JP4508765B2 - Peptide immobilization method - Google Patents

Description

  The present invention relates to a method for producing a peptide chip used in a substance interaction analysis system. More specifically, the present invention relates to a method for producing a peptide chip capable of increasing a binding signal between substances and reducing non-specific effects by using a specific low molecular weight compound as a cross-linking agent.

  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, when a biomolecule such as a peptide is immobilized via a large molecule such as dextran, the influence of dextran or the like is large, which may adversely affect the measurement of the properties of the peptide.
Anal. Biochem. 198, 268, 1991

  An object of the present invention is to provide a peptide chip that suppresses non-specific adsorption of biomolecules and increases the binding signal in a target substance.

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. The substrate of the chip having an amino group on the surface and the compound represented by the formula (I):

（式中、Ｒは水素原子またはＳＯ₃ ^-(1/nＭⁿ⁺)（Ｍⁿ⁺はｎ価のカチオンを示す。ｎは１又は２を示す。）である）
を反応させてチップの基板にマレイミド基を導入する工程、チオール基を有するペプチドと該マレイミド基を反応させてペプチドを固定化する工程、必要に応じてさらに基板上に残存するマレイミド基をブロッキングする工程を含む、ペプチドを固定化したチップの製造方法。
２．チップ表面が金であることを特徴とする項１記載の方法。
３．チップの基板が透明基板であることを特徴とする項１又は２に記載の方法。
４．２種類以上のペプチドが同一チップ上に固定化されることを特徴とする項１〜３のいずれか記載の方法。
５．固定化されるペプチドの少なくとも一方の末端がシステイン残基であり、該システイン残基のＳＨ基がマレイミド基と反応することを特徴とする項１〜４のいずれかに記載の方法。
６．チップが表面プラズモン共鳴（ＳＰＲ）解析に用いられることを特徴とする項１〜５のいずれかに記載の方法。
７．下記式

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2).
The step of introducing a maleimide group into the substrate of the chip by reacting, the step of reacting the peptide having a thiol group with the maleimide group to immobilize the peptide, and blocking the maleimide group remaining on the substrate as necessary A method for producing a chip on which a peptide is immobilized, comprising a step.
2. Item 2. The method according to Item 1, wherein the chip surface is gold.
3. Item 3. The method according to Item 1 or 2, wherein the substrate of the chip is a transparent substrate.
Item 4. The method according to any one of Items 1 to 3, wherein two or more kinds of peptides are immobilized on the same chip.
5). Item 5. The method according to any one of Items 1 to 4, wherein at least one terminal of the peptide to be immobilized is a cysteine residue, and an SH group of the cysteine residue reacts with a maleimide group.
6). Item 6. The method according to any one of Items 1 to 5, wherein the chip is used for surface plasmon resonance (SPR) analysis.
7). Following formula

（式中、「Substrate」はチップの基板を示し、「Peptide」はチップに固定化されるペプチド（結合に関与するCys残基を除く）を示し、S(Cys)は該ペプチドのCys残基のSH基に由来するSであることを示す。）
で表される構造を有するペプチドを固定化したチップ。
８．S(Cys)が、ペプチドの末端のCys残基のSH基に由来するSである項７に記載のチップ。

(Where “Substrate” indicates the substrate of the chip, “Peptide” indicates the peptide immobilized on the chip (excluding the Cys residue involved in binding), and S (Cys) is the Cys residue of the peptide. It is S derived from the SH group of
The chip | tip which fix | immobilized the peptide which has a structure represented by these.
8). Item 8. The chip according to Item 7, wherein S (Cys) is S derived from the SH group of the Cys residue at the end of the peptide.

  By using the low molecular weight compound in the present invention as a cross-linking agent, it is possible to obtain a measurement system using an accurate peptide chip that improves the target signal and further suppresses nonspecific adsorption.

