+

WO2004065624A1 - Dispositif de mesure et procede de detection d'une sequence d'adn - Google Patents

Dispositif de mesure et procede de detection d'une sequence d'adn Download PDF

Info

Publication number
WO2004065624A1
WO2004065624A1 PCT/AT2004/000022 AT2004000022W WO2004065624A1 WO 2004065624 A1 WO2004065624 A1 WO 2004065624A1 AT 2004000022 W AT2004000022 W AT 2004000022W WO 2004065624 A1 WO2004065624 A1 WO 2004065624A1
Authority
WO
WIPO (PCT)
Prior art keywords
electrode
measuring
electrolyte
measuring arrangement
dna sequence
Prior art date
Application number
PCT/AT2004/000022
Other languages
German (de)
English (en)
Inventor
Kriemhilt Roppert
Gert Fränzl
Original Assignee
Sy-Lab Vgmbh
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 Sy-Lab Vgmbh filed Critical Sy-Lab Vgmbh
Priority to EP04704149A priority Critical patent/EP1590483A1/fr
Publication of WO2004065624A1 publication Critical patent/WO2004065624A1/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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • 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

Definitions

  • the present invention relates to a measuring arrangement and a method using a measuring arrangement for the detection of a DNA sequence by means of impedance spectroscopy, which measuring arrangement comprises a gold electrode, the surface of which is covered by a soap-assembled monolayer (SAM) made of identical molecules, which on the side facing away from the electrode Side with oligonucleotides which are complementary to the DNA sequence to be detected and which hybridize with it, are covalently coupled, a hybridization buffer forming the electrolyte, a reference electrode, optionally a counter electrode and means for applying a direct and alternating voltage component, for setting a measuring frequency and for measuring the impedance of the working electrode / electrolyte system.
  • SAM soap-assembled monolayer
  • a standard method is the selective amplification of one for e.g. a microorganism-specific section of the genomic DNA by means of a PCR reaction, subsequent gel electrophoresis and optical detection of the separated fragments after staining with ethidium bromide. The detection takes place via the known fragment size.
  • a more precise, also common procedure is the so-called "Southern Blotting" method, in which the DNA separated in the gel is transferred to a special membrane after the PCR.
  • the detection is carried out by hybridization with a probe, for example with a fluorophore or an enzyme is marked so that an optical evaluation can be carried out.
  • a so-called "real-time PCR” is a more complex method, in which the amplification of the specifically amplified DNA fragment can be followed online by means of fluorescence.
  • Sensors are a group of devices that are designed to enable easy handling of complex samples. They consist of a transducer on or generally in the immediate vicinity of which there is a sensitive layer. If the substance to be detected binds or comes into direct interaction with this sensitive layer, there is a change that can be converted into an easily measurable, e.g. optical or electrical signal is converted.
  • the group of biosensors makes use of the interaction of biomolecules.
  • Electrochemical detection systems are generally considered to be both more sensitive and less expensive.
  • Known approaches to detect DNA as specifically and sensitively as possible with the aid of an electrochemical sensor are based on the use of electrodes based on mercury.
  • the toxicity of the metal used and the resulting disposal problem are disadvantageous here.
  • Another, often used approach uses electrochemically measurable intercalators which must have specificity either for the hybridized double strand or the non-hybridized single strand of the DNA to be detected.
  • these substances are also often toxic and mostly lack selectivity and reproducibility.
  • DNA probes labeled with various ferrocenes or more general "electron transfer moieties" e.g. WO 01/06016 A.
  • WO 01/06016 A general "electron transfer moieties”
  • the second type of electrode relies on using only a single working electrode and evaluating the capacitance at the electrode / solution interlayer.
  • the associated measuring principle is based on the theory of the electrolytic double layer, which can in principle be regarded as consisting of two conductive media, one of which is formed by the electrode and the second by the electrolyte. These two "phases" are separated from each other by the above-mentioned insulating layer. Changes in this layer (for example due to the binding of the analyte) cause a change in the capacitive measurement signal.
  • the size and thus the measurability of the signal also depends on the type of analyte as from the assignment with this.
  • a further layer is formed.
  • the catcher molecule has an affinity for a protein or a complementary DNA sequence in the solution and thereby binds to it. In all of these cases, the total capacity is reduced according to the theory.
