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WO2007079887A1 - Procédé de détermination hautement sensible de la présence d'acides nucléiques dans un milieu aqueux - Google Patents

Procédé de détermination hautement sensible de la présence d'acides nucléiques dans un milieu aqueux Download PDF

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Publication number
WO2007079887A1
WO2007079887A1 PCT/EP2006/011971 EP2006011971W WO2007079887A1 WO 2007079887 A1 WO2007079887 A1 WO 2007079887A1 EP 2006011971 W EP2006011971 W EP 2006011971W WO 2007079887 A1 WO2007079887 A1 WO 2007079887A1
Authority
WO
WIPO (PCT)
Prior art keywords
aqueous medium
nucleic acids
electrodes
dna
highly sensitive
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2006/011971
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German (de)
English (en)
Inventor
Holger MÜHLBERGER
Werner Hoffmann
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Karlsruher Institut fuer Technologie KIT
Original Assignee
Forschungszentrum Karlsruhe GmbH
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 Forschungszentrum Karlsruhe GmbH filed Critical Forschungszentrum Karlsruhe GmbH
Publication of WO2007079887A1 publication Critical patent/WO2007079887A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • the invention relates to a method for the highly sensitive detection of the presence of nucleic acids in an aqueous medium.
  • nucleic acids such as DNA (deoxyribonucleic acid), RNA (ribonucleic acid) or m-RNA (messenger ribonucleic acid) which are found in an aqueous medium, e.g. are located in a capillary, predominantly optically detected.
  • LIF laser-induced fluorescence
  • US 2005/136466 A1 discloses a method for carrying out impedance measurements on DNA, wherein an ionically labeled sample adheres in an electric field to a complementary DNA segment and subsequently an enzyme dissolves the ionic label. Thus, an electrical conductivity is generated in the solution, which is detected by means of an impedance measurement between a pair of electrodes.
  • WO 97/32039 A1 discloses a method and a device for the quantitative determination of nucleic acids in solution, wherein the measurement of the electrical conductivity in direct contact with the electrode trolyte takes place.
  • a measuring cell with a volume of at least 10 ⁇ l is used.
  • this method obviously shows problems with sensitivity and reproducibility of the detection.
  • WO 02/46357 A1 discloses the detection of nucleic acids by a measuring method in which a measuring volume via electrodes, which are separated by a thin insulating layer from the measuring volume, with an electromagnetic wave having a wide frequency range extending at least in the GHz range , is charged.
  • a disadvantage is the complex coplanar waveguide arrangement for coupling in the frequency range and the requirement of a very expensive network analyzer for detecting the nucleic acids.
  • No. 6,139,394 B1 discloses a method for detecting biological materials, wherein an electric field is coupled into a measuring volume within the aqueous medium via electrodes.
  • the disadvantage of this is that the electrodes are in direct contact with the aqueous medium in which the biological material is located, as a result of which interfering interactions such as electrolysis, biofouling or corrosion of the electrodes can occur.
  • the method according to the invention for the highly sensitive detection of the presence of nucleic acids in an aqueous medium comprises the steps a) to c) explained in detail below.
  • an aqueous medium containing the nucleic acids to be detected is first introduced into a measuring volume which has a volume of from 10 ⁇ l to 1 ⁇ l, preferably from 10 ⁇ l to 1 ⁇ l.
  • the present method is preferably useful for the highly sensitive detection of the presence of deoxyribonucleic acids (DNA), ribonucleic acids (RNA) such as transfer ribonucleic acid (t-RNA), messenger ribonucleic acid (m-RNA), ribosomal ribonucleic acid (r-RNA), small nuclear Ribonucleic acid (sn-RNA), one of its natural or synthetic derivatives or one of its fragments having a molecular weight of at least 10,000 g / mol, preferably of at least 50,000 g / mol, which are present in an aqueous medium.
  • RNA deoxyribonucleic acids
  • t-RNA transfer ribonucleic acid
  • m-RNA messenger ribonucleic acid
  • r-RNA ribosomal ribonucleic acid
  • sn-RNA small nuclear Ribonucleic acid
  • the nucleic acids are preferably present in an aqueous medium in a concentration of at most 1 ⁇ g / ⁇ l, more preferably of at most 0.1 ⁇ g / ⁇ l.
  • the measurement volume in which the aqueous medium containing the nucleic acids to be detected is applied according to step b) with an electromagnetic wave of a fixed frequency, for which a value of 10 kHz to 10 MHz, preferably from 100 kHz to 10 MHz , is elected.
  • the electromagnetic wave is introduced into the measuring volume via one or more pairs of electrodes which are separated from the aqueous medium by an insulating layer whose thickness preferably has a value of from 0.1 ⁇ m to 1 mm, particularly preferably from 10 ⁇ m to 250 ⁇ m are attached.
  • a measuring voltage is preferably applied to the electrode pairs of from 0.1 V to 1000 V, particularly preferably from 10 V to 1000 V, in particular from 100 V to 250 V.
  • a field strength of the electric field in the measuring volume is thereby preferably from 1 V / mm to 200 kV / mm, more preferably produced from 100 V / mm to 10 kV / mm, in particular from 200 V / mm to 1 kV / mm.
  • step c) the impedance is recorded and evaluated at one, two, three or even more fixed values in response to the system of nucleic acids and aqueous solution when it is exposed to the electromagnetic wave.
  • the nucleic acids are in an electrolyte-containing gel matrix, which simultaneously serves for the separation of the nucleic acids.
