JP2016075511A - Electrochemical detection method of specimen using aptamer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a quick and quantitative detection method and detection means with respect to various specimens, and to provide a method for creating means for detecting a specimen quickly and specifically.SOLUTION: There are provided a method and means for detecting a specimen. To put it concretely, this invention is related to a method, a kit and a sensor, for detecting a specimen by using aptamer bonding specifically to the specimen and polymerizing electrochemically therewith. A creation method of such aptamer is also provided.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method and means for detecting a test object. Specifically, the present invention relates to a method, a kit, and a sensor that detect an analyte using an aptamer that specifically binds to the analyte and electrochemically polymerizes. The present invention also relates to a method for producing such an aptamer.

  Detection of a test object such as a virus is routinely performed in fields such as medicine, food, and the environment. For example, detection of viruses including influenza virus is currently performed mainly by immunochromatography. This method has the problem that it is qualitative and time-consuming and cannot be quantitatively measured.

  Electrochemical sensing is also used, and the most successful example is a glucose sensor (for example, Patent Document 1). In addition to such enzyme detection, sensors based on antibodies or aptamers have been developed. While enzyme detection is limited to specific substrates, these sensor probes can be prepared for any target. For example, many antibodies and aptamers against influenza have been reported (Non-patent Document 1). However, antibodies and aptamers alone bind to influenza and have no other function. Also, since antibodies and aptamers have unique binding affinity, the detection process requires conjugation with a signal moiety and a washing process.

On the other hand, a sensor using a conductive polymer (a polymer colored when polymerized) has been reported (Non-patent Document 2). For example, Non-Patent Document 3 reports the design and development of a novel label-free DNA sensor based on conductive poly (3,4-ethylenedioxythiophene) for direct detection and quantification of target ssDNA. Yes. Non-Patent Document 4 synthesizes a poly (3,4-ethylenedioxythiophene) (PEDOT) / iron oxalate composite for a photoelectrochemical sensor of dissolved oxygen by electrochemical polymerization. Non-Patent Document 5 constructs Ag nanoparticle (NP) modified PEDOT nanotubes (NTs) using metal ion reduction mediated vapor deposition polymerization (VDP) for chemical sensors. Ag NPs / PEDOT NTs efficiently sense NH ₃ due to increased surface area and improved conductivity due to oxidation of PEDOT by Ag NPs.

Japanese Unexamined Patent Publication No. 2014-6155

Woo, H.M. et al., Antiviral Res. 100 (2): 337-345, 2013 Sikes, H.D. et al., Nature Materials 7 (1): 52-56, 2008 Krishnamoorthy, K. et al., Chem. Commun. 7: 820-821, 2004 Bencsik, G. et al., Analyst, 135 (2): 375-380, 2010 Park, E. et al., J. Mater. Chem. 22: 1521-1526, 2012

  An object of the present invention is to provide a rapid and quantitative detection method and detection means for various specimens. Another object of the present invention is to provide a method for producing a means for detecting a test object quickly and specifically.

  As a result of intensive studies to solve the above problems, the present inventor detects a target simply and quickly without requiring complicated steps by providing an aptamer serving as a target sensor probe with a binding function and a signal function. It has led to the development of methods and means that can. Specifically, a target-specific aptamer having a monomer group that can be electrochemically polymerized as a signal moiety and becomes conductive when it becomes a polymer is selected, and the interaction with the target is used to change the polymerization behavior of the aptamer. It was found that the target can be detected quantitatively.

