JPH10182693A - Control of orientation of molecules - Google Patents

Abstract

PROBLEM TO BE SOLVED: To control orientation of functional molecules of a protein, etc., useful as a probe, etc., for chemical sensors and scanning microscopes by selectively reacting a specific functional group on functional molecular surface with a functional group previously produced on a substrate. SOLUTION: When functional molecules comprising protein molecules such as film protein molecules or antibody molecules are bonded in a state oriented to a substrate, a functional group previously produced on the substrate is selectively reacted with a specific functional group such as an N-ended amino group, a C-ended carboxyl group or a specific thiol group on the functional molecule surface by using a proper crosslinking and condensing agent to provide the objective functional film suitable for making a chemical sensor highly functinal, a filter, a separated and purified carrier, a probe for scanning probe microscope, biochemical element and controlled in orientation of the functional molecule to the substrate. Furthermore, a same kind or different kind of functional molecule is selectively bonded through a specific functional group to the functional molecule fixed on a substrate by the resultant controlled orientation to build a multifilm layer structure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a multifunctional material comprising a substrate and functional molecules attached thereto, which is used for a chemical sensor, a filter, a separation / purification carrier, a probe of a scanning probe microscope, a biochemical element and the like. It relates to functionalization or high functionality. Specifically, the present invention provides a simple method for increasing the degree of orientation of functional molecules to be attached onto a substrate, and improving the quantitativeness and selectivity of a functional material. Further, according to the method of the present invention, it is possible to easily realize multilayering of a plurality of types of functional molecules on a substrate, so that energy / information conversion and information amplification on the surface of the functional material can be efficiently performed, and therefore, high sensitivity of the functional material can be achieved. It becomes possible.

[0002]

2. Description of the Related Art In the field of biotechnology, medical science, or clinical examination, in the detection, quantification, and qualitative analysis of biomolecules or derivatives thereof, intermolecular interactions such as antigen-antibody reactions or selective decomposition of substrates by enzymes are used. In many cases, the specific interaction of is used. However, in the prior art, molecules to be detected or molecules to be detected are randomly attached to a substrate or subjected to a reaction in a solution state. On the other hand, with the advance of technology, it has become possible in principle to measure the distribution of receptor molecules localized on the surface of living cells, or the state of activity, and the like. In fact, genetic recombination technology has made it possible to introduce labeled residues that emit fluorescence by replacing specific residues exposed on the surface of protein molecules with highly reactive cysteine. By observing with a microscope, the distribution of the receptor molecule localized on the surface of a living cell, or the state of activity or the like is measured. Applying specific interaction between molecules as another means to measure the distribution of receptor molecules or the active state, etc., by increasing the functionality of sensors and probe substrates of scanning probe microscopes There is a movement to try (E.-L. Florin et al., SCIENCE, Vol. 264, pp. 415-417 (1994)). Until now, protein conjugates have been produced by causing a cross-linking reaction within protein molecules or by cross-linking between protein molecules or between proteins and nucleic acids or other high molecular compounds (Protein Tailoring for Food). and Medical Use
s, ed. by RE Feeney et al. Marcel Dekker, Inc.,
New York (1986)), which analyzes the relative positions of residues in molecules or the relative topological relationships between molecules, and aims at stabilizing proteins and adding new functions. And was not aimed at controlling the orientation of molecules.

[0003]

[Problem to be solved]
It has become possible to introduce a special residue or the like for labeling using a newly generated thiol group by substituting a specific residue exposed on the surface of a protein molecule with cysteine. However, many steps such as gene construction, mass production by genetic recombination, and purification are required. Although these techniques are extremely powerful in principle, they each have inherent difficulties, and are often not always easy to implement.

On the other hand, in order to improve the selectivity of the sensor or probe surface and the quantitativeness of the detected value, a conventional method (E.
L. Florin et al., SCIENCE, Vol. 264, pp. 415-417 (19
In 94)), only the detection-side molecule was non-specifically attached to the probe surface, and the orientation of the molecule was random.
Therefore, the quantification was poor with respect to the distribution of the molecules to be detected, the strength of the interaction, and the like.

