WO2003083134A1 - Detecteur permettant une determination qualitative et quantitative d'oligomeres et polymeres (bio)organiques, procede d'analyse associe et procede de production du detecteur - Google Patents

Detecteur permettant une determination qualitative et quantitative d'oligomeres et polymeres (bio)organiques, procede d'analyse associe et procede de production du detecteur Download PDF

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Publication number
WO2003083134A1
WO2003083134A1 PCT/DE2003/001090 DE0301090W WO03083134A1 WO 2003083134 A1 WO2003083134 A1 WO 2003083134A1 DE 0301090 W DE0301090 W DE 0301090W WO 03083134 A1 WO03083134 A1 WO 03083134A1
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Prior art keywords
electrodes
electrode
sensor
attraction
detection
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PCT/DE2003/001090
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German (de)
English (en)
Inventor
Alexander Frey
Christian Paulus
Roland Thewes
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Infineon Technologies Ag
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Publication of WO2003083134A1 publication Critical patent/WO2003083134A1/fr

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    • 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
    • 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
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors

Definitions

  • the invention relates to a sensor and a method for the qualitative and quantitative determination of (bio) organic oligomers and polymers in solutions, and a method for producing the sensor.
  • the invention relates in particular to such a sensor and such a method which are suitable for the qualitative and quantitative determination of biopolymers in an electronic manner.
  • Another method for analyzing an electrolyte for existing DNA strands with a predetermined sequence is known from [2].
  • the DNA strands are marked with the desired sequence and based on the
  • the existence of fluorescent properties of the labeled molecules is determined.
  • light in the visible wavelength range is radiated onto the electrolyte and that light detected by the electrolyte, in particular by the DNA strand to be detected.
  • On the basis of this fluorescence behavior it is determined whether or not the DNA strand to be detected is contained in the electrolyte with the corresponding predetermined sequence.
  • This procedure is very complex, especially since a very precise knowledge of the fluorescence behavior of the corresponding labeled DNA strand and a very precise adjustment of the detection means for detecting the emitted light beams are required. This procedure is therefore expensive, complicated and very sensitive to interference, which means that the measurement result can easily be falsified.
  • Analytes can be measured directly via an electrical signal.
  • FIG. 1 a A method for the quantitative and qualitative determination of biopolymers by means of an electrode arrangement is known from [1].
  • Figures la and lb show a sensor described in [1].
  • the sensor 200 has two electrodes 201, 202 made of gold, which are embedded in an insulator layer 203 made of insulator material. Electrode connections 204, 205 are connected to the planar electrodes 201, 202, to which the electrical potential applied to the electrode 201, 202 can be supplied. DNA probes 206 immobilized according to the so-called gold-sulfur coupling are located on each electrode 201, 202 (cf. FIG. 1 a).
  • the analyte 207 to be examined is applied to the electrodes 201, 202 and consists for example of an electrolytic solution of the biopolymers to be detected.
  • the analyte 207 contains DNA strands 208 with a sequence that is complementary to the sequence of the DNA probe molecules, these DNA strands 208 hybridize with the DNA probe molecules 206 (cf. FIG. 1b).
  • sequences of DNA strands complementary to the sequences of the probe molecules can be added in the usual way, i.e. Base pairing over
  • Hybridization of a DNA probe molecule 206 and a DNA strand 208 only takes place if the sequences of the respective DNA probe molecule 206 and the corresponding DNA strand 208 are complementary to one another.
  • a DNA probe molecule of a given sequence is only able to bind a specific one, namely the DNA strand with a complementary sequence, i.e. to hybridize.
  • the substance to be detected thus hybridizes with immobilized capture molecules and thus changes the electrical environment in the vicinity of the sensor surface. This change can be evaluated by impedance measurements and represents a measure of the concentration of the substance in the analyte.
  • the electrical parameter which is evaluated according to the method known from [1], is the capacitance between the electrodes or the impedance of the two electrodes.
