EP1003905A1 - Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire - Google Patents

Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire

Info

Publication number
EP1003905A1
EP1003905A1 EP98940857A EP98940857A EP1003905A1 EP 1003905 A1 EP1003905 A1 EP 1003905A1 EP 98940857 A EP98940857 A EP 98940857A EP 98940857 A EP98940857 A EP 98940857A EP 1003905 A1 EP1003905 A1 EP 1003905A1
Authority
EP
European Patent Office
Prior art keywords
electrochemical
enzyme
substrate
nucleic acid
reporter system
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Withdrawn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
EP98940857A
Other languages
German (de)
English (en)
Inventor
Robert D. Univ. of Southern California MACPHEE
Clive R. Univ. of Southern California TAYLOR
Rainer Fraunhofer Institut HINTSCHE
Rene Fraunhofer Institut SEITZ
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.)
Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung eV
University of Southern California
Original Assignee
Fraunhofer Institute for Silicon Technology
University of Southern California
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
Priority claimed from US09/105,538 external-priority patent/US6682648B1/en
Application filed by Fraunhofer Institute for Silicon Technology, University of Southern California filed Critical Fraunhofer Institute for Silicon Technology
Publication of EP1003905A1 publication Critical patent/EP1003905A1/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Withdrawn legal-status Critical Current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
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • 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
    • G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • 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

