WO2016175049A1 - Novel method for detecting protein or pathogen - Google Patents

Abstract

Provided is a novel method for detecting a protein or a pathogen. The present invention pertains to a conductive diamond electrode wherein an element recognizing a protein or a pathogen is immobilized on the surface, and a method for detecting a protein or a pathogen using the conductive diamond electrode. More specifically, the present invention pertains to a diamond electrode wherein an element recognizing a virus or a protein thereof is immobilized on the surface, and a method for detecting a virus using the diamond electrode.

Description

Novel detection method of protein or pathogen

The present invention relates to a conductive diamond electrode having an element for recognizing a protein or pathogen immobilized on the surface, and a protein or pathogen detection method using the same. More specifically, the present invention relates to a conductive diamond electrode having an element recognizing a virus or a protein thereof immobilized on the surface, and a virus detection method using the same.

Conventionally, it has been desired to detect target pathogens, pathogenic bacteria, viruses and their proteins with high sensitivity. For example, influenza virus (IFV) may be a pandemic, and rapid and accurate detection of influenza virus is required. Currently used IFV detection methods include immunochromatography using antibodies as IFV recognition devices, detection by sugar chain arrays using sugar chains, RT-PCR using genes, and hemagglutination assays using red blood cells. and so on. However, these are time consuming and costly and require specialized knowledge and skills.

Grabowska et al. Reported an IFV detection method using hybridization (Non-patent Document 1). This method uses a sensor in which two different oligonucleotide probes are immobilized on the surface of the Au electrode via gold thiol bonds, thereby simultaneously detecting both hemagglutinin (HA) and neuraminidase (NA) oligonucleotide targets. Is possible. Kamikawa et al. Reported IFV detection using magnetic nanoparticles covered with electroactive polyaniline and modified with H5N1-type HA monoclonal antibody on the surface (Non-patent Document 2). In this method, nanoparticles interacting with H5N1 virus are collected from serum with a strong magnetic force (MPC), and the amount of virus binding is quantified by electrochemical measurement.

Sakurai et al. Reported a rapid and highly sensitive type classification of seasonal influenza using fluorescent immunochromatography (Non-patent Document 3). The immunochromatography method, which is a method for detecting IFV used in clinical practice, has a detection sensitivity of 1000 pfu and is difficult to detect at the early stage of infection. In the same document, Sakurai et al. Captured viruses in a sandwich type using both a virus supplement antibody and a detection antibody labeled with a sensitizer, and further performed detection using a densitometric analyzer and a fluorescence immunochromatography analyzer. It is reported that the improvement of the sensitivity 100 times that of the prior art has been achieved. However, complementary oligonucleotides for hybridization and antibodies to be immobilized on a substrate require enormous labor and cost for production, and have storage stability problems.

Hassen et al. Have reported the quantification of influenza A virus using electrochemical impedance spectroscopy (Non-patent Document 4). In this document, influenza A virus is detected using a device in which an antibody-sugar chain-removed avidin-thiol structure is immobilized on the gold electrode surface.

On the other hand, boron-doped diamond electrodes have excellent characteristics as compared with other conventional electrode materials such as glassy carbon and platinum electrodes, and have attracted attention in recent years. In addition to the well-known properties of diamond, which have high thermal conductivity and extremely high hardness, boron-doped diamond electrodes have a wide potential window, small background current, high adsorption resistance, and are chemically inert. Has attractive properties. Boron-doped diamond electrodes are physically and chemically stable and have excellent durability.

As a sensor equipped with a diamond electrode, a diamond electrode capable of accurately quantifying catechol or a catechol derivative and a sensor equipped with the diamond electrode have been reported (Patent Document 1). In this document, oxalic acid is electrochemically detected using a 4-pentenoic acid-modified diamond electrode.

A highly sensitive detection method is desired for not only viruses but also various pathogenic bacteria and pathogens without using antibodies or expensive equipment.

JP2007-292717 (Patent No. 4978858)

Grabowska et al., Anal. Chem., 85, 10167-10173 (2013) Kamikawa et al., Biosens Bioelectron, 26, 1346-1352 (2010) Sakurai et al., PLOS ONE DOI: 10.1371 / jounal.pone.0116715, 1-13 (2015) Hassen et al., Electrochim. Acta 56, 8325-8328 (2011)

The object of the present invention is to provide an apparatus and method for detecting proteins or pathogens quickly and with high sensitivity, which solves the conventional problems.

In order to solve the above problems, the present inventors have intensively studied a method for detecting proteins and pathogens. As a result, the present inventors have found that the influenza virus can be detected quickly and with high sensitivity by immobilizing the element recognizing the influenza virus on the surface of the boron-doped diamond electrode and performing electrochemical measurement, The present invention has been completed.

That is, the present invention includes the following.
[1] A conductive diamond electrode having an element for recognizing protein or pathogen immobilized on the surface.
[2] Elements that recognize proteins or pathogens are influenza virus, DNA virus, RNA virus, double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded RNA (+) strand virus, 1 Single-stranded RNA (-) strand virus, single-stranded RNA reverse transcription virus, double-stranded DNA reverse transcription virus, norovirus, rotavirus, rubella virus, measles virus, RS virus, herpes virus, hepatitis virus, adenovirus, foot-and-mouth disease virus , Recognizes rabies virus, human immunodeficiency virus, mycoplasma, botulinum, pertussis, tetanus, diphtheria, cholera, shigella, anthrax, pathogenic Escherichia coli, staphylococci, salmonella, welsh, or cereus 2. The electrode according to 1, wherein the electrode
[3] The electrode according to 1 or 2, wherein the element that recognizes the protein or pathogen has a peptide that recognizes the pathogen protein.
[4] Protein or pathogen recognition element is influenza virus hemagglutinin protein (HA), influenza virus neuraminidase (NA), M1 protein or M2 protein, mycoplasma bacterial P1 protein, membrane antigen protein or ribosomal protein L7 / L12 Botulinum toxin, pertussis toxin, tetanus toxin, diphtheria toxin, Clostridium perfringens alpha toxin, cholera toxin, verotoxin, anthrax toxin, Escherichia coli enterotoxin, staphylococcal enterotoxin, salmonella enterotoxin, or cereus enterotoxin The electrode according to any one of 1 to 3, which has a peptide to be recognized.
[5] The element that recognizes the protein or pathogen has a molecule that recognizes the protein or pathogen and a linker part, and the molecule that recognizes the protein or pathogen is immobilized on the surface of the diamond electrode via the linker part. 5. The electrode according to any one of 1 to 4.
[6] A device for detecting a protein or pathogen, comprising the electrode according to any one of 1 to 5.

[7] A method for detecting a protein or a pathogen using the electrode according to any one of 1 to 5 or the apparatus according to 6.
[8] The detection method according to 7, wherein the protein or pathogen is detected by cyclic voltammetry measurement or electrochemical impedance measurement.
[9] A method for producing a conductive diamond electrode for protein or pathogen detection, comprising a step of immobilizing a molecule that recognizes a protein or pathogen on the surface of the conductive diamond electrode to form an element that recognizes the protein or pathogen.
[10] The production method according to 9, comprising a step of immobilizing a linker molecule on the surface of the diamond electrode and a step of linking the linker molecule and a molecule that recognizes a protein or pathogen.

This specification includes the disclosure of Japanese Patent Application No. 2015-093132 which is the basis of the priority of this application.

The protein or pathogen can be detected quickly and with high sensitivity using the apparatus of the present invention. Compared to other electrochemical detection methods, the diamond electrode has an inactive surface, which can suppress nonspecific adsorption of proteins, etc., and has low noise and a wide potential window, enabling highly sensitive detection. . In addition, the present invention can detect proteins or pathogens with high sensitivity without using expensive molecules such as antibodies.

A scheme for solid phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer is shown. The schematic diagram of a hemagglutinin (HA) protein-peptide interaction and cyclic voltammetry (CV) measuring method is shown. The detection of HA protein by CV measurement using peptide unmodified (left) and peptide modified (right) diamond electrodes is shown. Detection of influenza virus (IFV) by CV measurement using peptide unmodified (left) and peptide modified (right) diamond electrodes. The detection of HA protein by electrochemical impedance (EIS) measurement using a peptide-modified diamond electrode is shown. The result of the target HA protein and the control protein BSA in EIS measurement using a peptide-modified diamond electrode is shown. The square is HA (R ² = 0.989) and the circle is BSA (R ² = 0.784). The detection of IFV by EIS measurement using a peptide-modified diamond electrode is shown. The measurement of HA protein by CV measurement using a peptide-unmodified (left) and a peptide-modified (right) glassy carbon electrode is shown. The measurement of IFV by CV measurement using a peptide-unmodified (left) and a peptide-modified (right) glassy carbon electrode is shown. The measurement of HA protein by EIS measurement using a glassy carbon electrode modified with a peptide is shown. The left is the Nyquist plot, the center is the R _ct value at each concentration, and the right is the comparison result of the R _ct values of HA and BSA. The result of IFV detection by ELISA method using peptide s2 (1-5) is shown. The result of the detection of IFV by a peptide modification electrode is shown. The left is the IFV H1N1 strain, and the right is the IFV H3N2 strain. Circle symbols indicate peptide-presented BDDs, and square symbols indicate control peptides. The vertical axis represents ΔR _ct value with respect to the R _ct value measured at 0 pfu (ΔR _ct / R _ct (0 pfu)), and the horizontal axis represents IFV (pfu) in logarithm. The HPLC chart of purified peptide (4mer) is shown.

Hereinafter, the present invention will be described in detail.

The present invention provides a conductive diamond electrode having an element for recognizing a protein or pathogen immobilized on the surface (hereinafter sometimes simply referred to as the electrode of the present invention). Moreover, this invention provides the protein or pathogen detection apparatus provided with this electrode as a working electrode. Here, “recognizing” a protein or pathogen means that the element interacts, binds, or associates with the protein or pathogen. When the element recognizes a protein or pathogen, when an electric potential is applied, an electric current flows due to an electrochemical reaction. By measuring this current, a recognized protein or pathogen can be detected. As used herein, “detection” includes qualitative detection and quantitative detection.
Apparatus of the Present Invention The apparatus for detecting a protein or pathogen of the present invention has a working electrode, a reference electrode (also referred to as a reference electrode), and a counter electrode. Moreover, the apparatus of this invention has a voltage application part, a current measurement part, and arbitrary recording means. In one embodiment, the apparatus of the present invention further includes a potentiostat and an AC transmitter and a lock-in amplifier connected thereto. This apparatus can perform electrochemical impedance measurement (AC impedance measurement).

