WO2003083134A1

WO2003083134A1 - Sensor for the quantitative and qualitative determination of (bio)organic oligomers and polymers, corresponding analysis method, and method for the production of said sensor

Info

Publication number: WO2003083134A1
Application number: PCT/DE2003/001090
Authority: WO
Inventors: Alexander Frey; Christian Paulus; Roland Thewes
Original assignee: Infineon Technologies Ag
Priority date: 2002-04-03
Filing date: 2003-04-02
Publication date: 2003-10-09
Also published as: DE10214719A1

Abstract

The invention relates to at least one detection electrode on which scavenger molecules are immobilized in order to be hybridized with (bio)organic oligomers and polymers that are to be determined and at least two attraction electrodes on which no scavenger molecules are located. The detection electrode is arranged between the second electrodes such that an analyte which is applied to the sensor arrangement and possibly contains the chemical compounds that are to be detected is moved across the first electrode by modifying the electric fields at the second electrodes according to the type and size of the electric fields.

Description

description

Sensor for the qualitative and quantitative determination of (bio) organic oligomers and polymers, analysis methods for this as well as methods for manufacturing the sensor

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.

In medical analysis, substances must be detected and quantified that are only contained in a solution in very low concentrations. An example of this is DNA analysis. Some optical methods are known which use fluorescence labels which are bound to the DNA to be detected. After hybridization of these labeled macromolecules with matching capture or probe molecules bound to a sensor surface, the concentration of the substance in the analyte can be determined by irradiation with short-wave light and measurement of the fluorescence intensity.

Another method for analyzing an electrolyte for existing DNA strands with a predetermined sequence is known from [2]. Here, the DNA strands are marked with the desired sequence and based on the

The existence of fluorescent properties of the labeled molecules is determined. For this purpose, 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.

As an alternative to optical processes, work is carried out on electrical processes in which the concentration of the

Analytes can be measured directly via an electrical signal.

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).

To the pyrimidine bases adenine (A), guanine (G), thymine (T), or cytosine (C), sequences of DNA strands complementary to the sequences of the probe molecules can be added in the usual way, i.e. Base pairing over

Hybridize hydrogen bonds between A and T or between C and G.

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. Thus, 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.

In the case of hybridization, 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.

To improve the measurement accuracy, it is known from [4] to use a large number of electrode pairs 201, 202 and to connect them in parallel, these being arranged in a toothed manner and a so-called

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.

It is also known from affinity chromatography ([3]) to use immobilized small molecules, in particular ligands of high specificity and affinity, to specifically bind peptides and proteins, for example enzymes, in the analyte. These optical and electrical methods require the hybridization of the substances to be examined to immobilized capture molecules on a sensor surface. Since the concentration of the DNA in the analyte is very low, it may take a very long time until all the DNA molecules in the solution have hybridized to suitable capture molecules by diffusion processes alone. In order to characterize these substances using microsensors in a short time, it is therefore necessary to concentrate the substance to be analyzed in the area of the sensor surface.

A known method of this type is electrophoresis. Here, 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

Sensor surface increased.

In the case of migration of uncharged, dielectric particles in the inhomogeneous electric field by induction of dipole moments, one speaks of dielectrophoresis.

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.

Due to the direct contact of the electrodes to the electrically conductive medium, electrolysis takes place, which changes the pH value in the vicinity of the electrodes and can also lead to gas formation at high current density. This effect can be reduced by applying AC voltage. However, since in particular biopolymers (bio-macromolecules) such as DNA are sensitive to changes in the pH value in the basic, WO 99/38612 uses a so-called “permeation layer” as a protective layer over the

Electrodes suggested. 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

To provide (bio) organic oligomers and polymers, which allows these determinations with greater sensitivity and / or speed. 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.

Accordingly, the sensor, the method for its production (production method) and the method for the qualitative and quantitative determination of (bio) organic oligomers and polymers (analysis method) according to the independent claims have been provided

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

A) with at least one first electrode on which capture molecules for hybridization with (bio) organic oligomers and polymers to be determined (“chemical compounds”) are immobilized (detection electrodes),

B) with at least two second electrodes on which there are no capture molecules (attraction electrodes),

• wherein the first electrode is arranged between the second electrodes, such that by

Change of the electric fields at the second electrodes a analyte applied to the sensor possibly containing the chemical compounds to be detected, in particular biopolymers, is moved over the first electrode depending on the type and size of the electric fields, so that qualitative and / or quantitatively determining (bio) organic oligomers and polymers can hybridize with the capture molecules.

The invention also relates to a method for the qualitative and quantitative determination of

(Bio) organic oligomers and polymers using the sensor according to the invention, wherein

a. this sensor, which comprises an electrode arrangement, at the attraction electrodes (B) and detection electrodes

(A) are present, is contacted with a solution containing the substance to be analyzed, b. 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.

Since very different (bio) organic oligomers and polymers can be determined qualitatively and quantitatively with the sensor according to the invention, general instructions for the construction of the sensor according to the invention and the implementation of the invention are given below

(Analysis) procedure given. To do this, the different electrical fields and their influence on the concentration of the molecules near the electrodes is considered depending on the electrode geometry.

Electrodes with a small diameter can be used to generate an inhomogeneous electric field for dielectrophoresis

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.

Two independent, competing processes can be observed in di- / electrophoresis. On the one hand, this is the diffusion of the molecules, which leads to their uniform distribution within the solution. On the other hand, migration due to the electrical force on the charged particles and the permanent and induced dipole of the macromolecule. The influence of the gravity field with density differences, which leads to particle transport by convection, is neglected here.

The electrical force F _e χ on a particle with the charge q, a permanent dipole moment m ₀ and a polarizability α in the electrical field is neglecting the gravitational field:

Fei = (+ ( ^m o + OCE) V) E. (1)

The frictional force of the moving molecule (e.g. a biopolymer such as DNA) acts on this electrical force

Counter solvent. The inertial force and the gravitational force are neglected here. For a spherical particle with radius rp applies to the friction force Ff _r :

Ffr = 6πηrpv, (2)

η is the viscosity of the surrounding medium and v is the velocity of the particle (molecule) in the field. After switching on the field, an equilibrium between electrical force and friction force is established, so that a drift velocity v ^ results as follows:

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 η of the solution in the following way:

D = kT / (6 π η r _D ). (5) In summary, the following differential equation results for the description of the spatial concentration distribution c:

In the following, the concentration profile is calculated depending on the properties of the dissolved molecules and the geometry of the electric field. In the following, only the case of 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 electric field E

The solution to this differential equation depends on the type of electric field present. In classical electrophoresis, constant fields are used, i.e. the following applies:

U _ E = E _n ex = - ex 7)

° d

for a field between two electrodes at a distance d and the voltage U. In this case the differential equation is reduced to: c = - qU

(8) dx kTd

One solution to this differential equation is:

-qUx c (x) = c ₀ e kTd. (9)

This means that the concentration of the substance to be analyzed is at a distance

KTD

P = (10) qU

has fallen from the one electrode surface to the 1 / e-th part, that is to say there is a usable concentration in a layer of thickness p around the electrode. 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.

b) Radially symmetrical electric field E

In the case of electrodes with a cylindrical geometry, the electrical field has the following dependence on the distance r from the center electrode:

E (r) = H. (Ü) r _? r In - U is the voltage applied to the capacitor, ri and X2 the radii of the inner and outer electrodes of the cylinder. If this field is inserted into the differential equation, no analytical solution can be found. For the special case of a charged particle without dipole moment (m = 0, α = o), the following solution results:

The concentration is at a distance

dropped to the 1st part.

c) Spherical electric field E

A spherically symmetrical electric field decreases with the square of the distance to the center of the sphere:

E (r) = ^, (14)

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. For the special case of a charged particle without dipole moment (m = 0, α = o), the following solution results:

So the concentration is at a distance of

dropped to the 1st part.

Depending on the electrode geometry, the electrical properties of the solute and the solvent as well as the applied voltage, 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

Sensor surface and is therefore advantageous for accelerating the measurement. 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

(d = r ₀ = r ^ = lμm, r ₂ = 2μm). The voltage was chosen to be IV, the initial concentration to lpM (represented in FIG. 2 by: • - • - • - • - • - •), the temperature to 37 ° C. and the charge number of the particles 20. 2 is the Recognize the concentration of the analyte in the immediate vicinity of the electrode surface. The concentration of the substance typically drops to the value of the initial concentration after only 10 nm. Concentration over several orders of magnitude can be observed on the electrode surface. The concentration is more pronounced if electrodes with a small radius of curvature are used instead of parallel electrodes (homogeneous field) (peak effect). In Fig. 2 the differently marked lines mean the concentration curve before

Electrodes depending on the electrode symmetry:

: Spherical symmetry

: Cylindrical symmetry

. -. -. -. -. -. -. -. - .--. : Homogeneous field

According to one embodiment of the invention, the sensor is monolithically integrated in a chip.

In another embodiment of the invention, 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.

The features of the sensor according to the invention are described in more detail below with reference to the figures.

Figure 1 shows a sensor described in [1], with a

Electrode arrangement with which in particular biopolymers can be determined qualitatively and quantitatively. 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.

Figure 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.

In the electrode arrangement of the sensor according to the invention there is at least one first electrode (A) on which catcher molecules for hybridization are also to be determined

(Bio) organic oligomers and polymers are immobilized

(Detection electrodes).

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

Coating the detection electrodes with the immobilized capture molecules the structure of the sensors according to the invention.

In a preferred embodiment of the invention, 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.

In a preferred embodiment of the sensor according to the invention, 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. In the event that biopolymers and in particular proteins or peptides are to be determined with the sensor according to the invention, the first molecules (first capture molecules) and the second molecules (second capture molecules, the first and second molecules being different)

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.

However, 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.

In the context of this description, a probe or capture molecule is understood to mean both a ligand and a DNA probe molecule.

If 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. In order to detect a DNA strand of a predetermined second sequence by means of second molecules on the second 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. In addition to the at least one electrode (A), the sensor according to the invention has at least two second electrodes (B) on which there are no catcher molecules (attraction electrodes).

In the sensor according to the invention, 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.

For this purpose, a first electrode (A) is generally located directly between the two second electrodes (B), preferably halfway. In a preferred embodiment of the invention, a plurality of first and second electrodes are used. Here, 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

(A) and preferably has at least 2 attraction electrodes (B). In a preferred embodiment of the invention, 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

2 area from 100 to 10000 μm. The attraction electrodes (B)

2 preferably each have an area of 0.1 to 100 μm.

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). As many small-area or as narrow as possible attraction electrodes should be used in order to be able to build up large electric fields. 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 μm.

In principle, 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

Spot diameter in the order of 100 μm, which results in the minimum area of the detection electrode. As an alternative to spotting, electrochemical methods for immobilizing the capture molecules can also be used, such as

TM, for example, proposed by the Nanogen company. In this case, the size of the detection electrodes is only given by the process-related minimum structure widths or the detection limits of the detection method.

Different geometries are possible for the sensors according to the invention or for the electrode arrangements comprising them.

The electrodes (A) and (B) used in the electrode arrangements can have different two- or three-dimensional structures.

For example, punctiform / round electrodes (button electrodes) (cf. FIG. 7) 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 lμm x lμm).

Furthermore, 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.

In one embodiment of the sensor according to the invention, which is particularly suitable for the determination of uncharged or slightly charged oligomers or polymers, 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.

For the determination of charged oligomers or polymers, however, it is advantageous if the electrodes (A) and (B) are planar.

Regardless of the type of molecule, it is preferred that 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

Immobilization of capture molecules is suitable. Furthermore, electrically conductive electrodes can be used as detection electrodes, which are coated with a (non-conductive) dielectric.

The attraction electrodes are mentioned in all

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].

Furthermore, different arrangements of the parallel connection of electrodes can be provided in the electrode arrangement, for example 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.

If 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. In the event that 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.

In the event that ligands are used as probe molecules, 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.

Alternatively, 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.

For example, known alkoxysilane derivatives can be used, such as 3-glycidoxypropylmethyloxysilane,

3-acetoxypropyltrimethoxysilane,

3-aminopropyltriethoxysilane,

4- (hydroxybutyramido) propyltriethoxysilane,

• 3-N, N-bis (2-hydroxyethyl) aminopropyltriethoxysilane, or other related materials which are capable of forming a preferably covalent or van der Waals bond with the surface of the silicon oxide at one end and the other end at the other to immobilizing probe molecule to offer a chemically reactive group such as an isocyanate, alkylsilane, epoxy, acetoxy, amine or hydroxyl radical for the reaction.

If 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”).

Thus, on a silicon substrate, as is produced for known CMOS processes, in which there are already integrated circuits and / or electrical connections for the electrodes to be formed, 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.

Furthermore, 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 ,

Alternatively, a vapor deposition process or a sputtering process can also be used to apply the insulator layer.

In a further step an 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.

In a further process step, 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.

In a further step, openings are etched into the gold layer, so that gaps form. For wet etching the

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.

Due to the columnar growth of the gold, generally of the metal, during the vapor deposition onto the adhesive layer, an anisotropic etching attack is achieved, so that the surface removal of the gold occurs approximately in a ratio of 1: 3. By etching the gold layer, 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.

After the metal electrodes have a sufficient width and the distance between the gold electrodes that have formed is reached, the wet etching is ended. Usually, due to the porous vapor deposition, 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.

Instead of a gold layer, other noble metals, such as platinum, titanium or silver, 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.

In the event that the adhesive layer in the open gaps between the metal electrodes is to be removed, this is also done in a self-adjusting manner by using the gold electrodes as an etching mask.

Compared to the known interdigital electrodes, 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. In other manufacturing methods for the sensor according to the invention, 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. Alternatively, a sputtering process or a CVD process can be used to apply the gold layer to the etching stop layer. In general, 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).

Using photolithography technology, 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.

If a biosensor array described in the following is to be produced with a multiplicity of electrodes, 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, ie the lacquer layer, corresponds essentially to the height of the electrodes to be produced. After application of the lacquer layer and exposure, which specifies the corresponding lacquer structures, 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.

In the method according to the invention for the qualitative and quantitative determination of (bio) organic oligomers or polymers using the electrode arrangement of the sensor according to the invention, 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.

In a preferred embodiment of this method, a voltage source is coupled to at least two second electrodes, with which a polarity reversible voltage can be applied to the second electrodes.

In one embodiment of the method according to the invention, in 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).

In the analysis method according to the invention, 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.

To build a closed circuit, 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. Depending on the measurement method, it may also be necessary to provide a reference electrode on the sensor. This is particularly necessary for types of detection based on electrochemical methods (eg redox recycling).

Instead of switching the electrodes on and off alternately, particles with permanent charge such as DNA also the polarity of the electrodes can be switched. In this embodiment of the invention, in addition to the attractive effect of the potential of the new electrodes, there is also the repulsive effect of the old 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.

In the method according to the invention, the electrodes are switched on alternately or the polarity is reversed, regardless of the shape of the individual electrodes.

According to the invention, not only can two electrode groups with attraction electrodes (B) be operated alternately, but 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. In sub-step b) of the method according to the invention, 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

Provide chip.

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.

In addition to the simplified control of the chip, an integration of these functions is particularly advantageous because, according to this embodiment, a high integration density can be achieved. Several sensor units that have been equipped with different catcher sequences are usually integrated on one chip. The described structure consisting of attraction and detection electrodes is contained within each of these sensor units.

If the control circuits are integrated on the chip, 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.

In the method according to the invention, after a washing process which removes all unbound (bio) organic oligomers or polymers, in 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.

For more sensitive detection, after the

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 following information is given as an example for biopolymers as substances to be analyzed. In the event that 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.

In particular, 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.

In the event that the probe molecules are low molecular weight ligands, they can, if unbound, also be removed enzymatically.

For this purpose, the ligands are covalently connected to the electrode via an enzymatically cleavable compound, for example via an ester compound.

In this case, for example, a carboxyl ester hydrolase (esterase) 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. In contrast, 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.

Usually, 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

Capture molecules. It should be noted that the fifth measurement is also optional.

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.

If the difference between the values of a first electrical measurement and a second electrical measurement is greater than the predefined threshold value, 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.

In general, 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.

As part of the first capacitance measurement, 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.

If the difference value between these capacitance values is greater than a predetermined threshold value, this means that DNA strands were contained in the electrolyte which are either hybridized with the DNA probe molecules. In this case, a corresponding output signal is output by the measuring device to the user of the measuring device.

According to the invention, 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.

Alternatively, 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.

Alternatively, this can be ensured by applying a sufficiently large negative electric field, which ensures that the negatively charged DNA probe molecules do not adhere to the reference electrodes. Each reference electrode is coupled to an electrical reference connection.

In a further exemplary embodiment, a first takes place

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.

After removal of the single-stranded DNA probe molecules after any hybridization of the appropriate DNA probe molecules with a DNA strand of the predetermined sequence complementary to the respective DNA probe molecule, a second impedance measurement is carried out in a known manner. On the basis of the possibly changed impedance values, it is then determined whether a hybridization of probe molecules and DNA strands, each with a complementary sequence, has taken place. In addition to the previously described purely qualitative analysis of the substance mixture in the analyte, 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. The following publications are cited in this document:

[1] R. Hintsche et al. , Microbiosensors Using Electrodes Made in Si -Technology, Frontiers in Biosensorics, Fundamental Aspects, edited by F. W. Scheller et al. , Dirk Hauser Verlag, Basel, pp. 267-283, 1997

[2] N.L. Thompson, B.C. Lagerholm, Total Internal Reflection Fluoresence: Applications in Cellular Biophysics, Current Opinion in Biotechnology, Vol. 8, pp. 58-64, 1997

[3] P. Cuatrecasas, Affinity Chromatography, Annual Revision Biochem, Vol. 40, pp. 259-278, 1971

[4] P. van Gerwen, Nanoscaled Interdigitated Electrode Arrays for Biochemical Sensors, IEEE, International Conference on Solid-State Sensors and Actuators, Chicago, pp. 907-910, June 16-19, 1997

[5] WO 96/07917 AI

[6] WO 99/53093 AI

LIST OF REFERENCE NUMBERS

200 sensor

201 electrode

202 electrode

203 insulator layer

204 electrode connection

205 electrode connection

206 immobilized DNA probe molecules

207 analyte

208 strands of DNA

(A) first electrode

(B) second electrode

1 electrode

2 electrodes

Claims

claims

1. Sensor for the qualitative and quantitative determination of (bio) organic oligomers and polymers in solution, comprising an electrode arrangement

With at least one first electrode as a detection electrode on which capture molecules for hybridization with (bio) organic oligomers and polymers to be determined (“chemical compounds”) are immobilized (detection electrodes (A)),

• With at least two second electrodes as attraction electrodes, on which there are no capture molecules (attraction electrodes (B)), • The first electrode being arranged between the second electrodes in such a way that by changing the electrical fields on the second electrodes, one that is to be detected Analyte possibly containing chemical compounds, applied to the sensor arrangement depending on

The type and size of the electric fields is moved over the first electrode, so that (bio) organic oligomers and polymers contained in the analyte and to be determined qualitatively and / or quantitatively can hybridize with the capture molecules.

2. Sensor according to claim 1, wherein the (bio) organic oligomers and polymers are biopolymers.

3. Sensor according to claim 1 or 2, wherein the detection electrode (s) (A) a larger Circle radius of curvature than the attraction electrodes (B).

4. Sensor according to one of claims 1 to 3, in which at least two detection electrodes (A)

Catcher molecules are coated which differ in terms of their trapping characteristics for different molecules to be determined.

5. Sensor according to one of claims 1 to 4, wherein the number of attraction electrodes (B) is from 100 to 1000 and the number of detection electrodes (A) is from 100 to 1000.

6. Sensor according to one of claims 1 to 5, in which the detection electrodes (A) are planar.

7. Sensor according to one of claims 1 to 6, in which the detection electrodes (A) each

2 have the same surface area from 100 to 10000 μm.

8. Sensor according to one of claims 1 to 7, wherein the attraction electrodes (A) each

2 have the same surface area from 0.1 to 100 μm.

9. Sensor according to one of claims 1 to 8, in which the attraction electrodes (B) and detection electrodes (A) of the electrode arrangement are arranged at substantially the same distance from one another.

10. Sensor according to one of claims 1 to 9, wherein the electrodes consist of gold or contain gold and are arranged on a silicon chip.

11. Sensor according to one of claims 1 to 10, integrated in a chip.

12. Sensor according to claim 11, with one or more electronic circuits in the chip, which circuits provide electronic functions for the operation of the attraction electrodes in the chip.

13. A method for the qualitative and quantitative determination of (bio) organic oligomers or polymers in solution using an electrode arrangement according to claim 1, wherein

a. a sensor is contacted with a solution containing the substance to be analyzed, the

Sensor comprises an electrode arrangement, in which attraction electrodes (B) and detection electrodes (A) are present, b. on at least part of the attraction electrodes (B) such a time-changing electrical

Voltage is applied that at least two groups of attraction electrodes (B) at least temporarily 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 part of the substance to be analyzed with the catcher molecules the detection electrode (s) (A) hybridizes, and c. the (bio) organic oligomers or polymers to be determined are detected optically or electronically.

14. The method according to claim 13, in which a voltage source is coupled to the two second electrodes, with which a polarity reversal voltage can be applied to the second electrodes.