JP2014149199A - Peptide probe, and measuring method of specimen using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To search for a chemical material capable of being kept stably in a long term while holding a high material identification capability of a biological material, and build a measurement system using it and supply the system to a marketplace.SOLUTION: The peptide probe is used for measuring a surface plasmon resonance or magnetic sensing. The peptide probe includes: a peptide region having at least one specimen binding portion that applies a specific binding capacity to a specimen; and a linker region for fixing the peptide region onto a solid-phase carrier surface serving as a reaction field of the surface plasmon resonance or magnetic sensing. Here, the linker region is constituted by a molecular chain of a molecular length of 4-21. The measuring method of the specimen uses the peptide probe.

Description

  The present invention relates to a peptide probe and a method for measuring a test substance using the peptide probe.

  In immunochromatographic assays that are frequently used in clinical diagnoses such as pregnancy test drugs, natural physiologically active substances such as antibodies are applied to filter paper, membrane filters, etc., and the physiologically active substance is separated from the physiologically active substance. In general, measurement is performed by sandwiching a test substance with a substance and detecting the latter physiologically active substance. In clinical diagnosis using genes, the concept of measuring nucleic acid such as DNA, which is a test substance, on a filter paper or membrane filter is the same. Since the above-mentioned physiologically active substances and nucleic acids are in-vivo substances, their activities change due to changes in the external environment such as temperature and humidity. Normally, the activity decreases in the direction of attenuation, so in clinical diagnosis, the value is lower than the original signal. As a result, although it actually showed a positive reaction, it became a false negative judged to be negative, causing a problem of overlooking the presence of the disease.

  In order to prevent these problems, after fixing the biologically active substance or nucleic acid as a probe to a filter paper or the like, the activity is lowered to a certain level by drying once, and the fluctuation range of the activity is reduced in the long term. The preservation method of suppressing is generally performed. Normally, the activity of a physiologically active substance is attenuated to about 50% by drying, but thereafter, the activity fluctuation range for one year is stabilized at about 10 to 20%. However, it is inevitable that the activity gradually decreases even after one year. In recent years, the need for quantitative analysis has increased in the field of clinical diagnosis such as determining disease symptoms and the progress of healing, and there has been an urgent need to stabilize the activity of in vivo substances.

  In recent years, research and development have been conducted on methods for stabilizing in vivo substances such as physiologically active substances and nucleic acids over a long period of time. For example, methods for stabilizing proteins that suppress inactivation of proteins such as antibodies, antigens, and enzymes in solution have been reported (see, for example, Patent Documents 1 and 2). In Patent Document 1, by adding peptidylprolyl cis-trans isomerase to a protein-containing solution, the activity of the protein is improved by the peptidyl prolyl cis-trans isomerization action and the polypeptide chain folding action of this substance. It protects against decline. Patent Document 2 adds a peptide protease inhibitor such as leupeptin, antipain, and pepstatin in order to stably store for a long period of time while maintaining the specificity, affinity, and binding force of an antigen or antibody present in a solution state. Is. In addition, a method for improving the accuracy of diagnosis by diagnosing cancer using a plurality of biomarkers to absorb fluctuations in the activity of physiologically active substances and errors generated for each individual has been reported (for example, Patent Documents). 3).

  However, in any of the above-described techniques, fluctuations in the activity of biological substances such as physiologically active substances and nucleic acids are inevitably occurring events, and the objective is to reduce the activity fluctuation range as much as possible. It was. Therefore, it does not fundamentally solve the problem of fluctuations in the activity of biological substances, but has only a coping therapy character.

JP-A-11-29596 JP-A-6-273418 Special table 2012-502281 gazette

  An object of the present invention is to fundamentally solve the problem of long-term stability of a substance in the living body in a measurement system using the substance in the body such as an antibody. Conventionally, in-vivo substances, particularly antibodies, have high binding activity and specificity, and antibody production techniques are established techniques, so it can be said that the fundamental problem solution has been postponed. Therefore, an object is to search for a chemical substance that can be stably maintained for a long time while maintaining excellent substance discrimination ability of in-vivo substances, and to build a measurement system using the chemical substance and supply it to the market.

  Therefore, as a result of earnest research to solve the above problems, the inventors of the present application have used a peptide as a probe for detecting a test substance, thereby maintaining excellent substance discrimination ability of in vivo substances for a long period of time. A highly sensitive and highly accurate measurement system by finding that the peptide can be stably maintained, and by optimizing the molecular length of the linker when immobilizing the peptide on a solid phase carrier as a reaction field such as surface plasmon resonance or magnetic sensing Found that can be built. Furthermore, it has been found that further improvement in sensitivity and accuracy can be realized by optimizing the charge characteristics of the solid phase carrier surface and the linker, and the present invention has been completed based on these findings.

That is, the invention shown in the following [1] to [11] is provided.
[1] A peptide probe for surface plasmon resonance or magnetic sensing measurement,
A peptide region comprising at least one test substance binding site exhibiting specific binding ability to the test substance;
A linker region for immobilizing the peptide region on the surface of a solid phase carrier serving as a reaction field for surface plasmon resonance or magnetic sensing, wherein the linker region is composed of molecular chains having a molecular length of 4 to 21 Peptide probe.
[2] The linker region is composed of molecular chains having a molecular length of 8 to 21.
[3] When the surface of the solid support is positively or negatively charged, the linker region is composed of an uncharged molecular chain.
[4] The linker region is an alkyl chain or a peptide nucleic acid chain.
[5] When the surface of the solid phase carrier is in an uncharged state, the linker region is composed of an amphiphilic molecular chain.
[6] The linker region is a polyethylene glycol chain or a nucleic acid chain.
[7] The peptide region is composed of 4 to 11 amino acids.
[8] The peptide region is composed of 8 amino acids.
[9] The peptide region is a peptide chain comprising the amino acid sequence shown in SEQ ID NO: 1.
[10] A method for measuring a test substance by surface plasmon resonance or magnetic sensing,
A step of immobilizing the peptide probe on the surface of a solid phase carrier serving as a reaction field of the surface plasmon resonance or magnetic sensing via the linker region;
Contacting the test substance with the peptide probe immobilized on the surface of the solid phase carrier; and detecting the test substance bound to the peptide region of the peptide probe by the surface plasmon resonance or magnetic sensing. Process.
[11] The peptide region of the peptide probe is a peptide chain comprising the amino acid sequence shown in SEQ ID NO: 1.

