JP2005164348A - Method for observing interaction of biomolecules - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for observing the precise interaction of biomolecules without causing non-specific adsorption even in a sample in which various substances coexist, in particular in the measurement of surface plasmon resonance. <P>SOLUTION: A hetero difunctional group type hydrophilic polymer is bonded to a nonionic functional group introduced into a surface in a covalent bonding manner and the biomolecule is immobilized on the surface of a solid by the direct or indirect chemical bond or physical adsorption of the terminal of the hydrophilic polymer and the biomolecule. The interaction of the immobilized biomolecule with a biomolecule or a biomolecule aggregate is observed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biomolecule obtained by covalently immobilizing a heterobifunctional hydrophilic polymer to a nonionic functional group introduced on the surface and immobilizing the biomolecule on the surface via the hydrophilic polymer. How to observe the interaction

  Biomolecule interactions have been investigated as a means of analyzing the functions of biomolecules in vivo. As one of the means for examining the interaction, a method is generally used in which one molecule (A) of a binding pair is immobilized on a solid surface and the interaction with the molecule (B) to be measured is analyzed. Yes. However, if there is nonspecific adsorption of B to a molecule bound to the solid surface to immobilize A on the surface, or to the solid surface itself, even if the binding can be detected in some way, it is truly specific. In many cases, it is difficult to determine whether or not the connection is static.

  If B is a pure system, only non-specific adsorption of B may be taken into account, but if B is contained in body fluid components such as cell lysate, cell extract, or serum, various substances Is contained in the solution, there is a possibility of adverse effects when substances other than B adsorb nonspecifically.

  As a conventional technique, a means for blocking bovine serum albumin (BSA), milk casein or the like in contact with a solid surface has been considered as a means for suppressing nonspecific adsorption. However, blocking is merely physical adsorption, and the bonding strength to the surface may be weak and may gradually peel off. Further, the immobilized A may be buried by blocking, and the interaction may not be detected.

  Therefore, a means for reducing the nonspecific adsorption without using a blocking operation by using a molecule for immobilizing A as a hydrophilic substance has been studied. For example, in a method of immobilizing a nucleic acid molecule by a covalent bond, a method of providing a polyethylene glycol spacer when synthesizing a nucleic acid molecule is shown (for example, see Patent Document 1). Here, a polyethylene glycol spacer is provided between the target sequence and the immobilizing functional group, and an effect of suppressing nonspecific adsorption can be expected. However, no nonspecific adsorption of B is taken into account for molecules bound to the solid surface to immobilize A on the surface.

  First, in this method, a self-organized surface of alkanethiol is prepared, and an amino group is introduced on the surface. This amino group is closely packed on the surface. This is reacted with a crosslinking agent having a succinimide group and a maleimide group, and finally the nucleic acid molecule is covalently immobilized. Since all of the amino groups introduced by the self-assembled surface cannot react with the succinimide group of the cross-linking agent, a large amount of amino groups are present on the solid surface and have a positive charge. This results in non-specific adsorption when B has a negative charge or when the B solution contains a negative charge.

  The main chain of the crosslinking agent used is a cyclohexane ring, which is very hydrophobic. In this case, when B is a substance having a hydrophobic site, or when a hydrophobic substance is contained in the solution of B, there is a risk of causing nonspecific adsorption.

  US Patent discloses a method for suppressing non-specific adsorption by forming a sugar hydrogel on the surface and providing a functional group in the gel. Although it is a very effective method, since the penetration and diffusion rates of the target substance are different between the inside and outside of the gel, it is unsuitable as a method for strictly measuring the interaction kinetics in real time. In addition, sugar is one of biomolecules, and cannot be applied when a substance that interacts with sugar is contained in the sample. (For example, refer to Patent Document 2).

