JP4288591B2 - Method for producing peptide chip - Google Patents

Description

  The present invention relates to a method for producing a biochip capable of suppressing non-specific adsorption of biomolecules by blocking a hydrophilic polymer in a substance interaction analysis system using a peptide chip.

  In recent years, biochips used for biomolecule interaction analysis, profiling of expressed molecules, or diagnosis have attracted attention. The operation is facilitated by immobilizing the biomolecule on the substrate, and in some cases, the interaction of a very large number of substances can be analyzed. In particular, peptide arrays in which peptides with relatively small molecular weights are immobilized on a substrate have relatively few problems of denaturation such as proteins, and have strong combinatorial chemistry aspects. It has come to be widely used for searching and the like.

  However, it is the influence of nonspecific adsorption that becomes a problem when observing the interaction. Non-specific adsorption refers to a case where the target substance adsorbs non-specifically to molecules that do not interact with each other. As a result, a false positive determination is given, which is not preferable. In order to suppress nonspecific adsorption, biochips in which hydrophilic polymers such as dextran and polyethylene glycol (PEG) are immobilized on the surface have been developed. These hydrophilic polymers are known to have an effect of suppressing nonspecific adsorption of biomolecules.

  A biosensor in which non-specific adsorption is suppressed by forming a hydrophilic polymer gel matrix on the biosensor surface is shown. In this method, functional groups are introduced into the gel matrix, and biomolecules are immobilized by covalent bonds using the functional groups. Specifically, the carboxyl group of carboxymethyl dextran was activated with water-soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and the formed succinimide group and the biomolecule were successfully immobilized (Non-patent Document). 1). However, since the formed succinimide group does not react 100%, it is necessary to block the remaining succinimide. Here, succinimide groups are blocked using ethanolamine, and a technique using a hydrophilic polymer as a blocking material is not shown.

  A means for reacting a hydrophilic polymer on a substrate after immobilizing a biomolecule is known (Patent Document 1). However, here, the maleimide group on the chip is reacted with the thiol group of the target molecule to immobilize the target molecule on the chip, but the subsequent step of blocking the maleimide group is not included. For this reason, the maleimide group remains and there is a concern of causing nonspecific adsorption problems. Further, since a hydrophilic polymer that can react with the immobilized biomolecule is used, the biomolecule may be modified and denatured.

  The present invention provides a means for efficiently blocking functional groups used for immobilizing peptides as target molecules without effectively denaturing the peptides and effectively suppressing nonspecific adsorption.

Anal. Biochem. 198, 268, 1991 US Pat. No. 6,127,129

  An object of the present invention is to provide a biochip that suppresses nonspecific adsorption of biomolecules. In particular, the object is to obtain a chip that can accurately evaluate the interaction when used for analyzing an interaction with a peptide.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention.
1. A method for producing a peptide chip in which two or more kinds of peptides are immobilized on the same chip, wherein the maleimide group introduced on the gold surface on the chip and the thiol group present in or added to the peptide After the peptide is immobilized on the chip, the maleimide group remaining on the gold surface on the chip is reacted with a hydrophilic polymer having a thiol group to covalently bond the maleimide group remaining on the gold surface. A method for producing a peptide chip for use in surface plasmon resonance imaging analysis, characterized in that blocking is performed.
2. 2. The method according to 1, wherein a hydrophilic polymer having a thiol group only at the terminal is used.
3. 3. The method according to 1 or 2, wherein the hydrophilic polymer has a molecular weight of 400 or more.
4). 4. The method according to any one of 1 to 3, wherein the hydrophilic polymer has a molecular weight of 1000 or more.
5). The method according to any one of 1 to 4 , wherein the hydrophilic polymer is polyethylene glycol.
6). 6. The method according to any one of 1 to 5, wherein the peptide to be immobilized has a cysteine residue at the terminal.
7). A peptide chip for use in surface plasmon resonance imaging analysis, which is produced by the method according to any one of 1 to 6.