  According to the configuration of [1] to [9], a peptide probe for surface plasmon resonance or magnetic sensing measurement can be provided. Such a peptide probe uses a synthetic peptide as a detection site for a test substance. Therefore, it is possible to solve the problems of conventional biologically active substances such as proteins and in vivo substance probes such as nucleic acids, such as changes in the external environment and changes in activity over time. That is, the measurement results are not affected by changes in the external environment, and long-term storage stability can be maintained. As a result, it is possible to contribute to the construction of a measurement system that meets the requirements of the clinical site, such as quantitative analysis such as determining disease symptoms and the progress of healing in the clinical diagnosis field. The peptide probe is immobilized on the surface of the solid phase carrier via a linker region, and is configured by optimizing the molecular length of the linker region. Since the distance between the peptide region, which is a site for recognizing the test substance, and the surface of the solid phase carrier greatly affects the measurement sensitivity, it is important to optimize the length of the linker region. In other words, in the case of a solid-phase reaction system, the distance between the solid-phase carrier surface and the site for recognizing the test substance affects the measured signal intensity, and the sensitivity can be improved by optimizing the distance. Construction of a highly sensitive and highly accurate measurement system can be realized. In addition, since the peptide probe of the present invention can be chemically synthesized, there is no activity error between producers and lots like antibodies, and no impurities are mixed as a by-product during production. Therefore, background noise that hinders measurement of a test substance can be reduced, and can be used to construct a measurement system with high sensitivity, high accuracy, and high stability. In addition, the peptide probe of the present invention can be produced at low cost using a normal peptide synthesizer, etc., and has a long-term storage stability as described above, so that it can be prepared in large quantities and contributes to cost reduction of the measurement system. be able to.

  In particular, according to the configurations of [3] to [6] above, the linker region of the peptide probe of the present invention can optimize the charge characteristics corresponding to the charge characteristics of the solid phase carrier surface. Thereby, the further measurement sensitivity improves and it can contribute to realization of a highly accurate measurement system.

  Furthermore, according to the configuration of [7] to [8] above, by specifying the amino acid length of the peptide region of the peptide probe of the present invention, the peptide region which is a site for recognizing the test substance and the surface of the solid support The distance can be further optimized. By optimizing in this way, the measurement sensitivity can be further improved, and the construction of a highly sensitive and highly accurate measurement system can be realized.

  Moreover, according to the configuration of [9] above, it is possible to provide a peptide probe that enables highly sensitive and highly accurate measurement of the cancer metastasis marker nm23. Thereby, construction of a highly sensitive and highly accurate measurement system for obtaining knowledge about cancer metastasis can be realized.

  According to the above [10] to [11], a method for measuring a test substance can be provided. Such a measurement method uses the peptide probe of the present invention having a synthetic peptide as a detection site for a test substance, so that the test can be performed without being affected by changes in the external environment, changes in activity over time, or decreases in activity. The substance can be measured. In addition, since the peptide probe of the present invention can maintain long-term storage stability, there is a need in the clinical field for quantitative analysis such as determining the symptoms of disease and the progress of healing in the clinical diagnostic field. Can respond. Since the peptide probe of the present invention optimizes the molecular length of the linker region, the distance between the surface of the solid support and the site for recognizing the test substance is optimized, and the target is highly sensitive and accurate. The test substance can be measured. Furthermore, since the linker region of the peptide probe of the present invention has an optimized charge characteristic corresponding to the charge characteristic of the surface of the solid phase carrier, it can be expected to improve measurement sensitivity. The test substance can be measured with high accuracy. And since the peptide probe of the present invention can be chemically synthesized, there is no activity error between producers and lots like antibodies, and impurities are not mixed as a by-product during production. Background noise that hinders measurement of the test substance can be reduced, and the test substance can be measured with high sensitivity, high accuracy, and stability. In addition, the peptide probe of the present invention is advantageous in terms of cost because it can be produced at low cost using a normal peptide synthesizer or the like and has long-term storage stability.

  In addition, according to the configuration of [11] above, since the peptide probe of the present invention that enables highly sensitive and highly accurate measurement of the cancer metastasis marker nm23 is used, a measurement method for obtaining knowledge relating to cancer metastasis is provided. Can be established.

  The peptide probe of the present invention comprises a peptide region and a linker region. The peptide region is a region where the test substance can be specifically recognized and bound. The linker region is a region for immobilizing the peptide region on the solid phase carrier interposed between the peptide region and the solid phase carrier that performs an analytical reaction such as surface plasmon resonance or magnetic sensing.

  Here, the test substance is not particularly limited as long as it is a substance capable of specifically binding to the peptide region of the peptide probe. For example, various proteins, peptides, sugars, lipids, glycoproteins, nucleic acids, organic substances, inorganic substances and the like can be mentioned. Examples include in vivo cancer markers, hormones, neurotransmitters, growth factors, and the like. Further, specific cells, biological tissues, organs, bacteria, viruses, fungi, and the like that specifically bind to the peptide region can also be the test substance. In addition to in-vivo substances, chemical substances such as drugs, agricultural chemicals, food additives and environmental hormones can also be used as test substances. Specifically, examples of the test substance include cancer metastasis markers such as nm23, hormone markers such as PTH (parathyroid hormone), and the like.

  The peptide region is composed of peptide molecular chains, and includes a binding site that can specifically recognize and bind to the test substance using specific binding property to the test substance, that is, substance discrimination ability. There may be a single or a plurality of binding sites for the test substance existing in the peptide region, and it preferably has 1 to 3 binding sites. Therefore, it is not necessary that all of the peptide region is composed of amino acids having specific binding properties to the test substance, and the entire peptide region may exhibit specific binding properties to the test substance.

  The peptide region has a length of about 8 amino acids from the viewpoint of specific binding. If the peptide region is too short, the specific binding ability to the test substance cannot be maintained, and if it is too long, non-specific binding is induced. For example, the length is preferably 0 to 21 amino acids, more preferably 4 to 11 amino acids or 4 to 16 amino acids, and particularly preferably 8 amino acids. Thus, by specifying the amino acid length of the peptide region, the specific binding ability to the test substance can be sufficiently exerted, and the distance between the peptide region that is a site for recognizing the test substance and the solid phase carrier surface Can be optimized.

  The sequence of the peptide region is not particularly limited as long as it specifically recognizes the test substance and preferably has specific binding properties. The binding between the peptide region and the test substance is similar to the binding between an antigen and an antibody, the binding between an enzyme substrate and an enzyme, the binding between DNA and a DNA binding protein, a covalent bond, an ion There is no particular limitation as long as the peptide region can recognize the test substance, such as a bond, a hydrogen bond, and a coordinate bond. For example, when the test substance is an antigenic substance, a sequence that can recognize and bind to an epitope on the antigen can be used. On the contrary, when the test substance is an antibody, the epitope can be used for the peptide region. Further, when the test substance is an enzyme, the enzyme specifically recognizes and reacts with the sequence of the specific region of the target substrate protein, and thus the peptide sequence of the specific region can be used. Alternatively, the recognition site sequence of the receptor-ligand reaction may be used. Specifically, nm23 binding peptide (Arg-Arg-Leu-Thr-Ile-Arg-Asn-Gly: SEQ ID NO: 1) which is a cancer metastasis marker is exemplified, but is not limited thereto.