  • the change in the impedance or the capacitance is recorded here if only DNA probe molecules are present compared to the case where DNA probe molecules are hybridized with the DNA strands to be recorded. If a hybridization takes place, the value of the impedance between the electrodes 201 and 202 changes. This changed impedance is applied by applying an AC voltage with an amplitude of approximately 50 mV to the electrode connections 204, 205 and the resulting current by means of a connected one Measuring device (not shown) determined.
  • the capacitive component of the impedance between the electrodes 201, 202 decreases. This is due to the fact that both the DNA probe molecules 206 and the DNA strands 208, which possibly hybridize with the DNA probe molecules 206, do not are conductive and thus shield the respective electrodes 201, 202 to a certain extent electrically.
  • Interdigital electrode results The dimension of the electrodes and the distances between the electrodes are of the order of the length of the molecules to be detected, i.e. of DNA strands 208 or below, for example in the range of 200 nm and below.
  • a known method of this type is electrophoresis.
  • the analyte is exposed to a suitable, possibly electrical field, which enables the substances to be investigated to move in the solvent in a directed manner. This can take place either due to a permanent electrical charge of the substances (ion migration) or due to polarization effects (migration of induced or permanent dipoles in the inhomogeneous electrical field).
  • the electrode geometry and the choice of the applied voltages or currents and their frequencies can be used to generate suitable concentration gradients within the solution, preferably in such a way that the concentration of the substance to be analyzed is close to the
  • the analyte In the case of di- / electrophoresis, the analyte is usually present in an aqueous solution.
  • the electrodes for generating the electrical field must be in direct electrical contact with the analyte, because otherwise the electrical field within the aqueous solution would be very small due to the high dielectric constant of water and its high electrical conductivity.
  • WO 99/38612 uses a so-called “permeation layer” as a protective layer over the
  • This semipermeable layer is permeable to small molecules or ions such as H 2 O, NaCl, etc., so that current flow is still possible, whereas macromolecules such as DNA cannot penetrate to the electrode surface.
  • [5] discloses an automated molecular biological diagnostic system.
  • [6] discloses a method for carrying out reactions between at least two reactants in aqueous reaction mixtures.
  • the object of the invention was therefore to provide a sensor for the qualitative and quantitative determination of
  • the object of the invention was also to provide a method for the quantitative and qualitative determination of (bio) organic oligomers and polymers using this sensor.
  • the invention therefore relates to a sensor for the qualitative and quantitative determination of (bio) organic oligomers and polymers in solution, comprising an electrode arrangement
  • the invention also relates to a method for the qualitative and quantitative determination of
  • this sensor which comprises an electrode arrangement, at the attraction electrodes (B) and detection electrodes
  • a time-changing electrical voltage is applied to at least some of the attraction electrodes (B) in such a way that at least two groups of
  • Attraction electrodes (B) at least at times have a different voltage, which causes the substance to be analyzed to migrate at least once over the detection electrode (s) (A), so that at least some of them hybridize with the capture molecules on the detection electrode (s) , and c. the (bio) organic oligomers or polymers to be determined are detected optically or electronically.
  • Radius of curvature can be used. As a result of the peak effect, when a voltage is applied, very large field gradients occur within the solution, so that a concentration gradient can be built up within the solution even with uncharged particles.
  • the frictional force of the moving molecule acts on this electrical force
  • ⁇ is the viscosity of the surrounding medium and v is the velocity of the particle (molecule) in the field.
  • a concentration gradient leads to a so-called diffusion pressure, which drives the particles into areas of lower concentration.
  • the electrical force counteracts this diffusion force. The following then applies to the flow of particles through any surface in the balance of electrical force and diffusion pressure:
  • c is the concentration of the substance to be analyzed in the solvent and D is its diffusion constant.
  • the diffusion constant is related to the viscosity ⁇ of the solution in the following way:
  • the concentration profile is calculated depending on the properties of the dissolved molecules and the geometry of the electric field.
  • electrically charged particles is expediently discussed, since the effect of an inhomogeneous electric field on permanent or induced dipoles is several orders of magnitude smaller than its effect on charged particles.
  • DNA is present in aqueous solution as a polyanion, the charge number q of the effective charge increasing with the number of nucleotides contained in it.
