JP2006047014A - Nonspecific signal suppression method in peptide chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To acquire a peptide chip capable of suppressing nonspecific adsorption of a biomolecule in the peptide chip, and acquiring data having high reliability by blocking an unreacted functional group, especially when used for surface plasmon imaging measurement. <P>SOLUTION: In a manufacturing method of the peptide chip, a functional group A on a chip is reacted with a functional group B existing in or added to a peptide, to thereby immobilize the peptide comprising amino acid residues to the number of 5-60 on the chip, and then the functional group A remaining on the chip surface is reacted with a compound having a molecular weight of 1,000 or less and having a functional group C, to thereby block the functional group A in a covalently bonded state. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a biochip blocking method capable of suppressing nonspecific adsorption of biomolecules by blocking with a low molecular weight compound 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 non-specific 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 completely, 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 effectively blocking nonspecific adsorption by efficiently blocking a functional group used for immobilizing a peptide as a target molecule using a low molecular weight compound without denaturing the peptide. To do.
Anal. Biochem. 198, 268, 1991 US Pat. No. 6,127,129

  An object of the present invention is to provide a biochip blocking technique for suppressing nonspecific adsorption of biomolecules. In particular, it is to obtain a blocking means capable of accurately evaluating an interaction when used for analyzing an interaction with a peptide immobilized on a substrate.

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. After reacting the functional group (A) on the chip with the functional group (B) present in or added to the peptide to immobilize the peptide consisting of 5 to 60 amino acid residues on the chip, the chip surface A method for producing a peptide chip, wherein the functional group (A) remaining on the substrate is reacted with a compound having a functional group (C) and having a molecular weight of 1000 or less to block the functional group (A) covalently.
2. A method of 1, wherein a compound having a molecular weight of 1000 or less having a functional group (C) only at the terminal is used.
3. The method of 1 or 2 whose molecular weight of the compound which has a functional group (C) is 600 or less.
4). The method of 1 or 2 whose molecular weight of the compound which has a functional group (C) is 400 or less.
5. The method of 1 or 2 whose molecular weight of the compound which has a functional group (C) is 300 or less.
6). The method according to 1 or 2, wherein the compound having the functional group (C) has a structure represented by the formula (I) (wherein n represents an integer of 2 to 12, R represents an alkyl group).

7). 6. The method according to 6, wherein n in formula (I) is 2-6.
8). The method according to any one of 1 to 7, wherein the chip surface is gold.
9. 9. The method according to any one of 1 to 8, wherein the substrate of the chip is a transparent substrate.
10. The method according to any one of 1 to 9, wherein 10.2 or more types of peptides are immobilized on the same chip.
11. 11. The method according to any one of 1 to 10, wherein the chip is used for surface plasmon resonance (SPR) analysis.
12 Formula (I) (In the formula, n represents an integer of 2 to 12. R represents an alkyl group.)
A blocking reagent comprising the structure shown in 1.

13. 12. Twelve blocking reagents wherein n in formula (I) is 2-6.

  By adopting the peptide chip blocking method in the present invention, a highly accurate measurement system using a peptide chip in which nonspecific adsorption is suppressed can be obtained.

In the biochip production method of the present invention, the functional group (A) on the chip and the functional group (B) present in or added to the peptide are reacted to comprise 5 to 60 amino acid residues on the chip. After immobilizing the peptide, the functional group (A) remaining on the chip surface is reacted with a compound having a functional group (C) having a molecular weight of 1000 or less to block the functional group (A) covalently. This is a manufacturing method.
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 the amino acid residues is usually about 5 to 60 residues, and 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 (B) 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, for example. 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 one to several sequences consisting of a glycine residue and / or an alanine residue or a serine residue.

  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 or added to the peptide to be immobilized, and can also react with the functional group (C) of the low-molecular compound. .
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.
Preferred combinations of the functional group (A), the functional group (B), and the functional group (C) are shown below.

  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) is preferably present at the end of the low molecular compound, and more preferably at one end. If the functional group (C) is present in the main chain of the low-molecular compound or a side chain 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 react with the functional groups (A) and are blocked. 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 low molecular weight compound having a functional group (C) only at one end and a low activity methoxy group or hydroxyl group at the other end is preferable.

  In the present invention, the low molecular weight compound has a molecular weight of 1000 or less, preferably 600 or less, more preferably 400 or less, and still more preferably 300 or less. If the molecular weight exceeds 1000, steric hindrance is likely to be given to the immobilized target molecule, and the probability of adverse effects such as a decrease in signal in the target molecule is increased, which is not preferable.