That is, the present invention includes the following.
[1] A method for detecting a test substance in a test sample,
A step of bringing an aptamer that specifically binds to a test substance and undergoes electrochemical polymerization into contact with a test sample on or between the electrodes,
A method for detecting a test object, comprising: applying a voltage to an electrode and measuring a current.
[2] The method according to [1], wherein the test substance is at least one selected from the group consisting of viruses, bacteria, fungi, protists, natural molecules, and synthetic molecules.
[2-1] The method according to [1] or [2], wherein the test substance is an influenza virus.
[3] The method according to [1] or [2], wherein the aptamer is at least one selected from the group consisting of a protein aptamer, a peptide aptamer, a peptide nucleic acid aptamer, and a nucleic acid aptamer.
[3-1] The aptamer consists of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 and The method according to [1] or [2], comprising a peptide that specifically binds to influenza virus.
[4] The method according to any one of [1] to [3], wherein the aptamer includes an electrochemical polymerizable group having at least one selected from the group consisting of thiophene, aniline, pyrrole, and acetylene.
[4-1] The method according to any one of [1] to [3], wherein the aptamer includes an electrochemically polymerizable group having thiophene.
[5] The method according to [4], wherein the electrochemically polymerizable group is 3,4-ethylenedioxythiophene (EDOT).
[6] In any one of [1] to [5], when the current is increased as compared with a control sample not containing a test substance, the test sample contains the test substance. the method of.
[7] If the current is reduced compared to a control sample containing a test substance, indicating that the test sample contains no test substance or contains less test substance than the control sample. The method according to any one of [1] to [5].
[7-1] The method according to any one of [1] to [7], wherein the electrode is a gold electrode.
[8] An analyte detection kit comprising an aptamer that specifically binds to an analyte and electrochemically polymerizes.
[9] An analyte binding means including an aptamer that specifically binds to an analyte and electrochemically polymerizes;
A test object sensor comprising: a detection means including an electrode.
[10] A peptide aptamer that specifically binds to influenza virus and electrochemically polymerizes.
[10-1] A peptide comprising the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and A peptide aptamer that electrochemically polymerizes, comprising a peptide that specifically binds to it.
[11] An influenza virus detection kit comprising the peptide aptamer according to [10-1].
[12] Influenza virus binding means comprising the peptide aptamer according to [10-1],
An influenza virus sensor comprising: detection means including an electrode.
[13] A method for producing an aptamer that specifically binds to an analyte and undergoes electrochemical polymerization,
Preparing an aptamer containing an electrochemically polymerizable group;
A method for producing an aptamer, comprising the step of confirming that the aptamer does not undergo electrochemical polymerization in the presence of a test substance and undergoes electrochemical polymerization in the absence of the test substance.
[14] A method for producing an aptamer that specifically binds to an analyte and undergoes electrochemical polymerization,
Preparing a library composed of molecules containing electrochemically polymerizable groups;
Contacting a molecule of the library with a test substance and selecting a molecule that specifically binds to the test substance;
A method for producing an aptamer, comprising a step of confirming that a selected molecule does not undergo electrochemical polymerization in the presence of a test substance and undergoes electrochemical polymerization in the absence of the test substance.
[14-1] The method according to [14], wherein the aptamer is a peptide aptamer and the library is a peptide library by ribosome display.
[14-2] The method according to [13] or [14], wherein the test substance is at least one selected from the group consisting of viruses, bacteria, fungi, protists, natural molecules, and synthetic molecules.
[14-3] The method according to [13] or [14], wherein the test substance is an influenza virus.
[14-4] The method according to [14], wherein the molecule is at least one selected from the group consisting of proteins, peptides, peptide nucleic acids, and nucleic acids.
[14-5] The method according to [13] or [14], wherein the electrochemically polymerizable group has at least one selected from the group consisting of thiophene, aniline, pyrrole, and acetylene.
[14-6] The method according to [14-5], wherein the electrochemically polymerizable group is 3,4-ethylenedioxythiophene (EDOT).
[15] An aptamer produced by the method according to [13] or [14].

  The present invention provides a rapid and quantitative detection method and detection means for a test object. In addition to the ability of the aptamer to bind to the test object, it also has a signal function to detect the test object without requiring a washing process or a detection process using expensive measuring instruments (SPR or QCM). Is possible. Therefore, the method, kit, and sensor according to the present invention are useful in fields such as medical care, food, and environment where detection of a test object is required.

It is the schematic which shows the detection principle of the test object by the aptamer which electrochemically polymerizes. It is a schematic diagram showing the principle of in vitro selection (in vitro selection) using ribosome display and misacylated tRNA. It is the graph which compared the affinity with respect to the influenza virus of the synthetic | combination peptide. It is the result of having investigated the biological specificity with respect to the influenza virus of the selected EDOT-peptide aptamer by the dot blot. An example of an experimental setup for electrochemical detection is shown. (a) Overall view of actual experimental setup, (b) Corresponding schematic, (c) Partial enlarged image of (a). FIG. 6 is a graph showing the average peak current from square wave voltammograms after polymerization and deposition of various concentrations of peptide # 7 on a gold electrode by cyclic voltammetry. (A) Graph showing average peak current from square wave voltammograms after polymerization and deposition of analyte on gold electrode by cyclic voltammetry. Various concentrations of inactivated influenza virus were detected in the presence of 0.1 mg / mL (1) or 0.2 mg / mL (2) peptide # 7. (B) Graph showing average peak current from square wave voltammogram after polymerization and deposition of analyte on gold electrode by cyclic voltammetry. The results for gelatin (3) and ovalbumin (4) as negative controls are shown. FIG. 6 is a graph showing the integrated density of fluorescent images from various concentrations of FITC-labeled peptide # 7 after polymerization and deposition on gold-coated silicon chips by cyclic voltammetry. An outline of a method for detecting a virus using a peptide aptamer that electrochemically polymerizes is shown.

Hereinafter, the present invention will be described in detail.
The present invention relates to a method for detecting an analyte using an aptamer that specifically binds to the analyte and undergoes electrochemical polymerization. In the present invention, “detection” not only detects the presence / absence of a test substance in a test sample, but also quantitatively detects the test substance, and determines the abundance ratio of the test substance. Including.

  The principle of the present invention is shown in FIG. As shown in FIG. 1, the aptamer used in the present invention can specifically bind to a test substance and undergoes electrochemical polymerization. In the absence of the test substance, the aptamer is electrochemically polymerized and deposited on the surface of the working electrode, and this acts as an insulating layer, so that the current decreases even when a voltage is applied to the electrode. On the other hand, when the analyte is present, the aptamer binds to the analyte, thereby reducing the number of free aptamer molecules, and the electrochemical polymerization of the aptamer on the electrode is inhibited in a concentration-dependent manner. As a result, the insulating layer is not formed, and the current increases when a voltage is applied to the electrode. Therefore, it becomes possible to detect the test substance in the test sample by bringing the aptamer into contact with the test sample on or between the electrodes and measuring the current in the electrode. This principle provides an object-specific electrical sensor.

  As the aptamer used in the present invention, any molecule can be used as long as it specifically binds to an analyte and undergoes electrochemical polymerization. For example, the aptamer is a protein aptamer (such as an antibody), a peptide aptamer, a peptide nucleic acid aptamer, a nucleic acid aptamer (DNA, RNA, DNA / RNA hybrid), or the like.