  • a homogeneous field only has an effect on charged particles, e.g. DNA as a polyanion. If the particles only have a dipole moment, they are aligned in the homogeneous field, but do not experience any translational force.
  • the electrical field has the following dependence on the distance r from the center electrode:
  • the concentration is at a distance
  • r 0 is the radius of curvature of the spherical surface and U is the applied voltage. No general solution can be found here either.
  • the substance to be analyzed can be concentrated in the vicinity of the electrode. Since the detection electrodes are usually adjacent to the attraction electrodes and are therefore spatially separated, the concentration can also mean that, for example, the DNA does not come close to the capture molecules in order to hybridize with them. On the other hand, however, a strong electric field promotes rapid concentration of the analyte on the
  • FIG. 2 shows the typical course of the local concentration of the analyte as a function of the distance to the electrode surface during electrophoresis.
  • the possible electrode geometries described above were investigated for a typical highly integrated system
  • Electrodes depending on the electrode symmetry are Electrodes depending on the electrode symmetry:
  • the senor is monolithically integrated in a chip.
  • one or more electronic circuits are / are integrated in the chip, which circuits provide electronic functions for the operation of the attraction electrodes in the chip.
  • Figure 1 shows a sensor described in [1], with a
  • FIG. 2 shows concentration profiles calculated for electrodes with different geometry for molecules to be determined.
  • Figure 3 shows a sensor according to the invention with a
  • Electrode arrangement in which electrodes (A) alternate in space (also in the following)
  • Detection electrodes and electrodes (B) (hereinafter also called attraction electrodes) are arranged adjacent to the sensor surface.
  • the smaller attraction electrodes 1, 2 differ from one another in the electrical field applied to them.
  • Catcher molecules are located on the larger detection electrodes.
  • FIG. 4 shows the sensor according to the invention from FIG. 3, the molecule to be detected (e.g. DNA) being enriched in the area of the attraction electrodes 2.
  • the molecule to be detected e.g. DNA
  • FIG. 5 shows the sensor of Figure 4 after switching off the
  • Attraction electrodes (2) The particle clouds move to the attraction electrodes (1) and pass through the larger detection electrodes, on which capture molecules are immobilized.
  • Figure 6 shows the sensor of Figure 5 after switching off the attraction electrodes (2) and long operation of the attraction electrodes (1). It is not shown here that when the particle cloud passed over the detection electrodes, some (DNA) molecules were hybridized and the number of particles for a renewed migration to the attraction electrode (1) thus decreased.
  • FIG. 7 shows a plan view of an electrode arrangement of a sensor according to the invention with point electrodes 1 and 2.
  • FIG. 8 shows a plan view of an electrode arrangement with strip-shaped electrodes 1 and 2.
  • the electrodes (A) are thus coated according to the invention with so-called capture molecules for the (bio) organic oligomers and polymers to be determined.
  • the nature of the oligomer or polymer to be determined determines the nature of the capture molecules and via that
  • the (bio) organic oligomers and polymers are biopolymers.
  • Biopolymers are to be understood in particular as proteins, peptides and DNA strands of a given sequence.
  • the (bio) organic oligomers and polymers are in solution for determination and not in a gel like one
  • Polyacrylamide gel An aqueous solution is preferred.
  • At least two detection electrodes (A) are coated with capture molecules which differ in terms of their capture characteristics for different molecules to be determined.
  • Differentiate catcher molecules in their binding or capture characteristics ligands used, for example active substances with a possible binding activity, which bind the proteins or peptides to be detected to the respective electrode on which the corresponding ligands are arranged.
  • enzyme agonists or enzyme antagonists pharmaceuticals, sugars or antibodies or another molecule which has the ability to specifically bind proteins or peptides can be considered as ligands.
  • a probe or capture molecule is understood to mean both a ligand and a DNA probe molecule.
  • DNA strands of a predetermined sequence are to be used as biopolymers, which are to be detected by means of the electrode arrangement, then DNA strands of a predetermined first sequence with DNA scavengers (probes) can be used as molecules with the sequence complementary to the first sequence as the first Molecules are hybridized on the first electrode.