  • Fig. 1 is a schematic presentation describing a pair of ELISA methods which exemplify the use of optical and electrochemical reporter systems in the immunochemical art.
  • Enzyme/substrates which may be used with the present invention include, but are not limited to, ⁇ -galactosidase/p- aminophenyl- ⁇ -D-galactopyranoside, ⁇ -galactosidase/p- aminophenyl- ⁇ -D-galactopyranoside, ⁇ -glucosidase/p- aminophenyl- ⁇ -D-glucopyranoside , ⁇ -glucosidase/p-aminophenyl- ⁇ -D-glucopyranoside, -mannosidase/p-aminophenyl- ⁇ -D- mannopyranoside, ⁇ -mannosidase/p-aminophenyl- ⁇ -D- mannopyranoside, acid phosphatase/p-aminophenylphosphate, alkaline phosphatase/p-aminophenylphosphate, and phosphodiesterase Il/p-aminophenyl
  • antigens refer to substances which can be recognized by in vivo or in vitro immune elements, and are capable of eliciting a cellular or humoral immunologic response.
  • electrochemically active reporter utilized in the embodiment is specified as para-aminophenol (generated by the action of a beta-galactosidase conjugate in conjunction with a specific substrate) , it should be noted that the invention is generally applicable to molecules capable of redox recycling, and enzyme systems capable of generating such reporters.
  • FIG. 1 a schematic presentation describing a pair of enzyme-linked immunoassays which exemplify and compare the use of optical and electrochemical reporter systems is shown.
  • each member of the paired immunoassays is first coated with HIV p24 as an antigenic substrate .
  • Each of the immunoassay pairs is then incubated with serum from HIV positive volunteers which contain antibodies to p24, thereby affording complexation of anti p24 antibodies with a target antigen substrate.
  • Each member of the paired immunoassays is then incubated with goat anti-human immunoglobulin conjugated to biotin.
  • one of the paired immunoassays is treated with 1) avidin/horseradish peroxidase conjugate and the other paired immunoassays is treated with beta galactosidase/avidin conjugate producing matched immunoassays with optical and electrochemical reporters respectively.
  • the quantity of p-aminophenol produced is indicative of the concentration of antibody in the specimen and can be measured using an array of interdigitated micro electrodes when the p-aminophenol reacts at the interdigitated anodes and cathodes thereof in a redox process repeatedly alternating between the p-aminophenol and quinoneimine .
  • an enzyme substrate for example, p-aminophenol- ⁇ -galactopyranoside is allowed to react with the labeling enzymes to produce a large quantity of p-aminophenol which when subjected to redox recycling on the interdigitated array of anodes and cathodes of a microelectronic sensor will recycle between p-aminophenol and quinoneimine, thus delivering a strong electrical signal indicative of the concentration of antibody present in the specimen.
  • an enzyme substrate for example, p-aminophenol- ⁇ -galactopyranoside is allowed to react with the labeling enzymes to produce a large quantity of p-aminophenol which when subjected to redox recycling on the interdigitated array of anodes and cathodes of a microelectronic sensor will recycle between p-aminophenol and quinoneimine, thus delivering a strong electrical signal indicative of the concentration of antibody present in the specimen.
  • Washing buffer solutions were applied consisting of Dulbecco's phosphate buffered saline solution of pH 7.4 (DPBS free of calcium and magnesium) obtained from BioWhittaker and containing 137 mM of sodium chloride, 3 mM of potassium chloride, a mM of sodium hydrogen phosphate and 1.5 mM of potassium dihydrogen phosphate.
  • Antibody was diluted in a buffer consisting of DPBS with 1% (weight/volume) of bovine serum albumin, heat shock quality (Jackson) and 0.01% (w/v) Tween 20 (10% w/v) (Boehringer) .
  • Blocking buffer and solution was prepared from DPBS with 5% (w/v) of bovine serum albumin as above and 0.01% (w/v of Tween 20 (10% w/v) (Boehringer) .
  • an enzyme mediated electrochemical reporter system utilizing the IDA previously described, was applied to an analytical method for the detection and quantitation of nucleic acid fragments.
  • the example used to illustrate the system's general applicability in this field compared the use of the novel electrochemical reporter system to a commercially available and FDA approved polymerase chain reaction (PCR) methodology for the quantitation of HIV RNA in human plasma.
  • PCR polymerase chain reaction
  • a pair of HIV Monitor' kits produced by Roche Molecular Systems (Branchberg, NJ) were each applied per instructions contained in the package insert, to a series of five plasmas from HIV seronegative volunteers and nine plasmas obtained from HIV patients. For one kit, all preparation, amplification and detection systems were processed entirely per the instructions contained in the package insert.
  • the present invention which utilizes thin-film microelectrode arrays and redox recyclable reporter molecules is also applicable to the examination of blood, body fluids, or tissues for the purpose of detecting and measuring the following: 1) free or complex immunoglobulins associated with health or disease (infection or autoimmunity) ; 2) the presence of antigens related to normal and abnormal developmental processes, (infectious or autoimmunity); 3) presence or absence of specific nucleotide sequences associated with normal or abnormal genetic development, malignancy, or infectious disease.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Electrochemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un système reporteur agissant au point final d'une réaction en biologie moléculaire et en chimie immunologique, couplé à des molécules électrochimiquement actives susceptibles d'un recyclage redox, ou couplé à des enzymes capables d'engendrer une production proportionnelle de ces molécules détectées et/ou quantifiés par amperiométrie, en association avec une micropuce en silicium possédant des électrodes en métal noble disposées selon un alignement matriciel avec un espacement étroit entre elles.
EP98940857A 1997-08-12 1998-08-12 Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire Withdrawn EP1003905A1 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US5546697P 1997-08-12 1997-08-12
US55466P 1997-08-12
US5575997P 1997-08-14 1997-08-14
US55759P 1997-08-14
US10553998A 1998-06-26 1998-06-26
US105539 1998-06-26
US105538 1998-06-26
US09/105,538 US6682648B1 (en) 1997-08-12 1998-06-26 Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures
PCT/US1998/016714 WO1999007879A1 (fr) 1997-08-12 1998-08-12 Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire

Publications (1)

Publication Number Publication Date
EP1003905A1 true EP1003905A1 (fr) 2000-05-31

Family

ID=27489724

Family Applications (1)

Application Number Title Priority Date Filing Date
EP98940857A Withdrawn EP1003905A1 (fr) 1997-08-12 1998-08-12 Systeme reporteur electrochimique permettant de detecter les dosages immunologiques analytiques et procedures de biologie moleculaire

