EP1055004A2 - Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides - Google Patents
Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotidesInfo
- Publication number
- EP1055004A2 EP1055004A2 EP99915484A EP99915484A EP1055004A2 EP 1055004 A2 EP1055004 A2 EP 1055004A2 EP 99915484 A EP99915484 A EP 99915484A EP 99915484 A EP99915484 A EP 99915484A EP 1055004 A2 EP1055004 A2 EP 1055004A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- electrode
- alloy
- hybridization
- dna
- metal
- 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- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- 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
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/22—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance
- G01N27/221—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating capacitance by investigating the dielectric properties
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
- G01N29/02—Analysing fluids
- G01N29/036—Analysing fluids by measuring frequency or resonance of acoustic waves
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
-
- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2291/00—Indexing codes associated with group G01N29/00
- G01N2291/02—Indexing codes associated with the analysed material
- G01N2291/025—Change of phase or condition
- G01N2291/0256—Adsorption, desorption, surface mass change, e.g. on biosensors
- G01N2291/0257—Adsorption, desorption, surface mass change, e.g. on biosensors with a layer containing at least one organic compound
Definitions
- the present invention relates to a device with which the hybridization of oligo- and / or polynucleotides can be detected.
- the invention relates to an arrangement with which various oligonucleotides and / or polynucleotides can be detected by means of electrochemical measurements.
- the analysis of biomolecular interactions is a versatile tool to determine the binding of, for example, receptor-ligand pairs without radioactive labeling.
- Hybridization is generally understood to mean the formation, under certain conditions, of base-pairing double-stranded nucleic acids from two completely separate, single-stranded nucleic acid molecules.
- DNA-DNA hybridization and DNA-RNA hybridization.
- Various hybridization techniques are known from the prior art, for example saturation hybridization, plus-minus hybridization, sandwich hybridization, fatigue hybridization, competition hybridization. These hybridization techniques are divided into liquid hybridization and filter hybridization.
- both reactants are in solution, and in filter hybridization, one of the reactants is bound to a filter, which is then incubated in a solution that contains the second reactant.
- blocking reagents are usually added to the hybridization solution.
- SPR surface plasmon resonance method
- Sensitive surfaces used in the SPR are described in EP 0 589 687.
- a disadvantage of these sensitive surfaces, however, is that they can only be used for this SPR method, since very good conductivity properties of the metal are a prerequisite for detecting the changes in the refractive index.
- Metals other than the metals gold, silver, aluminum, copper described here have a lower conductivity and are therefore too bad for the SPR process. With this method, too, it is not possible to carry out a large number of simultaneous verifications with a single device or arrangement.
- Another object is to provide an arrangement with which it is possible to detect the hybridization of different oligo- and / or polynucleotides in one and / or several method steps.
- a device which is characterized in that it has an electrode made of a metal or a metal (alloy) with a sensitive surface which comprises a monolayer or a monolayer receptor which or which is immobilized on this electrode surface.
- HS-X, S X, XSSY or XSi (R 3 )
- X and Y are arbitrary molecular fragments, in particular functional
- the detection method is characterized in that changes on or within the sensitive surface of the electrode, that is to say the hybridization of complementary or partially complementary oligo and / or polynucleotide sequences, are detected by means of electrochemical measurement methods, in particular capacitive measurement methods.
- the electrode and / or electrode arrangement according to the invention can therefore be used, depending on the use, as a biosensor, chemosensor, DNA probe or as an array with any number of identical or different electrodes.
- This use has the advantage that the device according to the invention can be used directly in a sample medium without the need for lengthy cleaning steps for the substances to be detected. It is also possible to use metal layers of any thickness or geometry.
- oligonucleotides and / or polynucleotides in particular deoxyribonucleic acids (DNA), ribonucleic acids (RNA), synthetic polyamide or peptide nucleic acids (PNA), nucleic acids that are completely or partially complementary to one another, as well as to detect the interactions between antibodies and antigens.