  • DNA (capture) probe molecules are used as second molecules which have a sequence that is complementary to the second sequence of the DNA strand.
  • the sensor according to the invention has at least two second electrodes (B) on which there are no catcher molecules (attraction electrodes).
  • the first electrode (A) is arranged between the second electrodes (B) in such a way that by changing the electric fields on the second electrodes, an analyte possibly containing the molecules to be determined (e.g. biopolymers) and applied to the sensor depending on Art and size of the electric fields is moved over the first electrode, so that (bio) organic oligomers and polymers contained in the analyte, which can be determined qualitatively and / or quantitatively, can hybridize with the capture molecules.
  • an analyte possibly containing the molecules to be determined (e.g. biopolymers) and applied to the sensor depending on Art and size of the electric fields is moved over the first electrode, so that (bio) organic oligomers and polymers contained in the analyte, which can be determined qualitatively and / or quantitatively, can hybridize with the capture molecules.
  • a first electrode (A) is generally located directly between the two second electrodes (B), preferably halfway.
  • a plurality of first and second electrodes are used.
  • the attraction and detection electrodes are generally arranged alternately on the sensor surface, the distance between the electrodes preferably being essentially the same size (cf. FIG. 3).
  • Attraction electrodes (B) and detection electrodes (A) of the electrode arrangement are thus preferably arranged at substantially the same distance from one another.
  • An electrode arrangement is used in the sensor according to the invention, which preferably has at least 1 detection electrode
  • the number of attraction electrodes (B) is from 100 to 1000 and the number of detection electrodes (A) from 100 to 1000.
  • the detection electrodes (A) preferably each have one
  • the electrodes (A) and (B) can have the same or different sized surfaces.
  • the electrodes (A) preferably have a larger surface area than the electrodes (B).
  • the minimal dimensions of the electrodes in turn result from the properties of the semiconductor process used to manufacture them. Usual minimum widths of metallic structures are currently in the range from 100 nm to 1 ⁇ m.
  • the smallest possible area of the detection electrodes is desirable, since a large number of detection units can then be integrated on a given chip area.
  • the area of the detection electrodes results on the one hand from the detection sensitivity of the measuring device, ie the more sensitive the detection can be (electrical or optical), the smaller the detection electrode can be.
  • Another constraint is the technology for applying the capture molecules. If the state-of-the-art microspotting technology (process similar to inkjet printing) is used, the minimum area of the detection electrodes results from the diameter of the spot. In this case the
  • the size of the detection electrodes is only given by the process-related minimum structure widths or the detection limits of the detection method.
  • the electrodes (A) and (B) used in the electrode arrangements can have different two- or three-dimensional structures.
  • punctiform / round electrodes are suitable as electrodes (A) and / or (B).
  • Pointed electrodes are to be understood here as those which correspond to the
  • Manufacturing process have minimal dimensions. (In a current process, these are, for example, electrodes with dimensions of l ⁇ m x l ⁇ m).
  • the electrodes (A) and / or (B) can be used as planar electrodes in the form of strips (see FIG. 8), rings or other planar surfaces, or as three-dimensional electrodes, for example in the form of cylinders, hemispheres, etc. are, which can be designed symmetrically or asymmetrically.
  • the electrode geometry is of particular importance for the qualitative and quantitative determination of uncharged Particles. Since uncharged molecules can only exert a force on the permanent or induced dipoles in an inhomogeneous electric field, electrodes with a small radius of curvature should preferably be used for their determination.
  • the electrodes (B) therefore have a smaller radius of curvature than the electrodes (A). It is preferred here if the electrodes (A) are planar and the electrodes (B) are pointed or round.
  • the electrodes (A) and (B) are planar.
  • the detection electrodes (A) are planar.
  • the detection electrodes do not necessarily have to be electrically conductive structures when optical detection methods are used. In this case, a surface made of a material is sufficient
  • electrically conductive electrodes can be used as detection electrodes, which are coated with a (non-conductive) dielectric.
