DE10227042B4 - Method for detecting, quantifying and / or characterizing an analyte - Google Patents

Abstract

Method for detecting, quantifying and / or characterizing an analyte (10) contained in a first fluid, comprising the following steps:
a) contacting and incubating the analyte (10) each having a first (20) and second probe (22) having an affinity for the analyte (10), wherein the affinity of the first probe (20) is determined by a specific affinity to at least a first Binding site (12) of the analyte (10) is effected and the incubation is carried out under conditions under which the first (20) and the second probe (22) bind to the analyte (10),
b) marking the first probe (20) by at least one electrochemically specifically detectable first marker (24), at least if it is not already detectable specifically electrochemically,
C) marking the second probe (22) by at least one electrochemically detectable second marker (26), at least if it is not already specifically detectable electrochemically,
d) separating the first (20) and second probe (22) bound to the analyte (10), ...

Description

The The invention relates to a method for detecting, quantifying and / or characterizing an analyte in a liquid.

With electrochemical processes can redox-active analytes are investigated and specified. The implementation the analyte takes place at a working electrode. To the powerless Measuring the voltage, a reference electrode is used. The over the Working electrode flowing Current or the voltage which is between the working and the reference electrode drops will over controlled a counter electrode. The electrochemical detection of Analytes can be potentiometric or amperometric. In a potentiometric measurement protocol is the over the working and reference electrode decreasing voltage measured time-dependent. While This measurement can be a controlled current waveform applied. In the case of a constant current, this measurement is called constant current chronopotentiometry designated. The study of adsorbed on a working electrode or complexed analytes using constant current chronopotentiometry is also called constant current potentiometric stripping analysis (KSPSA). The cathodic constant current potentiometric stripping analysis includes a method in which by applying a positive Stromes successive analytes are oxidized. From these voltage-dependent oxidation reactions can Conclusions on be pulled oxidizable analytes. The Anodic Constant Current Potentiometric Stripping Analysis includes a method in which a negative current is applied, to draw conclusions about reducible analytes from the obtained voltage curve shut down to be able to.

In an amperometric measurement protocol is the voltage which between the reference and the working electrode drops, via a counter electrode after changed according to a given protocol. At the same time, the current flowing across the working electrode is measured. Depending on applied voltage can redox-active analytes are reduced or oxidized. By an evaluation the course of the current can draw conclusions the analytes are pulled. An electrochemical measuring method with a stationary working electrode, in which a current-voltage characteristic is recorded is also as voltammetry and the associated graphic representation referred to as a voltammogram. For very sensitive measurements of redox processes became special measurement protocols developed, which takes place in addition to the redox processes the Measurement influencing capacitive processes are largely suppressed. A very efficient measurement protocol is Differential Pulse Voltammetry (DPV).

From the US 6,100,045 For example, a method for detecting an analyte is known. The analyte is labeled with a redox-active substance and bound to a solid phase. The solid phase may be magnetic microparticles. The solid phase is located near an electrode. By means of the redox-active label, the binding of the analyte to the solid phase is detected as an amperometric signal via the electrode. Above all, the known method can provide information about the presence of the analyte, but hardly about its properties. Furthermore, it is not very sensitive. For its implementation, a relatively complicated device is required.

From the US 5,871,918 It is known to hybridize an analyte, eg a DNA, with a capture probe bound to an electrode. After contacting the analyte with a reporter probe, the electrochemical detection of the hybridization takes place. For this purpose, the bases of a reporter probe after binding to the target DNA are reacted with transition metal complexes to electrochemically detectable markers. In this case, bases of the target DNA are converted and generate an electrochemical signal upon detection. This limits the detection sensitivity of this method.

From the US 6,346,387 B1 is just as a method as from the US 5,871,918 known.

Out Authier et al., (2001), Anal. Chem. 73, pages 4450-4456 it is known, a nucleic acid as a target DNA nonspecifically on an inner surface of a To bind vessel. The target DNA is labeled with a gold particle labeled oligonucleotide hybridized as a reporter probe. The reporter probe is electrochemically used detected by single-use microband electrodes. This method has numerous disadvantages. The use of a Vessel to Binding the target DNA makes it impossible to concentrate the target DNA in a small volume. That limits the Detection sensitivity. Furthermore, the non-specific binding of substances, in particular the reporter probe, to the inner surface of the Vessel to one high background signal and an interaction with the electrochemical Lead detection.

Out Marrazza G. et al., Clin. Chem. (2000), Vol. 46 (1), pages 31 to Figure 37 is a method for the electrochemical detection of PCR products known. This is a immobilized on an electrode capture DNA with PCR-amplified DNR under hybridization conditions in contact brought. The hybridization reaction at the electrode surface becomes using chronopotentiometric stripping analysis using followed by daunomycin as an indicator.

Out Sawada, K. et al., Analytical Biochemistry (2000), Vol. 286, p 59 to 66 is a method for detecting triplet expansion known. The method is based on the detection of triplet expansion in the patient genome by hybridization with a digoxigenin-labeled Probe. The probe will do this for used a Southern hybridization. Tied probe is going through Binding of an alkaline phosphatase labeled anti-digoxigenin Fab fragment and then Detection of alkaline phosphatase by a chemiluminescent reaction detected. The number of triplet repetitions will depend on the intensity of the process generated signal due to a linear relationship between the intensity and the number of triplet repeats.