To solve these problems, the present invention first provides a simple method for introducing a special residue into a specific functional molecule, and controls the orientation of the functional molecule with respect to the substrate by using this method. It provides a method. Hereinafter, the present invention will be described in detail using a protein as an example of a functional molecule.

[0006]

In order to expose the only amino group on the protein molecule to the surface of the molecule, first, all amino groups on the surface of the protein molecule are protected by a reaction such as acetylation, and then all carboxyl groups are protected. The groups are protected by reactions such as carbamylation. Then, when an acyl amino acid releasing enzyme is allowed to act on the modified protein, only the N-terminal acylated amino acid is removed by the action of the enzyme, and a new N-terminal is exposed as the only free amino group in the modified protein.

[0007] On the other hand, a similar technique can be applied to a case where only a carboxyl group is exposed on the surface of a protein molecule. That is, all amino groups and all carboxyl groups on the surface of the protein molecule are protected in advance in the same manner as in the above step. Then, carboxypeptidase Y, which is an exopeptidase, is allowed to act. In general, carboxypeptidase releases C-terminal L amino acids sequentially, but in particular, carboxypeptidase Y does not require a free α-carboxyl group unlike other carboxypeptidases, and also uses an amino acid having a modified carboxyl group as a substrate (Biochemistry Experiment Course 1, Protein Chemistry II, p.20
5 (1984) Tokyo Kagaku Doujin). As a result, some amino acids are continuously released from the C-terminal side of the modified protein, and the C-terminal is exposed as the only free carboxyl group in the modified protein. Carboxypeptidase Y has substrate specificity ((Biochemistry Laboratory Course 1, Protein Chemistry II, p.205 (1984)
(Tokyo Kagaku Dojin)) and the reaction progress is restricted by steric hindrance. Therefore, when a protein polypeptide chain as a functional molecule is a membrane protein embedded in a membrane, the reaction starts from the C-terminal to the peptide chain. When the protein has an SS bond, the reaction is stopped immediately before the SS bond, so that the protein is not digested indefinitely because the reaction stops immediately before the SS bond.

The termination of the reaction of carboxypeptidase Y due to steric hindrance such as SS also enables the creation of new special residues. That is, a pair of thiol groups can be generated by reducing, under mild conditions, a protein from which residues immediately before SS binding have been removed by carboxypeptidase Y.

[0009] Since only amino groups, carboxyl groups, or thiol groups can be generated on the surface of all protein molecules by the above-described steps, the amino groups, carboxyl groups, or thiol groups can be appropriately formed between the active groups on the substrate to which the active groups have been previously introduced. Cross-linking condensing agent (edited by the Biochemical Society of Japan,
New Chemistry Experiment Course 1, Protein IV, Structure-function relationship, 211-21
By linking using 4 pages, Tokyo Chemical Dojin (1991)), aligned molecules can be aligned on the substrate. In addition, by combining the above processes with proteins having only one amino group and carboxyl group or thiol group, the same or different proteins can be formed on the substrate without forming non-specific crosslinks in the molecule. A multilayer film can be constructed.

The protecting groups for amino, carboxyl, or thiol groups on the surface of the protein molecule are deprotected after forming a substrate-protein or protein-protein complex, and naturally-occurring amino, carboxyl, Alternatively, it is also possible to return to a thiol group. That is, the protection of the amino group can be reversibly modified by maleylation, trifluoroacetylation, nitrotroponylation, acetoacetylation, or the like (Biochemical Experiment Course 1, Protein Chemistry I).
V, pp. 10-34 (1977), Tokyo Kagaku Dojin), and the carboxyl group can be reversibly modified by methyl esterification using methanol-hydrochloric acid (Biochemical Experiment Course 1, Protein Chemistry IV, p. 46 (1977), Tokyo Chemical Dojin), and the thiol group can be reversibly modified with tetrathionate (New Chemistry Laboratory Course 1, Protein
IV, pp. 110-111 (1991) Tokyo Chemical Doujin). In addition, carboxypeptidase Y used to expose a free carboxyl group on the C-terminal side of the modified protein is a peptide bond,
Since amino acids esters, amides, anilides and the like can be hydrolyzed, proteins having a carboxyl group protected by these can be deprotected by the action of carboxypeptidase Y.