  The low molecular compound is not particularly limited in its chemical structure, but it is not preferable to use a compound having an ionic functional group because it aims to suppress nonspecific adsorption. Sometimes. 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. Specifically, those having a repeating structure of an ethylene glycol chain as shown in the formula (I) are more preferable.

In the formula (I), n represents an integer of 2 to 12, but a range of 2 to 6 is more preferable, and a range of 3 to 5 is still more preferable. If the value of n exceeds 12, it is not preferable because the influence of steric hindrance due to the increase in the molecular weight as described above tends to increase.
The alkyl group represented by R is a linear or branched alkyl group having 1 to 4 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and t-butyl. Preferable examples include methyl and ethyl, particularly methyl group.

  The peptide chip blocking method of 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 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 (particularly a gold thin film) that 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. The 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]
(Synthesis of blocking agent)
FIG. 1 shows a synthetic reaction scheme. Tetraethylene glycol monomethyl ether (TEG-OH; manufactured by Tokyo Chemical Industry) (1 equivalent) and pyridine (2 equivalents) were dissolved in dichloromethane, and thionyl chloride (4 equivalents) was gradually added dropwise thereto. The system was then stirred overnight at room temperature. Next, it was washed with water, 0.1 mM hydrochloric acid, water, 0.1 mM NaHCO ₃ (hydrochloric acid and NaHCO ₃ three times each), and dichloromethane was removed by evaporation under reduced pressure to obtain TEG-Cl. The product was confirmed by elemental analysis. The elemental analysis was performed by a differential heat conduction method using a Yanaco CNH coder MT5 type (manufactured by Yanagimoto Seisakusho), and the results are shown in Table 2. The synthesis yield was 66%.

  The obtained TEG-Cl (1 equivalent) was dissolved in DMF, thiourea (2 equivalents) was added, and the mixture was heated at reflux for 2 hours (about 100 ° C. in the presence of nitrogen gas). Thereafter, NaOH (3 equivalents) was added, and the mixture was further heated to reflux for 2 hours. The reaction mixture was extracted with dichloromethane, the dichloromethane was removed by evaporation under reduced pressure, and the mixture was purified by a column packed with silica gel (silica gel 60 manufactured by Kanto Chemical; particle size 63-200 mm, 70-230 mesh ASTM) (developing solvent: chloroform). The product (TEG-SH) was confirmed by elemental analysis and a method using a thiol quantification reagent. The results of elemental analysis were as shown in Table 3. The synthesis yield was 60%.