From the US 5,695,933A For example, another method for detecting extensive nucleotide repeat in genomic DNR is known. In this case, isolated genomic DNA is brought into contact with oligonucleotides that are ligatable with one another and complementary to the nucleotide repeat, so that hybridization takes place. The hybridized oligonucleotides are ligated together to form a multimer of the hybridized oligonucleotides. The genomic DNA is separated from the hybridized multimer by denaturation. Subsequently, the hybridization and ligation is repeated until an adequate amount of multimers has been generated for detection.

Out WO 93/20230 is a polarographic electrochemical process known for detecting DNA hybridization. In this process becomes an electrode first with single-stranded DNA contacted and a polarographic signal for this single DNA measured. Subsequently For example, an oligonucleotide probe is subjected to hybridization conditions added, excess reagent removed and a polarographic signal detected. By comparison the two detected signals can be determined if it is too a hybridization has come. With the procedure can only little information about the Properties of the examined DNA can be obtained.

task The invention is to the disadvantages of the prior art remove. In particular, it should be as quick and easy as possible feasible High sensitivity process for detecting, quantifying and / or characterizing an analyte be specified. The characterization may be e.g. in view on the length or size of the analyte respectively.

These The object is solved by the features of claims 1 and 2. Advantageous embodiments emerge from the features of claims 3 to 33.

• a) Inkontaktbringen und Inkubieren des Analyten mit jeweils einer eine Affinität zu dem Analyten aufweisenden ersten und zweiten Sonde, wobei die Affinität der erste Sonde durch eine spezifische Affinität zu mindestens einer ersten Bindungsstelle des Analyten bewirkt wird und das Inkubieren unter Bedingungen erfolgt, unter denen die erste und die zweite Sonde an den Analyten binden,
• b) Markieren der ersten Sonde durch mindestens einen elektrochemisch spezifisch nachweisbaren ersten Marker, zu mindest wenn sie nicht bereits elektrochemisch spezifisch nachweisbar ist,
• c) Markieren der zweiten Sonde durch mindestens einen elektrochemisch spezifisch nachweisbaren zweiten Marker, zumindest wenn sie nicht bereits elektrochemisch spezifisch nachweisbar ist,
• d) Absondern der an den Analyten gebundenen ersten und zweiten Sonde,
• e) Detektion eines durch die abgesonderte erste Sonde oder den ersten Marker verursachten ersten elektrochemischen Signals und eines durch die abgesonderte zweite Sonde oder den zweiten Marker verursachten zweiten elektrochemischen Signals und
• f) Nachweisen, Quantifizieren und/oder Charakterisieren des Analyten mittels eines Verhältnisses zwischen dem ersten und dem zweiten Signal.

According to the invention, a method is provided for detecting, quantifying and / or characterizing an analyte contained in a first fluid, comprising the following steps:

• a) contacting and incubating the analyte, each with a first and second probe having an affinity for the analyte, wherein the affinity of the first probe is caused by a specific affinity for at least a first binding site of the analyte and the incubation is under conditions, among which bind the first and the second probe to the analyte,
• b) marking the first probe by at least one electrochemically specifically detectable first marker, at least if it is not already detectable specifically electrochemically,
• c) marking the second probe by at least one electrochemically detectable second marker, at least if it is not already specifically detectable electrochemically,
• d) separating the first and second probes bound to the analyte,
• e) detecting a first electrochemical signal caused by the isolated first probe or the first marker and a second electrochemical signal caused by the second probe or the second marker
• f) detecting, quantifying and / or characterizing the analyte using a ratio between the first and second signals.

• a) Inkontaktbringen und Inkubieren des Analyten mit jeweils einer eine Affinität zu dem Analyten aufweisenden ersten Sonde, wobei die Affinität der erste Sonde durch eine spezifische Affinität zu mindestens einer ersten Bindungsstelle des Analyten bewirkt wird und das Inkubieren unter Bedingungen erfolgt, unter denen die erste Sonde an den Analyten bindet,
• b) Markieren der ersten Sonde durch mindestens einen elektrochemisch spezifisch nachweisbaren ersten Marker, zu mindest wenn sie nicht bereits elektrochemisch spezifisch nachweisbar ist,
• c) Markieren des Analyten durch mindestens einen elektrochemisch spezifisch nachweisbaren zweiten Marker, zumindest wenn der Analyt nicht bereits elektrochemisch spezifisch nachweisbar ist,
• d) Absondern des von der ersten Sonde gebundenen Analyten und der von dem Analyten gebundenen ersten Sonde,
• e) Detektion eines durch die abgesonderte erste Sonde oder den ersten Marker verursachten ersten elektrochemischen Signals und eines durch den abgesonderten Analyten oder den zweiten Marker verursachten zweiten elektrochemischen Signals und
• f) Nachweisen, Quantifizieren und/oder Charakterisieren des Analyten mittels eines Verhältnisses zwischen dem ersten und dem zweiten Signal.

Furthermore, a method for detecting, quantifying and / or characterizing an analyte contained in a first fluid is provided with the following steps:

• a) contacting and incubating the analyte, each with a first probe having an affinity for the analyte, the affinity of the first probe being effected by a specific affinity for at least a first binding site of the analyte, and incubating under conditions where the first probe binds to the analyte,
• b) marking the first probe by at least one electrochemically specifically detectable first marker, at least if it is not already detectable specifically electrochemically,
• c) labeling of the analyte by at least one electrochemically detectable second marker, at least if the analyte is not already detectable specifically electrochemically,
• d) separating the analyte bound by the first probe and the first probe bound by the analyte,
• e) detecting a first electrochemical signal caused by the secreted first probe or the first marker and a second electrochemical signal caused by the secreted analyte or second marker and
• f) detecting, quantifying and / or characterizing the analyte using a ratio between the first and second signals.