In place of the acylamino acid releasing enzyme, other various peptide chain-cleaving enzymes, for example, α-aminoacylpeptide hydrolases such as tripeptide aminopeptidase, dipeptide hydrolase, dipeptidyl peptide hydrolase, peptidyl dipeptide hydrolase Degrading enzymes, etc. (Enzyme Handbook, Asakura Shoten, pp.531-540, pp.811-812
(1982)).

On the other hand, in place of carboxypeptidase Y, other various peptide chain-cleaving enzymes such as serine carboxypeptidase, metal carboxypeptidase, cysteine carboxypeptidase and the like (enzyme handbook, Asakura Shoten, pp. 541-570, pp. 812) -813 (1982)).

Further, depending on the functional protein, papain,
It is also possible to release a small peptide by cleavage with a serine proteinase such as trypsin, thereby exposing a limited number of amino groups and carboxyl groups on the protein surface.

A functional layer having only one specific residue obtained by the above-described method is bonded to a reactive residue on a substrate, thereby constructing a molecular layer whose orientation with respect to the substrate is controlled. be able to. Reactive residues on the substrate
Various functional groups can be applied depending on the type of the linker used for linking. Particularly when a zero-chain-length linker such as a water-soluble carbodiimide is used, a carboxyl group is used when the special residue of the functional molecule is an amino group, and an amino group is preferable when the special residue is a carboxyl group. . When the special residue is a thiol group, for example, succinimidyl 4
-Various bivalent cross-linking reagents such as maleimidobutyrate (Shinsei Kagaku Experimental Course 1, Protein IV, Tokyo Chemical Dojin, pp.211-2
14 (1991)), it can be linked to an amino group or a thiol group on the substrate.

As the substrate, an inorganic material substrate such as mica or gold, a Langmuir-Blodgett film (hereinafter, referred to as an LB film) substrate, a membrane protein forming a stable two-dimensional crystal in a natural cell membrane (for example, a highly halophilic substrate) Purple membranes that form bacterial cell membranes) can be used.

As a method for introducing a reactive functional group into a substrate, in the case of mica, a newly cleaved surface is formed by LS-1420.
There is a method of introducing an amino group to the substrate surface by treating with a silane coupling agent such as (Shin-Etsu Chemical). A divalent crosslinking reagent such as succinimidyl 4-maleidobutylate having a succinimide group and a maleimide group in the amino group thus formed and an ω-thiolundecanoic acid having a thiol group and a carboxyl group (HS- (CH ₂ ) ₁₀ -COO
H) and the like can act to expose the carboxyl groups on the outermost surface. The thiol group can be exposed on the outermost surface of the substrate by reacting a divalent crosslinking reagent with a reagent having two or more thiol groups such as dithiothreitol. When gold is used as a substrate, a substrate having a thiol group and an amino group, such as cysteamine (HS- (CH ₂ ) ₂ -NH ₂ ), utilizing the property that clean gold easily adsorbs thiol groups. Or exposure to a reagent such as ω-thiolundecanoic acid having a thiol group and a carboxyl group (HS- (CH ₂ ) ₁₀ -COOH), or a reagent having two or more thiol groups such as dithiothreitol Thereby, an amino group, a carboxyl group, or a thiol group can be exposed on the outermost surface of the substrate. When an LB film is used as the substrate, a thiol group, an amino group, or a carboxyl group is added to both ends of the molecule of ₂₀ -mercaptoeicosan-1-ol (HS- (CH ₂ ) ₂₀ -OH) used as a lipid when preparing the LB film. Long-chain compounds having, for example, thiol eicosanoic acid (HS- (C
By adding a small amount of H ₂ ) ₂₀ -COOH) or a long-chain compound having two or more thiol groups, an amino group, a carboxyl group, or a thiol group can be generated on the substrate surface.
When a protein is used as the substrate, an amino group and a carboxyl group in the main chain or side chain, and a thiol group in the side chain can be used as a reactive functional group.