  In addition, a molecule with a functional group for immobilizing biomolecules at the end of a spacer with a hydrophilic polymer and a functional group that can be directly bonded to the solid surface at the other end is designed for interaction observation. Has succeeded. However, it is difficult for this substance alone to be densely packed on the surface, and it is mixed with other hydrophilic group terminal substances and immobilized on the surface. In order to suppress non-specific adsorption, hydrophilic group terminal substances are mixed at a high ratio, and the density at which biomolecules are immobilized on the surface is lowered, resulting in a problem of low measurement sensitivity. In addition, the designed molecules are not common and are difficult to obtain. (For example, see Non-Patent Documents 1 and 2)

  In US Pat. No. 5,629,213, an anionic substance is introduced into a metal substrate, poly-L-lysine, which is a polycation, is immobilized thereon, a substance specific to the molecule (B) is immobilized thereon, and surface plasmon resonance method Is disclosed. In this method, a metal substrate is densely filled with an anionic substance, and poly L lysine is then laminated so that the surface has a strong positive charge. In the example, streptavidin is immobilized on this surface, and a biotinylated antibody is immobilized, and the molecule (B) is detected. However, when the molecule (B) is contained in the cell extract or body fluid, the ionic substance contained in the cell extract or body fluid is not added to the poly-L lysine layer or the anionic substance densely introduced onto the substrate. There is a risk of specific adsorption. In this case, it is difficult to detect substances in the cell extract or body fluid. (See Patent Document 3)

  JP 2002-520620 also discloses an array in which an antibody is immobilized on a substrate. Here, the concept of analyzing a cell extract is also shown, and the concept of using a polymer for a single layer (organic thin layer) that is densely packed is also shown. Although the organic thin layer is described as having a functional group (for example, polyethylene glycol (PEG)) that reduces nonspecific binding of molecules on the surface, the PEG is introduced after the introduction of the nonionic functional group. No specific method for immobilizing is described (Patent Document 4).

International Publication No. 00/67028 US Patent 5436161 US Pat. No. 5,629,213 Special table 2002-520620 Sigal et. al. Anal. Chem. 68 (1996) 490-497 Jung et. al. Langmuir 16 (2000) 9421-9432

  The present invention makes it possible to immobilize a biomolecule by using a heterobifunctional hydrophilic polymer as a cross-linking agent in a nonionic functional group and suppress nonspecific adsorption.

  The present invention relates to a biomolecule obtained by covalently immobilizing a heterobifunctional hydrophilic polymer to a nonionic functional group introduced on the surface and immobilizing the biomolecule on the surface via the hydrophilic polymer. It is to provide a method for observing the interaction.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. A heterobifunctional hydrophilic polymer is covalently bonded to a nonionic functional group introduced on the surface, and the end of the hydrophilic polymer and a biomolecule are directly or indirectly bonded chemically or physically. 1. Method for observing the interaction between an immobilized biomolecule and a biomolecule or an assembly of biomolecules by immobilizing the biomolecule on the solid surface by mechanical adsorption 2. The method according to 1, wherein the nonionic functional group is one selected from a hydroxy group, a tosyl group, a tresyl group, a succinimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, and an epoxy group. 3. The method according to 1 or 2, wherein the heterobifunctional hydrophilic polymer has a molecular weight of 1,000 or more and 10,000 or less. 4. The method according to any one of 1 to 3, wherein the repeating unit of the heterobifunctional type hydrophilic polymer is ethylene glycol. The functional group of the heterobifunctional hydrophilic polymer is selected from the group consisting of amino group, carboxyl group, sulfonated succinimide group, maleimide group, thiol group, aldehyde group, vinyl group, isocyanate group, epoxy group, hydrazide group, and azide group. 5. The method according to any one of 1 to 4, which is two selected. 6. The method according to any one of 1 to 5, wherein the solid surface is a flat substrate. 7. The method according to any one of 1 to 6, wherein the solid surface is a thin gold layer on a flat substrate. 8. The method according to any one of 1 to 7, wherein a biomolecule is immobilized using a heterobifunctional hydrophilic polymer on a gold surface into which a nonionic functional group has been introduced on the surface using a gold-sulfur bond. 9. The method according to any one of 1 to 8, wherein a plurality of biomolecules are immobilized in an array. 10. The method according to any one of 1 to 9, wherein the number of biomolecules to be immobilized is 5 or more. 11. The method according to any one of 1 to 10, wherein the biomolecule to be immobilized is an antibody. 12. The method according to any one of 1 to 11, wherein the interaction is observed by a surface plasmon resonance method. The method according to any one of 1 to 12, wherein the interaction is observed by a surface plasmon resonance imaging method