  By adopting the method for producing a peptide chip of the present invention, a peptide chip in which nonspecific adsorption is suppressed can be easily obtained.

  The biochip production method of the present invention relates to a method of immobilizing a peptide on a chip by reacting the functional group (A) on the chip with the functional group (B) present in or added to the peptide. This is a method for producing a peptide chip, in which a functional group (A) that is not used in an immobilization reaction and remains on the chip is covalently blocked by reacting a hydrophilic polymer having the functional group (C). . Therefore, the blocking method by physical adsorption or ion adsorption is not included in the present invention. Moreover, regarding the peptide to be immobilized, there may be only one type, or two or more types. Here, the peptide refers to a generally used meaning, and two or more amino acids are linked by peptide bonds. The number of amino acid residues is not particularly limited, but is usually about 5 to 60 residues, more preferably about 10 to 25 residues. Depending on the purpose of analysis, amino acids in which one or several residues among the amino acid residues are chemically modified may be included. Further, although not particularly limited, it is preferable that at least one peptide having a function as a substrate for a specific enzyme is included.

  Here, the case where the functional group is present in the peptide specifically refers to a state in which at least one cysteine residue is present in the amino acid sequence of the peptide to be immobilized. In this case, the thiol group that the cysteine residue has corresponds to the functional group (B). The cysteine residue is an amino acid necessary for the peptide to be immobilized to have its original function, even if it exists as an essential residue as an amino acid sequence necessary for the peptide to function. It may be a case where it is further added to the sequence. The position of the cysteine residue in the peptide to be immobilized is not particularly limited, but it is preferably added to at least one end, more preferably only to one end. When a cysteine residue is added to one end, only a cysteine residue may be added, but a spacer is used in order to increase the efficiency of interaction with a substance that acts by increasing the degree of freedom of the immobilized peptide. As an amino acid sequence of 1 to several residues may be further added. The amino acid sequence of the spacer portion is not particularly limited, but it is particularly preferable to add a sequence having 1 to several glycine residues.

In addition, when a functional group is added to a peptide, one compound having a compound other than an amino acid corresponding to the functional group (B) such as a thiol group with respect to the peptide to be immobilized. It means a state in which any of the above amino acid residues is chemically bonded. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

The functional group (A) can react with the functional group (B) present in the peptide to be immobilized or added, and can also react with the functional group (C) of the hydrophilic polymer. it can.
Functional group (A), functional group (B), functional group (C) is not particularly limited, carboxyl group, amino group, succinimide group, sulfated succinimide group, maleimide group, epoxy group, thiol group, Examples include an aldehyde group, a vinyl group, a tosyl group, a tresyl group, an acetic anhydride group, an isocyanate group, an isothiocyanate group, an acyl azide group, a chlorinated sulfuric acid group, and an imide ester group. Here, the functional group (A) and the functional group (B), and the functional group (A) and the functional group (C) are a reactive pair. Functional group (A) and functional group (B), functional group (A) and functional group (C) are basically different functional groups, but functional group (B) and functional group (C) are the same. May be different functional groups.

  The means for introducing the functional group (A) onto the chip is not particularly limited. Examples thereof include a self-assembled surface method for aligning molecules on the substrate surface, a method of introducing using a reaction reagent, and a means for coating a substance having a functional group on a chip. Also included are means for introducing the functional group (A) using a cross-linking agent starting from the functional group introduced on the surface.

The functional group (C) in the hydrophilic polymer is preferably present at the polymer end, and more preferably at one end. If the functional group (C) is present in the main chain of the polymer or a side chain that is branched from the main chain, there is a risk of steric hindrance to the immobilized target molecule, which is not preferable. Further, when a large number of functional groups (C) are present in the main chain or side chain, the functional groups (C) still remain even if they are blocked by reacting with the functional groups (A). The possibility that the functional group (C) also causes nonspecific adsorption cannot be denied. Therefore, it is preferable that the functional group (C) exists only at the terminal. More preferably, a hydrophilic polymer having a functional group (C) only at one end and a low activity methoxy group or hydroxyl group at the other end is preferable.