As described above, the present invention has been described by taking a protein as an example of a functional molecule. However, the functional molecule is not limited to a protein. The present invention can be applied to all kinds of oligopeptides, amino groups, carboxyl groups, or thiol groups, or to all organic compounds having some of them.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to FIG. Example 1 1 mg (about 70 nmol) of anti-horse kidney ferritin goat antibody (1) was dissolved in 1 ml of half-saturated sodium acetate solution,
While stirring on ice, add 1 μl of acetic anhydride in large excess.
Add 10 times every 10 minutes (total about 100 micromol)
Functional groups inside the molecule acetylate only reactive amino groups on the surface of the molecule without modification. After reacting for 2 hours, the acetylated anti-horse kidney ferritin goat antibody (2) was desalted and purified by ultrafiltration membrane or gel filtration. Then freeze-dry and remove protein
Dissolve in 0 mM sodium acetate solution (pH 4.8), add 200 μl
Of 400 mM water-soluble condensing agent (N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride) (hereinafter abbreviated as EDC) and 2
00 μl of 100 mM N-hydroxysuccinimide) (hereinafter NHS
), 14 mg (approximately 125 micromol) of aminomethanesulfonic acid, which is in a large excess with respect to the carboxyl group of the protein, is added.
The reaction was allowed to proceed for hours, and the carboxyl group was carbamylated. The product was desalted and purified by ultrafiltration membrane or gel filtration in the same manner as after the acetylation reaction, and then lyophilized to remove water. As a result of carrying out the reaction under the non-denaturing condition of such a protein, an anti- equine kidney ferritin goat antibody (3) in which only the amino group and the carboxyl group on the molecular surface were protected was obtained. Then, half of this was added to 500 μl of 50 mM phosphate buffer.
(pH 7.2), and 0.05 units of an acyl amino acid releasing enzyme (Takara Shuzo) was added thereto, followed by reaction at 37 ° C. for 2 hours. As a result, a molecular species (4) having a free amino group only at the N-terminus was generated on the molecular surface. On the other hand, the remaining half of the molecular species (3) was added to 1 ml of 50 mM phosphate buffer (pH 6.5).
And 2.5 µg of carboxypeptidase Y (Oriental Yeast Co., Ltd.), an endopeptidase that releases amino acids one by one from the C-terminal side of the polypeptide chain, and reacted at 37 ° C for 2 hours to obtain molecular species (5) occurred.

Carboxypeptidase Y generally does not proceed further if there is a continuous sequence of glycine in the polypeptide chain. When an SS bond exists, the reaction is forcibly terminated immediately before the SS bond exists. More generally, by controlling the concentration of carboxypeptidase Y and the duration of the action, the length of the polypeptide to be removed can be controlled. Molecular species (4), (5) thus produced
After purification in the same manner as in the previous step, 100 μg of each was dissolved together in 1 ml of 50 mM phosphate buffer (pH 6.5), and the above 200 μl of 40 μl was dissolved.
Condensation with 0 mM EDC and 200 μl of 100 mM NHS gave molecular species (6). In this example, the same kind of molecule (4),
Although (5) is coupled, coupling of different types of protein molecules can be realized using exactly the same steps. The amino-, carboxyl-, or thiol-protecting groups on the surface of the protein molecule are deprotected after forming a substrate-protein or protein-protein complex, and are naturally-occurring amino, carboxyl, or thiol groups. It is also possible to return to the original. That is, regarding the protection of the amino group, maleylation, trifluoroacetylation,
Reversible modification by nitrotroponylation, acetoacetylation, etc. is possible (Biochemistry Experiment Course 1, Protein Chemistry IV, pp.10-34 (1977) Tokyo Chemical Dojin), and methanol-hydrochloric acid was used for the carboxyl group. Reversible modification by methyl esterification is possible (Biochemical Experiment Course 1,
Protein Chemistry IV, p.46 (1977), Tokyo Kagaku Dojin), and the thiol group can be reversibly modified with tetrathionate (New Chemistry Laboratory Course 1, Protein
IV, pp. 110-111 (1991) Tokyo Chemical Doujin). The substances to be reacted in the above-described steps are not limited to proteins. In addition, the method for generating only one amino group is not limited to cleavage by acylamino acid releasing enzyme, and similar effects can be obtained by using other various peptide chain cleavage enzymes. Further, the method for generating only one carboxyl group in the target molecule is not limited to carboxypeptidase, and similar effects can be obtained using other various peptide chain-cleaving enzymes. In this example, acetylation was used to protect the amino group, but in addition to the acylation such as formylation, succinylation, and maleylation, modification with various protecting groups is also possible. The action of carboxypeptidase Y may be such that the function of the target protein is not impaired. For example, in the case of an antibody molecule, digestion does not need to reach the Fv region in order to maintain selective antigen binding ability. In fact, no matter how long the reaction is performed with a large amount of enzyme, it is near the hinge region of the antibody molecule
Digestion stops shortly before SS binding. Therefore, the antibody molecule is digested in advance with the papain protease, and the whole molecule is Fab
And Fc, which is then treated with carboxypeptidase, and the thiol group can be released by reduction. This can be used for the ligation reaction.