The working electrode of the present invention has a substrate, a diamond layer on the substrate, and a modification layer on the diamond layer. The diamond layer on the substrate is a conductive diamond doped with a small amount of impurities. That is, a conductive diamond electrode is used for the working electrode of the present invention. This conductive diamond electrode is preferably doped with a small amount of impurities. By doping with impurities, desirable properties as an electrode can be obtained. Examples of impurities include boron (B), sulfur (S), nitrogen (N), oxygen (O), and silicon (Si). For example, diborane, trimethoxyborane and boron oxide are used to obtain boron in a source gas containing a carbon source, sulfur oxide and hydrogen sulfide are obtained to obtain sulfur, oxygen or carbon dioxide is obtained to obtain oxygen, Ammonia or nitrogen can be added to obtain nitrogen, and silane or the like can be added to obtain silicon. In particular, a conductive diamond electrode doped with boron at a high concentration is preferable because it has advantageous properties such as a wide potential window and a small background current compared to other electrode materials. Therefore, in the present invention, a boron-doped diamond electrode will be described below as an example. A conductive diamond electrode doped with other impurities may be used. In this specification, unless otherwise specified, a potential and a voltage are used synonymously and are interchangeable. In this specification, the conductive diamond electrode may be simply referred to as a diamond electrode, and the boron-doped diamond electrode may be referred to as a BDD electrode.

The electrode portion of the working electrode of the present invention has a diamond layer in which 0.01 to 8% w / w boron raw material mixed diamond is deposited on the substrate surface. The size of the substrate is not particularly limited, but a substrate having an area capable of measuring a sample solution in mL units or μL units is preferable. For example, the substrate may have a diameter of 1 to 10 cm and a thickness of 0.1 mm to 5 mm. The substrate Si substrate, a glass substrate or a quartz substrate such as SiO _2, ceramic substrate such as Al ₂ O _3, tungsten, may be a metal such as molybdenum. All or part of the surface of the substrate can be a diamond layer.

The size of the electrode part of the conductive diamond electrode of the present invention can be appropriately designed depending on the measurement target. For example, the electrode portion can be a surface having an area of, for example, 0.1 cm ² to 10 cm ² , 0.2 cm ² to 5 cm ² , or 0.5 cm ² to 4 cm ² . All or part of the diamond layer can be used for electrochemical measurements. A person skilled in the art can appropriately determine the area and shape of the electrode portion according to the measurement target.

The electrode part of the working electrode of the present invention has a diamond layer in which the surface of the Si substrate is deposited with diamond mixed with a high boron raw material (0.01-8% w / w boron raw material as raw material preparation). A preferable boron raw material mixing rate is 0.05 to 5% w / w, particularly preferably about 0.3% w / w. After the diamond layer is formed on the substrate, a modification layer can be provided thereon.

The deposition process of the diamond mixed with boron material on the substrate may be performed at 700 to 900 ° C. for 2 to 12 hours. The conductive diamond thin film is produced by a usual microwave plasma chemical vapor deposition (MPCVD) method. That is, a substrate such as a silicon single crystal (100) is set in a film forming apparatus, and a film forming gas using a high purity hydrogen gas as a carrier gas is allowed to flow. The deposition gas contains carbon and boron. When plasma is generated by applying microwaves to a film deposition system that flows high-purity hydrogen gas containing carbon and boron, carbon radicals are generated from the carbon source in the film-forming gas, and are formed on the Si single crystal. While maintaining the sp ³ structure and depositing while mixing boron, a diamond thin film is formed.

The film thickness of the diamond thin film can be controlled by adjusting the film formation time. The thickness of the diamond thin film can be, for example, 100 nm to 1 mm, 1 μm to 0.1 mm, 1 μm to 100 μm, 2 μm to 20 μm, and the like.

The conditions for the vapor deposition treatment of boron-doped diamond on the substrate surface may be determined according to the substrate material. As an example, the plasma output can be 500 to 7000 W, for example, 3 kW to 5 kW, preferably 5 kW. When the plasma output is within this range, the synthesis proceeds efficiently, and a high-quality conductive diamond thin film with few by-products is formed.

The above electrodes are disclosed in Japanese Patent Application Laid-Open No. 2006-98281, Japanese Patent Application Laid-Open No. 2011-152324, Japanese Patent Application Laid-Open No. 2015-172401, and the like, and can be manufactured according to the descriptions in these documents.

The conductive diamond electrode of the present invention has high thermal conductivity, high hardness, chemically inert, wide potential window, low electric capacity, low background current, and excellent electrochemical stability. ing.

The device of the present invention is equipped with a triode electrode. The resistance on the reference electrode side is set high, and no current flows between the working electrode and the reference electrode. Although a counter electrode is not specifically limited, For example, a silver wire and a platinum wire can be used. The reference electrode is not particularly limited, but a silver-silver chloride electrode (Ag / AgCl) is preferable from the viewpoint of stability and reproducibility. Unless otherwise specified in this specification, the measured voltage is measured with respect to a silver-silver chloride electrode (+0.199 V vs standard hydrogen electrode (SHE)). The size and position of the working electrode, counter electrode, and reference electrode in the sensor can be designed as appropriate, but the working electrode, counter electrode, and reference electrode are all designed and arranged so that they can be contacted simultaneously with the measurement sample. Is done.

A silver-silver chloride electrode used as a reference electrode has a structure in which an AgCl-coated silver wire (Ag / AgCl) is immersed in an aqueous solution containing chloride ions (Cl ⁻ ). The Ag / AgCl reference electrode of the present invention is not particularly limited as long as it has a larger surface area than the working electrode (conductive diamond electrode).

The shape of the sensor part of the apparatus of the present invention is not particularly limited as long as the working electrode, the counter electrode, and the reference electrode are all arranged so as to be in contact with the object to be measured.

The conductive diamond electrode of the present invention has an element that recognizes a protein or pathogen immobilized on the surface thereof. In some embodiments, immobilization means that the element is connected to the surface of the diamond electrode by a covalent bond. The layer on which the element is fixed may be referred to as a modification layer. When the modified layer is brought into contact with the target protein or pathogen, the element recognizes the protein or pathogen. At this time, when a potential is applied, a current flows, and the protein or pathogen in the sample can be detected by measuring the current.

As the pathogen detected by the electrode or apparatus of the present invention, any pathogen may be used as long as it is recognized by the element on the surface of the conductive diamond electrode of the present invention and current flows. Examples of pathogens include viruses and pathogenic bacteria. Pathogenic bacteria include, but are not limited to, Mycoplasma, Clostridium botulinum, Bordetella pertussis, Tetanus, Diphtheria, Vibrio cholerae, Shigella, Bacillus anthracis, pathogenic Escherichia coli, Staphylococcus, Salmonella, Clostridium perfringens, and Bacillus cereus. Absent.

As the virus detected by the electrode or device of the present invention, any virus may be used as long as it is recognized by the element on the surface of the conductive diamond electrode of the present invention and current flows. Detected viruses include DNA viruses, RNA viruses, double-stranded DNA viruses, single-stranded DNA viruses, double-stranded RNA viruses, single-stranded RNA (+) strand viruses, and single-stranded RNA (-) strand viruses. Single-stranded RNA reverse transcription virus, double-stranded DNA reverse transcription virus, such as influenza virus, norovirus, rotavirus, rubella virus, measles virus, RS virus, herpes virus, hepatitis virus, adenovirus, foot-and-mouth disease virus, rabies virus, And human immunodeficiency virus. Influenza viruses include A, B, C, avian influenza viruses and various subtypes thereof.

The protein detected by the electrode or apparatus of the present invention may be any protein as long as it is recognized by the element on the surface of the conductive diamond electrode of the present invention and current flows. In certain embodiments, the protein to be detected is one having a tryptophan residue. In certain embodiments, the protein to be detected is a protein derived from the pathogen, pathogenic bacterium, or virus described above. Preferably, the virus-derived protein can be a virus surface protein. For example, if the detection target is an influenza virus, the element may recognize hemagglutinin (HA) or neuraminidase (NA) protein derived from the influenza virus. Examples of the protein to be detected further include M1 protein and M2 protein of influenza virus. When the detection target is Mycoplasma bacterium, the element can be recognized by Mycoplasma bacterium-derived P1 protein, membrane antigen protein, or ribosomal protein L7 / L12. In certain embodiments, the protein to be detected is a toxin protein. Toxin proteins include exotoxin, botulinum toxin, pertussis toxin, tetanus toxin (tetanospasmin), diphtheria toxin, alpha toxin of C. perfringens, cholera toxin, verotoxin, anthrax toxin (PA, EF, or LF), and Enterotoxins derived from Escherichia coli, Staphylococcus, Salmonella, Bacillus cereus, and the like, but are not limited thereto.

The element for recognizing the protein or pathogen of the present invention may be any element as long as a current flows through the conductive diamond electrode of the present invention when interacting with the target protein or pathogen.

An element that recognizes the protein or pathogen of the present invention is schematically shown below. The element has one end immobilized on the surface of a conductive diamond electrode and the other end has a molecule that recognizes a protein or pathogen. Further, the element may optionally have a linker molecule.
Electrode- (L) -R
[In the formula, Electrode is a conductive diamond electrode, L is an optional linker, and R is a molecule that recognizes a protein or pathogen. ]

In one embodiment, the element for recognizing the protein or pathogen of the present invention has a peptide that specifically interacts with the target protein or pathogen. Explaining in the above formula, the molecule R that recognizes a protein or pathogen may have a peptide. For example, a peptide having a length of 4 amino acids or more, 5 amino acids or more, 6 amino acids or more, 7 amino acids or more, 8 amino acids or more, 9 amino acids or more, 10 amino acids or more, or 15 amino acids or more can be used. For example, a peptide having a length of 100 amino acids or less or 50 amino acids or less, such as 40 amino acids or less, such as 30 amino acids or less, such as 20 amino acids or less, can be used. The device can have one or more of the peptides. For example, the peptide s2 (1-5) (SEQ ID NO: 1) that binds to the HA protein of influenza virus greatly increases HA binding by dendrimerization, and in the case of s2 (1-5) alone in the 4-branch type About 750 times higher HA binding activity is obtained. Such a dendrimerized protein-binding peptide can be used in the device of the present invention.