  • a simple system of such a sensor is described for example in DE 39 23 420 A.
  • a sensor based on a semiconductor electrode is known, the electrode being covered with a polymeric, relatively disordered layer to which an antigen has been coupled as a capture molecule (Sibai, A. et al., Sens. Act. B31 (1996), S. 125-130).
  • an increase in capacity is measured in this sensor, which is based on the possibility of infiltration into the polymer layer and the charge exchange and charge deposition taking place there.
  • 91-93 describe a sensor for the impedance spectroscopic detection of a specific DNA sequence or of horseradish peroxidase, consisting of a gold electrode with a “soap assembled” bilayer made of a surface-active substance, to which one to be detected DNA sequence complementary oligonucleotide or a horseradish peroxidase antibody was coupled.
  • the impedance measurement was carried out in a frequency range of 1-10 kHz, with different measurement effects being achieved for both analytes.
  • the object of the invention is to provide a measuring arrangement and a method which enable rapid, specific and inexpensive detection of DNA sequences, in particular of such biological samples.
  • the detection of the DNA sequences should only require a small amount of work and should avoid the use of dangerous or harmful substances. In particular, to Reduction of the workload and the denaturation step can be omitted.
  • a measuring arrangement of the type mentioned at the outset which is characterized in that the ionic strength of the electrolyte is in a range from 0.005 to 0.15 mol / 1 and the means for setting the measuring frequency is designed such that a measuring frequency is adjustable between 17 kHz and 24 kHz.
  • the invention is based on the discovery that the behavior of a sensor, the basic structure of which corresponds to that of a capacitive sensor based on a gold electrode with a SAM, with the property that the sensor has a phase angle between -86.5 ° and -90 at 20 Hz ° no longer corresponds entirely to that of a capacitive sensor, but can only be assigned to it more than related if components of the measuring arrangement are used differently from conventional systems.
  • the model of the independent layer which generally corresponds to the functioning of a capacitive sensor, is not applicable to the invention. This is immediately evident in the increase in capacity as a result of a detectable, specific hybridization. In the case of a purely capacitive sensor, the measurement signal would have had to consist of a reduction in capacitance (although possibly very small and therefore possibly not measurable).
  • the equivalent circuit also differs significantly from that of a purely capacitive sensor: while purely capacitive sensors are adequately characterized by an equivalent circuit consisting of a resistor and a capacitance, the invention shows that it is necessary to determine the capacitance by means of a “constant phase element ( CPE) to replace.
  • CPE constant phase element
  • RE reference electrode, e.g. Calomel, Ag / AgCl
  • the structure of the working electrode used in the measuring arrangement according to the invention corresponds to that of known capacitive sensors.
  • a gold electrode is “self-assembled” with a layer of insulating, ie non-conductive, molecules that form a dense monolayer (SAM).
  • SAM dense monolayer
  • the gold electrodes used in the invention are characterized in that, in the raw state, at least the surface area which can come into direct contact with the electrolyte consists of high-purity gold (over 99% purity).
  • This surface can consist of solid gold, but the gold can also be in a relatively thin layer on insulating material, e.g. Glass, plastic or silicone can be applied, also with one or more intermediate layers, which only serve as an adhesion promoter between the insulating material and the gold, but can also influence the properties of the electrode.
  • insulating material e.g. Glass, plastic or silicone can be applied, also with one or more intermediate layers, which only serve as an adhesion promoter between the insulating material and the gold, but can also influence the properties of the electrode.
  • More complicated designs regarding the layer structure and geometry of the electrode are also possible. It is also possible to combine several electrodes on one chip and thus build up an electrode array.
  • a Seif Assembled Monolayer is a layer of organic or inorganic molecules that is only one molecule thick (“monolayer") and of the same or a few types (“seif assembled mixed monolayer”), but usually not more than two different ones Molecular types, of similar molecules, which arrange themselves through a lateral interaction to a relatively dense layer.
  • Such Layers can be produced on gold surfaces, for example, with the aid of molecules whose end and / or head groups have sulfur atoms (for example in the form of thiols, sulfides, disulfides and the like) which have a high affinity for gold.
  • X is a functional group selected from the group consisting of carboxy, amino, hydroxy, aldehyde, Keto and Azo group.