- DNA deoxyribonucleic acids
- RNA ribonucleic acids
- PNA synthetic polyamide or peptide nucleic acids
- nucleic acids that are completely or partially complementary to one another, as well as to detect the interactions between antibodies and antigens.
- viruses, phages, prions, microorganisms and the like can be detected.
- any solid conductive or semiconductive substance preferably a metal such as Ag, Au, Pt, Pd, an alloy, in particular Au / Pd, Ag / Pd, Au / Cr, a semiconductor, for example GaAs, Si, is suitable as the material for the electrode , Ge.
- a metal such as Ag, Au, Pt, Pd, an alloy, in particular Au / Pd, Ag / Pd, Au / Cr, a semiconductor, for example GaAs, Si
- an electrode made of a piece of silicon wafer that is first provided with a titanium-palladium or chrome layer as an adhesion promoter and then with an alloy of palladium and gold in a ratio of at least 65% Au and up to 35% Pd, is coated. The electrode coated in this way is then treated with 16-mercaptohexadecanoic acid.
- the complementary or partially complementary oligo- and / or polynucleotide sequences of the sequences to be detected are then immobilized on this alkylthiol-coated electrode surface.
- This immobilization can be done by different methods.
- the electrode then has the following structure after chemical coupling:
- the immobilization of avidin must take place on the activated terminal COOH groups of 16-mercaptohexadecanoic acid and the polynucleotide sequence is linked to the sensitive surface by the avidin-biotin binding.
- the electrode then has the following structure after chemical coupling:
- the method is then carried out in such a way that the hybridization of the sequences to be detected can be detected by means of electrochemical measurements, in particular the change in capacitance on and within the sensitive surface of the electrode.
- the hybridization can be detected using other electrochemical methods, for example impedance measurements, voltammetry, measurement of non-linear impedance properties, chronoamperometry and chronopotentiometry. All of these methods can be carried out both with and without a marker.
- the measurement of the nonlinear impedance properties is preferably understood to mean the measurement of the second and third harmonic, as well as that of the capacitive current, which are very sensitive to electrical or mechanical changes to the receptor layer react. These methods can be used both for the single electrode and for an electrode array.
- the detection of the hybridization of oligo and / or polynucleotides is also possible with the help of fluorescent markers and intercalation dyes.
- fluorescent markers and intercalation dyes In order to avoid the problem of quenching on the metal surface of the electrode, it is necessary to create a certain distance between the marker and the metal surface of the electrode.
- a receptor bound to the metal surface of the electrode for example a DNA double strand with an intercalated marker / fluorescent dye, is removed from the electrode surface by the electrically controlled desorption which releases the thiol-metal bond of the underlying dielectric base layer.
- the resulting fluorescence / marker staining can be detected with a fluorimeter or fluorescence microscope. This method is also used for oligo- or polynucleotide single strands or other receptors, the marker binding covalently or adsorbently to the molecules.
- a buffer system consisting of 10 mM Tris-HCl, 1 M NaCl, 1 mM EDTA (TE buffer) is just as suitable for the hybridization reaction as a buffer system made of 5 mM imidazole, 100 mM NaCl, pH 7.1 ( Imidazole buffer).
- buffers based on phosphate or acetate can also be used.
- FIG. 2 the capacitive detection of the hybridization of different complementary polynucleotide fragments, the complementary strand being covalently bound to an ⁇ -functionalized alkylthiol layer on a gold alloy
- FIG. 3 shows the kinetics of DNA hybridization when a certain DNA concentration is added
- FIG. 4 shows the total change in capacitance, divided into the individual steps of sensor preparation and application
- FIG. 5 shows the change in capacity during hybridization when using the different immobilization techniques described above
- Figure 7 shows the detection of bacteriophages when adding different bacteriophage concentrations.
- FIG. 1 shows the change in capacity as a function of the time during the immobilization of biotinylated polynucleotide sequences, avidin initially being immobilized on the activated terminal COOH groups of 16-mercaptohexadecanoic acid, so that the polynucleotide sequence through the avidin-biotin binding with the sensitive surface of the Link electrode.
