WO2008097190A1 - Procédés pour détecter électriquement un acide nucléique par l'intermédiaire d'une paire d'électrodes - Google Patents

Procédés pour détecter électriquement un acide nucléique par l'intermédiaire d'une paire d'électrodes Download PDF

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WO2008097190A1
WO2008097190A1 PCT/SG2007/000037 SG2007000037W WO2008097190A1 WO 2008097190 A1 WO2008097190 A1 WO 2008097190A1 SG 2007000037 W SG2007000037 W SG 2007000037W WO 2008097190 A1 WO2008097190 A1 WO 2008097190A1
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nucleic acid
molecule
target nucleic
sample
acid molecule
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Zhiqiang Gao
Yi Fan
Xiantong Chen
Jinming Kong
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors

Definitions

  • the present invention relates to methods of electrically detecting a target nucleic acid molecule by means of an electrode pair.
  • nucleic acids The detection and quantification of nucleic acids is a fundamental method not only in analytical chemistry but also in biochemistry, food technology or medicine.
  • the most frequently used methods for determining the presence and concentration of nucleic acids include the detection by autoradiography, fluorescence, chemiluminescence or bioluminescence as well as electrochemical and electrical techniques.
  • nucleic acid biosensors based on the electrical detection are able to provide high performance in terms of accuracy and sensitivity as well as low-cost, miniaturised readout unit and thus exempt from the problems encountered in the optical detection system, hi addition, combining with microfabrication technology and microelectronics, microscale electrical detection-based biosensors can be easily constructed, such as silicon nanowire biosensors 1.
  • Mirkin and co- workers also put forward a successful sample of electrical detection of DNAs with paired microelectrodes separated by a microgap and gold nanoparticle-conjugated oligonucleotide detection probes (Park et al., 2002, supra).
  • a technique for the specific detection of a selected nucleic acid well established in the art is based on the hybridisation between a nucleic acid capture probe and a target nucleic acid. Typically the respective nucleic acid capture probe is immobilised onto a solid support, and subsequently one of the above mentioned detection methods is employed.
  • Sensitivity and selectivity are the two important issues being constantly addressed in the evaluation of nucleic acid detection systems.
  • optical transduction engages not only highly precise and expensive equipment but also complex algorithms to interpret the data.
  • off-chip target amplifications also significantly increase the cost of the procedures and often lead to sequence-dependent quantification bias.
  • in situ signal amplification strategies such as rolling circle amplification (RCA), T7 DNA polymerase, branched DNA technology, catalysed reporter deposition, dendritic tags, enzymatic amplification and chemical amplification
  • RCA rolling circle amplification
  • T7 DNA polymerase T7 DNA polymerase
  • branched DNA technology branched DNA technology
  • catalysed reporter deposition catalysed reporter deposition
  • dendritic tags enzymatic amplification and chemical amplification
  • the invention provides methods of electrically detecting a target nucleic acid molecule by means of a pair of electrodes.
  • the electrodes are arranged at a distance from one another. Furthermore the pair of electrodes is arranged within a sensing zone.
  • a first respective method includes immobilising on an immobilisation unit a peptide nucleic acid (PNA) capture molecule.
  • the PNA capture molecule has a nucleotide sequence that is at least partially complementary to at least a portion of a strand of the target nucleic acid molecule.
  • the immobilisation unit is arranged within the sensing zone.
  • the method further includes contacting the immobilisation unit with a solution suspected to comprise the target nucleic acid molecule.
  • the method also includes allowing the target nucleic acid molecule to hybridise to the PNA capture molecule on the immobilisation unit. Thereby the method includes allowing the formation of a complex between the PNA capture molecule and the target nucleic acid molecule.
  • the method includes adding a polymerisable positively chargeable precursor.
  • the polymerisable positively chargeable precursor has an electrostatic net charge that is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the polymerisable positively chargeable precursor associates to the complex formed between the PNA capture molecule and the target nucleic acid molecule.
  • the polymerisation of the precursor can be carried out by means of a suitable reactant molecule.
  • the method further includes adding a suitable reactant molecule, thereby initiating the polymerisation of the polymerisable positively chargeable precursor. Thereby an electroconductive polymer (i.e. generally a conducting polymer) is formed from the polymerisable precursor.
  • This electroconductive polymer is associated with the complex formed between the PNA capture molecule and the target nucleic acid molecule. Further, the method includes determining the presence of the target nucleic acid molecule based on an electrical characteristic of a region in between the electrodes. The electrical characteristic is influenced by the electroconductive polymer. [0012] According to a particular embodiment, the method includes exposing the reactant molecule to a suitable catalyst.
  • the catalyst may be light, a metal chloride, a metal bromide, a metal sulphate or an enzyme.
  • the reactant molecule may be a substrate molecule for the catalyst.
  • a second respective method includes immobilising on an immobilisation unit a nucleic acid capture molecule.
  • the nucleic acid capture molecule has a nucleotide sequence that is at least partially complementary to at least a portion of a strand of the target nucleic acid molecule.
  • the immobilisation unit is arranged within the sensing zone.
  • the method further includes contacting the immobilisation unit with a solution suspected to comprise the target nucleic acid molecule.
  • the method also includes allowing the target nucleic acid molecule to hybridise to the nucleic acid capture molecule on the surface of the immobilisation unit. Thereby the method includes allowing the formation of a complex between the nucleic acid capture molecule and the target nucleic acid molecule.
  • the method includes adding a polymerisable positively chargeable precursor.
  • the polymerisable positively chargeable precursor has an electrostatic net charge that is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the polymerisable positively chargeable precursor associates to the complex formed between the nucleic acid capture molecule and the target nucleic acid molecule.
  • the polymerisation of the precursor can be carried out by means of a suitable reactant molecule.
  • the method further includes adding a suitable substrate molecule.
  • the method also includes adding an enzyme attached to a probe nucleic acid molecule.
  • the probe nucleic acid molecule is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • the detection probe hybridises to a portion of the target nucleic acid that is different from the portion to which the nucleic acid capture molecule hybridises.
  • the method thereby includes allowing the probe nucleic acid molecule to hybridise to the target nucleic acid molecule.
  • the method thereby also includes catalysing the polymerisation of the polymerisable positively chargeable precursor.
  • an electroconductive polymer i.e. generally a conducting polymer
  • This electroconductive polymer is associated with the complex formed between the nucleic acid capture molecule and the target nucleic acid molecule.
  • the method includes determining the presence of the target nucleic acid molecule based on an electrical characteristic of a region in between the electrodes. The electrical characteristic is influenced by the electroconductive polymer.
  • the invention provides a kit for electrically detecting of a target nucleic acid molecule.
  • a kit in a first embodiment includes a pair of electrodes.
  • the electrodes are arranged at a distance from one another. Furthermore the pair of electrodes is arranged within a sensing zone.
  • the respective kit further includes an immobilisation unit.
  • the immobilisation unit is arranged within the sensing zone.
  • the kit also includes a PNA capture molecule.
  • This PNA capture molecule has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • the kit also includes a polymerisable positively chargeable precursor.
  • the electrostatic net charge of the polymerisable positively chargeable precursor is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the kit includes a suitable reactant molecule.
  • the kit also includes a catalyst.
  • a kit in a second embodiment, includes a pair of electrodes.
  • the electrodes are arranged at a distance from one another. Furthermore the pair of electrodes is arranged within a sensing zone.
  • the respective kit further includes an immobilisation unit.
  • the immobilisation unit is arranged within the sensing zone.
  • the kit also includes a nucleic acid capture molecule. This nucleic acid capture molecule has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • the kit also includes a polymerisable positively chargeable precursor. The electrostatic net charge of the polymerisable positively chargeable precursor is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the kit includes a suitable substrate molecule.
  • the kit includes an enzyme attached to a probe nucleic acid molecule.
  • the probe nucleic acid molecule is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • Figure 1 depicts a (nano)gapped microelectrodes array that may be used in a method of the present invention.
  • the microarray of Fig. IA includes 10x10 pairs of nanogapped microelectrodes fabricated on 1.2 x 1.2 cm 2 silicon wafer. It was fabricated as a 10x10 array on a silicon chip with 500 nm coating of SiC>2 by standard photolithography.
  • Fig. IB depicts a schematic illustration of the pairs of nanogapped microelectrodes.
  • a power source can be connected via contacting pads (11) so that by applying an electric current an electric field between the microelectrodes can be generated.
  • FIG. 1C shows a SEM (JEOL- 6000 Field emission scan electron microscope) image of the nanogapped microelectrodes.
  • the chip included 100 pairs of interlocking comb-like microelectrodes (gold 15 nm, titanium 10 nm) with 150-200 fingers, each 700 nm wide and 200 ⁇ m long, and with a 300-n ⁇ n gap (12) (appearing dark on the photo).
  • Parallel connection potentiates the sensitivity of e.g. resistance measurements.
  • Figure 2 depicts a schematic representation of an exemplary method of the present invention.
  • a PNA capture molecule (3) is immobilised on an immobilisation unit (5).
  • the immobilisation unit is contacted with a solution suspected or known to include a target nucleic acid molecule (2).
  • the two nucleic acid molecules hybridise.
  • the polymerisable positively charged precursor molecule aniline and the reactant molecule hydrogen peroxide, as well as an optional catalyst, are added.
  • the polymerisable positively charged precursor molecule associates to the complex of PNA capture molecule (3) and target nucleic acid molecule (2).
  • the positively charged precursor polymerises, forming an electroconductive polymer.
  • An electrical characteristic of a region in between a pair of electrodes (1) can be used to determine the presence of the target nucleic acid molecule (2).
  • Figure 3 depicts a schematic representation of a further exemplary method of the present invention.
  • a nucleic acid capture molecule (4) is immobilised on an immobilisation unit (5).
  • the immobilisation unit is contacted with a solution suspected or known to include a target nucleic acid molecule (2).
  • the two nucleic acid molecules hybridise.
  • An enzyme attached to a probe nucleic acid molecule (6) is added.
  • the probe nucleic acid molecule (6) hybridises to the target nucleic acid molecule (2) at a portion different from the portion where the nucleic acid capture molecule (4) hybridises.
  • the polymerisable positively charged precursor molecule aniline and the reactant molecule hydrogen peroxide are added.
  • the polymerisable positively charged precursor molecule associates to the complex of nucleic acid capture molecule (4), target nucleic acid molecule (2) and probe nucleic acid molecule (6).
  • the positively charged precursor polymerises, forming an electroconductive polymer.
  • An electrical characteristic of a region in between a pair of electrodes (1) can be used to determine the presence of the target nucleic acid molecule (2).
  • FIG. 4 illustrates examples of an electrode arrangement (1) and an immobilisation unit (5), on which a nucleic acid capture molecule (4) is immobilised.
  • Fig. 4A Three ring-shaped electrodes (1) are provided.
  • the immobilisation unit (5) is arranged in such a way that its location partly overlaps with the region between two of the electrodes (1).
  • Fig. 4B Two interdigital electrodes (1) are provided.
  • the immobilisation unit (5), on which a nucleic acid capture molecule (4) is immobilised is arranged in vicinity to the electrodes (1) such that the electrical characteristics of the region within electrode areas opposing each can be influenced by a complex formed between an immobilised nucleic acid molecule and the electrically conducting polymer.
  • Fig. 4A Three ring-shaped electrodes (1) are provided.
  • the immobilisation unit (5) is arranged in such a way that its location partly overlaps with the region between two of the electrodes (1).