In the present embodiment, the steps of preparing a dimer are described, but by repeating the same steps, a multimer of a heterologous protein can be constructed. Regarding the crosslinking method between molecules, a thiol group, an imidazole group, a guanidino group, or the like can be used by using a polyvalent crosslinking reagent in addition to an amino group and a carboxyl group.

Another embodiment of the present invention will be described with reference to FIG. Example 2 Bacteriorhodopsin (hereinafter abbreviated as bR, molecular weight 26 kD), which is a membrane protein consisting of 248 amino acids, constituting the cell membrane of the extremely halophilic bacterium Halobacterium salinarium is embedded in a lipid double membrane as shown in (7). (N. Grigo
rieff et al., J. Mol. Biol., Vol. 259, pp. 393-421 (1
996)). The bR membrane fraction obtained by culturing Halobacterium salinarium was modified in the same manner as in Example 1 to obtain a molecular species (9) in which the amino group was acetylated and the carboxyl group was carbamylated. That is, 50 nmol in 1 ml of half-saturated sodium acetate solution
The bR membrane fraction was suspended in acetic anhydride while stirring on ice.
Add 10 μl each 10 minutes, and acetylate only the reactive amino groups on the molecule surface without modifying the amino groups embedded in the membrane. After reacting for 2 hours, the acetylated bR membrane fraction (8) was collected as a precipitate by centrifugation at 12,000 G for 30 minutes, and the precipitate was washed with 1 ml of a 10 mM sodium acetate solution (pH 4.8). Then, this was suspended in 1 ml of 10 mM sodium acetate solution (pH 4.8), and 200 μl of 400 mM EDC and 200 ml of 100 mM NHS were added thereto, and further 14 mg of aminomethanesulfonic acid was added, and the mixture was stirred at room temperature. For 2 hours to carbamyl the carboxyl group to obtain 40 nmol of modified bR (9). After the product is bisected and the membrane fraction collected by centrifugation as above,
Wash with 1 ml of 50 mM phosphate buffer (pH 7.2), then 1 ml
In 50 mM phosphate buffer (pH 7.2). The other precipitate was washed with 1 ml of 50 mM phosphate buffer (pH 6.5) and then resuspended in 1 ml of 50 mM phosphate buffer (pH 6.5). Next, 0.05 units of acylamino acid releasing enzyme (Takara Shuzo) was added to the former, and reacted at 37 ° C. for 2 hours. As a result, a molecular species (10) having an amino group capable of attacking from the outside of the molecule only at the N-terminus was generated. On the other hand, 2.5 μg of carboxypeptidase Y (Oriental yeast) was added to the latter, and reacted at 37 ° C. for 2 hours to produce a molecular species (11).
In the reaction with carboxypeptidase Y, since the digestive action did not reach the polypeptide chain embedded in the lipid membrane, the length of the generated polypeptide chain was shortened by about 20 amino acids as compared with the natural bR. Then, 10 nmol of the molecular species (10) and 10 nmol of (11) prepared in this way were purified by the above-mentioned centrifugation operation, suspended in 1 ml of pure water, and 200 μl of 400 mM EDC and 200 μl were suspended in the same manner as in the carbamylation reaction. Of 1
To the mixture was added 00 mM NHS, and the mixture was reacted at room temperature for 2 hours with stirring to obtain a dimer bR (12). FIG. 6 shows the results of electrophoresis of the condensation reaction product. In the case of the modified bR, about 50% of the dimer was formed. On the other hand, in the case of unmodified bR, the trimer was the main component. Further, by comparing the molecular weights of the monomers, it can be seen that the modified bR is shorter than the unmodified bR by a certain length. In the case of bR, the N-terminus of molecular species (10) protrudes from the cell membrane surface by about 10 amino acids, so that condensation is possible even when the C-terminus of the binding partner is located deep in the molecule. In order to increase the efficiency, various bivalent cross-linking reagents that are longer linkers than EDC (edited by the Biochemical Society of Japan, Lectures on Experimental Chemistry 1, Protein IV, Structure-Function Relationship, pp.211-214, Tokyo Chemical Dojin (1991)) It is also possible to use. Regarding the crosslinking method between molecules, a thiol group, an imidazole group, a guanidino group, or the like can be used by using a polyvalent crosslinking reagent in addition to an amino group and a carboxyl group.