- FIG. 2 shows the detection of DNA fragments on the sensitive surface.
- a biosensor with the structure Au (alloy) -S- (CH) ⁇ 5 -CO-NH-DNA is used and the degree of DNA hybridization is monitored at different concentrations of complementary DNA by the change in capacity. This change in capacity indicates the level of DNA hybridization.
- the complete hybridization of a 22mer on a 24mer fragment is achieved within a short time.
- the graphic representation of the change in capacity in FIG. 3 shows the course of the hybridization of polynucleotides to the activated sensitive surface of the biosensor with the structure Pd / Au (alloy) -S- (CH 2 ) ⁇ 5 -CO-NH-avidin-biotin polynucleotide , wherein the kinetics of the hybridization is represented as a change in capacity if constant amounts of complementary or partially complementary polynucleotide sequences to the biosensor with the structure Pd / Au (alloy) -S- (CH)
- FIG. 4 schematically shows the course of the changes in capacitance when the biosensor with the structure Pd / Au (alloy) -S- (CH 2 ) 15 -CO-NH-avidin-biotin polynucleotide is produced step by step.
- FIG. 7 shows the change in capacitance when detecting bacteriophages with a sensor of the structure Au-S- (CH 2 ) ⁇ 5 -CO-NH-Antibodies when various bacteriophage concentrations are added. Each symbol shown represents a series of measurements.
- thiol-modified oligonucleotides can also be adsorbed onto gold first and the gaps in the self-assembling monolayer can be filled with stable alkylthiols. Simultaneous coating with HS spacer oligonucleotide and alkylthiols in a certain ratio also produces the monolayer, which is required for the detection method by means of a change in capacity. In this case, the immobilization is simplified and the hybridization described above can be carried out immediately.
- the hybridization and immobilization can be controlled by changes in potential. It was found that the potential applied has an influence both on the immobilization of oligonucleotide strands and bacteriophages and on the hybridization of oligonucleotides. Already by Kelley, S.O. et al. It was described in Langmuir 14 (1998), 6781-6784 that double-stranded DNA helices, depending on the applied potential, orient themselves differently on the electrode surface due to their negatively charged phosphoric acid building blocks, depending on the electrode potential. The orientation of the DNA can therefore be controlled in order to achieve an optimal configuration for the hybridization of the nucleic acid molecules on the electrode surface.
- Biosensor or a DNA probe is provided with which it is possible by means of measuring the change in capacity, the sequences of DNA, RNA, PNA, gene segments, genetic defects, changes in the sequences in certain gene areas, effects of antibodies / antigens. It could also be shown that the detection of many different fragments is possible with an electrode array.
- the electrode according to the invention with the sensitive surface is suitable for use in chemical, medical or biological analysis.
- the device settings (20 Hz, +300 mV) were optimized for a two-electrode configuration for sensitive electrodes with a surface area of approx. 1 to 2 mm 2 .
- lower frequencies are required for an optimized measurement, whereas smaller electrodes behave in reverse.
- the following example describes the production of a sensor for the detection of hybridization of polynucleotide sequences.
- the sensitive electrodes are prepared as follows:
- Silicon wafer pieces with a size of 3.20 mm ⁇ 10.02 mm and a thickness of 450 ⁇ m were produced in a generally customary sputtering process or vapor deposition process with an electrode measuring 1.56 mm ⁇ 1.56 mm (reactive surface) and a feed line 10 ⁇ m wide and 6.65 mm long.
- the electrode was made up of titanium and palladium layers (bonding agent, each 50 nm thick) and a covering layer of gold alloy (200 nm).
- a silver wire was soldered onto the upper end of the leads as a contact point for the measuring system. First, the wafers were completely immersed in chloroform pa for 30 minutes.
- the wafer was immersed in a 1: 1 (v / v) mixture of chloroform and methanol and treated in an ultrasound bath for 10 minutes. Analogous to the chloroform used, ethanol (99%) and a 1: 1 (v / v) mixture of ethanol and methanol can also be used in this cleaning step.