  • the type and composition of the gel matrix are selected such that the impedance with and without analyte differ as clearly as possible.
  • nucleic acids can be detected electrically, after they have been separated by a suitable method such as electrophoresis of their size.
  • a suitable method such as electrophoresis of their size.
  • the invented The process according to the invention is therefore highly sensitive, because, for example, it is already possible to work with small amounts of nucleic acids in the measurement volume in capillary electrophoresis.
  • labein of the nucleic acids can be dispensed with.
  • Detection takes place in real time as the detector has short response times. To avoid electrochemical interactions with the analyte, the measurement takes place without contact, since the pairs of electrodes are arranged so that they can not come into contact with the aqueous medium. Since the analysis system can also be made using a non-transparent material such as e.g. the polymer PEEK can be produced, it is advantageous that the measurement is independent of the optical properties of the material.
  • the sensor is cost-effective, in particular if the device is designed such that the detection of the analyte in a plurality of measurement volumes takes place almost simultaneously by means of multiplexing.
  • FIG. 1 Schematic representation of the structure of a device for carrying out the method according to the invention
  • FIG. 2 Impedance spectrum (amount) of DNA in different dilution in water
  • FIG. 3 Impedance spectrum of DNA in different dilutions in an electrolyte (BGE)
  • FIG. 4 Impedance spectrum of a 1: 20,000 DNA diluted in water in FIG
  • Fig. 1 shows schematically the construction of an apparatus for carrying out the method for the highly sensitive detection of the presence of nucleic acids such as DNA in an aqueous medium.
  • the capillary 1 the aqueous medium in which the nucleic acids to be detected are introduced introduced.
  • the nucleic acids are subjected to high frequency, which, by suitable means 4 such.
  • a high-frequency impedance measurement radio-frequency transmitter and receiver is applied to a pair of electrodes 3, 3 'arranged outside the capillary 1.
  • the recording of the impedance is carried out by the means 4.
  • the electrode pair 3, 3 ' is separated by an insulating layer 5 of the capillary 1, in which the aqueous medium is.
  • Figure 2 shows the magnitude of the complex impedance (i.e., reciprocal of conductivity) of DNA in different dilutions with water when exposed to an electromagnetic wave of a frequency of 10 kHz to 1 MHz.
  • FIG. 3 shows the impedance spectrum of DNA, which is diluted to different degrees in an electrolyte (referred to here for short as BGE).
  • BGE electrolyte
  • the impedance spectrum in FIG. 4 shows at 1: 20,000 DNA diluted in water at frequencies below about 100 kHz, in particular below 10 kHz, large differences in the impedance to the electrolyte water, which are significantly above the measurement inaccuracy. Even with 1: 200,000 DNA fragments diluted in water (values not shown here), the impedance difference was still 8.5% at 10 kHz. Also, in the study of DNA in gel matrix systems, as shown in Fig. 6, large differences in the impedance between the gel matrix with and without analyte were observed.
  • composition of the electrolyte-containing gel matrix in such a way that a curvature is observed in the impedance spectrum with analyte, which is opposite to the curvature of the impedance spectrum without analyte.
  • the measuring cell has been optimized for conductivity measurement.
  • experiments were carried out to measure the conductivity of DNA in a gel matrix with optimized parameters (measuring cell 50 ⁇ m ⁇ 50 ⁇ m ⁇ 250 ⁇ m, insulation layer to the electrodes 40 ⁇ m, electrode spacing 250 ⁇ m, voltage of the measuring signal ⁇ 200 V, frequency 3.8 MHz) carried out.
  • the signal differences to the gel matrix without DNA are plotted in FIG. Even with a dilution of the DNA from 1 to 200,000, which corresponds to a DNA concentration of 0.005 ⁇ g / ⁇ l, there are still very large signal differences.
  • a DNA ladder by which DNA fragments of different lengths are understood, was separated electrophoretically and detected highly sensitively by means of the optimized measuring cell.
  • the total DNA concentration could be reduced to 0.0005 ⁇ g / ⁇ l, which corresponds to a dilution of 1 to 2 million.
  • Fig. 8 shows the electropherogram of the separation.
  • the signal peak at 1 minute can be assigned to the fragment with the lowest concentration of 0.0005 ⁇ g / ⁇ l; the signal peak at 2.7 minutes is assigned to the fragment with the highest concentration of 19% of the total concentration.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détermination hautement sensible de la présence d'acides nucléiques dans un milieu aqueux. Elle a pour objet de permettre la détection à la fois simple et hautement sensible de la présence d'acides nucléiques sans que les électrodes interagissent de manière gênante avec le milieu aqueux. Cet objet est réalisé par un procédé qui comprend les étapes suivantes : a) introduire le milieu aqueux contenant les acides nucléiques dans un volume de mesure (2) d'une capacité de 10 fl à 1 µl ; b) appliquer au volume de mesure (2) une onde électromagnétique de fréquence fixe, choisie dans la plage de 10 kHz à 10 MHz, par le biais d'au moins une paire d'électrodes (3, 3') séparée du milieu aqueux par une couche isolante (5) ; et c) mesurer l'impédance.
PCT/EP2006/011971 2005-12-22 2006-12-13 Procédé de détermination hautement sensible de la présence d'acides nucléiques dans un milieu aqueux Ceased WO2007079887A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102005061487.6 2005-12-22
DE200510061487 DE102005061487A1 (de) 2005-12-22 2005-12-22 Verfahren zum Nachweis eines Analyten in Form von Fragmenten einer Nukleinsäure und Vorrichtung zur Durchführung des Verfahrens