  According to the present invention, a heterobifunctional hydrophilic polymer is covalently immobilized on a nonionic functional group introduced on the surface, and the biomolecule is immobilized on the surface via the hydrophilic polymer. Provides a method of observing the interaction.

  The present invention is described in detail below. The present invention discloses a method of immobilizing a biomolecule using a heterobifunctional hydrophilic polymer as a crosslinking agent on a nonionic functional group introduced on the surface and observing the interaction between the molecules.

  As a method for analyzing the interaction between biomolecules, a method of immobilizing one molecule (A) of a binding pair on a solid surface and analyzing the interaction with the molecule (B) to be measured is generally used. It has been. However, if there is non-specific adsorption of the molecule (B) to the molecule bound to the solid surface to immobilize the molecule (A) on the surface or to the solid surface itself, the binding can be detected in some form. Even so, it is often difficult to determine whether the binding is really specific.

  In the present invention, the functional group introduced into the solid surface for immobilizing the molecule (A) on the surface is nonionic. Examples of the functional group introduced on the surface include a hydroxy group, a tosyl group, a tresyl group, a succinimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, and an epoxy group. Since they are nonionic, there is no fear of electrostatic nonspecific adsorption to the surface even if they remain unreacted on the surface.

 Some of the nonionic functional groups listed above have high binding reaction activity with respect to other functional groups, but they are not a big problem because they are easily blocked by covalent bonds. On the other hand, when a large number of ionic functional groups are present on the surface, blocking is often difficult, which is not preferable. Therefore, if the functional group that is densely introduced into the solid surface is ionic, non-reactive ionic functional groups and molecules (B) are adsorbed nonspecifically, or the substance in the solution containing the molecules (B) Adsorption is not preferable because it may affect the measurement.

 The method for introducing a functional group on the surface is not particularly limited. For example, when the solid substrate is a metal, an alkane having a metal-binding functional group at one end and a nonionic functional group at the other end. Functional groups can be introduced on the surface using substances. More specifically, it is possible to introduce a nonionic functional group by forming a self-organized surface of alkanethiol on the gold surface. In the case of a glass substrate, a functional group can be easily introduced using a silane coupling agent.

 In the present invention, a heterobifunctional hydrophilic polymer is covalently bonded to a nonionic functional group. The polymer needs to be hydrophilic in order to suppress nonspecific adsorption. The term “hydrophilic” as used herein means that the polymer has water solubility or a property of swelling with water, and the polymer is defined as a substance having 3 or more monomer repeating units. The molecular weight of the polymer is preferably 1,000 or more in order to function as a sufficient spacer. However, the cross-linking agent requires a certain molar concentration, and if the molecular weight is too large, the number of grams of the cross-linking agent required for immobilization increases, and therefore the molecular weight is preferably 10,000 or less.

  The polymer material is not particularly limited, but polyethylene glycol having ethylene glycol as a repeating unit is preferable from the viewpoint of hydrophilicity and chain flexibility.

  The functional group at one end of the crosslinking agent reacts with the functional group present on the surface, but the functional group present at the other end preferably remains unreacted. If both ends of the cross-linking agent react with a functional group on the surface, a loop is formed on the surface and there is no functional group for immobilizing a biomolecule. After one end of the cross-linking agent reacts and is immobilized on the surface, the biomolecule is immobilized using another functional group present at the opposite end. Accordingly, a heterobifunctional polymer having two different functional groups is preferable.