- the gold alloy wafer was immersed in a hot 3: 1 (v / v) mixture of concentrated sulfuric acid and 30% hydrogen peroxide solution for 5 minutes.
- the electrodes were thoroughly rinsed with ultrapure water (Millipore: Mili.QPlus-185; 18.2 M ⁇ cm "1 ) and dried. All glass and Teflon devices were cleaned in an analogous manner before use. The electrodes are then coated as described below:
- a chloroform solution containing 5 mM 16-mercaptohexadecanoic acid was used for the coating.
- the surface of the electrode was coated with alkylthiol by immersing the wafer in the unstirred coating solution at room temperature for 15 hours.
- the electrode was rinsed with chloroform for 15 seconds to remove excess molecules adhering to the surface. After drying in a stream of nitrogen and washing several times with ultrapure water, the electrode was installed in the measuring cell.
- the measurement is carried out as follows:
- the wafer plate with the alkylthiol-coated electrode was attached together with an AG / AgCl reference electrode (surface approx. 1 cm) to a Teflon holder, which serves as a cover for the measuring cell (Eppendorf cup 1.5 ml) and an opening for the addition and removal of Owns liquids.
- the cell was filled with electrolyte (100 mM KC1, pH 5.1, approx. 200 to 300 ⁇ l) until the reactive surface of the gold electrode and the reference electrode (Ag / AgCl) were completely immersed.
- a magnetic stirrer was used for constant mixing.
- a lock-in amplifier with an integrated sine generator generated a constant sine signal with a frequency of 20 Hz and an amplitude of 10 mV.
- the lock-in amplifier is also used to register the capacitive current.
- a DC voltage is applied via a voltage generator.
- the adsorption of molecules on the electrode surface was measured by changing the capacitance.
- the measurement signal was recorded on an xt recorder and transferred to the computer via a 16 bit AD converter.
- the absolute capacitance of the coated electrodes was determined at an electrical potential of +300 mV. This is followed by the immobilization of the oligo- and / or polynucleotide sequences:
- the immobilization of polynucleotide sequences was carried out in two different ways.
- the terminal COOH group of mercaptohexadecanoic acid was activated first.
- the activation was achieved, for example, with N-hydroxysuccinimide.
- the coated electrodes were immersed in 5 ml of dioxane and the solution was stirred for 10 minutes. After adding 50 mg of dicyclohexylcarbodiimide and 25 mg of N-hydroxysuccinimide, the solution was stirred for a further 4 hours. After cleaning the activated electrode with dioxane and methanol, it was placed in the measuring cell.
- the change in capacity when adding an amino-functionalized DNA sequence indicates the immobilization rate.
- water-soluble carbodiimide EDC is used as a reagent, immobilization can take place directly in the measuring cell without external activation.
- biotinylated polynucleotide sequences the immobilization of avidin to the activated terminal COOH groups of mercaptohexadecanoic acid is necessary, the polynucleotide sequence being linked to the surface by rinsing and changing the buffer by the avidin-biotin binding.
- the detection of the hybridization is carried out as follows:
- the detection of DNA requires the construction of a sensitive surface as described above.
- the measurement is carried out in an Eppenorf Cup containing 350 ⁇ l buffer (pH 7.1; 100 mM NaCl; 5 mM imidazole).
- the addition of non-complementary DNA and RNA shows little or no change in capacity.
- Different amounts of a complementary 22-mer DNA fragment are added to a biosensor of the structure Au (alloy) -S- (CH) ⁇ 5 -CO-NH-DNA and the hybridization is followed by changing the capacity (FI
- nucleotide double strand is immobilizing a nucleotide double strand on the electrode first.