In one embodiment, an element for recognizing a protein or pathogen of the present invention has a peptide that recognizes a protein derived from the above-mentioned pathogen, pathogenic bacterium, or virus. The amino acid sequence of such a peptide can be obtained from publicly known literatures or publicly known databases such as GenBank. The peptide may be optionally modified and may be dendrimerized as described above.

In one embodiment, the element that recognizes the protein or pathogen of the present invention has a peptide that recognizes influenza virus. As the peptide for recognizing influenza virus, any known peptide can be used. For example, International Publication No. 2007/105565 pamphlet, International Publication No. 2010/024108 pamphlet, Japanese Patent Application Laid-Open No. 2010-209052 (these are referred to by reference). The contents of which are incorporated into the present specification), but are not limited thereto. Examples of peptides that recognize influenza virus include peptides having the amino acid sequences of SEQ ID NOs: 1 to 4. Peptides having these sequences as partial sequences are also envisaged. The peptide may be appropriately modified. These are merely examples, and other peptides that recognize viruses, pathogens, and pathogens can also be used in the present invention.

In one embodiment, the chain length of the peptide recognizing influenza virus used in the device of the present invention may be an amino acid chain length of 30 or less, 20 or less, for example 15 or less. In certain embodiments, the chain length of a peptide that recognizes influenza virus used in the device of the present invention may be 4 or more amino acid chains long. In this specification, the term “peptide” used in the “peptide recognizing pathogen or virus” or “peptide recognizing influenza virus” used in the device of the present invention does not include an antibody or its antigen-binding domain. And

A peptide that recognizes a protein or pathogen is immobilized on the conductive diamond electrode of the present invention by an appropriate chemical method. In certain embodiments, the peptide may be immobilized on the diamond electrode via a linker molecule. In addition to known protein recognition peptides, equivalents having equivalent functions are envisaged and can be used in the present invention. The peptide can be chemically synthesized.

The element for recognizing the protein or pathogen of the present invention may be immobilized on the surface of the diamond electrode using any appropriate method. In some embodiments, the molecule of interest can be linked to the electrode surface by electrolytic grafting. In some embodiments, a photoinduced radical reaction may be used for immobilization.

If desired, a linker molecule can be used for immobilizing the device. The following are examples of molecules for forming the linker moiety.
AL ₁ -B (-P)
[Wherein A is a functional group capable of reacting with a diamond electrode, L ₁ is a linker moiety, B is a functional group capable of reacting with a molecule that recognizes a protein or pathogen, and P may optionally be present. Represents a protecting group. ]

In this case, the molecule is first linked to the electrode surface by an appropriate reaction such as electrolytic grafting. That is, the functional group A is reacted to connect the electrode and L ₁ . When the functional group B is protected by the protecting group P, the functional group B can be presented by performing a deprotection reaction thereafter. The molecule R that recognizes the protein or pathogen can then be linked to the linker moiety using any suitable ligation reaction.

Examples of molecules R that recognize proteins or pathogens are given below.
CR ₁
[Wherein R ₁ represents a moiety that recognizes a protein or a pathogen, and C represents a functional group capable of reacting with the functional group B. ]

The linking reaction between the linker moiety and the molecule R that recognizes the protein or pathogen (linking reaction between the functional group B and the functional group C) is the Husgen cycloaddition reaction, the Gracer reaction, its coupling, Suzuki / Miyaura coupling reaction Etc., and those that form a covalent bond are preferred, but not limited thereto.

The functional group A capable of reacting with the diamond electrode can be a diazo group, amino group, carboxy group, carbonyl group, aldehyde group, hydroxyl group, nitro group, and the like. The linker portion L ₁ may have an aromatic ring, a carbocycle, a heterocyclic ring, such as a phenyl group, a naphthyl group, a polyether group, a polyethylene glycol group, or a hydrocarbon group. These may optionally be further substituted with an appropriate substituent such as an alkyl group, an aryl group, a halogen group, or a hydroxyl group. In addition, the functional group B and the functional group C may be an alkynyl group, a boranyl group, a boryl group, a halogenated aryl group, an azide group, or the like. Further, when the Husgen cycloaddition reaction is used for the ligation reaction, the functional group B may have an alkynyl group and the functional group C may have an azide group. The functional group B may optionally be protected by a protecting group P. Protecting groups include trisubstituted silyl groups such as triisopropylsilyl group, trimethylsilyl group, butyldiphenylsilyl group, dimethylcumylsilyl group, benzyl group, lower alkoxycarbonyl group, halogeno lower alkoxycarbonyl group, benzyloxycarbonyl group, acetyl group Groups, acyl groups such as benzoyl groups, triphenylmethyl groups, tetrahydropyranyl groups, and the like, but are not limited thereto.

In some embodiments, A can be a diazo group, L ₁ can be a phenylene group, B can be an alkynyl group, and P can be a triisopropylsilyl group. The alkynyl group B can be presented by reacting A, immobilizing it on the electrode surface and deprotecting it. In certain embodiments, a molecule R that recognizes a protein or pathogen may have a peptide group as R ₁ and an azide group as functional group C. A molecule R that recognizes a protein or pathogen when azide group (functional group C) of R is reacted with an alkynyl group (functional group B) presented on the electrode surface to form a 1,2,3-triazole ring. Is immobilized on the electrode surface.

About a measuring method The target protein or pathogen can be detected using the electrode or apparatus of the present invention. When the electrode of the present invention is brought into contact with a sample, the element on the electrode surface recognizes a protein or pathogen. At this time, when a potential is applied to the electrode, current can be observed. This can be measured by cyclic voltammetry or electrochemical impedance measurement.

Cyclic voltammetry Cyclic voltammetry is performed using a technique that varies (sweeps) the potential. Specifically, the electrode potential is swept from the initial potential (E _i ) to the reversal potential (E _λ ) at the sweep speed (v), then reversed, and the current obtained when returning to E _i is observed. A current-potential graph (cyclic voltammogram) can be obtained by setting the initial potential E _i to a potential at which no electrode reaction occurs and the inversion potential E _λ to a potential at which the electrode reaction is diffusion-controlled. The initial potential, sweep speed, inversion potential, and the like can be set as appropriate.

In this case, for a protein or pathogen sample with a known concentration or amount, a peak current value is determined, a calibration curve in which the relationship between the concentration or amount and the peak current density is plotted is prepared, From the peak current value, the concentration or amount of the protein or pathogen contained in the sample can be calculated.

Electrochemical impedance measurement For electrochemical impedance measurement, a potentiostat with an AC transmitter connected is used. A constant DC potential is applied to the electrode using a potentiostat, and an AC potential of ± 5 to 10 mV is superimposed and applied using an AC transmitter. Also, an AC wave having the same phase as the AC input from the transmitter to the potentiostat is input to the lock-in amplifier. As a result, the flowing current is a combination of the direct current and the alternating current, and the lock-in amplifier compares the alternating current component of the current with the alternating current from the transmitter, and outputs the impedance and the phase difference between the two. The AC frequency from the transmitter is changed little by little, and complex plane plots are performed based on the impedance and phase difference obtained at each frequency.

In this case, for a protein or pathogen sample at a known concentration or amount, the electrode impedance (charge transfer resistance R _ct ) is determined by a Nyquist plot, and a calibration curve plotting the relationship between the concentration or amount and R _ct is created. The concentration or amount of the protein or pathogen contained in the sample can be calculated from the _Rct value of the measurement sample.

Chronoamperometry The electrochemical measurement using the apparatus of the present invention can also be performed by chronoamperometry. In chronoamperometry, the potential of the working electrode is stepped, and the change in current over time is measured. Chronoamperometry measurement can be performed by applying a constant step potential such as 0.1 to 3.0 V, 0.5 to 2.5 V, or the like.

In this case, chronoamperometry measurement is performed at a constant applied voltage on a protein or pathogen sample having a known concentration or amount. A calibration curve is created by recording the current value at a fixed time after voltage application and plotting the relationship between the concentration or amount of the protein or pathogen sample and the current value. Next, the current value at the predetermined time after the voltage application is measured for the measurement sample, and this is compared with the calibration curve, thereby calculating the concentration or amount of the protein or pathogen sample in the test sample solution.

The test sample to be measured by the method of the present invention is not particularly limited, but for example, any solution, for example, a solution that may be contaminated with biological samples, drinking water, and pathogens such as viruses or toxins. Is mentioned. Examples of the biological sample include saliva, sputum, tears, body fluid, blood, cell lysate, and the like. The origin of the sample is not particularly limited, and examples thereof include animals, mammals, mice, rats, humans, chicken eggs, and cells. Samples can be 1 μL to 10 mL, 10 μL to 1 mL, 20 μL to 0.1 mL, and the like. The sample may be concentrated or subjected to measurement as it is.

In the present invention, an aqueous solvent is mainly used as a solvent for measurement. The solution to be measured usually contains a supporting electrolyte. The supporting electrolyte is an ionic substance and is not particularly limited, and examples thereof include phosphate buffered saline (PBS), potassium nitrate, and sodium sulfate. Preferably the supporting electrolyte is PBS.

In the present specification, “detection” of a protein means that the target protein can be specifically measured in a protein mixed with a protein other than the target protein or a virus or bacteria. Further, “detection” of a pathogen means that the target pathogen can be specifically measured with respect to a sample containing a protein other than the target pathogen or a virus or bacteria.

In this specification, the viral load is expressed by the number of plaque forming units (pfu). In an embodiment, the pfu when describing the amount of influenza virus is that when using the influenza virus A / PR / 8/34 (H1N1) strain. The pfu measurement method can be estimated by measuring plaques formed when influenza virus is added to canine kidney-derived epithelial cells MDCK cells cultured in a monolayer. This is an index for convenience, and those skilled in the art can appropriately determine the corresponding pfu when using different influenza virus strains. Further, the pfu unit may be appropriately converted to the ng unit. In certain embodiments, the device of the present invention can detect influenza viruses with high sensitivity, for example, can detect influenza viruses of 2 pfu or more, 5 pfu or more, 10 pfu, 20 pfu or more, or 40 pfu or more, 1000 pfu or less. .

The following examples are intended for illustration only and are not intended to limit the technical scope of the present invention. Unless otherwise specified, the reagents are commercially available, or are obtained or prepared according to methods commonly used in the art and known literature procedures.
Abbreviations In this specification, the following abbreviations may be used.
IFV: Influenza virus
HA: Hemagglutinin
EIS: Electrochemical impedance spectroscopy
CV: Cyclic voltammetry
DMF: N, N-dimethylformamide
DCM: Dichloromethane
PyBOP: Benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate
DIEA: Diisopropylethylamine
PIP: Piperidine
TFA: trifluoroacetic acid
THPTA: Tris (3-hydroxypropyltriazolylmethyl) amine
TIS: Triilopropylsilane
TIPS-Eth-Ar-N ₂ ⁺ BF ₄ ⁻ : 4-((Triisopropylsilyl) ethynyl) benzenediazonium tetrafluoroborate α-CHCA: α-cyano-4-hydroxycinnamic acid
THF: tetrahydrofuran
TBAF: Tetra-n-butylammonium fluoride
Fmoc: 9-fluorenylmethyloxycarbonyl group
AFM: Atomic force microscope

Materials and compounds Unless otherwise specified, commercially available compounds and chemicals were used without further purification. Those skilled in the art can modify the described procedures without departing from the spirit of the invention.

Example 1 Synthesis of Peptide and Linker A bi-branched peptide dendrimer (ARLPR) ₂ -K-KN ₃ having a terminal introduced with azidolysine Lys (N ₃ ) was synthesized using the Fmoc solid phase method. Peptide ARLPR (SEQ ID NO: 1) is a hemagglutinin-binding peptide (Japanese Patent Laid-Open No. 2006-68020). In peptide synthesis by the solid phase method, a deprotection reagent is added to the peptide resin (solid phase support on which an amino acid is immobilized), and the N-α protecting group is removed. The activated amino acid is reacted therewith, and the chain length of the peptide is successively increased. The carboxy terminus of the peptide was amidated to eliminate unwanted charges. In addition, a branched structure was created by introducing Lys into the C-terminal side of the peptide. FIG. 1 shows a scheme for solid-phase synthesis of an azide group-introduced s2 (1-5) peptide dendrimer.

(A) Introduction of Lys (N ₃ ) into resin by manual synthesis Fmoc-NH-SAL resin (Watanabe Chemical, 0.59 mmol / g) 169 mg (0.1 mmol) was charged into a reaction column (PD-10, Amersham). To this was added 2 mL of DMF, and after gently shaking, DMF was removed from the bottom of the column with an aspirator. This operation was repeated three times to swell the resin.

2 mL of 20% (v / v) PIP / DMF was added to the reaction column, and after 1 minute, the solvent was removed with an aspirator. Similarly, 2 mL of 20% PIP / DMF was poured and shaken for 15 minutes to remove the solvent. The operation of pouring 2 mL of DMF and removing with an aspirator was repeated 4 times to wash the resin. Thereby, deprotection was performed.

Fmoc-Lys (N ₃ ) -OH (EUROGENTEC GROUP ANA SPEC, 53100-F025) 117 mg (0.3 mmol), PyBOP 156 mg (0.3 mmol), DMF 2 mL, DIEA 0.11 mL (0.6 mmol) were added to the reaction column for 1 hour. Shake. Thereby, a coupling reaction was performed.

After 1 hour, the reaction solution was removed with an aspirator, and 2 mL of DMF was poured into the reaction column, shaken gently, and then removed with the aspirator four times (washing). Furthermore, the operation of adding 2 mL of DCM and removing with an aspirator was repeated 4 times. The reaction column was placed in a desiccator and dried with a vacuum pump for 1 hour. When the sample was sufficiently dry, it was stored at 4 ° C.

(B) Quantification of amino acid introduction rate Fmoc-Lys (N ₃ ) -NH-SAL resin 20 mg introduced in (A) is accurately weighed into a sample tube, and 2 mL of 20% (v / v) PIP / DMF is added, The reaction was allowed to proceed for 20 minutes at room temperature. The supernatant was diluted 50 times with DMF, and the absorbance at 301 nm was measured. DMF was used for the measurement of the blank, and the amino acid introduction rate was calculated from the following formula from the molar extinction coefficient ε = 7800 of the Fmoc group at 301 nm.
Amino acid introduction rate (mol / g)
= (A ₃₀₁ , 1/50-A ₃₀₁ , DMF) x 50/7800 x (2 x 10 ^-3 ) x (1000/20)
= (0.644-0) × 0.641 × 10 ^-3
= 0.413 mmol / g
As a result of quantifying the amino acid introduction rate of the Fmoc-Lys (N ₃ ) -NH-SAL resin prepared in (A) above, the introduction rate was 0.413 mmol / g.

(C) Peptide elongation PD-10 empty columns (17-0435-01, Amersham Biosciences) was charged with 242 mg (0.05 mmol) of Fmoc-Lys (N ₃ ) -NH-SAL resin, and the following (C-1) Peptide elongation reaction was carried out by the procedure of (C-4) (the amount of resin at which the peptide was 0.1 mmol was used).

(C-1) Deprotection of Fmoc Group The resin with the first amino acid introduced was weighed onto a 121 mg (0.05 mmol) reaction column. After adding 2 mL of DMF to the reaction column and shaking lightly, DMF was removed from the lower part of the column with an aspirator. The operation of pouring 2 mL of DMF into the reaction column and removing it with an aspirator was repeated 4 times. 2 mL of 20% (v / v) PIP / DMF was poured into the reaction column, and after 1 minute, it was removed with an aspirator. Next, 2 mL of 20% (v / v) PIP / DMF was poured, shaken for 15 minutes, and removed. Thereafter, the operation of pouring 2 mL of DMF and removing it with an aspirator was repeated 5 times to wash the resin.

(C-2) Coupling Fmoc-AA-OH (each 0.3 mmol), PyBOP 157 mg (0.3 mmol), DMF 2 mL, DIEA 0.11 mL (0.6 mmol) were added to the reaction column, and shaken for 40 minutes. Amino acids were introduced in the order of lysine (Lys) → arginine (Arg) → proline (Pro) → leucine (Leu) → arginine (Arg) → alanine (Ala) from the C-terminal side. After 40 minutes, the reaction solution was removed with an aspirator, and 2 mL of DMF was poured into the reaction column and shaken lightly, and then removed with the aspirator four times. The amino acids used are shown in Table 1.

(C-3) Kaiser Test A reagent for the Kaiser test (code number 2590077, domestic chemistry) was used to determine whether the coupling reaction had been completed. Take about 1 mg of resin with a spatula, put in a microtube, add 10 μL each of reagents (1) ninhydrin / ethanol, (2) phenol / ethanol, and (3) KCN / pyridine, cover, and heat for about 1 minute with a dryer. did. Since the unreacted amino group remains when the color of the resin or solution becomes blue, the coupling reaction was performed once again with the same amino acid. When it became colorless or yellow, it was judged that the reaction was completed, and the above operations (C-1) to (C-3) were repeated until all amino acids were coupled.

(C-4) Deprotection After all extension reactions, pour 2 mL of 20% PIP / DMF onto the PD-10 column, stir gently with a spatula, remove 20% PIP / DMF with an aspirator, and 2 mL of 20% PIP / DMF. The Fmoc group was deprotected by removing it after pouring and shaking for 15 minutes. After adding 2 mL of DMF and removing with an aspirator 5 times, and adding t-butyl methyl ether and removing with an aspirator 2 times, cover the PD-10 column with aluminum foil and parafilm, and vacuum dry for 3 hours. It was.

(D) Cutting out (Cleavage and deprotection from resin)
In order to cut out the peptide from the synthesized resin, a cocktail solution was prepared with the composition shown in Table 2 and cut out. Since the azide group is easily decomposed by the reduction action of thiol, a cocktail solution having a thiol-free composition was used (see P. E. Schneggenburger et al., J. Pept. Sci., 16, 10-14 (2010)).

1 mL of the above cocktail solution was placed in a reaction vessel containing the peptide and left on ice for 2 hours under light-shielded conditions to prevent decomposition of the azide group by heat and light (usually left at room temperature for 8 hours without light) . After 2 hours, the reaction container was uncovered, pressurized from the upper mouth of the container, the contents of the reaction container were dropped into a 15 mL centrifuge tube, and washed twice with 200 μL of TFA. 2 mL of cold diethyl ether (peroxide free) was added, and after confirming that precipitation was possible, the volume was increased to 10 mL, and the mixture was vortexed. Subsequently, after centrifugation at 3500 rpm for 1 minute, the supernatant was removed, and the volume was again increased to 10 mL with cold diethyl ether. This operation was repeated a total of 5 times. By blowing N ₂ gas remaining peptide pellets centrifuge tube was volatilized cold diethyl ether (crude peptide).

(E) High performance liquid chromatography (HPLC)
To the crude peptide powder remaining in the 15 mL centrifuge tube, 300 μL of AN (acetonitrile) and 100 μL of Milli-Q (registered trademark) water were added and completely dissolved (400 μL of 75% acetonitrile). A syringe and a 0.45 μm filter (Millex-LH, 4 mm, code SLLH H04 NL, Millipore) were connected to remove insoluble matters, and collected in a 1.5 mL tube. Again, 400 μL of 75% acetonitrile was added to the centrifuge tube and washed again, and collected in another 1.5 mL tube.