  The molecular weight of the hydrophilic polymer is preferably 400 or more, and more preferably 1,000 or more. This is because the higher the molecular weight / repeating unit, the stronger the effect of suppressing nonspecific adsorption. However, a molecular weight of 50,000 or more is not preferable because it may cause steric hindrance to the immobilized target molecule and adversely affect it.

The hydrophilic polymer means a compound having a repeating unit having a property of being soluble in water or swelling in water, and may be a synthetic product or a natural product.
Specifically, as the hydrophilic polymer, for example, polyethylene glycol, polyvinyl alcohol, poly (meth) acrylic acid, poly (meth) acrylate, poly (meth) acrylamide, polyethyleneimine, polyvinylpyrrolidone, carboxylic acid Or the salt, the sulfonic acid, the monomer containing the salt, or polyester, polyurethane, carboxymethylcellulose which copolymerized hydrophilic parts, such as polyethyleneglycol, and polysaccharides, such as chitosan, carrageenan, and glucomannan, are mentioned.

  However, since the hydrophilic polymer in the present invention is intended to suppress nonspecific adsorption, a compound having an ionic functional group may not be preferable. Among these, those having no reactive moiety such as OH groups, carboxylic acids and salts thereof, amines, and imines such as polyethylene glycol, poly (meth) acrylamide, and polyvinylpyrrolidone are preferable, and polyethylene glycol is most preferable. Polyethylene glycol (sometimes referred to as polyoxyethylene) is highly hydrophilic and has a high effect of suppressing nonspecific adsorption because there is no reactive functional group.

  The peptide chip in the present invention can be applied to various uses, and is particularly suitable for optical detection methods such as surface plasmon resonance (SPR), sum frequency generation (SFG), localized plasmon resonance (LPR), and ellipsometry. Demonstrate the effect. Among these, it is particularly useful in an analysis system using SPR. In a general chip or array, detection by a labeling means using a fluorescent substance or a radioisotope is used as a method for detecting an interaction. In this case, it is possible to detect only whether or not the label substance is finally bound, and even if a substance not related to the interaction is adsorbed non-specifically, it is not erroneously detected. Therefore, if the interacting target substance is not non-specifically adsorbed to the negative control, it can be determined that the measurement is accurate.

  However, in the label-free optical detection method, any substance adsorbed on the chip is detected as a signal. That is, it is difficult to distinguish nonspecific adsorption of a substance that is not a measurement target from specific adsorption. Therefore, since a means for suppressing the non-specific adsorption more severely is required, the blocking method by the covalent bond of the present invention is very effective.

  In an ELISA method or an interaction analysis method using a label substance, physical adsorption by bovine serum albumin or casein is generally selected as a blocking method. Although the physical adsorption method is easy, it is not stable and may be detached from the chip surface over time. In the above optical detection method, even desorption of a blocking agent is detected, so that blocking by a covalent bond is necessary.

  In SPR, SFG, LPR, and ellipsometry, a metal substrate is used. In the present invention, the material of the substrate is preferably gold which is very stable in acid, alkali, organic solvent and the like. In fact, gold is a material frequently used in the above optical detection method. Although not preferred for gold, metals such as silver, platinum, copper, and aluminum can be used. Further, the material supporting gold is preferably transparent, and more preferably transparent glass. This is because transparent glass is not only easily available, but also very suitable for measuring SPR and LPR. Although not preferred for glass, a relatively transparent plastic material such as polycarbonate, polyester such as polyethylene terephthalate, or acrylic resin such as methyl methacrylate can also be used.

