WO2006033400A1

WO2006033400A1 - Dna detection method and reporter probe

Info

Publication number: WO2006033400A1
Application number: PCT/JP2005/017501
Authority: WO
Inventors: Shinichiro Sue; Hideo Katayama
Original assignee: Daikin Industries, Ltd.
Priority date: 2004-09-22
Filing date: 2005-09-22
Publication date: 2006-03-30
Also published as: JPWO2006033400A1

Abstract

An electrode where a capture probe (28a) is connected to the surface of a carbon electrode (21), a reporter probe (28c) labeled with an L-proline dehydrogenation enzyme (26), and an L-proline (25) serving as a substrate reactive with the L-proline dehydrogenation enzyme (26) are used. A target DNA (28b) is connected to the capture probe (28a) and the reporter probe (28c). By measuring the current value of the limiting oxidation current flowing through the carbon electrode (21) because of the reaction between the L-proline dehydrogenation enzyme (26) with which the reporter probe (28c) is labeled and the L-proline (25), the target DNA (28b) is detected.

Description

Specification

DNA detection method and reporter probe

Technical field

[0001] The present invention relates to target DNA by binding a target DNA using an electrode in which a capillary probe is bound to the surface of a conductor and a reporter probe labeled with an enzyme, and measuring the current value. Background Art Related to DNA Detection Method and Reporter Probe

[0002] Conventionally, in a notch-type system, a combination of two types of oxidoreductases and a coenzyme to quantitate a substrate (for example, a substance in a living body) with high sensitivity and measure enzyme activity. Has been established (for example, see Non-Patent Document 1).

[0003] When DNA is detected by sandwich hybridization using this method, an enzyme is labeled on the reporter probe, and electron exchange based on the enzyme reaction is measured. According to this method, it is necessary to denature the target DNA into a single strand for hybridization, but in order to increase the efficiency of hybridization, a high temperature of 90 ° C or higher is required. Is required.

[0004] On the other hand, L-proline dehydrogenase derived from Thermococcus profundus DS M9503, a hyperthermophilic bacterium, was purified and the activity of this L-proline dehydrogenase was demonstrated. (For example, see Non-Patent Document 2.)

Non-patent literature 1: Biochemistry experiment course, 5th V, Enzyme research method (Tokyo Chemical Doujin) P121-129 Non-patent literature 2: Haruhiko Sakuraba, Yoshinori Takamatsu, Takenon Satomura ), Ryusm Kawakami, Toshihisa Ohshima, “Applied and Environmental Microbiology” (USA), 2001, 6th 7 卷, ρ · 1470-1475

Disclosure of the invention

[0005] When DNA is detected using the above method, the heat-resistant enzyme that has been used conventionally is Since the enzyme was inactivated at a high temperature near 90 ° C, it was necessary to introduce the reporter probe labeled with the enzyme after reducing the temperature of the reaction solution to about 70 ° C. In other words, regardless of the efficiency of the hybridization, the reporter probe labeled with an enzyme had to be introduced after the temperature of the sample solution had dropped to a temperature at which the enzyme was not inactivated.

[0006] The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a DNA detection method and a reporter probe for detecting DNA with higher sensitivity and efficiency. .

[0007] The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a DNA detection method and a reporter probe for detecting DNA with higher sensitivity and efficiency. .

[0008] The DNA detection method of the present invention comprises using a capillary probe, a report probe labeled with a thermostable enzyme, and a substrate that reacts with the thermostable enzyme, and the captive probe and the reporter probe. The target DNA is bound to the target DNA, and the target DNA is detected electrochemically by a reaction with the thermostable enzyme labeled with the reporter probe and the substrate. According to this, a reporter probe labeled with a thermostable enzyme can be put into a high-temperature reaction solution that is a temperature at which the thermostable enzyme is active. Accordingly, since the reporter probe can be introduced into the reaction solution at a higher temperature, the efficiency of hybridization is increased, and the target DNA can be detected with higher sensitivity and efficiency.

[0009] In this DNA detection method, an electrode is formed by binding the capillary probe to the surface of a conductor, target DNA is bound to the capillary probe and the reporter probe, and the reporter probe is labeled. The target DNA can be detected by measuring the value of the current flowing through the electrode by the reaction between the thermostable enzyme and the substrate. According to this, a higher value can be obtained by measuring the value of the current flowing through the electrode using an electrode formed by binding the above-described captive probe to the surface of a conductor and a reporter probe labeled with a thermostable enzyme. Target DNA can be detected with sensitivity and efficiency. [0010] In this DNA detection method, the thermostable enzyme can be an enzyme derived from a hyperthermophilic bacterium. According to this, a reporter probe labeled with a thermophilic enzyme derived from a hyperthermophilic bacterium can be introduced into a high-temperature reaction solution that is a temperature at which the thermophilic enzyme derived from the hyperthermophilic bacterium remains active. . Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0011] In this DNA detection method, the enzyme derived from the hyperthermophilic bacterium can be L-proline dehydrase. According to this, a reporter probe labeled with an L-proline dehydrogenase derived from a hyperthermophilic bacterium can be introduced into a high-temperature reaction solution at which the L proline dehydrogenase is active. . Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0012] In this DNA detection method, the L proline dehydrogenase comprises Thermococcus' Puff Fungus, I'nermococcus profundus, Thermococcus heptonovifus, Pyrococcus furiosus, and It can be derived from an organism selected from a group force consisting of Pyrococcus horikoshi OT-3. According to this, a reporter probe labeled with L proline dehydrogenase derived from each of these organisms can be introduced into the reaction solution at a temperature at which the L proline dehydrogenase is active. For this reason, after the target DNA is denatured into single-stranded DNA, the reporter probe can be reacted with the target DNA at a high temperature. Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0013] In this DNA detection method, the reporter probe labeled with the thermostable enzyme can be introduced into a solution containing the target DNA during heat treatment for denaturing the target DNA. According to this, the capillary probe fixed to the electrode and the reporter probe labeled with the thermostable enzyme can coexist with the target DNA at the time of denaturation of the target DNA for hybridization. As a result, the efficiency of hybridization can be increased, and target DNA can be detected with higher sensitivity and efficiency. In addition, processing can be performed with a simpler operation.