Still another embodiment will be described with reference to FIGS. Example 3 An organic material that exposes an amino group on the surface or an inorganic material (13) modified so that the amino group is exposed on the surface,
By coupling a protein molecule (5) or a protein complex having a carboxyl group released only to a specific site, the protein molecule or protein complex can be aligned on the substrate with a certain orientation (14). That is, the first embodiment
After preparing the Fab fragment (3) of the anti-equine kidney ferritin goat antibody in which the carboxyl group was released in the same manner as described in
After irradiating this and the cleaved mica with water vapor plasma to activate the surface, aminopropyltriethoxysilane (H ₂ N (C
H ₂ ) ₃ Si (OC ₂ H ₅ ) ₃ ) was reacted with steam and connected to a substrate having an amino group exposed on the mica surface using EDC. The method of exposing the amino group on the mica substrate is an alternative method.
LS-1420 (H ₂ N (CH ₂ ) ₃ Si (OCH ₃ ) ₃ , Shin-Etsu Chemical) at room temperature
After reacting for an hour, treat at 100 ° C for 10 minutes and then in water for 1 minute.
It can also be obtained by irradiating with time ultrasonic waves. The antibody-modified substrate thus prepared is immersed in a horse kidney ferritin solution and Fab
After binding to X-ray fluorescence X-ray interferometry (YCSasaki et al.,
The distance from the substrate to the center of the ferritin molecule, that is, the iron atom core, was measured by SCIENCE, Vol. 263, pp. 62-64 (1994). FIG. 7 shows a principle diagram (a) of X-ray interference and an observation pattern (b). Here, θt is an extraction angle of the X-ray, and θc is a critical angle of total reflection of the detected X-ray. The relationship between the distance Z between the substrate and the light-emitting atom of the fluorescent X-ray and the optical path difference Δ between the direct light and the reflected light is Δ = 2Z · sin (θt), so that two optical paths are obtained from the interference fringes observed on the detection screen. The phase difference between the differences, that is, the distance Z between the substrate and the emission point of the fluorescent X-ray can be determined. In the present embodiment, an interference pattern was obtained by using a primary X-ray excitation method, ie, a method of irradiating a white X-ray generated by irradiating an electron beam on a copper counter-cathode with the above-mentioned sample, as an excitation method of iron as a labeled luminescent atom. As a result of the measurement, it was found that the distance from the substrate to the iron atom was about 13 nm. This value is based on X-ray crystallography (RJ Polja
Natl. Acad. Sci. USA, 70 (12), Pa.
rt I, pp.3305-3310 (1973)), electron beam structure analysis (G. Pre
cigoux et al., Acta Cryst. D50, pp.739-743 (1994))
The size of the Fab molecule and ferritin molecule obtained from the results of 8 nm × 5 nm × 4 nm and 10 nm × 10 nm × 1 respectively
The distance from the C-terminus of the Fab molecule predicted from 0 nm to the iron atom core at the center of the ferritin molecule was 13 nm (= 8 nm + 10 nm / 2). As a result, it was found that the Fab fragments of the anti-equine kidney ferritin goat antibody were aligned almost perpendicularly to the substrate. This method is based on the type of functional group exposed on the surface of the molecule to be bound on the substrate and various divalent cross-linking reagents (edited by The Biochemical Society of Japan, Lecture 1 on New Chemistry, Protein IV, Structure-Function Relationship, pp.211-214, By changing the combination with that of Tokyo Chemical Dojin (1991), the type of functional group generated on the substrate can be a thiol group other than an amino group or a carboxyl group.