  As a method of forming the metal substrate, a method of coating a thin metal layer is preferable. A method for coating the metal is not particularly limited, but generally, a vapor deposition method, a sputtering method, an ion coating method, or the like is selected. In order to provide an optical detection method, it is necessary to control the thickness of the thin metal layer at the nano level. The thickness of the thin metal layer is not particularly limited, but is generally selected in the range of 30 nm to 80 nm. In order to suppress peeling of the thin metal layer, a chromium layer or a titanium layer of 0.5 nm to 10 nm may be coated on the substrate in advance.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not particularly limited to the examples.

[Example 1]
(Peptide immobilization)
4 armPEG (SUNBRIGHT PTE-100SH manufactured by NOF Corporation) whose terminal functional group is a thiol group was dissolved in a mixed solution of 7 ml of ethanol: water = 6: 1 at a concentration of 1 mM. The molecular weight of 4armPEG is 10,000, which is a molecule in which four PEG chains having approximately the same length from the center are present, and is very hydrophilic. In addition, all four ends of PEG are thiol groups, and particularly exhibit metal binding properties to gold.

  A gold-deposited slide in which 3 nm of chromium was vapor-deposited on an SF15 glass slide of 18 mm square and 2 mm thickness and gold was vapor-deposited at 45 nm was immersed in the 4 armPEG thiol solution for 3 hours to bond 4 armPEG thiol to the entire gold substrate.

  A photomask was placed on the slide and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove 4 armPEG thiol in the UV irradiation part. The photomask has 16 square holes of 500 μm square (consisting of 4 × 4 patterns), and the pitch between the centers of the holes is designed to be 1 mm. UV light is transmitted through the holed portion of the photomask, and the slide is irradiated and patterned. In the part that was not irradiated, 4armPEG remains and functions as a reference part as a background part of the chip.

  It was immersed in a 1 mM ethanol solution of 8-Amino-1-Octanethiol, Hydrochloride (8-AOT, manufactured by Dojindo Laboratories) for 1 hour to form a self-organized surface of 8-AOT on the UV irradiation part. A heterobifunctional polyethylene glycol (NHS-PEG-MAL, manufactured by Nektar) having a succinimide (NHS) group and a maleimide (MAL) group at the end of a molecular weight of 3,400 is phosphate buffer (20 mM phosphate, 150 mM NaCl, It was dissolved in pH 7.2) at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. Since the amino group of 8-AOT and the NHS group of NHS-PEG-MAL reacted and the MAL group remained unreacted, a maleimide group could be introduced to the surface via PEG.

  A substrate peptide of cSrc kinase, which is one of tyrosine kinases, was immobilized on the surface obtained as described above. The amino acid sequence of the substrate peptide is shown in Table 1. Substrate 1 has an amino acid sequence that is phosphorylated by cSrc kinase. In substrate peptide 2, the tyrosine residue in substrate peptide 1 is phosphorylated in advance. Each substrate peptide was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) at 1 mg / ml, and 0.1 μl of each substrate peptide solution was manually prepared in the arrangement shown in FIG. Spotting was performed one by one. Then, the immobilization reaction was carried out by allowing to stand at room temperature for 16 hours in a wet environment. The maleimide group formed on the surface of the chip reacts with the thiol group possessed by the cysteine residue at the terminal of the substrate peptide, so that the substrate peptide can be covalently immobilized on the surface.

(Blocking of unreacted maleimide groups)
After washing the substrate peptide-immobilized surface with a phosphate buffer, in order to block unreacted maleimide groups, PEG thiol (the functional group at one end is a thiol group and the other functional group is a methoxy group) Nippon Oil & Fat SUNBRIGHT MESH-50H) was dissolved in a phosphate buffer (20 mM phosphoric acid, 150 mM NaCl, pH 7.2) at a concentration of 10 mg / ml, and 250 μl was poured onto the chip and allowed to react for one hour. The molecular weight of the PEG thiol used here is 5,000, the hydrophilicity is very high, and the effect of suppressing nonspecific adsorption can be expected.