  The present invention also provides a method for producing an aptamer that specifically binds to such an analyte and undergoes electrochemical polymerization.

  The method for producing an aptamer of the present invention comprises a step of preparing an aptamer containing an electrochemically polymerizable group, and the aptamer does not electrochemically polymerize in the presence of a test substance, and is electrochemical in the absence of the test substance. A step of confirming polymerization. For example, if an aptamer that specifically binds to the analyte is already known, prepare an aptamer containing an electrochemically polymerizable group by binding an electrochemically polymerizable group to any part of the aptamer molecule. can do.

  When an aptamer for a test substance is not known or an aptamer with higher affinity is required, it can be produced by in vitro selection (so-called in vitro selection or in vitro evolution). Specifically, this method comprises a step of preparing a library composed of molecules containing an electrochemically polymerizable group, contacting a molecule of the library with a test substance, and selecting a molecule that specifically binds to the test substance. And a step of confirming that the selected molecule is not electrochemically polymerized in the presence of the test substance and is electrochemically polymerized in the absence of the test object. An example of in vitro selection is shown in FIG. According to this method, an aptamer containing an electrochemically polymerizable group can be easily obtained.

  In order to obtain an aptamer that undergoes electrochemical polymerization, an electrochemical polymerizable group is used. An electrochemically polymerizable group means a group or moiety that can be polymerized electrochemically. Such groups include, but are not limited to, thiophenes such as 3,4-ethylenedioxythiophene (EDOT), 3-methylthiophene, 3-ethylthiophene, 3-butylthiophene, 3-hexylthiophene, Examples include monomers, dimers, and multimers such as 3-octylthiophene, bisthiophene; aniline, pyrrole, and acetylene.

  First, a library composed of molecules containing such electrochemical polymerizable groups is prepared. A library can be appropriately prepared by those skilled in the art depending on the type of molecule used. For example, in the case of producing a peptide aptamer, a ribosome display system (for example, JP 2003-102495 A, JP 2008-271903 A), a phage display system (for example Smith, GP et al., Science 228: 1315-1317, 1985). In brief, the ribosome display system transcribes the cDNA contained in the cDNA library into mRNA in vitro, and translates the mRNA in vitro to a polypeptide in the form of a complex of mRNA, polypeptide and ribosome. By translating the mRNA, the polypeptide is brought into contact with the specimen while maintaining the correlation between the mRNA and the polypeptide, and the polypeptide (ie, mRNA) that specifically binds to the specimen is selected and analyzed. It is a technology that can. To display peptides containing electrochemically polymerizable groups in a ribosome display system, in vitro translation is performed using a misacylated tRNA that carries an amino acid with an electrochemically polymerizable group and has an anticodon corresponding to the amber codon. Preferably it is done. Methods for introducing unnatural amino acids into polypeptides are known (eg, Taira, H. et al., Biochem. Biophys. Res. Commun. 2008, 374, 304-308, Noren, CJ et al., Science 244: 182-188, 1989, Japanese Patent No. 4914704, etc.), peptides containing electrochemically polymerizable groups can be prepared by any method. Thereby, the peptide library comprised from the peptide containing an electrochemically polymerizable group can be prepared.

  In addition, when a nucleic acid aptamer such as DNA or RNA is produced, a DNA library containing a random sequence, a genomic DNA library, a cDNA library, an mRNA library, or the like can be constructed. Incorporation of an electrochemically polymerizable group into a molecule in a library is also known in the art, and a nucleic acid containing an electrochemically polymerizable group by a transcription or amplification reaction using a nucleotide substrate to which an electrochemically polymerizable group has been added. A library composed of molecules can be prepared.

  Subsequently, the molecules of the library are brought into contact with the test object, and molecules that specifically bind to the test object are selected.

  In the present invention, the detection target, that is, the type of the test object is not particularly limited. For example, a test substance can be any material factor, specifically a naturally occurring molecule such as an amino acid, peptide, oligopeptide, polypeptide, protein, nucleic acid, lipid, carbohydrate (such as a sugar), steroid, glyco Peptides, glycoproteins, proteoglycans, etc .; synthetic analogs or derivatives of naturally occurring molecules, such as peptidomimetics, nucleic acid molecules (antisense nucleic acids, etc.); created using non-naturally occurring molecules, such as combinatorial chemistry techniques Low molecular organic compounds (inorganic and organic compound libraries, combinatorial libraries, etc.); and mixtures thereof. Further, the test object may be a single substance, a complex composed of a plurality of substances, a transcription factor, or the like. Further, the test object may be an individual such as a virus, a bacterium, a fungus, or a protist. Further, the test object may be a gas factor such as carbon, nitrogen, or oxygen.

  Conditions for bringing the molecules of the library into contact with the analyte are not particularly limited, and either a liquid phase system or a solid phase system can be used. A person skilled in the art can appropriately determine the conditions for contact according to the type of test substance, the type and form of the molecules in the library (such as the presence or absence of immobilization). For example, library molecules may be added to a solution containing the test substance and brought into contact with each other, or the library molecules may be brought into contact with the immobilized test substance.

  The binding between the molecules of the library after contact and the analyte can be determined according to any method known in the art. For example, when using an immobilized test substance, the library molecules that did not bind to the test substance are removed by washing or the like, and the library molecules are released from the remaining test substance. Bound library molecules can be obtained. Alternatively, the library molecules can be labeled (eg, fluorescently labeled) and contacted with the analyte, and then the library molecules bound to the analyte can be selected based on the label.