A further embodiment of the present invention will be described with reference to FIGS. 1, 3 and 4. Example 4 As shown in Example 3, alignment of a functional group at a specific site on a molecular surface on a substrate as a clue can be applied to a molecular complex. That is, the complex molecule (6) produced by the process shown in FIG. 1 is left on the surface of the molecular complex only by the same reaction as that used to generate FIGS. 1 (3) to (5). Is exposed to obtain a molecular species (15). Then, as shown in FIG. 3, the molecular species (16) is obtained by condensation with a functional group on the substrate surface. Actually, a dimer was prepared from the Fab fragment of the anti-horse kidney ferritin goat antibody shown in Example 3, and this was combined with the amino group exposed on the surface of the gold deposited on the mica substrate using EDC. When the sample thus connected was analyzed by X-ray fluorescence interferometry similar to that used in Example 3, it was confirmed that the distance from the substrate to the center of the ferritin molecule was constant within a range of 1 nm or less. In this embodiment, an example of a dimer molecule has been described. However, the same process can be repeated for a multimer of a trimer or more to modify the surface of a substrate with a multilayer molecule. This method changes the type of functional group exposed on the surface of the molecule to be bonded to the substrate and the various types of bivalent crosslinking reagents to change the type of functional group generated on the substrate to a thiol group other than an amino group. And a carboxyl group.

Another embodiment of the present invention will be described with reference to FIG. Example 5 In the step of forming a multilayer film of functional molecules on a substrate, as shown in Example 4, in addition to the step of immobilizing the functional molecules to be multilayered on the substrate after multimerizing the functional molecules, FIG. As shown in the figure, only the functional group at the specific site of the functional molecule (14) already immobilized on the substrate is activated (17), and the functional molecule of the heterogeneous functional molecule also activated at the specific site. By linking (5), a multilayer film structure (16) of functional molecules can be constructed. Actually, the Fab fragment of the anti-equine kidney ferritin goat antibody shown in Example 3 was connected to the amino group exposed on the surface of the gold deposited on the mica substrate using EDC (14). After treating the modified Fab fragment immobilized in the above with an amino acid releasing enzyme to release an amino group at the N-terminus, a separately prepared modified Fab fragment (5) having a carboxyl group only at the C-terminus is ligated. Multiple layers of proteins can be constructed on a substrate. The substrate on which one layer of protein was adsorbed
It can also be constructed according to the LB method. Ferritin molecules were adsorbed on the protein multilayer film thus constructed, and the distance from the substrate to the center of the ferritin molecule was confirmed to be constant within 1 nm or less by the fluorescent X-ray interference method described in Examples 3 and 4. did it. By combining a GTP-binding protein as the first layer protein and a seven-transmembrane receptor protein as the second layer protein, a highly sensitive sensor capable of amplifying ligand binding information on a substrate can be constructed. In the present embodiment, an embodiment regarding the construction of a two-layer structure of a protein is shown. However, by repeating the same steps for various kinds of materials, it is possible to modify the surface of a substrate with a multilayer body having a three-layer structure or more. Regarding the crosslinking method between molecules, a thiol group, an imidazole group, a guanidino group, or the like can be used by using a polyvalent crosslinking reagent in addition to an amino group and a carboxyl group.