  • Embodiments of the detection electrodes and measuring methods according to the invention for concentrating the analyte Substances are used on the sensor surface and thus to accelerate the measuring process.
  • the electrode arrangement can be a plate electrode arrangement or an interdigital electrode arrangement as known from [1].
  • the electrodes can be configured as cylindrical elements, which are each arranged concentrically around one another and are electrically isolated from one another, for example by means of a suitable dielectric, so that an electric field between the electrodes Forms electrodes.
  • the sensor according to the invention is generally in the form of a chip as a so-called sensor chip, which essentially consists of silicon.
  • the detection electrodes (A) and attraction electrodes (B) preferably consist of chemically inert, electrically conductive substances such as gold, platinum, palladium or alloys thereof. In principle, however, any electrically conductive substance is suitable as an electrode material.
  • the electrodes are made of gold, covalent connections between the electrodes and the probe molecules are preferably made, the sulfur being in the form of a sulfide or a thiol to form a gold-sulfur coupling.
  • the sulfur being in the form of a sulfide or a thiol to form a gold-sulfur coupling.
  • DNA probe molecules are used as probe molecules, such sulfur functionalities are part of a modified nucleotide which is incorporated at the 3 'end or at the 5' end of the DNA strand to be immobilized by means of phosphoramidite chemistry during an automated DNA synthesis process , The DNA probe molecule is thus immobilized at its 3 'end or at its 5' end.
  • the sulfur functionalities are formed by one end of an alkyl linker or an alkylene linker, the other end of which has a chemical functionality suitable for the covalent connection of the ligand, for example a hydroxyl radical, an acetoxy radical or a succinimidyl ester radical.
  • these electrodes can also be made from silicon oxide. These can be coated with a material that is suitable for immobilizing the probe molecules on them.
  • alkoxysilane derivatives can be used, such as 3-glycidoxypropylmethyloxysilane,
  • a probe molecule to be immobilized reacts with such an activated group, it is immobilized on the surface of the coating on the electrode via the selected material as a kind of covalent linker.
  • the electrodes (A) and / or (B) are generally arranged on a silicon chip.
  • the electrode arrangement or these supplementary electrical circuits can be produced, for example, by photolithography and various deposition techniques (e.g. “vapor deposition”).
  • an insulator layer which also serves as a passivation layer, has a sufficient thickness, for example in one Thickness of 500 nm can be applied by means of a CVD process.
  • the insulator layer can be made of silicon oxide SiO 2 or silicon nitride Si3N4.
  • tungsten vias contact holes filled with tungsten are formed for the electrical contacting of electrical components through the insulator layer.
  • the electrode arrangement of the biosensor is defined on the insulator layer by means of photolithography. Then, using a dry etching process, for example reactive ion etching (RIE), steps are produced in the insulator layer, ie etched, for example at a minimum height of approximately 100 nm. The height of the steps must be sufficiently large for a subsequent self-adjusting process for forming the metal electrode ,
  • RIE reactive ion etching
  • a vapor deposition process or a sputtering process can also be used to apply the insulator layer.
  • auxiliary layer e.g. a thickness of 10 nm, made of titanium on the step-shaped insulator layer.
  • the auxiliary layer can have tungsten and / or nickel chromium and / or molybdenum.
  • a gold layer with a thickness of approximately 500 to approximately 2000 nm is applied.
  • the thickness of the gold layer should be sufficient so that the gold layer grows porous and columnar.
  • openings are etched into the gold layer, so that gaps form.
  • TM openings can be an etching solution of 7.5 g Super Strip 100 (brand name of Lea Ronal GmbH, Germany) and 20 g
  • KCN can be used in 1000 ml of water H2O.
  • the gaps become dependent on the Duration of the etching process formed. This means that the duration of the etching process determines the base width, ie the distance between the gold electrodes that form.
  • the wet etching is ended.
  • the etching takes place much faster in the direction parallel to the surface of the insulator layer than in the direction perpendicular to the surface of the insulator layer.