[0014] The reporter probe of the present invention is used for target DNA binding to a capillary probe. A reporter probe that binds further, is labeled with a thermostable enzyme, and is used for electrochemically detecting the target DNA by a reaction with the thermostable enzyme and a substrate. According to this, a reporter probe labeled with a thermostable enzyme can be put into a high-temperature reaction solution that is a temperature at which the thermostable enzyme maintains its activity. Accordingly, since the reporter probe can be introduced into the reaction solution at a higher temperature and temperature, the efficiency of hybridization can be increased, and target DNA can be detected with higher sensitivity and efficiency.

[0015] In this reporter probe, the reporter probe is a target that binds to the cap probe of an electrode in which the cap probe is bonded to a conductor surface.

The target DNA can be detected by further binding to DNA and measuring the value of the current flowing through the electrode by the reaction with the thermostable enzyme and the substrate. According to this, a higher sensitivity can be obtained by measuring the value of the current flowing through the electrode using an electrode formed by binding the above-described captive probe to the surface of a conductor and a reporter probe labeled with a thermostable enzyme. And target DNA can be detected efficiently

[0016] In this reporter probe, the thermostable enzyme can be an enzyme derived from a hyperthermophilic bacterium. According to this, a reporter probe labeled with a thermophilic enzyme derived from a hyperthermophilic bacterium can be put into a high-temperature reaction solution that is a temperature at which the thermophilic enzyme derived from the hyperthermophilic bacterium remains active. it can. Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0017] In this reporter probe, the enzyme derived from the hyperthermophilic bacterium can be L_proline dehydrogenase. According to this, a reporter probe labeled with L-proline dehydrogenase derived from a hyperthermophilic bacterium can be put into a high-temperature reaction solution at which the activity of L_proline dehydrogenase is maintained. it can. Therefore, the efficiency of hybridization is improved, and target DNA can be detected with higher sensitivity and efficiency.

[0018] In this reporter probe, the L_proline dehydrogenase is Thermococcus' phlophanta, 'Hermococcus profundus) ^ ir ~ Mokok Blades' 7 卜 Nofifs (Thermoco ecus peptonophilus) (Pvrococcus furiosus), Pirococca It can be said that it is derived from the organism selected from the group power consisting of T. 3 (Pyrococcus horikoshi OT-3). According to this, a reporter probe labeled with L_proline dehydrogenase derived from each of these organisms can be introduced into the reaction solution at a temperature at which this L_proline dehydrogenase is active. For this reason, after the target DNA is denatured into single-stranded DNA, the reporter probe can be reacted with the target DNA at a high temperature. Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing one embodiment of the present invention.

FIG. 2 is a schematic diagram showing a biosensor used in one embodiment of the present invention.

FIG. 3 is a schematic diagram showing one embodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0020] In the DNA detection method of the present invention, target DNA is detected using a reporter probe labeled with a thermostable enzyme, which is the reporter probe of the present invention. Here, the thermostable enzyme labeled on the reporter probe will be described first, and then the electrode used in one embodiment of the DNA detection method of the present invention will be described, and then the DNA of the present invention will be described. An embodiment of the detection method will be described.

[0021] (Thermolytic enzyme labeled on the reporter probe)

The reporter probe of the present invention and the reporter probe used in the DNA detection method of the present invention are labeled with a thermostable enzyme. The thermostable enzyme may be either an artificially synthesized enzyme or a natural enzyme. Examples of natural heat-resistant enzymes include enzymes obtained from hyperthermophilic bacteria.