  If necessary, the above-described library preparation, contact with the specimen, and selection of the molecules of the library bound to the specimen may be repeated, thereby specifically binding to the specimen. The aptamer to do is refined more.

  Next, the electrochemical polymerization of the selected molecule is confirmed. Specifically, it is confirmed that the selected molecule does not electrochemically polymerize in the presence of the test object, and does electrochemical polymerization in the absence of the test object. For example, before and after bringing the selected molecule into contact with the test object, a voltage is applied on or between the electrodes to which a solution containing the selected molecule and the electrolyte is dropped, and the current is measured. Any compound can be used as the electrolyte, and for example, potassium ferricyanide can be used. The current can be measured by any method known in the art, and for example, cyclic voltammetry, square wave voltammetry, photocurrent spectroscopy, or the like can be used. If the molecule is electrochemically polymerized, the current decreases, and if the molecule is not electrochemically polymerized, there is no change in current or the decrease in current is small.

  In this way, an electrochemical polymerization aptamer for a desired analyte can be selected. The aptamer selected by the selection as described above can be produced by a known method. For example, in the case of a peptide aptamer, the target aptamer is produced by a solid phase synthesis method or the like, and in the case of a nucleic acid aptamer, an amplification reaction (PCR or the like), an oligonucleotide synthesis method or the like. Introduction of the electrochemically polymerizable group into the aptamer can be performed by a method known in the art. Preferably, the electrochemically polymerizable group can be introduced simultaneously with the production of the aptamer. For example, in the case of a peptide aptamer, a peptide aptamer is produced using a protected amino acid to which an electrochemically polymerizable group is bonded in a solid phase synthesis method, or as described above, an electrochemically polymerizable group is bonded. A peptide aptamer containing an electrochemically polymerizable group can be prepared by producing a peptide aptamer by in vitro translation using a misacylated tRNA carrying an amino acid and having an anticodon corresponding to an amber codon. In the case of a nucleic acid aptamer, an electrochemically polymerizable group was added by producing a nucleic acid aptamer by a transcription or amplification reaction using a nucleotide substrate to which an electrochemically polymerizable group was added, or by a solid phase synthesis method. By producing a nucleic acid aptamer using nucleotides, a nucleic acid aptamer containing an electrochemically polymerizable group can be prepared.

  The inventor selected an influenza virus as a test substance and attempted in vitro selection of peptide aptamers. As a result, the present inventors were able to obtain a peptide aptamer that specifically binds to the influenza virus and electrochemically polymerizes. In this peptide aptamer, as shown in Example 4 described later, in the presence of the influenza virus which is a test substance, binding to the influenza virus becomes steric hindrance, and electrochemical polymerization is inhibited. Current increases. On the other hand, in the absence of influenza virus, the peptide aptamer undergoes electrochemical polymerization under electrochemical conditions, and the current is interrupted. The present invention also provides such peptide aptamers.

  Specifically, the peptide aptamer according to the present invention is a peptide aptamer that undergoes electrochemical polymerization, including a peptide having an amino acid sequence represented by HAAXHATPTSG (wherein X represents EDOT-linked aminophenylalanine; SEQ ID NO: 5). . The peptide aptamer according to the present invention is a peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 and specifically binding to influenza virus. A peptide aptamer that undergoes electrochemical polymerization.

  The peptide aptamer according to the present invention can be produced by a known method, for example, a peptide solid phase synthesis method, a method using an in vitro transcription / translation system, a gene recombination method, or the like. The introduction of the electrochemically polymerizable group into the peptide aptamer can be performed as described above, by producing a peptide aptamer using a protected amino acid to which an electrochemically polymerizable group is bound in a solid phase synthesis method, or As described above, the peptide aptamer is produced by in vitro translation using a misacylated tRNA carrying an amino acid to which an electrochemically polymerizable group is bound and having an anticodon corresponding to the amber codon. Peptide aptamers containing groups can be prepared. Whether or not a peptide having an amino acid mutation specifically binds to an influenza virus can also be determined according to any method known in the art, for example, the method described above.

  In the test object detection method according to the present invention, the aptamer described above is first brought into contact with the test sample on or between the electrodes.

  The test sample is not particularly limited as long as it is a sample suspected of containing the test object to be detected, and may be a liquid, solid or gas sample, or a mixed sample thereof. Test samples specifically include biological fluid samples (saliva, nasal discharge, blood, urine, etc.), cell or tissue samples (oral swabs, etc.), environmental samples (seawater, river water, industrial water, soil, etc.) To do. The liquid sample can be used as it is, or diluted or concentrated with a solvent. The solid sample may be suspended in a solvent, homogenized by a pulverizer or the like, or a supernatant obtained by stirring with a solvent may be used. For example, a stick such as a cotton swab may be moistened with sterile water and the measurement location may be wiped, then rinsed in a solvent, and the resulting liquid may be used as a sample. Examples of the solvent include distilled water, physiological saline, phosphate buffer, Tris buffer, and the like. Furthermore, it is also possible to use a filter obtained by filtering a sample with a filter and capturing sample components (cells, bacteria, viruses, etc.). Such a filter is not particularly limited as long as it has a size capable of capturing the test object.