  • noble metals such as platinum, titanium or silver
  • these materials can also be used, since these materials likewise have holding areas or can be coated with a suitable material for holding immobilized DNA probe molecules or generally for holding probe molecules, and a columnar one Show growth on evaporation.
  • the structure according to this exemplary embodiment has the particular advantage that the self-adjusting opening of the gold layer over the edges means that the distance between the
  • Electrodes is not tied to a minimal resolution of the manufacturing process, ie the distance between the electrodes can be kept very narrow.
  • a substrate is assumed, for example a silicon substrate wafer, on which a metallization is already provided as an electrical connection, an etching stop layer made of silicon nitride Si 3 N 4 having already been applied to the substrate.
  • a metal layer for example a gold layer, is applied to the substrate by means of a vapor deposition process.
  • a sputtering process or a CVD process can be used to apply the gold layer to the etching stop layer.
  • the metal layer has the metal from which the electrode to be formed is to be formed.
  • An electrically insulating auxiliary layer made of silicon oxide Si0 2 is applied to the gold layer by means of a CVD method (alternatively by means of a vapor deposition method or a sputtering method).
  • a lacquer structure is formed from a lacquer layer, for example a cuboid structure, which lacquer structure corresponds to the shape of the electrode to be formed.
  • a lacquer structure is produced by means of photolithography, the structure of which corresponds to the electrodes to be formed, which form the sensor.
  • the lateral dimensions of the lacquer structure formed thus correspond to the dimensions of the sensor electrode to be produced.
  • the thickness of the lacquer structure corresponds essentially to the height of the electrodes to be produced.
  • the lacquer layer is removed in the areas which are not "developed", that is to say unexposed, for example by ashing or by wet chemical means.
  • the sensor is contacted with a solution containing the substance to be analyzed, the sensor comprising an electrode arrangement in which attraction electrodes (B) and Detection electrodes (A) are present, a time-changing electrical voltage is applied to at least some of the attraction electrodes (B) in such a way that at least two groups of attraction electrodes (B) at least temporarily have a different voltage, which causes the substance to be analyzed migrates at least once over the detection electrode (s) (A), ie passes the area of the detection electrode so that at least a part of the substance to be analyzed is held on the detection electrodes by the capture molecules.
  • a voltage source is coupled to at least two second electrodes, with which a polarity reversible voltage can be applied to the second electrodes.
  • step b which can also be referred to as a “concentration step”
  • a suitable potential is first applied to every second attraction electrode (B), so that the substance to be examined accumulates there (see Fig. 4).
  • the remaining attraction electrodes (B) are not connected, so that their potential results from the bath voltage and no attraction is exerted on the analyte. Then the previously used ones
  • Attraction electrodes switched off and the previously unused switched on.
  • the analyte which is already in the vicinity of one electrode, is now attracted to the other electrodes and passes through the detection electrodes of the sensor (see FIGS. 5 and 6).
  • a time-changing electrical voltage is applied to at least some of the attraction electrodes (B) such that at least two groups of attraction electrodes (B) have a different voltage, at least temporarily.
  • the electrical voltage can differ in terms of the sign, the level of the voltage and the duration.
  • the use of alternating voltage between the attraction electrodes (B) is also suitable. If a suitable AC voltage is applied to the electrodes, the probability of the analyte being in the vicinity of the detection electrodes increases and the measurement duration is reduced accordingly.
  • a counter electrode to the attraction electrodes is of course necessary, which must be in contact with the analyte.
  • This counter electrode is preferably designed as a single large surface electrode, which only has to be present once per sensor array, but can also be designed in the form of several partial electrodes.
  • a further embodiment of the invention consists in that the attraction electrodes (B) are first operated in such a way that the substance to be analyzed is concentrated in their vicinity. The voltage is then switched off, as a result of which the attracted molecules can diffuse into the surrounding areas of the detection electrodes (A), which are occupied by capture molecules, and can hybridize if there is a chemical match.
  • the electrodes are switched on alternately or the polarity is reversed, regardless of the shape of the individual electrodes.
  • the method can also be carried out with the aid of a plurality of electrode groups which are appropriately switched on or to which alternating signals are applied with a suitable phase position.