Of the The term "analyte" includes in particular single or double stranded nucleic acids, Proteins and other biomolecules. The first or second probe may be monoclonal or monoclonal polyclonal antibodies, Antibody fragments, Receptors, nucleic acids or ligands act. Under "nucleic acids" in the sense of this In addition to DNA and RNA, the invention also includes nucleic acid analogs such as peptide nucleic acids (PNA) and Hybrid and chimera from DNA and RNA and analogues of nucleic acids.

"Specific" in terms of the electrochemical detectability means that the first or second probe, the analyte, or the first or second marker, respectively electrochemically, i. by a redox reaction, separated from each other and optionally other, e.g. more in the first fluid contained, substances can be detected. That can be done by a specific Signal possible or that the signal is different from others Signals, in particular background or interference signals to be differentiated can. This is e.g. by a differential detachment of the first probe and / or purification processes possible.

To the specific evidence can the first and / or second probe and / or the analyte in each case at least one electrochemically detectable marker or a be marked electrochemically detectable marker group. By Labeling with multiple markers or marker groups can be significantly more intense Signals are generated as having an electrochemically self-detectable Analyte or an electrochemically self-detectable probe. The sensitivity and the specificity of the method thereby increased become. Even if the analyte or probe without label electrochemical are detectable, such as Nucleic acids which are a purine base, like guanine or adenine, they can be labeled. The Marking can be done by having an electrochemically detectable Marker over an affinity molecule, e.g. Avidin, on one of the analyte or the first or the second Probe arranged thereon corresponding affinity molecule, e.g. Biotin binds. By an affinity molecule is meant a molecule which has a specific high affinity to a corresponding one having another affinity molecule. The marking steps lit. b and lit. c have to lit before step. e, but can in each case before or after step lit. a or lit. d be performed.

The Binding of the second probe may be specific or nonspecific. One from nucleic acid Existing analytes are preferred under a specific binding understood a sequence-specific hybridization. It may be It also involves a sequence-specific binding of a protein. The Binding one from nucleic acid existing first or second probe to a nucleic acid consisting of Analytes can also be over a Hoogsteen base pair, which forms a triplex structure, respectively. This is preferably done with a binding of PNA one from double-stranded DNA existing analytes. As a second probe, e.g. a sequence independent the analyte-binding nucleic acid indicator, like a DNA intercalator.

By separating is meant a substantial separation of the bound from the unbound first or second probes or the bound from the unbound analyte. It can be done, for example, by degrading unbound first or second probes or the unbound analyte or by centrifuging, binding or washing. The separation of the bound from the unbound first probe and the analyte bound from the first probe from the unbound analyte can also be done in one step, eg by centrifuging or binding the formed complex of analyte and first probe and then washing. During washing, the complex is taken up in another liquid. The Detection of the first and second signals may be simultaneous or sequential and with one and the same or different electrochemical methods. The detection optionally also comprises a quantification of the signals. This can also include a mathematical operation, in particular an integration.

The Method is particularly well suited to pass a nucleic acid through her length to characterize. If the second signal, for example, by causes a DNA intercalator as the second probe and has the DNA a specific binding site for a first probe, so gives the relationship the first signal to the second signal information about the length of the nucleic acid. Farther can the length of nucleic acids with yourself often repeating sequences, for example, be determined by the first probe specific to one occurring only once in the molecule Sequence binds during the second probe specific to the repeating sequences binds. The first signal can be for the second signal represent an internal standard which, in particular if an expected first signal is known, a differentiation a specific portion of a non-specific portion of the second Signals allowed. This results in a high sensitivity or specificity of the method reached. Unspecific second signals may e.g. by not completely separate unbound second probes are caused.

Preferably For example, an electrode used for detection will not match the first one liquid brought into contact. This can reduce the sensitivity of the process clearly increased because in the first fluid, such as. Blood serum, substances contained by non-specific bind to the electrode and nonspecific signals during detection can generate. Unspecific background signals can thus be largely avoided become. This can be achieved, for example, by the analyte before step lit. e separated from the first liquid and in a second liquid is transferred. Preferably, the analyte prior to performing step lit. a from the first liquid separated and transferred to a second liquid. Thereby can in the first liquid contained substances that are attached to the first or second probe bind, break down or in other ways disrupt their function. Farther can Separate substances that interfere with the detection. The can e.g. Be a similar substance generate electrochemical signal as the first or second probe. It can also be substances that have the function of electrochemical detection used electrodes interfere. For separation, the analyte, in particular specific, of a Catcher molecule bound become.

The Binding of the analyte to the scavenger molecule can be achieved by a, preferably sequence-specific, hybridization done. The catcher molecule can, in particular before the binding of the analyte, at a first or second surface be immobilized. This will separate the analyte from the first liquid simplified. By immobilizing the capture molecules, the analyte can in one small volume to be enriched. This increases the detection sensitivity of the procedure.

The Catcher molecule can be one Nucleic acid, an analog of a nucleic acid, in particular a peptide nucleic acid (PNA), an antibody or be a receptor. Preferably, the capture molecule has an affinity molecule, in particular Streptavidin, avidin or biotin, or a biotinylated oligonucleotide on. This affinity molecule can either serve the catcher molecule at the first or second surface to immobilize or to bind the analyte.

