Classifications

• • 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
• • 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
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
  • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

• the invention relates to a method for detecting macromolecular biopolymers by means of an electrode arrangement.
• Fig.la and Fig.lb show such a (bio) sensor as described in [2].
• the sensor 100 has two electrodes 101, 102 made of gold, which are embedded in an insulator layer 103 made of insulator material. Electrode connections 104, 105 are connected to the electrodes 101, 102, from which the electrical potential applied to the electrode 101, 102 can be tapped.
• the electrodes 101, 102 are arranged as planar electrodes.
• DNA probe molecules 106 are immobilized on each electrode 101, 102 (cf. Fig.la). The immobilization takes place according to the so-called gold-sulfur coupling.
• the analyte to be examined for example an electrolyte 107, is applied to the electrodes 101, 102.
• DNA strands 108 are contained in the electrolyte 107 with a sequence that is complementary to the sequence of the DNA probe molecules, these DNA strands 108 hybridize with the DNA probe molecules 106 (FIG. 1b).
• Hybridization of a DNA probe roller 106 and a DNA strand 108 only takes place if the sequences of the respective DNA probe molecule 106 and the corresponding DNA strand 108 are complementary to one another. If this is not the case, no hybridization takes place. Thus, a DNA probe molecule of a given sequence is only able to bind, ie hybridize, a specific one, namely the DNA strand with a complementary sequence.
• the hybridization changes the capacitance between the electrodes in the sensor described above. This change in capacity is used as a measurement for the detection of DNA molecules.
• a further procedure for examining the electrolyte with regard to the existence of a DNA strand with a predetermined sequence is known from [7].
• the DNA strands are labeled with the desired sequence and their existence is determined on the basis of the reflective properties of the labeled molecules.
• light in the visible wavelength range is radiated onto the electrolyte and the light reflected by the electrolyte, in particular by the DNA strand to be detected, is detected. Due to the reflection behavior, i.e. in particular on the basis of the detected, reflected light rays, it is determined whether or not the DNA strand to be detected with the correspondingly predetermined sequence is contained in the electrolyte.
• Detection means for detecting the reflected light rays required so that the reflected light rays can be detected at all.
• affinity chromatography cf. [8]
• immobilized small molecules in particular ligands of high specificity and affinity
• ligands of high specificity and affinity to generate peptides and proteins, e.g. Enzymes to bind specifically in the analyte.
• DNA serves as a template for the formation of a conductive silver wire between two
• [12] also discloses a method and a device for identifying a biopolymer sequence on solid surfaces.
• a first biopolymer applied to a solid substrate is brought into contact with a second biopolymer affine to it.
• An affinity sensor for the detection of specific molecular binding events is also known from [13], which in particular e.g. should be used in DNA microarray tests.
• the invention is based on the problem of specifying an alternative method for detecting macromolecular biopolymers which is simple in design and has a high detection sensitivity.
• an electrode arrangement which has a first and a second electrode.
• Both the first electrode and the second electrode are provided with capture molecules that can bind macromolecular biopolymers.
• capture molecules can be single type molecules or also first and second type molecules, i.e. Catcher molecules different
• a first electrical measurement is also carried out on the electrodes.
• a solution to be examined is brought into contact with the electrode arrangement, wherein the solution can contain the macromolecular biopolymers to be detected.
• macromolecular biopolymers to be detected contained in the solution to be examined are bound to the capture molecules on the first and second electrodes.
• the electrode arrangement is also brought into contact with a reagent for increasing the conductivity of macromolecular biopolymers which binds to the macromolecular biopolymers and gives them increased electrical conductivity.
• a second electrical measurement is then carried out on the electrodes and the macromolecular biopolymers become dependent on that Comparison of the results of the two electrical measurements on the electrodes recorded.
• the present method is based on the knowledge that generally non-conductive or only weakly conductive macromolecular biopolymers are made electrically conductive by the addition / binding of a reagent which increases the conductivity of the biopolymers, and the macromolecular biopolymers that are now to be detected as conductive, as a “conductivity bridge "are used between two electrodes, this conductivity bridge influencing the flow of the current flowing between the electrodes.