The present invention is characterized in that a peptide is immobilized on a chip via a thiol group.
The thiol group (SH) is preferably derived from a cysteine residue in the peptide.
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.
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 terminal, more preferably only one terminal (N terminal or C terminal). 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. Alternatively, an amino acid sequence of 1 to several residues may be further added between the cysteine residue and the target peptide. 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 and / or alanine residues or serine residues.

  In addition, a compound having a thiol group is chemically bonded to an immobilized peptide at any one or more amino acid residues and bonded to a maleimide group via the thiol group of the added compound. Good. The bonding position of the compound is not particularly limited, but is preferably bonded to any terminal amino acid residue.

In the present invention, the compound Succinimidyl 4- [N-- represented by the formula (I) in which R is H is used as a heterobifunctional crosslinking agent having a succinimide (NHS) group and a maleimide (MAL) group for immobilization of peptides. maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SMCC.) or R is _{^{SO 3 - (1 / nM n}} +) (M, n is as defined above) compound shown in formula (I) wherein It is characterized by using Sulfosuccinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate (hereinafter referred to as SSMCC) as a crosslinking agent. In addition, the crosslinking agent used in the present invention does not only indicate a compound having the same structure as that of the compound represented by formula (I) but also includes an analogized compound as long as its function is not impaired. In the present invention, both SMCC and SSMCC can be applied. However, from the viewpoint of solubility in water, it is more preferable to use SSMCC when the reaction is carried out in an aqueous system such as a buffer solution.

（式中、Ｒは水素原子またはＳＯ₃ ^-(1/nＭⁿ⁺)（Ｍⁿ⁺はｎ価のカチオンを示す。ｎは１又は２を示す。）である）
1/nＭは、Ｈ，Ｎａ，Ｋ，Ｌｉ，Ｃｓ等のアルカリ金属(ｎ＝１)、ＮＨ₄，Ｎ(Ｍｅ)₄等のＣ₁〜Ｃ₄のアルキル基で置換されていてもよい四級アンモニウム（ｎ＝１）、1/2Ｃａ，1/2Ｍｇ，1/2Ｂａ等のアルカリ土類金属（ｎ＝２）等が挙げられ、好ましくはアルカリ金属、より好ましくはＮａである。

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2).
1 / nM may be substituted with an alkali metal (n = 1) such as H, Na, K, Li, or Cs, or a C _{1 to} C ₄ alkyl group such as NH ₄ or N (Me) _4. Examples include alkaline earth metals (n = 2) such as quaternary ammonium (n = 1), 1 / 2Ca, 1 / 2Mg, 1 / 2Ba, and the like, preferably an alkali metal, and more preferably Na.

  By applying the cross-linking agent of the formula (I) as described above, the efficiency of immobilizing the peptide on the chip is significantly higher than that of the high-molecular weight cross-linking agent, so that the binding efficiency with the target substance is also improved. As a result, the signal resulting from the binding becomes clearer. Moreover, there is almost no problem with non-specific effects, and the so-called S / N ratio can be increased.

In order to form the maleimide surface by introducing the SMCC or SSMCC onto the chip, a functional group reactive with the succinimide group at the other end of the SMCC or the sulfonic acid succinimide group at the other end of the SSMCC Specifically, it is necessary to introduce an amino group on the chip in advance. The means for introducing an amino group on 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 an amino group using a cross-linking agent starting from the functional group introduced on the surface.
The first step of the method of the present invention is to react a chip substrate having an amino group with a compound of formula (I), and to have a succinimide moiety (a sulfonic acid group or a salt thereof) of the compound of formula (I). May be eliminated) to form an amide bond, and a maleimide group is introduced into the substrate.
The reaction may be carried out by using an aqueous solution of the crosslinking agent of the formula (I) in an amount of 1 mol to an excess of 1 mol of amino group on the substrate (when R is a sulfonic acid or a salt thereof) or an organic solvent (when R is a hydrogen atom) ) The solution can be applied on the substrate or immersed in an aqueous solution or organic solvent solution of the crosslinking agent of formula (I). The reaction proceeds advantageously by, for example, reacting for about 1 to 24 hours at a temperature of about 10 to 60 ° C. (preferably room temperature).
Next, in the second step, a peptide having a thiol group and a maleimide group introduced through an amide bond are reacted with a substrate derived from the compound of formula (I). The reaction is performed by using an excess amount of a peptide having a thiol group with respect to 1 mol of the maleimide group and reacting in a solvent at a temperature of about 10 to 60 ° C. (preferably room temperature) for about 1 to 24 hours. Proceed with advantage. Solvents include water, water-containing organic solvents (water-containing alcohols such as water-containing ethanol, water-containing methanol, and water-containing propanol, water-containing THF, water-containing acetone, and water-containing THF), water-miscible organic materials such as lower alcohols, DMF, dimethylacetamide, and DMSO. Examples are solvents.

