WO2004044570A1 - Procede de detection d'hybridation - Google Patents

Procede de detection d'hybridation Download PDF

Info

Publication number
WO2004044570A1
WO2004044570A1 PCT/JP2003/012499 JP0312499W WO2004044570A1 WO 2004044570 A1 WO2004044570 A1 WO 2004044570A1 JP 0312499 W JP0312499 W JP 0312499W WO 2004044570 A1 WO2004044570 A1 WO 2004044570A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid probe
dna
hybridization
stranded
Prior art date
Application number
PCT/JP2003/012499
Other languages
English (en)
Japanese (ja)
Inventor
Takashi Terasawa
Masahiro Kadosaki
Megumi Makimura
Satoshi Fujiki
Katsumi Tanino
Akira Nakagawa
Takashi Mizuhara
Masanori Mizushima
Morihito Nakada
Original Assignee
Toyama Prefecture
Cosel Co., Ltd.
Tateyama Kagaku Industry Co., Ltd.
Toyo Kako Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toyama Prefecture, Cosel Co., Ltd., Tateyama Kagaku Industry Co., Ltd., Toyo Kako Co., Ltd. filed Critical Toyama Prefecture
Priority to AU2003266711A priority Critical patent/AU2003266711A1/en
Publication of WO2004044570A1 publication Critical patent/WO2004044570A1/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors

Definitions

  • the present invention relates to a method for detecting hybridization between a nucleic acid of interest and a nucleic acid such as a gene of interest contained in a gene sample, and specifically detecting a specific gene or the like present in the sample. . Background art
  • genes Genetic information imprinted on genes (DNA) is expressed as proteins or enzymes via messenger RNA. By the action of these proteins and enzymes, biosynthesis and metabolism of various compounds necessary for maintaining life are performed. Thus, organisms exist as a dynamic equilibrium system of various substances controlled by genes. As a result of the human genome project, it was revealed that the total number of human genes was about 30,000. If any abnormality or change occurs in these genes, for example, deletion or duplication, the characteristics, type and amount of the produced protein will change, resulting in an imbalance in the biological system and causing disease . Therefore, by detecting a known gene that causes a disease, it is possible to identify or prevent a disease. Such a diagnosis is called gene diagnosis.
  • Genetic diagnosis allows diagnosis and prediction before the phenotypic change of the disease, that is, before the onset of the disease, in the latent period of the disease, or at an extremely early stage.
  • genetic diagnosis for hereditary diseases does not depend on the organ or tissue being analyzed. This is particularly important in fetal diagnosis, and it is possible to make a diagnosis simply by collecting amniotic fluid from a pregnant woman and examining fetal cells floating in the amniotic fluid.
  • Patent Document 1 Japanese Patent Application Laid-Open No. Hei 6-77099
  • Patent Document 2 Japanese Patent No. 25733443
  • This gene detection method a gene sample extracted from a sample and denatured to a single strand is reacted with a single-stranded nucleic acid probe having a base sequence complementary to the target gene to be detected, and then This is a gene detection method for detecting the presence of the target gene by detecting the nucleic acid probe hybridized with the gene.
  • the nucleic acid probe is immobilized on an electrode surface or an optical fiber tip, and a double-stranded recognizer that specifically binds to a double-stranded nucleic acid and is electrochemically or photochemically active.
  • a double-stranded form of the nucleic acid probe and the target gene is obtained.
  • a single-stranded oligo DNA is immobilized on an electrode, and a dye that specifically interacts with the double-stranded DNA to determine whether or not it becomes double-stranded DNA when the sample DNA is dropped. Is detected from an electrochemical redox reaction.
  • the measurement of the oxidation, reduction potential and peak current value of the intercalating dye used is affected by the sweep speed of the potential, and moreover, the measurement is repeated because the dye is oxidized in almost one measurement. Can not. As a result, measurement accuracy is reduced.
  • the dye may be immobilized on the single-stranded oligo DNA or the electrode, and it may be difficult to detect whether or not the dye has become double-stranded DNA by electrochemical measurement of the dye.
  • the present invention is to solve the problem in the above method, in which a nucleic acid probe such as a single-stranded DNA is immobilized on an electrode, and a double-stranded DNA or the like is added when a nucleic acid sample such as a sample DNA is added.
  • This method electrochemically measures whether or not to form a double-stranded DNA, which does not require the use of a dye, enables repeated measurement, and forms the double-stranded DNA with higher accuracy.
  • An object of the present invention is to provide a method for detecting hybridization capable of detecting chromosomes. Disclosure of the invention
  • the present invention that solves the above-mentioned problems is achieved by coexisting a single-stranded nucleic acid probe immobilized on an electrode surface and a single-stranded nucleic acid sample, and then includes the nucleic acid probe and the nucleic acid sample.
  • Detection of hybridization with nucleic acid A method for detecting the hybridization between the target nucleic acid and the nucleic acid probe, which is performed by measuring the AC impedance of the electrode having the probe immobilized thereon.
  • nucleic acid probe is DNA, RNA, or PNA
  • the nucleic acid sample is a gene sample, and the target nucleic acid is a target gene;
  • the target nucleic acid has a nucleotide sequence complementary to a nucleic acid probe
  • the target nucleic acid has at least one mismatch with the nucleic acid probe; in particular, the target nucleic acid has one mismatch with the nucleic acid probe; Measuring the AC impedance after coexisting for hybridization, then bringing the temperature to the range of 80 to 88 ° C, and then returning to room temperature; and
  • the measurement of the AC impedance is preferably performed using a comb-shaped counter electrode.
  • FIG. 1 shows a plan view of a measuring apparatus including a comb-shaped counter electrode.
  • FIG. 2 shows an enlarged view of a comb-shaped counter electrode.
  • FIG. 3 is an explanatory diagram of an AC impedance measuring method.
  • FIG. 4 is an explanatory diagram of an equivalent circuit used for AC impedance measurement.
  • FIG. 5 shows the results of AC impedance measurement in the example.
  • FIG. 6 shows the results of AC impedance measurement (expansion of l to 100 Hz) in the example.
  • a single-stranded nucleic acid probe immobilized on the electrode surface is prepared.
  • the nucleic acid probe can be, for example, DNA, RNA, or PNA.
  • the nucleic acid probe can have, for example, a base sequence complementary to a target nucleic acid such as a target gene to be detected.
  • the base sequence of such a single-stranded nucleic acid probe is determined automatically once the target nucleic acid such as the target gene to be detected is determined, and such a single-stranded nucleic acid probe can be obtained by, for example, a chemical synthesis method.
  • a chemical synthesis method can be prepared. Examples of the chemical synthesis method include, but are not limited to, a phosphoramidite method, a phosphoric acid triester method, and an H-phosphonate method.
  • the number of bases of the single-stranded nucleic acid probe is not particularly limited, but may be, for example, in the range of 20 to 100, and may be appropriately determined in consideration of the number of bases of the target gene to be detected. Can decide.