• the conductivity bridge which can also be understood as a" molecular short circuit "between two electrodes, is in principle formed by only a single molecule can, the present method has a higher detection sensitivity than the known methods, namely a sensitivity of a single molecule of the macromolecular biopolymers to be detected.
• the resistance or the current flow is preferably determined in the electrical measurements on the electrodes.
• a single type of molecule can be used as a capture molecule in the process described here, e.g. a double-stranded nucleic acid with a defined nucleic acid sequence.
• the capture molecules are at least first and second
• Capture molecules for example at least two oligonucleotides with a different nucleic acid sequence (which therefore have different binding specificities) or two antibodies which can bind different surface areas (epitopes) of a macromolecular biopolymer.
• detection means both the qualitative and quantitative detection of macromolecular biopolymers in an analyte to be examined. This means that the term "capture” also includes determining the absence of macromolecular biopolymers in the analyte.
• a reagent for increasing the conductivity of macromolecular biopolymers is understood here to mean a reagent which is able to bind to macromolecular
• biopolymers preferably specifically, and which has a conductivity for the electric current that is higher than that of the macromolecular biopolymers to be detected.
• Such a reagent to increase conductivity is preferably a chemically reducible reagent, i.e. a reagent that can donate electrons, thereby lowering the oxidation state of at least one of the atoms of the reagent.
• the reagent preferably contains metal ions which are not only chemically reducible and can bind to macromolecular biopolymers, but are also easily soluble in solvents suitable for macromolecular biopolymers. Examples of such metal ions are silver, gold, copper or nickel ions or mixtures thereof, which act as cations on negatively charged
• nucleic acids as the biopolymers to be detected, such cations are bound to the negatively charged phosphate backbone of the nucleic acids. If proteins are to be recorded, such cations can be bound via the side chains of acidic amino acids such as aspartate or glutanate.
• reagent for increasing the conductivity are soluble polymers or oligomers which conduct electricity and which are positively charged in the conducting state.
• suitable substituted polypyrroles for example with 2 to 10 thiophene units, for example 6 thiophene units.
• Substituents which impart the solubility of these polymers or oligomers in solvents compatible with macromolecular biopolymers, preferably in aqueous media are, for example, sulfonic acid or carboxylic acid groups which are linked to the aromatic backbone via alkylene units.
• the substitution is preferably carried out via the 3-position of the aromatic ring.
• the addition to the macromolecular biopolymers to be detected is preferably carried out via electrostatic interactions with charged groups or residues of the biopolymers.
• the reagent can also be bound to increase the conductivity by interactions with other areas of the nucleic acid, such as the small groove of the nucleic acids.
• the electrode arrangement is brought into contact with a reducing agent which reduces the reagent (bound to the macromolecular biopolymers) in order to increase the conductivity.
• a reducing agent which reduces the reagent (bound to the macromolecular biopolymers) in order to increase the conductivity.
• Known and common organic or inorganic reducing agents such as hydroquinone or hydrogen sulfite can be used for the reduction. If, on the other hand, the above-mentioned conductive polymers or oligomers are used, no chemical reduction of the reagent is necessary to increase the conductivity, since the reagent in its bindable form already conducts the electrical current.
• Electrode arrangement after immobilization of the macromolecular biopolymers to be detected with the reagent Increase the conductivity of macromolecular biopolymers. Rather, it is also possible to first bring the solution to be examined into contact with the reagent for increasing the conductivity and then to bind the biopolymers to be detected to the electrodes, ie to immobilize them.
• nucleic acids As macromolecular biopolymers, nucleic acids, oligonucleotides, proteins, peptides or complexes thereof, i.e. For example, complexes of nucleic acids and proteins are recorded.