[0022] The hyperthermophilic bacterium refers to a bacterium that can grow at a particularly high temperature (usually 85 ° C to 90 ° C or more) among thermophilic bacteria. From such hyperthermophilic bacteria, an enzyme that maintains its activity even at high temperatures (near 90 ° C) can be obtained. That is, an enzyme that is not completely inactivated at the denaturation temperature of the target DNA can be obtained from such a hyperthermophilic bacterium. An example of such an enzyme is L_proline dehydrogenase derived from a hyperthermophilic bacterium. In addition, the enzyme derived from a thermostable enzyme and a hyperthermophilic bacterium in the present invention is not limited to this. [0023] Examples of L_proline dehydrogenase derived from hyperthermophilic bacteria include Thermococcus profundus ^ -Momocus fever (Thermococc us peptonophilus), Pyrococcus pylorisus ( Pyrococcus iuriosus) ^ and L-proline dehydrogenase derived from Pyrococcus horikoshi OT-3, including Thermococcus proflmdus (Th ermococcus proflmdus) The incoming L_proline dehydrogenase is particularly preferred.

[0024] As L-proline dehydrogenase derived from hyperthermophilic bacteria, Thermococcus profondus DSM9503, Thermococcus peptonophilus, DSMl0d43, Pyrococcus furiosus (Pyrococcus fur s) L proline dehydrogenase derived from DSM3638 and Pyrococcus horikoshi 〇T-3 has been extracted. Among them, L proline dehydrogenase derived from Thermococcus profondus DSM9503 has been purified and examined under various conditions. The activity of L-proline dehydrogenase derived from this Thermococcus profondus DSM9503 is preferably 80 ° C for temperature and may show high activity at 90 ° C. Has been reported (see Non-Patent Document 2).

[0025] (Detection of DNA by electrochemical method)

In the DNA detection method of the present invention, a capillary probe, a reporter probe labeled with a thermostable enzyme, and a substrate that reacts with the thermostable enzyme are used. Then, the target DNA is bound to the capillary probe and the reporter probe, and the target DNA is detected electrochemically by a reaction with the thermostable enzyme labeled on the reporter probe and the substrate.

[0026] In the following, when DNA is detected by an electrochemical method, a target probe is detected by binding a probe probe to the surface of a conductor to form an electrode and measuring the value of the current flowing through the electrode. The case of doing will be described in detail.

[0027] (Electrode)

The electrode used in this embodiment can be used to measure the activity of L-proline dehydrinase labeled on a reporter probe.

[0028] As the conductor, any material that is usually used as an electrode is used. can do. For example, graphite, carbon, carbon fabric, etc .; metal or alloy such as aluminum, copper, gold, platinum, silver, SnO, InO, WO, TiO, etc., conductive oxidation

2 2 3 3 2

A single layer of various materials such as a product, or a laminated structure of two or more types. The film thickness, size, shape, etc. of the electrode are not particularly limited, and can be appropriately adjusted depending on the type of mediator, enzyme, etc. used, the performance of the biosensor to be obtained, and the like. For example, a rectangular shape having an area of about 0.:! To about 5 mm and about 1 to about 10,000 mm ² is suitable.

[0029] The conductor is laminated with a mediator. Note that the mediator may be present in the solution without being stacked on the conductor.

As a mediator, it functions as an electron transfer medium that can exchange electrons between the electrode and L proline dehydrogenase, and if it does not adversely affect the reaction of L-proline dehydrogenase, Anything can be used. For example, Phenol, alkali metal ferricyanide (potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, etc.) or alkyl substitutions thereof (methyl substitution, ethyl substitution, propyl substitution, etc.), phenazine One or more redox organic or inorganic compounds such as methosulfate, p-benzoquinone, 2,6 dichlorophenol, indophenol, methylene blue, β-naphthoquinone-4-potassium sulfonate, phenazine etsulfate, vitamins, viologen, etc. Combinations are listed. Of these, those that dissolve in water or water-soluble organic solvents (lower alcohols, etc.) and are easy to handle and those that have a stable function as an electron transfer medium are preferred. Etc. are preferably used.

[0030] As the electron acceptor in the reaction by L_proline dehydrogenase, for example, flavin adenine dinucleotide (FAD), ferredoxin and the like can be used.

The film thickness of the mediator laminated on the conductor is not particularly limited. The size of the conductor, the type of mediator to be used, the type and amount of enzyme in the enzyme layer described later, the type of enzyme to be measured, etc. Can be adjusted as appropriate. For example, about 0. lzm to l 000 xm can be mentioned. An appropriate method for laminating the mediator on the conductor can be selected as appropriate depending on the type of the mediator. For example, the mediator is dissolved in a solvent that does not hinder its function, such as water or a water-soluble organic solvent, Coating and drying methods, mediators mixed and dispersed in a suitable carrier, for example, high molecular compounds such as resins and proteins, and the carrier is formed into a thin film, alternating lamination method (present chemistry, 1997 1 Any of the thin film forming methods known in the art can be used, such as Mon, p20-25). Especially, the alternating lamination method mentioned later is preferable.

[0031] Note that the enzyme layer may be further laminated on a mediator laminated on the conductor, and depending on the type of the mediator and the enzyme, the enzyme layer may be arranged on the conductor together with the mediator. Also good. The thickness of the enzyme layer is not particularly limited, and can be adjusted as appropriate depending on the size of the electrical conductor, the use, the type of mediator, the type of enzyme to be measured, and the like. For example, 0.1 / im ~: about 1000 / im. Examples of the method of stacking the enzyme layer include the same method as stacking the mediator together with or on the mediator.