  • the immobilization of the electrode surface to the single-stranded nucleic acid probe can be performed, for example, by (1) introducing a thiol group to the end of the single-stranded nucleic acid probe, and via a gold-sulfur coordination bond between gold and sulfur. (2) Reactive functional groups such as amino group, carboxyl group, sulfone group, hapten molecule, biotin, avidin, etc. (See Japanese Patent Application Laid-Open No. 6-79099).
  • (3) the method of immobilizing a single-stranded nucleic acid probe on the electrode surface by physical adsorption (see Japanese Patent Application Laid-Open No. 5-199988), Immobilization of
  • the area of the region where the single-stranded nucleic acid probe is immobilized is, for example, ⁇ 4 to 6 ⁇ m, and the amount of the single-stranded nucleic acid probe immobilized on the electrode surface is 400 pmol to 6 It can be in the nmo 1 range. However, it is not limited to these ranges.
  • Examples of electrodes for immobilizing single-stranded nucleic acid probes include noble metal electrodes (for example, gold, platinum, palladium, and rhodium), carbon electrodes, and oxide electrodes (for example, titanium oxide, tin oxide, and manganese oxide). , Lead oxide, etc.) can be used. Further, the electrode for immobilizing the single-stranded nucleic acid probe can be, for example, a comb-shaped counter electrode.
  • FIG. 1 is a plan view of a measuring apparatus including a comb-shaped counter electrode
  • FIG. 2 is an enlarged view of the comb-shaped counter electrode. The apparatus shown in FIG.
  • the apparatus shown in FIG. 1 is provided with, for example, a resin cover except for the comb-shaped counter electrode portion, and the measurement solution supplied to the comb-shaped counter electrode portion is used as another component. Short-circuit with the comb-shaped counter electrode can be prevented.
  • the circular portion provided around the comb-shaped counter electrode in FIG. 2 is a hole for supplying a measuring solution provided on a resin force par.
  • a single-stranded nucleic acid sample is prepared.
  • Nucleic acid sun The pull can be, for example, a genetic sample.
  • nucleic acid samples other than gene samples include nucleic acids contained in blood, leukocytes, serum, urine, feces, semen, saliva, cultured cells, tissue cells, and the like.
  • a nucleic acid sample synthesized as a nucleic acid sample can also be used.
  • the single-stranded denatured gene sample can be obtained, for example, by the following method.
  • TE 10mM Tris / HCl (pH8.0), ImM EDTA
  • Tris (hydroxymethinole) amino methane A single-stranded nucleic acid probe immobilized on the electrode surface and a single-stranded nucleic acid sample are allowed to coexist for hybridization.
  • the nucleic acid sample is a gene sample.
  • the coexistence for the above hybridization can be performed, for example, by dropping a solution containing a single-stranded denatured gene sample onto a single-stranded nucleic acid probe immobilized on the electrode surface.
  • concentration of the gene sample in the solution containing the single-stranded denatured gene sample can be, for example, 10 to 100 / zmol / L.
  • a solution containing a single-stranded denatured gene sample needs to function as an electrolytic solution. Examples of such a solution include various buffers.
  • the buffer is a buffer containing EDTA (ethylenediaminetetraacetic acid), such as Tris buffer containing EDTA (TE buffer), Tris / acetate buffer containing EDTA (TAE buffer), or EDTA. Tris. Borate buffer (TBE buffer), standard sodium citrate buffer (SSC buffer) and the like.
  • EDTA ethylenediaminetetraacetic acid
  • TBE buffer Tris buffer containing EDTA
  • TBE buffer Tris. Borate buffer
  • SSC buffer standard sodium citrate buffer
  • the hybridization temperature is the melting temperature (Tm)
  • Tm melting temperature
  • Tm can be adjusted by salt temperature or formamide concentration.
  • 1% formamide usually lowers Tm by about 0.6 ° C.
  • the hybridization in the method of the present invention is suitably performed, for example, at a temperature of 37 to 72 ° C.
  • measure the AC impedance of the electrode on which the nucleic acid probe is immobilized The measurement of the AC impedance can be performed as shown in FIG. Figure 3 shows the concept of the method for measuring whether the single-stranded DNA (probe DNA) immobilized on the gold electrode hybridized with the single-stranded DNA dropped on the sample from the AC impedance (Z).
  • a gold electrode is provided on the surface of a glass substrate, and there is a region on which a nucleic acid probe is fixed. Then, an electrolytic solution containing the target gene is dropped into the region where the nucleic acid probe is immobilized, and the mixture is placed under predetermined hybridization conditions. Specifically, for example, 40 ⁇ L (10 ⁇ 1 / L) of probe DNA with an SH group attached to the 5fe end is fixed onto the gold electrode by dropping, and the sample DNA is also 10 ⁇ L. Add 40 ⁇ L of mo1 / L. The diameter of the TE solution on the gold electrode is 4 mm (12.6 mm 2 ).
  • a platinum electrode as a counter electrode is brought into contact with the electrolyte, and for example, an AC impedance between a frequency of 0.01 Hz and 1 MHz is applied between the gold electrode and the platinum electrode.
  • the measurement of the AC impedance can be performed at room temperature. For example, when measuring the effect of temperature on hybridization, the measurement temperature can be appropriately changed.
  • Figure 4 shows the measurement of the AC impedance (Z) between the two electrodes in contact with the electrolyte. 5 shows an electrical equivalent circuit in a constant.
  • the interface 1 shows the state when the probe DNA and the sample DNA hybridize.
  • the impedance of the electrode, the interface 1, the electrolyte (TE solution), the interface 2, and the counter electrode is reduced.
  • the equation in the figure indicates the reaction resistance at the electrode interface, the resistance of the electrolyte, the capacitance, etc., and Z w in the figure is the so-called Warburg impedance, which indicates the diffusion of species that react at the electrode interface.
  • the species that react electrically include the electric conduction process in the cell, the Faraday charge transfer process, and the double-layer charging process.
  • the impedances corresponding to these electrochemical processes behave the same as simple passive elements such as resistors and capacitors (apart from Z w).