• macromolecular biopolymers are understood here to mean nucleic acids such as DNA and RNA molecules or smaller nucleic acid molecules such as oligonucleotides with a length of, for example, approximately 10 to 40 base pairs (bp).
• the nucleic acids can be double-stranded, but can also have at least single-stranded regions or can be present as single strands overall, for example by preceding thermal denaturation or another type of strand separation for their detection.
• the sequence of the nucleic acids to be detected can be at least partially or completely predetermined, i.e. be known.
• Other macromolecular biopolymers that can be detected here are proteins or peptides. These can be made up of the 20 amino acids usually found in proteins, but can also contain naturally occurring amino acids or e.g. be modified by sugar residues (oligosaccharides) or contain post-translational modifications.
• complexes can also be used here to mean nucleic acids such as DNA and RNA molecules or smaller nucleic acid
• Nucleic acids and proteins are detected, such as those formed by a DNA (specific) binding protein such as a translation factor with a DNA molecule which has the corresponding recognition sequence. If proteins or peptides are to be detected as macromolecular biopolymers, the capture molecules (located on the electrodes) preferably provide ligands with a binding activity for the proteins to be detected or
• the proteins or peptides to be detected can bind to the electrodes on which the corresponding ligands are arranged.
• the capture molecules / ligands are in turn preferably linked to the electrodes by covalent bonds.
• Low-molecular enzyme agonists or enzyme antagonists pharmaceuticals, sugars or antibodies or any molecule which has the ability to specifically bind proteins or peptides can be considered as ligands for proteins and peptides.
• nucleic acids or oligonucleotides are detected using the method described here, they can be in both single-stranded and double-stranded form.
• DNA probe molecules are preferably used as capture molecules for nucleic acids, which is why the nucleic acids then have at least one single-stranded region accessible to hybridization.
• DNA probe molecules with a sequence which is (completely) complementary to the single-stranded region are preferably used.
• the DNA probe molecules can be oligonucleotides or also have longer nucleotide sequences as long as they do not form any of the intermolecular structures that hybridize the probe molecule with the one to be detected
• nucleic acid Prevent nucleic acid.
• DNA or RNA binding proteins or agents as capture molecules.
• a problem in the detection of macromolecular biopolymers is the fact that the biopolymers to be detected are usually not identical in any area of their secondary and / or tertiary structure.
• polypeptides and proteins basically have a different and unique spatial structure at every point / area (surface).
• Nucleic acids to be detected generally have a different base sequence at their two terms (ie the 3 X terminus and the 5 'terminus).
• the capture molecules used are at least first and second capture molecules.
• the first capture molecules are able to (specifically) bind a first region of a biopolymer to be detected, and the second catcher molecules are able to (specifically) bind a second region of a biopolymer to be detected. In this way, the formation of the conductivity bridge described here is ensured.
• area of a macromolecular biopolymer to be detected is understood in the sense of the invention to mean both an area which, as in the case of proteins, has a special three-dimensional (spatial) structure, or, as in the case of nucleic acids, in principle the same or very much can have a similar three-dimensional structure, have a nucleotide sequence which differs from the other regions, and consequently the capture molecules can be, for example, two antibodies, each of which recognizes a specific epitope of the protein to be detected, or an antibody which has an epitope on the to detecting protein and a peptide that is in the
• the at least first and second capture molecules are preferably each homogeneously distributed, i.e. in a uniform distribution, applied to the two electrodes. This ensures that the macromolecular biopolymers are bound to the electrodes by means of the capture molecules, regardless of the orientation they have in the solution to be detected.
• This uniform distribution can e.g. can be achieved by first producing a mixture of the capture molecules and then applying this mixture to the electrodes.
• the electrode arrangement can be made of a common substrate, e.g. Silicon or gallium arsenide exist, to which a gold layer and a silicon nitride layer have first been applied, and which has subsequently been structured using conventional lithography and etching techniques to produce the electrode arrangement (s).
• a common substrate e.g. Silicon or gallium arsenide exist
• gold layer and a silicon nitride layer have first been applied, and which has subsequently been structured using conventional lithography and etching techniques to produce the electrode arrangement (s).