  • Fig. 4B Two interdigital electrodes (1) are provided
  • FIG. 4 C depicts in top view an arrangement of two interdigital electrodes (1) that resembles the arrangement depicted in Fig. 4B.
  • the immobilisation unit (5) on which a nucleic acid capture molecule (4) is immobilised, is arranged below the electrodes (1).
  • Fig. 4D An array of electrodes (1) is provided. The array of electrodes (1) defines a region in between them.
  • the immobilisation unit (5) is arranged in vicinity to the electrodes (1), so that the nucleic acid capture molecule (4) is capable of taking an orientation in which its location partly overlaps with the region between two of the electrodes (1).
  • a nucleic acid molecule (not shown) hybridising with the nucleic acid capture molecule (4) will therefore typically take an orientation in which it is essentially located in the region defined by the array of electrodes (1).
  • Figure 5 shows examples of aromatic amines that may serve as a substrate molecule: A: Aniline, Chemical Abstracts No. 62-53-3; B: 3-methylaniline, Chemical Abstracts No. 108-44-1; C: 3,4- ⁇ yridinediamine, CAS-No. 54-96-6; D: 5-(5-oxazolyl)-3- pyridinamine, CAS-No. 893566-28-4; E: 1-aminodibenzofuran, CAS-No. 50548-40-8; F: 4- amino-fluoren-9-one, CAS-No. 4269-15-2; G: 4-amino-2-phenyl-indene-l,3(2H)-dione, CAS-No.
  • H 2-acetyl-4-amino-indene ⁇ l,3(2H)-dione, CAS-No. 25125-06-8;
  • I fluorine- 1,9-diamine, CAS-No 15824-95-0;
  • J l-amino-fluoren-9-ol, CAS-No. 6957-58-0;
  • K 9-anthraceneamine, CAS-No. 779-03-3;
  • L lO-phenyl-9-anthraceneamine, CAS-No 1718-54-3;
  • M 4-[[4-(aminomethyl)phenyl]methoxy]-2-pyrimidinamine, CAS-No.
  • N 4,l l-diamino-naphth[2,3-f]isoindole-l,3,5,10(2H)-tetrone, CAS-No. 128-81-4; O: pyrrole, CAS-No. 109-97-7; P: 3-methanamine- ⁇ yrrol, CAS-No. 888473-50-5; Q: na ⁇ ht[2,3- fjisoindole, CAS-No. 259-05-4; R: 2-(lH-indol-5-yl)-pyrrolo[2,3-b]pyridin-4-ol, CAS-No.
  • Figure 6 depicts a comparison of the conductivity of the space defined by the distance between a pair of electrodes used in the method of the present invention.
  • Blank A PNA capture molecule was immobilised on an immobilisation unit, but not exposed to a target nucleic acid molecule, and neither to the positively chargeable precursor aniline;
  • Control A PNA capture molecule was immobilised on an immobilisation unit and a solution that included a non-complementary DNA molecule contacted therewith.
  • the positively chargeable precursor aniline, horseraddish peroxidase and hydrogen peroxide were added.
  • the formed electroconductive polyaniline polymer was contacted with the enhancer reagent HCl (doped) before determining the presence of the target nucleic acid molecule.
  • Target DNA A PNA capture molecule was immobilised on an immobilisation unit and a solution that included a complementary DNA molecule contacted therewith.
  • the positively chargeable precursor aniline, horseraddish peroxidase and hydrogen peroxide were added.
  • the formed electroconductive polyaniline polymer was contacted with the enhancer reagent HCl (doped) before determining the presence of the target nucleic acid molecule.
  • Figure 7 depicts scanning electron microscope (SEM) images of chip surface for (A), hybridisation with non-complementary DNA and deposition with polyaniline and (B), hybridisation with complementary DNA and deposition with polyaniline. SEM was performed with JEOL 4000 field emission scan electron microscope.
  • Figure 8 illustrates the influence of the concentration of the polymerisable positively charged precursor molecule aniline on conductivity of polyaniline.
  • DNA was used as the target nucleic acid molecule for hybridisation at 10 pM.
  • Horse raddish peroxidase was used as a catalyst, and H 2 O 2 as the reactant molecule.
  • the concentration of horse raddish peroxidase was 1 ⁇ g/mL.
  • H 2 O 2 was used in the same concentration as aniline.
  • the incubation time was set to 40 min.
  • Figure 9 illustrates the influence of the concentration of the catalyst horse radish peroxidase (HRP) on the conductivity of polyaniline.
  • DNA was used as the target nucleic acid molecule for hybridisation at 10 pM and the concentration of aniline and H 2 O 2 was 1 ⁇ g/mL.
  • the incubation time was set to 40 min.
  • Figure 10 depicts an optimisation of the incubation time for polyaniline deposition.
  • DNA was used as the target nucleic acid molecule for hybridisation at 10 pM.
  • the catalyst horse radish peroxidase was used at 1 ⁇ g/mL and the concentration of aniline was 2 mM.
  • Figure 11 shows a linear relation between conductance and the concentration of the target nucleic acid molecule DNA.
  • DNA concentration ranged from 50 fM to 100 pM.
  • Polyaniline deposition was performed under the optimised conditions.
  • Figure 12 depicts discriminating between complementary and single base mismatched DNA.
  • the hybridisation with complementary and single base mismatched DNA sample was carried out at 10 pM and 100 pM. Polyaniline deposition was performed with the same procedure for both samples.
  • the methods of the present invention allow for the detection of any target nucleic acid molecule.
  • the term “detecting”, “detect” or “detection” refers to measurements which provide an indication of the presence or absence, either qualitatively or quantitatively, of a target nucleic acid molecule. Accordingly, the term encompasses quantitative measurements of the concentration of a target nucleic acid molecule in a sample, as well as qualitative measurements in which for instance different types of target nucleic acid molecules in a given sample are identified, or, as a further example, the behaviour of a particular nucleic acid molecule in a given environment is observed.
  • a target nucleic acid is typically considered to be detected if a value of a measurement of an electrical characteristic exceeds a given threshold value to a reference measurement. If the threshold value is reached in the measurement, then it is typically concluded that the target nucleic acid is absent (which however means detected in the context of the present invention) from the analyte that is investigated.
  • a first measurement is carried out at the electrodes before adding the polymerisable positively chargeable precursor, and a second measurement is carried out after having added the reactant molecule that initiates the polymerisation of the polymerisable positively chargeable precursor.
  • the first and the second measurement are then compared and the value of the measurement is the result of this comparison.
  • nucleic acid molecule refers to any nucleic acid in any possible configuration, such as single stranded, double stranded or a combination thereof.
  • Nucleic acids include for instance DNA molecules, RNA molecules, analogues of the DNA or RNA generated using nucleotide analogues or using nucleic acid chemistry, locked nucleic acid molecules (LNA), and protein nucleic acids molecules (PNA).
  • LNA locked nucleic acid molecules
  • PNA protein nucleic acids molecules
  • DNA or RNA may be of genomic or synthetic origin and may be single or double stranded. Such nucleic acid can be e.g.
  • a respective nucleic acid may furthermore contain non-natural nucleotide analogues and/or be linked to an affinity tag or a label.
  • nucleotide analogues are known and can be used in nucleic acids used In the methods of the invention.
  • a nucleotide analogue is a nucleotide containing a modification at for instance the base, sugar, or phosphate moieties.
  • a substitution of 2'-OH residues of siRNA with 2'F, 2'0-Me or 2'H residues is known to improve the in vivo stability of the respective RNA.
  • Modifications at the base moiety include natural and synthetic modifications of A, C, G, and T/U, different purine or pyrimidine bases, such as uracil-5-yl, hypoxanthin-9-yl, and 2-aminoadenin-9-yl, as well as non-purine or non-pyrimidine nucleotide bases.
  • Other nucleotide analogues serve as universal bases.
  • Universal bases include 3-nitropyrrole and 5-nitroindole. Universal bases are able to form a base pair with any other base. Base modifications often can be combined with for example a sugar modification, such as for instance 2'-O-methoxyethyl, e.g. to achieve unique properties such as increased duplex stability.
  • the target nucleic acid molecule may be included in any analyte/sample of any origin. It may for instance, but not limited to, be derived from human or non-human animals, plants, bacteria, viruses, spores, fungi, or protozoa, or from organic or inorganic material of synthetic or biological origin.
  • any of the following samples selected from, but not limited to, the group consisting of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a sewage sample, a ground water sample, an abrasion sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, an urine sample, a stool sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a cancer sample, a tumour sample, a tissue sample, a cell sample, a cell culture sample, a
  • a respective sample may have been pre-processed to any degree.
  • a tissue sample may have been digested, homogenised or centrifuged prior to being used with the device of the present invention.
  • the sample may furthermore have been prepared in form of a fluid, such as a solution.
  • Examples include, but are not limited to, a solution or a slurry of a nucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide, an amino acid, a protein, a synthetic polymer, a biochemical composition, an organic chemical composition, an inorganic chemical composition, a metal, a lipid, a carbohydrate, a combinatory chemistry product, a drug candidate molecule, a drug molecule, a drug metabolite or of any combinations thereof.
  • a sample may furthermore include any combination of the aforementioned examples.
  • the sample that includes the target nucleic acid molecule may be a mammal sample, for example a human or mouse sample, such as a sample of total mRNA.
  • the sample is a fluid sample, such as a liquid or a gas.
  • the sample is solid.
  • an extraction by standard techniques known in the art may be carried out in order to dissolve the target nucleic acid molecule in a solvent.
  • the target nucleic acid molecule, or the expected target nucleic acid molecule is provided in form of a solution for the use in the present invention.
  • the target nucleic acid molecule may be provided in form of an aqueous solution.
  • further matter may be added to the respective solution, for example dissolved or suspended therein.
  • an aqueous solution may include one or more buffer compounds.
  • buffer compounds include, but are not limited to, solutions of salts of phosphate, carbonate, succinate, carbonate, citrate, acetate, formate, barbiturate, oxalate, lactate, phthalate, maleate, cacodylate, borate, N-(2-acetamido)-2-amirio-ethanesulfonate (also called (ACES), N-(2- hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (also called HEPES), 4-(2-hydroxyethyl)- 1-piperazine-propanesulfonic acid (also called HEPPS), piperazine-l,4-bis(2-ethanesulfonic acid) (also called PIPES), (2-[Ms(hydroxymemyl)-memylamino]
  • buffers include, but are not limited to, triethanolamine, diethanolamine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris(hydroxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxymethyl)methane (also called BIS- TRIS), and N-[Tris(hydroxyriietliyl)-methyl]-glycine (also called TRICINE), to name a few.
  • a respective buffer may be an aqueous solution of such buffer compound or a solution in a suitable polar organic solvent.
  • One or more respective solutions may be used to accommodate the suspected target nucleic acid as well as other matter used, throughout an entire method of the present invention.
  • nuclease inhibitors may need to be added in order to maintain a nucleic acid molecule in an intact state. While it is understood that for the purpose of detection any matter added should not obviate the formation of a complex between the PNA capture molecule (or other nucleic acid capture molecule used in another method of the invention, see below) and the target nucleic acid molecule, for the purpose of carrying out a control measurement a respective agent may be used that blocks said complex formation.
  • the methods of the present invention allow detecting a target nucleic acid molecule by means of an electrode arrangement such as a pair of electrodes.