  The electrode is not particularly limited as long as it can perform electrochemical detection. For example, metal materials such as noble metals (gold (Au), silver (Ag), platinum (Pt), palladium (Pd), rhodium (Rh), iridium (Ir), ruthenium (Ru), etc.), copper (Cu) , Aluminum (Al), Tungsten (W), Molybdenum (Mo), Chromium (Cr), Titanium (Ti), Nickel (Ni), etc., alloys such as stainless steel, hastelloy, inconel, monel, duralumin, carbon materials, examples An electrode formed using carbon or graphite (graphene or the like) or an electrode such as a semiconductor element (transistor, FET, or the like) is used. The electrode may have a rod shape, a chip shape, a film shape, a film shape, or the like, and may be formed on an insulator substrate. The size of the electrode can be appropriately set according to the type of analyte to be detected, the detection purpose, etc., for example, 1 mm to 1 cm, or smaller, for example, a micrometer or nanometer size. Is possible.

  The contact between the aptamer and the test sample means that the test object existing in the test sample and the aptamer can be brought into close proximity so that they can be combined. In addition, contact conditions can be appropriately determined according to the type or state of the test object and the type and form of the aptamer. For example, it can be performed by an operation such as adding an aptamer to a test sample solution, or mixing a test sample immobilized on beads or the like with an aptamer solution. The contact between the test substance and the aptamer can be performed at an appropriate temperature (for example, 5 to 100 ° C.) and at an appropriate pH condition (for example, pH 3 to 12).

  Subsequently, a voltage is applied to the electrode and the current is measured. At that time, it is preferable that the test sample and the aptamer are filled with a solution containing an electrolyte so that the aptamer is electrochemically polymerized. Application of voltage and measurement of current can be performed by a known method, for example, with a current change measuring instrument connected to the electrode. The current change measuring instrument may be a measuring instrument having any circuit configuration as long as it is a measuring instrument capable of detecting the intra-electrode electron movement (current) caused by the voltage change. For example, a measuring instrument such as cyclic voltammetry or square wave voltammetry can be used to measure the current change. Here, it is preferable that after the voltage is applied to the electrode to cause electrochemical polymerization, the electrode is washed to wash away the liberated aptamer and the test sample, and then the current is measured.

  The measured current is compared to a control to determine the change in current. For example, the comparison is made with a current obtained using a control sample not containing a test substance, a control sample containing a test substance (preferably a control sample containing a test substance having a known concentration). Therefore, when the current is increased as compared with the control sample not including the test object, it indicates that the test sample includes the test object. On the other hand, if it is lower than the control sample containing the test substance, it indicates that the test sample does not contain the test substance or contains less test substance than the control sample. Preferably, the degree of change in current also depends on the concentration of the analyte, so the current is measured using a set of control samples containing known graded concentrations of the analyte to create a calibration curve Thus, it is possible to quantify the specimen.

  The above-described method for detecting a test object according to the present invention can be easily performed using a kit, a sensor, or a device. The test object detection kit according to the present invention includes an aptamer that specifically binds to the test object and undergoes electrochemical polymerization. The test object detection kit may include an instruction describing a procedure for detecting the test object and a standard (a calibration curve or the like) for quantifying the test object. One aspect of the test object detection kit of the present invention is an influenza virus detection kit. One example of the influenza virus detection kit of the present invention is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 5 or an SEQ ID NO: 5 as an aptamer that specifically binds to a test substance and undergoes electrochemical polymerization. A peptide aptamer that undergoes electrochemical polymerization, comprising a peptide that consists of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence shown, and that specifically binds to influenza virus.

  Further, the test object sensor or the test object detecting device according to the present invention includes a test substance binding means including an aptamer that specifically binds to the test substance and electrochemically polymerizes, and a detection means including an electrode. Are provided. One aspect of the test object sensor or the test object detection device of the present invention is an influenza virus sensor or an influenza detection device. An influenza virus sensor or an influenza detection device according to the present invention includes an influenza virus binding unit including the peptide aptamer and a detection unit including an electrode.

  Since the kit and device according to the present invention can detect a test substance easily, they are useful for various analyses.

  Hereinafter, the present invention will be described more specifically with reference to examples and drawings. However, the following examples do not limit the present invention.

[Example 1] Production of aptamer Ribosome as a selection system for incorporating a non-standard amino acid having 3,4-ethylenedioxythiophene (EDOT) as an electrochemically polymerizable group into a peptide aptamer to produce an aptamer that undergoes electrochemical polymerization Developed a display. Using this, aptamer selection in vitro (in vitro selection) was performed by the method shown in FIG.

(1) Synthesis of EDOT-aminophenylalanyl-tRNA First, in order to obtain a peptide aptamer to be electrochemically polymerized, tRNA having aminophenylalanine bound to EDOT was prepared. First, tBoc-ε-aminophenylalanine is coupled with 5'-O-phosphoryl-2'-deoxycytidylyl- (3'-5 ') adenosine (pdCpA) and EDOT is coupled with aminophenylalanyl-pdCpA I let you. Next, a DMSO solution of EDOT-succinimidyl ester is added to the DMSO solution and aminophenylalanyl-pdCpA (AF-pdCpA) (5 mM, 40 μL) in aqueous pyridine-HCl (5M, pH 5.0, 80 μL). Mixed. After 3 hours incubation at 37 ° C, EDOT-AF-pdCpA was run in reverse phase HPLC (Waters XTerra C18; 2.5 μm, 4.6 mm x 20 mm) in 0.1% trifluoroacetic acid at a flow rate of 1.5 mL / min for 10 minutes Purified using a linear gradient from 0% to 100% acetonitrile. By MALDI-TOFMS (Voyager, Applied Biosystems ), [MH] - give the value of 1010.20 at the calculated value 1010.83, confirmed the product.