  • the method according to the invention using a measuring arrangement of the type mentioned at the outset is characterized in that the electrolyte is introduced with an ionic strength in the range from 0.005 to 0.15 mol / 1, and the DNA sequence to be detected is isolated from a sample by methods known per se is amplified and optionally denatured, is added to the electrolyte, and after hybridization of the amplificates with the oligonucleotides immobilized on the working electrode at room temperature, the impedance measurement is carried out at a measuring frequency between 17 kHz and 24 kHz. A statistically unambiguous increase in the measurement signal suggests the presence of the DNA sequence to be detected.
  • a change in the conformation of some / many molecules is thus communicated to the molecules not directly affected by an event and leads to a change in the properties of the entire group as a whole.
  • Possible examples of a change in conformation could be, for example, a change in the dielectric constant of the layer concerned, a change in the “crystal structure” of the SAM or the entire layer, or a change in the angle of inclination of the thiols of the gold surface and associated with it a change in the thickness of the SAM.
  • the detection of the DNA sequence is carried out according to the invention as follows: Gold electrodes are covered by self-assembly with a layer of insulating, ie non-conductive molecules, for example thiols, which form a dense monolayer (SAM). At their end facing away from the electrode surface, these molecules have a functional group (for example a carboxy group) which, after complete assembly, enables the covalent coupling of a capture probe which is complementary to the DNA sequence to be detected (“capture probe”) by means of standard chemistry.
  • the DNA to be detected is isolated from a sample, for example from microorganisms in a food sample, purified if necessary, and amplified with specific primers in the course of a PCR (polymerase chain reaction).
  • the PCR products are then heat denatured and transferred in a shock at about -20 ° C on ice in order to obtain a single-stranded DNA.
  • the denaturation step is not necessary for some applications of the detection method.
  • the measurement is carried out using the technique of impedance spectroscopy in an electrolyte, which allows both a specific hybridization of the capture probe with the PCR fragment at room temperature and a detection of the change in conformation of the non-conductive layer due to the hybridization that has taken place.
  • the signal consists in an increase in the (pseudo) capacitance of the working electrode / electrolyte system as a result of a change in the conformation of the non-conductive layer.
  • FIG. 1 The structure of the working electrode and the course of the hybridization are shown schematically in FIG. 1.
  • FIG. 2 schematically illustrating part of an embodiment of the measuring arrangement according to the invention
  • FIGS. 3-5 and 8 a graphical representation of the measurement results (capacity versus time) of the Examples 1, 2, 3, 5 and 6 show
  • FIGS. 6 and 7 each show a block diagram in which the percentage increase in capacity for the measurements of example 4 is shown.
  • Gold electrodes were made to order by E & E-Electronics GmbH (Engerwitzdorf, AT). These electrodes in thin-film technology consist of a glass substrate on which a structure, as can be seen from FIG. 2, was applied by means of ⁇ i / Cr adhesion promoter.
  • the reaction zone has a diameter of approx. 700 ⁇ m; the glass substrate has a format of approx. 3 x 10 mm.
  • the length of the supply line and the exact dimensions of the contact area are not relevant to the invention.
  • the oligonucleotides used in the examples were obtained from VBC-Genomics. Modification of the electrodes with a thiol layer (SAM):
  • Electrodes were placed individually in a glass jar in approx. 700 ⁇ l absolute ethanol. (from Merck) 2 x 4 min in an ultrasonic bath (floating stand), the solution being changed in between. Then the electrodes were dried in a stream of nitrogen. First 1 ⁇ l of 30% hydrogen peroxide (ppA, Fluka) and then 3 ⁇ l of concentrated sulfuric acid (ACS reagent, Sigma) were pipetted onto the reaction zone of the electrode. After a reaction time of 1 to 2 minutes, the liquid was removed and washed with 2 ⁇ 700 ⁇ l of water per electrode (water for the molecular biology from Sigma, filtered through a 0.2 ⁇ m Norspritze filter from ⁇ algene).
  • ppA 30% hydrogen peroxide
  • ACS reagent concentrated sulfuric acid
  • the mixture was then dried over silica gel first in a stream of nitrogen and then in a vacuum desiccator (60 min).
  • the incubation was then carried out in 700 ⁇ l of a solution of 3.2 mg of 11-mercaptoundecanoic acid (Aldrich) in 100 ml of chloroform (Sigma, ACS-Reagent, Spectrophotometric Grade) in sealed glass vessels for at least 14 hours.
  • Multilayer layers were removed by washing twice with ethanol (approx. 700 ⁇ l each) and the electrodes were dried in a stream of nitrogen.