In A preferred embodiment includes the first or second probe or the analyte is an affinity molecule, in particular Biotin, avidin or streptavidin. The affinity molecules are to be chosen such that it can not come to bindings, which the execution of the inventive method prevent. In particular, it should not be about the Affini tätsmoleküle to bindings between the first and the second probe or to bindings of the first probe to the analyte. This makes it easy on Way possible, the analyte from the first fluid separate. Furthermore possible the affinity molecule binds a marker on the first or second probe or analyte or immobilizing the analyte. The analyte. can a, in particular a poly-T-end or a poly A-end having nucleic acid. The nucleic acid can be double or single stranded be educated. Also to a double-stranded nucleic acid a sequence-specific binding, e.g. by a Hoogsteen binding one out PNA existing first or second probe possible. The poly-T-end or allows the poly A end the binding of the analyte to a poly A sequence or a poly T sequence having a catcher molecule serving Nucleic acid. This allows the analyte to be easily separated from the liquid. Furthermore possible a poly-T end is a label with several osmium complexes. In In a particular embodiment of the method, the characterization takes place by determining the length the nucleic acid.

The analyte is preferably before or during step lit. a by means of a Nukleinsäurevielvielfältigungsre action, in particular a PCR, duplicated. This significantly increases the sensitivity of the procedure. The analyte is then essentially the product of the amplification reaction is detected, quantified and / or characterized. This also applies if the product does not completely conform to the analyte because of the primers used for the amplification reaction or if it is a product of the amplification reaction which is complementary to the analyte. In the case of PCR, the primer may be the capture molecule or the first or second probe. By PCR, specific binding sequences can be introduced into the analyte.

Of the Analyte may do so before the step lit. d, especially before the step lit. a are immobilized on the first or second surface. The can also over the binding to the scavenger molecule take place. This facilitates the separation from the first liquid and the separation according to lit. d, then, e.g. can be done by washing the first surface. The first surface can the surface one, especially superparamagnetic, particle. A superparamagnetic Particles can be easily separated by means of a magnetic field become. The particle can otherwise be separated by centrifugation become. Allows a concentration of the analyte. Furthermore, it is due to particles possible, a big binding surface provide. The particle preferably has a diameter from 10 nm to 100 μm, in particular 1 to 10 μm, on. This particle size makes it possible the particles in the liquid to suspend and thereby intensive contact of the analyte to produce with the first and possibly second probe.

In a preferred embodiment the analyte is going to be carried out of the step lit. a is immobilized on the first surface by means of a scavenger molecule. The catcher molecule can do this immobilized on the first surface before or after binding to the analyte become. Subsequently a washing step takes place in which the first liquid removed and replaced with a second liquid. The second liquid can be chosen this way be that optimal conditions for tying the first or the first and the second probe at step lit. a are given. Step lit. d is preferably carried out by the immobilized analyte with attached first or first and second probe for removal the unbound first or first and second probe washed becomes. In this case, the second liquid through a third liquid be replaced. The third liquid is preferably chosen so that the detection according to lit. e can be carried out under optimal conditions.

Preferably is the second surface a serving for detection, in particular an electrically conductive plastic or an electrically conductive Polymer, mercury, amalgam, gold, platinum, carbon or indium tin oxide containing electrode.

Prefers the second probe has a specific affinity for at least one specific second binding site of the analyte. From the relationship between The first and the second signal can then be information about the relationship gain the number of first to the number of second binding sites. Preferably the analyte has a known number of first binding sites and an unknown number of second binding sites. Characterizing can then determine the unknown number of second binding put through the relationship from bound first to bound second probe or the ratio of the first signal to the second signal. The signal ratio or the number of second binding sites can be determined with the length be correlated to the analyte. The analyte can be a DNA fragment which is due to a triplet expansion disease, such as fragile X syndrome, Huntington's disease, bulbar muscle atrophy, spinocerebellar ataxia type I, myotonic dystrophy or the Friedreich Ataxia, has repetitive sequences: Such Diseases are usually over one Polymerase chain reaction detected with subsequent Southern blot. In relation to to the method according to the invention, in which the repetitive sequences serve as second binding sites can, that is very expensive and takes a long time.

In a preferred embodiment, the first probe of the analyte and / or the analyte of the first or second surface between step lit, d and step lit. e, in particular by heat denaturation, chemical denaturation, enzymatic digestion or chemical degradation, released. The release from the surface allows concentrating the analyte or the first probe in a very small volume of liquid, thereby increasing the sensitivity of the method. It also allows close contact of the first probe or analyte with the electrode used for detection. Furthermore, it is thus possible to perform the detection without the electrode coming into contact with the first surface. As a result, possible disturbances of the electrochemical detection by the first surface can be avoided. It is also possible to use materials as the first surface which would normally disturb the electrochemical detection. For example, streptavidin-coated particles can be used as the first surface and cysteine-labeled PNA as the first probe, without the use of electrochemistry chemical detection of cysteine labeling by the cysteine residues of streptavidin disorders may occur. Furthermore, by releasing the first probe from the analyte, it is possible to detect the first and second signals separately. Thus, the implementation of the method is also possible if the first and the second signal are identical, for example because it is caused by identical markers.