  Here, the peptide is a substance suitable for the combinatorial chemistry approach. Therefore, the peptide region may be designed so as to be used for searching for a peptide chain having specific binding property to a test substance, sequence optimization, and the like. Alternatively, the peptide probe of the present invention may be constructed using a peptide chain identified by combinatorial chemistry as a peptide region.

  Here, the peptide chain is a compound in which two or more amino acids are linked by peptide bonds. As amino acids constituting the peptide chain, in addition to natural amino acids, chemically modified amino acids may be included. Chemical modification of amino acids may be performed on functional groups such as amino group which is N-terminal amino acid of lysine, imidazole group of histidine, indole nucleus of tryptophan, carboxyl group of aspartic acid and glutamic acid, hydroxyl group of serine and threonine. it can. For example, chemical modification such as acetylation of amino group, succinylation, guanidylation, ethoxyformylation of imidazole group, amidation of carboxyl group, ethyl esterification, etc. can be performed, but there are a wide variety of chemical modification techniques for amino acids Various techniques have been reported, and any of them can be used.

  Moreover, the peptide chain used by this invention may be comprised including the non-amino acid. In some cases, the bond adjacent to the non-amino acid is not a peptide bond. In this case, it is also referred to as a peptide in this specification. For example, the peptide chain used in the present invention may be a peptide nucleic acid chain having a nucleobase in the peptide backbone. By using the peptide nucleic acid chain, the peptide probe of the present invention can be used for nucleic acid detection.

  The peptide chain constituting the peptide region of the present invention can be synthesized using a known peptide synthesis technique. For example, a chemical synthesis technique such as a liquid phase synthesis method or a solid phase synthesis method can be used. In particular, a method using a solid phase synthesis technique using a fluorenylmethoxycarbonyl (hereinafter referred to as “Fmoc”) group or a tert-butoxycarbonyl (hereinafter referred to as “tBoc”) group is widely used. As a specific technique, for example, a suitably protected amino acid can be sequentially bound on a carrier by a desired amino acid sequence by various condensation methods. In general, amino acids that are synthesized from the C-terminal to the N-terminal and that protect all functional groups other than the α-amino group of the amino acid that becomes the C-terminal are condensed with amino acids that protect all functional groups other than the carboxy group. To form peptide bonds. Next, it can be carried out by deprotecting the amino group, condensing amino acids in which all functional groups other than the carboxyl group are condensed, and repeating the condensation reaction until the amino acid at the N-terminal is reached. The amino acid can be protected by a known technique. For example, a tBoc group or Fmoc group is used to protect an amino group, a short alkyl chain such as methyl or ethyl, or a 4-nitrobenzyl group is used to protect a carboxyl group. Various commercially available protected amino acids can also be used. For the condensation reaction, for example, carbodiimides such as dicyclohexylcarbodiimide method and diisopropylpropylcarbodiimide can be preferably used. In addition, a commercially available peptide automatic synthesizer can be used.

  The linker region is a region for immobilizing the peptide region on the surface of the solid phase carrier interposed between the above peptide region and a solid phase carrier that performs an analytical reaction such as surface plasmon resonance or magnetic sensing. It is. For this reason, the linker region preferably contains a site capable of being immobilized on the surface of the solid phase carrier and a site capable of introducing the peptide region in the molecule. And the peptide probe of this invention detects and measures a test substance using the specific binding property of the biological substance which a peptide area | region has. For this reason, the linker region must not exhibit a non-specific interaction with a contaminant substance or the like contained in a test sample other than the test substance.

  The structure of the linker region may be linear, branched or cyclic, but is preferably linear. In the case of branching and ringing, the positions and shapes of the branches and rings are configured so as not to cause steric hindrance in the reaction between the peptide region and the test substance.

  In surface plasmon resonance, magnetic sensing, etc. using a solid phase carrier as a reaction field, the distance between the peptide region, which is the site for recognizing the test substance, and the solid phase carrier surface greatly affects the measurement sensitivity. The length optimization is important. That is, in the case of a solid-phase reaction system, the distance between the surface of the solid-phase carrier and the site for recognizing the test substance affects the measured signal intensity, and the sensitivity can be improved by optimizing the distance. Measurement sensitivity decreases when the distance between the surface of the solid support and the site for recognizing the test substance increases, and conversely, if it is too short, recognition of the test substance by the peptide region causes steric hindrance and other factors. Sensitivity will decrease. Therefore, the length of the linker region is preferably 4 to 21, more preferably 7 to 21, or 8 to 21, and preferably 16 in the case of a linear linker. Particularly preferred.

  Further, when the linker region is configured as branched and cyclic, it is configured such that the distance from the solid phase carrier surface to the peptide region is as long as the linear linker described above. For example, in the case of a branched shape, the molecular length of the main chain is designed as the molecular length of the linker region without including the side chain.

  The configuration of the linker region is selected according to the charge characteristics of the solid support surface. When the surface of the solid phase carrier is positively or negatively charged, the linker region is constituted by an uncharged molecular chain. On the other hand, when the surface of the solid support is uncharged, the linker region is composed of amphiphilic molecular chains. Thus, by optimizing the charge characteristics, the reactivity between the test substance and the peptide region, and the signal detection intensity in surface plasmon resonance, magnetic sensing, and the like can be increased.

  The linker region can be prepared following the preparation of the peptide region, or the peptide region can be prepared after the preparation of the linker region. For example, it can be configured to sequentially synthesize using an automatic synthesizer such as a peptide synthesizer. On the other hand, a separately prepared linker region and peptide region may be linked. In that case, it can link through covalent bond, physical adsorption, ionic bond, etc. For example, what is necessary is just to link the reactive functional group of a linker area | region, and the reactive functional group of a peptide area | region. An appropriate spacer may be interposed at the time of connection. For example, since an amino group or a carbonyl group is used as a reactive functional group, an amino acid that does not participate in binding to a test substance may be added and used for linking. Further, a crosslinking method in which crosslinking is fixed with a polyfunctional crosslinking reagent having a divalent or higher functional group can also be used. As the crosslinking reagent, for example, glutaraldehyde, diisocyanate compound, bisdiazobenzidine and the like can be used. Then, the interaction of in vivo substances such as biotin-avidin bond can be used. The spacer is preferably selected so as not to change the charge characteristics of the linker region and the solid support surface.

  When the linker region is configured as an uncharged molecular chain, the linker region can be used without any particular limitation as long as the linker region has no charge when immobilized on the surface of the solid phase carrier. Here, uncharged means not charged with electricity. That is, it is a state in which it is electrically neutral, and refers to a state in which ionization, that is, transfer of electrons is not performed. For example, it can be configured as a hydrocarbon chain such as a chain hydrocarbon chain, an alicyclic hydrocarbon chain, or an aromatic hydrocarbon chain. Specifically, an ethylene chain such as an alkyl chain, an alkenyl chain, an alkyldienyl chain, and a polyenyl chain, an acetylene hydrocarbon chain such as an alkynyl chain, an aryl chain such as a cycloalkyl chain, a phenyl chain, and a naphthyl chain, An arylalkyl chain etc. can be mentioned. Further, it can be configured as a peptide nucleic acid (hereinafter referred to as “PNA”) chain, but is not limited thereto. In particular, an alkyl chain and a PNA chain are preferable.