The obtained EDOT-AF-pdCpA was obtained from mycoplasma without 3 'dinucleotide by chemical ligation method as previously reported (Taira, H. et al., Biochem. Biophys. Res. Commun. 2008, 374, 304-308). -Ligated to amber suppressor tRNA derived from Mycoplasma capricolum Trp ₁ tRNA (2-base shortened tRNA encoding amber codon). Purified EDOT-AF-tRNAs were frozen and stored at -80 ° C.

(2) Preparation of peptide library As a random sequence library, a random sequence library of DNA containing amber codons, that is, (VVN) ₃ TAG (VVN) ₇ (V represents G, C or A, N represents G, C, T Or represents A) (SEQ ID NO: 1). Specifically, a T7 RNA polymerase primer sequence, an Escherichia coli ribosome binding sequence, a SfiI restriction enzyme sequence, a protein linker sequence, and a plasmid (13Trx) having a ribosome stop sequence are used. Strand DNA (dsDNA) was prepared. Library sequences were obtained from Operon Co. Ltd. (Tokyo, Japan). The sequence is 5′-ATATGGCCATGCAGGCC (VVN) ₃ TAG (VVN) ₇ GGCCAGCTAGGCCAGTT-3 ′ (V represents G, C or A, N represents G, C, T or A equally) (SEQ ID NO: 2) Met. VVN contains only 12 natural amino acids other than hydrophobic amino acids (eg leucine, valine, tryptophan, etc.) and stop codons (TAG, TAA and TGA). This design is for high solubility in aqueous buffers and for incorporating EDOT molecules at the same position in the library sequence without unintentional additional incorporation of EDOT. Perform one cycle of polymerase chain reaction (PCR) using Takara Ex taq DNA polymerase (Takara Bio Inc., Otsu, Shiga, Japan) to prepare dsRNA, and prepare QIAquick PCR Purification kit (Qiagen, Valencia, CA, USA) And purified. The obtained dsDNA and 13Trx plasmid were digested with restriction enzymes (SfiI; New England Biolabs, Ipswich, MA, USA) and ligated (DNA Ligation Kit; Takara Bio). Finally, the double-stranded DNA library was prepared using new primers (New-T7-fp-rec-1: 5′-GTAATACGACTCACTATAGGCCGCGTCGACAATAA-3 ′ (SEQ ID NO: 3) and New-rp-fp-M13-NS: 5′- It was prepared by PCR using GATTACGCCAAGCTGAGTGAGA-3 ′ (SEQ ID NO: 4)). For in vitro transcription of random sequence DNA libraries, transcription was performed at 37 ° C. for 3 hours, and the product was subsequently treated with DNase. MRNA was purified using RNeasy kits (Qiagen).

  Subsequently, using the PURE system Classic II kit (Wako Pure Chemical Industries Ltd., Chuo-ku, Osaka, Japan), in vitro translation was performed in the presence of tRNA having EDOT-bound aminophenylalanine, and an EDOT-containing peptide library Was made. The TAG codon corresponds to EDOT-linked aminophenylalanine. The library is unable to release mRNA or translated peptides due to loss of stop codon. By maintaining at a low temperature, the EDOT-modified peptide that is stably bound as a ribosome-mRNA-peptide complex and displayed on the surface of the ribosome is produced. This is the so-called ribosome display system. The reaction was stopped by placing the mixture on ice for 10 minutes. This translation yield was approximately 10 times lower due to the addition of tRNA.

(3) In vitro selection (in vitro selection)
The translated library solution is immobilized on silica affinity beads (Sumitomo Bakelite co., Ltd, Tokyo, Japan) in a selection buffer (0.1% Tween 20, 50 mM Tris, 150 mM NaCl, 50 mM magnesium acetate). Affinity selection was performed by incubating with activated influenza virus A / California / 07/2009 (provided by H1N1, Denka Seiken co., Ltd, Tokyo, Japan) at 4 ° C. for 1 hour. The virus-immobilized beads are collected by centrifugation at 10,000 g for 5 minutes, and the unbound mRNA-ribosome-peptide complex is washed away at 4 ° C using a washing buffer (50 mM Tris, 150 mM NaCl, 50 mM magnesium acetate). It was. After incubation with ethylenediaminetetraacetic acid (EDTA) for 30 minutes at room temperature, mRNA was recovered from the bound mRNA-ribosome-peptide complex.

  The isolated mRNA was purified using the RNeasy kit. Reverse transcription (RT) -polymerase chain reaction (PCR) was performed to amplify DNA from the recovered mRNA. The DNA product was purified using QIAquick PCR purification kits (Qiagen). The isolated DNA was used as a template for the next round of selection (transcription and translation).

  After 6 rounds of the selection process, the selected DNA sequence was cloned and sequenced. We found 89 sequences from 92 clones.

[Example 2] Peptide synthesis In order to chemically synthesize three peptide sequences that were duplicated from the peptide sequences obtained in Example 1, and to confirm the binding to influenza as described later, fluorescein was added to the peptides. Added (Table 1).