[0025]

According to the present invention, a functional molecule or a functional molecule complex whose orientation is controlled can be constructed and immobilized on a substrate, so that a chemical sensor, a filter, a separation and purification carrier, A functional film suitable for enhancing the functions of a probe of a scanning probe microscope, a biochemical element, or the like can be constructed.

[Brief description of the drawings]

FIG. 1 is a process diagram of constructing a dimer by linking only amino groups or carboxyl groups generated on the surface of a water-soluble functional molecule.

FIG. 2 is a process diagram showing the construction of a dimer between a molecule having a unique amino group on the molecular surface of a protein having an integral membrane and a molecule having a unique carboxyl group on the surface.

FIG. 3 is a process chart for orienting a molecule having only one carboxyl group using a functional group on a substrate as an anchor.

FIG. 4 is a process chart for orienting a complex molecule having only one carboxyl group using a functional group on a substrate as an anchor.

FIG. 5 is a process chart for constructing a multilayer film after constructing a monolayer film of a molecule using a functional group on a substrate as an anchor.

FIG. 6 is a schematic diagram of an electrophoresis image showing a result of a dimerization reaction of an integral membrane protein.

FIG. 7 is a graph showing the principle of X-ray fluorescence interferometry and an analysis example of protein molecules arranged on a mica substrate.

[Explanation of symbols]

(1) Schematic diagram of a protein molecule exposing an amino group and a carboxyl group on the molecular surface. (2) Schematic diagram of a protein molecule in which only the amino group exposed on the molecule surface is modified. (3) Schematic diagram of a protein molecule in which an amino group and a carboxyl group exposed on the molecular surface are modified. (4) Schematic diagram of a protein molecule in which the N-terminal residue is exposed as the only amino group on the molecular surface. (5) Schematic diagram of a protein molecule in which the C-terminal residue is exposed as the only carboxyl group on the molecular surface. (6) Schematic diagram of a protein dimer bound at a specific site. (7) Schematic diagram of an integral membrane protein molecule exposing an amino group and a carboxyl group on the molecular surface. (8) Schematic diagram of an integral membrane protein molecule in which only the amino group exposed on the molecular surface is modified. (9) Schematic diagram of an integral membrane protein molecule in which an amino group and a carboxyl group exposed on the molecular surface are modified. (10) Schematic diagram of an integral membrane protein molecule in which the N-terminal residue is exposed as the only amino group on the molecular surface. (11) Schematic diagram of an integral membrane protein molecule in which the C-terminal residue is exposed as the only carboxyl group on the molecular surface. (12) Schematic diagram of an integral membrane protein dimer bound at a specific site. (13) A schematic view of a substrate having an amino group exposed on the surface. (14) Schematic diagram of a protein monolayer structure constructed by linking an amino group on a substrate and a C-terminal carboxyl group of a protein. (15) Schematic diagram of a protein multimer molecule in which the C-terminal residue is exposed as the only carboxyl group on the molecular surface. (16) Schematic diagram of a protein multi-layer structure constructed by linking an amino group on a substrate with a C-terminal carboxyl group of a protein multimeric molecule. (17) Schematic diagram of the monolayer structure of a protein molecule in which the N-terminal residue is exposed as the only amino group on the molecular surface.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 37/00 G01N 37/00 A // G01N 33/48 33/48 Z

Claims (5)

[Claims] In the step of orienting and bonding a functional molecule to a substrate, the functional group is formed by selectively reacting a functional group previously generated on the substrate with a specific functional group on the surface of the functional molecule. A method for controlling the orientation of a hydrophilic molecule with respect to a substrate. 2. The functional molecule is a protein molecule, and the specific functional group on the surface of the functional molecule is an N-terminal amino group, a C-terminal carboxyl group, or a specific thiol group.
The method described in. 3. The method according to claim 2, wherein the protein molecule is a membrane protein molecule. 4. The method according to claim 2, wherein the protein molecule is an antibody molecule. 5. A functional molecule immobilized on a substrate in a controlled orientation obtained by the method of claim 1 and a functional molecule of the same or different type via a specific functional group of the functional molecule. A method for constructing a multilayer structure of functional molecules by selectively binding molecules.