EP1264172A1 - Quantifier des molecules cibles contenues dans un liquide - Google Patents

Quantifier des molecules cibles contenues dans un liquide

Info

Publication number
EP1264172A1
EP1264172A1 EP01915041A EP01915041A EP1264172A1 EP 1264172 A1 EP1264172 A1 EP 1264172A1 EP 01915041 A EP01915041 A EP 01915041A EP 01915041 A EP01915041 A EP 01915041A EP 1264172 A1 EP1264172 A1 EP 1264172A1
Authority
EP
European Patent Office
Prior art keywords
biopolymers
electrode
measurement
liquid
step lit
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.)
Withdrawn
Application number
EP01915041A
Other languages
German (de)
English (en)
Inventor
Jürgen SCHÜLEIN
Björn GRASSL
Jörg HASSMANN
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.)
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Original Assignee
November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
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 November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin filed Critical November AG Novus Medicatus Bertling Gesellschaft fuer Molekular Medizin
Publication of EP1264172A1 publication Critical patent/EP1264172A1/fr
Withdrawn legal-status Critical Current

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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/004—Enzyme electrodes mediator-assisted
    • 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
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the invention relates to a method for the detection and / or quantification of target molecules contained in a liquid.
  • a chip for the detection of polynucleotide sequences is known from WO 96/01836.
  • a large number of miniaturized reaction fields are provided on the chip produced from a silicon substrate.
  • a probe is bound to each of the reaction fields.
  • hybridization occurs with one of the specified probes.
  • Hybridization can e.g. can be detected by a fluorophore label provided on the probe.
  • DE 198 08 884.1 describes a method for the detection of chemical substances using two interacting fluorophoric groups which are bound to a molecule. When the molecule attaches specifically to the chemical substance to be detected, the interaction between the fluorophoric groups is changed.
  • WO 99/47700 relates to a method for the detection of a target molecule by means of fluorescence. It is a provided with a fluorophoric group probe bound to a solid phase ge ⁇ . If the target sequence is present in the solution, a second fluorophoric group is bound in the vicinity of the first fluorophoric groups in such a way that a radiation-free energy transfer can take place between the two fluorophoric groups.
  • US 5,591,578 describes a method for the detection of polynucleotide sequences using redox indicators.
  • a probe that is complementary to the target polynucleotide sequence is covalently bound to an electrode.
  • Covalently redox-active transition metal complexes are bound to the probe.
  • a redox signal can be measured at the electrode.
  • DE 196 28 171 discloses a process for the purification and enrichment of charge-carrying first molecules which have a specific affinity for second molecules bound to an electrode.
  • a voltage protocol is carried out in such a way that the first molecules are enriched on the electrode.
  • the object of the invention is to eliminate the disadvantages of the prior art.
  • a sensitive, simple and inexpensive electrochemical method for the detection and / or quantification of small amounts of first biopolymers in a liquid is to be specified.
  • a method for the detection and / or quantification of first biopolymers contained in a liquid is provided, with the following steps:
  • the proposed method enables sensitive detection of first biopolymers in a liquid.
  • the use of electrodes provided with a plastic surface allows the method to be carried out inexpensively.
  • the method also enables the first biopolymers contained in the liquid to be quantified.
  • the first biopolymer can in particular be a single-stranded DNA or RNA complementary to the second biopolymer.
  • the second biopolymers are preferably covalently bonded to the plastic surface.
  • osmium tetroxide and bipyridine a particularly high sensitivity is achieved.
  • the plastic is an electrically conductive composite material, e.g. a composite of carbon fibers and polycarbonate.
  • the electrode as a whole is advantageously made of the plastic. Such electrodes can be manufactured in an inexpensive pressing process.
  • step lit. e performed one of the following measurements: DC voltage measurement, cyclovoltammetric measurement, chronoamperometric measurement, chronovoltammetric measurement. Furthermore, in step lit. e a differtial pulse voltammogram or an impedance spectrum are recorded. It is also possible in step lit. e to measure an AC signal in a phase-sensitive manner. A DC signal can be impressed on the AC signal. To quantify the first biopolymers, a peak of the measurement signal can be integrated. As
  • the distance between the peak height and the background can be used as a quantification variable.
  • the electrode can be removed after step lit. e be rinsed or heated.
  • a thermal denaturation of the first biopolymers can be achieved by heating the electrode.
  • Certain first biopolymers preferably bind at a predetermined temperature.
  • the specificity of the method can be increased further by heating or adjusting the temperature.
  • the specificity or the stringency can also be increased by a suitable adjustment of the pH in the liquid.
  • the first biopolymers are lit. a subjected to a polymerase chain reaction. This enables the detection of particularly small amounts of the first biopolymers.
  • the only figure shows a differtial pulse voltammogram of an unoccupied one with single-stranded oligonucleotides occupied and a working electrode with hybridized oligonucleotides after a treatment with osmium tetroxide and bipyridine.
  • the working electrodes each consist of carbon composite material, which is preferably composed of 30% carbon fibers and 70% polycarbonate.
  • Oligonucleotides with the sequence 5 * -GCC TTC CCA ACC ATT CCC TTA-3 ' were covalently bound to the surface of the working electrodes using carbodiimide using a standard method.
  • the occupancy density was 15 mol / mm 2 '.
  • the hybridization of the oligonucleotides was carried out in a buffered solution of 0.5-fold TBE (TRIS borate EDTA), 0.5M NaCl and 100 mol / ⁇ l of complementary oligonucleotides. After the hybridization, the working electrodes were washed stringently. An untreated, one with single-stranded oligonucleotides and one with hybridized oligonucleotides working electrode was immersed for 30 seconds in a solution of 2 mM Os0 and 13 mM bipyridine. The measurement was carried out using a platinum gene electrode and an Ag / AgCl reference electrode with an Autolab
  • the hybridization of the target oligonucleotides to the working electrode can be accelerated by applying a voltage.
  • the density of oligonucleotides on the surface of the working electrode can be increased by adding salt during the coating or by basic pretreatment of the surface. For example, an occupancy density of 85fmol / mm 2 can be achieved in a 10mM MgCl 2 solution. With a three-hour pretreatment of the surface in 5M NaOH, an occupancy density of 750fmol / mm 2 can be achieved.
  • the electrode after step lit. yours opposite voltage are applied.
  • a thermal denaturation of the first biopolymers can be achieved by heating the electrode.
  • the specificity of the method can also be increased by heating or adjusting the temperature of the electrode, because predetermined first biopolymers bind at a specific temperature.

Landscapes

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

Abstract

L'invention concerne un procédé permettant de détecter et/ou de quantifier de premiers biopolymères contenus dans un liquide, qui comprend les étapes suivantes : a) préparer une électrode comportant une surface en matière plastique et recouverte de seconds biopolymères présentant une affinité spécifique avec les premiers polymères à détecter ; b) mettre en contact ladite électrode avec le liquide ; c) appliquer un protocole de tension prédéfini à l'électrode, de manière à concentrer les premiers biopolymères sur les seconds biopolymères ; d) ajouter du tétroxyde d'osmium et de la bipyridine au liquide ; e) mesurer de signal d'oxydoréduction retombant au niveau de l'électrode.
EP01915041A 2000-03-01 2001-02-28 Quantifier des molecules cibles contenues dans un liquide Withdrawn EP1264172A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10009715 2000-03-01
DE10009715 2000-03-01
PCT/DE2001/000736 WO2001065246A1 (fr) 2000-03-01 2001-02-28 Quantifier des molecules cibles contenues dans un liquide

Publications (1)

Publication Number Publication Date
EP1264172A1 true EP1264172A1 (fr) 2002-12-11

Family

ID=7632936

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01915041A Withdrawn EP1264172A1 (fr) 2000-03-01 2001-02-28 Quantifier des molecules cibles contenues dans un liquide

Country Status (6)

Country Link
US (1) US20030175737A1 (fr)
EP (1) EP1264172A1 (fr)
JP (1) JP2003525449A (fr)
AU (1) AU2001242274A1 (fr)
CA (1) CA2401830A1 (fr)
WO (1) WO2001065246A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1713926B1 (fr) 2004-02-06 2012-08-01 Bayer HealthCare, LLC Especes oxydables servant de reference interne a des biocapteurs, et procede d'utilisation
WO2005106034A1 (fr) * 2004-04-29 2005-11-10 Agency For Science, Technology And Research Procede et dispositif permettant la detection d'acides nucleiques et/ou de polypeptides
MX2008000836A (es) 2005-07-20 2008-03-26 Bayer Healthcare Llc Amperimetria regulada.
AU2006297572B2 (en) 2005-09-30 2012-11-15 Ascensia Diabetes Care Holdings Ag Gated Voltammetry
MX2009004400A (es) 2006-10-24 2009-05-11 Bayer Healthcare Llc Amperimetria de decadencia transitoria.
WO2009076302A1 (fr) 2007-12-10 2009-06-18 Bayer Healthcare Llc Marqueurs de contrôle pour la détection automatique d'une solution de contrôle et procédés d'utilisation
US10876148B2 (en) 2018-11-14 2020-12-29 Element Biosciences, Inc. De novo surface preparation and uses thereof
US10704094B1 (en) 2018-11-14 2020-07-07 Element Biosciences, Inc. Multipart reagents having increased avidity for polymerase binding
US20200149095A1 (en) * 2018-11-14 2020-05-14 Element Biosciences, Inc. Low binding supports for improved solid-phase dna hybridization and amplification

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4123933A1 (de) * 1991-07-19 1993-01-21 Krupp Corpoplast Masch Vorformling, verfahren zur herstellung des vorformlings sowie verfahren zur beheizung des vorformlings
US5591578A (en) * 1993-12-10 1997-01-07 California Institute Of Technology Nucleic acid mediated electron transfer
US5871918A (en) * 1996-06-20 1999-02-16 The University Of North Carolina At Chapel Hill Electrochemical detection of nucleic acid hybridization
AU2365895A (en) * 1994-04-26 1995-11-16 Regents Of The University Of Michigan, The Unitary sandwich enzyme immunoassay cassette, device and method of use
DK0871773T3 (da) * 1995-06-27 2002-12-09 Univ North Carolina Elektrokemisk detektion af nukleinsyrehybridisering
US6060327A (en) * 1997-05-14 2000-05-09 Keensense, Inc. Molecular wire injection sensors
DE19725190A1 (de) * 1997-06-14 1998-12-17 Innova Gmbh Vorrichtungen mit integrierten Elektroden aus elektrisch leitfähigen Kunststoffen
AU4833899A (en) * 1998-06-24 2000-01-10 Therasense, Inc. Multi-sensor array for electrochemical recognition of nucleotide sequences and methods
JP2967197B1 (ja) * 1998-08-11 1999-10-25 九州大学長 遺伝子の特殊一本鎖核酸部位の検出用プローブ、遺伝子の特殊一本鎖核酸部位の検出方法およびその装置

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0165246A1 *

Also Published As

Publication number Publication date
AU2001242274A1 (en) 2001-09-12
CA2401830A1 (fr) 2001-09-07
US20030175737A1 (en) 2003-09-18
WO2001065246A1 (fr) 2001-09-07
JP2003525449A (ja) 2003-08-26

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