  Examples of functional groups at the end of the polymer include amino groups, carboxyl groups, succinimide groups, sulfonated succinimide groups, maleimide groups, thiol groups, aldehyde groups, vinyl groups, isocyanate groups, epoxy groups, hydrazide groups, and azide groups. it can. Among them, amino groups and carboxyl groups, and amino groups and maleimide groups are preferable in terms of reactivity, as the target functional groups that react with each other.

  After the heterobifunctional hydrophilic polymer is covalently immobilized on the nonionic functional group on the solid surface, the molecule (A) is immobilized on the surface using the functional group at one end of the polymer. It becomes. The bond between one end of the polymer and the molecule (A) is not particularly limited, and examples thereof include a covalent bond, a chelate bond, an ionic bond, and a hydrogen bond.

  Introducing functional groups and substances that bind to biomolecules after using the heterobifunctional type hydrophilic polymer of the present invention is also included in the present invention. For example, a substance containing a nitrilotriacetic acid (NTA) group is immobilized by a heterobifunctional polymer, and then a histidine tag protein is introduced to the surface with a nickel chelate bond, or biotin or streptavidin is heterobifunctional. A method of introducing a biotinylated biomolecule onto the surface after introduction by a crosslinking agent is conceivable.

 By the present invention, the molecule (A) is immobilized on the surface via the hydrophilic polymer, so that nonspecific binding is suppressed. However, the effect of the present invention is not limited to the suppression of non-specific binding. Since the molecule (A) is immobilized by taking a space from the substrate, it functions as a spacer, and it is expected that the influence of the substrate, for example, the influence of steric hindrance can be reduced. In addition, by adopting a flexible polymer chain as typified by polyethylene glycol, it is possible to give mobility to the immobilized molecule (A) and analyze the interaction in a state closer to the solution. It is expected to be.

  As the solid surface for immobilizing biomolecules, a flat substrate suitable for interaction analysis is preferable. For the planar substrate, a thin gold layer capable of analyzing the interaction by the surface plasmon resonance (SPR) method is very preferable. There is another reason why the surface of the gold thin layer is preferable, and it is possible to introduce a functional group on the surface using a gold-sulfur bond. That is, a functional group can be introduced on the surface by using a substance having both a thiol group or a disulfide group and a functional group serving as a starting point for the reaction. The present invention can be achieved by reacting the introduced functional group with a heterobifunctional type crosslinking agent.

  In addition, a plurality of substances to be immobilized are preferable because high-throughput analysis is possible, and it is preferable that three or more different biomolecules can be analyzed simultaneously. In order to immobilize a plurality of substances, it is preferable to immobilize the substances in an array, and the interaction can be analyzed at each immobilization site. The biomolecule to be immobilized is not particularly limited, and examples thereof include nucleic acids, proteins, peptides, sugar chains and the like. Among these, antibodies are preferable in that they are stable and many types can be easily secured.

  The substrate of the present invention has an effect of suppressing non-specific adsorption because the influence of electric charge is reduced and the molecule (A) can be immobilized via a hydrophilic polymer. Therefore, this method is suitable as a method for immobilizing biomolecules in the interaction analysis between biomolecules. As an interaction analysis method, an SPR method is preferable, and an SPR imaging method capable of analyzing an array is particularly preferable. The SPR method is a label-free interaction analysis method and is very suitable for analysis in which proteins bind to immobilized biomolecules. This is because the protein may lose its activity by labeling.

The molecule (B) is not particularly limited, but the present invention is suitable for a protein as described above. The solution containing (B) is not particularly limited, and examples thereof include buffer solutions, cell extracts, and body fluids such as serum and plasma. Since the present invention has an advantage of less nonspecific adsorption, it can be applied to analysis of substances in cell extracts and serum. The cell extract includes a cell disruption solution and a solution obtained by diluting them. Body fluid includes all liquid components obtained from living organisms, and examples include blood components and semen.
In the future, it can be expected that it can be applied to the analysis of substances present in cells and blood using an array in which antibodies are immobilized.

