WO2004023128A1 - Detection of target nucleic acid molecules by alteration of reaction of a redox species following hybridization with immoblized capture nucleic acid molecules - Google Patents

Detection of target nucleic acid molecules by alteration of reaction of a redox species following hybridization with immoblized capture nucleic acid molecules Download PDF

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Publication number
WO2004023128A1
WO2004023128A1 PCT/AU2003/001147 AU0301147W WO2004023128A1 WO 2004023128 A1 WO2004023128 A1 WO 2004023128A1 AU 0301147 W AU0301147 W AU 0301147W WO 2004023128 A1 WO2004023128 A1 WO 2004023128A1
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Prior art keywords
nucleic acid
acid molecule
redox species
apparatus according
capture nucleic
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PCT/AU2003/001147
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French (fr)
Inventor
Justin Gooding
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Unisearch Limited
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Priority to AU2002951231 priority Critical
Priority to AU2002951231A priority patent/AU2002951231A0/en
Priority to AU2002952940 priority
Priority to AU2002952941 priority
Priority to AU2002952941A priority patent/AU2002952941A0/en
Priority to AU2002952940A priority patent/AU2002952940A0/en
Application filed by Unisearch Limited filed Critical Unisearch Limited
Priority claimed from AU2003257260A external-priority patent/AU2003257260A1/en
Publication of WO2004023128A1 publication Critical patent/WO2004023128A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366Apparatus specially adapted for solid-phase testing
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6834Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Abstract

The invention relates to an apparatus for the detection of a target nucleic acid molecule, and to methods for making and using the apparatus. The apparatus includes a capture nucleic acid molecule immobilized on a surface, a redox species (e.g. a thiol group (mercaptohexanol), a redox intercalator etc.) and a reaction means (e.g. an electrode) for reaction with the redox species, wherein the capture nucleic acid molecule is capable of (i) moving the redox species and the reaction means into engagement, (ii) displacing the redox species and the reaction means from each other or (iii) permiting a reaction species (such as an ion in solution) to contact the redox species when the capture nucleic acid is hybridised to a target nucleic acid molecule, (i), (ii) or (iii) resulting in an alteration in the reaction of the redox species with the reaction means or the reaction species. The surface optionally includes a plurality of repellent molecules (such as mercaptoethanol, mercaptopropanol, or mercaptohexanol) which limit the adsorption of the capture nucleic acid molecule to the surface and repel suitably charged redox species.

Description

DETECTION OF TARGET NUCLEIC ACID MOLECULES BY ALTERATION OF REACTION OF A REDOX SPECIES FOLLOWING HYBRIDIZATION WITH IMMOBILIZED CAPTURE NUCLEIC

ACID MOLECULES

Field of the invention

The invention relates to an apparatus, such as a biosensor, for detection of a nucleic acid molecule, and to methods for making and using the apparatus. Background of the invention

The detection of a target nucleic acid molecule by an apparatus, such as a biosensor or the like, is typically achieved by reference to a hybridisation event. Such apparatus typically include a capture nucleic acid molecule having a nucleotide sequence that has complementarity with the sequence of a target nucleic acid molecule. In use, the apparatus is contacted with the sample for determination in conditions for permitting hybridisation of the capture nucleic acid molecule with the target nucleic acid molecule. Hybridisation of the target nucleic acid molecule with the capture nucleic acid molecule is then determined, to determine whether the sample includes the target nucleic acid molecule. Many apparatus for detection of a nucleic acid molecule detect the hybridisation event indirectly and this impinges on the fidelity of these apparatus. For example, some apparatus include means for detecting a labelled target nucleic acid molecule, such as a nucleic acid molecule labelled with a fluorophore. Hybridisation is determined by determining the presence of a fluorescent signal. Other apparatus include means for detecting phenomenon that occur at or subsequent to hybridisation, such as means for detecting evanescent waves, the reflectance of light, acoustic waves and electrochemical reactions where the different affinity of a redox species for single and double strands of DNA gives a difference signal. Unacceptable signal to noise ratio and/or detection of false positive signals are a consequence of indirect detection of a hybridisation event. Another consequence of indirect detection is that the hybridisation event and also, the denaturation of the target and capture nucleic acid molecule complex, cannot be monitored in situ, so that such apparatus cannot be used in applications where measurement of kinetics of hybridisation and denaturation is essential, such as in monitoring a polymerase chain reaction. One approach for determining a hybridisation event is that based on the detection of long range electron transfer, a phenomenon that is permitted when nucleic acid molecules including complementary sequences hybridise by perfect Watson-Crick base pairing to form a duplex. More particularly, it is believed that when such hybridisation occurs, a π electron pathway is formed that is sufficient for transferring electrons along the length of the duplex. As hybridisation is required for long range electron transfer to occur, the detection of an electric signal (for example, a current) associated with long range electron transfer demonstrates a hybridisation event, for example, between capture and target nucleic acid molecules, and accordingly, determines the presence of a target nucleic acid molecule in a sample.

One of the particularly advantageous characteristics of determining hybridisation events by detecting long range electron transfer is that a single nucleotide mismatch between nucleic acid molecules included in a duplex is sufficient for interrupting the π electron pathway and hence reducing or completely diminishing the electric signal that would otherwise be detected between nucleic acid molecules having complete sequence complementarity. Accordingly, determining a hybridisation event by reference to long range electron transfer allows not only the detection of a target nucleic acid molecule in a sample, but also for identifying whether the target nucleic acid molecule is one that has a nucleotide sequence that has perfect complementarity with the capture nucleic acid molecule, or one that has one or more regions of nucleotide sequence mismatch.

Long range electron transfer has been demonstrated by assembling duplexes including a capture nucleic acid molecule via an alkanethiol linker attached to the 5' end of the capture nucleic acid molecule onto a gold electrode surface, so that a densely packed film of duplexes is formed. When used for detecting a target nucleic acid molecule in a sample, the duplexes are denatured to provide a single stranded nucleic acid molecule (i.e. the capture nucleic acid molecule), before contact with the sample. A redox species capable of intercalating in a duplex, such as methylene blue or daunomycin, is then applied for intercalation of the species into a duplex formed by hybridisation of capture and target nucleic acid molecules, typically at a region distal to the 5' end of the capture nucleic acid molecule. Conditions are then provided to facilitate electron flow from the redox species, and where there is sequence complementarity between the strands of a duplex, along the π electron pathway, (ie along the duplex), through the linker and to the gold electrode surface for detection.

In the above described arrangement, the densely packed film is necessary for providing duplexes in an alignment at about 40° to the gold electrode surface. This limits the interaction of the capture nucleic acid molecule included in the duplex with the gold electrode surface. Such interactions tend to interfere with hybridisation of the capture nucleic acid molecule to a target nucleic acid molecule. Further, the densely packed film attempts to limit direct contact of a redox species that has not intercalated in a duplex with the electrode surface, an event that might otherwise occur when the redox species is applied. This is important for preventing the electrode from receiving a signal that has not been transmitted along the duplex. Accordingly, the densely packed film of duplexes seeks to limit the signals received by the gold electrode surface to those transmitted along a duplex as a consequence of hybridisation of capture and target nucleic acid molecules. There are a number of problems with the above described arrangement. For example, the dense packing of the duplexes is not optimal for hybridisation of a target nucleic acid molecule to the capture nucleic acid molecules in the film, because the target nucleic acid molecules tend to be repelled by the dense packing of the capture nucleic acid molecules that are provided when the duplexes are denatured prior to contact with the sample. Importantly, when one attempts to solve this problem by providing a less densely packed film, a background current is detected by the gold electrode surface. This limits the sensitivity of the arrangement for the detection of a target nucleic acid molecule, particularly where the intention is to determine whether a target nucleic acid molecule hybridised to the capture nucleic acid molecule includes one or more regions of sequence mismatch.

A limitation of many apparatus for detection of nucleic acid molecules is that they have limited compatibility with high through-put applications or point-of-care applications. Summary of the invention

The invention seeks to minimise some of the above limitations and/or to provide an improvement in the detection of nucleic acid molecules.

In a first aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species and a reaction means for reaction with the redox species. The apparatus is characterised in that the capture nucleic acid molecule is sufficient for arranging the redox species and the reaction means relative to each other for affecting reaction of the redox species with the reaction means, when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule.

As described herein, the inventor has found that the change in rigidity of a nucleic acid molecule that is associated with the formation of a double stranded nucleic acid molecule from a single stranded molecule, is sufficient for enabling a capture nucleic acid molecule immobilised by attachment to a surface, to arrange the redox species and the reaction means in relation to affect reaction of the redox species with the reaction means. In particular embodiments described herein, the detection of a signal, such as an electric current, resulting from such arrangement, permits the direct detection of the hybridisation of a target nucleic acid molecule to the surface -attached capture nucleic acid molecule, and hence, the improved detection of the target nucleic acid molecule.

In a second aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species, in use having a charge for permitting the redox species to be attracted to the reaction means, for contact with the reaction means and a reaction means for reaction with the redox species. The apparatus is characterised in that the capture nucleic acid molecule is sufficient for displacing the redox species and the reaction means from each other for limiting reaction of the redox species with the reaction means, when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule. In a third aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species and a reaction means for reaction with the redox species. The redox species is arranged relative to the reaction means for permitting reaction of the redox species with the reaction means, when the redox species is contacted with a reaction species capable of oxidising or reducing the redox species. The apparatus is characterised in that the capture nucleic acid molecule is sufficient for permitting a reaction species to contact the redox species, when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule. In a fourth aspect, the invention provides a method for determining whether a sample includes a target nucleic acid molecule. The method includes the following steps:

(a) contacting the sample with an apparatus according to the invention in conditions sufficient for permitting a target nucleic acid molecule to hybridise to the capture nucleic acid molecule; and (b) determining whether the redox species and the reaction means are arranged for reaction of the redox species with the reaction means; to determine whether the sample includes the target nucleic acid molecule.