  • the reaction resistance at the interface 1 the resistance of the double layer, the solution, and the like can be obtained from the equations in the figure. Also, by decoding the low-frequency side below Equation 1 OKHz, it is possible to obtain the diffusion information of ions at the fixed probe DNA interface. Therefore, at interface 1, a reaction occurs as to whether or not the probe DNA and the sample DNA hybridize. Therefore, the presence or absence of hybridization can be analyzed by the AC impedance measurement method. The results obtained by the impedance measurement are generally displayed as a Bode plot or a complex plane plot (Cole-Cole-plot).
  • the horizontal axis is the frequency and the vertical axis is the logarithm of the absolute value of the impedance, showing the relationship between the frequency and the absolute value of the impedance.
  • the solution resistance Rsol is obtained from the high frequency band (several hundred kHz or more), the double layer capacitance Cdl from the middle frequency band (several lOOmHz to several kHz), and the reaction resistance r from the low frequency band (several lOOmHz or less).
  • the complex plane display Cold-Cole-plot
  • the imaginary component z '' is plotted on the axis.
  • the measured frequency is added to the plotted point, and Rsol, Cdl, and r can be obtained from the complex plane display as in 1.
  • Rsol, Cdl, and r can be obtained from the complex plane display as in 1.
  • the detection of the hybridization will be described in detail in Examples.
  • the resistance value r is obtained from the results shown in the above port diagram display (for example, the results shown in FIG. 5), and the hybridization is detected. Since r fluctuates depending on the presence or absence of the hybridization, hybridization can be detected.
  • the result at the same frequency depends on the presence or absence of hybridization. Since the absolute value of the impedance fluctuates, the hybridization can be detected.
  • hybridization can be detected as follows.
  • the optimal conditions for hybridization depend on the nucleotide sequence, length, nucleic acid concentration, etc. of the nucleic acid probe used.
  • the hybridization temperature is about 25 ° C lower than the melting temperature, usually in the range of 37 to 72 ° C.
  • the temperature is brought to a range of 80 to 88 ° C., and then the AC impedance is measured, and the hybridization can be detected as follows.
  • the target gene having a mismatch with the nucleic acid probe is dissociated from the nucleic acid probe. Therefore, the measurement results of AC impedance before and after the above temperature treatment (resistance) Appears in the image, and the presence of the mismatch can be detected.
  • the optimal temperature within the range of 80 to 88 ° C. can be appropriately determined according to the length and the base sequence of the target gene and the nucleic acid probe. Example .
  • the base sequence of the oligo DNA used is as follows.
  • aO Trp64 (fat-reduced type) gtggccatcgcctggactccgagac (SEQ ID NO: 1)
  • bO Arg64 (fat storage type) gtggccatcgcccggactccgagac (SEQ ID NO: 2)
  • al DNA complementary to Trp64 caccggtagcggacctgaggctctg (SEQ ID NO: 3)
  • bl Arg64 and complementary DNA Only one central base differs. (One is a lean type and the other is an obese type due to the difference of only one base.)
  • the combination of a0-a1 is a hybridizing yarn, and the combination of aO-bl is a non-hybridizing combination.
  • the impedance was measured while automatically sweeping the frequency from 0.01 Hz to 1 MHz.
  • the impedance / gain-phase analyzer 1260 manufactured by Solartron was used as the AC impedance measuring device.
  • Figure 5 shows the measurement results.
  • Fig. 5 (1) shows the results when only the gold electrode was used, (2) shows the results when TrpSH (aO) was immobilized as the probe DNA on the gold electrode, and (3) immobilized TrpSH (aO) as the probe DNA.
  • (4) is the result of hybridization of b1 with a single base difference to a gold electrode on which TrpSH (aO) was immobilized as probe DNA.
  • Is the result of The results of the AC impedance measurement shown in Fig. 5 are shown in a Bode diagram, where the horizontal axis is the frequency and the vertical axis is the logarithm of the absolute value of the impedance, showing the relationship between the frequency and the absolute value of the impedance.
  • the resistance r can be obtained by extrapolating this graph to the low frequency side.
  • Table 1 The results are shown in Table 1.
  • r ( ⁇ ) indicates the reaction resistance (charge transfer resistance) at the gold electrode interface where the probe DNA is immobilized. The measurement was performed between the gold electrode and the TE solution both when using only the gold electrode and when DNA was fixed on the gold electrode.
  • table 1 From Table 1, the reaction resistance r at the gold electrode interface is 3.6 ⁇ for the gold electrode only (when no DNA is present) and 3.7 3. ⁇ for the probe DNA only.
  • the r when hybridized is 5.1 ⁇ , and the r for a mismatched junction that does not originally hybridize is 5.1 ⁇ .
  • mismatched junctions can be converted to single-stranded DNA by heat treatment at 80 to 88 ° C, and those that have hybridized normally cannot be converted to single-stranded DNA at this temperature.
  • the presence or absence of hybridization can be determined by measuring after performing the temperature treatment described above.
  • the presence / absence of hybridization and the presence / absence of mismatch can be determined without using a labeling substance used for identifying double-stranded DNA. .
  • Fig. 6 shows the result of Fig. 5 expanded for the frequency range of 1 to 10 OHz. From the results in Fig. 6, the presence or absence of Can be specified.
  • heat treatment was performed at 84-88 ° C for 10 minutes, then the temperature was returned to room temperature.
  • the results of AC impedance measurement also showed that the frequency ranged from 1 to 10 OHz. Then, it is possible to determine the presence or absence of a mismatch.
  • a nucleic acid probe such as a single-stranded oligo DNA is immobilized on an electrode, and it is determined whether a double-stranded DNA or the like is formed when a nucleic acid sample such as a sample DNA is added.
  • a method of chemical measurement that does not require the use of dyes, can be repeated, and can detect the formation of double-stranded DNA with higher accuracy.
  • a detection method can be provided.