[0032] In the electrode used in this DNA detection method, the above-mentioned mediator and enzyme layer may be laminated in layers with a polymer carrier. As the polymer carrier that can be used here, one that has a charge, one that is a polymer, and one that is water-soluble, preferably one that satisfies all properties is suitable. The charge may be positive or negative. Examples of the polymer carrier include various proteins (eg, enzymes, antibodies, receptor tanks, etc.), polypeptides (eg, polylysine, polyaspartic acid, polyglutamic acid, etc.), water-soluble synthetic polymer compounds (eg, polyethylene Amine, polyacrylic acid, carboxymethylenosenololose, polystyrene sulphonic acid, polydimethinoresinolylene monum chloride, etc.) and natural polymers (alginic acid and its salts, tragacanth gum, etc.).

[0033] The method of laminating the mediator and the like together with the polymer carrier is not particularly limited. The mediator or the like, which is not limited, is dispersed in the polymer carrier, or an appropriate solvent (water, buffer solution, water-soluble organic solvent) together with the polymer carrier Etc.) may be dissolved or dispersed, applied and dried, or an alternate lamination method may be used.

[0034] The alternate lamination method of laminating the mediator and enzyme layers (here, the method of laminating each layer in sequence) can be performed, for example, as follows.

First, the conductor surface is activated to be positively or negatively charged. Next, the molecules that make up the mediator, or the high molecular weight that binds the mediator to the dispersion / mediator. By charging the child carrier negatively or positively and bringing it into contact with the conductor surface, the mediator is stacked on the conductor surface through the interaction between positive and negative charges. If the required amount of mediator can be secured by such a single positive / negative interaction, only one layer as described above is required. However, when a larger amount of mediator is to be stacked on the conductor surface In this case, a plurality of mediator layers, for example, about 2 to 20 layers may be laminated by performing the above-described interaction a plurality of times. In this case, a carrier having the opposite charge (for example, the above-described polymer carrier) or a mediator or the like is laminated on the laminated mediator, and a molecule constituting the mediator is again added to the polymer carrier or the like. By repeating a series of operations of stacking on top, it is possible to realize the stacking of multiple mediators.

[0035] Further, in the electrode of the DNA measuring apparatus, in order to measure the activity of the enzyme to be measured, (1) the enzyme to be measured for activity may be laminated, or (2) An enzyme to be measured may be arranged separately from this electrode so as to come into contact. In the case of (1), lamination can be performed using the same lamination method as that for mediators and the like, and similarly, it is preferably formed by an alternate lamination method. In the case of (1), the enzyme to be measured is laminated on the conductor, so that, for example, the enzyme to be measured is compared with that supplied by the solution together with the substrate. There is an advantage that the current value generated due to the reaction can be obtained as a sufficiently high output, the measurement sensitivity becomes better, and the measurement error can be reduced. In the case of (2), for example, the enzyme to be measured may be brought into contact with the electrode in the form of a solution, suspension or dispersion, preferably in the presence of a substrate. In the case of (2), it may be a liquid containing the enzyme to be measured.

[0036] A DNA fragment is further immobilized on the electrode used in the present embodiment. This DNA fragment is a capillary probe for pairing with DNA for detection and / or quantification (hereinafter referred to as “target DNA”) using the complementarity of DNA.

[0037] The target DNA is a DNA fragment for detection and Z or quantification purposes, which is paired with the capture probe using the complementarity of DNA. In the DNA detection method of the present invention, a specific D having complementarity to the capillary probe and the reporter probe, respectively. The NA fragment is detected, and the DNA fragment to be detected corresponds to the target DNA. The collected DNA is amplified by the polymerase chain reaction (PCR) and used.

[0038] The reporter probe pairs with the target DNA by utilizing the complementarity of DNA. This report probe is labeled with an enzyme for detection and Z or quantification of the target DNA. In the present invention, this enzyme is L-proline dehydrogenase.

[0039] Then, by reacting the capture probe, the target DNA, and the reporter probe, the capture probe, the target DNA, and the reporter probe are bonded to the electrode on the J jet. That is, so-called hybridization of the target DNA is performed on the electrode. Then, by using an electrode to which the target DNA and the reporter probe are bound, indirect electron exchange via the mediator can be measured, for example, as a current value, thereby detecting and / or quantifying the target DNA. .

[0040] As a method of fixing the capillary probe on the electrode, all methods known in the art and methods according to them can be used. For example, streptavidin is laminated on the electrode, the 5 ′ end of the capillary probe is modified with biotin, and fixed by avidin-biotin interaction or a similar method.

[0041] The method using a DNA fragment (capture probe, reporter probe), that is, the DNA probe method and the DNA hybridization itself use all methods known in the art and methods based thereon. can do. Any commercially available DNA fragment can be used. For example, a Salmonella probe (see Rahn K., SA Deurandis, RC Clarke, SA McEwen, JE Galan, Lrinocchio, R. Curtiss and CL Gyles, Mol. Cell. Probes, 6, 271-279 (1992)). , Takara Biochemicals, Shigella and Enteroinvasive Escherichia coli (EIEC) Primer Set INV-1 & 2 for detecting invE gene, Shigella and Enteroinvasive Escherichia coli (EIEC) ipaH gene detecting Primer Set IPA-1 & 2, Collera toxin The gene detection _primer Set VCT-1 & 2 (both manufactured by Shimadzu Corporation) can be listed.

[0042] The electrode produced as described above usually contains a counter electrode, these electrode and counter electrode, and can contain a liquid, preferably a solution containing a substrate of the enzyme to be measured. Is placed in a reactor that is capable of further immersion in the reactor. It is suitable to be used in biosensors filled with solution each time. In such a biosensor, based on the result of cyclic voltammetry, for example, a constant potential having a predetermined potential difference is applied to both electrodes, and the current value generated due to the reaction of the enzyme to be measured. Can be measured (for example, measured as a limiting oxidation current value), and the activity of the enzyme to be measured can be determined. The current value can be detected by, for example, a current detection unit such as a potentiostat or an ammeter. In this embodiment, DNA is detected by measuring a current value using such a nanosensor.

[0043] In the DNA detection method of the present embodiment, the reporter probe is labeled with a thermostable enzyme, and a solution containing the substrate of the thermostable enzyme is accommodated in the reactor. Then, the electrode prepared as described above, the counter electrode, and the reference electrode are immersed in a solution containing the substrate, and a constant potential having a predetermined potential difference is applied to the electrode and the counter electrode, so that the heat resistance Measure the current value generated by the reaction between the enzyme and the substrate.

Hereinafter, the DNA detection method in the present embodiment will be specifically described with reference to the drawings. Although the case where the thermostable enzyme is an L proline dehydrogenase derived from a hyperthermophilic bacterium is described here, as described above, the thermostable enzyme in the present invention is not limited to this.

[0045] (Working electrode)

As shown in FIG. 1, the working electrode has a captive probe 28a immobilized on the carbon electrode 21 through a mediator 22 in a complementary manner to hybridize with the target DNA 28b. Note that the mediator 22 does not have to be fixed to the carbon electrode 21 but is present in the solution.

[0046] Then, the reporter probe 28c labeled with L-proline dehydrogenase 26 is obtained by binding the reporter probe 28c labeled with the capillary probe 28a, the target DNA 28b, and L_proline dehydrogenase 26. A coupled working electrode can be obtained. Here, when the DNA to be detected is present in the DNA sample solution, the single-stranded DNA obtained by modifying this DNA corresponds to the single-stranded target DNA 28b. In order to detect target DNA28b, it is necessary to detect target DNA28b in the DNA sample solution. It is necessary that there is enough DNA collected. For this reason, first, in order to obtain a sufficient amount of DNA for detection of the target DNA 28b, PCR (Polymerase Chain Reaction) is performed using the DNA sample solution to amplify the DNA in the sample solution.

[0047] Then, the DNA sample solution is heat-treated at 96 ° C for 30 seconds, for example, to denature the double-stranded DNA into single-stranded DNA. Then, a working electrode is inserted into the sample solution, and a reporter probe 28c labeled with L-proline dehydrogenase 26 is introduced. The reporter probe 28c labeled with the L proline dehydrogenase 26 may be lyophilized or a liquid reagent.

[0048] Here, in order to maintain the activity of L proline dehydrogenase 26 labeled on reporter probe 28c, the temperature of the reaction solution needs to be lower than the heat resistant temperature of L proline dehydrogenase 26. If the temperature of the reaction solution is lower than the heat-resistant temperature of L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium, reporter probe 28c labeled with L proline dehydrogenase 26 can be added to the reaction solution without further cooling. .

[0049] Here, when the target DNA 28b is present in the DNA sample solution, as shown in FIG. 1, the capillary probe 28a, the single-stranded target DNA 28b, and the reporter probe 28c are annealed. Here, when the detection target DNA is obtained, this corresponds to the detection target DNA force single-stranded target DNA 28b. Therefore, when the DNA to be detected is contained in the sample solution, the capillary probe 28a, the target DNA 28b, and the reporter probe 28c are bonded to the carbon electrode 21 in this order.

[0050] (Biosensor)

In this DNA detection method, DNA is detected using the above working electrode, a reference electrode and a counter electrode. That is, a biosensor having a working electrode, a reference electrode, and a counter electrode is used. A schematic diagram of this biosensor is shown in Figure 2.

[0051] As shown in FIG. 2, the biosensor 18 is connected to the working electrode 11 connected to the lead 11a, the counter electrode 14 also connected to the lead 14a, and the lead 12a in the reactor 15. The reference electrode 12 is accommodated. Then, in the reactor 15, as an electrolyte solution 13, a buffer solution containing L proline which is a substrate of L proline dehydrogenase labeled on the reporter probe (for example, For example, Tris buffer) is stored. Then, the working electrode, the reference electrode and the counter electrode are immersed in this buffer solution. A separator 16 is provided between the working electrode 11 and the counter electrode 14. In addition, an ammeter 17 is connected to the lead 11a as a current detector for measuring a current value generated in the working electrode 11.

[0052] By applying a potential of, for example, +5 OOmV to the working electrode 11 of the biosensor 18 with reference to the reference electrode 12, a substrate (L-proline) and an enzyme (L- Electron dehydrogenase) reacts and exchanges electrons through the mediator. As a result, the current flowing through the working electrode can be measured with an ammeter as the limiting oxidation current. Therefore, the current flowing through the working electrode 11 is defined as the limiting oxidation current by the transfer of electrons by L-proline in the electrolyte 13, L-loop phosphorus dehydrogenase and mediator labeled on the reporter probe, and the ammeter 17 Can be measured.

[0053] (Detection of target DNA by measuring current value)

Next, detection of the target DNA by measuring the current value using the biosensor 18 equipped with the electrode (working electrode 11) configured as described above will be described.

[0054] Here, when the DNA to be detected is contained in the sample solution, the capillary probe 28a, the target DNA 28b, and the reporter probe 28c are combined in this order on the working electrode 11. Yes. The reporter probe 28c is labeled with L proline dehydrogenase 26.

[0055] The current value is measured by applying a potential of +5 OOmV to the working electrode 11 of the biosensor 18 with reference to the reference electrode 12, and measuring the current flowing through the working electrode 11.

Here, when a potential is applied to the working electrode 11 of the biosensor 18, L-proline 25 and L-proline dehydrogenase 26 in the reaction solution react, and electrons are transferred via the mediator 22. Specifically, as shown in FIG. 1, when L-proline dehydrogenase 26 reacts with the substrate L proline 25 to produce Δ ¹ -pyrroline-15 carboxylic acid 27, mediator 22 By receiving hydrogen, it is converted to a reduced form. This reduced-type mediator 22 moves hydrogen and transmits and receives the electrons generated thereby to the carbon electrode 21.

Using this working electrode 11, counter electrode 14, and reference electrode 12, carbon of working electrode 11 Measure the current value of the current flowing through electrode 21 (limit oxidation current). For example, when a potential of +500 mV is applied to the working electrode 11 with reference to the reference electrode 12, L monoproline 25 and L-proline dehydrogenase 26 in the reaction solution react, and the mediator 22 is The current flowing through the working electrode 11 can be measured by the ammeter 17 as the limit oxidation current.

[0057] Even if only the electrolyte solution is used and the enzymatic reaction by L_proline dehydrogenase 26 does not occur, the base current is generated, but the oxidation-reduction reaction when the enzymatic reaction occurs The force S can be measured as a larger value compared to this base current.

[0058] Thus, by monitoring the electron transfer between the enzyme electrodes based on the enzyme reaction by L proline dehydrogenase 26, that is, by capturing a series of electron transfer as an electric current, the presence or absence or concentration of the target DNA is detected. Can be detected. Therefore, it is possible to detect DNA by sandwich hybridization assay that uses a reporter probe to obtain a signal by further binding to a capture probe that captures the target DNA and a captured target DNA. Become.

As described above, according to the present embodiment, the following effects can be obtained.

• In the above embodiment, the target DNA is bound to the capture probe and the reporter probe using the capture probe, the reporter probe labeled with the thermostable enzyme, and the substrate that reacts with the thermostable enzyme, and the reporter The target DNA is detected electrochemically by a reaction between a thermostable enzyme labeled on the probe and a substrate. As a result, the reporter probe labeled with the heat-resistant enzyme can be put into a high-temperature reaction solution at which the heat-resistant enzyme is active. Therefore, since the reporter probe can be introduced into the reaction solution at a higher temperature, the efficiency of hybridization can be increased, and the target DNA can be detected with higher sensitivity and efficiency.

[0060] In the above-described embodiment, a heat-promoting enzyme labeled with a reporter probe by binding a capture probe to a conductor surface to form an electrode, binding a target DNA to the capture probe and the reporter probe, and The target DNA is detected by measuring the value of the current flowing through the electrode due to the reaction with the substrate. This will allow you to Detecting target DNA more sensitively and efficiently by measuring the value of the current flowing through the electrode using an electrode constructed by bonding to the surface of a conductor and a reportable probe labeled with a thermostable enzyme be able to.

[0061] · In the above embodiment, a repo-tableb labeled with an enzyme derived from a hyperthermophilic bacterium is used. Since hyperthermophilic bacteria can obtain enzymes that are not completely inactivated at the denaturation temperature of the target DNA, according to this, the report probe is introduced into the reaction solution at the denaturation temperature of the target DNA. It becomes possible. For this reason, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0062] In the embodiment described above, an electrode in which a capillary probe 28a is bonded to the surface of the carbon electrode 21, and a reporter probe 28c labeled with L proline dehydrogenase 26 derived from a hyperthermophilic bacterium The L proline dehydrogenase 26, which is a substrate that reacts with the L proline dehydrogenase 26, is used. Then, the target DNA 28b is bound to the capillary probe 28a and the reporter probe 28c, and the limit oxidation that flows to the carbon electrode 21 based on the reaction by the L-proline dehydrogenase 26 and L proline 25 labeled on the reporter probe 28c. The target DNA28b is detected by measuring the current value of the current. According to this, reportable probe 28c labeled with L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium is introduced into the reaction solution at a high temperature at which this L-proline dehydrogenase 26 remains active. It becomes possible to do. Therefore, the efficiency of the hybridization is increased, and the target DNA can be detected with higher sensitivity and efficiency.

[0063] In the above-described embodiment, a reportable tab labeled with an enzyme derived from a hyperthermophilic bacterium is used. Since hyperthermophilic bacteria can obtain enzymes that are not completely inactivated at the denaturation temperature of the target DNA, according to this, the report probe is introduced into the reaction solution at the denaturation temperature of the target DNA. It becomes possible. For this reason, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0064] In the above-described embodiment, an electrode formed by coupling a capillary probe 28a to the surface of the carbon electrode 21, and a reporter probe 28c labeled with L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium. And L-proline 25, which is a substrate that reacts with L-proline dehydrogenase 26, is used. And for the probe probe 28a and the reporter probe 28c The target DNA28b is detected by binding the target DNA28b and measuring the current value of the limiting oxidation current flowing through the carbon electrode 21 based on the reaction with L-proline dehydrogenase 26 and L-proline 25 labeled on the reporter probe 28c. To do. According to this, the repotable probe 28c labeled with L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium is added to the reaction solution at a high temperature at which this L-proline dehydrogenase 26 maintains its activity. It becomes possible to input. Therefore, the efficiency of the hybridization is increased, and the target DNA can be detected with higher sensitivity and efficiency.

[0065] In the above embodiment, the reporter probe 28c labeled with L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium is used. According to this, reporter probe 28c labeled with L-proline dehydrogenase 26 derived from a hyperthermophilic bacterium is put into a high-temperature reaction solution at a temperature at which this L-proline dehydrogenase 26 remains active. can do. For this reason, it is possible to denature the target DNA into single-stranded target DNA28b and then react the reporter probe 28c with target DNA28b at a high temperature that keeps L-proline dehydrogenase 26 active. It becomes. Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0066] · In the above embodiment, Thermococcus profundus, Thermococcus peptonophilus, Pyrococcus iuriosus ^ and Pyrococcus' Tokosyo Tcc 3 L_proline dehydrogenase derived from an organism selected from the group consisting of O T_3) can be used. Therefore, a reporter probe labeled with L_proline dehydrogenase derived from each of these organisms can be introduced into the reaction solution at a temperature at which the L_proline dehydrogenase is active. For this reason, after the target DNA is denatured into single-stranded DNA, the reporter probe can be reacted with the target DNA at a high temperature. Therefore, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency.

[0067] In the above-described embodiment, when the target DNA is denatured for hybridization, a target probe is used as a capture probe immobilized on an electrode and a reporter probe labeled with L-proline dehydrogenase. Put into the contained solution. For this reason, the electrode The probe probe fixed to the target and the reporter probe labeled with L-proline dehydrogenase can coexist with the target DNA during denaturation of the target DNA for hybridization. As a result, the efficiency of hybridization is increased, and target DNA can be detected with higher sensitivity and efficiency. In addition, processing can be performed with simpler operations.

[0068] The embodiment described above may be changed to the following modes.

In the above embodiment, a captive probe is bonded to the surface of a conductor to form an electrode, and the target DNA is detected by measuring the value of the current flowing through the electrode. However, the present invention is not limited to this. For example, a capillary probe may be immobilized on a pH sensitive membrane, and a change in pH due to an enzyme reaction may be detected by a change in voltage, thereby detecting a target DNA. Hereinafter, an embodiment in this case will be described.

FIG. 3 shows a field effect transistor structure used in the DNA detection method of one embodiment of the present invention. This field effect transistor structure includes a p-type silicon substrate 31, a pH sensitive film 32, a counter electrode 34, a source electrode 35, a drain electrode 36, and an n ⁺ -type layer 37. The pH sensitive film 32 exhibits a pH response, and for example, Si N (silicon nitride film) or the like can be used. And

3 4

The pH sensitive membrane 32 and the counter electrode 34 are immersed in the electrolytic solution.

[0070] (pH sensitive membrane)

In this embodiment, as shown in the enlarged view at the bottom of FIG. 3, a capture probe 48a is fixed on the pH sensitive membrane 32. The capture probe 48a hybridizes in a complementary manner to the target DNA 48b. Then, as shown in FIG. 3, the heat-resistant enzyme 46 can be held in the vicinity of the pH sensitive membrane 32 by binding the reporter probe 48c labeled with the capillary probe 48a, the target DNA 48b, and the heat-resistant enzyme 46.

[0071] Here, since DNA is detected by detecting a change in pH by measuring a voltage, the reaction between thermostable enzyme 46 and substrate 45 needs to be accompanied by a change in pH. Therefore, in the present embodiment, as the thermostable enzyme 46, for example, an enzyme that changes pH in response to the reaction with the substrate 45, such as glucose oxidase, is used.

[0072] If the DNA to be detected is present in the DNA sample solution, this DNA was denatured 1 Double-stranded DNA corresponds to single-stranded target DNA48b. In order to detect the target DNA48b, the collected DNA must be present in the DNA sample solution sufficient for the detection of the target DNA48b. Therefore, first, in order to obtain a sufficient amount of DNA for detection of the target DNA48b, the DNA in the sample solution is amplified by performing PCR using the DNA sample solution.

[0073] Then, this DNA sample solution is heat-treated at 96 ° C for 30 seconds, for example, to denature the double-stranded DNA into single-stranded DNA. Then, the pH sensitive membrane 32 is immersed in this sample solution, and a reporter probe 48c labeled with a thermostable enzyme 46 is introduced. Here, in order to keep the activity of the thermostable enzyme 46 labeled on the reporter probe 48c, the temperature of the reaction solution needs to be lower than the thermostable temperature of the thermostable enzyme 46. If the temperature of the reaction solution is lower than the heat-resistant temperature of thermostable enzyme 46, reporter probe 48c labeled with thermostable enzyme 46 can be added to the reaction solution without further cooling.

[0074] When the target DNA 48b is present, as shown in FIG. 3, the capillary probe 48a, the single-stranded target DNA 48b, and the reporter probe 48c are annealed. Here, when the DNA to be detected is obtained, this corresponds to the target DNA48b having a single-strand DNA force to be detected. Therefore, when the DNA to be detected is contained in the sample solution, the capillary probe 48a, the target DNA 48b, and the reporter probe 48c bind to the pH sensitive membrane 32 in this order.

[0075] (Detection of target DNA by voltage measurement)

Next, detection of target DNA by measuring voltage using the field effect transistor configured as described above will be described.

First, the reaction solution is discarded, and the pH sensitive membrane 32 and the counter electrode 34 are immersed in a buffer solution that is an electrolytic solution. Here, when the DNA to be detected is contained in the Sampnore solution, on the pH sensitive membrane 32, the reporter probe 48c, which is detected by the capillar probe 48a, the target DNA 48b, and the thermostable enzyme 46, They are combined in this order. Therefore, in this case, the thermostable enzyme 46 is held in the vicinity of the pH sensitive membrane 32.

[0077] Then, when the substrate 45 that reacts with the thermostable enzyme 46 labeled on the reporter probe is introduced into the electrolyte, the substrate 45 and the thermostable enzyme 46 in the electrolyte react to react with the substrate 47 after the reaction. And the pH in the vicinity of the pH sensitive membrane 32 changes.

In the field effect transistor configured as described above, the interfacial potential between the pH sensitive film 32 and the electrolytic solution changes according to the change in pH. This change in interfacial potential is measured. For example, the gate current is measured while keeping the drain current and the source Z-drain voltage constant. Thus, the presence or concentration of the target DNA can be detected by detecting the change in pH by measuring the voltage.

Claims

The scope of the claims

[1] Using a capillary probe, a reporter probe labeled with a thermostable enzyme, and a substrate that reacts with the thermostable enzyme,

A target DNA is bound to the capillary probe and the reporter probe, and the target DNA is detected electrochemically by a reaction with the thermostable enzyme labeled on the reporter probe and the substrate. DNA detection method.

[2] An electrode is formed by binding the above-mentioned captive probe to a conductor surface,

The target DNA is bound by binding the target DNA to the captive probe and the reporter probe, and measuring the value of the current flowing through the electrode by the reaction with the thermostable enzyme labeled on the reporter probe and the substrate. 2. The DNA detection method according to claim 1, wherein the detection is performed.

[3] The DNA detection method according to claim 1 or 2, wherein the thermostable enzyme is an enzyme derived from a hyperthermophilic bacterium.

[4] The DNA detection method according to any one of [4], wherein the enzyme derived from the hyperthermophilic bacterium is L_proline dehydrogenase.

[5] The L_proline dehydrogenase is Thermococcus prof undus, ¹ to Mococ 7s' Thermococcus peptonophilus, Pyrococcus furiosus, and Ό The DNA detection method according to claim 4, wherein the DNA detection method is derived from an organism selected from the group consisting of Pyrococcus hori koshi OT-3.

[6] In the heat treatment for denaturation of the target DNA, the reporter probe labeled with the thermostable enzyme is put into a solution containing the target DNA. A DNA detection method according to 1.

[7] A reporter probe that further binds to a target DNA that binds to a capillary probe,

It is labeled with a thermostable enzyme,

A reporter probe which is used for electrochemically detecting the target DNA by a reaction between the thermostable enzyme and a substrate.

[8] The reporter probe further binds to a target DNA that binds to the capture probe of an electrode formed by binding a capture probe to the surface of a conductor.

The reporter probe according to claim 7, wherein the reporter probe is used to detect the target DNA by measuring a value of a current flowing through the electrode by a reaction between the thermostable enzyme and a substrate.

[9] The reporter probe according to claim 7 or 8, wherein the thermostable enzyme is an enzyme derived from a hyperthermophilic bacterium.

10. The reporter probe according to claim 9, wherein the enzyme derived from the hyperthermophile is L proline dehydrogenase.

[11] The L proline dehydrogenase is Thermococcus prof undus, Thermococcus peptonophims, Pyrococcus furiosus, and Pyrococcus T3. The reporter probe according to claim 10, wherein the reporter probe is derived from an organism selected from the group consisting of (Pyrococcus hori koshi OT-3).