- One of the two strands has at least one functional group that binds to the terminal groups of the monolayer via chemical coupling. If the nucleotide strand has e.g. a thiol group, it can be applied directly to the metal surface of the electrode. If the bound double strand is denatured by increasing the temperature or other methods, only the coupled single strand remains in an oriented form on the electrode surface. After changing the electrolyte solution and removing the free nucletide segments, the associated complementary strand can now be detected.
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- Health & Medical Sciences (AREA)
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Molecular Biology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Tropical Medicine & Parasitology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
L'invention concerne un dispositif permettant de détecter l'hybridation d'oligonucléotides et/ou de polynucléotides. L'invention concerne également un dispositif permettant de détecter différents oligonucléotides et/ou polynucléotides par des mesures électrochimiques.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19807339 | 1998-02-20 | ||
DE19807338 | 1998-02-20 | ||
DE19807338A DE19807338A1 (de) | 1998-02-20 | 1998-02-20 | Kapazitive Vorrichtung für die Detektion der Polynukleotid-Hybridisierung |
DE1998107339 DE19807339A1 (de) | 1998-02-20 | 1998-02-20 | Sensitive Oberfläche für Biosensor - Systeme |
PCT/DE1999/000460 WO1999042827A2 (fr) | 1998-02-20 | 1999-02-19 | Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1055004A2 true EP1055004A2 (fr) | 2000-11-29 |
Family
ID=26044047
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP99915484A Withdrawn EP1055004A2 (fr) | 1998-02-20 | 1999-02-19 | Dispositif de detection d'hybridations d'oligonucleotides et/ou de polynucleotides |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP1055004A2 (fr) |
WO (1) | WO1999042827A2 (fr) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE60029802T2 (de) | 1999-05-14 | 2007-01-18 | Iris Biotechnologies, Inc., Santa Clara | Reversibel immobilisierung von liganden an metalloberflächen, deren herstellung und verwendung als biosensoren |
EP1234182A2 (fr) * | 1999-06-30 | 2002-08-28 | Iris Bio Technologies | Hybridation d'adn cible avec des analogues d'acide nucleique immobilises |
DE10113355A1 (de) * | 2000-04-28 | 2001-09-13 | Vladimir M Mirsky | Kalibrierung und Erhöhung der Selektivität der Chemosensoren |
DE10048997A1 (de) * | 2000-09-27 | 2002-04-11 | Santron Ag | Vorrichtung zum Nachweis geladener Makromoleküle |
AT413214B (de) * | 2003-01-24 | 2005-12-15 | Sy Lab Vgmbh | Messanordnung und verfahren zur detektion einer dna-sequenz |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5846708A (en) * | 1991-11-19 | 1998-12-08 | Massachusetts Institiute Of Technology | Optical and electrical methods and apparatus for molecule detection |
GB9206671D0 (en) * | 1992-03-27 | 1992-05-13 | Cranfield Biotech Ltd | Electrochemical detection of dna hybridisation |
SE9703314D0 (sv) * | 1997-09-15 | 1997-09-15 | Sangtec Medical Ab | Capacity affinity sensor |
DE19751658A1 (de) * | 1997-11-21 | 1999-07-29 | Wolfbeis Otto S Prof Dr | Verfahren zur Bildung lateral organisierter Strukturen auf Trägeroberflächen |
DE19753056A1 (de) * | 1997-11-29 | 1999-08-05 | Wolfbeis Otto S Prof Dr | Künstliche Oberfläche mit vorbestimmter Affinität |
DE19901761A1 (de) * | 1999-01-18 | 1999-07-01 | Gerhard Dr Hartwich | Verfahren zur elektrochemischen Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen |
-
1999
- 1999-02-19 EP EP99915484A patent/EP1055004A2/fr not_active Withdrawn
- 1999-02-19 WO PCT/DE1999/000460 patent/WO1999042827A2/fr not_active Application Discontinuation
Non-Patent Citations (1)
Title |
---|
See references of WO9942827A3 * |
Also Published As
Publication number | Publication date |
---|---|
WO1999042827A2 (fr) | 1999-08-26 |
WO1999042827A3 (fr) | 1999-10-28 |
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