The principle of thiol quantification is shown in FIG. 2 (see Anal. Chem. Vol. 59, pp. 1509 (1987)), but DTNB (5,5′-Dithiobis (2-nitrobenzoic acid)). ; Produced by Dojindo Chemical Co., Ltd.), when an SH group is present, an S—S bond corresponding to the amount of the SH group is broken to generate 5-Mercapto-2-nitrobenzoic acid, and the absorbance of this thiol (λ _max = 412 nm) The SH group can be quantified. By adding TEG-SH, the absorbance at 412 nm derived from 5-Mercapto-2-nitrobenzoic acid increased and the presence of a thiol group could be confirmed. Under the conditions of DTNB 60 μM and TEG-SH 30 μM (final concentration), the absorbance after 3 minutes and 30 minutes after the addition was measured (solvent PBS). It was 32.1 μM as determined from the absorbance at 412 nm.
(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 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. 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. 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 3400 is phosphate buffer (20 mM phosphate, 150 mM NaCl; pH 7. 2) was dissolved 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 and the arrangement for immobilization thereof are shown in FIG. Substrate peptide B has an amino acid sequence that is phosphorylated by cSrc kinase. Substrate peptide C is a positive negative control substrate in which the tyrosine residue in substrate peptide B is phosphorylated in advance. Substrate peptide A is a negative control substrate in which the tyrosine residue in substrate peptide B is substituted with a phenylalanine residue. D shows the blank spot which does not fix | immobilize a peptide. Each substrate peptide was dissolved in a 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 as 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 phosphate buffer, phosphate buffer (20 mM phosphate, 150 mM NaCl) is used so that the TEG-SH has a concentration of 1 mM in order to block unreacted maleimide groups. Dissolved in pH 7.2), 250 μl was poured onto the chip and allowed to react at room temperature for 1 hour. As a comparative example, a phosphate buffer solution (20 mM phosphate) is used so that PEG thiol (SUNBRIGHT MESH-50H, manufactured by NOF Corporation) has a functional group at one end as a thiol group and the other functional group as a methoxy group. , 150 mM NaCl; pH 7.2), 250 μl was poured onto the chip, and reacted at room temperature for 1 hour. The molecular weight of PEG thiol used here is 5,000.
(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 allowed to flow through 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 converted into 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 and the result by SPR imaging are shown in FIG. The sensorgram plots the average value of the signal at four locations for each spot. For Background, the average value of signals obtained by using four arbitrarily selected points other than the spot portion of the substrate peptide as measurement points is plotted. When blocking treatment with TEG-SH was performed, it was observed that the antibody bound to the phosphorylated substrate peptide C and hardly bound to the blank D non-phosphorylated substrate peptides A and B. 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 site of the phosphorylated substrate peptide C and hardly binds to other portions. Thus, it can be confirmed that nonspecific adsorption is suppressed by blocking the functional group on the surface covalently with a hydrophilic polymer. On the other hand, when a blocking treatment with PEG-SH is performed, the antibody binding signal in the phosphorylated substrate peptide C is also very weak. This is considered to be due to the large molecular weight of PEG-SH, which inhibits antibody binding due to steric hindrance.
[Example 2]
(Peptide immobilization)
An array in which PKA (protein kinase A) including positive and negative controls and a cSrc substrate were immobilized in a pattern as shown in FIG. 5 was prepared. Peptide immobilization in Example 1 except that 8 × 12 96-point pattern photomasks were used for UV irradiation, and that spotting of the substrate peptide solution was not performed manually but using a MultiSPRinter spotter (manufactured by Toyobo). The array was prepared in the same manner as described above.
(Blocking of unreacted maleimide groups)
In the same manner as in Example 1, a comparative study was performed by performing treatment with TEG-SH and PEG-SH.
(Measurement by autoradiography)
Phosphorylation with PKA was performed on 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 30 minutes. The composition of the PKA solution was 1 μl of PKA catalyst subunit (Promega), 378 μl of 50 mM Tris-HCl buffer (pH 7.4), 20 μl of 1M magnesium chloride solution, and 1 μl of γ ³² P-ATP (Amersham Biosciences). Thereafter, the array was washed three times with PBS and water, and the surface of the array was dried, followed by an exposure treatment for 30 minutes to perform autoradiography measurement. Reading was performed using BAS1800II (manufactured by Fuji Photo Film) using a dedicated IP sheet.
(Observation results and discussion)
The measurement results are shown in FIG. When blocking treatment with TEG-SH was performed, RI uptake was confirmed only in the PKA substrate, and it was confirmed that only this substrate was specifically phosphorylated. On the other hand, when blocking treatment with PEG-SH was performed, non-specific RI uptake was confirmed even in spots other than the PKA substrate due to some factors when compared under the same conditions.

  By the method of the present invention, nonspecific adsorption on the peptide chip is suppressed, and more accurate measurement is possible. In addition, the processing method is easy, and it is expected to greatly contribute to the industry.

It is a figure which shows the synthetic scheme of TEG-SH. It is a figure which shows the principle of thiol fixed_quantity | quantitative_assay. FIG. 3 is a diagram showing an immobilization pattern of a substrate on the SPR array in Example 1. 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 autoradiography in Example 2. FIG.

Claims (13)

  After reacting the functional group (A) on the chip with the functional group (B) present in or added to the peptide to immobilize the peptide consisting of 5 to 60 amino acid residues on the chip, the chip surface A method for producing a peptide chip, wherein the functional group (A) remaining on the substrate is reacted with a compound having a functional group (C) and having a molecular weight of 1000 or less to block the functional group (A) covalently.   The method according to claim 1, wherein a compound having a functional group (C) only at the terminal and having a molecular weight of 1000 or less is used.   The method according to claim 1 or 2, wherein the compound having the functional group (C) has a molecular weight of 600 or less.   The method according to claim 1 or 2, wherein the compound having a functional group (C) has a molecular weight of 400 or less.   The method according to claim 1 or 2, wherein the compound having the functional group (C) has a molecular weight of 300 or less. The compound having a functional group (C) is represented by the formula (I) (wherein n represents an integer of 2 to 12).
R represents an alkyl group. The method according to claim 1, comprising the structure shown in FIG.

  The method according to claim 6, wherein n in formula (I) is 2-6.   The method according to claim 1, wherein the chip surface is gold.   9. The method according to claim 1, wherein the substrate of the chip is a transparent substrate.   The method according to any one of claims 1 to 9, wherein two or more kinds of peptides are immobilized on the same chip.   The method according to claim 1, wherein the tip is used for surface plasmon resonance (SPR) analysis. A blocking reagent comprising a structure represented by the formula (I) (wherein n represents an integer from 2 to 12; R represents an alkyl group).

  13. The blocking reagent according to claim 12, wherein n in the formula (I) is 2-6.

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