Publications (1)

Publication Number Publication Date
WO2007079887A1 true WO2007079887A1 (fr) 2007-07-19

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PCT/EP2006/011971 Ceased WO2007079887A1 (fr) 2005-12-22 2006-12-13 Procédé de détermination hautement sensible de la présence d'acides nucléiques dans un milieu aqueux

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DE (1) DE102005061487A1 (fr)
WO (1) WO2007079887A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046357A1 (fr) * 2000-10-26 2002-06-13 The Trustees Of Princeton University, Princeton University Procede et appareil de spectroscopie dielectrique de solutions biologiques
DE202005009960U1 (de) * 2005-06-24 2005-09-01 Forschungszentrum Karlsruhe Gmbh Messgerät zur Kapillarelektrophorese

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6169394B1 (en) * 1998-09-18 2001-01-02 University Of The Utah Research Foundation Electrical detector for micro-analysis systems
WO2001018246A1 (fr) * 1999-08-26 2001-03-15 The Trustees Of Princeton University Dispositifs electroniques microfluidiques et nanofluidiques permettant de detecter des modifications de la capacite de fluides et techniques d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002046357A1 (fr) * 2000-10-26 2002-06-13 The Trustees Of Princeton University, Princeton University Procede et appareil de spectroscopie dielectrique de solutions biologiques
DE202005009960U1 (de) * 2005-06-24 2005-09-01 Forschungszentrum Karlsruhe Gmbh Messgerät zur Kapillarelektrophorese

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ABAD-VILLAR EVA M ET AL: "Determination of biochemical species on electrophoresis chips with an external contactless conductivity detector.", ELECTROPHORESIS OCT 2005, vol. 26, no. 19, October 2005 (2005-10-01), pages 3609 - 3614, XP002423009, ISSN: 0173-0835 *
PARK ET AL.: "A Microfluidic Device With Integrated Impedance Detection for ?-DNA", MATERIAL RESEARCH SOCIETY SYMP. PROC., vol. 773, 2003, pages N6.5.1 - N6.5.6, XP002423164 *
PUMERA M ET AL: "New materials for electrochemical sensing VII. Microfluidic chip platforms", TRAC - TRENDS IN ANALYTICAL CHEMISTRY 2006 NETHERLANDS, vol. 25, no. 3, 10 October 2005 (2005-10-10), pages 219 - 235, XP002423010, ISSN: 0165-9936 *

Also Published As

Publication number Publication date
DE102005061487A1 (de) 2007-06-28

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