  When the linker region is configured as a hydrocarbon chain such as an alkyl chain, the molecular length is preferably 4 to 21, more preferably 7 to 21, or 8 to 21, and particularly preferably 16 in terms of carbon number. The alkyl chain can be produced by a method known in the art, and can be synthesized by, for example, hydrogenation of an alkene or hydrolysis of a Grignard reagent.

  A PNA chain is a molecular chain having a nucleobase in the peptide backbone. Specifically, N- (2-aminoethyl) glycine linked by peptide bonds is used as the backbone unit, and this is linked to the nucleic acid via a methylenecarbonyl group. It is a general term for molecular chains having a structure in which bases are bound. The PNA chain does not have a charge due to the phosphate group found in DNA and RNA. The molecular length is the number of N- (2-aminoethyl) glycine, preferably 4 to 21, more preferably 7 to 21, or 8 to 21, and particularly preferably 16. The PNA chain can be produced by a method known in the art, and for example, can be synthesized using the Fmoc method or the tBoc method in the same manner as peptide synthesis.

  When the linker region is configured as an amphiphilic molecular chain, the linker region can be used without particular limitation as long as it has amphipathic properties. Here, the amphiphilic molecular chain means one having a hydrophilic group and a hydrophobic group therein. A hydrophilic group means an atomic group in a molecule that easily forms a hydrogen bond with a water molecule. Generally, a group containing an atom such as oxygen, nitrogen, or sulfur, specifically, a hydroxyl group , Carboxyl group, amino group, sulfo group and the like. The hydrophobic group means an atomic group having a low affinity for water, and examples thereof include the above-described hydrocarbon groups. Examples of the amphiphilic molecule include alcohols in which one or several hydrogen atoms of the above-mentioned hydrocarbons are substituted with hydroxy groups, fatty acids in which one or several hydrogen atoms of the above-mentioned hydrocarbons are substituted with carbonyl groups, ammonia Or an amine in which one or several hydrogen atoms are substituted with the above-mentioned hydrocarbons, or a plurality of them may be polymerized. In particular, polyethylene glycol obtained by polymerizing a plurality of ethylene glycols, which are divalent alcohols (hereinafter referred to as “PEG”), is preferable. Further, it may be a nucleic acid chain which is a biopolymer in which nucleotides composed of a base, sugar and phosphate are linked by a phosphate ester bond. Nucleic acid chains are roughly classified into DNA chains and RNA chains depending on the type of sugar, and any of them can be used as a linker region, but DNA chains are preferred.

The PEG chain is a polymer compound having a structure in which ethylene glycol is polymerized. Here, the molecular length of the PEG chain means the number of polymerized ethylene glycol constituting the PEG chain. That is, the number of n in the structural formula (—CH ₂ —CH ₂ —O—) n of polyethylene glycol is the molecular length, and the molecular length is preferably 4 to 21, and 7 to 21 or 8 to 21. More preferably, 16 is particularly preferable. The PFG chain can be produced by a method known in the art, and can be synthesized, for example, by polymerizing ethylene oxide in the presence of an ion catalyst.

  In the DNA chain, nucleotide residues composed of 2-deoxyribose, purine or pyrimidine base, and ester-linked phosphate groups are 3 ', 5'-phosphodiester linked, and sugars and phosphate groups are linked alternately Polydeoxyribonucleotide. The bases contained in the DNA strand are adenine, guanine, cytosine, and thymine, but there is no restriction on the base. Further, it may contain a modified base subjected to modification such as methylation. The number of DNA strands is not particularly limited, and may be a single strand or multiple strands such as a double strand and a triple strand, but is preferably a single strand. The molecular length of the DNA chain is preferably 4 to 21, more preferably 7 to 21, or 8 to 21, particularly preferably 16, with the number of bases as the molecular length. The DNA can be produced by a method known in the art, and can be synthesized, for example, using a chemical synthesis method based on the phosphoramidite method or the like.

  The peptide probe used in the method of the present invention should be suitably used for an analysis system using a solid phase carrier as a reaction field, such as surface plasmon resonance (hereinafter may be abbreviated as “SPR”) or magnetic sensing. Can do.

  Here, SPR is a technique for analyzing molecular interactions using surface plasmons, and surface plasmons are special electromagnetic waves that propagate along the surface of a metal. Specifically, a peptide probe is fixed to a metal thin film side on a solid phase carrier, which is formed by sequentially laminating a metal thin film on one side of a transparent substrate and, if necessary, a thin film for immobilizing a peptide probe. Turn into. Then, the test sample is brought into contact. When the test substance is contained in the test sample, the peptide probe and the test substance are combined, and the apparent molecular weight changes. Due to this change in molecular weight, a change occurs in the surface plasmon resonance, which is detected as an optical change called a change in the refractive index of the reflected light. The test substance is analyzed based on this optical change. Here, the contact between the test sample and the solid phase allows the test sample to be continuously supplied by a continuous liquid feeding method.

  The principle of surface plasmon resonance will be described in detail. When light is incident at a total reflection angle or more from a light source, very little evanescent wave is generated at the interface between the transparent substrate and the metal thin film when the light is reflected. On the other hand, surface plasmons can also be generated in the metal thin film. Resonance occurs when the speed at which the evanescent wave is transmitted coincides with the speed at which the surface plasmon wave that can be generated is transmitted, the surface plasmon is induced, and the light energy is changed to the energy of the surface plasmon wave. As described above, since a part of the light energy is used for excitation of the surface plasmon, an apparatus using the change in the light energy that returns as reflected light is used. That is, a metal is vapor-deposited on the transparent substrate surface to produce a thin film, and the peptide probe of the present invention is immobilized. A test sample is flowed there, and the test substance and the peptide probe are brought into contact with each other. The metal thin film is irradiated with light of a certain wavelength before and after contact, and the time change of reflected light is observed. The reflected light is affected by the density of the material near the metal thin film due to the surface plasmon phenomenon. Therefore, the surface density increases due to the binding of the test substance. By detecting this, the test substance is detected. In addition, photochemical changes can be detected over time, and binding rate constants and dissociation rate constants can be calculated from analysis of binding and dissociation processes.

  The solid phase carrier is configured by sequentially laminating a metal thin film and, if necessary, a thin film for immobilizing a peptide on a transparent substrate. The shape of the solid phase is not particularly limited as long as it is a shape suitable for SPR, and may be a three-dimensional shape such as a flat plate shape, a spherical shape, or a cube. Any shape can be used. Preferably, it forms as a sheet form or plate shape. Moreover, the thickness of each layer can also be adjusted suitably.

  Any transparent insoluble material can be used as the transparent substrate, and it may be glass, silicon, or a synthetic polymer such as polycarbonate, cycloolefin polymer, polymethyl methacrylate, polyethylene terephthalate.