  • the analyte or the species to be detected is thus moved back and forth in a targeted manner via active sensor surfaces (detection electrodes (A)). This is achieved by several neighboring attraction electrodes (B), which are suitably operated electrically. This also results in significantly shorter measuring times for such sensors, for example if DNA is to be determined.
  • the attraction electrodes can either be passive
  • Electrodes are designed, that is, they can be contacted at the edge of the substrate and operated by means of an external voltage supply, or they are designed as active electrodes.
  • This preferred active embodiment includes in particular the monolithic integration of electronic components and thus one or more electronic circuits in the chip, which circuits have electronic functions for the operation of the attraction electrodes in the
  • the functions include, in particular, the switching functions for the application of the required electrical potentials for the attraction or repulsion of the charged particles, and the time control for switching over the potentials and for switching over from the attraction mode to the measurement mode.
  • each sensor can be independent of the surrounding, i.e. neighboring, sensors are operated and so the electrical boundary conditions as the applied
  • Attraction potentials the temporal sequence of the impulses, as well as the switch between attraction and detection mode can be set individually for the respective biological species.
  • step c) the hybridized (bio) organic oligomers or polymers to be determined on the detection electrodes (A) are optically or electronically detected proven in a manner known per se. This detection is preferably carried out electronically by means of impedance measurements.
  • Enrichment step b) of the method according to the invention optionally removes unbound (bio) organic oligomers and polymers, in particular biopolymers, from the respective electrode on which they are located.
  • the probe molecules are DNA strands
  • this is done, for example, enzymatically using an enzyme that selectively degrades single-stranded DNA.
  • the selectivity of the degrading enzyme for single-stranded DNA must be taken into account. If the enzyme selected for the degradation of non-hybridized DNA single strands does not have this selectivity, the hybridized double-stranded DNA to be detected may also be undesirably degraded.
  • DNA nucleases for example a nuclease from mung beans, the nuclease P1 or the nuclease S1 can be used to remove the unbound DNA probe molecules from the respective electrode.
  • the DNA polymerases which are capable of breaking down single-stranded DNA due to their 5 '- ⁇ 3' exonuclease activity or their 3 '-> 5' exonuclease activity can also be used.
  • probe molecules are low molecular weight ligands, they can, if unbound, also be removed enzymatically.
  • the ligands are covalently connected to the electrode via an enzymatically cleavable compound, for example via an ester compound.
  • a carboxyl ester hydrolase can be used to remove unbound ligand molecules.
  • This enzyme hydrolyzes the ester compound between the electrode and the respective ligand molecule that was not bound by a peptide or protein.
  • the ester compounds remain between the electrode and those Molecules that have entered into a binding interaction with peptides or proteins are intact due to the reduced steric accessibility that results from the molecular mass of the bound peptide or protein.
  • a first measurement is first optionally carried out without immobilized capture molecules.
  • a subsequent second measurement takes place after immobilization of the capture molecules, a subsequent third measurement after hybridization has taken place and a washing process has been carried out.
  • a fourth measurement is carried out after enzymatic removal of unbound capture molecules (if applicable).
  • a final fifth measurement is made after all have been completely removed
  • the values determined from the electrical measurements are then generally compared with one another. If the difference between certain measured values (e.g. measured capacitance values) is greater than a predetermined threshold value, it is assumed that biopolymers have bound to probe molecules, which has caused the change in the electrical signal at the electrodes.
  • the result is output that the corresponding biopolymers that specifically bind the first molecules or second molecules have been bound and the corresponding biopolymers are thus in were contained in the medium.
  • the first electrical measurement and the second electrical measurement can be realized by measuring the capacitance between the electrodes. Alternatively, the electrical resistance between individual electrodes can also be determined.
  • an impedance measurement can be carried out as the first electrical measurement and as the second electrical measurement, in the course of which both the capacitance between the electrodes and the electrical resistances are measured.
  • a reference capacitance value is usually determined and stored in a memory.
  • a second capacitance measurement takes place after the single-stranded DNA probe molecules have been removed from the respective electrode.
