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WO2006033400A1 - Procede de detection adn et sonde d’analyse - Google Patents

Procede de detection adn et sonde d’analyse Download PDF

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Publication number
WO2006033400A1
WO2006033400A1 PCT/JP2005/017501 JP2005017501W WO2006033400A1 WO 2006033400 A1 WO2006033400 A1 WO 2006033400A1 JP 2005017501 W JP2005017501 W JP 2005017501W WO 2006033400 A1 WO2006033400 A1 WO 2006033400A1
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WIPO (PCT)
Prior art keywords
probe
dna
reporter probe
target dna
enzyme
Prior art date
Application number
PCT/JP2005/017501
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English (en)
Japanese (ja)
Inventor
Shinichiro Sue
Hideo Katayama
Original Assignee
Daikin Industries, Ltd.
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Publication date
Application filed by Daikin Industries, Ltd. filed Critical Daikin Industries, Ltd.
Priority to JP2006536417A priority Critical patent/JPWO2006033400A1/ja
Publication of WO2006033400A1 publication Critical patent/WO2006033400A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • 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.
  • 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 ⁇ , ⁇ ⁇ 1470-1475
  • 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.
  • 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. .
  • 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. .
  • 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.
  • 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.
  • thermostable enzyme can be an enzyme derived from a hyperthermophilic bacterium.
  • 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.
  • the enzyme derived from the hyperthermophilic bacterium can be L-proline dehydrase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the efficiency of hybridization can be increased, and target DNA can be detected with higher sensitivity and efficiency.
  • processing can be performed with a simpler operation.
  • 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.
  • 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.
  • 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
  • the thermostable enzyme can be an enzyme derived from a hyperthermophilic bacterium.
  • 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.
  • the enzyme derived from the hyperthermophilic bacterium can be L_proline dehydrogenase.
  • 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.
  • the L_proline dehydrogenase is Thermococcus' phlophanta, 'Hermococcus profundus) ⁇ ir ⁇ Mokok Blades' 7 ⁇ 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.
  • 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.
  • target DNA is detected using a reporter probe labeled with a thermostable enzyme, which is the reporter probe of the present invention.
  • 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.
  • 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.
  • 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.
  • the enzyme derived from a thermostable enzyme and a hyperthermophilic bacterium in the present invention is not limited to this.
  • L_proline dehydrogenase derived from hyperthermophilic bacteria examples 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.
  • L-proline dehydrogenase derived from hyperthermophilic bacteria As L-proline dehydrogenase derived from hyperthermophilic bacteria, Thermococcusoirus DSM9503, Thermococcus peptonophilus, DSMl0d43, Pyrococcus furiosus (Pyrococcus fur s) L proline dehydrogenase derived from DSM3638 and Pyrococcus horikoshi ⁇ T-3 has been extracted. Among them, L proline dehydrogenase derived from Thermococcusexcellentus DSM9503 has been purified and examined under various conditions. The activity of L-proline dehydrogenase derived from this Thermococcusexcellentus DSM9503 is preferably 80 ° C for temperature and may show high activity at 90 ° C. Has been reported (see Non-Patent Document 2).
  • 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.
  • 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.
  • the electrode used in this embodiment can be used to measure the activity of L-proline dehydrinase labeled on a reporter probe.
  • any material that is usually used as an electrode is used. can do.
  • graphite, carbon, carbon fabric, etc . metal or alloy such as aluminum, copper, gold, platinum, silver, SnO, InO, WO, TiO, etc., conductive oxidation
  • 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.
  • the conductor is laminated with a mediator. Note that the mediator may be present in the solution without being stacked on the conductor.
  • Phenol alkali metal ferricyanide (potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, etc.) or alkyl substitutions thereof (methyl substitution, ethyl substitution, propyl substitution, etc.), phenazine
  • alkali metal ferricyanide potassium ferricyanide, lithium ferricyanide, sodium ferricyanide, etc.
  • 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, ⁇ -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
  • L_proline dehydrogenase for example, flavin adenine dinucleotide (FAD), ferredoxin and the like can be used.
  • FAD flavin adenine dinucleotide
  • ferredoxin ferredoxin
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the above-mentioned mediator and enzyme layer may be laminated in layers with a polymer carrier.
  • a 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.
  • polymer carrier examples 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.).
  • 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.
  • natural polymers al
  • 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.
  • 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.
  • the conductor surface is activated to be positively or negatively charged.
  • 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.
  • a carrier having the opposite charge for example, the above-described polymer carrier
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • it may be a liquid containing the enzyme to be measured.
  • 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.
  • 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.
  • 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.
  • 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.
  • this enzyme is L-proline dehydrogenase.
  • 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.
  • indirect electron exchange via the mediator can be measured, for example, as a current value, thereby detecting and / or quantifying the target DNA. .
  • the method using a DNA fragment 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.
  • 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)).
  • 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.
  • 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.
  • thermostable enzyme is an L proline dehydrogenase derived from a hyperthermophilic bacterium
  • thermostable enzyme in the present invention is not limited to this.
  • 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.
  • the mediator 22 does not have to be fixed to the carbon electrode 21 but is present in the solution.
  • 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.
  • the single-stranded DNA obtained by modifying this DNA corresponds to the single-stranded target DNA 28b.
  • 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.
  • 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.
  • 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.
  • 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. .
  • the capillary probe 28a, the single-stranded target DNA 28b, and the reporter probe 28c are annealed.
  • the detection target DNA 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.
  • 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.
  • 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.
  • 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
  • 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.
  • an ammeter 17 is connected to the lead 11a as a current detector for measuring a current value generated in the working electrode 11.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a target probe 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.
  • 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.
  • the present invention is not limited to this.
  • 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.
  • 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.
  • Si N silicon nitride film
  • the pH sensitive membrane 32 and the counter electrode 34 are immersed in the electrolytic solution.
  • 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.
  • 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.
  • 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.
  • Double-stranded DNA corresponds to single-stranded target DNA48b.
  • the collected DNA 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.
  • 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.
  • the pH sensitive membrane 32 is immersed in this sample solution, and a reporter probe 48c labeled with a thermostable enzyme 46 is introduced.
  • 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.
  • the capillary probe 48a, the single-stranded target DNA 48b, and the reporter probe 48c are annealed.
  • 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.
  • 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.
  • a buffer solution that is an electrolytic solution.
  • 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.
  • 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.
  • the gate current is measured while keeping the drain current and the source Z-drain voltage constant.
  • the presence or concentration of the target DNA can be detected by detecting the change in pH by measuring the voltage.

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Abstract

L’invention porte sur une électrode dans laquelle une sonde d’acquisition (28a) connectée à la surface d’une électrode de carbone (21), une sonde d’analyse (28c) marquée avec une enzyme de déshydrogénation de L-proline (26), et une L-proline (25) servant de substrat réactif avec l’enzyme de déshydrogénation de L-proline (26) sont utilisées. Un ADN cible (28b) est connecté à la sonde d’acquisition (28a) et la sonde d’analyse (28c). Afin de détecter L'ADN cible (28b), on mesure la valeur actuelle du courant d’oxydation limiteur s’écoulant dans l’électrode de carbone (21) à cause de la réaction entre l’enzyme de déshydrogénation de L-proline (26) avec laquelle la sonde d’analyse (28c) est marquée et la L-proline (25).
PCT/JP2005/017501 2004-09-22 2005-09-22 Procede de detection adn et sonde d’analyse WO2006033400A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS619300A (ja) * 1984-06-22 1986-01-16 Fujirebio Inc 耐熱性酵素を標識したポリヌクレオチド測定用試薬
WO1992008808A1 (fr) * 1990-11-14 1992-05-29 Siska Diagnostics, Inc. Detection non isotopique d'acides nucleiques utilisant une technique d'hybridation en sandwich base sur des supports en polystyrene et compositions prevues a cet effet
WO1999067628A1 (fr) * 1998-06-24 1999-12-29 Therasense, Inc. Ensemble multicapteur de reconnaissance electrochimique de sequences nucleotidiques, et procedes y relatifs
WO2000032813A1 (fr) * 1998-12-01 2000-06-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procede et systeme permettant de detecter des oligonucleotides dans un echantillon

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS619300A (ja) * 1984-06-22 1986-01-16 Fujirebio Inc 耐熱性酵素を標識したポリヌクレオチド測定用試薬
WO1992008808A1 (fr) * 1990-11-14 1992-05-29 Siska Diagnostics, Inc. Detection non isotopique d'acides nucleiques utilisant une technique d'hybridation en sandwich base sur des supports en polystyrene et compositions prevues a cet effet
WO1999067628A1 (fr) * 1998-06-24 1999-12-29 Therasense, Inc. Ensemble multicapteur de reconnaissance electrochimique de sequences nucleotidiques, et procedes y relatifs
WO2000032813A1 (fr) * 1998-12-01 2000-06-08 Yissum Research Development Company Of The Hebrew University Of Jerusalem Procede et systeme permettant de detecter des oligonucleotides dans un echantillon

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SAKURABA H ET AL: "Purification, Characterization, and Application of a Novel Dye-Linked L-Proline Dehydrogenase from a Hyperthermophilic Archaeon, Thermococcus profundus.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY., vol. 67, no. 4, April 2001 (2001-04-01), pages 1470 - 1475, XP002995063 *

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