(Measurement by SPR imaging method)
The peptide-immobilized chip thus obtained was set in an SPR imaging device (MultiSPRinter: manufactured by Toyobo Co., Ltd.), and 50 mM Tris-HCl buffer (pH 7.4) was flowed into the flow cell as a running buffer at a rate of 100 μl / min. . After confirming that the signal from the SPR was stabilized, a phosphorylated tyrosine antibody PT66 (manufactured by Sigma) was injected into the cell in the SPR device to act. The antibody used was a 2000-fold diluted solution with the above running buffer. When the signal rise reached a plateau state, the running buffer was fed again for washing. The change of the SPR signal at that time was observed. In addition to the spot site of each substrate, the signal change was also observed in the background.
Analysis by SPR imaging was also examined. At the time of the SPR analysis, an image obtained by imaging the surface of the array with a CCD camera is taken every 2 seconds, and from the image taken at the time after the antibody reaction, the image at the time before the reaction is obtained from the image calculation processing software Scion Image. By performing a subtraction process using (made by Scion Corp.), it is possible to show how the antibodies in the entire array are bound.

(Observation results and discussion)
The graph of signal change is shown in the upper part of FIG. The sensorgram plots the mean value of the signal at 8 locations. For Background, the average value of the signals obtained with 8 arbitrarily selected points other than the spot portion of the substrate peptide as measurement points is plotted. It was observed that the antibody bound to phosphorylated substrate peptide 2 and hardly bound to non-phosphorylated substrate peptide 1. The background portion in this graph shows no change in signal, indicating that the antibody was hardly bound. As a result of SPR imaging, it has been observed that the antibody binds only to the spot portion of the substrate peptide 2 and hardly binds to other portions. Thus, by blocking the functional group on the surface covalently with a hydrophilic polymer, it was possible to easily obtain a peptide chip in which nonspecific adsorption was suppressed.

[Comparative Example 1]
(Peptide immobilization)
The same operation as in Example 1 was performed.
(Blocking of unreacted maleimide groups)
Here, the blocking operation was not performed, and the process proceeded directly to the next step (measurement by SPR imaging method).
The same operation as in Example 1 was performed.

(Observation results and discussion)
The graph of signal change is shown in the lower part of FIG. Here, the tendency that the antibody is bound to the phosphorylated substrate peptide 2 and the antibody is not bound to the non-phosphorylated substrate peptide 1 is the same as in Example 1, but an increase in signal is observed at the background portion in this graph. ing. In addition, from the results of SPR imaging, a part where the influence of nonspecific adsorption is suggested in the background portion is partially recognized.

  Nonspecific adsorption is suppressed in the peptide chip produced by the method of the present invention, and accurate measurement is possible. In addition, the manufacturing method is easy, and it is expected to greatly contribute to the industrial world.

SPR analysis results in Example 1 and Comparative Example 1

Explanation of symbols

1 cSrc
2 p-cSrc
bg bakground

Claims (7)

A method for producing a peptide chip in which two or more kinds of peptides are immobilized on the same chip, wherein the maleimide group introduced on the gold surface on the chip and the thiol group present in or added to the peptide After the peptide is immobilized on the chip, the maleimide group remaining on the gold surface on the chip is reacted with a hydrophilic polymer having a thiol group to covalently bond the maleimide group remaining on the gold surface. A method for producing a peptide chip for use in surface plasmon resonance imaging analysis, characterized in that blocking is performed. The method according to claim 1, wherein a hydrophilic polymer having a thiol group only at the terminal is used.   The method according to claim 1 or 2, wherein the molecular weight of the hydrophilic polymer is 400 or more. The method according to claim 1, wherein the hydrophilic polymer has a molecular weight of 1000 or more. The method according to any one of claims 1 to 4 , wherein the hydrophilic polymer is polyethylene glycol. The method according to any one of claims 1 to 5, wherein the peptide to be immobilized has a cysteine residue at the terminal. A peptide chip for use in surface plasmon resonance imaging analysis, which is produced by the method according to claim 1.

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