  • a capacitance value is determined by means of the second capacitance measurement, which is compared with the reference capacitance value.
  • an impedance measurement can be carried out with the sensor instead of the capacitance measurement.
  • a reference electrode is usually provided for each detection electrode in such a way that the DNA probe molecules do not adhere to these reference electrodes. This can be ensured by having a material for the reference electrodes is chosen, which does not allow sulfur binding.
  • undesired adhesion of the DNA probe molecules to the reference electrode can be prevented by not applying the coating material suitable for immobilizing the DNA probe molecules (see above) to the reference electrode in advance. This means that there are no chemically reactive groups on the reference electrode that would otherwise form a covalent bond with the DNA probe molecules and would therefore immobilize them there.
  • each reference electrode is coupled to an electrical reference connection.
  • Impedance measurement in the unoccupied state i.e. for example in a state without probe molecules on the later detection electrodes or with non-hybridized DNA probe molecules.
  • the measuring method according to the invention is also suitable for the quantitative measurement of the concentration of certain species in the analyte. The accuracy of the measurement depends on the measurement method used.
  • the device according to the invention for concentrating the analyte in the vicinity of the detection electrodes leads in any case to an acceleration of the entire measuring process and to an increase in measuring sensitivity, since by means of the concentration of the species to be detected on the sensor surface and the electrically driven movement across the detection electrodes, potentially find all the binding partners to be detected in the analyte and thus the effect to be measured is maximal.
  • An advantage of the sensor and method according to the invention is that it enables rapid qualitative and quantitative determination of (bio) organic oligomers and polymers, in particular DNA, with increased sensitivity.
  • a further advantage of the sensor and method according to the invention is that even with strong electrical fields and thus large concentration gradients, it is ensured that the substance to be analyzed comes close to the capture molecules.

Abstract

L'invention concerne au moins une électrode de détection sur laquelle sont immobilisées des molécules de captation destinées à l'hybridation avec des oligomères et polymères (bio)organiques à déterminer. L'invention concerne également au moins deux électrodes d'attraction sur lesquelles ne se trouve aucune molécule de captation. L'électrode de détection est placée entre ces deux électrodes de telle manière que la modification les champs électriques des deux électrodes permet à un analyte de se déplacer au-dessus de la première électrode (électrode de détection). Cet analyse contient éventuellement les composés chimiques à détecter et est déposé sur le système de détection en fonction de la nature et de l'importance des champs électriques.
PCT/DE2003/001090 2002-04-03 2003-04-02 Detecteur permettant une determination qualitative et quantitative d'oligomeres et polymeres (bio)organiques, procede d'analyse associe et procede de production du detecteur WO2003083134A1 (fr)

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DE2002114719 DE10214719A1 (de) 2002-04-03 2002-04-03 Sensor zur qualitativen und quantitativen Bestimmung von (bio)organischen Oligomeren und Polymeren, Analyseverfahren hierzu sowie Verfahren zur Herstellung des Sensors
DE10214719.1 2002-04-03

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WO2006002617A1 (fr) 2004-06-30 2006-01-12 Siemens Aktiengesellschaft Ensemble detecteur plan, reseau de detecteurs et procede pour produire un ensemble detecteur plan
WO2008020364A2 (fr) * 2006-08-14 2008-02-21 Koninklijke Philips Electronics N. V. Capteur biochimique
WO2009132667A1 (fr) * 2008-04-30 2009-11-05 Micronas Gmbh Procédé permettant de déceler et/ou de déterminer la concentration d'un ligand

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GB0404135D0 (en) * 2004-02-25 2004-03-31 Univ Cranfield Impedimetric DNA hybridisation sensor
DE102004025580A1 (de) * 2004-05-25 2005-12-22 Infineon Technologies Ag Sensor-Anordnung, Sensor-Array und Verfahren zum Herstellen einer Sensor-Anordnung
US10209212B2 (en) 2016-02-15 2019-02-19 Infineon Technologies Ag Sensor arrangement for particle analysis and a method for particle analysis

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