  • electrode as used herein is employed in its conventional sense, thereby referring to an object that is capable of serving as an electric conductor, through which an electrical current or voltage may be brought into and/or out of a medium in contact with the electrode.
  • an electrode is one of at least two terminals of an electrically conducting medium.
  • electrode arrangement or “pair of electrodes” as used herein refers to any number of electrodes of two or higher. Accordingly, two or more electrodes are provided in the method (as well as the kits, see below) of the invention. The electrodes are arranged at a distance from one another. In embodiments where two electrodes are provided, the two electrodes may for instance be separated by a gap.
  • the two electrodes of this pair of electrodes may face each other across the gap.
  • the two electrodes are at least essentially parallel.
  • the electrodes may be of any desired dimension and shape. They may for example have the shape of a flat, arched, concave or convex slab. In some embodiments they may have the shape of a ring (for an example see Green, BJ, & Hudson, J.L., Phys. Rev. E (2001), 63, 026214; see also Fig. 4A).
  • interdigital electrodes are provided, which typically include a digitlike or f ⁇ ngerlike pattern of parallel in-plane electrodes (see Mamishev, A. V., Proc.
  • an array of electrodes may be provided. If desired, one or more floating electrodes may be used. In some embodiments the electrodes that are provided are of similar size, for example of identical size.
  • the distance between the two or more electrodes may be of any dimension, as long as the change of an electrical characteristic of the respective region can be determined in the method of the present invention (see below), so that a detection of a target nucleic acid molecule can be carried out.
  • the distance at which the electrodes are arranged may be identical between each of the respective electrodes. In other such embodiments the distance at. which the electrodes are arranged may be identical between some of the respective electrodes. In yet other embodiments where more than two electrodes are provided, each distance at which two electrodes are arranged may be different from another distance at which two electrodes are arranged.
  • the distance at which the electrodes are arranged may be in a range that corresponds to the length of the target nucleic acid molecule. It is noted in this regard that for instance a linearised chromosome may have a length of up to 1.5 m (http://hypertextbook.com/facts/ 1998/StevenChen.shtml). As a further illustration, already Watson and Crick were able to determine the distance between the two strands of DNA as 2 nanometres. From their DNA model the vertical rise per base pair along the axis of a DNA molecule can be calculated to be 0.34 nm.
  • the distance at which the electrodes are arranged is of the same or a smaller length than the length of the target nucleic acid molecule.
  • the target nucleic acid molecule is capable of spanning the respective gap.
  • a respective distance, e.g. a gap may for instance have a with selected in the range of about 0.5 nm to about 10 ⁇ m, such as a range of about .
  • a distance of 30 nm may be selected, which would roughly correspond to a length of a linear nucleic acid of about 100 bp. (Such an estimate can be made based on the known helical pitch of ideal A, B and Z DNA for example.
  • B DNA for example, has a height of 0.34 nm per helical turn and base pair so that 10 base pairs (bp) bridge a distance of 3.4 nm).
  • the distance with can be determined empirically for longer non linear nucleic acids; a distance of 200 nm, may roughly correspond to a length of a nucleic acid of about 2000 to 5000 bp.
  • a target nucleic acid molecule may be of for instance 100 - 500 nm, which is for example of a sufficient size of a nucleic acid molecule to includes exemplary genes.
  • the PNA capture molecule may in such an embodiment be immobilised in vicinity to the region in between the electrodes, or even within the respective region. In embodiments where this region in between the electrodes is defined by a small distance separating the electrodes, such as e.g.
  • the sensing zone is usually a region or aperture into which the target nucleic acid molecule is caused to be located.
  • the sensing zone may be a region or aperture to which the target nucleic acid molecule is caused to flow or into which the target nucleic acid molecule is disposed.
  • the sensing zone is defined by the zone in which an electric field of the pair of electrodes is effective.
  • a capture molecule (see below) is used that is capable of associating with the target nucleic acid molecule.
  • the capture molecule is immobilised on the immobilisation unit, generally on a surface or a part of a surface of an immobilisation unit.
  • the respective surface (or surface part) of the immobilisation unit is arranged within the sensing zone.
  • at least a part of the respective surface of the immobilisation unit is arranged in a zone where an electric field of the pair of electrodes is effective.
  • at least a part thereof is included in the region defined by the distance between the (or some of the)electrodes (see e.g. Fig. 2 or Fig 4D).
  • the surface of the immobilisation unit may be of any material as long as an electrical detection can be carried out.
  • the surface of the immobilisation unit may include or consist of an electric insulator. It maybe desired to select the material of the immobilisation unit in order to immobilise a nucleic acid thereon (see also below).
  • the surface of the immobilisation unit, or a part thereof, may also be altered, e.g. by means of a treatment carried out to alter characteristics of the solid surface. Such a treatment may include various means, such as mechanical, thermal, electrical or chemical means.
  • the surface properties of any hydrophobic surface can be rendered hydrophilic by coating with a hydrophilic polymer or by treatment with surfactants.
  • Examples of a chemical surface treatment include, but are not limited to exposure to hexamethyldisilazane, trimethylchlorosilane, dimethyldichlorosilane, propyltrichlorosilane, tetraethoxysilane, glycidoxypropyltrimethoxy silane, 3-aminopropyltriethoxysilane, 2-(3,4- epoxy cyclohexyl)ethyltrimethoxysilane, 3-(2,3-epoxy propoxyl)propyltrimethoxysilane, polydimethylsiloxane (PDMS), ⁇ -(3,4-epoxycyclohexyl)ethyltrimethoxysilane, poly(methyl methacrylate) or a polymethacrylate co-polymer, urethane, polyurethane, fluoropolyacrylate, poly(methoxy polyethylene glycol raethacrylate), poly(dimethyl acryl
  • the surface may for instance be coated with an electroconductive polymer, such as polypyrrole (Wang, J., et al., Anal. Chem. (1999) 71, 18, 4095-4099; Wang, J., et al., Anal. CMm.
  • an electroconductive polymer such as polypyrrole (Wang, J., et al., Anal. Chem. (1999) 71, 18, 4095-4099; Wang, J., et al., Anal. CMm.
  • polythiophene polyaniline, polyacetylene, poly(N-vinyl carbazole), or a copolymer such as a copolymer of pyrrole and thiophene or a copolymer of juglone and 5-hydroxy-3-thioacetic-l,4-naphthoquinone (Reisberg, S., et al., Anal. Chem. (2005) 77, 10, 3351 -3356).
  • the surface is a surface of a carbon paste electrode, it may for example be modified with carboxyl groups by mixing stearic acid with the paste.
  • the linking molecule ethylenediamine may for instance be immobilised on a respective electrode in order to facilitate the subsequent immobilisation of a capture molecule (see below).
  • the electrical characteristic of the region in between the electrode arrangement must be influenced by the electrically conducting polymer associated with the target nucleic acid, should this polymer be formed.
  • the immobilisation unit or at least the surface or a part of the surface thereof, is located in vicinity to the electrodes of the electron pair.
  • the (immobilised) complex of the nucleic acid molecule in particular a nucleic acid molecule with a size of several thousands or more base pairs
  • the electrically conducting polymer may, for example, swing by Brownian motion with their flexible parts into the distance in between the electrodes.
  • the electrical interaction between the electrically conducting polymer and an electrical field applied at the electrodes can alone also be sufficient to influence the electrical characteristics in the gap in between the electrodes in a detectable manner.
  • the respective immobilisation surface of the immobilisation unit is arranged within the respective region defined by the distance between the (or some of the) electrodes.
  • the immobilisation surface is included on one of the electrode (e.g. a detection electrode).
  • a respective detection electrode may for example be used for the detection of an electric signal in the method of the present invention (see below).
  • a respective detection electrode may be used for the generation of an electric field.
  • the surface is conductively connected to an electrode.
  • One method of the invention includes immobilising on the immobilisation unit, or at least the surface or a part of the surface thereof, a peptide nucleic acid (PNA) capture molecule.
  • PNA peptide nucleic acid
  • a PNA molecule is a nucleic acid molecule in which the backbone is a pseudopeptide rather than a sugar. Accordingly, PNA generally has a charge neutral backbone, in contrast to DNA or RNA. Nevertheless, PNA is capable of hybridising at least complementary and substantially complementary nucleic acid strands, just as e.g. DNA or RNA (to which PNA is considered a structural mimic).
  • the PNA capture molecule may be of any suitable length.
  • the PNA capture molecule or other nucleic acid capture molecule has a nucleic acid sequence of a length of about 7 to about 30 bp, for example a length of about 9 to about 25 bp, such as a length of about 10 to about 20 bp.
  • the PNA capture molecule used in the present invention has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • the respective nucleotide sequence of the PNA capture molecule may for example be 70, for example 80 or 85, including 100 % complementary to another nucleic acid sequence.
  • the respective nucleotide sequence is substantially complementary to at least a portion of the target nucleic acid molecule.
  • “Substantially complementary” as used herein refers to the fact that a given nucleic acid sequence is at least 90, for instance 95, such as 100 % complementary to another nucleic acid sequence.
  • nucleotide sequence is the complement of another nucleotide sequence if all of the nucleotides of the first sequence are complementary to all of the nucleotides of the second sequence.
  • the respective nucleotide sequence will specifically hybridise to the respective portion of the target nucleic acid molecule under suitable hybridisation assay conditions, in particular of ionic strength and temperature.
  • more than one PNA capture molecule may be immobilised. This may for instance be desired in order to broadly screen for the presence of any of a group of selected target nucleic acid sequences.
  • the use of more than one PNA capture molecule may also be desired for the detection of the same target nucleic acid molecule via different recognition sequences, e.g., the 5'- and 3'-termini thereof, which enhances the likelihood to detect even a few copies of a target nucleic acid molecule in a sample.
  • Any target nucleic acid molecule that is capable of hybridising to at least a part of the PNA-capture molecule can be detected by the method of the present invention.
  • the skilled artisan will appreciate that the method of the present invention allows the detection of small numbers of target nucleic acid molecules, including a single target nucleic acid molecule (see below).
  • the present method thus redundantises the need of conventional nucleic acid detection methods of amplifying nucleic acid molecules prior to detection, in cases where it is desired to detect such low numbers of target molecules.
  • the target nucleic acid molecule that can be detected may be of any length.
  • the target nucleic acid molecule includes a predefined sequence
  • the target nucleic acid molecule furthermore includes at least one single-stranded region.
  • the PNA capture molecule can directly form Watson-Crick base pairs with the target nucleic acid molecule, without the requirement of separating complementary strands of the target nucleic acid molecule.
  • the target nucleic acid molecule, or a region thereon that includes a predefined sequence is provided or suspected to be in double strand form
  • the respective nucleic acid duplex may be separated by any standard technique used in the art, for instance by increasing the temperature (e.g.
  • multiple respective PNA capture molecules may be used, each of which being at least partially complementary to e.g. a selected portion of the target nucleic acid molecule (see also below). It is of course also possible to detect a plurality of nucleic acid molecules by using a plurality (that means at least two) different capture molecules each being complementary to a specific target nucleic acid. As an illustrative example, if dengue virus nucleic acid is to be detected using the present invention, four different capture molecules each of which is specific for each of the four dengue virus sub-strains can be immobilised on sensing units being arranged in different locations of an interdigitated electrode arrangement.
  • the PNA capture molecule may be immobilised on the immobilisation unit at any stage during the present method of the invention. As two examples, it may be immobilised at the beginning of the method or before adding a polymerisable positively charged precursor (see below). In typical embodiments it is immobilised before performing an electrical measurement (see below).
  • the PNA capture molecule may be immobilised by any means. It may be immobilised on the entire surface, or a selected portion of the surface of the immobilisation unit. In some embodiments the PNA capture molecule is provided first and thereafter immobilised onto the surface of the immobilisation unit. An illustrative example is the mechanical spotting of the PNA capture molecule onto the surface of tihe immobilisation unit.
  • This spotting may be carried out manually, e.g. by means of a pipette, or automatically, e.g. by means of a micro robot.
  • the polypeptide backbone of the PNA capture molecule may be covalently linked to a gold surface via a thio- ether-bond.
  • the surface of the immobilisation unit may be activated prior to immobilising the PNA capture molecule thereon, for instance in order to facilitate the attachment reaction (see also above).
  • the respective surface may for example be modified with aminophenyl or aminopropyl silanes.
  • 5'-succinylated PNA capture molecules (or in other methods of the invention other nucleic acid capture molecules) may be immobilised thereon by carbodiimide-mediated coupling, hi some embodiments the surface may for instance be coated with an electroconductive polymer, such as polypyrrole (Wang, J., et al., Anal. Chem. (1999) 71, 18, 4095-4099; Wang, J., et al, Anal.
  • a linking moiety such as an affinity tag may be used to immobilise the PNA capture molecule.
  • an affinity tag include, but are not limited to biotin, dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), calmodulin binding peptide (CBP), FLAG'-peptide, the T7 epitope (Ala-Ser-Met-Thr-Gly- Gly-Gln-Gln-Met-Gly), maltose binding protein (MBP), the HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp of herpes simplex virus glycoprotein D, the hemagglutinin (HA) epitope of the sequence Tyr-Pro-Tyr-Asp-Val-Pro
  • nucleotide sequences may differ to such an extent that the sequence of the nucleotide tag is not capable of hybridising to the sequence of any portion of the target nucleic acid molecule.
  • an oligonucleotide tag may for instance be used to hybridise to an immobilised oligonucleotide with a complementary sequence.
  • a further example of a linking moiety is an antibody.
  • an antibody- nucleic acid conjugate may be used as the PNA-capture molecule.
  • Avidin or streptavidin may for instance be employed to immobilise a biotinylated nucleic acid, or a biotin containing monolayer of gold may be employed
  • the PNA capture molecule may be locally deposited, e.g. by scanning electrochemical microscopy, for instance via pyrrole-oligonucleotide patterns (e.g. Fortin,
  • the PNA capture molecule may be directly synthesised on the surface of the immobilisation unit, for example using photoactivation and deactivation.
  • any remaining PNA capture molecule, or molecules, that were not immobilised may be removed from the immobilisation unit.
  • Removing an unbound PNA capture molecule may be desired to avoid subsequent hybridisation of such PNA molecule with the target nucleic acid molecule, which might reduce the sensitivity of the present method.
  • Removing an unbound PNA capture molecule may also be desired to avoid a nonspecific binding of such PNA molecule to any matter present in a sample used, which might for instance alter the conductivity of such matter (e.g., reducible metal cations), which might interfere with the results of the electrical measurement (see also below).
  • An unbound capture molecule may for instance be removed by exchanging the medium, e.g. a solution that contacts the surface of the immobilisation unit.
  • a blocking agent may be immobilised on the surface of the immobilisation unit.
  • This blocking agent may serve in reducing or preventing non-specific binding of matter included in the solution expected to include the target nucleic acid molecule. It may also serve in reducing or preventing non-specific binding of any other matter, such as a molecule or solution that is further added to the immobilisation unit when carrying out the method of the invention.
  • the blocking agent may be added together with the PNA capture molecule or subsequently thereto.
  • Any agent that can be immobilised on the immobilisation unit and that is able to prevent (or at least to significantly reduce) the non-specific interaction between molecules, the detection of which is undesired, and the PNA capture molecule is suitable for that purpose, as long as the specific interaction between the PNA capture molecule and the target nucleic acid molecule is not prevented.
  • agents are thiol molecules, disulfides, thiophene derivatives, and. polythiophene derivatives.
  • An illustrative example of a useful class of blocking reagents include thiol molecules such as 16-mercaptohexadecanoic acid, 12-mercaptododecanoic, 11-mercaptodecanoic acid or 10-mercaptodecanoic acid.
  • derivative refers to a compound which differs from another compound of similar structure by the replacement or substitution of one moiety by another.
  • Respective moieties include, but are not limited to atoms, radicals or functional groups.
  • a hydrogen atom of a compound may be substituted by alkyl, carbonyl, acyl, hydroxyl, or amino functions to produce a derivative of that compound.
  • Respective moieties include for instance also a protective group that may be removed under the selected reaction conditions.
  • the present method of the invention further includes contacting the immobilisation unit with the solution expected to include the target nucleic acid molecule (cf. Fig. 2).
  • the immobilisation unit may for example be immersed in a solution, to which the solution expected to include the target nucleic acid molecule is added. In some embodiments both such solutions are aqueous solutions, hi one embodiment the entire method is carried out in an aqueous solution.
  • the method further includes allowing the target nucleic acid molecule to hybridise to the PNA capture molecule on the immobilisation unit. As already indicated above, thereby the formation of a complex between the PNA capture molecule and the target nucleic acid molecule is allowed (cf. Fig. 2).
  • the conditions are chosen so that the target nucleic acid molecules can either bind simultaneously or consecutively to their respective capture molecules.
  • the analyte/solution expected to include the target nucleic acid molecule may be removed. This may for example be desired in order to remove any negatively charged molecules, such as nucleic acid molecules that were present in the solution expected to include the target nucleic acid molecule.
  • any extraction used if any; cf.
  • removing components of the respective solution may improve the signal-to-noise ratio of the electrical measurement performed in the method of the invention (see below).
  • Washing or rinsing etc. of the immobilisation unit
  • Any matter known to be present in a sample used may also be removed by a suitable method that does not dissolve the complex formed between PNA capture molecule and target nucleic acid molecule, such as enzymatically.
  • a suitable method that does not dissolve the complex formed between PNA capture molecule and target nucleic acid molecule, such as enzymatically.
  • this may be accomplished by an enzyme, which selectively breaks down single-stranded DNA, such as mung bean nuclease, nuclease Pl or nuclease Sl.
  • the method of the present invention also includes providing a polymerisable positively chargeable precursor.
  • the method further includes adding the polymerisable positively chargeable precursor (cf. Fig. 2).
  • the polymerisable positively chargeable precursor has an electrostatic net charge that is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the target nucleic acid molecule is negatively charged in neutral and acidic conditions, in particular where the target nucleic acid molecule is DNA or RNA, due to the backbone of alternating sugar and phosphate molecules in this type of nucleic acid. Accordingly, conditions are selected where the polymerisable positively chargeable precursor has a positive net charge.
  • the pK value of a selected precursor molecule serves as a valuable guidance in choosing a suitable pH range or pH value for a solution, in which the precursor molecule is positively charged.
  • This pH range or value is obtained by way of adjusting (e.g. titrating) or by providing the target nucleic acid — and thus typically the immobilisation unit as well - in a solution of the desired pH.
  • the target nucleic acid molecule may for example be included in a solution of a pH value selected in the range of about 1.5 to about 8.0, such as in the range of about 1.7 to about 7.0, in the range of about 1.9 to about 6.0, or in the range of about 2.0 to about 5.5.
  • the pH value may thus for instance be selected to be about 3.0 or about 4.0.
  • the pH may also be adjusted to be in a respective range (e.g.
  • the polymerisable positively chargeable precursor associates to the target nucleic acid molecule, and thus to the complex formed between the PNA capture molecule and the target nucleic acid molecule. Therefore, the polymerisation of the polymerisable precursor can be carried out by means of a suitable reactant molecule. The respective polymerisation will accordingly involve, including start at, a precursor that is associated to the target nucleic acid molecule.
  • Any positively chargeable precursor may be used as long as it can be polymerised in the presence of the complex between the PNA capture molecule and the target nucleic acid molecule, without dissolving or degrading the respective complex in such a manner that the target nucleic acid molecule is no longer associated to the surface of the immobilisation unit.
  • the polymerisable positively chargeable precursor may be an aromatic amine (see e.g. Fig. 5 for examples), such as aniline.; pyridineamine (e.g. 2-pyridineamine, 3-pyridineamine or 4-pyridineamine), pyrrole, imidazole or a derivative thereof.
  • an aniline derivative may also be of the general formula
  • R 1 and R 2 may independently selected from the group consisting of H, aliphatic, cycloaliphatic, aromatic, arylaliphatic, and arylcyclo aliphatic hydrocarbyl groups, comprising 0 - 5 heteroatoms, i.e. atoms that differ from carbon, for example 0 - 3 heteroatoms, selected from the group N, O, S, and Si.
  • R 1 and R 2 may optionally be linked so as to define an aliphatic, cycloaliphatic, aromatic, arylaliphatic, or arylcycloaliphatic hydrocarbyl bridge.
  • a compound is selected that includes a negatively chargeable moiety (or functional group, e.g.
  • aliphatic means, unless otherwise stated, a straight or branched hydrocarbon chain, which may be saturated or mono- or polyunsaturated. An unsaturated aliphatic group contains one or more double and/or triple bonds.
  • the branches of the hydrocarbon chain may include linear chains as well as non-aromatic cyclic elements.
  • the hydrocarbon chain which may, unless otherwise stated, be of any length, and contain any number of branches. Both the main chain as well as the branches may furthermore contain heteroatoms as for instance N, O, S, Se or Si.
  • alicyclic means, unless otherwise stated, a nonaromatic cyclic hydrocarbon moiety, which may be saturated or mono- or polyunsaturated.
  • the cyclic hydrocarbon moiety may be substituted with nonaromatic cyclic as well as chain elements.
  • the main chain of the cyclic hydrocarbon moiety may, unless otherwise stated, be of any length and contain any number of non-aromatic cyclic and chain elements.
  • Both the cyclic hydrocarbon moiety and the cyclic and chain substituents may furthermore contain heteroatoms, as for instance N, O, S, Se or Si.
  • aromatic means, unless otherwise stated, a planar cyclic hydrocarbon moiety of conjugated double bonds, which may be a single ring or include multiple fused or covalently linked rings.
  • the main chain of the cyclic hydrocarbon moiety may, unless otherwise stated, be of any length and contain any number of heteroatoms, as for instance N, O and S.
  • arylaliphatic is meant a hydrocarbon moiety, in which one or more aryl groups are attached to or are substituents on one or more aliphatic groups.
  • arylaliphatic includes for instance hydrocarbon moieties, in which two or more aryl groups are connected via one or more aliphatic chain or chains of any length, for instance a methylene group.
  • aliphatic alicyclic
  • aromatic arylaliphatic
  • aliphatic alicyclic
  • aromatic arylaliphatic
  • Substituents my be any functional group, as for example, but not limited to, amino, amido, azido, carbonyl, cyano, isocyano, dithiane, halogen, hydroxyl, nitro, seleno, silyl, silano, thio, thiocyano, and trifluoromethyl.
  • the present method of the invention further includes providing a suitable reactant molecule that is capable of undergoing a reaction with the positively chargeable precursor that results in polymerisation of the latter.
  • the reactant molecule may also be able to initiate the polymerisation of the polymerisable positively chargeable precursor.
  • the reactant molecule may be an oxidant.
  • a suitable oxidant examples include, but are not limited to, a ruthenium tris(bipyridinium) complex, a persulfate (such as ammonium persulphate or tetrabutylammonium persulphate), a peroxide (see also below), hydrogen tetrachloroaurate (auric acid, HAuCl 4 ), a chromate, a dichromate, a manganate, a permanganate, oxygen, ozone, nitrogen oxide, a halogene, a chlorite, a chloride, a perchloride, a chlorate, a iodate, a nitrate, a sulfoxide and osmium tetroxide.
  • a ruthenium tris(bipyridinium) complex examples include, but are not limited to, a persulfate (such as ammonium persulphate or tetrabutylammonium pers
  • the method of the present invention also includes adding the reactant molecule (cf. Fig. 2).
  • the addition of a suitable reactant molecule initiates the polymerisation of the polymerisable positively chargeable precursor (see. Fig. 2).
  • a suitable reactant molecule initiates the polymerisation of the polymerisable positively chargeable precursor (see. Fig. 2).
  • aniline or a suitable derivative thereof is selected as the respective precursor and an oxidant is selected as the reactant molecule
  • the polymerisation may start according the following reaction scheme (I) and subsequently continue respectively:
  • a respective electroconductive form of the depicted reaction product polyaniline according to any of schemes (I) to (III) is also termed to be in the "emeraldine" oxidation state in the art (see e.g. Huang, J. et al, Chem. Eur. J. (2004), 10, 1314-1319).
  • the polymerisation typically occurs at the para position as shown above (scheme I, scheme III).
  • the hybridised anionic target nucleic acid molecule may serve as a template, providing a local environment that guides para-coupling of aniline molecules.
  • Previous studies on enzymatically catalysed polymerisation of aniline have shown that the polymerisation of aniline is catalysed by peroxidases, horse-radish peroxidase for example, under mild conditions (Liu, W., et al., J. Am. Chem. Soc.
  • the polymerisation may start according the following reaction scheme (IV) and subsequently continue respectively:
  • the anionic target nucleic acid molecule which forms a complex with the PNA capture molecule, typically serves as a template for the polymerisation of the positively chargeable precursor. As a consequence, the polymerisation occurs exclusively at the target nucleic acid molecule.
  • a suitable initiator may be added.
  • An initiator is generally matter such as a molecule that can generate radical species under mild conditions. Usually it can also promote radical polymerisation reactions.
  • Examples of a respective initiator include, but are not limited to a radical initiator, such as a halogen molecule, an azo compound, a persulfate molecule, a peroxydisulfate ((SO 3 ) 2 ⁇ 2 2 ⁇ ) and a peroxide compound, such as an organic peroxide compound.
  • a radical initiator such as a halogen molecule
  • an azo compound such as a persulfate molecule, a peroxydisulfate ((SO 3 ) 2 ⁇ 2 2 ⁇ )
  • a peroxide compound such as an organic peroxide compound.
  • Two illustrative examples of a halogen molecule are chlorine or bromine.
  • Three illustrative examples of an azo compound are 2,2'-azobis(2- amidinopropane), 2,2'-azobis(2 J 4-dimethylvaleronitrile) and azobis(isobutyramidine).
  • a persulfate molecule Two illustrative example of a persulfate molecule are ammonium persulfate and potassium persulfate. Where desired, a persulfate molecule may also be combined with a thiosulfate (e.g. sodium thiosulfate, Na 2 S 2 O 3 ), a bisulfite, a dithionite, or an ascorbic acid molecule.
  • a thiosulfate e.g. sodium thiosulfate, Na 2 S 2 O 3
  • a bisulfite e.g. sodium thiosulfate, Na 2 S 2 O 3
  • a bisulfite e.g. sodium thiosulfate, Na 2 S 2 O 3
  • a bisulfite e.g. sodium thiosulfate, Na 2 S 2 O 3
  • a bisulfite e.g. sodium thiosulfate, Na 2 S
  • an organic peroxide compound examples include, but are not limited to, benzoyl peroxide, cumenyl peroxide, dicumyl peroxide, cumene hydroperoxide, diphenyl peroxide, bis-(tert-butyl)peroxide, bis(tert-butylperoxysuccinyl) peroxide, a lipid peroxide such as lauroyl peroxide, diacetyl peroxide or trifmoroacetyl peroxide, bis(o-iodophenylacetyl) peroxide, ethyl peroxide, 2-naphthyl peroxide, 2-naphthoyl peroxide, butyl hydroperoxide, tert-butyl hydroperoxide, vinyl hydroperoxide, cyclohexyl hydroperoxide, trifluoromethyl peroxide, 2,5-bis(l-methylethyl)phenyl-hydroperoxide, l,5
  • a suitable catalyst may furthermore be provided and the reactant molecule be exposed thereto.
  • a catalyst include, but are not limited to, light, a metal halide such as a metal chloride or a metal bromide, a metal sulphate, and an enzyme or an enzyme-conjugate.
  • suitable metal halides include, but are not limited to a ferrous halide, e.g. ferrous chloride, a lithium halide, e.g. lithium chloride, a copper halide, e.g.
  • a copper(I) halide or a copper(II) halide such as CuCl 2 or CuBr 2
  • a molybdenum halide such as MoCl 5
  • an indium halide such as (NELj) 2 IrCl 6
  • a manganese chloride such as MnCl 2
  • a nickel halide such as NiCl 2
  • a titanium chloride such as titanium trichloride (TiCl 3 )
  • an aluminium halide such as aluminium chloride.
  • a respective catalyst may for example be added to the immobilisation unit, on which the PNA capture molecule is immobilised. It may, for instance, be added after the polymerisable positively chargeable precursor has associated to the complex formed between the PNA capture molecule and the target nucleic acid molecule. It may for instance also be added together with the reactant molecule or subsequently thereto.
  • the catalyst may for instance be selected in such a way that the reactant molecule is a substrate for the catalyst.
  • any enzyme may be used that is capable of catalysing the polymerisation of the positively chargeable precursor.
  • the enzyme may for instance be selected in such a way that the reactant molecule is a substrate for the enzyme.
  • an oxidoreductase enzyme may be selected.
  • an oxidoreductase enzyme include, but are not limited to, a peroxidase, an oxidase, a dehydrogenase, a monooxygenase, a hydroxylase, a dioxygenase, or a hydrogenase.
  • a respective peroxidase enzyme may for instance be a haem peroxidase enzyme.
  • haem peroxidase enzyme include, but are not limited to, horseradish peroxidase, cytochrome c peroxidase, glutathione peroxidase, myeloperoxidase, thyroid peroxidase, eosinophil peroxidase, lactoperoxidase, ascorbate peroxidase, peroxidasin, prostaglandin H synthase, a bacterial catalase-peroxidase such as E. coli catalase-peroxidase, M.
  • tuberculosis catalase-peroxidase or Bacteroides fragilis catalase-peroxidase, lignin peroxidase, plant ascorbate peroxidase, Haem chloroperoxidase, manganese peroxidase, stigma specific peroxidase, Euphorbia characias latex peroxidase, Arthromyces ramosus peroxidase, sorghum grain peroxidase SPC4, soybean peroxidase, Phanerochaete chrysosporium manganese-dependent peroxidase, lacrimal gland peroxidase, or any combination thereof.
  • an oxidase enzyme examples include, but are not limited to, the oxygen oxidase laccase, glucose oxidase, lactase oxidase, pyruvate oxidase, tyrosinase or any combination thereof.
  • a respective peroxidase enzyme may also be a non-haem peroxidase enzyme such as for instance bromoperoxidases BPO-Al, BPO-A2 or BPO-B, the non-haem chloroperoxidase from Pseudomonas fluorescens, or the non-haem extracellular peroxidase from Thermomonospora fusca BD25.
  • a secreted mixture or an extract of enzymes may also be used as a catalyst where desired.
  • the so-called "white-rot fungi” produces lignin-modifying extracellular enzymes (LME) that include two peroxidase enzymes (lignin peroxidase and Mn peroxidase), an oxidase enzyme laccase and an oxidase enzyme aryl alcohol oxidase.
  • LME lignin-modifying extracellular enzymes
  • a respective crude extract may be used as a catalyst in a method of the present invention (cf. also Curvetto, N.R., et al., Biochemical Engineering Journal (2006) 29, 3, 191-203).
  • suitable catalysts include haematin (Curvetto et al., 2006, supra) and haemoglobin.
  • the oxidant is a peroxide such as hydrogen peroxide
  • the catalyst is horse radish peroxidase.
  • Horse radish peroxidase has previously been reported (Liu, W., et al., J. Am. Chem. Soc. (1999) 121, 71-78; Nagarajan, R., et al., Macromolecules (2001) 34, 3921-3927) to catalyse the oxidative polymerisation of aniline in the presence of the oxidant hydrogen peroxide (H 2 O 2 ).
  • This polymerisation can also be carried out in the presence of DNA, which expedites the polymerisation reaction (Nickels, P.
  • the oxidant is molecular oxygen (O 2 ) and the catalyst is laccase (e.g. isolated from Coriolus hirsutus). Laccase C. hirsutus has been reported to possess a higher operational stability than horse radish peroxidase under acidic conditions in the polymerisation of aniline (Karamyshev, A. V. et al., Enzyme and Microbial Technology (2003) 33, 5, 556-564).
  • the catalyst is in solution or in suspension.
  • the catalyst is coupled to a detection probe.
  • the detection probe may for example be a label that emits light of a certain wavelength upon irradiation (e.g. fluorescein or a fluorescent protein), thus enabling an optical control measurement to verify the presence of the catalyst.
  • the detection probe may also be a probe nucleic acid molecule.
  • the catalyst is an enzyme or an enzyme-conjugate to which a probe nucleic acid molecule is coupled.
  • the probe nucleic acid molecule may be any nucleic acid of any length, for example of about 5 to about 250 bp, such as about 5 to about 100 bp. Such a nucleic acid molecule is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • the detection probe which is attached to the catalyst, hybridises to a respective portion of the target nucleic acid different from the portion to which the capture nucleic acid molecule hybridises. Accordingly, the detection probe directs the catalyst to the target nucleic acid and associates it thereto.
  • an optical detection may also be performed or enhanced by means of an optically amplifying conjugated polymer, e.g. in a F ⁇ rster energy transfer system (Gaylord, B. S., et al., Proc. Natl. Acad. Sd. USA (2005) 102, 34-39; Gaylord, B.S., et al., J Am. Chem. Soc.
  • a cationic polythiophene may be added, which changes its color and fluorescence in the presence of single-stranded or double-stranded nucleic acid molecules (Ho, H.A., et al., J. Am. Chem. Soc. (2005) 127, 36, 12673 -12676).
  • the polymerisation of the polymerisable positively chargeable precursor forms an electroconductive polymer.
  • this electroconductive polymer is likewise associated with the complex formed between the PNA capture molecule and the target nucleic acid molecule.
  • the present inventors have found that the electroconductive polymer is usually robustly bound to the surface of the immobilisation unit. For example extensive washing and potential cycling thereafter produced no noticeable changes in subsequent electrical measurements. Where desired, further reactions, washing steps and additional detection methods etc. may therefore be performed.
  • the electroactivity of the electroconductive polymer allows ultrasensitive electrochemical detection of target nucleic acid molecules as explained in the following.
  • the distance between the two electrodes is of a dimension that is comparable to the length of the target nucleic acid molecule, hi some embodiments the target nucleic acid molecule even spans the entire gap.
  • the electroconductive polymer formed allows the transfer of electrons along the target nucleic acid molecule. In some embodiments it even allows the transfer of electrons between the two electrodes.
  • the electroconductive polymer may for example include moieties such as functional groups (e.g. amino groups) that are capable of accepting and/or donating electron density, thus allowing the flow of electrons between or from/to the two electrodes.
  • the present invention thus uses the dynamic growing process of an electroconductive polymer as a means to both generate and amplify an electrical signal.
  • a longer electroconductive polymer generally includes more moieties, e.g. functional groups such as amino groups that are capable of accepting and/or donating electron density. Accordingly, a longer polymer generally has a higher capacity of allowing the flow of electrons.
  • the obtained polymer will typically furthermore include branches of various degree rather than form a purely linear product.
  • Such branches also amplify signals, including signals from the respective growing ends of the polymer. Furthermore typically a plurality of polymerisable positively chargeable precursors associates to one target nucleic acid molecule, such that multiple polymer chains start at a single target nucleic acid molecule.
  • the present invention thus includes a highly efficient signal amplification and transduction route.
  • the polymer associated to the target nucleic acid is electroactive, the current generated from it nevertheless directly correlates to the concentration of nucleic acid " in the sample solution.
  • the combination of highly efficient polymerisation, guided- deposition, and electrical detection thus provides a generic platform for ultrasensitive detection of nucleic acids.
  • using horseradish peroxidase as a catalyst, hydrogen peroxide as a reactant molecule and aniline as a positively chargeable precursor a linear correlation between the number of target nucleic acid molecules and the detected signal was observed (see Fig. 11).
  • the detection limit was in this example about 40 fM.
  • the catalytic and cumulative nature of the system causes the signal (see below) to increase with increasing incubation time before sampling.
  • the signal is only limited by capacity of the selected system in terms of the amounts of reactant molecule and polymerisable precursor, and where applicable, the amount of catalyst in the system.
  • a longer incubation period produces a higher signal and a lower detection limit (see e.g. Fig. 10).
  • an enhancer reagent may furthermore be added.
  • the electroconductive polymer is in such embodiments contacted with the respective enhancer reagent, typically before determining the presence of the target nucleic acid molecule.
  • a respective enhancer molecule serves in further enhancing a signal (see below) caused by the electroconductive polymer.
  • an acid or base may be used as an enhancer molecule.
  • suitable acids include, but are not limited to HCl (hydrochloric acid), HBr (hydrobromic acid), H2SO4 (sulphuric acid) and HCIO4 (perchloric acid) (G ⁇ k, A., et al, Int. J. Polym. Anal. Charact.
  • a respective enhancer molecule may also be an oxidant, such as for example a halobenzoquinone (e.g. o-chloranil or o-bromanil).
  • a halobenzoquinone e.g. o-chloranil or o-bromanil
  • Such an oxidant may cause an oxidation according to scheme (V) or even form a covalent bond with the electroconductive polymer as observed by Kang et al. (1990, supra).
  • the formation of a respective exemplary reaction product may be illustrated by the following scheme (VII):
  • Examples of further enhancer molecules include, but are not limited to an organic solvent such as chloroform, ethanol and benzene (G ⁇ k et al., 2006, supra), a pesticide such as methylparathion, paraquat, bentazon or glyphosate and a cyclodextrine compound such as ⁇ -cyclodextrine acid sulphate.
  • an organic solvent such as chloroform, ethanol and benzene (G ⁇ k et al., 2006, supra)
  • a pesticide such as methylparathion, paraquat, bentazon or glyphosate
  • a cyclodextrine compound such as ⁇ -cyclodextrine acid sulphate.
  • the electroconductive polymer changes an electrical characteristic of a region in between the electrodes.
  • an enhancer reagent may be used to enhance this change of a respective electrical characteristic.
  • the polymer may for instance change the electric field, change the conductivity or resistance of a medium in the electric field, obtain a charge, transfer charge or conduct a current.
  • the detection technique that may be employed in the method of the present invention is thus selected to be based on the measurement of a respective electrical characteristic. It may thus for instance be based on an impedance or a capacitance measurement. A change of a value of the electrical characteristic, such as a change in electric current, may also be detected as a function of time.
  • a respective signal of the electroconductive polymer is detected in the method of the present invention.
  • a detection according to the invention may or instance include a measurement of a conductance, a voltage, a current, a capacitance or a resistance.
  • conductance may be measured by linear cyclic voltammetry, square wave voltammetry, normal pulse voltammetry, differential pulse voltammetry and alternating current voltammetry.
  • the immobilisation unit, or at least a part of the surface thereof may be exposed to an electric field. In this case the electroconductive polymer immobilised thereon via its association to the complex of analyte molecule and capture molecule is likewise exposed to the respective electric field.
  • an electric field is generated, which may in some embodiments be a symmetric or a homogenous electric field.
  • the electric filed may for example be an external field. It may also be generated at at least one electrode of the pair of electrodes.
  • the result obtained is then compared to that of a reference measurement (or control measurement), hi a respective reference measurement PNA capture molecules unable to bind the target nucleic acid to be detected may for instance be used.
  • a control PNA capture molecule is a PNA molecule having a sequence not complementary to any portion of the target nucleic acid molecule (see e.g. Fig. 12).
  • a further example of a reference measurement which may also be performed on the same sample during analysis, is determining the respective electrical characeristic in the absence (or before) adding the respective polymerisable precursor. Yet a further example of a reference measurement (that may also be performed on the same sample during analysis) is determining the respective electrical characeristic in the absence (or before) adding the respective reactant molecule. If the two measurements of the respective electrical characteristic, i.e. "sample” and "control" measurement, differ in such a way that the difference between the values determined is greater than a pre-defined threshold value (see below), the sample solution contained the relevant target nucleic acid molecule.
  • a respective threshold value may for instance be determined in a calibration experiment, e.g.
  • the method is designed in such a way that a reference measurement and a measurement for detecting a target nucleic acid molecule are performed simultaneously. This may for instance be done by carrying out a reference measurement only with a control medium and, at the same time, a measurement with the sample solution expected to contain the target nucleic acid to be detected. Likewise, a respective control measurement with a PNA capture molecule that is not complementary to any portion of the target nucleic acid molecule may be carried out in parallel to a measurement for detecting a target nucleic acid molecule.
  • a threshold value in relation to a reference measurement is defined (see above).
  • This threshold value may be a fixed value of a respective electrical characteristic, the change of which is expected in the presence of the target nucleic acid molecule. It may also be a fixed value of a parameter that is caused by the electrical characteristic.
  • the presence or absence of a target nucleic acid molecule is then determined by means of comparison of the value detected, typically in relation to the reference measurement, with the threshold value. If the obtained value exceeds the threshold value, then it is inferred that the target nucleic acid molecule is present in the sample, and if appropriate in what concentration.
  • a detected value is below the threshold value it is inferred that no target nucleic acid molecule is present, hi the same way the rate of change of a parameter more than a predetermined threshold value can be used to indicate the presence of a target nucleic acid molecule in a sample.
  • the present method also allows detecting more than one target nucleic acid molecule simultaneously or consecutively in a single measurement.
  • a substrate including a plurality of immobilisation units as described above may for example be used, wherein different types of PNA capture molecules, each of which exhibiting (specific) is capable of hybridising to at least a portion of a particular target nucleic acid molecule, are immobilised on the surfaces of the immobilisation units.
  • the method according to the invention may be carried out by using virtually any electrode arrangement known in the art that includes a pair of electrodes.
  • Such an electrode arrangement may include a detecting or working electrode and a reference electrode (see also above).
  • the electrodes may be a conventional metal electrode (gold electrode, silver electrode etc.) or an electrode made from polymeric material or carbon.
  • An electrode arrangement may also include a common silicon or gallium arsenide substrate, to which a gold layer and a silicon nitride layer have been applied, and which has subsequently been structured by means of conventional lithographic and etching techniques to generate the electrode arrangement(s).
  • the method according to the present invention also allows detecting more than one type of analyte simultaneously or consecutively in a single measurement.
  • a substrate comprising a plurality of electrode pairs with immobilisation units as disclosed herein may be used, wherein different types of capture molecules, each of which exhibiting (specific) binding affinity for a particular analyte to be detected, are immobilised on the electrodes of the individual electrode arrangements.
  • An example of an electrode arrangement which may be used for carrying out the present method, as well as any other method according to the invention, is a conventional interdigitated electrode. Consequently, an arrangement provided with a plurality of interdigitated electrodes, i.e. an electrode array, can be employed for parallel or multiple determinations.
  • Another usable electrode arrangement is an electrode arrangement in the form of a trench or a cavity, which is formed, for example, by holding regions such as, for example, a gold layer on which the capture molecules capable of binding the analytes are immobilised being located on two opposite side walls.
  • Another method of the invention includes immobilising on the immobilisation unit a nucleic acid capture molecule that has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • This capture molecule may be any nucleic acid as long as it is capable of hybridising to at least a portion of the sequence of the target nucleic acid molecule.
  • the capture molecule may for example be a DNA, RJSfA or PNA molecule.
  • a respective nucleic acid molecule may be immobilised by any means, as long as it can hybridise to the target nucleic acid molecule thereafter. Examples of immobilising a nucleic acid capture molecule have already been explained above.
  • an ionisable thiol compound such as 2-dimethylaminoethanethiol hydrochloride may be covalently linked to the surface of the immobilisation unit. This modification allows nucleic acid molecules to bind through electrostatic interactions.
  • more than one nucleic acid capture molecule may be immobilised on the immobilisation unit. This may for instance be desired in order to broadly screen for the presence of any of a group of selected nucleic acid sequences. This may also be desired to allow for the simultaneous or consecutive detection of different analytes such as two or more genomic DNAs, each of them having binding specificity for one particular type of capture molecule. In some embodiments similar nucleic acid sequences, e.g. a number of nucleic acid sequences that are partially or substantially complementary to a selected target nucleic acid molecule, may be immobilised in order to enhance the likelihood of detecting the respective target nucleic acid molecule.
  • a further selectivity may be introduced by the selection of the nucleic acid molecule used that is attached to the enzyme added (see below). Furthermore, in this manner the detection of the same target nucleic acid molecule via different recognition sequences can be achieved, e.g., the 5'- and 3 '-termini of a nucleic acid molecule, which enhances the likelihood to detect even a few copies of a target nucleic acid molecule in a sample.
  • the present method also includes contacting the surface of the immobilisation unit with a solution expected to include the target nucleic acid molecule (see Fig. 3), and allowing the target nucleic acid molecule to hybridise to the nucleic acid capture molecule on the. immobilisation unit (see Fig. 3), thereby allowing the formation of a complex between the nucleic acid capture molecule and the target nucleic acid molecule (cf. above).
  • the present method also includes adding a polymerisable positively chargeable precursor (see Fig. 3).
  • this polymerisable positively chargeable precursor has an electrostatic net charge that is complementary to the electrostatic net charge of the target nucleic acid molecule, it associates with the complex formed between the nucleic acid capture molecule and the target nucleic acid molecule (see above).
  • the present method further includes adding a suitable substrate molecule (see Fig. 3).
  • a suitable substrate molecule may be used that is suitable as a substrate for the enzyme used (see above for examples) in the present method.
  • the enzyme may be a peroxidase enzyme, for instance a haem peroxidase, hi such embodiments the substrate will typically be a peroxide.
  • Any peroxide that can, at least to a certain degree, be dissolved in a selected solution used in this method or the method described above, such as an aqueous solution, may be used in the present invention.
  • a suitable peroxide examples include, but are not limited to, hydrogen peroxide, nitrogen peroxide, magnesium peroxide, calcium peroxide, zinc peroxide, benzoyl peroxide, cumenyl peroxide, dicumyl peroxide, diphenyl peroxide, bis(tert-butylperoxysuccinyl) peroxide, a lipid peroxide such as lauroyl peroxide, diacetyl peroxide or trifluoroacetyl peroxide, bis(o-iodophenylacetyl) peroxide, ethyl peroxide, 2-naphthyl peroxide, 2-naphthoyl peroxide, butyl hydroperoxide, vinyl hydroperoxide, cyclohexyl hydroperoxide, trifluoromethyl peroxide, 2,5-bis(l-methyl- ethyl) ⁇ henyl-hydroperoxide, 1 ,5-dimethyl-6,
  • the enzyme may also be an oxidase enzyme such as for instance laccase.
  • oxygen may for instance conveniently be added together with any aqueous solution used in the method of the invention, in which it is dissolved.
  • no additional measures need to be taken to add oxygen.
  • this may however be carried out by mechanical action, such as stirring, rotating or shaking.
  • the enzyme is attached to a probe nucleic acid molecule.
  • Any nucleic acid molecule may be used for this purpose that is at least partially complementary to at least a portion of the target nucleic acid molecule. Accordingly, the detection probe hybridises to a respective portion of the target nucleic acid different from the portion to which the capture nucleic acid molecule hybridises (cf. also above).
  • the catalyst such as an enzyme used in the method described above, may optionally also be attached to a probe nucleic acid molecule.
  • both the respective nucleic acid capture molecule and the target nucleic acid molecule may however be negatively charged under the assay conditions selected.
  • the target nucleic acid molecule may for instance be included in a solution of a pH value selected in the range of about 1.5 to about 8.0, such as in the range of about 1.7 to about 7.0, in the range of about 1.9 to about 6.0, or in the range of about 2.0 to about 5.5 (including a value of about 3.0 or about 4.0).
  • the pH may also be brought to a desired range or value, such as one of the aforementioned ranges, during or after adding the polymerisable positively chargeable precursor.
  • a desired range or value such as one of the aforementioned ranges
  • various DNA and RNA molecules will typically have a negatively charged backbone.
  • the positively chargeable precursor may bind to either nucleic acid molecule. Additional selectivity is however introduced into the present method by the use of an enzyme, which is attached to a probe nucleic acid molecule.
  • the present method also includes allowing the probe nucleic acid molecule to hybridise to the target nucleic acid molecule (see Fig. 3).
  • the two nucleic acid molecules i.e. the capture molecule and the analyte molecule
  • a complex is formed. It is understood that for the quantification of such a nucleic acid molecule a plurality of the respective capture molecules is usually required. Ih a suitable concentration range of the analyte molecule, where the method of the invention can be used to quantify a respective analyte molecule, generally an excess of capture molecules in comparison to analyte molecule is required.
  • one or more single-stranded nucleic acid capture molecules which do not form a complex with an analyte molecule, may remain.
  • the presence of such a nucleic acid capture molecule may interfere with the detection of the method of the present invention, hi particular where the nucleic acid capture molecule is a single-stranded DNA molecule or a single-stranded RNA molecule, such a remaining nucleic acid molecule may be removed from the surface.
  • the surface may be contacted with at least one enzyme with nuclease activity, in order to remove any nucleic acid capture molecule that has not hybridised to an analyte molecule. It may be desired to reduce or block nuclease activity that is directed against double-strands of nucleic acids in order to avoid a reduction of detection signal, caused by the degradation of complexes of capture molecule and analyte.
  • an enzyme may be selected that selectively degrades single-stranded nucleic acids. Examples of such enzymes include, but are not limited to, mung bean nuclease, nuclease Pl (e.g. from fungi), nuclease Sl (e.g.
  • CEL I nuclease e.g. from plants
  • recJ exonuclease e.g. from E. coli
  • DNA polymerase capable of degrading single-stranded DNA due to its 5'-> 3' exonuclease activity
  • DNA polymerase capable of degrading single-stranded DNA due to its 3'-> 5' exonuclease activity.
  • the present method also includes catalysing the polymerisation of the polymerisable positively chargeable precursor. Since the enzyme, which catalyses the polymerisation, is associated with the complex formed between the nucleic acid capture molecule and the target nucleic acid molecule (via the probe nucleic acid molecule), the polymerisation usually starts at the target nucleic acid molecule. At this location the polymerisation accordingly also progresses.
  • the target nucleic acid/enzyme adduct accordingly catalyses the polymerisation of the precursor and guides the deposition of electroconductive polymer at the immobilisation unit in the sensing zone.
  • the electroconductive polymer which is formed from the polymerisable precursor, is thus associated with the complex formed between the nucleic acid capture molecule and the target nucleic acid molecule.
  • the present method further includes determining the presence of the target nucleic acid molecule based on an electrical characteristic of a region in between the electrodes. This electrical characteristic is influenced by the electroconductive polymer (supra).
  • the method may for instance include performing an electrical measurement of an electric signal caused by the electroconductive polymer. Where applicable, the method may include quantifying the amount of target nucleic acid present (supra).
  • the methods according to the present invention may be a diagnostic method for the detection of one or more target genes.
  • the target gene may be involved in or associated with a disease or a state of the human or animal body that requires prophylaxis or treatment.
  • the method of the invention may be combined with other analytical and preparative methods.
  • the target nucleic acid may in some embodiments for instance be extracted from matter in which it is included. Examples of other methods that may be combined with a method of the present invention include, but are not limited to isoelectric focusing, chromatography methods, electrochromatographic, electrokinetic chromatography and electrophoretic methods.
  • kits for electrically detecting a target nucleic acid molecule which may for instance be diagnostic kits.
  • a respective kit includes a pair of electrodes, which are arranged at a distance from one another, for example separated by a gap. The pair of electrodes is arranged within a sensing zone.
  • a kit according to the present invention furthermore includes an immobilisation unit. The surface of the immobilisation unit is arranged within the sensing zone. As explained above, the sensing zone may for example be defined by the zone in which an electric field of said pair of electrodes is effective.
  • kits according to the present invention also includes a PNA capture molecule, which has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule (see above).
  • the kit also includes a polymerisable positively chargeable precursor. Illustrative examples of suitable precursors have been given above and are also found in Fig. 5. As explained above, the electrostatic net charge of the polymerisable positively chargeable precursor is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the kit also includes a suitable reactant molecule, such as an oxidant.
  • the kit may also include a suitable initiator, for example a halogen molecule, an azo compound, a persulfate molecule, and a peroxide compound.
  • a suitable initiator for example a halogen molecule, an azo compound, a persulfate molecule, and a peroxide compound.
  • the kit may include a catalyst, e.g. a metal chloride, a metal bromide, a metal sulphate or an enzyme (e.g. a peroxidase enzyme or an oxidase enzyme).
  • the catalyst may for example be in solution or coupled to a probe nucleic acid molecule.
  • the reactant may be a substrate molecule for the catalyst.
  • a respective kit may furthermore include means for immobilising the capture molecule to the surface of the immobilisation unit.
  • a nucleic acid capture molecule included in the kit may have a moiety that allows for, or facilitates, an immobilisation on a respective immobilisation unit.
  • the kit may also include a linking molecule.
  • 6-mercapto-l-hexanol may be included in the kit.
  • the PNA capture molecule may then be 5'-C 6 H 12 SH-modified (see above for examples).
  • kits according to the present invention also includes a nucleic acid capture molecule, which has a nucleotide sequence that is at least partially complementary to at least a portion of the target nucleic acid molecule (see above).
  • the kit further includes a polymerisable positively chargeable precursor, e.g. an aromatic amine.
  • the electrostatic net charge of the polymerisable positively chargeable precursor is complementary to the electrostatic net charge of the target nucleic acid molecule.
  • the kit also includes a substrate molecule, such as a peroxide or oxygen dissolved in a solution. As indicated above, oxygen dissolves in aqueous solutions, so that any aqueous solution included in the kit may be the source of this substrate molecule.
  • the kit includes an enzyme attached to a probe nucleic acid molecule (see above for examples).
  • the probe nucleic acid molecule is at least partially complementary to at least a portion of the target nucleic acid molecule.
  • a respective kit may furthermore include means for immobilising the capture molecule to the surface of the immobilisation unit as described above.
  • a respective kit may be used to carry out a method according to the present invention. It may include one or more devices for accommodating the above components before, while carrying out a method of the invention, and thereafter. As an illustrative example, it may include a microelectromedical system (MEMS).
  • MEMS microelectromedical system
  • the present invention is of particular significance in the design and manufacture of ultrasensitive non-labelling nucleic acid biosensors and biosensor arrays.
  • the nucleic acid-guided deposition of an electroconductive polymer such as polyanilme combined with efficient biocatalysis offers a very attractive alternative to hybridisation-based nucleic acid biosensors.
  • the skilled artisan will further appreciate that the present invention also allows for the fabrication of simple, low-cost, and portable electrical nucleic acid detection devices, providing fast, cheap and simple solutions for e;g. molecular diagnosis, particularly for cancer diagnosis, point-of-care, and field uses. For instance, point-of-care applications require systems that are portable, robust and easy to use, coupled with a reliable assay.
  • PNA capture probe and oligonucleotides were obtained from Eurogentec and used as received.
  • Amino-terminated peptide nucleic acid (PNA) capture probes (N- » C: NH 2 -AAC CAC ACA ACC TAC TAC CTC A) (SEQ ID NO: 1), DNA, 5'-TGA GGT AGT AGG TTG TGT GGT T-3' (SEQ ID NO: 2), Homo sapiens microRNA let-7b, corresponding to of SwissProt-Acc.-No AJ421727; single base mismatched DNA, 5'-TGA GGT AGT AGG ATG TGT GGT T-3' (SEQ ID NO: 3), non-complementary DNA, 3'-ACAAGACA TGGTTTTTCCCCCCATCA AAGGAAT-5' (SEQ ID NO: 4).
  • Biosensor fabrication [0123] The microelectrodes (Au 15 nm, Ti 10 nm) with 500 nm gaps were fabricated as 10x10 arrays on Si wafer with 500 nm coating of SiO 2 by the photolithography method, as displayed in Fig. 1.
  • the chips were first cleaned with chloroform and acetone in sequence thoroughly to remove any possible organic contaminants. After rinsing with IM NaOH, followed by thoroughly washing with water, the chips were activated in oven at 120 0 C for 20 min. The silanisation of the chips were carried out by soaking the chips in ethanol containing 2 % 3-aminopropyltriethoxysilane and 1 % water (v/v). Then the chips were washed with ethanol and allowed to dry under a mild stream of nitrogen before aging at 120 °C for 20 min. The bifunctional coupling agent phenylendiisothiocyanate was employed to hyphenate PNA capture molecules to the amino-modified chips.
  • a washing step utilising ultrapure water and methanol was applied to remove unreacted probes.
  • the surface was deactivated with a dimethylformamide solution (25 ml) containing aminoethanol (0.1 ml), diisopropylethylarnine (0.65 ml) over a period of 2 h.
  • the slides were subsequently washed with dimethylformamide, acetone, and water, and dried with nitrogen. Slides were not fully dried between washing steps.
  • FIG. 2 and Fig 3. A schematic illustration of the sensing procedure was displayed in Fig. 2 and Fig 3.
  • the hybridisation was performed with a 10 mM Tris-HCl, 1.0 niM EDTA and 0.1 M NaCl (TE) buffer at room temperature for 6 min in a commercially available hybridisation chamber. After hybridisation, the chips were thoroughly rinsed with blank hybridisation buffer (see above) to remove the unhybridised DNA.
  • Deposition of polyaniline was accomplished as described earlier (Liu, W. et al., J. Am. Chem. Soc.
  • the conductivity achieved for the respective biosensor exposed to 10 pM target DNA was 0.36 nS considering the width of the gap was 500 nm), which was at the same level as that for DNA-polyaniline pellets [Nagarijan, R., et al., J. Macromol. Sci.-Pure Appl. Chem. (2001) A38, 1519-1537] while was as about one order of magnitude lower then that of polyaniline deposited with L-DNA as the template [Liu & Kumar, 1999, supra].
  • a scanning electron microscope characterisation was performed for the chips exposing to control sample and the complementary DNA sample. The images were shown in Fig. 7. It could be seen that, after polyaniline deposition, the chip surface with complementary DNA hybridised was rough with a polymer network. While the control one showed no visible change. The morphology of the polyaniline deposited was somewhat different from the wire-like polymer found in using aligned long DNA strands as the templates for polymerisation. The possible reason might be the DNA sample used here was relatively short with only 22 bases long (estimated strand length of 7 ⁇ 8 nm) and randomly spread.
  • an acidic buffer solution at pH 4.0 was selected as the background to perform the polyaniline synthesis and deposition in order to facilitate the electrostatic interactions between aniline and the phosphate groups and also provide adequate activity of HRP [Liu et al., 1999, supra; Gaylord, B. S., et al., Proc. Natl.
  • the optimisation of aniline concentration was performed by varying the aniline concentration in the mixture solution for polymerisation from 0.5 to 5mM while keeping the HRP concentration and incubation time constant.
  • the aniline concentration increased from 0.5 to 5 mM, the conductivity of the resulted polyaniline formed in the nanogaps first increased and then achieved a steady value at 2 mM. It is known that the rate of enzyme catalyzing reactions increases with the increasing substrate concentration until the substrate concentration is high enough to saturate the enzyme.
  • 2 mM of aniline was selected to perform polyaniline deposition, which was also high enough to facilitate the longer polymer chain growth and avoid forming short chain segments [Gaylord et al., 2005, supra].
  • the concentration of HRP was also optimised in the range of 0.1 to 2.5 ⁇ g/mL; the results were depicted in Fig. 9.
  • the conductivity of the resulted polyaniline increased with the increasing of the HRP concentration in the range of 0.1 ⁇ 1.0 ⁇ g/mL, and further increasing the HRP concentration did not result in continuous increasing in conductivity.
  • the reaction rate is linearly correlated with the enzyme concentration when the substrate concentration is high enough.
  • the incubation time of polyaniline deposition was also optimised. As displayed in Fig. 10, the longer the incubation time, the higher the conductivity could be achieved. However, an increase of the background noise was shown after 45 min of incubation. The reason might be the increase of the deposition of the polyaniline synthesised in the solution onto the nanogaps. Thus, an incubation time of 40 min was selected to ensure highest signal/noise ratio.
  • the complementary DNA can be detected in a dynamic range of 50 fM ⁇ 100 pM with the regression coefficient R 2 as 0.976, as depicted in
  • Fig. 11 The detection limit achieved was 40 fM. Comparing with the gold nanoparticle labeling and silver enhance method for detection of DNAs [Liu & Kumar, 1999, supra], the sensitivity of the present assay was one order of magnitude higher.
  • microRNA let-7b sequence of SwissProt-Acc.-No AJ421727
  • TE buffer 0.48 and 0.40 ⁇ g/ ⁇ L, respectively and direct taken for hybridisation. Both samples gave the positive conductive changes and the results were normalised with respect to total RNA.
  • let-7b found in HeIa cell and lung cancer cell total RNA samples was 2.34 ⁇ 0.29> ⁇ 10 7 copies and 2.53 ⁇ 0.33xl0 7 copies, respectively.
  • the results were consistent with previously published work [Gao, Z. Q., Yang, Z. C, Anal. Chem. (2006) 78, 1470-1477].
  • the precision of the assay was also satisfactory with the RSDs better than 16 %, which provided satisfactory accuracy to distinguish slight microRNA expression differences. Therefore, this assay is promising in microRNA analysis.

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Abstract

L'invention concerne des procédés pour détecter électriquement une molécule d'acide nucléique cible. Une paire d'électrodes est agencée à une distance l'une de l'autre et à l'intérieur d'une zone de détection. Une molécule de capture d'APN, possédant une séquence de nucléotides qui est au moins partiellement complémentaire à une partie de l'acide nucléique cible, est immobilisée sur une unité d'immobilisation. La molécule d'acide nucléique cible s'hybride et forme un complexe avec cette molécule de capture. Un précurseur polymérisable pouvant être chargé positivement, muni d'une charge nette électrostatique complémentaire à l'acide nucléique cible, est ensuite ajouté. Ce processeur s'associe au complexe formé entre la molécule de capture d'ANP et l'acide nucléique cible. Après l'ajout d'une molécule de réactif appropriée, la polymérisation est déclenchée. Le polymère électroconducteur qui est formé influence les caractéristiques électriques de la zone située entre les électrodes, en permettant la détection de la présence de la molécule d'acide nucléique cible.
PCT/SG2007/000037 2007-02-05 2007-02-05 Procédés pour détecter électriquement un acide nucléique par l'intermédiaire d'une paire d'électrodes Ceased WO2008097190A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012009578A3 (fr) * 2010-07-14 2012-04-05 The Curators Of The University Of Missouri Détection de molécules individuelles d'acides nucléiques facilitée par des nanopores
WO2013057096A1 (fr) 2011-10-17 2013-04-25 Westfaelische Wilhelms-Universitaet Muenster Évaluation du risque de lemp et méthodes associées
WO2014125267A1 (fr) * 2013-02-14 2014-08-21 Eluceda Limited Dosage pour détecter un analyte d'acide nucléique dans un échantillon biologique
US9732379B2 (en) 2013-03-15 2017-08-15 The Curators Of The University Of Missouri Encoded nanopore sensor for multiplex nucleic acids detection
CN111316096A (zh) * 2017-02-08 2020-06-19 Essenlix公司 生物/化学材料提取和测定
US20200400602A1 (en) * 2013-12-12 2020-12-24 Altratech Limited Capacitive sensor and method of use
CN113801912A (zh) * 2021-10-18 2021-12-17 上海交通大学医学院附属仁济医院 一种基于核酸介导的酶级联用于肌氨酸的检测方法
US11834697B2 (en) 2017-09-15 2023-12-05 Oxford University Innovation Limited Electrochemical recognition and quantification of cytochrome c oxidase expression in bacteria
US12553078B2 (en) 2017-09-20 2026-02-17 Altratech Limited Diagnostic device and system

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6018018A (en) * 1997-08-21 2000-01-25 University Of Massachusetts Lowell Enzymatic template polymerization
WO2002081739A2 (fr) * 2001-04-09 2002-10-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Analyse non enzymatique a liaison liposomique avec electrodes en reseau etroitement espacees (nel-ela) pour detecter et quantifier des acides nucleiques
US20050079533A1 (en) * 1997-08-21 2005-04-14 Samuelson Lynne A. Enzymatic template polymerization
WO2005040403A1 (fr) * 2003-10-29 2005-05-06 Agency For Science, Technology And Research Procede de detection d'analytes par l'intermediaire d'un dispositif bicouche analyte/activateur polymere
US20060160100A1 (en) * 2005-01-19 2006-07-20 Agency For Science, Technology And Research Enzymatic electrochemical detection assay using protective monolayer and device therefor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6018018A (en) * 1997-08-21 2000-01-25 University Of Massachusetts Lowell Enzymatic template polymerization
US20050079533A1 (en) * 1997-08-21 2005-04-14 Samuelson Lynne A. Enzymatic template polymerization
WO2002081739A2 (fr) * 2001-04-09 2002-10-17 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Analyse non enzymatique a liaison liposomique avec electrodes en reseau etroitement espacees (nel-ela) pour detecter et quantifier des acides nucleiques
WO2005040403A1 (fr) * 2003-10-29 2005-05-06 Agency For Science, Technology And Research Procede de detection d'analytes par l'intermediaire d'un dispositif bicouche analyte/activateur polymere
US20060160100A1 (en) * 2005-01-19 2006-07-20 Agency For Science, Technology And Research Enzymatic electrochemical detection assay using protective monolayer and device therefor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LE FLOCH ET AL.: "Ferrocene-Functionalized cationic polythiophene for the label-free electrochemical detection of DNA", ADVANCED MATERIALS, vol. 17, 2005, pages 1251 - 1254 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012009578A3 (fr) * 2010-07-14 2012-04-05 The Curators Of The University Of Missouri Détection de molécules individuelles d'acides nucléiques facilitée par des nanopores
US9395353B2 (en) 2010-07-14 2016-07-19 The Curators Of The University Of Missouri Nanopore-facilitated single molecule detection of nucleic acid
US9574228B2 (en) 2010-07-14 2017-02-21 The Curators Of The University Of Missouri Nanopore-facilitated single molecule detection of nucleic acids
US10273527B2 (en) 2010-07-14 2019-04-30 The Curators Of The University Of Missouri Nanopore-facilitated single molecule detection of nucleic acids
WO2013057096A1 (fr) 2011-10-17 2013-04-25 Westfaelische Wilhelms-Universitaet Muenster Évaluation du risque de lemp et méthodes associées
WO2013057092A1 (fr) 2011-10-17 2013-04-25 Westfaelische Wilhelms-Universitaet Muenster Méthodes d'évaluation du risque de lemp et appareil associé
WO2014125267A1 (fr) * 2013-02-14 2014-08-21 Eluceda Limited Dosage pour détecter un analyte d'acide nucléique dans un échantillon biologique
US9732379B2 (en) 2013-03-15 2017-08-15 The Curators Of The University Of Missouri Encoded nanopore sensor for multiplex nucleic acids detection
US11796498B2 (en) * 2013-12-12 2023-10-24 Altratech Limited Capacitive sensor and method of use
US20200400602A1 (en) * 2013-12-12 2020-12-24 Altratech Limited Capacitive sensor and method of use
US12487238B2 (en) * 2013-12-12 2025-12-02 Altratech Limited Capacitive sensor and method of use
CN111316096B (zh) * 2017-02-08 2023-08-11 Essenlix公司 生物/化学材料提取和测定
CN111316096A (zh) * 2017-02-08 2020-06-19 Essenlix公司 生物/化学材料提取和测定
US11834697B2 (en) 2017-09-15 2023-12-05 Oxford University Innovation Limited Electrochemical recognition and quantification of cytochrome c oxidase expression in bacteria
US12553078B2 (en) 2017-09-20 2026-02-17 Altratech Limited Diagnostic device and system
CN113801912A (zh) * 2021-10-18 2021-12-17 上海交通大学医学院附属仁济医院 一种基于核酸介导的酶级联用于肌氨酸的检测方法

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