• the distance between the two electrodes can be made variable, depending on the type of structuring technique used and the type of macromolecules to be detected. In general, the distance between the electrodes is about 5 nanometers (nm) to 100 or several 100 nanometers. Smaller distances in the range from approx. 5 nm to approx. 30 or 40 nm are smaller for the detection
• the preferred electrode spacing can be estimated from the size of the biopolymer.
• nucleic acids the 3D structure of which is generally known, can be based on the known helical pitch of ideal A, B and Z DNA (cf. [10]), which e.g. for B-DNA is 0.34 nm per helical turn and base pair, it can be approximately assumed that 10 base pairs (bp) bridge a distance of 3.4 nm and thus the distance between the electrodes is estimated.
• the catcher molecules can be lengthened or shortened if necessary become.
• Electrode distance to be determined purely empirically without knowledge of the 3-dimensional dimension of the macromolecular biopolymer to be detected.
• capture molecules which do not have sufficient conductivity per se, but which are made conductive by modifications.
• a negatively charged spacer can be used to bind the hormone to the electrodes.
• a plurality of pairs of electrodes can also be used, each pair having only one capture molecule or at least first and second Capture molecules is provided, which specifically binds one of the biopolymers to be detected.
• a conventional interdigital electrode for example, can be used as the electrode arrangement for carrying out the method described here.
• one with several interdigital electrodes i.e. an electrode array, provided biosensor.
• Another electrode arrangement that can be used is a
• Electrode arrangement in the form of a trench or a cavity. This is formed, for example, by holding areas such as e.g. there is a gold layer on which the capture molecules that can bind the macromolecular biopolymers are immobilized.
• a first electrical measurement is carried out on the electrodes in a first method step.
• the capture molecules can already be attached to the immobilization agent, but do not have to. Any technique known for this purpose can be used to apply the capture molecules. If a multiple determination is to be carried out, this can be done
• a medium for example an electrolyte, is brought into contact with the electrode arrangement. This takes place in the
• the macromolecular biopolymers can bind to the capture molecules.
• the conditions are chosen so that they can bind to their corresponding capture molecule at the same time or one after the other.
• unbound catcher molecules can be removed from the electrodes on which they are located.
• the capture molecules are nucleic acid (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 as well.
• 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.
• DNA polymerases which are due to their
• DNA degradation can be used.
• the ligands are ligands, they can, if unbound, also be removed enzymatically.
• the ligands are enzymatically cleavable
• connection covalently connected to the electrodes 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.
• ester connections between the electrode and those molecules which have entered into a binding interaction with peptides or proteins remain intact due to the reduced steric accessibility which occurs as a result of the space filling of the bound peptide or protein.
• Removal of the unbound capture molecules is optional. However, it can have the advantage that the measurement signal obtained e.g. is not influenced by capture molecules which (like oligonucleotides) are also able to bind reagents to increase the conductivity of the macromolecular biopolymers such as reducible metal cations.
• Catcher molecules are brought into contact with the electrode arrangement with a reagent for increasing the conductivity of macromolecular biopolymers which binds to the macromolecular biopolymers and gives them electrical conductivity. Doing so is also sufficient
• a second electrical measurement is then carried out on the electrodes.
• the values determined from the • first and second electrical measurements are then compared with one another. If the measured values of the measured variable used differ in such a way that the difference between the determined values is greater than a predetermined threshold value, it is assumed that macromolecular biopolymers have bound to capture molecules or generally to the electrodes, and thereby the change in the intensity of the signal received at the receiver has been caused.
• the result is output that the corresponding macromolecular biopolymers that specifically bind a capture molecule have been bound and thus that the corresponding macromolecular biopolymers are contained in the medium were.