  The signal evaluation method in the surface plasmon resonance is not particularly limited, but it is conceivable to take the result of signal difference (before and after change) before and after flowing the cell extract or body fluid on the basis of the running buffer. Further, it is possible to take a change (change during inflow) immediately after flowing the cell extract or body fluid. If there is a lot of nonspecific adsorption, it may be effective to observe changes during inflow after nonspecific adsorption by nucleic acids or lipids in cell extracts and body fluids is completed at a very fast rate. is there.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
[Example]
After vapor-depositing 3 nm of chromium on a transparent glass substrate made of SF15 having a thickness of 2 mm and 18 mm × 18 mm, gold was vapor-deposited by 45 nm. The thickness of the deposition was monitored with a crystal oscillator. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 8-Hydroxy-1-Octanethiol (8-HOT, manufactured by Dojindo Laboratories) for 16 hours to form a self-organized surface of 8-HOT. By this operation, a nonionic hydroxy group can be introduced on the substrate surface.

0.5 g of Tosyl Chloride (manufactured by Tokyo Chemical Industry) was dissolved in 5 ml of anhydrous acetone, and neutralized by adding 1 ml of pyridine. The gold substrate on the surface of the hydroxy group was immersed in this solution for 10 minutes to tosylate the hydroxy group. A heterobifunctional polyethylene glycol (NH ₂ -PEG-COOH, manufactured by Nektar Therapeutics) having an amino group and a carboxyl group at the end of a molecular weight of 3,400 on a tosylated surface is mixed with a phosphate buffer (20 mM phosphate, 150 mM NaCl). , PH 7.2) at 10 mg / ml and reacted on the gold surface for 2 hours. Since the tosyl group formed on the surface reacted with the amino group of NH ₂ -PEG-COOH and the carboxyl group remained unreacted, the carboxyl group could be introduced to the surface via PEG.

  The gold substrate was immersed in a phosphate buffer solution of 0.2M water-soluble carbodiimide (EDC: manufactured by Nacalai Tesque) and 0.05M N-hydroxysuccinimide (manufactured by Nacalai Tesque) for 1 hour to succinimidize the carboxyl group. .

  An anti-actin antibody (manufactured by Santa Cruz), anti-KOD antibody (manufactured by Toyobo), anti-streptavidin antibody (manufactured by Vector Laboratories) using a MultiSPRinter automatic spotter (manufactured by Toyobo Co., Ltd.) on the obtained succinimidized surface ) Was spotted at a concentration of 200 μg / ml and reacted for 2 hours to prepare an array. The spot size is about 500 μm in diameter. The chemical formula of the above surface reaction is shown in FIG.

After washing the surface on which the antibody is immobilized with a phosphate buffer, a gold slide is placed on a phosphate buffer solution of mPEG-NH ₂ (manufactured by Nektar Therapeutics) at a concentration of 10 mg / ml to block unreacted succinimide groups. It was immersed for 15 hours and washed with a phosphate buffer to obtain an antibody array.

  As a substance to be measured, actin abundant in the cell extract was selected. As a cell, mouse Thymus (thymus) was used. The thymus was excised from the mouse, and a proteinase inhibitor was added using a mammalian cell lysis reagent CelLytic M (manufactured by SIGMA), mechanically disrupted, and centrifuged to collect the supernatant. Finally, it was filtered through a 0.45 μm filter to obtain a Thymus extract.

  The gold chip on which the antibody array was formed was set in a MultiSPRinter SPR imaging device (manufactured by Toyobo Co., Ltd.), and a phosphate buffer was flowed into the flow cell. After confirming that the signal from the SPR was stabilized, a solution obtained by diluting the Thymus extract 10-fold with a phosphate buffer was poured into the SPR apparatus for about 15 minutes, and then washed with a phosphate buffer. The signal change at that time is shown in FIG. As a result, the SPR signal was greatly increased in the spot where the anti-actin antibody was immobilized. An increase in signal was also observed at other spots, but the level was small. The increase in signal while flowing a 10-fold diluted solution of Thymus extract (10 × Thymus) is largely due to the bulk effect of the components in the cell extract. Even after rinsing with phosphate buffer, the signal is somewhat increased in all spots. This difference in signal is due to non-specific adsorption, and non-specific adsorption cannot be completely suppressed.