  • the lead of the electrode (1) was carefully stripped with insulating tape (2) so that only the reaction zone (3) can come into contact with the electrolyte (4).
  • the contact area of the electrode (1) was inserted into a small gold clip located in a Teflon head.
  • the Teflon head has recesses through which a platinum wire used as counter electrode (5) and a salt bridge (6), which leads to the reference electrode, can come into contact with the electrolyte (4), as well as an internally arranged, conductive connection (7) of the gold clip outwards (see Fig. 2).
  • the salt bridge (6) consists of a curved thin glass tube, which can be made, for example, from a Pasteur pipette. This was filled with a hot saturated KC1 solution (approx.
  • concentration is preferably in a range from 0.02 ⁇ SSC to 1 ⁇ SSC, in particular from 0.05 ⁇ SSC to 0.5 ⁇ SSC.
  • a saturated calomel electrode (SCE) coupled via the salt bridge described above serves as the reference electrode.
  • the measurement was carried out with a potentiostat / galvanostat PGSTAT30 with FRA2 module from Ecochemie (NL) in the three-electrode configuration.
  • the potential range preferably corresponds to 200 to 300 mV.
  • the amplitude was 10 mV, and the detection frequency set was 18,828 Hz.
  • Nucleotide mix 200 ⁇ M each of dATP, dGTP, dCTP; 600 ⁇ M dUTP (Genexpress, Genecraft)
  • Electrodes were prepared analogously to example 1.
  • the capacity increase for the complementary PCR fragment and the negative control of the PCR are 6.5% and 2.3%, respectively.
  • Fig. 4 (a, b) and from a comparison with Fig. 3 (a, b) it can be seen that the difference between positive and negative reactions in PCR products, i.e. Hybridization or no hybridization, has a lower ratio than for pure oligonucleotides.
  • the signal-to-noise ratio is a sufficient 280%. A clear assignment of the signals is thus guaranteed.
  • a signal increase of at least 3.3% indicates clear detection of the specific hybridization with a PCR fragment.
  • N-uracil-N-glycosilase Röche
  • This enzyme breaks down any contamination in the form of PCR products.
  • the N-uracil-N-glycosilase used is heat-labile and is consequently inactivated in the course of the PCR reaction, but remains in the solution.
  • Fig. 5 (a, b).
  • the capacity increase is 6.7% for the positive reaction and 4.1 for the negative reaction. This corresponds to a signal-to-noise ratio of 163%.
  • the key figures are therefore not as good as in Examples 1 and 2. Further experiments have shown, however, that a capacity increase of at least 5.4% is a clear indication of a specific hybridization. Carrying the negative control is not necessary for the flawless detection as in the previous example.
  • Example 3 The procedure corresponds to that of Example 3 with the exception of heat denaturation, which was omitted in the present example.
  • the measurement results show no significant difference from Example 3. It follows that the denaturation of the PCR fragments is not necessary for a given amount of PCR fragment and the sensitivity achieved, since that small percentage of the PCR fragments which is present as a single strand is for the detection is sufficient.
  • Example 5 Specific detection of germs in different inoculated meat samples
  • Mashed mixed pork and beef were purchased from a butcher and kept refrigerated until used. The same applies to the onion sausage.
  • the bacterial count of the overnight cultures used for artificial contamination, the total bacterial count and the bacterial count of the coliforms in the non-vaccinated meat were determined impedometrically using a BacTrac 4300 analysis system according to the manufacturer's specifications (SY-LAB Engineering, Neupurkersdorf, AT). The measurement method was calibrated for the strains used with the plate count method (number of colonies on plates). Coliforms were determined to be approximately 2.8 x 10 2 bacteria / ml, the total number of bacteria was approximately 1.6 x 10 4 bacteria / ml. With 1-10 inoculated germs there is an approximately 1000 to 10,000-fold excess of the background flora, with 10-100 an approximately 100 to 1000-fold excess of other germs. Isolation of DNA from meat samples:
  • the electrode preparation largely corresponds to example 1, but the electrodes were dried over silica gel orange over silica gel instead of one hour for two hours.
  • the terminal carboxy group was activated by 3 ⁇ l instead of 5 ⁇ l EDC solution of the same concentration and immobilized was 1 ⁇ l instead of 0.5 ⁇ l “SLT1-CPPCR” oligonucleotide with 5 ′ aminolink, dissolved in water for molecular biology with a concentration of 0.5 ⁇ g / ⁇ l.
  • the results are shown in Fig. 6 in the form of a block diagram.
  • the percentage increase in capacity for onion sausage was 6.88% and 6.72%, that of the negative control only 4.8%.
  • the difference between a specific and a non-specific reaction is smaller than in the case of the spotted one, but the slight variation in the measurement results shows that here too one can speak of an absolutely reliable distinction between positive and negative samples.