• the method can be designed in such a way that a reference measurement and a measurement for the detection of macromolecular biopolymers is carried out. This happens, for example, in the way that a
• Reference measurement is only carried out with the medium, and at the same time a measurement with the medium which contains (or not also) the macromolecular biopolymers to be recorded, if, for example, a qualitative proof is desired.
• Figures la and lb a sketch of two planar electrodes, by means of which the existence of DNA strands to be detected in an electrolyte ( Figure la) or their non-
• Figure 2 is a sketch of an electrode arrangement, which for
• Figures 4a to 4e different process states of a method for the detection of proteins according to a further embodiment of the invention.
• FIG. 2 shows a sectional view of a trench-shaped electron arrangement 200 which can be used for the method disclosed here.
• Electrode arrangement 200 are applied to an insulating substrate 201, for example a silicon oxide substrate, a gold layer 202 and a silicon nitride 203.
• an insulating substrate 201 for example a silicon oxide substrate, a gold layer 202 and a silicon nitride 203.
• trench shape 204 is formed, the
• Electrode is formed by the opposite side walls 205 and 206.
• the first electrode 205 is provided with a first electrical connection 207 and the second electrode is provided with a second electrical connection 208.
• first electrical connection 207 and the second electrode is provided with a second electrical connection 208.
• Multiple measurement of suitable sensor can have, for example, a plurality of trenches arranged in parallel.
• FIG. 3a shows a section of an electrode arrangement 300 with an insulating substrate 301, a first layer 302, a silicon nitride layer 303, a first electrode 305 and a second electrode 306, the first electrode 305 and the second electrode 306 being made of gold.
• the electrode arrangement forms a trench 304.
• electrodes 305 and 306 can also be made of silicon oxide. These can be coated with a material that is suitable for immobilizing the capture molecules on them.
• alkoxysilane derivatives can be used, such as
• a capture 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.
• DNA probe molecules 307, 308 are applied to the immobilized areas of the electrodes 305, 306 as capture molecules. With the gold electrodes shown here, the immobilization takes place e.g. about the gold-sulfur coupling.
• First DNA probe molecules 307 are applied to the first electrode 305, the nucleotide sequence of which is complementary to a predetermined first DNA sequence of a nucleic acid to be detected.
• Second DNA probe molecules 308 are applied to the second electrode 306, the nucleotide sequence of which is complementary to a predetermined second DNA sequence of the nucleic acid to be detected.
• This embodiment thus represents an example in which first and second capture molecules with different specificity are used.
• a first electrical measurement is carried out on the electrodes either before or after the immobilization of the DNA probe molecules.
• the electrode is preferably used
• Resistance or current flow is determined.
• a reference value e.g. for the resistance, determined and stored in a memory (not shown).
• adenine (A), guanine (G), thymine (T), or cytosine (C) can be added to the sequences of Probe molecules hybridize complementary sequences of DNA strands in the usual way, ie by base pairing via hydrogen bonds between A and T or between C and G.
• 3a also shows an electrolyte 309 which is brought into contact with the electrodes 305, 306 and the DNA probe molecules 307, 308.
• the electrode arrangement 300 in the event that the electrolyte 309 contains a DNA molecule 310 which has a predetermined first sequence and a predetermined second sequence, each of which is complementary to the sequence of the first DNA probe molecule 307 or of the second DNA molecule 308.
• the DNA molecule can be single-stranded, as indicated in FIG. 3, or double-stranded.
• the DNA strand 310 to be detected hybridizes to the first DNA probe molecule 307 via the first predetermined sequence and to the second DNA probe molecule 308 via the second predetermined sequence.
• the hybridization can take place spontaneously, but also in the case of double-stranded nucleic acid molecules 310, e.g. by thermal denaturation or by induction of a fluid movement perpendicular to the electrodes, as described in [9].
• the result after hybridization has taken place is the formation of a DNA “bridge” between the electrodes.
• a biochemical process for example by adding DNA nucleases to the electrolyte 309, hydrolyzes non-hybridized single-stranded DNA probe molecules 307 or 308 (see FIG. 3b). Should capture single-stranded DNA should be dispensed with, however, if this makes it possible for the single strand 310 to be detected to also be dismantled.