  There is a possibility that a maleimide group remains in the chip on which the obtained peptide is immobilized. Although this residual maleimide group may be left as it is, it is desirable to further react with a compound having a thiol group to remove the residual maleimide group. This reaction condition can be carried out under the same conditions as those for the reaction with the peptide having a thiol group in the second step except that a less polar organic solvent can be used as the solvent.

  The peptide chip immobilization method in the present invention can be applied to various uses. In general, it is also effective in detection systems using fluorescent materials, chemiluminescent materials, radioactive materials, etc., which are often used as detection means in arrays, but in particular surface plasmon resonance (SPR), sum frequency generation (SFG), localization It is extremely effective for optical detection methods such as plasmon resonance (LPR) and ellipsometry. 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 non-specific adsorption more severely is required, the immobilization method 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, it is preferable to perform blocking by covalent bond in order to detect even the elimination of the blocking agent. In particular, when blocking the surface of the unreacted maleimide group, a compound having a thiol group is preferably used, and a PEG (polyethylene glycol) derivative is particularly preferably used.

  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. 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.

    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.

  The SPR imaging method using this SPR is a method for monitoring the state of interaction between substances by irradiating a polarized light beam over a wide range and analyzing the reflected image by making full use of image processing technology or the like. It is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.

  In the SPR imaging method, in order to analyze the reflected image, a means for irradiating the chip with a polarized light beam in a wide range and sufficiently securing the illuminance of the light beam is required. The brighter the illuminance of the polarized light flux, the more preferable is the sensitivity of the sensor.

  The type of the light source is not particularly limited, but it is preferable to use light including near infrared light that makes the change in the SPR resonance angle particularly sensitive. Specifically, a white light source capable of irradiating light over a wide range such as a metal halide lamp, a mercury lamp, a xenon lamp, a halogen lamp, a fluorescent lamp, and an incandescent lamp can be used. A halogen lamp that is sufficiently high, has a simple light power supply and is inexpensive is particularly preferable.

  A normal white light source has a defect that light and dark unevenness occurs in the filament portion. If the light from the light source is irradiated as it is, light and dark unevenness occurs in the image obtained by reflection, and it becomes difficult to evaluate screening and morphological changes. Therefore, as a means for uniformly irradiating the chip with light, a method of making the light into parallel light after passing through the pinhole is preferable. The means for passing a pinhole is preferable as a means for obtaining a light beam having a uniform brightness, but there is a drawback that the illuminance decreases when light is passed through the pinhole as it is. Therefore, as a means for ensuring sufficient illuminance, it is preferable to use a method in which a convex lens is installed between the pinhole and the light source, and condensed to pass through the pinhole.

Since the white light source is radiated light, it needs to be converted into parallel light using a convex lens before condensing. Parallel light can be obtained by installing a light source near the focal length of the convex lens. By installing another convex lens and installing a pinhole in the vicinity of the focal length of the lens, it is possible to pass the condensed light through the pinhole. The light that intersects and passes through the pinhole is converted into parallel light by the CCTV lens for the camera, and the cross-sectional area of the parallel light beam obtained at that time is preferably adjusted to 10 to 1000 mm ² . This method enables a wide range of screening and morphological observation.