  As the metal thin film, gold, silver, copper, platinum, aluminum, or the like can be used, and these may be used alone or in combination. From the viewpoint of durability such as oxidation, gold is preferable. The formation of the metal thin film may be performed using any known method. For example, the metal thin film is formed on the substrate by sputtering, vapor deposition, electroplating, electroless plating, or the like. Can do.

  And it is preferable to optimize suitably the charge characteristic of the surface of a solid-phase carrier according to the structure of a linker area | region. When the linker region is uncharged, it is configured to be positively or negatively charged, and when the linker region is amphiphilic, it is configured to be uncharged. Therefore, when laminating a thin film for immobilizing a peptide probe or performing a surface treatment, it is necessary to consider the charge characteristics.

  Here, the surface of the solid phase carrier charged positively or negatively means that the surface of the solid phase carrier is charged with electricity. Positively charged refers to a state having a positive charge, and negatively charged refers to a state having a negative charge. Uncharged means that the surface of the solid support is not charged. Therefore, it is a state where the surface of the solid phase carrier is electrically neutral, and refers to a state where ionization, that is, transfer of electrons is not performed.

  When a thin film for immobilizing a peptide probe is laminated, as described above, the thin film affects the charge characteristics on the surface of the solid phase carrier, and therefore, it is made of a material having desired charge characteristics. When the thin film is uncharged, it can be composed of any insoluble material having uncharged characteristics. Examples include polysaccharides in which neutral sugars such as dextran and cellulose are polypolymerized, synthetic polymer polymers such as polystyrene, polyethylene, polypropylene, polycarbonate, and polyethylene terephthalate, and combinations thereof, but are not limited thereto. Is not to be done. When the thin film is positively or negatively charged, it can be composed of any insoluble material having positive or negative charging characteristics. Therefore, specifically, those containing functional groups such as a carboxyl group, an amino group, a sulfone group, and a phosphate group are exemplified. Accordingly, a material obtained by introducing a functional group such as a carboxyl group, an amino group, a sulfone group, or a phosphoric acid group into a part of the above-mentioned uncharged material can be suitably used. Examples include polysaccharides such as carrageenan, dextran sulfate, xanthan gum, carboxymethyl cellulose, synthetic polymers such as polyacrylamide, polystyrene sulfonic acid, polyamide, etc., and combinations thereof, but are not limited thereto. Absent.

  The thin film for immobilizing the peptide probe may be configured as a self-assembled monomolecular film. A self-assembled monolayer is a monolayer formed by spontaneously binding, accumulating and regularly organizing organic molecules on the surface of a solid support such as a metal surface such as gold, platinum, silver or copper. . As organic molecules forming such a self-assembled monolayer, organic molecules containing a thiol group, a disulfide group, a selenol group, an isocyanide group, and the like are known, and alkanethiol is particularly widely used. In order to facilitate the immobilization of peptide probes, self-assembled monolayers into which various functional groups are introduced are known, and they can be suitably used.

  The peptide probe is immobilized on the solid phase carrier surface by immobilizing the linker region on the solid phase carrier surface, as long as it does not affect the specific binding properties and charge characteristics of the peptide probe to the test substance. Any known method can be used. For example, it is possible to use a carrier binding method that is immobilized through covalent bonding, physical adsorption, or ionic bonding, but the covalent bonding method is preferable. Examples of the covalent bond method include, but are not limited to, a diazo method, an acid azide method, an isocyanate method, a bromocyan method, a direct condensation method of a carbonyl group and an amino group, and a disulfide bond method. And it is preferable to fix | immobilize using reactive functional groups, such as an amino group, a carboxyl group, a hydroxyl group, a sulfone group, a phosphoric acid group, a thiol group, an imidazole group of a linker area | region and a solid-phase carrier surface. In addition, these functional groups can be activated during immobilization, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (WSC) and N-hydroxysuccinimide (NHS). The group may be activated.

  An appropriate spacer may be interposed upon immobilization. For example, since an amino group, a carbonyl group, or the like is used as a reactive functional group, an amino acid that does not participate in binding to the test substance may be added and used for the connection. Further, a crosslinking method in which crosslinking is fixed with a polyfunctional crosslinking reagent having a divalent or higher functional group can also be used. As the crosslinking reagent, for example, glutaraldehyde, diisocyanate compound, bisdiazobenzidine and the like can be used. Then, the interaction of in vivo substances such as biotin-avidin bond can be used. The spacer is selected so as not to change the charge characteristics of the linker region and the solid support. Furthermore, it is also possible to use a polymer having a network structure such as a polysaccharide, a conductive polymer, a redox polymer, a photocrosslinkable polymer, a comprehensive method in which it is sealed in a semipermeable membrane such as a dialysis membrane, etc. it can. Moreover, you may use combining these.

  Various known SPR devices can be used. For example, a device using a Kretschmann mechanism can be used. A commercially available product can be used as the SPR device, for example, Biacore 1000 or the like. The structure of the SPR device will be described with an example. A solid support on which a metal thin film and a thin film capable of immobilizing a peptide are sequentially arranged on a transparent substrate, and a side of the transparent substrate on which the metal thin film is not formed are described. Measures the intensity of the light reflected at the interface between the transparent substrate and the metal thin film, and the light source capable of entering light at various angles with respect to the arranged prism, the interface between the transparent substrate and the metal thin film And a photodetector.

  Magnetic sensing is a technique for analyzing the interaction of substances by detecting the magnitude and direction of magnetism and geomagnetism generated by magnetic substances and currents. For example, a magnetic sensor or the like can be used. Specifically, a peptide probe is immobilized on a solid support. When the test sample is brought into contact, if the test substance is contained in the test sample, the peptide region of the peptide probe and the test substance are combined. Then, the test substance is analyzed by detecting the magnetic signal intensity from the test substance. The detection of the magnetic signal intensity from the test substance can be performed, for example, by labeling the test substance with magnetic particles, and a substance having specific binding property to the test substance, such as a peptide or an antibody, can be detected. It can be carried out by labeling with magnetic particles.

Here, the magnetic particle means a material containing a magnetic substance magnetized in a magnetic field and formed as a particle. Magnetic materials are classified into ferromagnetic materials, diamagnetic materials, paramagnetic materials, superparamagnetic materials, ferrimagnetic materials, and the like. Any one that can detect a magnetic signal and does not inhibit the specific binding property between the peptide probe and the test substance, the charge characteristics of the peptide probe and the solid phase carrier, and the functions of other measurement reagents can be used. For example, ferromagnetic metals such as iron, cobalt, nickel and gadolinium, alloys of the above ferromagnetic metals such as iron-nickel and iron-cobalt, alloys containing ferromagnetic metals, magnetite (Fe ₃ O ₄ ), maghemite ( Examples thereof include ferromagnetic metal oxides represented by various ferrites such as γ-Fe ₂ O ₃ ) and intermediates thereof. In particular, ferrites are preferably used from the viewpoint of chemical stability and reactivity to a magnetic field, and ferrites added with metals such as cobalt, manganese, nickel, and zinc can also be suitably used.

  There are no particular restrictions on the shape and size of the magnetic particles, as long as they do not interfere with the specific binding properties of the peptide probe and the test substance, the charge characteristics of the peptide probe and the solid phase carrier, and the functions of other measurement reagents. A suitable shape and size is selected. Examples of the shape include a spherical shape, an ellipsoidal shape, a granular shape, a cubic shape, a columnar shape, a rod shape, a plate shape, a needle shape, a fiber shape, and a lump shape, and the shape is preferably formed into a uniform shape. The magnetic particles are preferably configured as nanoparticles or microparticles, and particularly preferably magnetic nanoparticles. As a specific average core particle size, for example, 1 nm to 3 μm is preferable, 10 nm to 150 nm is more preferable, and 15 nm to 30 nm is particularly preferable.

  The magnetic particles may be composed of only a magnetic substance, or may be configured such that the magnetic substance is distributed on the whole or the surface of the particle, or may be enclosed inside the particle. For example, a polymer or the like in which a magnetic substance is uniformly distributed may be formed into particles, and can be prepared by coating a polymer or the like with a core in which the magnetic substance is uniformly distributed. . The polymer can be appropriately selected according to the measurement mode regardless of whether the polymer is a hydrophilic polymer or a hydrophobic polymer. Specifically, high molecular polymers such as polystyrene, silica gel, gelatin, and polyacrylamide can be preferably used. Moreover, you may comprise so that functional groups may be expressed by introduce | transducing functional groups, such as an amino group, a carboxyl group, a tosyl group, and a hydroxyl group, on the particle | grain surface. In preparation, it is preferable to prepare so that the magnetization per particle is uniform.

  For labeling with magnetic particles, any known labeling technique can be used as long as the stability and functionality of the substance having specific binding properties to the magnetic particles and the analyte to be labeled can be maintained. For example, a physical adsorption method and a chemical bonding method are exemplified. The physical adsorption method can be performed by bringing the magnetic particles and the trapping substance into contact with each other in a water-soluble solvent such as water, physiological saline, or various buffer solutions. Examples of the chemical bonding method include labeling by formation of a covalent bond by a diazo method, an acid azide method, an isocyanate method, a bromocyan method, or the like. Further, a method of immobilizing using a cross-linking reagent such as a multifunctional reagent having two or more functional groups such as glutaraldehyde can also be used. Then, as described above, the functional group on the capture substance is chemically bonded to the functional group such as amino group, carboxyl group, tosyl group, hydroxyl group and the like introduced so as to express the functionality on the surface of the magnetic particle. It can also be made. In addition, the magnetic particles and the capture substance can be chemically bonded via a reactive side chain (spacer) having an appropriate chain length, and a protein such as albumin or protein A. In particular, a biotin-avidin (streptavidin) binding method having strong affinity and specificity can be used.

  Magnetic signals from the magnetic particles are detected by magnetic detection means such as a magnetic sensor. The magnetic signal intensity is proportional to the amount of the test substance. Accordingly, it is preferred that the magnetic detection means can be configured to generate an electrical signal in response to magnetism from the magnetic particles.

  The solid phase carrier that serves as a reaction field between the test substance and the peptide probe and serves as a magnetic signal detection surface is not particularly limited in shape, and may be a three-dimensional shape such as a flat plate shape, a spherical shape, or a cube. Good. Preferably, it is configured as a flat plate shape laminated in a plane. Preferably it is configured as an array. For example, it is preferable to form a plurality of wells, compartments, chambers, channels and the like on the surface of the solid phase carrier.

  And the material which comprises a solid-phase carrier can be manufactured with synthetic compounds, such as inorganic compounds, such as a silicon | silicone, a silica, and a titanium oxide, acrylamide, a polystyrene, and a polycarbonate. And antiferromagnetic layer mainly composed of PtMn, NiMn, IrMn, PdMn, PdPtMn, RhMn, etc., mainly Ta, Ru, Cr, Rh, Ir, Au, Ag, Cu, Zr, Pt, Mo, W, etc. A nonmagnetic layer as a component, a ferromagnetic layer mainly composed of NiFe, Co, CoFe, or the like, a seed layer, an insulator layer, or the like may be laminated, and can be appropriately selected according to the detection method of the magnetic signal. And it is preferable to optimize suitably the charge characteristic of the surface of a solid-phase carrier according to the structure of a linker area | region. When the linker region is uncharged, it is configured to be positively or negatively charged, and when the linker region is amphiphilic, it is configured to be uncharged. In particular, when a thin film for immobilizing a peptide is laminated or when a surface treatment is performed, it is necessary to consider the charge characteristics. About this structure, it can carry out similarly to the case of SPR.

  The peptide probe can be immobilized on the surface of the solid phase carrier according to the immobilization method in SPR. Also, during the immobilization, the surface of the solid phase carrier can be immobilized as necessary to suppress nonspecific binding. It is also possible to perform processing such as blocking.

  Magnetic detection is preferably performed by a magnetic sensor, and the magnetic sensor is not limited as long as it can detect magnetic signals from magnetic particles on the sensor surface. For example, a Hall element, magnetic impedance (magneto-impedance) : MI) elements, magnetoresistive elements such as magnetoresistive (MR) elements and giant magnetoresistive (GMR) elements, tunnel magnetoresistive (TMR) elements, superconducting quantum interference elements (TMR) elements Superconductivity Quantum Interface Device (SQUID) or the like can be used. As a measurement method, any known technique such as magnetic susceptibility measurement or magnetic relaxation measurement can be used. In particular, it is a GMR element that is a magnetoresistive element, and uses a GMR sensor as described in SX Wang et al., Biosensors and Bioelectronics, 2010, Vol. 25, No. 9, pages 2051 to 2057, etc. It is preferable. For example, it can be configured to detect the magnetization of the magnetic particle by utilizing the change in the resistance value of the magnetic sensor due to the change in the magnetic field. The presence of an external magnetic field magnetizes the magnetic particles on the sensor, and the magnetized magnetic particles induce a change in the resistance of the magnetic sensor. The presence of magnetic particles can be detected by detecting the resistance change amount of the magnetic sensor.

  As described above, the peptide probe of the present invention uses a synthetic peptide as a detection site for a test substance. Therefore, it is possible to solve the problems of conventional biologically active substances such as proteins and in vivo substance probes such as nucleic acids, such as changes in the external environment and changes in activity and decrease in activity over time. That is, the measurement results are not affected by changes in the external environment, and long-term storage stability can be maintained. As a result, it is possible to contribute to the construction of a measurement system that meets the requirements of the clinical site, such as quantitative analysis such as determining disease symptoms and the progress of healing in the clinical diagnosis field. The peptide probe is immobilized on the surface of the solid phase carrier via a linker region, and is configured by optimizing the molecular length of the linker region. Since the distance between the peptide region, which is a site for recognizing the test substance, and the surface of the solid phase carrier greatly affects the measurement sensitivity, it is important to optimize the length of the linker region. In other words, in the case of a solid-phase reaction system, the distance between the solid-phase carrier surface and the site for recognizing the test substance affects the measured signal intensity, and the sensitivity can be improved by optimizing the distance. Construction of a highly sensitive and highly accurate measurement system can be realized. In addition, since the peptide probe of the present invention can be chemically synthesized, there is no activity error between producers and lots like antibodies, and no impurities are mixed as a by-product during production. Therefore, background noise that hinders measurement of a test substance can be reduced, and can be used to construct a measurement system with high sensitivity, high accuracy, and high stability. In addition, the peptide probe of the present invention can be produced at low cost using a normal peptide synthesizer, etc., and has a long-term storage stability as described above, enabling mass preparation and contributing to cost reduction of the measurement system. be able to.

  The peptide probe of the present invention can be used in a method for detecting a test substance contained in a test sample, and the method for measuring the test substance also forms part of the present invention.

  Specifically, the method for measuring a test substance of the present invention is a method for detecting a test substance by the above-described surface plasmon resonance or magnetic sensing, and (1) a reaction field for surface plasmon resonance or magnetic sensing. A step of immobilizing the peptide probe of the present invention on a solid phase carrier by the linker region, (2) a step of bringing the test substance into contact with the peptide probe immobilized on the solid phase carrier, and (3) the above The method includes a step of detecting a test substance bound to the peptide region of the peptide probe by surface plasmon resonance or magnetic sensing. Each step can be performed as described above.

  Here, the test sample containing the test substance applied to the method of the present invention is not particularly limited as long as it is a sample that may contain the test substance to be measured. For example, animal-derived blood, plasma, serum, cerebrospinal fluid, amniotic fluid, milk, sweat, urine, saliva, sputum, feces, tissue, cell culture, and other biological samples, plant roots, stems, leaves, flowers, Examples include plant-derived samples such as fruits, environmental samples such as soil, groundwater, river water, and lake water, and foods such as vegetables, meat, eggs, and processed foods. These samples can be used as measurement targets as they are, but pretreatments such as separation, extraction, and purification of a test substance may be performed. Therefore, the peptide probe of the present invention and the method for measuring a test substance using the peptide probe can be used in various technical fields, particularly in the medical field such as clinical diagnosis, the food field, and the environmental field.

  As described above, the method for measuring a test substance of the present invention uses the peptide probe of the present invention having a synthetic peptide as a detection site for a test substance, thereby changing the external environment, fluctuation of activity over time, A test substance can be measured without being affected by a decrease in activity or the like. In addition, since the peptide probe of the present invention can maintain long-term storage stability, there is a requirement in the clinical field such as quantitative analysis such as determining disease symptoms and the progress of healing in the clinical diagnosis field. Can respond. Since the peptide probe of the present invention optimizes the molecular length of the linker region, the distance between the surface of the solid support and the site for recognizing the test substance is optimized, and the target is highly sensitive and accurate. The test substance can be measured. Furthermore, since the linker region of the peptide probe of the present invention has an optimized charge characteristic corresponding to the charge characteristic of the surface of the solid phase carrier, it can be expected to improve measurement sensitivity. The test substance can be measured with high accuracy. And since the peptide probe of the present invention can be chemically synthesized, there is no activity error between producers and lots like antibodies, and impurities are not mixed as a by-product during production. Background noise that hinders measurement of the test substance can be reduced, and the test substance can be measured with high sensitivity, high accuracy, and stability. In addition, the peptide probe of the present invention is advantageous in terms of cost because it can be produced at low cost using a normal peptide synthesizer or the like and has long-term storage stability.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, it should be clearly understood that the present invention is not limited to the following examples, and various embodiments are possible.

Example 1. Effect of linker region molecular length on reaction activity (SPR)
In this example, the effect of the molecular length of the linker region of the peptide probe of the present invention on the reaction activity was examined by comparing the strength of SPR.

(reagent)
A peptide probe having the following peptide region and linker region was synthesized by a peptide synthesizer and used.
For the peptide region, nm23-binding synthetic peptide, which is a cancer metastasis marker, was used. The sequence information of the nm23-binding synthetic peptide is as follows.
nm23-binding synthetic peptide: Arg-Arg-Leu-Thr-Ile-Arg-Asn-Gly (SEQ ID NO: 1)
As the linker region, 5, 8, 12, 16, 21, 25 molecular length PEG chains were used. As for the molecular length of the PEG chain, the number of n in the structural formula (—CH ₂ —CH ₂ —O—) n of the PEG chain was defined as the molecular length.

(Method)
A peptide probe in which the nm23-bonded synthetic peptide and each molecular length of PEG were linked was immobilized on a gold thin film of an SPR solid phase carrier. Upon immobilization, the gold thin film was treated with 1-mercaptoundecanoic acid and 1-mercaptododecanol (1:10) to form a self-assembled film on the gold thin film, and a water-soluble carbodiimide / PEG-attached peptide (10: 1 ) Immobilized by adding the mixture. Subsequently, the binding rate constant of each peptide probe was determined by SPR. Biacore 1000 (Biacore) was used as the SPR device, and PBS was used as the developing solution. As a test sample, nm23 / PBS was added at a flow rate of 200 μg / ml and measured at 25 ° C.

(result)
The results are shown in Table 1. Table 1 shows the relative activity value at each molecular length, assuming that the activity when the molecular length of the linker region is 16 is 100%.

As a result of Table 1, it was confirmed that the peptide probe of the present invention showed high reactivity when the molecular length of the linker region was 8-21. In the case of a solid-phase reaction system, the distance between the surface of the solid-phase carrier and the site that recognizes the test substance affects the measured signal intensity, and the measurement sensitivity and accuracy can be improved by optimizing the distance. Can understand.
However, since the reactivity is affected by the measurement system, the molecular length of the linker region may be optimized according to the measurement system actually used.

Example 2 Evaluation of Heat Resistance and Storage Stability of Peptide Probe In this example, the heat resistance and storage stability of the peptide probe of the present invention were evaluated by comparison with general antibodies. This example was also evaluated by SPR as in Example 1.

(reagent)
A peptide probe having the following peptide region and linker region was used.
As a peptide region, a nm23-binding synthetic peptide (SEQ ID NO: 1), which is a cancer metastasis marker, was used, and a 6-molecule long PEG chain was used as a linker region.
As a comparative example, a general antibody against nm23, nm23-H1 Antibody (NM301) (product number sc-465, manufactured by Santa Cruz) monoclonal antibody was used. Here, a general antibody is a protein antibody produced by the immune response of an animal.