• 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 degraded undesirably, which would lead to a falsification of the measurement result.
• one of the following substances can be added to remove the single-stranded DNA probe molecules 306 and 307 on the two electrodes:
• DNA polymerases that 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 for this purpose.
• the electrode arrangement 300 is brought into contact with a reagent for increasing the conductivity of macromolecular biopolymers which binds to the macromolecular biopolymers and gives them electrical conductivity.
• This reagent is, for example, silver ions 311 dissolved in an alkaline medium, as described in [9]. The resulting binding of the silver ions 311 to the DNA molecules shown in FIG. 3c takes place through the exchange of the sodium ions bound to the phosphate backbone.
• the silver ions 311 bound to the DNA molecules are reduced to form the conductivity bridge.
• small silver nuclei can first be formed on the DNA using a basic hydroquinone solution and then the DNA can be converted into a “wire” completely covered with metallic silver by adding an acidic “developer solution” of hydroquinone and silver ions become.
• Such a “wire” is shown in FIG. 3d.
• a second electrical measurement e.g. a second measurement of resistance.
• the second resistance measurement is used to determine a value for the resistance, which is compared with the reference value.
• a corresponding output signal is output by the measuring device to the user of the measuring device.
• FIG. 4 shows a further embodiment of the present method, in which a protein, more precisely a DNA-binding protein such as a transcription factor, for example, is detected as a biopolymer to be detected with the aid of the electrode arrangement 400.
• the electrode arrangement 400 has an insulating substrate 401, a first layer 402, a silicon nitride layer 403, a first electrode 405 and a second electrode 406.
• the first electrode 405 and the second electrode 406 are in turn made of gold.
• the electrode arrangement also forms a trench 304.
• double-stranded nucleic acid molecules 407 which have a recognition sequence for the DNA-binding protein (FIG. 4 a).
• the immobilization of the nucleic acid molecules 407 on the two electrodes 405 and 406 takes place via the gold-sulfur coupling.
• thiol groups are each added to the 3 'termini of nucleic acid 407, which is possible, for example, as described in [9], by enzymatically extending nucleic acid 407 with oligonucleotides that have disulfide groups at the 3' end (cf. Fig. 3).
• nucleic acid molecule 407 serving as the capture molecule itself via a first and a second oligonucleotide which is attached to the first electrode 405 and to the second electrode 406, i.e. via two further capture molecules to bind to the two electrodes 405 and 406.
• a first electrical measurement is carried out on the electrodes either before or after immobilization of the DNA probe molecules 407, two of which are not shown in FIG.
• Electrode connections on the first and second electrodes 405 and 406 and a connected measuring device preferably the resistance or the current flow is determined, and then a reference value, for example for the resistance, is determined in the course of the first electrical measurement and stored in a memory (not shown) is saved.
• This single strand can be removed in a further biochemical process step, suitable,
• Single-strand specific nucleases such as nuclease P1, which is mentioned in the exemplary embodiment described with reference to FIG. 3, can be used.
• nuclease P1 which is mentioned in the exemplary embodiment described with reference to FIG. 3
• capture molecules 407 to which the protein to be detected 409 is bound remain after this treatment, or, e.g. no such protein was present in the electrolyte 409, no capture molecules 407 (FIG. 4e).
• Electrode assembly 400 contacted with a reagent to increase the conductivity of macromolecular biopolymers such as silver ions dissolved in an alkaline medium. After binding to the complex of capture molecule 407 and protein 409 to be detected, these are reduced to form a conductivity bridge, as also described above (cf. FIGS. 3d, 3e).
• a second electrical measurement is carried out, the presence or absence of the protein to be detected being concluded by comparing the measurement value obtained in the process.
• the method according to this second exemplary embodiment does not only bind proteins such as a DNA Protein can be detected, but also complexes of macromolecular biopolymers such as nucleic acid / protein complexes.

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