It is possible, too, the first and second probes from the analyte or the first probe from the analyte and the analyte from the first or second surface, respectively release separately and then detect. Also This differentiates between identical signals the causative molecule possible. Furthermore, a concentration in a small volume of liquid is possible.

alternative it is also possible the first marker and / or the second marker, in particular each separately from each other, from the first and / or second probe or analyte release and then detect. The release can be through enzymatic digestion or chemical degradation. For example could the first and / or the second marker in each case via a nucleic acid sequence with a specific restriction site to the first or second probe or analyte. A specific release is then possible by restriction enzymes.

The first and / or second probe may be a nucleic acid or an analog of a Nucleic acid, in particular a peptide nucleic acid (PNA), his. Preferably, the first and / or second probe bind sequence specific, in particular by hybridization, to the analyte. The probe can In this case, for example, be a nucleic acid which is complementary to a Sequence of the analyte is. The probe may also be around an antibody which is a particular amino acid sequence in one of a Protein detects existing analytes.

The first or second signal can each be from one through the first or second markers caused catalytic water release caused. Preferably, the first and / or the second marker is reversible reducible or oxidizable. The first or the second marker can an osmium complex, a nanogold particle, a cysteine, ferrocenyl, daunomycin, Benzoquinone, naphthoquinone, anthraquinone or p-aminophenol group or a dye, in particular indophenol, thiazine or phenazine, exhibit.

Prefers the first or the second probe is marked by several markers. This makes it possible to get one Probe for different electrochemically different detectable analytes to use. Preferably, the first or second probe has a linear primary structure on, at one end of the marker is arranged. By the terminal arrangement the marker becomes a danger of influencing the interaction minimized between the first or the second probe with the analyte.

The Detection of the first and second signals may be at the same electrode and / or by the same electrochemical detection methods. The detection is preferably carried out by means of cathodic stripping voltammetry (CSV), square wave voltammetry, cyclic voltammetry or Chronopotentiometry. The detection can be done by means of a reversible Redox process or a catalytic hydrogen evolution take place.

following The invention will be explained in more detail with reference to embodiments. It demonstrate:

1 a schematic representation of the labeling of a first probe by osmium tetroxide and 2,2'-bipyridine,

2a . b DPV voltammograms to determine the detection limit,

3a . b . c schematic representation of the detection of an analyte consisting of nucleic acid by hybridization with an osmium-labeled first probe,

4 DPV voltammogram for detecting the analyte consisting of nucleic acid by hybridization with an osmium-labeled first probe,

5a . b Voltammograms of a Chronopotentiometric Stripping Analysis (CPSA) for Bestim the sensitivity of cysteine PNA probes,

6a - f schematic representation of a method according to the invention with a first and a second probe,

7a - f schematic representation of a method according to the invention with a sequence-specific first and a sequence-unspecific second probe,

8a - f schematic representation of a method according to the invention, wherein a first probe and the analyte are used to generate electrochemical signals, and

9a - G a schematic representation of the determination of the number of specific for a triplet disease reversible sequences.

To produce an osmium labeled probe, a 97mer with the sequence 5-AAA AAA AAA AAA AAA AAA AAA ATT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT C 3 (SEQ ID NO: 1) from the attached Sequence Listing with 2 mmol / l OsO ₄ , 2 mmol / l 2,2'-bipyridine in 100 mmol / l Tris-HCl, pH 7.0, for 180 minutes at 37 ° C incubated. Subsequently, free OsO ₄ and bipyridine have been removed by dialysis against deionized water for 5 hours. Schematically, the mark is in 1 shown. An oligonucleotide having a first sequence 9 complementary to a portion of an analyte comprising nucleic acid and a second sequence containing many thymine residues T is treated with osmium tetroxide and 2,2'-bipyridine. The thymine residues form specific osmium-containing complexes Os, which can be detected as electrochemical markers.

To determine the detection limit of the probe thus prepared, 3 .mu.l of a 10, 25, 50, 100, 250 and 500 ng / ml of the osmium-labeled probe in Britton Robinson buffer (0.04 mol / l H ₃ BO ₃ , 0 , 04 mol / l H ₃ PO ₄ and 0.04 mol / l CH ₃ COOH mixed with 0.2 mol / l NaOH), pH 3.9 by means of differential pulse voltammetry (DPV) with a scan rate of 10 mV / s, an amplitude of 50 mV / s, an absorption time of 240 s and a scan time of 120 s to 240 s. The result is in the 2a to be taken from the DPV voltammogram shown. The curves 36 . 38 . 40 . 42 . 44 and 45 correspond respectively to 500, 250, 100, 50, 25 or 10 ng / ml of the osmium labeled probe. 2 B shows a DPV voltammogram generated by the same procedure with 3 μl of a solution containing 250 μg / ml osmium-labeled probe 46 and a no-probe DPV voltammogram 47 , The detection limit of the osmium-labeled probe is therefore below 250 pg / ml. That corresponds to 25 attomol probe.

In 3a is an analyte consisting of nucleic acid 10 with a poly A strand (A) _n at one end, which has one with poly-T (T) _n as a scavenger molecule 8th coated particles is brought into contact. The analyte 10 binds to the particle 18 by hybridization of (A) _n with (T) _n . The particle-bound analyte is labeled with an osmium-labeled first probe 20 brought into contact ( 3b ). The first probe 20 has one to the unhybridized portion of the analyte 10 complementary sequence. Under the chosen conditions, the first probe binds 20 to the analyte 10 , Unbound first probe 20 is removed by washing the particles. Subsequently, the bound first probe 20 from the analyte 10 released by heat treatment ( 3c ). The first probe 20 can be detected electrochemically by its osmium label.