(Method)
The peptide probe in which the above-mentioned nm23-bonded synthetic peptide and PEG chain were linked was immobilized on a gold film of an SPR solid phase carrier. Connection and immobilization conditions and the like were performed according to the procedure of Example 1. A comparative monoclonal antibody was also immobilized on a thin gold film (Comparative Example). Then, in order to evaluate heat resistance and storage stability, after standing at 25 ° C. and 50 ° C. for 0 hour, 1 day, 1 week, 2 weeks and 1 month, the binding rate constant was determined by SPR. The SPR device and measurement conditions were performed in accordance with the procedure of Example 1.

(result)
The results are shown in Table 2. Table 2 shows the relative activity values of the antibody and the peptide probe of the present invention under each condition, assuming that the antibody activity at 25 ° C. for 0 hour is 100%.

  From the results in Table 2, the peptide probe of the present invention is inferior to the signal intensity of the antibody only at the initial peak at 25 ° C. for 0 hour, but can maintain the activity under other conditions and realize a stable measurement system. I can understand. In particular, the antibody activity was remarkably reduced at 50 ° C., but no activity fluctuation was observed with the peptide probe. That is, it can be understood that the peptide probe of the present invention can maintain long-term storage stability without being affected by changes in the external environment.

Example 3 Effect of linker region molecular length on reaction activity (magnetic sensing)
In this example, the influence of the molecular length of the linker region of the peptide probe of the present invention on the reaction activity was examined by comparing the magnetic intensity by magnetic sensing.

(reagent)
A peptide probe having the following peptide region and linker region was synthesized by a peptide synthesizer and used.
As a peptide region, a cancer metastasis marker nm23-binding synthetic peptide (SEQ ID NO: 1) was used.
As the linker region, 0, 4, 7, 11, 16, 21, and 25 molecular length PEG chains and 0, 4, 7, 11, 16, 21, and 25 molecular length alkyl chains were used, respectively. In the PEG chain, the number of n in the structural formula (—CH ₂ —CH ₂ —O—) n of the PEG chain was defined as the molecular length, and the alkyl chain was defined as the molecular length of carbon.

(Method)
The magnetic strength was measured using a GMR sensor as the magnetic sensor. Here, the GMR sensor used was constructed based on the description of SX Wang et al., Biosensors and Bioelectronics, 2010, Vol. 25, No. 9, pp. 2051-2057.

First, after cleaning the surface of the GMR sensor with alcohol, the impurities were completely decomposed by plasma irradiation (1000 W, 1 minute). Subsequently, the Si surface of the sensor surface was chemically modified to an amino group by immersing the sensor surface in 2% APTES / ethanol at 68 ° C. After the GMR sensor was sufficiently dried, a peptide probe in which the above-mentioned nm23-bonded synthetic peptide and PEG chain or alkyl chain were linked was applied to the GMR sensor and fixed. Specifically, the size of one GMR sensor was 100 μm × 100 μm, and 10 ¹¹ to ¹² peptides were immobilized thereon. And 80 sensors are mounted on one chip, and the above-mentioned different linker types are fixed at four locations. Subsequently, at room temperature of 25 ° C., 150 μl of 10 ng / ml nm23 antigen / PBS was added and reacted for 2 hours, followed by washing (0.1% BSA, 0.05% Tween20 / PBS), and 1 μg / ml biotin-labeled anti-antibody. 150 μl of nm23 antibody / PBS was added and reacted for another 1 hour. After washing, 100 μL of streptavidin-labeled magnetic nanoparticle solution was added and reacted, and the intensity of the magnetic signal was measured 15 minutes after the reaction.

(result)
The results are shown in Table 3. Table 3 shows the magnetic signal intensities of the peptide probes of each linker type and each molecular length, and is an average value for four places. The signal intensity was calculated according to the following formula.

[Equation 1]
Magnetic signal strength (ppm) ≒ △ Changed magnetic resistance (Ω) / Multiplied magnetic resistance (Ω)
Note: Since the changing magnetic resistance is about μΩ and the magnetic resistance applied to the sensor is about 2 kΩ, the unit of magnetic signal intensity is “ppm”.

  From the results in Table 3, it can be understood that the alkyl chain has a slightly higher reaction efficiency than PEG. Since the GMR sensor surface, which is a solid phase carrier, is aminated and the solid phase carrier surface is positively charged, it is appropriate to select an uncharged alkyl chain as the linker region. Is guided. Moreover, it was confirmed that the peptide probe of the present invention showed high reactivity when the molecular length of the linker region was 4-21. In the case of magnetic sensing using a GMR sensor, the closer the distance from the sensor surface to the magnetic nanoparticles, the higher the sensitivity, and even if the molecular length of the linker region is shorter than the SPR of Examples 1 and 2. It seemed to be good, and the result of this example was consistent with it.

  The peptide probe of the present invention and the method for measuring a test substance using the peptide probe can be used in various technical fields where measurement of the test substance is required, particularly in the medical field such as clinical diagnosis, It can be used in the food and environmental fields.

Claims (11)

A peptide probe for surface plasmon resonance or magnetic sensing measurement,
A peptide region comprising at least one test substance binding site exhibiting specific binding ability to the test substance;
A linker region for immobilizing the peptide region on the surface of a solid phase carrier serving as a reaction field of the surface plasmon resonance or magnetic sensing, wherein the linker region is composed of a molecular chain having a molecular length of 4 to 21 Peptide probe.   The peptide probe according to claim 1, wherein the linker region is composed of a molecular chain having a molecular length of 8 to 21.   The peptide probe according to claim 1 or 2, wherein when the surface of the solid phase carrier is positively or negatively charged, the linker region is composed of an uncharged molecular chain.   The peptide probe according to claim 3, wherein the linker region is an alkyl chain or a peptide nucleic acid chain.   The peptide probe according to claim 1 or 2, wherein the linker region is composed of an amphiphilic molecular chain when the surface of the solid phase carrier is in an uncharged state.   The peptide probe according to claim 5, wherein the linker region is a polyethylene glycol chain or a nucleic acid chain.   The peptide probe according to any one of claims 1 to 6, wherein the peptide region is composed of 4 to 11 amino acids.   The peptide probe according to claim 7, wherein the peptide region is composed of 8 amino acids.   The peptide probe according to any one of claims 1 to 8, wherein the peptide region is a peptide chain comprising the amino acid sequence shown in SEQ ID NO: 1. A method for measuring a test substance by surface plasmon resonance or magnetic sensing,
A step of immobilizing the peptide probe according to any one of claims 1 to 9 via the linker region on the surface of a solid phase carrier serving as a reaction field of the surface plasmon resonance or magnetic sensing;
Contacting the test substance with the peptide probe immobilized on the surface of the solid phase carrier; and detecting the test substance bound to the peptide region of the peptide probe by the surface plasmon resonance or magnetic sensing. A method for measuring a test substance comprising a step.   The method for measuring a test substance according to claim 10, wherein the peptide region of the peptide probe is a peptide chain comprising the amino acid sequence represented by SEQ ID NO: 1.