Specifically, the selected peptides # 07, # 81 and # 93 were synthesized by a general Fmoc solid phase method at RIKEN Brain Science Institute. Fmoc-aminophenylalanyl EDOT was synthesized to incorporate EDOT into selected peptide sequences. A ΔTIS cleavage solution containing trifluoroacetic acid: phenol: thioanisole: ddH ₂ O: ethanedithiol in the ratio 85: 5: 4: 4: 2 (v / v) for the cleavage of these peptides from peptide synthesis resins Was used to avoid reduction of the thiophene moiety.

[Example 3] Biological specificity The affinity of the synthesized peptides was tested in the same manner as (3) of Example 1 and compared as shown in FIG. Of the three peptide aptamers, only # 7 showed binding affinity for influenza virus.

  The biological specificity of binding of the selected EDOT-aptamer, peptide # 7, to inactivated influenza virus was further confirmed by dot blot. First, a PVDF membrane (Immobilon-P Transfer Membrane, pore size: 0.45 μm) was immersed in methanol, and then immersed in a blotting buffer (25 mM Tris, 192 mM glycine, 20% (v / v) ethanol). 2 μL of inactivated influenza virus A / California / 07/2009 (provided by H1N1, Denka Seiken co., Ltd, Tokyo, Japan) (128 HA units / μL of milliQ after placing the membrane on filter paper to avoid drying) Diluted 1: 1, 1: 2, 1: 4 and 1: 8 ratios) on the membrane, respectively, while 2 μL of 1.5 mg / mL gelatin and 2 μL of 1.5 mg / mL bovine as negative controls Serum albumin (BSA, Sigma-Aldrich) was added dropwise at the same dilution ratio. After drying, the membrane was again immersed in methanol followed by blotting buffer. Then immerse in blocking buffer (5% w / v ECL Advance blocking agent in TBS-T buffer (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4)), shake at room temperature for 1 hour, Nonspecific binding was blocked. Washed briefly with TBS-T buffer and placed on parafilm to keep wet and 100 μL of 0.2 mg / mL FITC-labeled peptide # 7 was dropped onto the membrane to cover the entire area. Incubate at room temperature in the dark for 1 hour, wash 3 times with TBS-T buffer (5 minutes each), and then obtain an image of the membrane surface using Molecular Imager FX (Bio-Rad Laboratories, Tokyo, Japan) at RIKEN Brain Science Institute did.

  The results are shown in FIG. FIG. 4 shows that peptide # 7 can specifically bind to influenza virus. Negative controls gelatin and BSA did not bind to influenza virus.

[Example 4] Electrochemical detection by sensor Using peptide # 7 having the highest binding, polymerization of peptide # 7 was examined. That is, it was examined whether there was a current change according to the influenza concentration based on the principle shown in FIG.

(1) Manufacture of sensor As shown schematically in Fig. 5, a gold (Au) electrode (OD: 6 mm, ID: 3 mm; ALS Co., Ltd, Tokyo, Japan) was used as the working electrode, and platinum ( A Pt) wire and an Ag / AgCl electrode (RE-1S, ALS Co., Ltd, Tokyo, Japan) were used as a counter electrode and a reference electrode, respectively. (A) of FIG. 5 is an overall view of an actual experimental setting, (b) is a corresponding schematic diagram, and (c) is a partially enlarged image of (a). The gold electrode was first mechanically polished using a 6 μm polishing diamond followed by a 0.05 μm polishing alumina slurry and then thoroughly rinsed with milliQ.

(2) Electrochemical detection The experiment was carried out using autolab (AUTOLAB PGSTAT128N, Eco Chemie BV, Utrecht, Netherlands). As an analyte, a stock solution of peptide # 7 was prepared with milliQ and subsequently diluted to the desired concentration with 0.1 M Tris-HCl buffer (pH 7.0).

After the electrodes were installed (FIG. 5), 20 μL of the analyte was dropped onto the gold electrode so that the analyte touched all the electrodes. Cyclic voltammetry (0 to 1.2 V vs. Ag / AgCl, scan rate 0.1 V / s) was performed for 6 cycles to polymerize peptide # 7 at various concentrations to form a polymer film on the gold electrode. The gold electrode was then immersed in milliQ and gently shaken to remove the remaining analyte solution. Air-dry and drop 20 μL of 5 mM K ₃ [Fe (CN) ₆ ] in 0.1 M Tris-HCl buffer (pH 7.0) onto a gold electrode, and square wave voltammetry ((0.05V, 1s; 0.45V, 1s ), 5 cycles), and the current was detected. The measured values at 6s, 8s and 10s time points are averaged and shown in FIG.

  FIG. 6 shows the average peak current from square wave voltammograms after polymerization and deposition of various concentrations of peptide # 7 on the gold electrode by cyclic voltammetry. This result shows that the current decreased with increasing peptide concentration and eventually saturated (FIG. 6).

  Subsequently, as an analyte, a stock solution of peptide # 7, gelatin and ovalbumin (albumin from chicken egg white, Sigma-Aldrich) was prepared with milliQ, followed by the desired concentration with 0.1 M Tris-HCl buffer (pH 7.0). Dilute to In addition, the concentration of peptide # 7 was fixed at 0.1 mg / mL and 0.2 mg / mL, respectively, and various concentrations of influenza virus were added to the analyte. The results of performing the same electrochemical process as above are shown in FIGS. 7A and 7B.