In a fifth aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule, a surface for receiving an electric signal, a plurality of repellent molecules arranged on the surface for limiting adsorption of the capture nucleic acid molecule to the surface, a linker for linking the capture nucleic acid molecule to the surface to permit an electric signal to be received by the surface from the linker, and a redox species, in use having a charge for permitting the redox species to be repelled by the plurality of repellent molecules from the surface.

In sixth aspect, the invention provides a method for determining whether a sample includes a target nucleic acid molecule. The method includes the following steps:

(a) contacting the sample with an apparatus according to the fifth aspect to permit intercalation of the redox species into a duplex formed by hybridisation of the capture nucleic acid molecule with a target nucleic acid molecule;

(b) providing conditions for permitting transmission of an electric signal from an intercalated redox species along a duplex to the linker; and (c) determining whether an electric signal is received by the surface from the linker, to determine whether the sample includes a target nucleic acid molecule.

Brief description of the drawings

Figure 1. Schematic representation of an embodiment of the invention showing the secondary structure of the capture nucleic acid molecule when bound to a target nucleic acid molecule.

Figure 2. Label free apparatus before and after hybridisation with complementary target nucleic acid molecule 2 (SEQ ID NO:2).

Figure 3. Label free apparatus before and after addition of non- complementary target nucleic acid molecule 5 (SEQ ID NO: 5).

Figure 4. DAD-labelled apparatus before and after hybridisation with complementary target DNA 2 (SEQ ID NO:2), and after recovery of single stranded biorecognition element.

Figure 5. {Rh(phi)2(bpy)]3+- labelled apparatus before and after hybridisation with complementary target DNA 2 (SEQ ID NO:2).

Figure 6. Schematic representation of the DNA recognition layer and subsequent hybridization and intercalation of AQS which allows electrochemical detection of hybridisation.

Figure 7. OSWV of (i) probe ssDNA/MCH modified electrode before hybridisation, (ii) after hybridized to 4 μM complementary target DNA 2 (SEQ ID

NO:2). OSWNs were obtained 0.05M phosphate buffer (pH7.0) at step of 4mN, pulse amplitude of 25mN and frequency of 10 Hz. Figure 8. Plot of current signal versus the logarithm to base 10 of the concentration of target DNA 2 (SEQ ID NO:2) as measured using the same OSWV conditions outlined in the caption for figure 2.

Figure 9. OSWV of (i) probe ssDNA/MCH modified electrode before hybridisation, (ii) after hybridized to 4 μM noncomplementary target DNA 5 (SEQ ID

NO:5). OSWNs were obtained 0.05M phosphate buffer (pH 7.0) at step of 4mN, pulse amplitude of 25mN and frequency of 10 Hz.

Figure 10. OSWN of (i) probe ssDΝA/MCH modified electrode before hybridisation, (ii) after hybridized to 4 μM noncomplementary target DΝA (SEQ ID ΝO:3). OSWNs were obtained 0.05M phosphate buffer (pH 7.0) at step of 4mN, pulse amplitude of 25mN and frequency of 10 Hz.

Figure 11. OSWN of (i) probe ssDΝA/MCH modified electrode before hybridisation, (ii) after hybridized to 4 μM noncomplementary target DΝA 4 (SEQ ID ΝO:4). OSWNs were obtained 0.05M phosphate buffer (pH 7.0) at step of 4mN, pulse amplitude of 25mN and frequency of 10 Hz.

Detailed description of the embodiments

According to the apparatus of the first aspect of the invention, the capture nucleic acid molecule may arrange the redox species and the reaction means relative to each other for affecting reaction of the redox species and the reaction means, by bringing the redox species and the reaction means into engagement, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule. Accordingly, in this embodiment, the change in the rigidity of the capture nucleic acid molecule that occurs as a consequence of hybridisation to the target nucleic acid molecule is sufficient for permitting the capture nucleic acid molecule to move a redox species that is attached to, or arranged in relation to, the capture nucleic acid molecule, into engagement with a reaction means. Alternatively, the change in rigidity of the capture nucleic acid molecule is sufficient for permitting the capture nucleic acid molecule to move a reaction means that is attached to, or arranged in relation to, the capture nucleic acid molecule, into engagement with a redox species. The redox species and reaction means may be moved into engagement by the capture nucleic acid molecule, by being brought into direct contact with each other, or via contact with another molecule. The detection of a signal, such as an electric current, resulting from such arrangement, detects the hybridisation of the capture nucleic acid molecule to the target nucleic acid molecule. Thus, the capture nucleic acid molecule may arrange the redox species and the reaction means relative to each other for affecting reaction of the redox species and the reaction means, by bringing the redox species and the reaction means into engagement, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule.

Alternatively, the capture nucleic acid molecule may arrange the redox species and the reaction means relative to each other for affecting reaction of the redox species and the reaction means, by displacing the redox species and the reaction means from each other, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule, h this embodiment, the change in rigidity of the capture nucleic acid molecule that occurs as a consequence of the hybridisation to the target nucleic acid molecule is sufficient for permitting the capture nucleic acid molecule to move a redox species attached to, or arranged in relation to, the capture nucleic acid molecule, apart from the reaction means. Alternatively, the change in rigidity of the capture nucleic acid molecule is sufficient for permitting the capture nucleic acid molecule to move a reaction means attached to, or arranged in relation to, the capture nucleic acid molecule, apart from the redox species. The detection of a signal, such as an electric current, resulting from such arrangement, detects the hybridisation of the target nucleic acid molecule to the capture nucleic acid molecule. Thus, the capture nucleic acid molecule may arrange the redox species and the reaction means relative to each other for affecting reaction of the redox species with the reaction means, by displacing the redox species and the reaction means from each other, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule.

Where the capture nucleic acid molecule arranges the reaction means and redox species relative to each other for affecting reaction of the redox species and reaction means, typically, the redox species, in use has a charge for permitting the redox species to be attracted to the reaction means sufficient for contact with the reaction means. According to the invention, improved or limited reaction of the redox species and the reaction means is contemplated when the capture nucleic acid molecule arranges the redox species and the reaction means relative to each other for affecting reaction of the redox species with the reaction means. Thus, in one embodiment, the capture nucleic acid molecule arranges the redox species and the reaction means relative to each other for limiting reaction of the redox species and reaction means, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule. In another embodiment, the capture nucleic acid molecule arranges the redox species and reaction means relative to each other for improving reaction of the redox species and reaction means, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule.

The reaction means is typically arranged on the surface to which the capture nucleic acid molecule is attached. Accordingly, where the capture nucleic acid molecule arranges the redox species and reaction means for affecting reaction of the redox species and reaction means, by bringing the redox species and reaction means into engagement, the redox species is typically arranged on or attached to the capture nucleic acid molecule, and the redox species is brought by the capture nucleic acid molecule to engage with the reaction means. Further where the capture nucleic acid molecule displaces the redox species and reaction means for affecting reaction of the redox species and reaction means, the redox species is arranged on or attached to the capture nucleic acid molecule, and the capture nucleic acid molecule displaces, or in other words, removes the redox species from the reaction means. Thus, the reaction means may be arranged on the surface to which the capture nucleic acid molecule is attached.

Notwithstanding the above, it will be recognised that, in a particular arrangement, the reaction means may be arranged other than on the surface to which the capture nucleic acid molecule is attached, and in particular, the reaction means may be arranged on or attached to the capture nucleic acid molecule. For example, the reaction means may be brought into engagement with or displaced from the redox species by the capture nucleic acid molecule, to affect the reaction of the redox species and reaction means, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule. In this particular arrangement, the redox species may be arranged on the surface to which the capture nucleic acid molecule is attached.

As described herein the reaction means may provide the surface to which the capture nucleic acid molecule is attached and typically so when the redox species is arranged on or attached to the capture nucleic acid molecule. In this embodiment, when not hybridised to the target nucleic acid molecule, the capture nucleic acid molecule is arranged on the reaction means for permitting reaction of the redox species and reaction means, for example by permitting contact of the redox species and surface, and when hybridised to a target nucleic acid molecule, the redox species is displaced from the surface, for affecting reaction of the redox species with the surface.

The apparatus may be adapted for compatibility with a detector for detecting a signal, such as an electric current resulting from the arrangement of the redox species and reaction means. Alternatively the apparatus may further include detector means for detecting the signal. In one embodiment, the surface for attachment of the capture nucleic acid molecule is a detector for detecting a signal resulting from the arrangement of the redox species and reaction means. Preferred surfaces are those that include a substance capable of reacting with a redox species and include gold, glassy carbon, platinum, silver, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt. As described above, the redox species may be arranged on the capture nucleic acid molecule, for example, the redox species may be adsorbed to the backbone of the capture nucleic acid molecule. Alternatively, the redox species may be attached to the capture nucleic acid molecule, for example by a covalent bond. Typically, the redox species is attached to the terminus of the capture nucleic acid molecule. In a second aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species, in use having a charge for permitting the redox species to be attracted to the reaction means, for contact with the reaction means and a reaction means for reaction with the redox species. The apparatus is characterised in that the capture nucleic acid molecule is sufficient for displacing the redox species and the reaction means from each other for limiting reaction of the redox species with the reaction means, when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule.