  However, in this example, it is considered that the specific adsorption of actin present in the cell extract to the anti-actin antibody can be detected. Table 1 shows the signal difference results (before and after change) before and after flowing 10 × Thymus, based on the condition in which the phosphate buffer is flowing. Table 1 also shows the difference in signal (change during inflow) after 1 minute and 15 minutes after starting to flow 10 × Thymus. The signal change in the anti-actin antibody is very large compared to the other antibodies, whether it is a back-and-forth change or a change during inflow.

  Next, FIG. 7 shows changes in SPR signal when 10 × Thymus added with KOD (manufactured by Toyobo Co., Ltd.) at a concentration of 100 ng / ml was passed through this antibody array. It can be said that the signal rise in the anti-KOD antibody is very large and KOD can be detected. Furthermore, FIG. 8 shows changes in SPR signal when 10 × Thymus added with streptavidin (SA: Vector Laboratories) at a concentration of 100 ng / ml was passed. It can be said that the signal rise in the anti-SA antibody is very large and SA can be detected. Thus, it was possible to detect the substance in a cell extract.

[Comparative example]
After vapor-depositing 3 nm of chromium on a transparent glass substrate made of SF15 having a thickness of 2 mm and 18 mm × 18 mm, gold was vapor-deposited by 45 nm. The thickness of the deposition was monitored with a crystal oscillator. The substrate on which gold was deposited was immersed in a 1 mM ethanol solution of 7-Carboxy-1-Heptanethiol (7-CHT, manufactured by Dojindo Laboratories) for 16 hours to form a 7-CHT self-assembled surface. By this operation, a carboxyl group having a negative charge is introduced to the substrate surface.

  The gold substrate was immersed in a phosphate buffer solution of 0.2M water-soluble carbodiimide (EDC: manufactured by Nacalai Tesque) and 0.05M N-hydroxysuccinimide (manufactured by Nacalai Tesque) for 1 hour to succinimidize the carboxyl group. .

  An anti-actin antibody (manufactured by Santa Cruz), anti-KOD antibody (manufactured by Toyobo), anti-streptavidin antibody (manufactured by Vector Laboratories) using a MultiSPRinter automatic spotter (manufactured by Toyobo Co., Ltd.) on the obtained succinimidized surface ) Was spotted at a concentration of 200 μg / ml and reacted for 2 hours to prepare an array. The spot size is about 500 μm in diameter. The chemical formula of the above surface reaction is shown in FIG.

After washing the surface on which the antibody is immobilized with a phosphate buffer, a gold slide is placed on a phosphate buffer solution of mPEG-NH ₂ (manufactured by Nektar Therapeutics) at a concentration of 10 mg / ml to block unreacted succinimide groups. It was immersed for 15 hours and washed with a phosphate buffer to obtain an antibody array. In this case, since the carboxyl groups densely introduced to the surface by the self-assembled surface formation remain unreacted, a negative charge remains on the substrate.

  The gold chip on which the antibody array was formed was set in a MultiSPRinter SPR imaging device (manufactured by Toyobo Co., Ltd.), and a phosphate buffer was flowed into the flow cell. After confirming that the signal from the SPR was stabilized, a solution obtained by diluting the Thymus extract 10-fold with a phosphate buffer was poured into the SPR apparatus for about 15 minutes, and then washed with a phosphate buffer. The signal change at that time is shown in FIG. Although a result of a large increase in the SPR signal was obtained at the spot on which the anti-actin antibody was immobilized, an increase in the signal was also observed at other spots, and it was difficult to distinguish them. Table 1 shows the signal difference results (before and after change) before and after flowing 10 × Thymus, based on the condition in which the phosphate buffer is flowing. Table 1 also shows the difference in signal (change during inflow) after 1 minute and 15 minutes after starting to flow 10 × Thymus. Although the signal change in the anti-actin antibody is larger than that of the other antibodies, it is difficult to judge whether the difference is small. The increase in signal in antibodies other than actin can be considered as non-specific adsorption due to substrate charging.