Country Status (5)

Country Link
EP (1) EP1003905A1 (fr)
JP (1) JP2001512691A (fr)
AU (1) AU8903998A (fr)
CA (1) CA2300268A1 (fr)
WO (1) WO1999007879A1 (fr)

Families Citing this family (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6358752B1 (en) * 1996-09-27 2002-03-19 Cornell Research Foundation, Inc. Liposome-enhanced test device and method
US6391558B1 (en) 1997-03-18 2002-05-21 Andcare, Inc. Electrochemical detection of nucleic acid sequences
US6682648B1 (en) 1997-08-12 2004-01-27 University Of Southern California Electrochemical reporter system for detecting analytical immunoassay and molecular biology procedures
DE19916921A1 (de) * 1999-04-14 2000-10-19 Fraunhofer Ges Forschung Elektrisches Sensorarray
AU2907201A (en) * 1999-12-09 2001-06-18 Motorola, Inc. Methods and compositions relating to electrical detection of nucleic acid reactions
US6518024B2 (en) 1999-12-13 2003-02-11 Motorola, Inc. Electrochemical detection of single base extension
US6824669B1 (en) 2000-02-17 2004-11-30 Motorola, Inc. Protein and peptide sensors using electrical detection methods
FR2805545B1 (fr) * 2000-02-24 2002-05-17 Argene Sa Procede electrochimique de detection d'acides nucleiques
US7223330B2 (en) 2000-03-30 2007-05-29 Siemens Aktiengesellschaft Biosensor, biosensor array and method for detecting macromolecular biopolymers with a biosensor
US7455975B2 (en) 2000-04-14 2008-11-25 Esa Biosciences, Inc. Electrochemical detection of nucleic acid sequences
US6562209B1 (en) 2001-04-19 2003-05-13 Northrop Grumman Corporation Automated computer controlled reporter device for conducting imunnoassay and molecular biology procedures
US6803202B2 (en) * 2001-10-23 2004-10-12 Northrop Grumman Corporation Combinational strategy for identification of biological agents
EP1376111A1 (fr) * 2002-06-24 2004-01-02 Universite Catholique De Louvain Procédé et dispositif de détection à haute sensibilité d'ADN et autres sondes
DE102008025680A1 (de) 2008-05-29 2009-12-03 Siemens Healthcare Diagnostics Gmbh Analyseeinrichtung und Verfahren zum Redoxcycling ohne Potentiostat
DE102008025992B4 (de) 2008-05-30 2011-01-27 Siemens Healthcare Diagnostics Gmbh Titerplatte und Verfahren zur Detektion eines Analyten
DE102008027038A1 (de) * 2008-06-06 2009-12-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren zum Detektieren von chemischen oder biologischen Species sowie Elektrodenanordnung hierfür
CN109705177A (zh) * 2018-12-24 2019-05-03 郑州安图生物工程股份有限公司 一种底物及其制备方法和应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4840893A (en) * 1983-12-16 1989-06-20 Medisense, Inc. Electrochemical assay for nucleic acids and nucleic acid probes
US4963245A (en) * 1986-05-02 1990-10-16 Ciba Corning Diagnostics Corp. Unitary multiple electrode sensor
US5391272A (en) * 1992-03-06 1995-02-21 Andcare, Inc. Electrochemical immunoassay methods
DE4318519C2 (de) * 1993-06-03 1996-11-28 Fraunhofer Ges Forschung Elektrochemischer Sensor
DE19541033C1 (de) * 1995-11-03 1997-06-26 Inst Chemo Biosensorik Elektrochemisches Verfahren zur quantitativen Bestimmung von Bindungsproteinen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9907879A1 *

Also Published As

Publication number Publication date
WO1999007879A1 (fr) 1999-02-18
CA2300268A1 (fr) 1999-02-18
AU8903998A (en) 1999-03-01
JP2001512691A (ja) 2001-08-28

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