  • the results of the measurements are shown in FIG. 7 in the form of a block diagram.
  • Example 6 Detection of undenatured, complementary PCR products vs. non-complementary, non-denatured PCR products
  • the electrodes were prepared as in Example 5.
  • the PCR information for the complementary SLT1 fragment corresponds to Example 2.
  • the non-complementary PCR fragment SLT2 was generated analogously with the following primers:
  • the percentage increase in capacity was 7.64% for the complementary SLT1 fragment and 4.66% for the non-complementary SLT2 fragment. This corresponds to a signal-to-noise ratio of 164%.
  • the measurement curves are shown in Fig. 8 (a, b). It can be seen that a complementary and a non-complementary PCR fragment can be clearly distinguished.
  • the capacity increases of the complementary PCR fragment are in the same range as that of the complementary PCR products of the meat experiment (example 5).
  • the reaction to the non-complementary SLT2 fragment corresponds to the reaction of the water control in the meat experiment.
  • the measuring arrangement according to the invention and the method according to the invention have considerable advantages over the prior art. Both the hybridization and the detection of the hybridization are extremely fast. The determination of the presence of sequences complementary to the immobilized oligonucleotides (“capture probe”) is completed in 5 minutes at the latest Time span of just over 10 minutes after completion of the PCR. This significantly minimizes the time-related advantage of, for example, RT-PCR through online detection. In comparison, the method according to the invention is far less complex both in terms of material consumption and in terms of apparatus, and is therefore less expensive. An already very rapid concept based on a dot-plot hybridization system with enzyme-labeled probes requires at least 150 minutes between the end of the PCR and the detection of the hybridization.
  • the specificity of the method according to the invention is so high that no washing processes are necessary, i.e. the workload is reduced to a minimum.
  • Hybridization also takes place at room temperature (elevated temperatures are usually necessary for specific hybridizations).
  • detection using the method or measuring arrangement according to the invention does not require any handling of toxic chemicals, such as Intercalators, which is becoming increasingly important in an age of heightened sensitivity to the topic of occupational safety.
  • the DNA can be isolated quickly and without cost. After amplification by the PCR, the DNA fragment to be detected can be immediately subjected to a measurement with the measuring arrangement according to the invention or according to the method of the invention without further purification or even just denaturation.
  • the detection limit using the example of an EHEC detection is around or below 10 germs per 25 g meat.
  • the measuring arrangement according to the invention and the measurement itself are relatively simple in terms of apparatus and are therefore extremely inexpensive for specific detection. This point is particularly noticeable in comparison to the equipment and thus financial expenditure in the area of microarrays or real-time PCR.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nanotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Composite Materials (AREA)
  • Materials Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un dispositif de mesure permettant de détecter une séquence d'ADN par spectroscopie d'impédance. Ce dispositif de mesure comprend une électrode en or dont la surface est recouverte d'une monocouche auto-assemblée (SAM) constituée de molécules identiques, lesquelles sont couplées de façon covalente, sur le côté opposé à l'électrode, avec des oligonucléotides qui sont complémentaires vis-à-vis de la séquence d'ADN à détecter et s'hybrident avec celle-ci. Ledit dispositif comprend par ailleurs un tampon d'hybridation formant l'électrolyte, une électrode de référence, éventuellement une contre-électrode, ainsi que des moyens pour appliquer une composante continue et une composante alternative, pour régler une fréquence de mesure et pour mesurer l'impédance du système électrode de travail/électrolyte. L'invention se caractérise en ce que la force ionique de l'électrolyte est comprise entre 0,005 et 0,15 mol/l et en ce que les moyens pour régler la fréquence de mesure sont conçus de telle sorte qu'une fréquence de mesure comprise entre 17 kHz et 24 kHz puisse être définie. Ce dispositif de mesure permet de détecter des séquences d'ADN de façon rapide, spécifique et économique, y compris à partir d'échantillons réels.
PCT/AT2004/000022 2003-01-24 2004-01-22 Dispositif de mesure et procede de detection d'une sequence d'adn WO2004065624A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP04704149A EP1590483A1 (fr) 2003-01-24 2004-01-22 Dispositif de mesure et procede de detection d'une sequence d'adn