The inventor has found that the change in rigidity of a nucleic acid molecule, that is associated with the formation of a double stranded nucleic acid molecule from a single stranded molecule, is sufficient for enabling a capture nucleic acid molecule, immobilised by attachment to a surface, to displace a redox species and a reaction means such as a gold electrode, from each other, for limiting reaction of the redox species with the reaction means. More specifically, as in use, the redox species has a charge for attracting the redox species to the reaction means, reaction of the redox species and the reaction means occurs and can be detected. One finding of the inventor is that the change in rigidity of a nucleic acid molecule that is associated with formation of a double stranded nucleic acid molecule from a single stranded nucleic acid molecule, is sufficient to displace such a redox species from the reaction means and so at least limit or inhibit reaction of the redox species and the reaction means. Accordingly, the direct detection of the hybridisation of a target nucleic acid molecule to the surface-attached capture nucleic acid molecule, and hence, the improved detection of the target nucleic acid molecule occurs when a signal, such as an electric current resulting from such arrangement of the redox species and reaction means, is detected. According to the apparatus of the second aspect of the invention, the capture nucleic acid molecule displaces the redox species and the reaction means from each other when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule. Typically, the capture nucleic acid molecule displaces the redox species from the reaction means, when hybridised to the target nucleic acid molecule. The detection of a signal, for example, an electric current resulting from such arrangement of the redox species and the reaction means, indicates hybridisation of the capture nucleic acid molecule to the target nucleic acid molecule. In summary, in one embodiment, the capture nucleic acid molecule displaces the redox species from the reaction means for limiting reaction with the reaction means, when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule. The reaction means is typically arranged on the surface to which the capture nucleic acid molecule is attached. The redox species is typically arranged on or attached to the capture nucleic acid molecule so that the capture nucleic acid molecule displaces the redox species from the reaction means, when hybridised to a target nucleic acid molecule.

The reaction means may provide the surface to which the capture nucleic acid molecule is attached. Preferred surfaces are those that include a substance capable of reacting with a redox species and include gold, glassy carbon, platinum, silver, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.

Where the reaction means provides the surface to which the capture nucleic acid molecule is attached, when not hybridised to the target nucleic acid molecule, the capture nucleic acid molecule is arranged on the reaction means for permitting reaction of the redox species with the reaction means, for example, by permitting contact of the redox species and the surface. When hybridised to a target nucleic acid molecule, the redox species is displaced from the surface, to at least limit the reaction between the redox species and the surface.

Where the reaction means is glassy carbon and provides a surface for attachment of the capture nucleic acid molecule, the capture nucleic acid molecule may be attached via amine groups on 2' or 3' carbons on the ribose or deoxyribose ring.

The apparatus may be adapted for compatibility with a detector for detecting a signal, such as an electric current resulting from the arrangement of the reaction means and the redox species. An electrode is one example of such a detector. Alternatively, the apparatus may further include a detector for detecting a signal resulting from the arrangement of the redox species and reaction means. In one embodiment, the surface for attachment of the capture nucleic acid molecule is a detector for detecting a signal.

Where the detector is an electrode, typically the electrode includes gold, for detection of an electric current that is characteristic of a reaction between the redox species and the reaction means. Examples of other electrodes include those including or consisting of platinum, silver, glassy carbon, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.

As described above, the redox species may be arranged on the capture nucleic acid molecule, for example, the redox species may be adsorbed to the backbone of the capture nucleic acid molecule. Examples of redox species for adsorption to the backbone of the capture nucleic acid molecule include ferrocenes, diaminodurene, derivatives of transition metals, hydro quinone, anthraquinone, reducible and oxidisable organic salts, cobaltocenes, hexacyanides, ethidium, porphyrins, Rh(phi)2(bpy') where phi is 9,10- diimine phenathrenequinone and bpy' is butyric acid 4' methylbipyridine. Methods for adsorbing a redox species to the backbone of a nucleic acid molecule are described further herein.

Typically, the redox species is attached to the capture nucleic acid molecule, for example by a covalent bond. Typically, the redox species is attached to the terminus of the capture nucleic acid molecule. The selection of redox species for attachment to the capture nucleic acid molecule is dependent on the nature of the reaction means for reaction with the redox species. An example of a suitable redox species for attachment to the capture nucleic acid molecule is R_ (phi)2(bpy') where phi is 9,10-diimine phenathrenequinone and bpy' is butyric acid 4' methylbipyridine. This species is typically attached via an amine linker. The attachment of a redox species to a capture nucleic acid molecule is described further herein.

In a third aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species and a reaction means for reaction with the redox species. The redox species is arranged relative to the reaction means for permitting reaction of the redox species with the reaction means, when the redox species is contacted with a reaction species capable of oxidising or reducing the redox species. The apparatus is characterised in that the capture nucleic acid molecule is sufficient for permitting a reaction species to contact the redox species, when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule. The inventor has found that the change in rigidity of a nucleic acid molecule that is associated with the formation of a double stranded nucleic acid molecule from a single stranded molecule, is sufficient for enabling a capture nucleic acid molecule immobilised by attachment to a surface to permit contact of a reaction species, including, for example, an ion, with the redox species for reaction of the redox species with the reaction means.

Accordingly, the detection of a signal such as an electric current resulting from reaction of the redox species with the reaction means enables the direct detection of the hybridisation of a target nucleic acid molecule to the surface-attached capture nucleic acid molecule, and hence, the improved detection of a target nucleic acid molecule. According to the third aspect of the invention, when the capture nucleic acid molecule is not hybridised to the target nucleic acid molecule, the capture nucleic acid molecule is arranged for limiting contact of a reaction species, for example, an ion in a solution such as an electrolyte, and the redox species for at least limiting reaction of the redox species with the reaction means. The change in rigidity of the capture nucleic acid molecule that occurs as a consequence of hybridisation to the target nucleic acid molecule affects the arrangement of the capture nucleic acid molecule so that the redox species is exposed to a reaction species sufficient for the redox species to react with the reaction means. The detection of a signal such as an electric current resulting from the reaction of the redox species with the reaction means detects the hybridisation of the capture nucleic acid molecule to the target nucleic acid molecule.

Typically the redox species is arranged on the reaction means. For example, as described herein, a redox species such as a species including a thiol group, may be attached to a reaction means such as a gold surface.

Typically the redox species includes a thiol group for reaction with the reaction means. Alkanethiols are useful as a redox species, especially mercaptopropanol, mercaptoethanol and mercaptohexanol. The alkanethiol may include a terminal group selected from the group consisting of alkyl, hydroxyl, acetyl, carboxyl, amine, aldehyde, succinimide ester, ketone, glycol, ester ether and sulphonate. The alkanethiol may include 2, 3, 6, 9, 12 or 15 carbon atoms. The apparatus may be adapted for compatibility with a detector for detecting a signal such as an electric current. Alternatively, the apparatus may further include a detector for detecting an electric current. In one embodiment, the surface for attachment of the capture nucleic acid molecule is a detector for detecting an electric current. In accordance with the above embodiments, the detection of an electric current that is characteristic of the reaction of the redox species with the reaction means detects the hybridisation of the capture nucleic acid molecule to the target nucleic acid molecule.

The detector is typically an electrode. Examples of suitable electrodes include those including or consisting of gold, platinum, silver, glassy carbon, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.

With regard to the apparatus of the first, second and third aspects of the invention, the inventor recognises that particular combinations of a capture nucleic acid molecule and a surface for attachment of the capture nucleic acid molecule thereto, may result in adsorption of the capture nucleic acid molecule to the surface and hence limit hybridisation efficiency. For example, where the surface selected for attachment of the capture nucleic acid molecule is gold, extensive chemical bonding between the capture nucleic acid molecule and the surface may adsorb the capture nucleic acid molecule so as to essentially preclude hybridisation of the capture nucleic acid molecule with the target nucleic acid molecule.

In an embodiment of the first, second and third aspect of the invention, the apparatus further includes means for limiting adsorption of the capture nucleic acid molecule to the surface to which the capture nucleic acid molecule is attached. Such means are particularly useful for enhancing hybridisation efficiency, and so improve detection of a nucleic acid molecule in a sample. The means may include a plurality of molecules for limiting the formation of a chemical bond between the capture nucleic acid molecule and the surface. Additionally, or alternatively, the means for limiting adsorption of the capture nucleic acid molecule to the surface may include a plurality of molecules for repelling the capture nucleic acid molecule from the surface. Alkanethiol molecules are particularly useful for this purpose because the thiol group of such molecules displace a nucleic acid molecule from a surface such as gold. Further the alcohol terminal at the end opposite from the end of the molecule including the thiol group has a negative dipole and accordingly, is capable of repelling a negatively charged back-bone of a nucleic acid molecule from a surface to which the nucleic acid molecule is attached. Examples of alkanethiol molecules useful for limiting adsorption of the capture nucleic molecule to the surface include mercaptopropanol, mercaptoethanol and mercaptohexanol. Alkanethiol molecules useful in this embodiment of the invention may include 2, 3, 6, 9, 12 or 15 carbon atoms. Methods for attachment of alkanethiol molecules to a surface to which the capture nucleic acid molecule is attached, for the purpose of limiting adsorption of the capture nucleic acid molecule to the surface, are described herein.