• 1. Analyt: 5'-CTT TT CCT TCT CAA AAA AAA AAA AAA AAA AAA (SEQ ID NO: 2)
• 2. Sonde I mit komplementärer Sequenz zum Analyten: 5'-TTT TTT TTT TGA GAA GGA AAA AG (SEQ ID NO: 3)
• 3. Sonde II ohne komplementäre Sequenz zum Analyten: 5'-TTT TTT TTT TAG AGA AAG GGA AA (SEQ ID NO: 4)

The proof can be carried out, for example, with the following components:

• 1. Analyte: 5'-CTT TT CCT TCT CAA AAA AAA AAA AAA AAA AAA (SEQ ID NO: 2)
• 2. Probe I with complementary sequence to the analyte: 5'-TTT TTT TTT TGA GAA GGA AAA AG (SEQ ID NO: 3)
• 3. Probe II without complementary sequence to the analyte: 5'-TTT TTT TTTAGAGAGAAGGGA AA (SEQ ID NO: 4)

The Thymine residues of the probes I and the probe used for the control II are labeled with osmium tetroxide and 2,2'-bipyridine as described above Service.

To immobilize the analyte, 40 μl of superparamagnetic particles immobilized with oligo-T-25meres from Dynal, Norway are added to 40 μl of a 10 μg / ml of the analyte in 0.1 mol / l NaCl, 0.05 mol / l phosphate buffer, pH 7.4 (incubation buffer) containing solution. The suspension is for 30 Incubated for a few minutes at room temperature. The particles are washed twice in 100 μl of incubation buffer and taken up in a volume of 40 μl.

to Hybridization of the analyte with the probe will be 40 μl of the 400th incubation buffer containing ng / ml of the probe to the particles given and for Incubated for 30 minutes at room temperature. Unbound probes will be by washing four times in 100 μl Incubation buffer removed. The bound probe is heated at 85 ° C released and by separating the supernatant containing the probe apart. For electrochemical detection of osmium-labeled Probe will be 3 μl of the supernatant by DPV with an absorption time of 2 minutes in Britton-Robinson buffer, pH 3.87 analyzed. To determine a non-specific binding the analyte with the probe II in a separate approach under identical conditions were brought into contact and incubated.

This in 4 shown DPV voltammogram shows a clear signal 48 the probe I and a very weak signal 50 the probe used for the control II.

to Determination of sensitivity Chronopotentiometric Stripping Analysis (CSPA) when used of cysteine PNA probes are different concentrations of cysteine PNA (cys-PNA) in 5 mmol / l phosphate buffer, pH 7.0 for absorbed an absorption time of 7 minutes on an electrode Service.

5a Figure 4 shows a DPV plot with the hydrogen peak curves taken in 0.2 M ammonium phosphate buffer, pH 8.5, at -2 μA stripping current 52 . 54 and 56 each 0.375, 0.180 and 0.094 ng / ml cys-PNA. The detection limit of cys-PNA was below 0.09 ng / ml. This corresponds to 36 attomol cys-PNA in 1 μl. 5b shows a DPV diagram of Brdicka's signal curves 58 . 60 and 62 each of 0.750, 0.375 and 0.187 ng / ml cys-PNA in 1 mmol / l Co ³⁺ , 0.1 mol / l ammonium phosphate buffer, pH 9.47. The detection limit of cys-PNA was below 0.19 ng / ml.

6a - f show a schematic representation of a method according to the invention, in which a first 20 and a second probe 22 sequence-specific to the analyte 10 tie.

6a shows an analyte 10 with a singular first binding site 12 and three repetitive second binding sites 14 , The analyte 10 is at a first surface 16 of a particle 18 immobilized ( 6b ). The analyte 10 comes with the first probe 20 and the second probe 22 brought into contact ( 6c ). The first probe 20 has an affinity for the first binding site 12 and the second probe 22 an affinity for the second binding sites 14 on. The first probe 20 is with a first electrochemical marker 24 and the second probe 22 with a second electrochemical marker 26 marked.

Under the chosen conditions, the first probe binds 20 to the first binding site 12 and the second probe 22 to the second binding sites 14 ( 6d ). Unbound probes are removed by washing. The. Probes are released from the analyte and detected with a second electrode surface 27 brought into contact ( 6e ). That through the marker 24 caused signal Si1 and that through the marker 26 caused signal Si2 are measured ( 6f ) and in relation to each other. The ratio of Si 2 to Si 1 corresponds to the ratio of the number of second binding sites 14 to the first binding site 12 ,

7a - f show a schematic representation of the method according to the invention, wherein a first probe 20 sequence specific and a second probe 22 sequence-unspecific to the analyte 10 binds.

7a shows an analyte 10 , which is a singular first binding site 12 having. The analyte 10 is at a first surface 16 of a particle 18 immobilized ( 7b ). The analyte 10 comes with the first probes 20 and second probes 22 brought into contact ( 7c ). The first probe 20 has a binding affinity to the first binding site 12 on. The second probe 22 has a sequence-unspecific affinity for the analyte 10 on. The second probe 22 may be, for example, a DNA intercalator. The first probe 20 is through a first electrochemical marker 24 and the second probe 22 through a second electrochemical marker 26 marked.

Under the chosen conditions bind the first probe 20 and the second probe 22 to the analyte 10 ( 7d ). Unbound first 20 and second probe 22 are removed by washing. Bound first 20 and the second probe 22 are released from the analyte and with a second surface serving as a detection electrode 27 brought into contact ( 7e ).