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ATA95/2003 2003-01-24
AT952003A AT413214B (de) 2003-01-24 2003-01-24 Messanordnung und verfahren zur detektion einer dna-sequenz

Publications (1)

Publication Number Publication Date
WO2004065624A1 true WO2004065624A1 (fr) 2004-08-05

Family

ID=32739100

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AT2004/000022 WO2004065624A1 (fr) 2003-01-24 2004-01-22 Dispositif de mesure et procede de detection d'une sequence d'adn

Country Status (3)

Country Link
EP (1) EP1590483A1 (fr)
AT (1) AT413214B (fr)
WO (1) WO2004065624A1 (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
CN103343164A (zh) * 2013-06-17 2013-10-09 武汉轻工大学 产志贺毒素大肠杆菌多重pcr检测方法、试剂盒及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19610115A1 (de) * 1996-03-14 1997-09-18 Fraunhofer Ges Forschung Detektion von Molekülen und Molekülkomplexen
WO1998031839A2 (fr) * 1997-01-21 1998-07-23 President And Fellows Of Harvard College Detection de proprietes electroniques de molecules biologiques sur des surfaces
WO1999014596A1 (fr) * 1997-09-15 1999-03-25 Ab Sangtec Medical Detecteur d'affinite a effet capacitif
WO1999042827A2 (fr) * 1998-02-20 1999-08-26 Wolfbeis, Otto, Samuel Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides
WO2000062047A1 (fr) * 1999-04-14 2000-10-19 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Mosaique de capteurs electriques

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001042508A2 (fr) * 1999-12-09 2001-06-14 Motorola, Inc. Procedes et compositions se rapportant a la detection electrique des reactions d'acides nucleiques
AU2000263067A1 (en) * 2000-08-07 2002-02-18 Azur Environmental Ltd. Method of an apparatus for the detection of analytes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19610115A1 (de) * 1996-03-14 1997-09-18 Fraunhofer Ges Forschung Detektion von Molekülen und Molekülkomplexen
WO1998031839A2 (fr) * 1997-01-21 1998-07-23 President And Fellows Of Harvard College Detection de proprietes electroniques de molecules biologiques sur des surfaces
WO1999014596A1 (fr) * 1997-09-15 1999-03-25 Ab Sangtec Medical Detecteur d'affinite a effet capacitif
WO1999042827A2 (fr) * 1998-02-20 1999-08-26 Wolfbeis, Otto, Samuel Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides
WO2000062047A1 (fr) * 1999-04-14 2000-10-19 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Mosaique de capteurs electriques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VAGIN MIKHAIL YU ET AL: "Surfactant bilayers for the direct electrochemical detection of affinity interactions.", 15 May 2002, BIOELECTROCHEMISTRY (AMSTERDAM, NETHERLANDS) 15 MAY 2002, VOL. 56, NR. 1-2, PAGE(S) 91 - 93, ISSN: 1567-5394, XP002280352 *