The capture nucleic acid molecule of the apparatus of the first, second and third aspect of the invention may be any type of nucleic acid molecule that is capable of hybridising to a target nucleic acid molecule and that is capable of undergoing the change in rigidity that is associated with the formation of a double stranded nucleic acid molecule from a single stranded molecule, when attached to a surface. Examples of capture nucleic acid molecules include deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, molecules including both deoxyribonucleic and ribonucleic acids and peptide nucleic acid (PNA) molecules. As the persistence length of a single stranded DNA is about 1 nm, which correlates to about 3 nucleotides, the capture nucleic acid molecule is more than 3 nucleotides in length. Typically, for the purpose of providing a sufficient stringency for hybridisation to the target nucleic acid molecule, the capture nucleic acid molecule is considerably more than 3 nucleotides in length and typically, at least 8 nucleotides in length. Methods for selection of an appropriate length for the capture nucleic acid molecule are described ftirther herein. It will be understood that the nucleotide sequence of the capture nucleic acid molecule need not be entirely complementary to the sequence of the target nucleic acid molecule, provided that the capture nucleic acid molecule is capable of hybridising with the target nucleic acid molecule by Watson-Crick base pairing to form a duplex that has a persistence length that is greater than the persistence length of the capture nucleic acid molecule in the single stranded form. Accordingly, the capture nucleic acid molecule may include nucleotide sequence that is identical to the complementary strand of the target nucleic acid molecule. Alternatively, the capture nucleic acid molecule may include nucleotide sequence that is homologous to the complementary strand of the target nucleic acid molecule. Further the capture nucleic acid molecule may includes one or more regions of nucleotide sequence that is identical, or homologous, to the complementary strand of the target nucleic acid molecule. The level of identity of the capture nucleic acid molecule to the complementary strand of the target nucleic acid molecule that is necessary for detection of that target nucleic acid molecule with the apparatus of the invention can be determined using standard techniques known to the skilled addressee. Examples are described further herein. Thus, in summary, in one embodiment, the capture nucleic acid molecule is identical to the complementary strand of the target nucleic acid molecule.

In accordance with the first, second and third aspect of the invention, the capture nucleic acid molecule is typically attached to the surface via a 5' or 3' terminus of the molecule. The capture nucleic acid molecule may be directly attached to the surface, or indirectly attached via a linker. Examples of linkers useful for this purpose include linkers including a thiol group at 3 to 6 carbon atoms. Other linkers are known to the skilled addressee. Methods for attachment of the capture nucleic acid molecule to the surface are described further herein. With regard to the fifth aspect of the invention, as described herein, the inventor has determined how to maximise the efficiency of hybridisation of the capture nucleic acid molecule to a target nucleic acid molecule in a sample so that a hybridisation event can be detected based on long range electron transfer, without loss of sensitivity. Indeed, in the invention described herein, the sensitivity is substantially improved, to the extent that no signal is detected when the capture nucleic acid molecule is single stranded (i.e. when the capture nucleic acid molecule has not hybridised to a target nucleic acid molecule). This means that one can more clearly distinguish a low stringency hybridisation event (i.e. hybridisation resulting from the presence of a target nucleic acid molecule in a sample that has limited sequence complementarity with the capture nucleic acid molecule) from a circumstance where the capture nucleic acid molecule has not hybridised to a target nucleic acid molecule.

The improved sensitivity of the method and apparatus of the invention for detecting a hybridisation event between the capture and target nucleic acid molecules is provided by a redox species that has a charge for permitting the redox species to be repelled by the plurality of repellent molecules under the conditions applied for transfer of electrons from the redox species through the DNA duplex, in particular, the conditions described in step (b) of the method of the invention described above. In particular, in experiments leading up to the invention, the inventor found that where a redox species includes a charge that is opposite to the charge on a plurality of alkanethiol molecules arranged on an electrode, a redox species is attracted to the alkanethiol molecules and this increases the likelihood of a redox species that has not intercalated into a duplex, directly contacting the surface and causing the surface to detect a signal. It is believed that by selecting a redox species according to the invention, the redox species that do not intercalate (for example, because a capture nucleic acid has not bound to a target nucleic acid molecule to form a duplex) are repelled by the plurality of repellent molecules, and accordingly, repelled from the surface. A consequence of repelling the redox species that have not intercalated in a duplex from the surface is that the only signal that can be detected by the surface is the signal that is the result of long range electron transfer through the duplex.

The efficiency of hybridisation of the capture nucleic acid molecule to a target nucleic acid molecule in the method and apparatus of the invention is improved by a redox species that in use has a charge for repelling the redox species from the plurality of repellent molecules. More particularly, as the charge on the redox species forces the redox species away from the surface under the conditions for transfer of electrons from the redox species, this avoids the need for forming a densely packed film of duplexes for arranging capture nucleic acid molecules sufficient to sterically block a redox species from directly contacting the surface.

Another important advantage is that according to the invention, the amount of target nucleic acid molecule in a sample can be detected. This is an advance over apparatus that include a densely packed film of duplexes. Such apparatus require that each capture nucleic acid molecule (which as described above are generated by denaturation of the duplex) must be hybridised with target nucleic acid molecule, otherwise a redox species that has not intercalated may contact the electrode. Accordingly, the apparatus and method of the invention provide for quantitative detection of target nucleic acid molecules in a sample, whereas apparatus that include a densely packed film of duplexes can only detect the presence or absence of the target nucleic acid molecule.

A further advantage of the plurality of repellent molecules is that they limit adsorption of the capture nucleic acid molecule to the surface. Such adsorption would limit the capacity of the capture nucleic acid molecule to hybridise to a target nucleic acid molecule. Accordingly, a consequence of providing a redox species that in use has a charge for repelling the species from the plurality of repellent molecules on the surface is that the capture nucleic acid molecules may be linked to the surface at a relatively low density and so are more freely available for hybridisation with a target nucleic acid molecule.

In summary, the provision of a redox species that has a charge for repelling the species from the repellent molecules under the conditions for transfer of electrons from the redox species through the DNA duplex provides the method and apparatus of the invention with improved efficiency for hybridisation of a capture nucleic acid molecule to a target nucleic acid molecule and improved sensitivity for detection of the hybridisation event.

Accordingly, in one aspect, the invention provides an apparatus for detecting a target nucleic acid molecule. The apparatus includes: (a) a capture nucleic acid molecule for hybridising to a target nucleic acid molecule;

(b) a surface for receiving an electric signal;

(c) a plurality of repellent molecules arranged on the surface for limiting adsorption of the capture nucleic acid molecule to the surface; (d) a linker for linking the capture nucleic acid molecule to the surface to permit an electric signal to be received by the surface from the linker; and

(e) a redox species, in use having a charge for permitting the redox species to be repelled by the plurality of repellent molecules from the surface. h accordance with the fifth aspect of the, invention, typically, the redox species, in use, has a negative charge. The redox species is typically one selected from the group consisting of negatively charged variants of anthraquinone (e.g. disulfonic acid anthraquinone or sulfonic acid anthraquinone), napthoquinone (e.g. l,2-napthoquinone-4- sulfonic acid), porphyrins (e.g. 21H,23H-Porphine-2,18-dipropanoic acid), phenazines (e.g. Benzo[a]phenazine-5-sulfonic acid), phthalocyanines (e.g. copper phthalocyanine

3,4',4", 4"'-tetrasulfonic acid), acridities (e.g. L-glutaminyl-L-glutaminyl-L-seryl-L- isoleucyl-L-α-glutamyl-L-glutaminyl-L-leucyl-L-α-glutamyl-N 1 - [3 - [(6-chloro-2- methoxy-9-acridinyl)amino]propyl]- (9CI) ), phenothiazines (e.g. Phenothiazine-2-acetic acid) and quinolones (e.g. oxolinic acid) Where the plurality of repellent molecules has a positive charge (for example, in the conditions provided in step (b) of the method of the invention), the redox species is typically one selected from the group consisting of positively charged variants of anthraquinones (e.g. doxorubicin), metal complexes containing intercalating ligands (such as 9,10-diimine phenathrenequinone or chrysene) po hyrins (e.g. Copper(II) tetrakis(4-N-methylpyridyl)porphyrin) , phthalocyanines (e.g. Cuprolinic Blue), acridines

(e.g. Lucigenin), phenothaizines (e.g. methylene blue), phenazines (e.g. safranine T) and phenanthridines (e.g. ethidium)

In one embodiment, the redox species in use has a charge for repelling the species from the surface sufficient for inhibiting contact of the redox species with the surface. Typically, the redox species is arranged on the apparatus in a form so that the redox species can be solubilised to permit intercalation of the species into a duplex formed between capture and target nucleic acid molecules, when the apparatus is contacted with a sample. Typically the redox species is arranged on the surface of the apparatus. Methods for arranging the redox species on the apparatus to permit solubihsation of the species when contacted with a sample are known to one skilled in the art. For example, the redox species may be adsorbed onto the surface in powder form, or dried on the surface or attached to the capture nucleic acid molecule.

As described above, an electric signal may be conducted along the π electron pathway of the duplex in conditions such as those provided in step (b) where there is sequence complementarity between capture and target nucleic acid molecule nucleotide sequences. It is believed however that in appropriate conditions, an electric signal may be conducted across a region including a single G-A mismatch. Accordingly, the capture nucleic acid molecule may include a nucleotide sequence that is identical to the complementary strand of the target nucleic acid molecule or one that is homologous to the complementary strand of the target nucleic acid molecule in as much as the sequence of the capture nucleic acid molecule differs from the complementary strand of the target nucleic acid molecule by about 1 or 2 nucleotide mismatches.