That through the marker 24 caused first signal Si1 and that through the marker 26 caused second signal Si2 are measured ( 7f ) and in relation to each other. The ratio corresponds to the ratio of the first binding site 12 to the total length of the analyte 10 ,

In 8a is an analyte consisting of nucleic acid 10 with a singular first binding site 12 and a certain number of guanine residues G are shown. The analyte 10 is at a first surface 16 of a particle 18 immobilized ( 8b ). He will come with a first probe 20 , which with an electrochemical marker 24 is labeled and has a specific affinity for the first binding site 12 has been brought into contact ( 8c ). Under the chosen conditions, the first probe binds 20 to the first binding site 12 ( 8d ). Unbound first probe 20 is removed by washing. To the analyte 10 bound first probe 20 and the guanine residues G are from and / or from the analyte 10 , for example, by acid hydrolysis, released and with a second serving as a detection electrode surface 27 brought into contact ( 8e ). The signal Si generated by G and that through the marker 24 generated signal Si2 are measured ( 8f ) and in relation to each other. The ratio of signals Si1 to Si2 corresponds to the numerical ratio of the first binding site 12 to the total number of G in the analyte 10 ,

In 9a is a double-stranded DNA fragment 11 containing an unknown number of triplet disease specific repetitive second binding sites 14 containing sequences and a singular one first binding site 12 having containing sequence. The DNA fragment is coated with a first, at its 5 'end a poly-A-portion pA having primer 28 and a second primer 30 brought into contact. The first 28 and second primer 30 each bind to a DNA strand of the DNA fragment 11 and frame the second binding sites 14 and the first binding site 12 one ( 9b ).

A replication reaction becomes an amplification product 32 generates, which the second binding parts 14 and the first binding site 12 and the poly A moiety at a 5 'end ( 9c ). The amplification product 32 is due to denaturation of its counterpart 33 separated and with a poly-T probe as a capture molecule pT having superparamagnetic particles 18 brought into contact. In this case, the poly-A moiety pA binds specifically to the poly-T probe pT ( 9d ).

That to the particle 18 bound amplification product 32 comes with a first probe 20 which has a specific affinity for the first binding site 12 and a second probe 22 which has a specific affinity to the second binding sites 14 has brought into contact. The first probe 20 is with an electrochemical marker 24 and the second probe 22 with an electrochemical marker 26 marked ( 9e ).

Under the chosen conditions, the first probe binds 20 to the first binding site 12 and the second probe 22 to the second binding sites 14 ( 9f ). The excess of unbound first 20 and second probe 22 is removed by washing.

Bound first 20 and second probes 22 are from the amplification product 32 released and with a detection electrode 27 brought into contact ( 9g ). Through the markers 24 and 26 , caused electrochemical signals are detected separately. The number of repetitive sequences is determined by the ratio of the markers 24 and 26 determined generated signals. The method is thus suitable for detecting and identifying a triplet disease.

SEQUENCE LISTING

Claims (33)

Method for detecting, quantifying and / or characterizing an analyte contained in a first fluid ( 10 ) comprising the following steps: a) contacting and incubating the analyte ( 10 ) each having an affinity for the analyte ( 10 ) first ( 20 ) and second probe ( 22 ), wherein the affinity of the first probe ( 20 ) by a specific affinity for at least a first binding site ( 12 ) of the analyte ( 10 ) and the incubation is carried out under conditions in which the first ( 20 ) and the second probe ( 22 ) to the analyte ( 10 ), b) marking the first probe ( 20 ) by at least one electrochemically detectable first marker ( 24 ), at least if it is not already detectable specifically electrochemically, C) marking the second probe ( 22 ) by at least one electrochemically detectable second marker ( 26 ), at least if it is not already detectable electrochemically, d) separating the analytes ( 10 ) bound first ( 20 ) and second probe ( 22 ), e) detection of a secreted by the first probe ( 20 ) or the first marker ( 24 ) first electrochemical signal (Si1) and one by the second probe ( 22 ) or the second marker ( 26 Second electrochemical signal (Si2) and f) detection, quantification and / or characterization of the analyte ( 10 ) by means of a ratio between the first (Si1) and the second signal (Si2). Method for detecting, quantifying and / or characterizing an analyte contained in a first fluid ( 10 ) comprising the following steps: a) contacting and incubating the analyte ( 10 ) each having an affinity for the analyte ( 10 ) first probe ( 20 ), where the affinity of the first probe ( 20 ) by a specific affinity for at least a first binding site ( 12 ) of the analyte ( 10 ) and the incubation is carried out under conditions under which the first probe ( 20 ) to the analyte ( 10 ), b) marking the first probe ( 20 ) by at least one electrochemically detectable first marker ( 24 ), at least if it is not already detectable electrochemically, c) labeling the analyte ( 10 ) by at least one electrochemically detectable second marker ( 26 ), at least when the analyte ( 10 ) is not already detectable electrochemically specifically, d) separating the from the first probe ( 20 ) bound analytes ( 10 ) and that of the analyte ( 10 ) bound first probe ( 20 ), e) detection of a secreted by the first probe ( 2O ) or the first marker ( 24 ) first electrochemical signal (Si1) and one by the secreted analyte ( 10 ) or the second marker ( 26 Second electrochemical signal (Si2) and f) detection, quantification and / or characterization of the analyte ( 10 ) by means of a ratio between the first (Si1) and the second signal (Si2). A method according to claim 1 or 2, wherein an electrode used for detection ( 27 ) is not brought into contact with the first liquid. Method according to one of the preceding claims, wherein the analyte ( 10 ) before step lit. e, in particular before step lit. a, is separated from the first liquid and transferred into a second liquid. Method according to one of the preceding claims, wherein the analyte ( 10 ), in particular specifically, by a scavenger molecule (pT, (T) _n ) is bound. Method according to one of the preceding claims, wherein the scavenger molecule (pT, (T) _n ) at a first ( 16 ) or second surface is immobilized. Method according to one of the preceding claims, wherein the capture molecule (pT, (T) _n ) is a nucleic acid, an analog of a nucleic acid, in particular a peptide nucleic acid (PNA), an antibody or a receptor. Method according to one of the preceding claims, wherein the capture molecule (pT, (T) _n ) has an affinity molecule, in particular streptavidin, avidin or biotin, or a biotinylated oligonucleotide. Method according to one of the preceding claims, wherein the first ( 20 ) or second probe ( 22 ) or the analyte ( 10 ) contains an affinity molecule, in particular biotin, avidin or streptavidin. Method according to one of the preceding claims, wherein the analyte ( 10 ) is a nucleic acid, in particular a poly-T end (pT, (T) _n ) or a poly A end (pA, (A) _n ). Method according to one of the preceding claims, wherein the characterization is done by determining the length of the nucleic acid. Method according to one of the preceding claims, wherein the analyte ( 10 ) before or during step lit. a is amplified by means of a nucleic acid amplification reaction, in particular a PCR. Method according to one of the preceding claims, wherein the analyte ( 10 ) before step lit. d, in particular before step lit. a, at the first ( 16 ) or second surface is immobilized. Method according to one of the preceding claims, wherein the first surface ( 16 ) the surface of a, in particular superparamagnetic, particle ( 18 ). The method of claim 14, wherein the particle ( 18 ) has a diameter of 10 nm to 100 .mu.m, in particular 1-10 .mu.m. Method according to one of the preceding claims, wherein the second surface of a serving for detection, in particular an electrically conductive plastic or an electrically conductive polymer, mercury, amalgam, gold, platinum, carbon or indium-tin oxide containing electrode ( 27 ). Method according to one of the preceding claims, wherein the second probe ( 22 ) has a specific affinity for at least one specific second binding site ( 14 ) of the analyte ( 10 ) having. Process according to the preceding claim, wherein the analyte ( 10 ) a known number of first binding sites ( 12 ) and an unknown number of second binding sites ( 14 ) having. Method according to one of the preceding claims, wherein the analyte ( 10 ) is a DNA fragment resulting from a triplet expansion disease such as fragile X syndrome, Huntington's disease, bulbar muscle atrophy, spinocerebellar ataxia type I; of myotonic dystrophy or Friedreich's ataxia, has repetitive sequences. Method according to one of the preceding claims, wherein the first probe ( 20 ) of the analyte ( 10 ) and / or the analyte ( 10 ) from the first ( 16 ) or second surface between step lit. d and step lit. e, in particular by heat denaturation, chemical denaturation, enzymatic digestion or chemical degradation, is released. Method according to one of the preceding claims, wherein the first ( 20 ) and the second probe ( 22 ) of the analyte ( 10 ) or the first probe ( 2 0 ) of the analyte ( 10 ) and the analyte ( 10 ) from the first ( 16 ) or second surface are each released separately and then detected. Method according to one of the preceding claims, wherein the first marker ( 24 ) and / or the second marker ( 26 ), in particular separately from each other, from the first ( 20 ) and / or second probe ( 22 ) or the analyte ( 10 ) are released and then detected. Method according to one of the preceding claims, wherein the first ( 24 ) and / or the second marker ( 26 ) are released by enzymatic digestion or chemical degradation. Method according to one of the preceding claims, wherein the first ( 20 ) and / or second probe ( 22 ) is a nucleic acid or an analog of a nucleic acid, in particular a peptide nucleic acid (PNA). Method according to one of the preceding claims, wherein the first ( 20 ) and / or second probe ( 22 ) sequence-specific, in particular by hybridization, to the analyte ( 10 ) binds. Method according to one of the preceding claims, wherein the first (Si1) or second signal (Si2) in each case from one through the first 24 ) or second marker ( 26 ) caused catalytic hydrogen release. Method according to one of the preceding claims, wherein the first ( 24 ) and / or the second marker ( 26 ) is reversibly reducible or oxidizable. Method according to one of the preceding claims, wherein the first ( 24 ) or second markers ( 26 ) has an osmium complex, a nanogold particle, a cysteine, ferrocenyl, daunomycin, benzoquinone, naphthoquinone, anthraquinone or p-aminophenol group or a dye, in particular indophenol, thiazine or phenazine. Method according to one of the preceding claims, wherein the first ( 20 ) or second probe ( 22 ) by several markers ( 24 . 26 ) is marked. Method according to one of the preceding claims, wherein the first ( 20 ) or second probe ( 22 ) has a linear primary structure at one end of which the marker ( 24 . 26 ) is arranged. Method according to one of the preceding claims, wherein the detection of the first (Si1) and the second signal (Si2) the same electrode and / or by the same electrochemical detection method. Method according to one of the preceding claims, wherein detection by cathodic stripping voltammetry (CSV), Rectangular wave voltammetry, cyclic voltammetry or chronopotentiometry takes place. Method according to one of the preceding claims, wherein the detection by means of a reversible redox process or a catalytic hydrogen evolution takes place.