparent glass or a transparent plastic on which gold is deposited is used as a substrate. 4. The biochip according to claim 1, wherein the biochip is used for analyzing a reaction to a biomolecule or a biomolecule aggregate immobilized on the chip using a surface plasmon resonance (SPR) phenomenon. The biochip according to any one of the above. The biochip is for immobilizing a plurality of biomolecules or biomolecule aggregates on the chip and analyzing a plurality of reactions simultaneously by SPR imaging. Biochip. The biochip according to any one of claims 1 to 5, wherein the substance serving as a starting point of the immobilization site is at least one selected from the group consisting of biotin, glutathione, streptavidin, amylose, and an antibody. The functional group of the substance having a functional group as a starting point of the immobilization site is at least one selected from the group consisting of a carboxyl group, an amino group, a thiol group, a maleimide group, a succinimide group, and a nitrosotriacetic acid group. The biochip according to any one of claims 1 to 6, wherein the biochip is used. The biochip according to any one of claims 1 to 7, wherein the hydrophilic polymer immobilized on the background portion is polyethylene glycol. The biochip according to claim 8, wherein the molecular weight of the polyethylene glycol is 1,000 or more.

Images