  When the interaction is monitored, the polarized light beam is applied to the opposite surface of the metal thin film on which the substance or the substance aggregate is fixed. The polarized light beam is applied to the opposite surface of the metal thin film on which the substance or substance aggregate is fixed, and the reflected light beam is obtained. The reflected light beam from the metal thin film passes through a near-infrared wavelength optical interference filter and transmits only light in the vicinity of a certain wavelength before being photographed by a CCD camera.

  The center wavelength of the optical interference filter is preferably 600 to 1000 nm with high SPR sensitivity. The wavelength width of the wavelength at which the transmittance of the optical interference filter is half that at the maximum is called a half-value width, but the smaller the half-value width, the sharper the wavelength distribution is, and more specifically, the half-value width is preferably 100 nm or less. An image photographed by the CCD camera through the optical interference filter is taken into a computer, and a change in the brightness of a certain part can be evaluated in real time, and the whole image can be evaluated by image processing. Thus, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.

  The chip for SPR used in the present invention preferably comprises a metal substrate in which a metal thin film is formed on a transparent substrate, and directly or indirectly, chemically or physically on the metal thin film, a substance or a substance. A slide on which the assembly is fixed is used. The material of the substrate is not particularly limited, but it is preferable to use a transparent material. Specific examples include glass or plastics such as polyethylene terephthalate (PET), polycarbonate, and acrylic. Of these, glass is particularly preferable.

The thickness of the substrate is preferably about 0.1 to 20 mm, and more preferably about 1 to 2 mm. In order to achieve the object of evaluating the reflection image from the metal thin film, the SPR resonance angle is as small as possible, and there is no fear that the image to be photographed will be fuzzy, and the analysis is easy. Therefore, the refractive index n _D of the transparent substrate or the transparent substrate and the prism in contact with the transparent substrate is preferably 1.5 or more.

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 almost 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 96 square holes of 500 μm square (consisting of 8 × 12 patterns), and the pitch between the hole centers is designed to be 1 mm. The portion of the photomask with a hole is transparent to UV light and is irradiated onto the slide for patterning. 4 armPEG remains in the part that was not irradiated 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. SSMCC (Pierce) was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 0.4 mg / ml and reacted with 8-AOT on the gold surface for 15 minutes. The amino group of 8-AOT and the NHS group of SSMCC reacted to introduce an unreacted MAL group on the surface.

On the surface obtained as described above, as shown in the lower part of FIG. 1, a peptide (PKA) consisting of an amino acid sequence serving as a substrate for protein kinase A, a negative control (nPKA; serine residue of protein kinase A substrate) Alanine residue), protein kinase A substrate positive control (pPKA; serine residue phosphorylated), cSrc kinase substrate peptide (cSrc) consisting of phosphate buffer (20 mM phosphate) Acid, 150 mM NaCl; pH 7.2) was dissolved at 1 mg / ml, and spotted 10 nl at a time using a MultiSPRinter spotter (manufactured by Toyobo). 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 array 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) NOF SUNBRIGHT MEH -50H) phosphate buffer so as to 1mM concentration (20 mM phosphate, 150 mM NaCl; dissolved in pH 7.2), issued Note the 250μl onto the chip, and reacted at room temperature for one hour It was. The molecular weight of PEG thiol used here is 5,000.
(Detection of PKA phosphorylation by SPR analysis)
Phosphorylation by PKA was performed using the array that had been blocked as described above. 400 μl of PKA solution was dropped on the array and reacted at 30 ° C. for 4 hours. The composition of the PKA solution was 1 μl of PKA catalyst subunit (manufactured by Promega), 375 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, and 4 μl of 10 mM ATP. Thereafter, the array was washed three times with PBS and water, and the surface of the array was dried. Then, the array was set in an SPR imaging device (MultiSPRinter: manufactured by Toyobo), and 50 mM Tris-HCl buffer (pH 7.4) was used as a running buffer. Was flowed into the flow cell at a rate of 100 μl / min. After confirming that the signal from SPR was stabilized, phosphorylated serine antibody PSR-45 (manufactured by Sigma) was injected into the cell in the SPR device to act. The antibodies were allowed to act in order from the one with the highest dilution ratio using solutions diluted 4000 times, 2000 times and 1000 times 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.
(Observation results and discussion)
The result of the sensorgram which graphed the SPR signal change is shown in FIG. The sensorgram plots the average value of the signal for each spot. For Background, the average value of signals obtained by using arbitrarily selected locations other than the spot portion of the substrate peptide as a measurement point is plotted. A very strong increase in antibody binding signal was confirmed with respect to the positive control, and a certain level of increase in binding signal was also observed in the PKA substrate. On the other hand, almost no signal change is observed for the negative control, cSrc substrate, blank, and background. Therefore, by this method, phosphorylation of the PKA substrate can be detected very specifically.
[Comparative Example 1]
After introducing 8-AOT on the gold surface, instead of SSMCC, a heterobifunctional polyethylene glycol having a succinimide (NHS) group and a maleimide (MAL) group at the end with a molecular weight of 3400 (NHS-PEG-MAL, manufactured by Nektar) Except for the point of acting), all were examined in the same manner as in Example 1. NHS-PEG-MAL was dissolved in phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7.2) at 10 mg / ml and reacted with 8-AOT on the gold surface for 2 hours. The results are shown in FIG.

  In this case, the binding signal itself is weak as a whole. Furthermore, non-specific signals have been confirmed in addition to PKA substrates, which is an undesirable result in terms of specificity.

  By the method of the present invention, it is possible to amplify the binding signal in the peptide chip, while suppressing non-specific adsorption, and more accurate measurement is possible. The processing method is also very easy, and is expected to contribute greatly to the industry.

It is a figure which shows the result of the SPR analysis in Example 1. It is a figure which shows the result of the SPR analysis in the comparative example 1.

Claims (6)

(1) A substrate of a chip having an amino group on the gold surface, and a compound represented by the formula (I) in an amount from 1 mol to an excess amount relative to 1 mol of the amino group:

(In the formula, R is a hydrogen atom or SO ₃ ⁻ (1 / nM ^{n +} ) (M ^{n +} represents an n-valent cation; n represents 1 or 2))
A process of introducing maleimide groups into the chip substrate by reacting
(2) a step of immobilizing the peptide by reacting an excess amount of a peptide having 5 to 25 amino acid residues and having a thiol group from 1 mol with respect to 1 mol of the maleimide group, and (3) a substrate Blocking the remaining maleimide group with polyethylene glycol (PEG) terminated with a thiol group,
A chip manufacturing method for detecting a phosphorylation reaction of a peptide by surface plasmon resonance (SPR) analysis. The method according to claim 1, wherein the substrate of the chip is a transparent substrate. The method according to claim 1 or 2, wherein two or more kinds of peptides are immobilized on the same chip. At least one terminal of the peptide is a cysteine residue;
Thiol group of the cysteine residue, the method according to any one of claims 1 to 3, characterized in that reacts with the maleimido group introduced into the substrate. The peptide includes a portion serving as a substrate for a phosphorylase, and a spacer portion,
The spacer part is:
5. The method according to claim 1, wherein the amino acid sequence is composed of (a) an amino acid sequence of 1 to several residues; and (b) is located between a portion serving as a substrate for a phosphorylase and the cysteine residue. the method according to item 1. The chip, by detecting antibodies binding signals by surface plasmon resonance (SPR) analysis, a chip for detecting the phosphorylation of a peptide, the method according to any one of claims 1 to 5.

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