Abstract

L'invention vise à mettre au point un procédé permettant de détecter une hybridation, selon lequel il est prévu d'immobiliser un échantillon d'acide nucléique (par exemple un oligo ADN à brin simple) sur une électrode, d'ajouter un échantillon d'acide nucléique (par exemple un spécimen d'ADN), puis de mesurer par voie électrochimique si un double brin, tel qu'un ADN à double brin, est formé ou non. Ledit procédé doit permettre de détecter avec une grande précision la formation double brin d'un ADN double brin, sans avoir recours à un quelconque colorant, ainsi que d'effectuer la mesure à plusieurs reprises. L'invention concerne un procédé de détection d'hybridation entre un acide nucléique cible et un échantillon d'acide nucléique, qui comprend les étapes suivantes : faire coexister un échantillon d'acide nucléique à brin simple immobilisé à la surface d'une électrode, avec un échantillon d'acide nucléique dénaturé en acide nucléique à brin simple, puis détecter l'hybridation de l'échantillon d'acide nucléique avec l'acide nucléique cible contenu dans l'échantillon d'acide nucléique décrit ci-dessus, par mesure de l'impédance du courant alternatif de l'électrode portant l'échantillon d'acide nucléique immobilisé dessus.
PCT/JP2003/012499 2002-11-14 2003-09-30 Procede de detection d'hybridation WO2004044570A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003266711A AU2003266711A1 (en) 2002-11-14 2003-09-30 Method of detecting hybridization

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2002-331059 2002-11-14
JP2002331059 2002-11-14

Publications (1)

Publication Number Publication Date
WO2004044570A1 true WO2004044570A1 (fr) 2004-05-27

Family

ID=32310613

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2003/012499 WO2004044570A1 (fr) 2002-11-14 2003-09-30 Procede de detection d'hybridation

Country Status (2)

Country Link
AU (1) AU2003266711A1 (fr)
WO (1) WO2004044570A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008139016A1 (fr) 2007-05-09 2008-11-20 Consejo Superior De Investigaciones Científicas Biocapteur impédancemétrique et ses applications
WO2011073481A1 (fr) 2009-12-15 2011-06-23 Consejo Superior De Investigaciones Científicas (Csic) Système et procédé multianalyte reposant sur des mesures impédimétriques

