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WO2010117341A1 - Biocapteur d'acide nucléique - Google Patents

Biocapteur d'acide nucléique Download PDF

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Publication number
WO2010117341A1
WO2010117341A1 PCT/SG2010/000139 SG2010000139W WO2010117341A1 WO 2010117341 A1 WO2010117341 A1 WO 2010117341A1 SG 2010000139 W SG2010000139 W SG 2010000139W WO 2010117341 A1 WO2010117341 A1 WO 2010117341A1
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Prior art keywords
nucleic acid
acid molecule
target
molecule
target nucleic
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English (en)
Inventor
Xiaojun Chen
Zhiqiang Gao
Hong Xie
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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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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

Definitions

  • the present invention refers to the field of biochemistry and in particular to technologies concerning immobilization of biomolecules and detection of nucleic acids.
  • Ultrasensitive detection of nucleic acids at detection levels of femtomolar concentration provides promising applications in diagnosis of genetic and infectious diseases, forensic investigation and environmental monitoring. In particular, a rapid, sensitive and accurate detection of the influenza virus is of great importance for both clinical and public health purposes.
  • State of the art techniques for detection of influenza viruses in clinical laboratories include virus culture, rapid antigen detection, serological tests and real-time polymerase chain reaction (also called RT-PCR).
  • RT-PCR real-time polymerase chain reaction
  • RT-PCR can be sensitive and specific for detection of subtypes of influenza virus, the bulky and high cost equipment, as well as easy photobleaching of the fluorescence labels used, has limited its application. [0005] It is therefore an object of the present invention to provide alternative methods to detect biomolecules with properties that overcome at least some of the above described disadvantages.
  • the present invention is directed to a method for the electrochemical detection of a target nucleic acid molecule, wherein the method comprises:
  • the agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and being capable of binding to the target nucleic acid molecule to bind to the target nucleic acid molecule;
  • ⁇ contacting the detection electrode with a metallic nanoparticle precursor material and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material; ⁇ allowing the substrate compound and the metallic nanoparticle precursor material to react with the molecule capable of transferring electrons to the metallic nanoparticle precursor material to form metallic nanoparticles capable of depositing to the sensing region of the detection electrode;
  • the present invention is directed to a method for the electrochemical detection of a target nucleic acid molecule, wherein the method comprises:
  • ⁇ contacting the detection electrode comprising the complex between the capture nucleic acid molecule and the target nucleic acid molecule with a target probe, wherein the target probe is capable of hybridizing to a second region of the target nucleic acid;
  • the agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target probe of the second complex to bind to the target probe of the second complex so that the agent binds to the target nucleic acid molecule via the target probe;
  • ⁇ allowing the substrate compound and the metallic nanoparticle precursor material to react with the molecule capable of transferring electrons to the metallic nanoparticle precursor material to form metallic nanoparticles capable of depositing in the sensing region of the detection electrode; ⁇ detecting a change in the electrical current in case of formation of the metallic nanoparticles which deposited in the sensing region of the detection electrode to determine the presence or absence of the target nucleic acid molecule.
  • the present invention provides a kit for electrochemically detecting a target nucleic acid molecule.
  • the kit can include an electrode arrangement comprising a detection electrode.
  • the kit can further include a capture nucleic acid molecule.
  • the capture nucleic acid comprises a nucleotide sequence that is at least partially complementary to at least a first portion of the target nucleic acid molecule.
  • the kit can further include a target probe.
  • the target probe comprises a nucleotide sequence that is at least partially complementary to at least a second portion of the target nucleic acid molecule.
  • the kit can further include an agent.
  • the agent comprises a molecule capable of transferring electrons to a metallic nanoparticle precursor material and being capable of binding to the target nucleic acid molecule, wherein the molecule capable of transferring electrons to a metallic nanoparticle precursor material is capable of reducing the metallic nanoparticle precursor material.
  • the kit can further include a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material is capable of accepting an electron to form a metallic nanoparticle.