Cited By (3)

* 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
CN103343164A (zh) * 2013-06-17 2013-10-09 武汉轻工大学 产志贺毒素大肠杆菌多重pcr检测方法、试剂盒及应用

Also Published As

Publication number Publication date
AT413214B (de) 2005-12-15
EP1590483A1 (fr) 2005-11-02
ATA952003A (de) 2005-05-15

Similar Documents

Publication Publication Date Title
Bu et al. Ultrasensitive detection of pathogenic bacteria by CRISPR/Cas12a coupling with a primer exchange reaction
Bhatnagar et al. Chitosan stabilized gold nanoparticle mediated self-assembled gliP nanobiosensor for diagnosis of Invasive Aspergillosis
Izadi et al. Fabrication of an electrochemical DNA-based biosensor for Bacillus cereus detection in milk and infant formula
Ali et al. Development of an advanced DNA biosensor for pathogenic Vibrio cholerae detection in real sample
DE3513168A1 (de) Biosensor bestehend aus einem halbleiter auf silizium oder kohlenstoffbasis (elektronischer teil) und nukleinbasen (od. anderen biol. monomeren)
Sun et al. A highly selective and sensitive electrochemical CS–MWCNTs/Au-NPs composite DNA biosensor for Staphylococcus aureus gene sequence detection
DE10154291A1 (de) Verfahren zum Nachweis von Nukleinsäuren in Form eines Trockenschnelltestes
CN108760853A (zh) 一种检测牛奶中卡那霉素残留的适配体传感器的制备方法
EP1376128A1 (fr) Procédé et appareil de détection impedimetrique d'un ou de plusieurs analytes dans un échantillon
Aoki et al. 384-Channel electrochemical sensor array chips based on hybridization-triggered switching for simultaneous oligonucleotide detection
Cao et al. An ultrasensitive biosensor for virulence ompA gene of Cronobacter sakazakii based on boron doped carbon quantum dots-AuNPs nanozyme and exonuclease III-assisted target-recycling strategy
DE68909445T2 (de) Methode zur Bestimmung genetischer Sequenzen und Testsatz hierfür.
Feng et al. A supersensitive MSPQC bacterium sensor based on 16S rRNA and “DNA-RNA switch”
EP1456417A2 (fr) Procede de determination d'analytes du type acide nucleique
DE10256898B4 (de) Elektrochemischer Nachweis von Nukleinsäuren
Plácido et al. Novel strategies for genetically modified organism detection
AT413214B (de) Messanordnung und verfahren zur detektion einer dna-sequenz
Dabhade et al. Miniature lab-made electrochemical biosensor: A promising sensing kit for rapid detection of E. coli in water, urine and milk
CN107922978A (zh) 用于性别和类别识别的电化学dna生物传感器
DE10228785A1 (de) Detektion von toxischen Algen
DE102013000682B3 (de) Verfahren zur Detektion von Molekülen
EP1055004A2 (fr) Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides
DE10227042B4 (de) Verfahren zum Nachweisen, Quantifizieren und/oder Charakterisieren eines Analyten
EP1489407A1 (fr) Méthode et dispositif pour la mesure de la conductivité électronique des substances a analyser avec des marqueurs électro-actifs
O'Riordan et al. Amplification-free, highly sensitive electrochemical DNA-based sensor for simultaneous detection of stx1 and stx2 genes of Shiga toxin-producing E. coli (STEC)

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 BW 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 JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA 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): BW 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
WWE Wipo information: entry into national phase

Ref document number: 2004704149

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 90202004

Country of ref document: AT

WWP Wipo information: published in national office

Ref document number: 2004704149

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2004704149

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载