An ODS-3 column ((i) φ4.6 × 250 mm, (ii) φ20 × 250 mm, GL Science) was used for HPLC, and the flow rate was 1 mL / min for (i) and 10 mL / min for (ii). 20 μL of peptide solution (co-wash solution) was injected, and elution conditions determined using the column (i) are shown below. At this time, fractions were collected every 30 seconds and analyzed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).
Solvent: A… 0.1% TFA / H ₂ O B… 0.1% TFA / AN
Gradient: B conc. 20% (10 minutes) + B conc. 20 → 60% (20 minutes) + B conc. 100% (10 minutes)
Wavelength: 220 nm

Under the determined elution conditions, the peptide solution was subjected to HPLC using the column (ii), and the fraction containing the peak was collected every 15 seconds, and the fraction containing the target peptide was collected using MALDI-TOF MS, The product was isolated by lyophilization.

(F) MALDI-TOF MS
MALDI-TOF MS was measured using Ultraflex ™ (Bruker Daltonics). An N ₂ laser (337 nm) was used as the laser light source. As a matrix, α-cyano-4-hydroxycinnamic acid (α-CHCA, Sigma) was used. α-CHCA was suspended in 0.1% TFA / AN (3: 2, v / v) at a rate of 10 mg / mL, irradiated with ultrasonic waves, centrifuged, and this supernatant was used. For the calibration, a peptide calibration standard (code number 206195, Bruker) diluted according to the protocol was used.

4 μL of α-CHCA solution and 2 μL each of the fraction solution after HPLC were put into a 2 mL tube and pipetted, and 2 μL was placed on a MALDI plate and air-dried. Calibration samples were also performed in the same manner. For Ultraflex ™, measurements were made in positive ion mode using reflectron mode.

The synthesized bifurcated peptide dendrimer (Lot. 140607) was confirmed by HPLC and MALDI-TOF MS. As a result of HPLC analysis, a sharp single peak was detected in 20 to 25 minutes, and it was confirmed that the target product had a high purity of 98% or more. In addition, the analysis results with MALDI-TOF MS showed an error of 0.1% or less, confirming that it was the target product (Exact Mass: 1486.94, calculation [M + H] ⁺ 1487.95, measurement [M + H] ⁺ 1487.95; calculation [M + Na] ⁺ 1509.84, measurement [M + Na] ⁺ 1509.98). The yield of this peptide was 7.7 mg, the yield was 10.8%, and the purity was> 98%. From the above results, it was judged that the target azide group-introduced peptide dendrimer was obtained.

Linker molecules _{TIPS-Eth-Ar-N 2} + BF ₄ ^- synthetic diamond linker molecule _{TIPS-Eth-Ar-N 2} + BF used in the presentation of the alkynyl group to the electrode ₄ of the ^- (4 - ((triisopropylsilyl) ethynyl ) Benzenediazonium tetrafluoroboric acid). First, the terminal alkyne and aryl halide are cross-coupled by the coupling, thereby obtaining an alkynylated aryl (aromatic acetylene) (see S. Anderson, Chem. Eur. J. 7, 4706-4714 (2001)). ). Palladium, copper and base were used as the catalyst. Furthermore, the amino group was diazoniumized for electrolytic grafting.

Experimental method (A) Synthesis of TIPS-Eth-Ar-NH ₂ by its coupling

The water in the three-necked flask was evaporated with a heat gun, vacuumed and then filled with argon (Ar). 4-Iodoaniline 1.0 g (4.57 mmol) and triethylamine 10 mL (71.7 mmol, d = 0.726 g / cm ³ ) were added. After evacuation and Ar filling were repeated 3 times each, degassing was performed, and while circulating Ar, copper iodide 8.9 mg (0.05 mmol), palladium acetate 20.3 mg (0.09 mmol), triphenylphosphine 50.6 mg (0.19 mmol) ) In order and added to a three-necked flask. Further, 1.2 mL (5.35 mmol, d = 0.813 g / cm ³ ) of triisopropylsilylacetylene was added, charged with Ar, and reacted at room temperature with stirring overnight. The reagents used are shown in Table 3.

Celite filtration A filter paper was placed on the Kiriyama funnel, and Celite was spread on the funnel. The reaction solution in the flask was filtered and washed with hexane. The reaction solid that could not be removed from the wall surface was subjected to ultrasonic treatment, dissolved or suspended in hexane, and filtered. The eggplant flask containing the filtrate was put on an evaporator, and the filtrate was concentrated (about 2 mL).

Silica gel column chromatography As a developing solvent, hexane: ethyl acetate = 10: 1 was used. Silica (Silica gel 60, Merck) 75 cc was dispersed in a developing solvent and packed in a column. The concentrated filtrate was gently and uniformly added on the sea sand, and the cock was opened and collected in a test tube.

Thin layer chromatography (TLC)
TLC of the fraction collected in the test tube was performed. Dichloromethane: hexane = 1: 1 was used as a developing solvent. TLC plate (TLC silica gel 60 F ₂₅₄ , (105714, Merck Millipore)) was spotted with a solution of iodoaniline (R _f = 0.29) as a raw material in dichloromethane and a solution before silica gel column chromatography. Confirmed by post-UV irradiation. Next, the solution in the test tube was spotted on the TLC plate and developed in order, and the test tube sample in a range where the reaction product (R _f = 0.39) could be confirmed was collected in an eggplant flask and concentrated by an evaporator (about 2 mL). The eggplant flask containing the concentrated reaction solution (TIPS-Eth-Ar-NH ₂ ) was depressurized with a pump and vacuum dried for 1 hour. After measuring the yield, ¹ H-NMR measurement was performed.

(B) TIPS-Eth-Ar -N 2 + BF 4 by diazotization ^- Synthesis of

TIPS-Eth-Ar-NH by diazotization of _{2 TIPS-Eth-Ar-NH} 2 + BF 4 - Scheme of Synthesis advance NaNO ₂ 0.4 g of (6.0 mmol) was dissolved in H ₂ O in 2 mL, refrigerated at 4 ° C. I left it. The eggplant flask containing TIPS-Eth-Ar-NH ₂ (1.1 g, 4.0 mmol) obtained in (A) was placed in a bath containing methanol (MeOH) and liquid nitrogen, and the temperature in the flask was −5 ° C. It cooled until it fell down. 4 mL of H ₂ O was added thereto and mixed well with a stirrer until dispersed, and then 3.36 mL of HBF ₄ (16 mmol, d = 1.4 g / cm ³ ) was added. ₂ mL of a precooled NaNO ₂ aqueous solution was added dropwise while maintaining the temperature in the flask at −5 to −10 ° C. After stirring at −5 ° C. for 20 minutes, the mixture was returned to room temperature and stirred for 20 minutes. The reagents used are shown in Table 4.

Suction Filtration NaBF ₄ 4 mg (36.4 nmol) was dissolved in 80 mL of H ₂ O to prepare a 5% NaBF ₄ aqueous solution. The 5% NaBF ₄ aqueous solution, MeOH, diethyl ether, and H ₂ O used as the washing solution were cooled in advance at 4 ° C. The filter paper was placed on the Kiriyama funnel, and the filter paper was applied with H ₂ O while sucking. All the reaction liquid was filtered and washed with H ₂ O. A 5% NaBF ₄ solution was washed in this order, followed by MeOH and diethyl ether, and the remaining reaction product was recovered by sonication. After the powder on the filter paper was dried in a desiccator under reduced pressure for 1 hour, the yield was measured.

The yield and yield of the product, and the results of analysis by ¹ H-NMR are shown below.
(A) TIPS-Eth-Ar-NH ₂
Yield: 1.01 g (4.0 mmol, Lot. 140801)
Yield: 87%
¹ H-NMR (400 MHz, CDCl ₃ , TMS): δ (ppm) = 7.28 (2H, d), 6.58 (2H, d), 3.78 (2H, s), 1.11 (2H, s)

(B) TIPS-Eth-Ar -N 2 + BF 4 -
Yield: 0.11 g (0.32 mmol, Lot. 140826)
Yield: 8%
¹ H-NMR (400 MHz, CDCl ₃ , TMS): δ (ppm) = 8.58 (2H, d), 7.79 (2H, d), 1.11 (2H, s)

From the above ¹ H-NMR analysis, regarding TIPS-Eth-Ar—NH ₂ , the proton ratios of the respective peaks matched, and thus the target compound could be synthesized. The _{TIPS-Eth-Ar-N 2} + BF 4 - relates, synthetic and peaks of 3.77ppm derived amino group disappears, since the proton ratio of each of the other peaks were consistent likewise desired compound Was confirmed.

Example 2 Production of Boron Doped Diamond (BDD) Electrode Briefly, a diamond film was synthesized on a Si substrate by chemical vapor deposition using microwave plasma. Methane was used as the carbon source and trimethylborane was used as the boron source. The concentration of trimethylborane to be doped in the raw material was 0.3% w / w. The surface morphology was characterized using a scanning electron microscope. The quality of the thin film was confirmed by Raman spectroscopy. The BDD electrode produced in this way was used. This will be specifically described below.

Preparation of conductive diamond thin film by vapor phase synthesis method (A) Pretreatment of Si substrate Si substrate (diameter 5cm, thickness 1mm) is placed in a petri dish containing diamond powder so that the mirror surface is facing down, and Si substrate is used for 20 minutes. The substrate surface was scratched by rotating it manually. Thereafter, the Si substrate was dipped in a beaker containing 2-propanol and cleaned by ultrasonic irradiation for 20 minutes. Finally, the solvent was volatilized with N ₂ gas and dried.

(B) Synthesis of diamond film on Si substrate Diamond film synthesis on Si substrate by chemical vapor deposition (CVD method, chemical vapor deposition method) using microwave plasma is performed by Plasma Deposition System (AX6500, Sekitechno). TRON Co., Ltd.). Four types of gases were used: methane, trimethylborane, hydrogen, and oxygen. The boron concentration was set to 0.3% w / w and reacted for 5 hours. Table 7 shows the synthesis conditions.

Raman spectroscopy Using a confocal Raman optical microscope for 532 nm (ST-BX51, Seki Technotron Co., Ltd.), the chemical bonding state of the substrate surface after film formation was analyzed. Naphthalene was used for calibration, and laser light was irradiated 5 times for 5 seconds. In order to confirm that the film was formed uniformly, analysis was performed at any nine locations on the diamond electrode.

As a result, a boron-boron bond peak was observed in the vicinity of 520 cm ⁻¹ . In the vicinity of 1333 cm- ¹ , a Raman peak of carbon with sp ³ structure was observed. On the other hand, no Raman peak of carbon with sp ² structure was observed in the vicinity of 1560 cm ⁻¹ . From this, it was judged that a high-purity diamond film was uniformly synthesized.

SEM Observation The surface and cross-sectional shape of the diamond film were measured using a scanning electron microscope FE-SEM (JSM-7600F, JEOL Ltd.). A diamond electrode (Lot. 140530) ultrasonically cleaned with ultrapure water, ethanol, and acetone for 5 minutes each was cut into 0.5 cm squares and fixed to a sample stage with silicon grease (Shin-Etsu Chemical Co., Ltd.). The acceleration voltage was set to 5.0 kV for surface observation and 2.0 kV for cross-sectional observation. SEM observation confirmed the synthesis of polycrystalline BDD.

Example 3 Modification of Boron Doped Diamond (BDD) A scheme for the presentation of alkynyl groups by electrolytic grafting is shown below.

(A) Preparation of electrode and solution The cell, O-ring, Pt wire, and diamond electrode were sonicated in the order of H ₂ O, then ethanol (EtOH), and then acetone for 5 minutes each. First, 3.87 g (0.01 mol) of TBA · PF ₆ was weighed into a volumetric flask, and acetonitrile (AN) was added to prepare a 100 mM tetrabutylammonium hexafluorophosphate (TBAPF ₆ ) solution. Cells were assembled as shown in FIG. The three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used for CV measurement.

(B) Reaction between linker molecule and diamond electrode (electrolytic grafting)
Volumetric flask _{TIPS-Eth-Ar-N 2} + BF 4 - to 18.6 mg (0.05 mol) weighed and, 10mM TIPS-Eth-Ar- N 2 + BF 4 dissolved in an electrolytic solution TBAPF ₆ / AN 5mL (100mM) ^- solution was prepared. This whole quantity was put into the cell. The control PC was set as shown in Table 8, and cyclic voltammetry (electrolytic grafting) was performed 5 times at -0.7 to + 0.6V.

(C) Deprotection of triisopropylsilyl (TIPS) group After completion of measurement, first put 9.5 mL of THF in the cell, and then add 0.5 mL of THF solution of TBAF (1 mol / L), and let stand for 20 minutes. Was deprotected (see YR Leroux et al., J. Am. Chem. Soc., 132, 14039-14041 (2010)).

Immobilization of azide group-introduced peptide An alkynyl group-presenting diamond electrode was sonicated with Milli-Q (registered trademark) water, EtOH, and acetone for 5 minutes each. A reaction solution was prepared so that the peptide concentration was 0.1 mM with the composition shown in Table 9 (peptide charge amount 100 times), and a reaction solution was further diluted to 0.1 nM with MeOH: H ₂ O = 1: 1. (Peptide charge 0.01 times).

A diamond electrode was immersed in each reaction solution and allowed to react while shaking at room temperature for 24 hours. After 24 hours, the reaction solution was removed and sonicated with Milli-Q (registered trademark) water, EtOH, and acetone for several seconds each. The electrode was dried by blowing N ₂ gas, and stored in a sealed container containing silica gel at 4 ° C.

As the electrode after the reaction, a 1 mM K ₃ [Fe (CN) ₆ ] / Na ₂ SO ₄ aqueous solution was prepared, and cyclic voltammetry (CV) measurement was performed on each electrode for 3 cycles to confirm the surface state. In addition, CV measurement was performed for 3 cycles of PBS, which is a solvent for HA and IFV, and changes in the background were confirmed. In addition, the contact angle was observed with these electrodes, and the wetting characteristics of the surface were examined.

Example 4 Detection of hemagglutinin protein (HA) and influenza virus (IFV) using boron-doped diamond (BDD) electrode Cyclic voltammetry (CV) measurement method Measurement of HA protein using unmodified and peptide-modified BDD electrodes Shows the schematic diagram of HA-peptide interaction and CV measurement method. A three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used. The left is the interaction with H1HA (15 minutes), the right is the measurement in PBS solution.

First, a cell was assembled with a peptide-immobilized electrode (peptide charge x 0.01), and the background was measured for 3 cycles with PBS. Thereafter, about 60 μL of 500 nM HA / PBS was added to the cell so that the electrode portion was immersed, and the cells were allowed to interact for 30 minutes. After 30 minutes, the HA solution was removed and washed with PBS three times. The cells were filled with PBS, and cyclic voltammetry measurement was performed for 3 cycles. These operations were performed again at different locations on the electrode, and the interaction was observed at a total of two locations. The measurement conditions shown in the table below were used.

In the same manner as above, 50 to 500 nM HA solution diluted with PBS was allowed to interact for 30 minutes each. CV measurement was performed in 3 cycles each while washing the electrode with PBS before changing the concentration. Measurement conditions were as shown in Table 10.

The results are shown in FIG. The left side of FIG. 3 shows the measurement of HA in the solution when using a diamond electrode without peptide modification. An increase in current density was observed in the 500 nM HA solution. The right side of FIG. 3 shows a case where a peptide-modified diamond electrode is used. In the first cycle, a significant increase in current density was observed compared to the unmodified diamond electrode, and HA could be detected. From the second cycle onward, it can be seen that the current density is not much different from that of PBS alone, and most of the HA protein can be detected in the first cycle.

Measurement of IFV using unmodified and peptide-modified BDD electrodes Cells were assembled with peptide-immobilized electrodes (peptide charge x 0.01), and the background with PBS was measured for 3 cycles at 3 locations. Thereafter, 1 mL (200 pfu) of a 200 pfu / mL virus solution was added to adjust the electrode part so that it was immersed and allowed to interact for 15 minutes. After 15 minutes, the virus solution was removed and the cells were washed with PBS three times. The cells were filled with PBS, and cyclic voltammetry measurement was performed at three positions for 3 cycles each.

The results are shown in FIG. The left side of FIG. 4 shows IFV measurement in solution when using a diamond electrode not modified with peptide. An increase in current density was observed in the 200 pfu IFV solution. The right side of FIG. 4 shows the result of using a peptide-modified diamond electrode. A marked increase in current density was observed compared to an unmodified diamond electrode, and IFV could be detected. In this way, IFV could be detected with high sensitivity.

Electrochemical impedance (EIS) measurement method HA and IFV detection by EIS measurement using peptide-modified BDD electrodes The conditions for impedance measurement were based on SK Arya et al., Sens. Actuators, B, 194, 127-133 (2014). .

First, a redox substance solution was prepared. K ₃ [Fe (CN) ₆ ] 0.164 g (0.5 mmol) and K ₄ [Fe (CN) ₆ ] 0.211 g (0.5 mmol) were weighed separately in a volumetric flask, dissolved in 50 mL of PBS, and 10 mM K ₃ [ Fe (CN) ₆ ] / PBS and 10 mM K ₄ [Fe (CN) ₆ ] / PBS were prepared. These were mixed at 1: 1 (v / v) to give 5 mM [Fe (CN) ₆ ] ^{3- / 4-} / PBS.

Next, the control PC was set as shown in Table 11. Since the initial potential (E) Start) uses the potential (natural potential) already generated when the working electrode, counter electrode, and reference electrode are attached, it was input as needed while checking the value displayed on the control PC. Sampling number (Frequency), frequency domain (Frequency Scan), amplitude (Amplitude), etc. were determined from S. K. Arya et al., Sens. Actuators, B, 194, 127-133 (2014).

Similar to the CV measurement, using the three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl), each concentration of HA solution can be exchanged while the cells are assembled as shown in the schematic diagram of FIG. Acted. First, a cell was assembled with a peptide-immobilized electrode (peptide charge × 100), 5 mM [Fe (CN) ₆ ] ^{3− / 4−} / PBS was added, and the background was measured for 3 cycles. Thereafter, about 60 μL of an HA solution diluted with PBS (−) was added to the cell with PBS to adjust so that the electrode portion was immersed, and allowed to interact for 15 minutes. After 15 minutes, the solution was removed and washed with PBS three times, and 5 mM [Fe (CN) ₆ ] ^{3− / 4−} / PBS was filled in the cell and measured for 3 cycles. This operation was performed with HA solution (5 to 500 nM, 1 to 100 μg / mL, respectively), IFV solution (1 to 140 pfu), and bovine serum albumin (BSA) solution (5 to 500 nM).

The results of HA EIS measurement using a diamond electrode are shown in FIGS. HA could be detected specifically. In FIG. 6, there was no correlation between concentration and response for the irrelevant protein BSA (control), whereas the response for HA was well proportional to the concentration. From these results, HA could be detected with high sensitivity and specificity.

FIG. 7 shows the results of IFV EIS measurement using a diamond electrode. R _ct increases linearly with 40pfu or less, could be detected IFV concentration-dependent manner. In addition, it was possible to detect with high sensitivity even in the low viral load region of 0-40pfu.

Comparative Example 1 Detection of hemagglutinin protein (HA) and influenza virus (IFV) using a glassy carbon (GC) electrode Modification of the glassy carbon (GC) electrode will be described below.

(1) Presentation of alkynyl group (electrolytic grafting)
Onto the surface of the glassy carbon (GC) electrode, the linker molecule _{TIPS-Eth-Ar-N 2} + BF 4 - Immobilization was carried out as in the case of the diamond electrode surface. As a pretreatment of the GC electrode, hydrogen termination of the electrode surface was performed to increase the surface reactivity. This was done by irradiating plasma on both sides of the GC using the microwave plasma CVD apparatus used for diamond electrode synthesis.
(A) Preparation of electrode and solution The cell, O-ring, Pt wire, and GC electrode were sonicated with H ₂ O and acetone for 5 minutes each. First, 3.87 g (0.01 mol) of TBAPF ₆ was weighed into a volumetric flask, and AN was added to prepare a 100 mM TBAPF ₆ solution. Cells were assembled as shown in FIG. The three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used for CV measurement.
(B) Reaction between linker molecule and GC electrode (electrolytic grafting)
Volumetric flask _{TIPS-Eth-Ar-N 2} + BF 4 - to have weighed 18.6mg (0.05mol), 10mM TIPS- Eth-Ar-N 2 + BF 4 dissolved in an electrolytic solution TBAPF ₆ / AN5mL (100mM) ^- A solution was made. This whole quantity was put into the cell. Under the measurement conditions shown in Table 12, cyclic voltammetry was performed for 5 cycles at −0.7 to +0.6 V.

(C) Deprotection of TIPS group After the electrografting of (B) above, 9.5 mL of THF and 0.5 mL of TBAF in THF (1 mol / L) were added, and left for 20 minutes to deprotect the TIPS group.

Immobilization of linker molecules by electrolytic grafting was confirmed by cyclic voltammogram. Only in the first cycle, a cyclic voltammogram in which a reduction peak was observed in the vicinity of -0.3 V (vs. Ag / AgCl) could be measured. Thus, the linker molecule could be fixed on the glassy carbon electrode by electrolytic grafting.

Furthermore, CV measurement with ferrocene, a redox substance, was performed before and after fixing the linker molecule and deprotecting the TIPS group. From this, it was confirmed that deprotection was performed in addition to the immobilization of the linker molecule.

(2) Immobilization of peptides (Husgen cycloaddition reaction)
The GC electrode presenting an alkynyl group was sonicated with Milli-Q (registered trademark) water, EtOH, and acetone for 5 minutes each. Reaction solutions were prepared so that the amount of peptide charged was 100 times (peptide concentration: 0.1 μM) or the amount of peptide charged was 0.01 times (peptide concentration: 0.01 nM) (see Table 13). A GC electrode was immersed in each reaction solution and reacted at room temperature with shaking for 24 hours. After 24 hours, the reaction solution was removed and sonicated with Milli-Q (registered trademark) water, EtOH, and acetone for several seconds each. The electrode was dried by blowing N ₂ gas, and stored under reduced pressure in a desiccator.

Detection of HA using an unmodified glassy carbon (GC) electrode and a modified GC electrode In the CV measurement of an HA solution and an IFV solution at an unmodified diamond electrode, 1.0 V (vs Ag / AgCl) depends on the concentration of either solution. An increase in the oxidation current value was observed. Furthermore, in HA, an oxidation peak thought to be derived from amino acids also appeared in the vicinity of 0.8 V (vs Ag / AgCl). Therefore, CV measurement was performed on a glassy carbon (GC) electrode using the same sample, and the difference between the electrodes was compared.

(1) CV measurement of HA using unmodified GC electrode Unmodified GC electrode, cell, O-ring and Pt line were sonicated for 5 minutes each with Milli-Q (registered trademark) water and acetone. The cell was assembled, PBS was filled in the cell, measurement was performed under the conditions shown in Table 14, and the electrode was cleaned (cleaning treatment). Next, about 3 mL of PBS (−) was added to the cell, and 5-cycle CV measurement was performed under the measurement conditions shown in Table 14. Prepare approximately 3 mL each of H1 type HA (A / New Caledonia / 20/99 (H1N1)) solution serially diluted with PBS (-), 50, 125, 250, 375, 500 nM (10, 25, 50, (75, 100 μg / mL) in order of CV measurement at each concentration for 3 cycles.

Measured conditions for the HA solution were 0V (vs Ag / AgCl), sweeping started (E Start), 0V to 1.0V (Vertex 1,2), and 1 cycle (N Scans) measurement. E Steps represents the data acquisition width.

(2) CV measurement using peptide-modified glassy carbon electrode First, a cell was assembled with a GC electrode having a peptide charge of 0.01 times, and the background was measured with PBS for 3 cycles. Thereafter, 50 μL of 500 nM HA / PBS was added to the cell so that the electrode portion was immersed, and the cells were allowed to interact for 30 minutes. After 30 minutes, the HA solution was removed and washed 3 times with PBS, and the cell was filled with PBS to perform CV measurement. These operations were performed at several locations on the electrode, and the interaction was observed.

The results of the above (1) and (2) are shown in FIG. The left side of FIG. 8 is a cyclic voltammogram of the HA solution at an unmodified GC electrode. The unmodified GC electrode had a large background, and the oxidation current derived from HA as observed with the diamond electrode could not be observed. The right side of FIG. 8 shows the measurement results of HA when using a peptide-modified GC electrode. Even when comparing before HA interaction (PBS) and after interaction, there was no significant change in cyclic voltammogram. In the GC electrode, the background of PBS was larger than that of the diamond electrode (about 0 to 10 μA / cm ^{2 for the} diamond electrode), and it is considered that the oxidation current derived from HA was buried in the background and was not detected.

Detection of IFV using unmodified glassy carbon (GC) electrode and modified GC electrode
(1) IFV CV measurement using an unmodified GC electrode Dispense 10 μL each of IFV (A / PR / 8/34 (H1N1)) that has been frozen and stored, and dissolve in 2.5 mL of PBS. 4000 pfu / mL. After measuring the background with PBS for 3 cycles under the conditions shown in Table 14, the concentration was greatly changed to 20 pfu, 200 pfu, and 4000 pfu (10 ¹ to 10 ³ orders), and 2.5 mL each was put into the cell. Cycled.

(2) IFV CV Measurement Using Peptide-Modified GC Electrode First, a cell was assembled with electrode 1-3 (× 0.01 peptide charge), and the background was measured with PBS for 3 cycles. Thereafter, about 50 μL of IFV / PBS was added to the cell so that the electrode portion was immersed, and the cells were allowed to interact for 30 minutes. After 30 minutes, the IFV solution was removed, and the cells were washed with PBS three times. The cells were filled with PBS, and cyclic voltammetry measurement was performed for 3 cycles.

The results of the above (1) and (2) are shown in FIG. The left side of FIG. 9 is a cyclic voltammogram of the IFV solution at an unmodified GC electrode. The unmodified GC electrode had a large background, and the oxidation current derived from IFV as observed with the diamond electrode could not be observed. The right side of FIG. 9 shows IFV measurement results when using a peptide-modified GC electrode. No significant oxidation current was observed in the range of 0 to 1.0 V (vs Ag / AgCl). Even when comparing before IFV interaction (PBS) and after interaction, there was no significant change in the cyclic voltammogram. It is considered that the background of the PBS electrode was larger than that of the diamond electrode (about 0 to 10 μA / cm ^{2 for the} diamond electrode), and the oxidation current derived from IFV was buried in the background and was not detected.

HA detection by EIS measurement using peptide-modified GC electrode A solution of redox substance [Fe (CN) ₆ ] ^{3− / 4−} / PBS was prepared. Table 15 shows the EIS measurement conditions.

Similarly to the CV measurement, a three-electrode method (working electrode: diamond electrode, counter electrode: Pt, reference electrode: Ag / AgCl) was used, and HA solutions of various concentrations were allowed to interact while the cells were assembled. First, cells were assembled with a GC electrode (peptide charge amount 100 times), 5 mM [Fe (CN) ₆ ] ^{3- / 4-} / PBS was added, and the background was measured for 3 cycles. Thereafter, 50 μL of 5 nM HA / PBS was added to the cell with PBS to adjust so that the electrode portion was immersed, and allowed to interact for 15 minutes. After 15 minutes, the HA solution was removed and washed with PBS three times. The cells were filled with 5 mM [Fe (CN) ₆ ] ³⁻⁴⁻ / PBS and measured for 3 cycles. This operation was performed at HA concentrations of 5, 50, 125, 250, 375, and 500 nM (1, 10, 25, 50, 75, and 100 μg / mL, respectively).

The results are shown in FIG. The left side of FIG. 10 shows a Nyquist plot in which the impedance at each frequency is plotted. From this, the charge transfer resistance _Rct is approximated as a semicircular radius, and the analysis result is shown in the center of FIG. Also in EIS measurement using a GC electrode, the _Rct value increased depending on the HA concentration (left and center in FIG. 10). However, the increase in R _ct value did not become linear (right side of FIG. 10). Furthermore, the same experiment was performed with BSA, and the state of change in _Rct value was compared with HA (right side of FIG. 10). There was no significant difference from the interaction with BSA, and the _Rct value was higher in BSA at low concentrations. From the above, it was not possible to distinguish HA and BSA significantly with a peptide-immobilized electrode using a GC electrode.

Comparative Example 2 Detection of influenza virus by ELISA Peptide s2 (1-5) was used as an influenza virus recognition device. Briefly, a peptide lipid in which a lipid was bound to a peptide having 5 amino acid residues (SEQ ID NO: 1) was synthesized. A peptide lipid membrane was formed using this peptide lipid. The interaction between this peptide lipid membrane and influenza virus was evaluated by ELISA.

First, a 5-residue peptide (SEQ ID NO: 1) introduced with an azide group and DPPE (dipalmitoylphosphatidylethanolamine) introduced with an alkynyl group are coupled by a Huisgen cycloaddition reaction to form a peptide lipid (hereinafter referred to as pep-DPPE). Was synthesized.

Next, a peptide-immobilized membrane was prepared using the synthesized pep-DPPE. Lipid molecules such as pep-DPPE and dioleoylphosphatidylcholine (hereinafter DOPC) were spread on the water surface using a Langmuir trough and compressed. 1-palmitoyl-2-oleoylphosphatidylcholine (hereinafter referred to as POPC) -coated mica was submerged to produce a peptide-immobilized membrane, and the formation of the peptide-immobilized membrane was observed by atomic force microscopy (AFM) in the liquid phase. confirmed. This lipid was accumulated on a plastic plate to form a peptide-immobilized membrane, where influenza virus interacted at room temperature for 1 hour, and the interaction between the peptide lipid membrane and influenza virus was evaluated by ELISA and PCR.

Pep-DPPE / DOPC (50:50) mixed membrane (peptide-immobilized membrane) prepared on a plastic plate interacts with H1N1 and H3N2 influenza viruses for 1 hour at room temperature, and then primary antibody and HRP-labeled secondary antibody Then, the absorbance was measured.

The results are shown in FIG. The vertical axis represents the absorbance at 492 nm, and the horizontal axis represents the viral load (pfu). From FIG. 11, when H1N1 interacted with 1600 pfu (left) and H3N2 interacted with 440 pfu (right), a difference from the control was observed. That is, in the ELISA measurement using the peptide s2 (1-5) (SEQ ID NO: 1), the detection limit of H1N1 virus was 1600 pfu and the detection limit of H3N2 virus was 440 pfu.

Example 5 Detection of IFV using a BDD electrode
1. Preparation of peptide modified BDD electrode and Lys modified BDD electrode
(1) Peptide-modified BDD electrode A diamond electrode presenting an alkynyl group produced by the method described in Example 3 was immersed in a click reaction solution prepared with the composition shown in the table below, and the reaction was performed while shaking overnight at room temperature. I let you. In the table, TBTA was a reaction accelerator, the peptide was 1 μM (× 100), and the solvent was water only. Under these conditions, the peptide immobilization density on the BDD electrode surface was 3.6 pmol / cm ² . Thereafter, the reaction solution was removed, and sonication was performed in Milli-Q (registered trademark) water for several seconds, and then the electrode was dried by blowing N ₂ gas, and stored at 4 ° C. in a sealed container containing silica gel.

(2) Lys-modified BDD electrode A click reaction solution containing Fmoc-Lys- (N ₃ ) (MW: 390.0) at the same concentration instead of the peptides listed in Table 16 was prepared. A diamond electrode presenting an alkynyl group was immersed in this click reaction solution and reacted while shaking overnight at room temperature. Thereafter, about 5 mL of 20% PIP / DMF was added for de-Fmoc treatment, and then the reaction solution was removed, followed by sonication for several seconds each in DMF and Milli-Q (registered trademark) water. Next, N ₂ gas was blown onto the substrate and dried, and the obtained electrode was stored at 4 ° C. in a sealed container containing silica gel.
2. EIS Measurement Method EIS measurement was performed according to the procedure described in Example 4. The EIS measurement conditions are as shown in Table 11. EIF measurement was performed on IFV H1N1 and IFV H3N2 as an IFV solution (1 to 140 pfu).
3. Results The results are shown in FIG. The left is the result of IFV H1N1 subtype, and the right is the result of IFV H3N2 subtype. When IFIS EIS measurement was performed using the peptide-modified BDD electrode prepared by the above procedure, signals were observed in both H1N1 and H3N2 subtypes, and viruses could be detected effectively over a wide range of virus pfu. .

From these results, a person skilled in the art understands that any virus capable of binding to a peptide can be similarly detected using the method and apparatus of the present invention.

Example 6 IFV detection using a 4-branched modified peptide-modified electrode Next, a 4-branched peptide was used. The structure of the 4-branched peptide ((ARLPR) ₂ -K) ₂ -KN ₃ is as follows.

Method for synthesizing 4-branched peptide A 4-branched peptide ((ARLPR) ₂ -K) ₂ -KN ₃ was synthesized by the following procedure. Residue elongation was performed using an automatic peptide synthesizer PSSM-8 system (Shimadzu Corporation). 13 mg (5 μmol) of Fmoc-Lys (N ₃ ) —NH—SAL-resin (amino acid introduction rate 0.38 mmol / g) was added to the reaction vessel, and a reaction vessel insert was inserted. Reagents (Fmoc-AA-OH, HOBt / DMF, NMM / DMF, PIP / DMF) and a reaction vessel were set, and amino acid elongation was performed.

After the amino acid elongation, the reaction solution was removed with an aspirator, and 1 mL of DMF was poured into the reaction column, shaken gently, and then washed with the aspirator for 4 times. Furthermore, the operation of pouring 1 mL of methanol and removing with an aspirator was repeated 5 times, and the operation of pouring 1 mL of t-butyl methyl ether and removing with an aspirator was repeated twice. The reaction column was shielded from light with aluminum foil and parafilm and vacuum dried for 3 hours.

<Cleavage>
First, a cocktail solution was prepared with a composition of 950 μL of TFA, 25 μL of TIS (triisopropylsilane), and 25 μL of H ₂ O. This was placed in a reaction column containing the peptide, and allowed to react for 2 hours on ice under light-shielded conditions. Thereafter, the lid of the reaction column was removed, the pressure was applied from the top of the container, the contents of the reaction column were dropped into a 15 mL centrifuge tube, and washed twice with 200 μL of TFA. After adding 2 mL of cold diethyl ether (without peroxide) and confirming that precipitation was possible, the volume was raised to 10 mL and stirred by vortexing. Subsequently, after centrifugation at 3500 rpm for 1 minute, the supernatant was removed, and the volume was again made up to 10 mL with cold diethyl ether. This operation was repeated 5 times. N ₂ gas was blown onto the peptide pellet remaining in the centrifuge tube to remove cold diethyl ether, and a crude peptide was obtained.

<Mass purification>
Using an ODS-3 (φ20 × 250 mm) column for fractionation, fractionation was performed under the following elution conditions determined from the analysis results by HPLC. At this time, the flow rate was 10 mL / min and the recovery was 0.25 min / tube.
Gradient: B conc. 0% / 0 → 10 min, 0 → 100% / 10 → 30 min, 100% / 31 → 50 min (cleaning)

For each fraction (0.25 min / tube) collected by HPLC, it was confirmed whether the target peptide was obtained using a MALDI-TOF MS analyzer. The fraction containing the target peptide was lyophilized and subjected to final analysis using HPLC and MALDI-TOF MS apparatus (UltraflexTM, Bruker Daltnics) to determine the yield. The result of HPLC is shown in FIG. Yield: 5.7 mg (total for two batches, 1.9 μmol), yield: 19%, purity:> 97%, measured mass 2932.30 ([M + H] +, theoretical value 299.991).

1. Preparation of peptide-modified BDD electrode Next, the peptide-modified BDD electrode was obtained in the same manner as in Example 5 using the 4-branched peptide ((ARLPR) ₂ -K) ₂ -KN ₃ obtained as described above. Was made. The reaction between the 4-branched peptide and the BDD electrode was carried out under the conditions shown in Table 16. However, ((ARLPR) ₂ -K) ₂ -KN ₃ was used at 1 μM instead of (ARLPR) ₂ -K-KN ₃ .
2. EIS measurement method EIS measurement was performed according to the procedure described in Example 4 using the obtained 4-branched peptide-modified BDD electrode. The EIS measurement conditions are as shown in Table 11. EIF measurement was performed on IFV H1N1 as an IFV solution (1 to 140 pfu).
3. Results Even when EIS measurement of IFV was performed using a 4-branched peptide-modified BDD electrode, a signal was observed in the same manner as in Example 5, and influenza virus could be detected.

Summary As described above, 20 pfu influenza virus and 3 pfu influenza virus could be detected by using the apparatus of the present invention. Non-specific binding of proteins existing in the living body such as albumin was not observed, and high-sensitivity detection was possible without using a sensitizer-labeled antibody that was essential in the conventional method. When a GC electrode was used as a comparative example, the noise was so great that influenza virus binding could not be detected. As another comparative example, when the ELISA method was used, the detection limit of influenza virus was 1600 pfu or 440 pfu. From the results of these comparative examples, the usefulness of the conductive diamond electrode of the present invention was shown.

Brief Description of Sequence SEQ ID NO: 1 Peptide s2 (1-5) (ARLPR)
Sequence number 2 peptide s2 (ARLPRTMVHPKPAQP)
Sequence number 3 GLAMAPSVGHVRQHG
SEQ ID NO: 4 GLAMAPSVGHVRQHG (however, the serine residue in the sequence is modified with N-acetylgalactosamine through an O-glycoside bond)

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (10)

A conductive diamond electrode with an element that recognizes proteins or pathogens immobilized on its surface. Elements that recognize proteins or pathogens are influenza virus, DNA virus, RNA virus, double-stranded DNA virus, single-stranded DNA virus, double-stranded RNA virus, single-stranded RNA (+) strand virus, single-stranded RNA (-) Strand virus, single-stranded RNA reverse transcription virus, double-stranded DNA reverse transcription virus, norovirus, rotavirus, rubella virus, measles virus, RS virus, herpes virus, hepatitis virus, adenovirus, foot-and-mouth disease virus, rabies virus , An element that recognizes human immunodeficiency virus, Mycoplasma, Clostridium botulinum, Bordetella pertussis, Tetanus, Diphtheria, Vibrio cholerae, Shigella, Bacillus anthracis, pathogenic Escherichia coli, Staphylococcus, Salmonella, Clostridium perfringens, or Bacillus cereus The electrode according to claim 1. The electrode according to claim 1 or 2, wherein the element that recognizes a protein or pathogen has a peptide that recognizes a pathogen protein. Elements that recognize proteins or pathogens include influenza virus hemagglutinin protein (HA), influenza virus neuraminidase (NA), M1 protein or M2 protein, mycoplasma bacterial P1 protein, membrane antigen protein or ribosomal protein L7 / L12, botulinum toxin Pertussis toxin, tetanus toxin, diphtheria toxin, alpha toxin of C. perfringens, cholera toxin, verotoxin, anthrax toxin, enterotoxin from Escherichia coli, enterotoxin from staphylococci, enterotoxin from Salmonella or enterotoxin from Bacillus cereus The electrode according to any one of claims 1 to 3, comprising: The element that recognizes a protein or pathogen has a molecule that recognizes the protein or pathogen and a linker moiety, and the molecule that recognizes the protein or pathogen is immobilized on the surface of the diamond electrode via the linker moiety. Item 5. The electrode according to any one of Items 1 to 4. An apparatus for detecting a protein or pathogen, comprising the electrode according to any one of claims 1 to 5. A method for detecting a protein or a pathogen using the electrode according to any one of claims 1 to 5 or the apparatus according to claim 6. The detection method according to claim 7, wherein the protein or pathogen is detected by cyclic voltammetry measurement or electrochemical impedance measurement. A method for producing a conductive diamond electrode for protein or pathogen detection, comprising a step of immobilizing a molecule that recognizes a protein or pathogen on the surface of the conductive diamond electrode to form an element that recognizes the protein or pathogen. The production method according to claim 9, comprising a step of immobilizing a linker molecule on the surface of the diamond electrode and a step of linking the linker molecule and a molecule that recognizes a protein or pathogen.

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