Notwithstanding the above, it will be understood that an electric signal may be conducted through the duplex in the circumstance that there is one or more regions of substantial sequence mismatch between the capture and target nucleic acid molecules and where the redox species intercalates so as to define with the linker, a region of sequence complementarity between the capture and target nucleic acid molecules.

Typically, for the purpose of providing a sufficient stringency for hybridisation to the target nucleic acid molecule, the capture nucleic acid molecule is considerably more than 3 nucleotides in length and typically, at least 8 nucleotides in length. Methods for selection of an appropriate length for the capture nucleic acid molecule are described further herein.

The capture nucleic acid molecule of the apparatus may be any type of nucleic acid molecule that is capable of hybridising to a target nucleic acid molecule. Examples of capture nucleic acid molecules include deoxyribonucleic acid (DNA) molecules, ribonucleic acid (RNA) molecules, molecules including both deoxyribonucleic and ribonucleic acids and peptide nucleic acid (PNA) molecules.

As described above, one function of the plurality of repellent molecules is to at least limit adsorption of the capture nucleic acid molecule to the surface to which the capture nucleic acid molecule is linked. This enhances hybridisation efficiency, and so improves detection of a target nucleic acid molecule in a sample. In this regard, typically, the repellent molecules limit the formation of a chemical bond between the capture nucleic acid molecule and the surface. Additionally, or alternatively, the repellent molecules may repel the capture nucleic acid molecule from the surface and so limit adsorption of the capture nucleic acid molecule to the surface.

Importantly, the repellent molecules in use have sufficient charge character for the redox species to be repelled from the surface to which the repellent molecules are attached. In other words, when in use, the redox species has a negative charge, the plurality of repellent molecules has a negative charge sufficient for repelling the redox species from the surface.

Alkanethiol molecules are particularly useful as repellent molecules in the method and apparatus of the invention. In the conditions of step (b) of the method of the invention, these molecules may have a negative charge character sufficient for repelling redox species with a negative charge character from the surface. Further, the thiol group of alkanethiol molecules is sufficient for displacing a nucleic acid molecule from a surface such as gold. Further, the alcohol terminal at the end opposite from the end of the molecule including the thiol group has a negative dipole and accordingly, is capable of repelling a negatively charged back-bone of a nucleic acid molecule from a surface to which the nucleic acid molecule is linked. Examples of alkanethiol molecules useful in the method and apparatus of the invention as repelling molecules include mercaptopropanol, mercaptoethanol and mercaptohexanol. Alkanethiol molecules useful in this embodiment of the invention may include 2, 3, 6, 9, 12 or 15 carbon atoms. Methods for attachment of alkanethiol molecules to a surface to which the capture nucleic acid molecule is attached are described herein.

In accordance with the invention, the capture nucleic acid molecule is typically attached to the surface via a 5' or 3' terminus of the molecule. The capture nucleic acid molecule may be directly attached to the surface, or indirectly attached via a linker. Examples of linkers useful for this purpose include linkers including a thiol group at 3 to 6 carbon atoms. Other linkers are known to the skilled addressee. Methods for attachment of the capture nucleic acid molecule to the surface are described further herein.

The apparatus may be adapted for compatibility with a detector for detecting an electric signal received by the surface, such as an electric current. Alternatively, the apparatus may further include detector means for detecting the signal. In one embodiment, the surface for receiving an electric signal is a detector means. A preferred detector means is an electrode. Preferably, the electrode includes a substance selected from the group consisting of gold, glassy carbon, platinum, silver, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.

The invention also provides a method for determining whether a sample includes a target nucleic acid molecule. The method includes the following steps:

(a) contacting the sample with an apparatus according to the first aspect to permit intercalation of the redox species into a duplex formed by hybridisation of the capture nucleic acid molecule with a target nucleic acid molecule;

(b) providing conditions for permitting transmission of an electric signal from an intercalated redox species along a duplex to the linker; and

(c) determining whether an electric signal is received by the surface from the linker, to determine whether the sample includes a target nucleic acid molecule.

The conditions for permitting transmission of an electric signal from an intercalated redox species through a duplex to the linker can be determined by one skilled in the art, having regard to the redox species used in the method. Examples of such conditions are described further herein. The conditions for permitting the capture nucleic acid molecule of the apparatus to hybridise with a target nucleic acid molecule in a sample to form a duplex can be determined by one skilled in the art. Further, the relevant considerations for providing these conditions, such as temperature, salt concentration and time for hybridisation, are described further herein. Further, the conditions for permitting a redox species to intercalate into a duplex formed by hybridisation of the capture and target nucleic acid molecules can be determined by one skilled in the art. Examples of these conditions where the redox species is anthraquinone, are further described below. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative embodiments of the invention.

The foregoing describes embodiments of the present invention and modifications, obvious to those skilled in the art can be made thereto, without departing from the scope of the present invention.

Examples

Example 1 General Approach

An embodiment of an apparatus according to the present invention is represented schematically in Figure 1. The apparatus is fabricated by preparing a capture nucleic acid molecule (a single stranded oligonucleotide) of between approximately 10 to 50 nucleotides in length, and preferably 20 nucleotides in length. At the 3 '-end, the oligonucleotide is attached to the surface of a gold electrode through a mercaptoalkyl linker. A secondary short-chain alkanethiol is allowed to self-assemble on the gold electrode. This is useful for limiting adsorption of the backbone of the oligonucleotide to the electrode surface and thus increases hybridisation efficiency. The persistence length of single stranded DNA is approximately 1 nm, while that of double stranded DNA at high salt concentration (>10 mM) is 45-50 nm. The oligonucleotide is approximately

6nm in length and so with defined flexibility. Upon hybridisation with the target DNA, the oligonucleotide becomes like a rigid rod, extending into solution approximately normal to the electrode surface. This may be detected elec trochemically.

For example, bulk gold electrodes are prepared by sealing polycrystalline gold wire (>99.99% gold, Aldrich) of 1 mm diameter in 4 mm diameter glass tubes, followed by attachment of nichrome wires for electrical connection to the back of the electrodes. The electrodes are first polished with 1.0 μm alumina slurry, followed by 0.3 and 0.05 μm alumina slurry on micro cloth pads (Buehler, Lake Bluff, IL, USA). After removal of the trace alumina from the surface by rinsing with Milli-Q (18 MΩ cm) water, and brief cleaning in an ultrasonic bath, the electrodes are cleaned by electrochemical etching in 0.05 M H2SO . This is achieved by cycling the electrode potential between -0.3 and +1.5

N versus a silver/ silver chloride reference electrode until a reproducible cyclic voltammogram is obtained. The gold electrodes are then modified immediately by immobilisation of the capture nucleic acid molecule thereon.

The capture nucleic acid molecule consists of a 20-mer oligonucleotide having a mercaptoalkyl linker at the 3' end. The 20-mer oligonucleotide is specific for the p53 gene, wherein the nucleotide sequence is: 5'-GGG GCA GTG CCT CAC AAC CT-3'

(SEQ ID ΝO:l). The oligonucleotide is attached to the gold substrate by self-assembly, via the thiol groups. In the self-assembly of thiols onto metal surfaces, exposure of the metal surface to a solution containing the terminal thiol species results in the thiol groups forming pseudocovalent bonds with the gold surface, to produce a monolayer. For attachment of the oligonucleotide, the cleaned gold electrode is exposed to a 4 μM solution of thiol oligonucleotide in 1 M phosphate buffer, pH 4.5, for 2V_ hours. After adsorption of the oligonucleotide, and rinsing with Milli-Q (18 MΩ cm) water, the electrode is immersed in a 0.1 M aqueous solution of an alkanethiol for 1 hour, rinsed in Milli-Q (18 MΩ cm) water and stored in a 0.3 M sodium chloride and 50 mM phosphate buffer, pH 7.0, until needed.

A reference electrode (a silver/ silver chloride electrode), counter electrode (platinum) and electrochemical instrumentation (BAS 100B potentiostat) are used during the operation of the apparatus. The electrochemistry is performed in a 0.3 M sodium chloride and 50 mM phosphate buffer, pH 7.0.

Hybridisation with target DNA is carried out in a 1 M sodium chloride and 10 mM Tris[hydroxtmethyl]aminomethane (TRIZMA) buffer, pH 7.0. The 20-mer target DNA is injected into the buffer solution and the apparatus is immersed in this for 2J hours. Example 2 Fabrication of a label-free DNA biosensor using a 2- mercaptoethanol (MCE) diluent layer and hybridisation to homogeneous complementary target DNA.

1. Construction of apparatus Polycrystalline gold electrodes are polished with 1.0, 0.3 and 0.05 μm alumina water slurries on polishing clothes. The electrodes are sonicated in an ultrasonic bath and then cleaned further by cycling between the potentials -0.3 V and + 1.5 V versus Ag/AgCl in 0.05 M H SO solution at a scan rate of 150 mVs"1 until a reproducible cyclic voltammograms are obtained. The gold electrodes are then modified immediately by immobilisation of the capture nucleic acid molecule thereon.

The capture nucleic acid molecule consisted of a 20-mer oligonucleotide having a 3-mercaptopropyl linker at the 3 '-end wherein the sequence is 5'-GGG GCA GTG CCT CAC AAC CT-3' (SEQ ID NO:l). The attachment of the oligonucleotide to the gold substrate is achieved by self-assembly, via the thiol groups at the 3 '-end of oligonucleotide. For attachment of thiolated oligonucleotide, the cleaned gold surface is exposed to a 4 μM solution of thiol oligonucleotide in immobilization buffer (I M KH2PO , pH 4.5) for 90 minutes and then rinsed thoroughly in Milli-Q (18 MΩ cm) water. The electrodes with oligonucleotide attached were then immersed in a lmM solution of 2-mercaptoethanol (MCE) for 60 minutes to form a diluent layer. The electrodes were subsequently rinsed thoroughly in Milli-Q (18 MΩ cm) water. The label- free apparatus is now ready for use.

2) Detection of tarfiet nucleic acid molecule

The apparatus was used with a reference electrode (Ag/AgCl electrode), counter electrode and electrochemical instrumentation (BAS 100B potentiostat). All potentials are quoted relative to the Ag/AgCl reference electrode. The oligonucleotide modified electrode is cycled in a potential range between 0 and +0.6 V and the current recorded. In the absence of the complementary strand to the nucleotide sequence of the oligonucleotide, no peak was observed in the cyclic voltammogram. Such cycling prior to hybridisation acts as a background to show that any current signal observed after hybridisation is caused by the actual hybridisation event.

Hybridisations were performed at room temperature and proceed for 150 minutes by exposing the electrode with oligonucleotide attached to 4 μM solutions of target nucleic acid molecule, i.e. a 20-mer oligonucleotide that includes a nucleotide sequence complementary to the nucleotide sequence of the oligonucleotide, i.e. 5'-AGG TTG TGA GGC ACT GCC CC-3'(SEQ ID NO:2). The potential of the oligonucleotide-modified electrode was cycled between 0 V and +0.6 V versus Ag/AgCl reference electrode, to monitor the hybridisation as it occurs. The appearance of an oxidation peak at +0.2 V and reduction peak at +0.15 V indicates that hybridisation has occurred. The faradaic currents observed were due to the electrochemistry of the gold-sulphur bond (i.e. from MCE), assisted by ions in the solution (Figure 2)

Example 3 Fabrication of a label-free DNA biosensor using 2- mercaptoethanol (MCE) diluent layer and hybridisation to homogeneous non-complementary target DNA.

An apparatus is prepared as described in Example 2, using a MCE diluent layer and with the same capture nucleic acid molecule.

To test the ability of the apparatus to accurately record specific hybridisation events, the capture nucleic acid molecule of the apparatus is hybridised, using the conditions described in Example 2. However the target nucleic acid molecule in the analyte solution is substituted for 20-mer non-complementary oligonucleotide including the nucleotide sequence, i.e. 5'-GGA TGG ACG AAG CGC TCA GG-3' (SEQ ID NO: 5).

Cycling of the potential of the sensing electrode is performed 150 minutes after injection of the target nucleic acid molecule. No current signal at +0.2 V (oxidation peak) and +0.15 V (reduction peak) were observed indicating the absence of hybridisation of the non-complementary sequences to the capture nucleic acid molecule.

Example 4 Fabrication of DNA biosensor using a non-specifically bound 3,6-diaminodurene (DAD) label, hybridisation to homogeneous complementary target DNA and denaturation of DNA duplex to recover single stranded oligonucleotide biorecognition element.

1) Construction of an apparatus for detecting p53 gene sequences

An apparatus is prepared as described in Example 1. The mercaptoalkyl linker at the 3' end of the capture nucleic acid molecule (an oligonucleotide) is a 6-mercaptohexyl linker. The secondary alkanethiol solution is an aqueous solution of 0.1 M 6- mercaptohexanol.

The apparatus is immersed in an aqueous solution of 10 mM DAD (3,6- diaminodurene), 10 mM EDC (N-ethyl-N-[dimethylaminopropyl] carbodiimide) and 10 mM Melm (1-methylimidazole), adjusted to pH 7.5 with hydrochloric acid, for 5 hours at

60°C in a humid chamber. This causes the DAD to non-specifically bind to the capture nucleic acid molecule. It is subsequently rinsed with 0.25% Tween20®, 5 x SSC buffer, preheated to 60°C and then with Milli-Q (18 MΩ cm) water at room temperature.

Electrochemistry is performed as described in Example 1 by cycling the potential between -0.2 and +0.6 N versus the silver/ silver chloride reference electrode. Due to the persistence length of the capture nucleic acid molecule, electrochemistry of the DAD is observed between -0.05 and +0.1 N (Figure 4).

2) Detection of p53 gene sequences in solution

Hybridisation is carried out as described in Example 1. Excess target DΝA (5 x 10"10 mol) is used so that 100% hybridisation occurs. Hybridisation causes the capture nucleic acid molecule extending into solution approximately normal to the electrode so that the DAD cannot come into contact with the electrode.

Electrochemistry is performed as described previously in this example. No electrochemistry is observed in the -0.05 to +0.1 V region (Figure 4). 3) Denaturation of target and capture nucleic acid molecule complex

Denaturation of the target and capture nucleic acid molecule complex is carried out at 70°C in Milli-Q (18 MΩ cm) water for 10 minutes. The apparatus is subsequently rinsed with Milli-Q (18 MΩ cm) water at room temperature. Electrochemistry is performed as described previously in this example. Electrochemistry of the DAD is observed between -0.05 and +0.1 N due to the removal of the target nucleic acid molecule from the capture nucleic acid molecule subsequent to denaturation (Figure 4). Example 5 Fabrication of DΝA rigidity biosensor with a redox label attached to the end of the probe oligonucleotide and using a 6- mercaptohexanol (MCH) diluent layer, hybridisation to homogenous complementary DΝA.

The capture nucleic acid molecule consisted of a 20-mer oligonucleotide having a 3-mercaptopropyl linker at the 3 '-end and an aminononyl linker at the 5 '-end. The capture nucleic acid molecule is attached to the gold substrate by self-assembly, via the thiol groups, h the self-assembly of thiols onto metal surfaces, exposure of the metal to a solution containing the terminal thiol species results in the thiol groups forming a pseudocovalent bond with the gold surface, to produce a monolayer. For attachment of thiol oligonucleotide the cleaned gold surface is exposed to 1 μM solution of thiol oligonucleotide in phosphate buffer (immobilization buffer, pH 6.7) for 150 minutes.

After rinsing with Milli-Q water, the self-assembly of thiols onto the gold electrode surface is achieved by incubating the electrodes in a solution of thiol such as mercaptohexanol (MCH) or mercaptoundecanol (MCU) for 30 minutes, rinsed in 18 MΩ water and stored in 18 MΩ water until ready for the redox-ac five label Rh(phi)2(bpy')3+.

For the attachment of the Rh(phi)2(bpy')3+ label, purified Rh(phi)2(bpy')3+ is prepared as described by Pyle, A. M.; Chiang, M. Y.; Barton, J. K. Inorg. Chem. 1990, 29, 4487-4495., activated to an N-succinimidyl ester and attached to the amine terminal of the immobilised biorecognition oligonucleotide. Briefly, the complex is dissolved in a pH 5.5 3-[Ν-Morpholino]propanesulfonic acid (MOPS) buffer solution containing 2mM l-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 5mM N- hydroxysuccinimide (ΝHS), and incubated therein for at least 2.5 h. The electrode is washed extensively in buffer solution. The apparatus is now ready for use. The apparatus developed in this protocol is believed to have a surface density of capture nucleic acid molecules of approximately 5 x 10"12 mol cm"2 of electrode. A reference electrode (a silver/ silver chloride electrode) counter electrode and electrochemical instrumentation (BAS 100B potentiostat) is used with the operation of the apparatus. All potentials are quoted relative to the Ag/AgCl reference electrode. The oligonucleotide modified electrode is cycled in a potential range between -0.6 and +0.6 N and the current recorded. In the absence of the complementary strand to the nucleotide sequence of the capture nucleic acid molecule, a large peak at approximately -0.3N was observed. Such cycling prior to hybridisation provides a background signal to be subtracted after hybridisation.

Hybridisation is monitored in a buffer solution containing 5 nM Tris(hydroxy methyl)amino methane buffer (pH 7.1) and 20 nM sodium chloride. The electrodes are incubated in the buffer solution, containing the 20-mer sequence of the target nucleic acid molecule, for at least 2.5 hours (at room temperature), to allow hybridisation. Once hybridisation has occurred there is also a concomitant diminution of the large redox peak at -0.3N (Figure 5). Note also the peaks associated with the labelless process of examples 2 and 3 can also be observed in Figure 5.

Example 6 Materials and methods

Materials: 6-Mercapto-l-hexanol (MCH) and 2,6-anthraquinonedisulfonic acid

(AQS) were purchased from Aldrich Chemicals (Sydney, Australia). Tris-HCl was purchased from Sigma (Sydney, Australia). Reagent grade K2HPO4, KH2PO4, NaCl, KC1, NaOH, HC1 and absolute ethanol were purchased from Ajax Chemicals Pty. Ltd. (Sydney, Australia). All reagents were used without further purification. All solutions were prepared using Milli-Q purified water. The 20-mer deoxyoligonucleotides were purchased from Genset Oligos Pacific Pty. Ltd (Sydney, Australia). Their base sequences are as follows: Thiolated DNA probe (20-base sequence DNA 1 (SEQ ID NO:l)): 5'-

GGGGCAGTGCCTCACAACCT-/ -(CH2)3-SH-3'

Complementary DNA target (20-base sequence DNA 2 (SEQ ID NO:2)): 5'- AGGTTGTGAGGCACTGCCCC-3 '

C-A mismatch DNA target (20-base sequence DNA 3 (SEQ ID NO:3)): 5'- AGGTTGTGAGGCCCTGCCCC-3' G-A mismatch DNA target (20-base sequence DNA 4 (SEQ JD NO:4)): 5'- AGGTTGTGAGGCGCTGCCCC-3 '

Non-complementary DNA target (20-base sequence DNA 5 (SEQ ID NO:5)): 5'- GGATGGACGAAGCGCTCAGG-3 ' All oligonucleotide stock solutions were prepared with lOmM Tris-HCl, (pH

8.00) and stored in a-80°C freezer until use.

Solution Preparation: Several buffers were used in this work. Immobilisation buffer contained 1 M KH2PO (pH 4.5); hybridisation buffer contained 1 M NaCl, 10 mM Tris-HCl (pH 7.0); phosphate buffer in which electrochemical experiments were performed contained 3 M NaCl, 50mM K2HPO4/ KH2PO4 (pH 7.0). The l.OmM MCH solution used was made by dissolving MCH in ethanol. 1 mM AQS stock solution contained 0.2 M KC1 and 50 mM KH PO4 (pH 5.5). The pH was adjusted with either NaOH or HC1 solution. Milli-Q water and all buffers were autoclaved.

Electrochemical measurements: All electrochemical measurements were performed with a BAS 100B electrochemical analyser (Bioanalytical System Inc.,

Lafayette, Indiana, USA). Osteryoung square wave voltammery (OSWV) were carried out with a conventional three-electrode system, including a bare or modified gold working electrode, a platinum auxiliary electrode and an Ag/AgCl/3.0 M NaCl reference electrode (from Bioanalytical System Inc., Lafayette, Indiana, USA). All potentials are reported versus Ag/AgCl reference at room temperature. The solution was degassed with Ar for approximately 15 min prior to data acquisition and was blanketed under Ar atmosphere during the entire experimental period. Osteryoung square wave voltammetry was conducted at a pulse amplitude of 25 mV, a step of 4mV and a frequency of 10 Hz. OSW voltammograms were measured between -250 mV and -650 mV. Electrode Preparation: Bulk gold electrodes were prepared by sealing polycrystalline gold wire (>99.99°/o gold, Aldrich) in 4 mm diameter glass tubes, followed by the attachment of nichrome wires for electrical connection to the back of the electrodes. The electrodes were first polished with 1.0 μm alumina, followed by 0.3 and 0.05 μm alumina slurry on micro cloth pads (Buehler, Lake Bluff, IL, USA). After removal of the trace alumina from the surface by rinsing with Milli-Q water and brief cleaning in an ultrasonic bath, the electrodes were further cleaned by electrochemical etching in 0.05 M H2SO4 by cycling the electrode potential between -0.3 and +1.5 N until a reproducible cyclic voltammogram was obtained. The cleaned gold electrodes were modified by incubation in 4 μM solutions of thiolated oligonucleotide 1 in immobilisation buffer for 150 min at room temperature. The modified gold electrodes were rinsed with water prior to adsorption of MCH monolayer. MCH monolayer was adsorbed by immersing the ssDΝA modified gold electrodes in 1 mM MCH ethanolic solution for 60 min, followed by rinsing with absolute ethanol. Figure 6 shows schematically the DΝA recognition layer and subsequent hybridization and intercalation of AQS which allows electrochemical detection of hybridisation.

Hybridisations were performed at room temperature by immersing the ssDΝA/MCH modified gold electrodes in 4 μM solutions of target DΝA in hybridisation buffer, followed by rinsing with phosphate buffer.

Detection of hybridisations was performed at room temperature by immersing the double-stranded DΝA (dsDΝA) covered surface (dsDΝA/MCH modified gold electrodes) in 1 mM AQS solution for overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution for the subsequent voltametric experiments by OSWN.

Example 7 Fabrication of DΝA biosensor using a 6-mercap tohexanol (MCH) insulating layer and hybridisation to homogeneous complementary target DΝA 2 The DΝA interface was fabricated as described above. Prior to hybridisation the single strand modified electrodes were exposed to 1 mM AQS, rinsed in phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry (OSWN) was used to measure the accumulation of AQS at the DΝA modified electrode surface. Figure 7(i) shows the OSWN response to AQS. It is clear from the voltammogram that no electrochemistry due to the AQS, characteristically observed in the potential window of -250 mV to -650 mN versus Ag/AgCl, was observed.

The electrode was transferred to a solution containing 4 μM of target DNA 2 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded

DNA (dsDNA) covered surface (dsDNA/MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. The OSWN current signal observed due to AQS is now clearly visible in figure 7(ii). The presence of this current signal indicates that a DΝA duplex has formed, that AQS has intercalated into the duplex and now long range electron transfer between the AQS and the electrode can occur.

Example 8 Fabrication of DΝA biosensor using a 6-mercaptohexanol (MCH) insulating layer and hybridisation to different concentration of homogeneous complementary target DΝA 2

The DΝA interface was fabricated as described above. Prior to hybridisation the single strand modified electrodes were exposed to 1 mM AQS, rinsed in phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry (OSWN) was used to measure the accumulation of AQS at the DΝA modified electrode surface. As with the previous example no electrochemistry due to the AQS, was observed.

The electrode was transferred to a solution containing 0.25 μM of target DΝA 2 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded

DΝA (dsDΝA) covered surface (dsDΝA/MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. An OSWN current signal observed due to AQS, similar to that seen in the previous example but of lower current magnitude was observed. The presence of this current signal indicates that a DNA duplex has formed, that AQS has intercalated into the duplex and now long range electron transfer between the AQS and the electrode can occur.

Subsequently a new electrode modified with probe DNA was transferred to a solution containing 0.5 μM of target DNA 2 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded DNA (dsDNA) covered surface (dsDNA MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. An increased OSWN current signal due to AQS was observed relative to the 0.25 μM target 2. The procedure was repeated for target 2 concentrations of 1, 2, 3, 4, 5, 6, and 10 μM. The variation in current response with the concentration of target is shown in figure 8. The ability to investigate solutions with different concentrations of target shows that the invention can be used to determine concentrations of target DΝA in solution and also show that complete hybridisation of the probe DΝA is not necessary for use of the apparatus.

Example 9 Fabrication of DΝA biosensor using a 6-mercaptohexanol (MCH) insulating layer and hybridisation to homogeneous noncomplementary target DΝA 5

The DΝA interface was fabricated as described above. Prior to hybridisation the single strand modified electrodes were exposed to 1 mM AQS, rinsed in phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry (OSWN) was used to measure the accumulation of AQS at the DΝA modified electrode surface. Figure 9(i) shows the OSWN response to AQS. It is clear from the voltammogram that no electrochemistry due to the AQS was observed.

The electrode was transferred to a solution containing 4 μM of the noncomplementary target DΝA 5 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded DΝA (dsDΝA) covered surface (dsDNA/MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. The OSWN clearly shows to electrochemistry due to AQS in figure 9(ii). The absence of this AQS current signal indicates that DΝA duplexes have not formed. Hence the AQS has not been able to intercalate and no long range electron transfer between the AQS and the electrode can occur. This example demonstrates the ability of the apparatus to differentiate between complementary and noncomplementary target sequences.

Example 10 Fabrication of DΝA biosensor using a 6-mercaptohexanol (MCH) insulating layer and hybridisation to homogeneous target DΝA 3 containing a C-A mismatch

The DΝA interface was fabricated as described above. Prior to hybridisation the single strand modified electrodes were exposed to 1 mM AQS, rinsed in phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry (OSWN) was used to measure the accumulation of AQS at the DΝA modified electrode surface. Figure 10(i) shows the OSWN response to AQS. It is clear from the voltammogram that no electrochemistry due to the AQS was observed. The electrode was transferred to a solution containing 4 μM of the noncomplementary target DΝA 3 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded DΝA (dsDΝA) covered surface (dsDΝA/MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. The OSWN shows a small current signal due to AQS, figure 10(ii). This current signal is significantly suppressed relative to when the DΝA modified electrode is exposed to complementary sequence 2 as seen in example 1. The suppression of this AQS current signal indicates that DΝA duplexes have formed, the AQS has been able to intercalate but the ability of long range electron transfer to occur between the electrode and the AQS has greatly diminished. This example demonstrates the ability of the apparatus to differentiate between complementary and C-A mismatched target sequences. Example 11 Fabrication of DNA biosensor using a 6-mercaptohexanol

(MCH) insulating layer and hybridisation to homogeneous target DNA 4 containing a G-A mismatch

The DNA interface was fabricated as described above. Prior to hybridisation the single strand modified electrodes were exposed to 1 mM AQS, rinsed in phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry (OSWN) was used to measure the accumulation of AQS at the DΝA modified electrode surface. Figure ll(i) shows the OSWN response to AQS. It is clear from the voltammogram that no electrochemistry due to the AQS was observed.

The electrode was transferred to a solution containing 4 μM of the noncomplementary target DΝA 4 in hybridisation buffer for two hours, followed by rinsing with phosphate buffer. Detection of hybridisations was performed at room temperature by immersing the double-stranded DΝA (dsDΝA) covered surface (dsDΝA/MCH modified gold electrodes) in 1 mM AQS solution overnight for the intercalation of AQS into the duplexes. After the accumulation, the modified gold electrodes were rinsed with phosphate buffer and then transferred to an AQS free phosphate buffer solution. Osteryoung square wave voltammetry was used for the measurement of intercalation and hence hybridisation. The OSWV shows a small current signal due to AQS, figure l l(ii). This current signal is significantly suppressed relative to when the DΝA modified electrode is exposed to complementary sequence 2 as seen in example 1. The current signal observed however is greater than that observed with the same concentration of target 3 with a C-A mismatch as seen in example 4. The suppression of this AQS current signal indicates that DΝA duplexes have formed, the AQS has been able to intercalate but the ability of long range electron transfer to occur between the electrode and the AQS has been diminished relative to a perfectly complementary sequence but enhanced relative to a C-A mismatch. As a G-A mismatch is as thermodynamically stable as a complementary sequence, and hence this is the most difficult of mismatches to detect, this example demonstrates the ability of the apparatus to differentiate between complementary target sequences and target sequences containing a single base pair mismatch. This differentiation is achieved without any elecfrocatalysis.

Claims

Claims
1. An apparatus for detecting a target nucleic acid molecule including a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species and a reaction means for reaction with the redox species, wherein the capture nucleic acid molecule is capable of arranging the redox species and the reaction means relative to each other to alter the reaction of the redox species with the reaction means when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule.
2. An apparatus according to claim 1 wherein the capture nucleic acid molecule arranges the redox species and the reaction means when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule, by moving the redox species and the reaction means into engagement.
3. An apparatus according to claim 2 wherein the capture nucleic acid molecule moves the redox species into engagement with the reaction means.
4. An apparatus according to claim 1 wherein the capture nucleic acid molecule arranges the redox species and reaction means relative to each other for improving reaction of the redox species and reaction means when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule.
5. An apparatus according to claim 1 wherein the reaction means is arranged on the surface to which the capture nucleic acid molecule is attached.
6. An apparatus according to claim 1 wherein the reaction means provides the surface to which the capture nucleic acid molecule is attached
7. An apparatus according to claim 5 wherein the reaction means includes a component selected from the group consisting of gold, glassy carbon, platinum, silver, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.
8. An apparatus according to claim 1 wherein the redox species is arranged on or attached to the capture nucleic acid molecule.
9. An apparatus according to claim 8 wherein the redox species is attached to the terminus of the capture nucleic acid molecule.
10. An apparatus according to claim 9 wherein the redox species is Rh(phi)2(bpy'), where phi is 9,10-diimine phenathrenequinone and bpy' is butyric acid 4' methylbipyridine.
11. An apparatus according to claim 1, further including detector means for detecting a signal associated with the reaction of the redox species with the reaction means.
12. An apparatus according to claim 1, further including means for limiting adsorption of the capture nucleic acid molecule to the surface to which the capture nucleic acid molecule is attached.
13. An apparatus according to claim 12 wherein the means for limiting adsorption of the capture nucleic acid molecule includes a plurality of alkanethiol molecules.
14. An apparatus according to claim 13 wherein the alkanethiol molecules are selected from the group consisting of mercaptopropanol, mercaptoethanol and mercaptohexanol.
15. An apparatus according to claim 1 wherein the capture nucleic acid molecule is selected from the group consisting of DNA, RNA, molecules including both DNA and RNA and PNA molecules
16. An apparatus according to claim 15 wherein the capture nucleic acid molecule is at least 8 nucleotides in length.
17. An apparatus according to claim 16 wherein the capture nucleic acid molecule includes a nucleotide sequence that is identical to the complementary strand of the target nucleic acid molecule.
18. An apparatus according to claim 17 wherein the capture nucleic acid molecule is attached to the surface via a 5' or 3' terminus of the molecule.
19. An apparatus for detecting a target nucleic acid molecule including a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a reaction means, a redox species in use having a charge to attract the redox species to contact the reaction means, wherein the capture nucleic acid molecule is capable of displacing the redox species and the reaction means from each other for limiting reaction of the redox species with the reaction means when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule.
20. An apparatus according to claim 19 wherein the capture nucleic acid molecule displaces the redox species from the reaction means for limiting reaction with the reaction means when the capture nucleic acid molecule is hybridised to the target nucleic acid molecule.
21. An apparatus according to claim 19 wherein the reaction means is arranged on the surface to which the capture nucleic acid molecule is attached.
22. An apparatus according to claim 19 wherein the reaction means provides the surface to which the capture nucleic acid molecule is attached.
23. An apparatus according to claim 22 wherein the reaction means includes a component selected from the group consisting of gold, glassy carbon, platinum, silver, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.
24. An apparatus according to claim 19 wherein the redox species is arranged on or attached to the capture nucleic acid molecule.
25. An apparatus according to claim 24 wherein the redox species is attached to the tenninus of the capture nucleic acid molecule.
26. An apparatus according to claim 25 wherein the redox species is diaminodurene.
27. An apparatus according to claim 19, further including detector means for detecting a signal associated with the reaction of the redox species with the reaction means.
28. An apparatus according to claim 19, further including means for limiting adsorption of the capture nucleic acid molecule to the surface to which the capture nucleic acid molecule is attached.
29. An apparatus according to claim 28 wherein the means for limiting adsorption of the capture nucleic acid molecule includes a plurality of alkanethiol molecules.
30. An apparatus according to claim 29 wherein the alkanethiol molecules are selected from the group consisting of mercaptopropanol, mercaptoethanol and mercaptohexanol.
31. An apparatus according to claim 19 wherein the capture nucleic acid molecule is selected from the group consisting of DNA, RNA, molecules including both DNA and RNA and PNA molecules
32. An apparatus according to claim 31 wherein the capture nucleic acid molecule is at least 8 nucleotides in length.
33. An apparatus according to claim 32 wherein the capture nucleic acid molecule includes a nucleotide sequence that is identical to the complementary strand of the target nucleic acid molecule.
34. An apparatus according to claim 33 wherein the capture nucleic acid molecule is attached to the surface via a 5' or 3' terminus of the molecule.
35. An apparatus for detecting a target nucleic acid molecule including a capture nucleic acid molecule for hybridising to a target nucleic acid molecule and being attached to a surface, a redox species and a reaction means for reaction with the redox species, wherein the redox species is arranged relative to the reaction means for reaction of the redox species with the reaction means when the redox species is contacted with a reaction species capable of oxidising or reducing the redox species, wherein the capture nucleic acid molecule permits a reaction species to contact the redox species when the capture nucleic acid molecule is hybridised to a target nucleic acid molecule and wherein the capture nucleic acid molecule limits contact of a reaction species with the redox species when the capture nucleic acid molecule is not hybridised to a target nucleic acid molecule.
36. An apparatus according to claim 35 wherein the redox species is arranged on the reaction means.
37. An apparatus according to claim 36 wherein the redox species includes a thiol group for reaction with the reaction means.
38. An apparatus according to claim 35 wherein the reaction means includes gold.
39. An apparatus according to claim 35, further including detector means for detecting a signal associated with the reaction of the redox species with the reaction means.
40. An apparatus according to claim 35 wherein the surface for attachment of the capture nucleic acid molecule is a detector for detecting an electric current associated with the reaction of the redox species with the reaction means.
41. An apparatus according to claim 40 wherein the detector is typically an electrode.
42. An apparatus according to claim 41 wherein the electrodes includes a component selected from the group consisting of gold, platinum, silver, glassy carbon, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.
43. An apparatus for detecting a target nucleic acid molecule including a capture nucleic acid molecule for hybridising to a target nucleic acid molecule, a surface for receiving an electric signal, a plurality of repellent molecules arranged on the surface for limiting adsorption of the capture nucleic acid molecule to the surface, a linker for linking the capture nucleic acid molecule to the surface to permit an electric signal to be received by the surface from the linker and a redox species in use having a charge for repelling the redox species from the plurality of repellent molecules on the surface.
44. An apparatus according to claim 43 wherein the redox species in use has a charge for repelling the species from the surface sufficient for inhibiting contact of the redox species with the surface.
45. An apparatus according to claim 43 wherein the redox species in use has a negative charge.
46. An apparatus according to claim 43 wherein the redox species is arranged on the apparatus in a form so that the redox species can be solubilised to permit intercalation of the species into a duplex formed between capture and target nucleic acid molecules, when the apparatus is contacted with a sample.
47. An apparatus according to claim 43 wherein the capture nucleic acid molecule has a sequence that is identical to the complementary strand of the target nucleic acid molecule.
48. An apparatus according to claim 43 wherein the capture nucleic acid molecule is at least about 8 nucleotides in length.
49. An apparatus according to claim 43 wherein the capture nucleic acid molecule is selected from the group consisting of DNA, RNA, molecules including both DNA and RNA and PNA molecules
50. An apparatus according to claim 43 wherein the repelling molecules are alkanethiol molecules
51. An apparatus according to claim 50 wherein the repelling molecules include mercaptopropanol, mercaptoethanol and mercaptohexanol.
52. An apparatus according to claim 43 wherein the capture nucleic acid molecule is typically attached to the surface via a 5' or 3' terminus of the molecule.
53. An apparatus according to claim 43, further including detector means for detecting a signal associated with the reaction of the redox species with the reaction means.
54. An apparatus according to claim 43 wherein the surface for attachment of the capture nucleic acid molecule is a detector for detecting an electric current associated with the reaction of the redox species with the reaction means.
55. An apparatus according to claim 54 wherein the detector is an electrode.
56. An apparatus according to claim 55 wherein the electrode includes a component selected from the group consisting of gold, platinum, silver, glassy carbon, vitreous carbon, carbon paste, carbon fibre, carbon black, pyrolytic carbon, indium tin oxide, iridium, tungsten, mercury, a metal oxide and an organic salt.
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