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0478319A1 (fr) * 1990-09-28 1992-04-01 Kabushiki Kaisha Toshiba Méthode pour la détection de gènes
WO1993022678A2 (fr) * 1992-04-23 1993-11-11 Massachusetts Institute Of Technology Procedes et appareil optiques et electriques de detection de molecules
WO2000039325A2 (fr) * 1998-12-23 2000-07-06 Institut für Physikalische Hochtechnologie e.V. Detecteur d'affinite pour deceler des evenements de liaisons moleculaires specifiques et son utilisation
WO2000077523A1 (fr) * 1999-06-10 2000-12-21 Motorola, Inc. Biocapteurs utilisant des polymeres conjugues a charge neutre
WO2001042508A2 (fr) * 1999-12-09 2001-06-14 Motorola, Inc. Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0478319A1 (fr) * 1990-09-28 1992-04-01 Kabushiki Kaisha Toshiba Méthode pour la détection de gènes
WO1993022678A2 (fr) * 1992-04-23 1993-11-11 Massachusetts Institute Of Technology Procedes et appareil optiques et electriques de detection de molecules
WO2000039325A2 (fr) * 1998-12-23 2000-07-06 Institut für Physikalische Hochtechnologie e.V. Detecteur d'affinite pour deceler des evenements de liaisons moleculaires specifiques et son utilisation
WO2000077523A1 (fr) * 1999-06-10 2000-12-21 Motorola, Inc. Biocapteurs utilisant des polymeres conjugues a charge neutre
WO2001042508A2 (fr) * 1999-12-09 2001-06-14 Motorola, Inc. Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008139016A1 (fr) 2007-05-09 2008-11-20 Consejo Superior De Investigaciones Científicas Biocapteur impédancemétrique et ses applications
US8608919B2 (en) 2007-05-09 2013-12-17 Consejo Superior De Investigaciones Científicas Impedimetric sensor and applications thereof
WO2011073481A1 (fr) 2009-12-15 2011-06-23 Consejo Superior De Investigaciones Científicas (Csic) Système et procédé multianalyte reposant sur des mesures impédimétriques
US9170227B2 (en) 2009-12-15 2015-10-27 Consejo Superior De Investigaciones Cientificas (Csic) Multi-analyte system and method based on impedimetric measurements

Also Published As

Publication number Publication date
AU2003266711A8 (en) 2004-06-03
AU2003266711A1 (en) 2004-06-03

Similar Documents

Publication Publication Date Title
ES2636664T3 (es) Microelectrodos nanoestructurados y dispositivos de biodetección que los incorporan
Wang et al. Magnetic bead-based label-free electrochemical detection of DNA hybridization
Wang et al. Amplified label-free electrical detection of DNA hybridization
WO2001057278A3 (fr) Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hela humaines ou d'autres cellules epitheliales humaines du col de l'uterus
US5656429A (en) Polynucleotide and protein analysis method using magnetizable moieties
WO2005018436A2 (fr) Procede de diagnostic, de pronostic et de traitement du syndrome metabolique
CN107589163A (zh) 一种用于mecp2突变基因检测的电化学传感器制备方法
KR100481115B1 (ko) 유전자 검출용 담체 및 인터페론 요법의 유효성을검출하기 위한 그 사용방법
US11505799B2 (en) Aptamers for measuring lipoprotein levels
CN104031990B (zh) 苯丙酮尿症ｐａｈ基因检测试剂盒
CN108845003A (zh) 一种通用型纳米孔检测传感器及检测方法
KR100423021B1 (ko) 혼합 인터칼레이터, 이를 이용한 디엔에이의 전기화학적검출방법 및 이를 위한 검출 키트
US20040058331A1 (en) Blood testing method, testing chip, and testing device
EP2473849A1 (fr) Puce de nitrure de silicium nanoporeuse pour l'analyse de profils d'expression génique
CN113621697A (zh) 引物组、包含该引物组的试剂盒以及该引物组的用途
WO2004044570A1 (fr) Procede de detection d'hybridation
CN108445067A (zh) 一种双信号无酶的信号放大rna纳米生物传感器、制备方法及其应用
CN111812166A (zh) 一种检测外泌体microRNA的电化学生物传感器及其制备方法
JP2004177399A (ja) ハイブリダイゼーションの検出方法
Diessel et al. Online resistance monitoring during autometallographic enhancement of colloidal Au labels for DNA analysis
US8691499B2 (en) Method of detecting molecules
WO2018014682A1 (fr) Procédé de détection d'un gène cible au moyen d'une technique de pcr combinée à une technique efirm
JP2001321198A (ja) 試料核酸断片の相補性の検定方法
JP2013013375A (ja) 電極上における核酸の増幅反応を伴う電気化学的検出方法
JP4040834B2 (ja) ２本鎖ｄｎａの分析方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase