DE10221091B4 - SECM for the detection of nucleic acid oligomer hybridization events - Google Patents

Abstract

A method of detecting nucleic acid oligomer hybridization events comprising the steps
a) providing a modified surface, the modification consisting in the attachment of at least one type of ligate oligonucleotide,
b) providing a sample with ligand oligonucleotides,
c) contacting a solution with the modified surface, wherein the solution contains at least one type of redox-active species,
d) contacting the sample with the modified surface,
e) detection of reduction and / or oxidation of the redox-active species by a suitable electrochemical method using a measuring electrode whose distance from the modified surface is at most 5 times the radius of its electrochemically active surface,
f) Comparison of the values obtained in step e) with reference values.

Description

technical area

The The present invention relates to a method for detecting nucleic acid oligomer hybridization events using Scanning Electrochemical Microscopy.

From the DE 199 17 052 For example, a method for the determination of chemical or biochemical analytes is known in which the diffusion coefficient of a redox-active species coupled to a specific recognition element is modulated by attaching a complementary recognition structure, and the modulation of the diffusion coefficient with an electrochemical process enhanced by recycling Redox species is detected.

The basis of this detection by means of an electrochemical process amplified by recycling of the redox species is the fact that an electrode which is polarized with the aid of a potentiostat to a suitable constant potential with respect to a reference electrode oxidizes or reduces a redox species in solution corresponding to the potential can. According to DE 199 17 052 As a result of the conversion of the redox species to a microelectrode, a concentration gradient arises from the volume of the solution in the direction of the electrode, which results in the construction of a hemispherical diffusion field in front of the microelectrode in which the redox species is transported by diffusion to the electrode surface. If the concentration of the redox species to be reacted at the location of the electrode equals zero, the diffusion mass transfer limits the conversion at the electrode, and the so-called diffusion limiting current is obtained as the maximum current. If a microelectrode, at which an oxidation or reduction reaction of a redox species dissolved in the diffusion proceeds, approaches a macroscopic, conductive surface so far that the hemispherical diffusion profile in front of the microelectrode is disturbed by this surface, it can, with a suitable potential at the macroscopic Electrode - the back reaction of the reaction taking place on the microelectrode proceed. As a result of this back reaction, the number of redox species to be reacted at the microelectrode is increased in the volume space of the diffusion profile in the time unit, so that as a consequence the diffusion limiting current increases.

The electrochemical detection method described above is referred to as "Electrochemical Scanning Microscopy (SECM)" and belongs to the class of scanning probe techniques in which a probe is scanned sequentially over a surface (sample) and the recorded quantity for each raster point results from the interaction between probe and sample , In the special case of the SECM, an ultramicroelectrode (UME) serves as a probe which is moved across the surface at a working distance on the order of the electrode tip radius in an electrolyte. The potential of the UME is adjusted via a potentiostat so that a dissolved redox-active species is converted in a diffusion-controlled manner. If one approaches the UME step by step to the surface and thereby detects the resulting current, two cases can be distinguished. If the surface is non-conductive, the approximation of the UME due to the disturbance of the previously hemispherical diffusion field causes a reduced mass transfer to the UME and thus a decrease in the diffusion limiting current. One speaks of the negative feedback ( 1a . 2a ). On the other hand, if a conductive surface is present and the reverse reaction of the redox-active species reacted at the UME takes place reversibly, the diffusion paths of the oxidized and reduced form of the redox-active species become smaller and smaller with decreasing distance and the resulting current consequently increases. We talk about redox recycling or positive feedback ( 1b . 2 B ). The current amplification by redox recycling is dependent on the concentration of the redox species, the heterogeneous electron transfer kinetics at the sample surface as well as the distance between UME and sample surface.

One Raster of the surface at a constant distance with detection of the current (Constant height mode) or at constant current with detection of the readjusted Microelectrode distance (Constant Current mode) thus allows the Topography of the surface or the electrochemical activity of the surface.

In disease diagnosis, toxicological testing, genetic research and development, as well as in the agricultural and pharmaceutical sectors, the sequential analysis of DNA and RNA is increasingly being used. In addition to the known serial methods with autoradiographic or optical detection are increasingly parallel detection methods using array technology using soge called DNA chips application. Even in the case of the parallel methods, the actual detection is based on optical, radiographic, mass spectrometric or electrochemical methods.

to DNA analysis on a chip becomes known on a surface of a library DNA sequences ("ligate oligonucleotides") in an ordered Grid fixed so that the position of each individual DNA sequence is known. Exist in the study solution DNA fragments ("ligand oligonucleotides"), their sequences are complementary to certain ligate oligonucleotides on the chip, so can the ligand oligonucleotides by detection of the corresponding hybridization events be identified (read) on the chip.

Although methods for the detection of nucleic acid oligomer hybridization events using chip technology are known in the meantime ( DE 199 26 457 . DE 100 49 527 ) there is still a need for alternative methods which offer advantages in particular with regard to the reliability and reproducibility of the measurement results.

presentation the invention

task Therefore, the present invention is a method for detection of nucleic acid oligomer hybridization events to create, which allows measurements with high reproducibility.

These The object is achieved by the method according to independent claim 1 and the kit according to independent claim 28 solved. Further advantageous details, aspects and embodiments of the present invention Invention will be apparent from the dependent claims, the Description, examples and figures.

DNA

Desoxyribonukleinsäure

RNA

Ribonukleinsäure

PNA

Peptidnukleinsäure (Synthetische DNA oder RNA, bei der die Zucker-Phosphat Einheit durch eine Aminosäure ersetzt ist. Bei Ersatz der Zucker-Phosphat Einheit durch die -NH-(CH₂)₂-N(COCH₂-Base)-CH₂CO- Einheit hybridisiert PNA mit DNA.)

A

Adenin

G

Guanin

C

Cytosin

T

Thymin

U

Uracil

Base

A, G, T, C oder U

Bp

Basenpaar

Nukleinsäure

Wenigstens zwei kovalent verbundene Nukleotide oder wenigstens zwei kovalent verbundene Pyrimidin- (z.B. Cytosin, Thymin oder Uracil) oder Purin-Basen (z.B. Adenin oder Guanin). Der Begriff Nukleinsäure bezieht sich auf ein beliebiges "Rückgrat" der kovalent verbundenen Pyrimidin- oder Purin-Basen, wie z.B. auf das Zucker-Phosphat Rückgrat der DNA, cDNA oder RNA, auf ein Peptid-Rückgrat der PNA oder auf analoge Strukturen (z.B. Phosphoramid-, Thio-Phosphat- oder Dithio-Phosphat-Rückgrat). Wesentliches Merkmal einer Nukleinsäure im Sinne der vorliegenden Erfindung ist es, dass sie natürlich vorkommende cDNA oder RNA sequenzspezifisch binden kann.

nt

Nukleotid

Nukleinsäure-Oligomer

Nukleinsäure nicht näher spezifizierter Basenlänge (z.B. Nukleinsäure-Oktamer: Eine Nukleinsäure mit beliebigem Rückgrat, bei der 8 Pyrimidin- oder Purin-Basen kovalent aneinander gebunden sind.).

ns-Oligomer

Nukleinsäure-Oligomer

Oligomer

Äquivalent zu Nukleinsäure-Oligomer.

Oligonukleotid

Äquivalent zu Oligomer oder Nukleinsäure-Oligomer, also z.B. ein DNA-, PNA- oder RNA-Fragment nicht näher spezifizierter Basenlänge.

Oligo

Abkürzung für Oligonukleotid.

Mismatch

Zur Ausbildung der Watson-Crick Struktur doppelsträngiger Oligonukleotide hybridisieren die beiden Einzelstränge derart, dass die Base A (bzw. C) des einen Strangs mit der Base T (bzw. G) des anderen Strangs Wasserstoffbrücken ausbildet (bei RNA ist T durch Uracil ersetzt). Jede andere Basenpaarung bildet keine Wasserstoffbrücken aus, verzerrt die Struktur und wird als "Mismatch" bezeichnet.

ss

Single strand (Einzelstrang)

ds

Double strand (Doppelstrang)

Oxidationsmittel

Chemische Verbindung (chemische Substanz), die durch Aufnahme von Elektronen aus einer anderen chemischen Verbindung (chemischen Substanz) diese andere chemische Verbindung (chemische Substanz) oxidiert.

Reduktionsmittel

Chemische Verbindung (chemische Substanz), die durch Abgabe von Elektronen an eine andere chemische Verbindung (chemische Substanz) diese andere chemische Verbindung (chemische Substanz) reduziert.

sulfo-NHS

N-Hydroxysulfosuccinimid

NHS

N-Hydroxysuccinimid

EDC

(3-Dimethylaminopropyl)-carbodiimid

HEPES

N-(2-Hydroxyethyl]piperazin-N'-[2-ethansulfonsäure]

Ligand-Oligonukleotid

Bezeichnung für Oligonukleotide, die von Ligat-Oligonukleotiden spezifisch gebunden werden.

Ligat-Oligonukleotid

Bezeichnung für Oligonukleotide, die von Ligand-Oligonukleotiden spezifisch gebunden werden.

Linker

Molekulare Verbindung zwischen zwei Molekülen bzw. zwischen einem Oberflächenatom, Oberflächenmolekül oder einer Oberflächenmolekülgruppe und einem anderen Molekül. In der Regel sind Linker als Alkyl-, Alkenyl-, Alkinyl-, Hetero-Alkyl-, Hetero-Alkenyl- oder Hetero-Alkinylkette käuflich zu erwerben, wobei die Kette an zwei Stellen mit (gleichen oder verschiedenen) reaktiven Gruppen derivatisiert ist. Diese Gruppen bilden in einfachen/bekannten chemischen Reaktionen mit dem entsprechenden Reaktionspartner eine kovalente chemische Bindung aus. Die reaktiven Gruppen können auch photoaktivierbar sein, d.h. die reaktiven Gruppen werden erst durch Licht bestimmter oder beliebiger Wellenlänge aktiviert. Bevorzugte Linker sind solche der Kettenlänge 1 – 20, insbesondere der Kettenlänge 1 – 14, wobei die Kettenlänge hier die kürzeste durchgehende Verbindung zwischen den zu verbindenden Strukturen, also zwischen den zwei Molekülen bzw. zwischen einem Oberflächenatom, Oberflächenmolekül oder einer Oberflächenmolekülgruppe und einem anderen Molekül, darstellt.

Spacer

Äquivalent zu Linker.

Mica

Muskovit-Plättchen, Trägermaterial zum Aufbringen dünner Schichten.

Au-S-(CH₂)₂-ss-oligo

Gold-Film auf Mica mit kovalent aufgebrachter Monolayer aus derivatisiertem Einzelstrang-Oligonukleotid. Hierbei ist die endständige Phosphatgruppe des Oligonukleotids am 3' Ende mit (HO-(CH₂)₂-S)₂ zum P-O-(CH₂)₂-S-S-(CH₂)₂-OH verestert, wobei die S-S Bindung homolytisch gespalten und dadurch je eine Au-S-R Bindung bewirkt wird.

Au-S-(CH₂)₂-ds-oligo

Au-S-(CH₂)₂-ss-oligo-Sparer hybridisiert mit dem zu ss-oligo komplementären Oligonukleotid.

E

Elektrodenpotential, das an der Arbeitselektrode anliegt.

i

Stromdichte (Strom pro cm² Elektrodenoberfläche)

SECM

Scanning Electrochemical Microscopy, Elektrochemische Rastermikroskopie.

UME

Ultramikroelektrode

Cyclovoltammetrie

Aufzeichnung einer Strom/Spannungskurve. Das Potential einer stationären Arbeitselektrode wird dabei zeitabhängig linear verändert, ausgehend von einem Potential, bei dem keine Elektrooxidation oder -reduktion stattfindet bis zu einem Potential, bei dem eine gelöste oder an die Elektrode adsorbierte Spezies oxidiert oder reduziert wird (also Strom fließt). Nach Durchlaufen des Oxidations- bzw. Reduktionsvorgangs, der in der Strom/Spannungskurve einen zunächst ansteigenden Strom und nach Erreichen eines Maximums einen allmählich abfallenden Strom erzeugt, wird die Richtung des Potentialvorschubs umgekehrt. Im Rücklauf wird dann das Verhalten der Produkte der Elektrooxidation oder -reduktion aufgezeichnet.

(Chrono)Amperometrie

Aufzeichnung einer Strom/Zeitkurve. Hierbei wird das Potential einer stationären Arbeitselektrode z.B. durch einen Potentialsprung auf ein Potential gesetzt, bei dem die Elektrooxidation oder -reduktion einer gelösten oder adsorbierten Spezies stattfindet und der fließende Strom in Abhängigkeit von der Zeit aufgezeichnet.

Chronocoulometrie

Aufzeichnung einer Ladungs/Zeitkurve. Hierbei wird das Potential einer stationären Arbeitselektrode z.B. durch einen Potentialsprung auf ein Potential gesetzt, bei dem die Elektrooxidation oder -reduktion einer gelösten oder adsorbierten Spezies stattfindet und die transferierte Ladung in Abhängigkeit von der Zeit aufgezeichnet. Die Chronocoulometrie kann folglich als Integral der Amperometrie verstanden werden.

Differential-Puls-Voltammetrie

Bei der Differential-Puls-Voltammetrie werden einer schrittweise anwachsenden Gleichspannungsrampe Rechteckpulse mit kleiner, konstanter Amplitude überlagert. Die Messungen des Stroms als Funktion der Spannung erfolgen dabei unmittelbar vor dem Puls und am Ende des Pulses. Aufgetragen werden die Differenzen der Strommesswertpaare gegen das Potential.

Square-Wave-Voltammetrie

Bei der Square-Wave-Voltammetrie wird einer schrittweise anwachsenden Gleichspannungsrampe eine rechteckförmige Wechselspannung mit kleiner, konstanter Amplitude und niedriger Frequenz überlagert. Zur Messung des Stroms als Funktion der Spannung werden jeweils mehrmals die Differenzen der Ströme bei maximaler und minimaler Rechteckspannung bestimmt.

The following abbreviations and terms are used in the context of the present invention:

DNA

deoxyribonucleic acid

RNA

ribonucleic acid

PNA

Peptide nucleic acid (synthetic DNA or RNA in which the sugar-phosphate moiety is replaced by an amino acid.) When the sugar-phosphate moiety is replaced by the -NH- (CH ₂ ) ₂ -N (COCH ₂ -Base) -CH ₂ CO- Unit hybridizes PNA with DNA.)

A

adenine

G

guanine

C

cytosine

T

thymine

U

uracil

base

A, G, T, C or U

bp

base pair

nucleic acid

At least two covalently linked nucleotides or at least two covalently linked pyrimidine (eg cytosine, thymine or uracil) or purine bases (eg adenine or guanine). The term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as the sugar-phosphate backbone of the DNA, cDNA or RNA, to a peptide backbone of the PNA or to analogous structures (eg, phosphoramid , Thio-phosphate or dithio-phosphate backbone). An essential feature of a nucleic acid according to the present invention is that it can bind naturally occurring cDNA or RNA sequence-specific.

nt

nucleotide

Nucleic acid oligomer

Nucleic acid of unspecified base length (eg nucleic acid octamer: A nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to each other.).

ns oligomer

Nucleic acid oligomer

oligomer

Equivalent to nucleic acid oligomer.

oligonucleotide

Equivalent to oligomer or nucleic acid oligomer, eg a DNA, PNA or RNA fragment of unspecified base length.

oligo

Abbreviation for oligonucleotide.

mismatch

To form the Watson-Crick structure of double-stranded oligonucleotides, the two single strands hybridize such that the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (in the case of RNA, T is replaced by uracil) , Any other base pairing does not form hydrogen bonds, distorts the structure, and is referred to as a "mismatch."

ss

Single strand (single strand)

ds

Double strand (double strand)

oxidant

Chemical compound (chemical substance) that oxidizes this other chemical compound (chemical substance) by absorbing electrons from another chemical compound (chemical substance).

reducing agent

A chemical compound that reduces this other chemical compound by releasing electrons to another chemical compound.

sulfo-NHS

N-hydroxy sulfosuccinimide

NHS

N-hydroxysuccinimide

EDC

(3-dimethylaminopropyl) carbodiimide

HEPES

N- (2-hydroxyethyl] piperazine-N '- 2-ethanesulfonic acid []

Ligand-oligonucleotide

Name for oligonucleotides that are specifically bound by ligate oligonucleotides.

Ligate oligonucleotide

Name for oligonucleotides that are specifically bound by ligand oligonucleotides.

left

Molecular connection between two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule. As a rule, linkers are commercially available as alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl chain, the chain being derivatized in two places with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partner. The reactive groups may also be photoactivatable, ie the reactive groups are activated only by light of specific or arbitrary wavelength. Preferred linkers are those of the chain length 1-20, in particular the chain length 1-14, wherein the chain length here is the shortest continuous connection between the structures to be connected, ie between the two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule , represents.

spacer

Equivalent to linker.

Mica

Muscovite platelets, carrier material for applying thin layers.

Au-S- (CH ₂ ) ₂ -ss-oligo

Gold film on mica with covalently applied monolayer of derivatized single-stranded oligonucleotide. Here, the terminal phosphate group of the oligonucleotide at the 3 'end with (HO- (CH ₂ ) ₂ -S) ₂ to the PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH esterified, the SS bond homolytically cleaved and thereby each an Au-SR bond is effected.

Au-S- (CH ₂ ) ₂ -ds-oligo

Au-S- (CH ₂ ) ₂ -ss-oligo saver hybridizes with the oligonucleotide complementary to ss-oligo.

e

Electrode potential applied to the working electrode.

i

Current density (current per cm ² electrode surface)

SECM

Scanning Electrochemical Microscopy, Electrochemical Scanning Microscopy.

UME

Ultramicroelectrode

Cyclic voltammetry

Recording of a current / voltage curve. The potential of a stationary working electrode is thereby linearly changed over time, starting from a potential at which no electrooxidation or reduction takes place up to a potential at which a species dissolved or adsorbed to the electrode is oxidized or reduced (ie current flows). After passing through the oxidation or reduction process, which generates an initially rising current in the current / voltage curve and, after reaching a maximum, a gradually decreasing current, the direction of the potential feed is reversed. The response then records the behavior of the products of electrooxidation or reduction.

(Chrono) amperometry

Recording a current / time curve. Here, the potential of a stationary working electrode is set, for example, by a potential jump to a potential at which the electro-oxidation or reduction of a dissolved or adsorbed species takes place and the flowing current is recorded as a function of time.

chronocoulometry

Recording a charge / time curve. Here, the potential of a stationary working electrode is set, for example, by a potential jump to a potential at which the electrooxidation or reduction of a dissolved or adsorbed species takes place and the transferred charge is recorded as a function of time. The chronocoulometry can thus be understood as the integral of amperometry.

Differential pulse voltammetry

In differential pulse voltammetry, rectangular pulses of small, constant amplitude are superimposed on a stepwise increasing DC voltage ramp. The measurements of the current as a function of the voltage take place immediately before the pulse and at the end of the pulse. Plotted are the differences of the current value pairs against the potential.

Square wave voltammetry

In square wave voltammetry, a stepwise increasing DC voltage ramp is superposed with a rectangular, small amplitude, constant amplitude, low frequency AC voltage. To measure the current as a function of the voltage, the differences of the currents at maximum and minimum square-wave voltage are determined several times in each case.

The The present invention provides a method for detecting nucleic acid oligomer hybridization events ready to take the steps of providing a modified surface, taking the modification in the attachment of at least one type of ligate oligonucleotide providing a sample with ligand oligonucleotides, Contacting a solution with the modified surface, being the solution contains at least one type of redox-active species, contacting the sample with the modified surface, detection of the reduction and / or oxidation of the redox-active species by a suitable Electrochemical process using a measuring electrode whose Distance from the modified surface is at most 5 times as large as the radius of their electrochemically active surface, and comparison of at The detection includes received throw with reference values.

The electrochemically active surface of the measuring electrodes used in the context of the present invention is usually circular ( 3 ). In this case, the term "radius of the electrochemically active surface" is self-explanatory. In the event that the electrochemically active surface is not circular, the term "radius of the electrochemically active surface" means half of the largest dimension of the part of the measuring electrode which is electrochemically active, that is to say in contact with the redox-active species. In the case of a measuring electrode with a square tip, that is half the diagonal length.

The process according to the invention utilizes the fact that the conversion of a redox-active species onto an electrode, that is to say the oxidation or reduction of the redox-active species, depends on the condition of the electrode surface. The binding of a chemical substance to the electrode surface causes alone by the consequent occupancy of the electrode surface modulation of the electron transfer kinetics of the redox-active substance and thus a change in the current. If the electrode surface is occupied, for example, with negatively charged molecules or molecule groups, then it is negative for example loading redox-active substances much harder to get to the electrode surface and there to be oxidized, as if an uncharged electrode surface is present. In this case, there is a dramatic decrease in the measured current. Likewise, it can of course lead to an increase in the current, for example, if a negatively charged redox-active substance is to migrate to an electrode to which a positively charged substance is bound. In any case, the interaction between the species bound to the electrode surface and the redox-active substance results in a modulation of the electron transfer kinetics and thus in a change in the current.

The detection method according to the present invention is based on a SECM measurement. As the measuring electrode approaches the modified surface, the hemispherical diffusion field of the redox-active species contained in the solution is disturbed. If a non-conductive modified surface is used, the approach of the measuring electrode causes a reduced mass transport to the measuring electrode and thus a decrease of the diffusion limiting current. Such negative feedback is in the 1a and 2a shown schematically. If a conductive surface is used and the reverse reaction of the redox-active species reacted on the measuring electrode takes place reversibly there, the diffusion paths of the oxidized and reduced form of the redox-active species become smaller and smaller with decreasing distance, and the resulting current consequently increases. This positive feedback is schematic in the 1b and 2 B shown.

Of the described effect is particularly evident in the case if the redox-active species is a species, which can be reversibly oxidized or reduced at the measuring electrode is.

From the 2 It becomes clear that an effect measured with corresponding reliability and accuracy only occurs when the distance of the measuring electrode from the modified surface is at most 5 times the radius of its electrochemically active surface. With decreasing distance, the effect caused by the disturbance of the diffusion profile of the redox active species dramatically increases. According to a preferred embodiment of the present invention, the distance of the measuring electrode from the modified surface is therefore at most 4 times, more preferably at most 3 times as large as the radius of the electrochemically active surface of the measuring electrode. Very particular preference is given to a distance of the measuring electrode from the modified surface which is at most 2 times, particularly preferably at most, exactly the same as the radius of the electrochemically active surface of the measuring electrode.

According to a further preferred embodiment of the present invention, a measuring electrode having a radius of the electrochemically active surface of from 0.05 to 250 μm, preferably from 0.5 to 50 μm, particularly preferably from 5 to 25 μm, is used. Typically, this is a microelectrode with a radius <100 .mu.m in an insulating sheath, which has a multiple diameter of the electrochemically active surface of the measuring electrode ( 3 ). Particularly preferably, a disk electrode is used as the measuring electrode, which is surrounded by an insulating jacket made of glass or polymers.

Out the size of the electrochemical active surface the measuring electrode results that, according to a preferred embodiment the present invention, the distance between the measuring electrode and modified surface while the measurement maximum 1250 microns is. Particularly preferred is the distance max. 500 μm, most preferably 100 .mu.m and in particular 50 microns.

The measuring electrode is approximated to the modified surface by means of displacement elements, such as stepper motor-driven or manual micrometer screws or piezostacks, so that the diffusion profile of the redox species in front of the microelectrode extends to the opposite modified surface ( 1b ). In the case of a conductive modified surface, the redox-active species is regenerated at this surface. This regeneration of the redox species leads to an amplification of the current which can be selected within wide limits by the distance of the measuring electrode as a function of the diffusion coefficient of the redox species reacted at the measuring electrode.

According to a particularly preferred embodiment of the present invention, a modified surface is used, to which at least two different types of ligate oligonucleotides are attached. However, it is of course possible for a very large number of types of ligate oligonucleotides to be bound to the surface, for example 100, 1000 or more than 100 000 different species. According to a particularly preferred embodiment of the present invention, each predominantly one type of ligate oligonucleotides is in a spatially substantially separated region of the modified surface tethered. By "spatially substantially separated regions" is meant areas of the surface which are predominantly modified by attachment of a particular type of ligate oligonucleotide. Only in areas where two such substantially separated regions adjoin one another can spatial mixing of different types of ligate oligonucleotides occur. It is very particularly preferred that in each case only one type of ligate oligonucleotide is bound in a spatially limited area of the modified surface.

The size of these spatially substantially separated areas is basically arbitrary. For technical reasons, preferably up to 10 cm ² large, substantially separated areas are preferably used. In the context of the present invention, spatially substantially separated regions of a size of 100 μm ² to ¹ 000 000 μm ² are particularly preferred.

are the different types of ligate oligonucleotides in spatially limited Tied to areas of modified surface, so can at the detection of the hybridization events using the measuring electrode the modified surface be scanned and so the hybridization events selectively for the different ones Types of Ligat oligonucleotides in the different spatially limited Areas are detected. In this context are arrays from test sites (spatially essentially separated regions) preferred by regions the surface are separated from each other, in which no ligate oligonucleotides to the surface are bound. Due to the spatial Distance between each test sites can be the measuring electrode easily be approximated accurately and clearly to a particular type of ligate oligonucleotide. Thus, uniquely assignable measurement results are obtained.

All it is particularly preferred in the context of the present invention to use an array of microelectrodes as a modified surface. In this case, in the area of a microelectrode, one becomes each Type of ligate oligonucleotide attached to the surface. Due to the spatial Distance between the individual microelectrodes can be the measuring electrode easily and accurately to a specific type of ligate oligonucleotide approximated become.

By the controllability of the individual microelectrodes can be the obtained measurement results very easily certain hybridization events assign.

In the most general embodiment The present invention, after contacting the Sample with the modified surface detected throw of the Current flow compared with already known reference values. These Reference values can in independent Experiments for each a certain set of parameters (distance of the measuring electrode from the modified surface, Concentration of the redox-active species in the solution, applied potential, Temperature, salt concentration, etc.).

According to one preferred embodiment of the present invention are the reference values before addition the sample is determined in a separate measurement. In this embodiment So after contacting the redox-active species takes place containing solution with the modified surface a detection of the reduction and / or oxidation of the redox-active Species by a suitable electrochemical method with the help a measuring electrode whose distance from the modified surface maximum 5 times as big like the radius of their electrochemically active surface. by virtue of this under exactly the same conditions of actual measurement conducted Reference measurement even more accurate and reliable values are detected.

According to the following described, particularly preferred embodiments of the present invention can be the separate implementation a reference measurement can be avoided. On the modified surface are in these cases Reference sites applied to which after addition of the sample a whole certain degree of association can be assigned. That at the Detection received signal is then on for that particular degree Association characteristic and can be used to normalize the signals of the test sites.

Namely, the present invention also encompasses methods in which a modified surface modified by attachment of at least two types of ligate oligonucleotides is used. The different types of ligate oligonucleotides are bound to the surface in spatially substantially separated regions. In the method preferred in the context of the present invention, before the sample is brought into contact with the modified surface, the addition of one type of ligand oligonucleotide to the sample takes place, wherein the ligand oligonucleotide is a binding partner with a high association cons aant to a specific type of ligate oligonucleotides, which is bound to the surface in a certain area (test site T ₁₀₀ ). The ligand oligonucleotide is then added in an amount to the sample, which is greater than the amount of ligand oligonucleotides, which is necessary in order to ligate oligonucleotides ₁₀₀ test sites to completely associate the T. The last step of this process is the comparison of the values obtained in the detection of the reduction and / or oxidation of the redox-active species with the value obtained for the T ₁₀₀ range. The value obtained for the region T ₁₀₀ thus corresponds to the value at complete association (100%).

According to a particularly preferred embodiment, a modified surface is used which has been modified by attachment of at least three types of ligate oligonucleotides. The different types of ligate oligonucleotides are bound to the surface in spatially substantially separated regions. In this case, at least one type of ligate oligonucleotide is bound to the surface in a specific region (test site T ₀ ), which is known to contain no binding partner with a high association constant in the sample, that is to say the corresponding complementary oligonucleotide is not present in the sample Sample occurs. In this particularly preferred method, before the sample is brought into contact with the modified surface, the addition of a ligand oligonucleotide to the sample takes place, wherein the ligand oligonucleotide is a binding partner with a high association constant to a specific type of ligate oligonucleotides certain area (test site T ₁₀₀ ) is bound to the surface. The ligand oligonucleotide is then added in an amount to the sample, which is greater than the amount of ligand oligonucleotides, which is necessary in order to ligate oligonucleotides ₁₀₀ test sites to completely associate the T. The last step of this process is the comparison of the values obtained in the detection of the reduction and / or oxidation of the redox-active species with the value obtained for the range T ₁₀₀ and the value obtained for the range T ₀ . The value obtained for the region T ₀ thus corresponds to the value in the absence of association (0%).

According to a very particularly preferred embodiment of the two methods described above, which manage without separate reference measurement, the addition of the sample to the modified surface is followed by the addition of at least one further type of ligand oligonucleotide to the sample, it being known that this ligand oligonucleotide is not included in the original sample. This further type of ligand oligonucleotide has an association constant> 0 to a type of ligate oligonucleotide which is bound to the surface in a certain range (test site T _n ). The ligand oligonucleotide is added to the sample in an amount such that upon contacting the sample with the modified surface, n% of the ligate oligonucleotides of the test site T _{n are} in associated form. The last step of this process is the comparison of the values obtained in the detection of the reduction and / or oxidation of the redox-active species with the value obtained for the region T ₁₀₀ , with the value obtained for the region T ₀ and with those for the ranges T _n obtained values. The value obtained for a given test site T _n thus corresponds to the value in the presence of n% ligate oligonucleotide / ligand oligonucleotide associates relative to the total number of ligate oligonucleotides corresponding to Art.

The amount of ligand oligonucleotides that must be contacted with the modified surface to effect n% association at the test site T _n can be determined by one skilled in the art by simple routine examinations. For this purpose, for example, after detection of the throw for T ₀ and T _100, a calibrated measurement is carried out in which the signal intensity of (different) detection labels is determined, with which the ligate oligonucleotides and the ligand oligonucleotides are equipped. The ratio of ligand oligonucleotide label signal to ligate oligonucleotide label signal corresponds to n%.

If a sufficient number of reference sites T _{n are} applied to the modified surface, a reference curve can be recorded with high accuracy. The standardization of the measurements of the actual test sites with the aid of this reference curve significantly improves the reproducibility of the analysis with the aid of the chip technology.

It It should be noted that finding a ligand oligonucleotide, that is not in the sample, does not cause any problems because even the most extensive genomes are still a sufficient choice to offer non-existent sequences. In the event that is not existing sequence of a present sequence only by a base differs, the Hybridisierungsschntt must be under stringent Conditions performed become. Preferably, however, sequences are used which are clearly that is, in several bases, of the sequences present in the sample differ. Particularly good results are achieved when for the test sites and for the reference sites oligonucleotides with the same or at least a similar one Number of bases to be used.

As already described above, in the inventive method exploited the fact that the turnover of a redox-active species on an electrode, ie the oxidation or reduction of the redox-active Species, depending on the condition of the electrode surface. Is the electrode surface partially or Completely with negatively charged molecules or molecular groups occupied, so it is for negatively charged redox - active substances are much more difficult for electrode surface to get there and be oxidized there, as if an uncharged one electrode surface is present. This effect becomes more apparent the larger the from the to the electrode surface bound molecules carried cargo and the larger the is charge carried by the redox active species. For this reason In the context of the present invention, redox-active species, which carry a high charge, compared to species with low charge and these in turn opposite uncharged compounds are preferred.

Besides should be mentioned Be that covering the surface with molecules or molecular groups Of course can be done in any density. It is obvious that the above-described effect of the difficult access of the redox-active Species to the surface the stronger occurs, the denser the surface with molecules or molecular groups is occupied. The same applies to to the surface bound ligate oligonucleotides. An upper limit of the density of Occupancy in this case is through the detection of hybridization events essential requirement of a certain amount of space around the ligate oligonucleotides which hybridization with the ligand oligonucleotides only possible power.

According to one another, particularly preferred embodiment of the present invention Invention are substantially uncharged ligate oligonucleotides used, which brings a very decisive advantage: Are to the electrode surface Ligat oligonucleotides bound, which is a negative per phosphate unit Carrying charge, as results in the binding of the complementary ligand oligonucleotide to the ligate oligonucleotides in a first approximation, a doubling of Number of charges per electrode surface. Become essentially uncharged ligate oligonucleotides bound to the electrode surface, see above increases with subsequent Binding of a complementary Ligand oligonucleotide, the charge per electrode surface, so to speak by a factor ∞. This dramatic change The charge density also has a dramatic impact on sales a negatively charged redox-active species at the electrode surface. The detected current decreases significantly and thus allows a simple and clear Proof of whether surface-bound, substantially uncharged ligate oligonucleotides complementary ligand oligonucleotides are bound or not.

Under a charged oligonucleotide is within the scope of the present invention an oligonucleotide that has a number of elementary charges wearing, which about the number of nucleotides that make up the oligonucleotide composed. As is known, are the individual nucleotides over Phosphate groups linked together. Because these phosphate groups usually simply negatively charged, results for the entire Oligonucleotide is a total charge that is essentially (namely to on the end of the nucleotide chain missing phosphate groups) of the number corresponds to nucleotides.

Under the term "im essentially uncharged " in the context of the present invention, a number of simply negative Understood charges (elementary charges) of the ligate oligonucleotide, the maximum of 50% of the number of nucleotides of the ligate oligonucleotide is. Preferred is the number of elementary charges of the ligate oligonucleotide maximum 40% of the number of nucleotides of the ligate oligonucleotide, especially preferably not more than 30%, in particular not more than 20% and especially preferably at most 10% of the number of nucleotides of the ligate oligonucleotide. The best results are obtained with fully uncharged ligate oligonucleotides achieved. The uncharged oligonucleotides according to the following structural formulas deliver particularly good results and are therefore part of the particularly preferred according to the invention.

The shown methylphosphonate oligonucleotide and the illustrated Methyl phosphate oligonucleotide are exemplary for Classes of compounds in which the methyl group is replaced by a any uncharged rest is exchanged, especially against Alkyl radicals such as e.g. Ethyl radicals. In addition, e.g. at the top phosphoamidates represented the NHR group e.g. be replaced by SR.

In principle, it is also possible to replace the entire phosphate group. The individual oxygen atoms can be replaced by S atoms (one to three substitutions), P by N, O by NH, etc. From the prior art, a variety of such compounds is known.

When Extreme case can be the complete one Exchange of sugar and the phosphate group are considered as it is e.g. in the case of the peptide oligonucleotides shown above Case is.

in the Substantially uncharged ligate oligonucleotides can be prepared by using the above uncharged backbones shown with "normal" loaded DNA backbones. Depending on the proportion of charged and uncharged sections result in essence uncharged "ligate oligonucleotides, which carry a number of elementary charges between 0 and maximum 50% of the number of nucleotides of the ligate oligonucleotide is. Of course can also mixed forms of the above, various uncharged ligate oligonucleotides be used. The backbone consists in this case of any Mixed forms of modified or unmodified nucleotides, which only satisfy the condition need to be "essentially uncharged".

Besides The present invention also relates to a kit for carrying out a Method for detecting nucleic acid oligomer hybridization events. The kit includes a modified surface, the modification in the attachment of at least one type of ligate oligonucleotide and an effective amount of a redox active species.

The surface

With The term "surface" is any carrier material designated, which is suitable directly or after appropriate chemical Modification ligate oligonucleotides covalently or via others to bind specific interactions. The solid support can made of conductive or non-conductive Material exist.

Under the term "conductive surface" is used with each carrier an electrically conductive Surface of any Thickness understood, especially surfaces of metals such as in particular Platinum, Palladium, Gold, Cadmium, Mercury, Nickel, Zinc, Silver, Copper, iron, lead, aluminum and manganese. In addition, can also any doped or non-doped semiconductor surfaces of any desired Thickness can be used. All semiconductors can find use as pure substances or as mixtures. As not restrictive Examples given here are carbon, silicon, Germanium, α-tin, Called Cu (I) and Ag (I) halides of any crystal structure. Also suitable are all binary Compounds of any composition and structure from the elements of the groups 14 and 16, the elements of the groups 13 and 15, as well as the elements of groups 15 and 16. In addition, ternary compounds any composition and any structure of the elements the groups 11, 13 and 16 or the elements of the groups 12, 13 and 16 are used. The names of the groups of the periodic table of elements refer to the 1985 IUPAC Recommendation.

When Materials of non-conductive surfaces become glass, modified Glass and polymers preferred. The modification may e.g. by silanization done and leads in all cases to functional groups that are suitable in coupling reactions to bind appropriately functionalized ligate oligonucleotides. This modification concludes Layer structures on the glass surface using polymers such as e.g. Dextran polymers showing a variation of layer thickness and surface finish allow, with. Further derivatization possibilities of the glass for ultimate attachment of the ligate oligonucleotides consists e.g. in applying a thin (about 10 - 200 nm) metallization layer, in particular a gold metallization layer, the additional with (thiol-functionalized) polymers, in particular dextrans can be. In addition, the glass can also be used after silanization Biotin functionalized (e.g., amino-functionalized glass surface) the silanization and coupling of the carboxylic acid biotin via EDC and NHS or via a Biotin active esters such as biotin-N-succinimidyl ester) or alternatively immobilized on dextran lysine or dextran Biotin coated become. The biotinylated glass surfaces thus produced are subsequently treated with avidin or streptavidin and can then bind to biotinylated ligate oligonucleotides be used.

at the array of microelectrodes used in the present invention are commercially available Components. The microelectrodes themselves are made of a conductive material while between the individual microelectrodes a non-conductive material is located, through which the individual microelectrodes both spatially as be electrically separated from each other. The microelectrodes can be active or passively controlled. In case of active control In particular, the use of CMOS structures in question.

Bonding the Ligate oligonucleotides to the surface

method for the immobilization of nucleic acid oligomers on a surface are known in the art. The ligate oligonucleotides may be e.g. covalently over Hydroxyl, epoxide, amino or Carboxy groups of the carrier material with naturally on the ligate oligonucleotide or by derivatization on Ligat oligonucleotide attached thiol, hydroxy, amino or Carboxyl groups to the surface be bound. The ligate oligonucleotide can be directly or via a Linker / spacer to the surface atoms or molecules a surface be bound. In addition, the ligate oligonucleotide by the usual in immunoassays Methods are anchored such. by using biotinylated Ligate oligonucleotides for non-covalent immobilization on avidin or streptavidin-modified surfaces. The chemical modification the ligate nucleic acid oligomers with a surface anchor group already in the course of automated solid phase synthesis or but introduced in separate reaction steps. This is the Nucleic acid oligomer directly or via a linker / spacer with the surface atoms or molecules of a surface linked to the type described above. This binding can be done in several ways (See, for example, WO 00/42217).

Ligand oligonucleotides

When Ligand oligonucleotides are called nucleic acid oligomers which specifically with the immobilized on a surface ligate oligonucleotides can interact to form a complex. Such an interaction forming a complex, the presence of one sets to one Ligand oligonucleotide complementary or nearly complementary Ligat oligonucleotide ahead. As a nucleic acid oligomer is under of the present invention, a compound of at least two covalently linked Nucleotides or at least two covalently linked pyrimidine (e.g., cytosine, thymine, or uracil) or purine bases (e.g., adenine or guanine) a DNA, RNA or PNA fragment. The term nucleic acid refers referring to any "backbone" of the covalently bound Pyrimidine or Purine bases, e.g. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone the PNA or analogous backbone structures, such as. a thio-phosphate, a dithio-phosphate, a phosphoramide, an alkyl phosphonate, an alkyl phosphate or a phosphoamidite backbone. Essential feature of a nucleic acid in the sense of the present invention Invention is the sequence-specific binding of naturally occurring DNA, RNA or their transcription or amplification products.

redox-active species

As the redox-active species transition metal complexes can be used which preferably have a negative total charge. In addition, however, the use of neutral transition metal complexes with negatively charged ligands is possible. In the present invention, transition metal complexes of iron, cobalt, nickel, copper, ruthenium, and osmium with negatively charged ligands are preferred. , Oxo (O ^2-), thio as ligands can, for example, cyano (CN-), thiocyanato (SCN-), hydroxo (OH), halogenido- (Fl ^-, Cl ^{- -} I, Br,) (S ^2- ) groups and similar, but also sterically more complex, with negative groups such as -COOH, -OS (OH) ₃ or -OPO (OH) ₂ modified ligands such as pyridyl (py), bipyridyl (bipy), cyclopentadienyl (cp) derivatives and the like. The last-mentioned ligands modified with negative groups are preferably used in solutions whose pH is above the pKa value of the particular compound, because this ensures that the particular transition metal complex in the solution is negatively charged.

Of course, all of the abovementioned ligands can occur in any desired mixed forms, ie a central atom of a transition metal complex can simultaneously carry different ligands. An arbitrarily selected example of such a complex is the compound ammonium tetranitro-diammine-cobaltate (III) NH ₄ [Co (NO ₂ ) ₄ (NH ₃ ) ₂ ].

In addition, quinones such as pyrroloquinolinoquinone, ubiquinone, anthraquinone, naphthoquinone or menaquinone and their derivatives modified with negative groups such as -COOH, -OS (OH) ₃ or -OPO (OH) ₂ can also be used as redox-active species.

in the Within the scope of the present invention, the transition metal complex is iron hexacyanoferrate particularly preferred.

electrochemical methods

When Detection method own electrochemical methods such as Cyclic voltammetry, chronoamperometry, chronocoulometry, differential pulse voltammetry, Square wave voltammetry or amperometry at constant potential.

at The cyclic voltammetry is a current / voltage curve recorded. The potential of a stationary Working electrode is thereby time-linearly changed, starting from a potential where no electro-oxidation or reduction takes place to a potential where a dissolved or oxidized or reduced to the electrode adsorbed species (ie current flows). After undergoing the oxidation or reduction process, the the current / voltage curve an initially rising current and generates a gradually decreasing current after reaching a maximum, the direction of the potential feed is reversed. In the return is then the behavior of the products of electrooxidation or reduction recorded.

at Chronocoulometry is the recording of a charge / time curve. Here, the potential of a stationary working electrode, e.g. by set a potential jump to a potential at which the electro-oxidation or -reduction of a solved one or adsorbed species takes place and the charge transferred dependent on recorded by the time. The chronocoulometry can therefore be understood as the integral of amperometry. The method of Chronocoulometry is e.g. in Steel, A.B., Herne, T.M. and Tarlov M.J .: Electrochemical Quantitation of DNA Immobilized on Gold, Analytical Chemistry, 1998, Vol. 70, 4670-4677 and references cited therein.

at The differential pulse voltammetry is gradually increasing DC voltage ramp Rectangular pulses with small, constant amplitude superimposed. The measurements of the current as a function of the voltage take place immediately before the pulse and at the end of the pulse. Be applied the differences of the current measuring value pairs against the potential.

at The square wave voltammetry is gradually increasing DC voltage ramp a rectangular AC voltage with small, superimposed on constant amplitude and low frequency. To measure the Current as a function of voltage are each several times the differences the streams determined at maximum and minimum square-wave voltage.

at (chrono) amperometric measurements are made by recording a Current / time curve. Here, the potential of a stationary working electrode e.g. by a potential jump to a potential, at the electrooxidation or reduction of a dissolved or Adsorbed species takes place and the flowing stream in dependence recorded by the time. Amperometric methods are in the WO 00142217 in detail shown.

Short description the drawings

The Invention is intended below with reference to embodiments in connection closer with the drawings explained become. Show it

1 Schematic representation of negative feedback ( 1a ) and positive feedback ( 1b ).

2 Schematic plot of the current at the UME against the quotient of the distance of the UME from the modified surface and the radius of the electrochemically active surface of the UME. 2a ) shows the course with negative feedback and 2 B ) the course with positive feedback.

3 Schematic representation of a measuring electrode used in a method according to the invention.

4 Schematic representation of the method according to the invention for determining the hybridization of ss-DNA to ds-DNA at predefined locations of the chip surface.

5 Results of a SECM measurement of a 20-mer oligonucleotide monolayer (5 mmol / l K ₄ Fe (CN) ₆ in 0.1 mol / l phosphate buffer pH 7; Pt microelectrode with diameter of 10 m μm; Scanning speed: 10 μm / s). The different shades of gray represent a current value according to the gray scale shown.

6 Three-dimensional representation of the measured values according to 5 ,

The 1 schematically shows an ultramicroelectrode (UME), which is moved over a surface at a certain working distance in an electrolyte. The potential of the UME is set via a potentiostat so that a dissolved redox-active species (Red / Ox) is converted in a diffusion-controlled manner. If one approaches the UME step by step to the surface and thereby detects the resulting current, two cases can be distinguished.

Is it a non-conductive (inactive) surface ( 1a ), the approximation of the UME by the disturbance of the previously hemispheric diffusion field causes a reduced mass transfer to the UME and thus a decrease in the diffusion limiting current. One speaks of the negative feedback ( 1a ). The 2a ) shows a schematic plot of the UME current versus the quotient of UME distance from the modified surface and radius of the UME electrochemically active surface in the case of negative feedback.

On the other hand, if there is a conductive (active) surface ( 1b ) and reversibly reverses the reaction of the redox-active species (red / ox) reacted at the UME, the diffusion paths of the oxidized and reduced form of the redox-active species become smaller and smaller with decreasing distance and the resulting current consequently increases. We talk about redox recycling or positive feedback ( 1b ). The 2 B ) shows a schematic plot of the UME current versus the quotient of UME distance from the modified surface and radius of the UME electrochemically active surface in the case of a positive feedback.

The 3 shows a schematic representation of a measuring electrode used in a method according to the invention. The electrochemically active surface of the measuring electrode has a radius <100 microns and is located in an insulating sheath (glass capillary), which has a multiple diameter of the electrochemically active surface.

The 4 shows a schematic representation of the method according to the invention for determining the hybridization of ss-DNA to ds-DNA at predefined locations of the chip surface. The redox mediator Fe (CN) ₆ ^{3- / 4-} (represented as Fe ^{2 + / 3 +} ), which is diffusion-limited at the microelectrode, is reduced with high electron-transfer kinetics, so that the local concentration of the reduced mediator in the diffusion field in front of the microelectrode is increased in the time average. A comparatively high current is detected (the dashed line in FIG 4 represents a current increasing in the vertical direction). About with ss-DNA modified sites of the surface is obtained both by Coulomb interactions, as well as by the steric blockage of the electrode surface modulation of the electron-transfer kinetics (in the case shown a slowing of Rereduktion the oxidized redox mediator at the microelectrode Fe (CN) ₆ ^{3- / 4-} ). A reduction of the measuring current is detected. After selective hybridization of the ligate oligonucleotides with the complementary ligand oligonucleotides results in a further blocking of the surface and thus a stronger modulation of the electron transfer rate of the freely diffusing redox mediator.

It a further reduction of the measuring current is detected.

In the 5 the results of a SECM measurement of a 20-mer oligonucleotide monolayer in 5 mmol / l K ₄ Fe (CN) ₆ , 0.1 mol / l phosphate buffer pH 7 are shown. A Pt microelectrode with a diameter of 10 μm and a scanning speed of 10 μm / s was used. The x, y coordinates approached when the surface is scanned with the microelectrode are assigned a current value corresponding to the gray scale scale shown. The hybridized spots can be clearly recognized by the significantly reduced current values.

The 6 shows a three-dimensional representation of the measurements of in 5 presented experiment. The three-dimensional representation illustrates the modulation of the electron transfer rate induced by hybridization of the ss oligonucleotides to ds oligonucleotides.

Ways to execute the invention

to execution In SECM measurements, different modified ligate nucleic acid oligomers were different Sequence with the immobilization techniques described above a carrier bound. With the arrangement of the ligate nucleic acid oligomers of known sequence at defined positions of the surface, a DNA array the hybridization event of any ligand nucleic acid oligomer or a (fragmented) ligand DNA, e.g. mutations in the ligand oligonucleotide and sequence specific evidence. These are on a surface the Surface atoms or molecules a defined area (a test site) with DNA / RNA / PNA nucleic acid oligomers known, but any sequence, as described above, linked. When Ligate nucleic acid oligomers and / or Ligand nucleic acid oligomers Nucleic acid oligomers (e.g., DNA, RNA or PNA fragments) of base length 3 to 70, preferably Length 8 used to 50, particularly preferably the length 10 to 30.

Becomes the surface thus provided with immobilized ligate oligonucleotides with one (as concentrated as possible) Test solution contacted with ligand oligonucleotide (s), it just happens in the case of hybridization, in which the solution contains ligand-nucleic acid oligomer strands, the to the surface bound ligate nucleic acid oligomers complementary or at least in a wide range are complementary.

To hybridization between ligate oligonucleotide and ligand oligonucleotide is measured in a SECM measurement (e.g., an amperometric measurement) the number of hybridization events in the different spatial Determined areas of the DNA chip. By comparing the obtained Values with the known reference values can be the degree of association for the individual test sites.

Example 1: Representation of amino- and thiol-modified oligonucleotides for anchoring on gold as ligate oligonucleotides

The synthesis of the oligonucleotides is carried out in an automatic oligonucleotide synthesizer (Expedite 8909, ABI 384 DNA / RNA synthesizer) according to the manufacturer's recommended synthesis protocols for a 1.0 μmol synthesis. Modifications to the 5 'position of the oligonucleotides are made with a 5 minute extended coupling step. The amino modifier C2 dT (Glen Research 10-1037) is incorporated into the sequences with the respective standard protocol. The preparation of 3'-thiol-modified ligate oligonucleotides (or HO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ OPO ₃ oligonucleotides) is carried out on 1-O-dimethoxytrityl-propyl disulfide-CPG carrier (Glen Research 20-2933) analogous to standard protocols, wherein the oxidation steps are carried out with a 0.02 mol / l iodine solution in order to avoid an oxidative cleavage of the disulfide bridge. The coupling efficiencies are determined photometrically or conductometrically online during the synthesis via the DMT cation concentration. The oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h. The purification of the oligonucleotides is carried out by means of RP-HPL chromatography according to standard protocols (mobile phase: 0.1 mol / l triethylammonium acetate buffer, acetonitrile), the characterization by means of MALDI-TOF MS.

Example 2: Preparation of the oligonucleotide electrode Au-S (CH ₂ ) ₂ -ss-oligo

The preparation of Au-S (CH ₂ ) ₂ -ss-oligo is subdivided into 2 sections, namely the preparation of the conductive surface and the derivatization of the surface with the ligate oligonucleotide in the presence of a suitable monofunctional linker (incubation step).

The support material for the covalent attachment of the double-stranded oligonucleotides forms an approximately 100 nm thin gold film on mica (muscovite platelets). For this purpose, freshly split mica is cleaned in an electric discharge chamber with an argon ion plasma and applied by electrical discharge gold (99.99%) in a layer thickness of about 100 nm. Subsequently, the gold film is freed from surface contamination with 30% H ₂ O ₂ /70% H ₂ SO ₄ (oxidation of organic deposits) and immersed in ethanol for about 20 minutes in order to displace oxygen adsorbed on the surface. After rinsing the surface with bidistilled water, a previously prepared 1 × 10 ^-4 molar solution of the (modified) oligonucleotide is applied to the horizontally mounted surface so that the complete gold surface is wetted (incubation step, see also below).

For incubation, a modified 20 bp single-stranded oligonucleotide of the sequence 5'-TAG CGG ATA ACA CAG TCA CC-3 'is used, which is attached to the phosphate group of the 3' end with (HO- (CH ₂ ) ₂ -S) ₂ PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH (see Example 1) is esterified. To a 5 × 10 ^-5 molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5 with 0.7 molar addition of TEATFB), an approximately 10 ^-5 to 10 ^-1 molar propanethiol solution (or another thiol or disulfide of appropriate chain length), completely wet the gold surface of a test site and incubate for 2 to 24 hours. During this reaction time, the disulphide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is homolytically cleaved. The spacer with Au atoms of the surface forms a covalent Au-S bond, resulting in a 1: 1 coadsorption of the ss oligonucleotide and the split-off 2-hydroxy-mercaptoethanol. The simultaneously present in the incubation solution, free propanethiol is also coadsorbed by formation of an Au-S bond (incubation step). Instead of the single-stranded oligonucleotide, this single strand may also be hybridized with its unmodified complementary strand.

Example 3: Alternative preparation of the oligonucleotide electrode Au-S (CH ₂ ) ₂ -ss-oligo

The alternative preparation of Au-S (CH ₂ ) ₂ -ss-oligo is subdivided into 3 sections, namely the representation of the conductive surface, the derivatization of the surface with the ligate oligonucleotide (incubation step) and the subsequent deposition of the thus modified electrode with a suitable monofunctional linker (Nachbelegungsschritt). The support material for the covalent attachment of the ligate oligonucleotides forms an approximately 100 nm thin gold film on mica (muscovite platelets), cf. Ex. 2.

For incubation, a modified 20 bp single-stranded oligonucleotide of the sequence 5'-TAG CGG ATA ACA CAG TCA CC-3 'is used, which is attached to the phosphate group of the 3' end with (HO- (CH ₂ ) ₂ -S) ₂ to the PO - (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH esterified. The gold surface of a test site is wetted with an approximately 5 × 10 ^-5 molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5) and incubated for 2-24 h. During this reaction time, the disulphide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is homolytically cleaved. In this case, the spacer with Au atoms of the surface forms a covalent Au-S bond, as a result of which a coadsorption of the ss oligonucleotide and of the cleaved 2-hydroxy-mercaptoethanol occurs (incubation step).

Subsequently, the thus modified gold electrode is completely wetted with a ca. 10 ^-5 to 10 ^-1 molar propanethiol solution (in water or buffer, pH 7 - 7.5) or with another thiol or disulfide (suitable chain length) and 2 - Incubated for 24 hours. The free propanethiol occupies the remaining free gold surface after the incubation step by forming an Au-S bond.

Claims (28)

Method for detecting nucleic acid oligomer hybridization events comprising the steps a) providing a modified Surface, wherein the modification in the attachment of at least one kind consists of ligate oligonucleotides, b) providing a Sample with ligand oligonucleotides, c) contacting one solution with the modified surface, being the solution contains at least one type of redox-active species, d) Contacting the sample with the modified surface, e) Detection of reduction and / or oxidation of the redox-active species by a suitable electrochemical process using a Measuring electrode whose distance from the modified surface maximum 5 times as big like the radius of their electrochemically active surface, f) Comparison of the values obtained in step e) with reference values. The method of claim 1, wherein after step c) and before step d), step c ₁ ) detection of reduction and / or oxidation of the redox-active species by a suitable electrochemical method using a measuring electrode whose distance from the modified surface at most 5 times is large as the radius of their electrochemically active surface is performed and in step f) the values obtained in step e) are compared with the reference values obtained in step c ₁ ). Method according to claim 1 or 2, wherein the distance the measuring electrode of the modified surface at most 4 times, preferably a maximum of 3 times as big like the radius of their electrochemically active surface. The method of claim 3, wherein the distance of Measuring electrode of the modified surface at most 2 times, preferably is just as big like her radius. Method according to one of the preceding claims, wherein a measuring electrode with a radius of the electrochemically active surface from 0.05 to 250 μm, preferably from 0.5 to 50 μm, more preferably from 5 to 25 microns is used. Method according to one of the preceding claims, wherein a disc electrode is used as the measuring electrode, which of an insulating jacket of glass or polymers is surrounded. Method according to one of the preceding claims, wherein as a detection method, a method selected from the group consisting from cyclic voltammetry, chronocoulometry, differential pulse voltammetry, Square wave voltammetry or in particular amperometry used becomes. Method according to one of the preceding claims, wherein as redox-active species one or more electrically charged compounds be used, in particular negatively charged transition metal complexes, especially preferably Cu, Fe, Ru, Os, Ni and Co transition metal complexes. Method according to one of the preceding claims, wherein the modified surface is a conductive surface. The method of claim 9, wherein the conductive surface is made of a metal, a metal alloy, a semiconductor, a binary compound the elements of groups 14 and 16, a binary compound of the elements of groups 13 and 15, a binary one Connection of elements of groups 15 and 16, a binary compound the elements of groups 11 and 17, a ternary compound of the elements of groups 11, 13 and 16 or a ternary compound elements of the Groups 12, 13 and 16 exists. Method according to one of the preceding claims, wherein as a modified surface an array of microelectrodes is used and in the range of one Microelectrode each have a kind of ligate oligonucleotides to the surface is connected: Method according to one of claims 1 to 8, wherein it is at the modified surface a non-conductive one surface is. The method of claim 12, wherein the non-conductive surface is made of Glass, modified glass or a polymer material. Method according to one of the preceding claims, wherein the attachment of the ligate oligonucleotides the surface covalently or by chemisorption or physisorption. Method according to one of claims 1 to 13, wherein the surface branched or unbranched moieties of any composition and chain length covalently or by chemisorption or physisorption are attached and the ligate oligonucleotides covalently attached to these moieties are. Method according to one of the preceding claims, wherein in step a) one by attachment of at least two different ones Species of ligate oligonucleotides modified surface provided becomes. The method of claim 16, wherein each predominantly a type of ligate oligonucleotides in a spatially limited range of modified surface is connected. The method of claim 16, wherein each only one Type of ligate oligonucleotides in a limited range of modified surface is connected. Method according to one of the preceding claims, wherein as step a), the step a) providing a modified surface, wherein the modification in the attachment of at least two types of ligate oligonucleotides and the different types of ligate oligonucleotides in spatially substantially separated areas are bound to the surface is carried out, b after step) and d before step) of step b ₁₎ addition of a ligand oligonucleotide to the sample, wherein the ligand oligonucleotide a binding partner having a high association constant of within a certain range T ₁₀₀ to is the surface-bound ligate oligonucleotide, wherein the ligand oligonucleotide is added in an amount greater than the amount of ligand oligonucleotide necessary to completely associate the ligate oligonucleotides of the T ₁₀₀ test sites is performed and in step f) the values obtained in step e) are compared with the value obtained for the range T ₁₀₀ . The method of claim 19, wherein as step a) the step a) providing a modified surface, wherein the modification consists in the attachment of at least three types of ligate oligonucleotides and the different types of ligate oligonucleotides in spatially substantially separated regions to the Surface are bound, wherein at least one kind of ligate oligonucleotide in a certain range T _{0 is} attached to the surface, and wherein in the sample no binding partner with high association constant to this ligate oligonucleotide is carried out is performed and in step f) in Step e) are compared with the value obtained for the region T ₁₀₀ and with the value obtained for the region T ₀ . The method of claim 19 or 20, wherein prior to step d), step b ₂ ) addition of at least one further type of ligand oligonucleotide to the sample, wherein the ligand oligonucleotide is not included in the sample provided in step b) and the ligand Oligonucleotide has an association constant> 0 to a Ligat oligonucleotide bound to the surface in a certain range T _n , wherein the ligand oligonucleotide is added in an amount such that after step d) n% of the ligate oligonucleotides in the range T _n in an associated form, and in step f) the values obtained in step e) are compared with the value obtained for the range T ₁₀₀ , the value obtained for the range T ₀ and the values obtained for the ranges T _n . Method according to one of the preceding claims, wherein the ligand oligonucleotides and / or the ligate oligonucleotides 3 to 70 bases, especially 8 to 50 bases, more preferably 10 to 30 bases. Method according to one of the preceding claims, wherein as ligate oligonucleotides substantially uncharged ligate oligonucleotides be used. The method of claim 23, wherein as substantially uncharged ligate oligonucleotides used ligate oligonucleotides which carry a number of elementary charges, the maximum 40% of the number of nucleotides of the ligate oligonucleotide is preferred maximum 30%. The method of claim 24, wherein as substantially uncharged ligate oligonucleotides used ligate oligonucleotides which carry a number of elementary charges, the maximum 20% of the number of nucleotides of the ligate oligonucleotide is preferred maximum 10%. Method according to one of the preceding claims, wherein used as ligate oligonucleotides uncharged ligate oligonucleotides become. Method according to one of claims 23 to 26, wherein as in the essentially uncharged ligate oligonucleotides or as uncharged ligate oligonucleotides oligonucleotides selected from the group consisting of methyl phosphonate oligonucleotides, methyl phosphate oligonucleotides, phosphoamidate oligonucleotides, peptide oligonucleotides and mixtures thereof are used. Kit to carry out a method according to any one of the preceding claims a modified surface, wherein the modification in the attachment of at least one kind of Ligat oligonucleotide, and an effective amount of a redox-active species.