  Next, FIG. 7 shows changes in SPR signal when 10 × Thymus added with KOD (manufactured by Toyobo Co., Ltd.) at a concentration of 100 ng / ml was passed through this antibody array. Although the signal rise in the anti-KOD antibody is large and it can be said that KOD can be detected, the signal increase is small compared to the Examples. Furthermore, FIG. 8 shows changes in SPR signal when 10 × Thymus added with streptavidin (SA: Vector Laboratories) at a concentration of 100 ng / ml was passed. The signal for the anti-SA antibody was also small when compared to the examples. The antibody immobilized by non-specific adsorption seems not to function sufficiently.

  According to the present invention, a heterobifunctional hydrophilic polymer is covalently immobilized on a nonionic functional group introduced on the surface, and a biomolecule is immobilized on the surface via the hydrophilic polymer, thereby making it non-specific. It is important to contribute to the industry by providing a method for observing precise biomolecule interactions without adsorption.

Surface chemical reaction in which a heterobifunctional PEG is reacted with a hydroxyl group to immobilize an antibody on the surface (Example) Example of change in SPR signal when 10 × Thymus was passed Example: Change in SPR signal when flowing 10 × Thymus with 100 ng / ml KOD added Changes in SPR signal when 10 × Thymus added with 100 ng / ml SA was added (Example) Surface chemical reaction that immobilizes antibody directly on carboxyl group (comparative example) Change in SPR signal when 10 × Thymus was passed (comparative example) Change in SPR signal when flowing 10 × Thymus with 100 ng / ml KOD added (Comparative example) Change in SPR signal when flowing 10 × Thymus added with 100 ng / ml SA (comparative example)

Claims (13)

A heterobifunctional hydrophilic polymer is covalently bonded to a nonionic functional group introduced on the surface, and the end of the hydrophilic polymer and a biomolecule are directly or indirectly bonded chemically or physically. Method for observing the interaction between immobilized biomolecules and biomolecules or biomolecule aggregates by immobilizing biomolecules on a solid surface by mechanical adsorption The method according to claim 1, wherein the nonionic functional group is one selected from a hydroxy group, a tosyl group, a tresyl group, a succinimide group, a thiol group, an aldehyde group, a vinyl group, an isocyanate group, and an epoxy group. The method according to claim 1 or 2, wherein the heterobifunctional hydrophilic polymer has a molecular weight of 1,000 or more and 10,000 or less. The method according to any one of claims 1 to 3, wherein the repeating unit of the heterobifunctional hydrophilic polymer is ethylene glycol. The functional group of the heterobifunctional hydrophilic polymer is selected from the group consisting of amino group, carboxyl group, sulfonated succinimide group, maleimide group, thiol group, aldehyde group, vinyl group, isocyanate group, epoxy group, hydrazide group and azide group. The method according to claim 1, wherein two are selected. The method according to claim 1, wherein the solid surface is a flat substrate. The method according to claim 1, wherein the solid surface is a thin gold layer on a flat substrate. The method according to any one of claims 1 to 7, wherein a biomolecule is immobilized using a heterobifunctional hydrophilic polymer on a gold surface into which a nonionic functional group is introduced on the surface using a gold-sulfur bond. The method according to claim 1, wherein a plurality of biomolecules are immobilized in an array. The method according to claim 1, wherein the number of biomolecules to be immobilized is 3 or more. The method according to any one of claims 1 to 10, wherein the biomolecule to be immobilized is an antibody. The method according to claim 1, wherein an interaction between substances contained in a cell extract or a body fluid is analyzed. The method according to claim 1, wherein the interaction is observed by a surface plasmon resonance method.

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