  FIG. 7A shows the average peak current from a square wave voltammogram after polymerization and deposition of the analyte on the gold electrode by cyclic voltammetry. Various concentrations of inactivated influenza virus (H1N1, Californian) were detected in the presence of a fixed concentration of peptide # 7 (upper graph: 0.1 mg / mL, fitted curve; lower graph: 0.2 mg / mL, direct plot). FIG. 7B shows the results obtained using gelatin and ovalbumin with 0.2 mg / mL peptide # 7 as a negative control. From FIG. 7A, it was found that at both concentrations, as the concentration of influenza virus increased, the current increased correspondingly. This indicates that peptide # 7 binds to influenza virus, reducing the concentration of effective (unbound) peptide # 7 and thus thinning the polymer membrane. As shown in FIG. 7B, such a change did not occur in the presence of a protein such as albumin or gelatin to which peptide # 7 does not bind. The flat curve shows that peptide # 7 was able to specifically detect influenza virus.

  In this way, a quantification protocol for influenza virus in the analyte was established, as shown in FIG.

[Example 5] Confirmation of polymerization The polymerization and deposition of peptide # 7 on the gold electrode surface were confirmed using FITC-labeled peptide # 7.

  In order to meet the measurement requirements of the Molecular Image FX system, a gold-coated silicon chip (5 mm × 10 mm) cut from a gold-coated silicon wafer (Sigma-Aldrich) was used as the working electrode instead of the rod-shaped gold electrode. The experimental settings and cyclic voltammetry for polymerization were the same as in Example 4. Specifically, 20 μL of 0, 0.5, 1 and 1.5 mg / mL FITC-labeled peptide # 7 in 0.1 M Tris-HCl buffer (pH 7.0) was dropped onto a gold-coated silicon chip, and a potentiostat (ALS / CH Six cycles of cyclic voltammetry (0-1.2 V vs. Ag / AgCl, scan rate 0.1 V / s) were performed using Instruments, Electrochemical Analyzer, model 700C, BAS. Thereafter, the chip was immersed in milliQ and gently shaken to remove the remaining FITC-labeled peptide # 7. After air drying, an image of the chip surface was obtained by Molecular Imager FX (Bio-Rad Laboratories, Tokyo, Japan) at RIKEN Brain Science Institute. Figure 8 was constructed using the integrated density of analyte spots by analyzing the fluorescence image with the software ImageJ64.

  FIG. 8 shows that the integrated density of the images increased as the concentration of FITC labeled peptide # 7 increased. This indicates that the peptide aptamer has been successfully polymerized and deposited on the working electrode surface.

  The present invention enables rapid and quantitative detection of a test object. Therefore, the present invention is useful in fields such as medicine, food, and environment where detection of a test object is required.

SEQ ID NO: 1 and 2: Artificial sequence (synthetic oligonucleotide)
SEQ ID NOs: 3 and 4: Artificial sequence (primer)
SEQ ID NOs: 5-7: Artificial sequence (synthetic peptide)

Claims (15)

A method for detecting an object in a sample,
A step of bringing an aptamer that specifically binds to a test substance and undergoes electrochemical polymerization into contact with a test sample on or between the electrodes,
A method for detecting a test object, comprising: applying a voltage to an electrode and measuring a current.   The method according to claim 1, wherein the test substance is at least one selected from the group consisting of a virus, a bacterium, a fungus, a protist, a natural molecule, and a synthetic molecule.   The method according to claim 1 or 2, wherein the aptamer is at least one selected from the group consisting of a protein aptamer, a peptide aptamer, a peptide nucleic acid aptamer, and a nucleic acid aptamer.   The method of any one of claims 1 to 3, wherein the aptamer comprises an electrochemically polymerizable group having thiophene.   The method of claim 4, wherein the electrochemically polymerizable group is 3,4-ethylenedioxythiophene (EDOT).   6. The method according to any one of claims 1 to 5, wherein the test sample contains a test substance when the current is increased compared to a control sample containing no test substance.   Claim 1--indicating that the test sample contains no test substance or less test substance than the control sample if the current is reduced compared to a control sample containing the test substance. 6. The method according to any one of 5 above.   A kit for detecting an analyte, comprising an aptamer that specifically binds to the analyte and electrochemically polymerizes. An analyte binding means comprising an aptamer that specifically binds to the analyte and electrochemically polymerizes;
A test object sensor comprising: a detection means including an electrode.   A peptide consisting of the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and specific to influenza virus A peptide aptamer that electrochemically polymerizes, including a peptide that binds.   An influenza virus detection kit comprising the peptide aptamer according to claim 10. An influenza virus binding means comprising the peptide aptamer according to claim 10;
An influenza virus sensor comprising: detection means including an electrode. A method for producing an aptamer that specifically binds to an analyte and undergoes electrochemical polymerization,
Preparing an aptamer containing an electrochemically polymerizable group;
A method for producing an aptamer, comprising the step of confirming that the aptamer does not undergo electrochemical polymerization in the presence of a test substance and undergoes electrochemical polymerization in the absence of the test substance. A method for producing an aptamer that specifically binds to an analyte and undergoes electrochemical polymerization,
Preparing a library composed of molecules containing electrochemically polymerizable groups;
Contacting a molecule of the library with a test substance and selecting a molecule that specifically binds to the test substance;
A method for producing an aptamer, comprising a step of confirming that a selected molecule does not undergo electrochemical polymerization in the presence of a test substance and undergoes electrochemical polymerization in the absence of the test substance.   An aptamer produced by the method according to claim 13 or 14.

Images