  • the kit can further include a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor solution.
  • FIG. 1 a general scheme of the method of detecting a nucleic acid molecule.
  • a detection electrode comprising capture nucleic acid molecules immobilized within a sensing region of the detection electrode is shown in Figure l(a). It is contacted with a solution comprising target nucleic acid molecules.
  • Each capture nucleic acid molecule can comprise a nucleic acid sequence capable of hybridizing with the target nucleic acid molecule.
  • the target nucleic acid molecules are allowed to hybridize with the capture nucleic acid molecules, thereby forming a first complex between the target nucleic molecule and the capture nucleic acid molecule as shown in Figure l(b).
  • the detection electrode which now comprises the first complex between the target nucleic acid and the capture nucleic acid, can contact with an agent (denoted by Ml) comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target nucleic acid molecule.
  • the agent can bind to the target nucleic acid molecule of the first complex as shown in Figure l(c).
  • the detection electrode is contacted with a metallic nanoparticle precursor material (denoted as NPM) and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material and substrate compound can react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material, in which the substrate compound is oxidized (denoted as o-substrate compound), and due to reduction of the metallic nanoparticle precursor material, metallic nanoparticles (denoted as NPM red.) are formed which can deposit on the detection electrode as shown in Figure l(d).
  • FIG. 2 shows an embodiment of the present invention.
  • a detection electrode comprising capture nucleic acid molecules immobilized within a sensing region of the detection electrode is shown in Figure 2(a). It is contacted with a solution comprising target nucleic acid molecules and target probes.
  • Each capture nucleic acid molecule can comprise a nucleic acid sequence capable of hybridizing to a first region of the target nucleic acid molecule.
  • the target nucleic acid molecules are allowed to hybridize with the capture nucleic acid molecules, thereby forming a first complex between the target nucleic molecule and the capture nucleic acid molecule.
  • the target probe can hybridize to a second region on the target nucleic acid to form a second complex as illustrated in Figure 2(b).
  • the detection electrode is then contacted with an agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target probe of the second complex (denoted as Ml).
  • the agent can bind with the target probe of the second complex so that the agent binds to the target nucleic acid molecule via the target probe, as shown in Figure 2(c).
  • the detection electrode is contacted with a metallic nanoparticle precursor material (denoted as NPM) and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material and substrate compound can react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material, in which the substrate compound is oxidized (denoted as o-substrate compound), and due to reduction of the metallic nanoparticle precursor material, metallic nanoparticles (denoted as NPM red.) are formed which can deposit on the detection electrode.
  • Fig. 3 shows fluorescence images of electrode surfaces which were immobilized with DNA capture probes and after hybridization with nucleic acids.
  • Figure 3 (a) is a fluorescence image of surface hybridized with a mixture of TAM- labeled totally mismatched (non-complementary) targets (Totally Mismatched Target 1 and 2 in Table 1).
  • Figure 3(b) is a fluorescence image of surface hybridized with complementary target DNA (Complementary Target 1 in Table 1). There is no noticeable fluorescence when the surface was hybridized with the totally mismatched TAM-labeled target DNA. However, after hybridizing with the complementary target DNA, strong red fluorescence was observed at excitation wavelength of 545 nm.
  • FIG. 4 shows cyclic voltammograms of electrodes made according to embodiments of the present invention.
  • Figure 4(a) shows cyclic voltammograms of a bare gold electrode (curve denoted by "Au/CuHCF") and a gold electrode coated with 11-mercapto-l-undecanic acid-DNA (MUA-DNA) (curve denoted by "Au/MUA-DNA/CuHCF”).
  • Two pairs of redox peaks were observed for the bare gold electrode, whereas only one pair of redox peak was observed for the MUA-DNA coated electrode.
  • Figure 4(b) shows cyclic voltammograms of a gold electrode which was exposed to totally mismatched (non-complementary) target (Totally Mismatched Target 2 in Table 1) (curve denoted by “Control”) and used as a control electrode, and a gold electrode hybridized with 100 fM target DNA (Complementary Target 1 in Table 1) (curve denoted by "100 fM”), both of which were subjected to GOx-catalyzed deposition of copper hexacyano ferrate nanoparticles.
  • a gold electrode hybridized with 100 fM target DNA shows a pair of redox current peaks at about 0.75 V.
  • Insert of Figure 4(b) shows a differential pulse voltammogram (DPV) of the electrode hybridized with 100 fM target DNA (Complementary Target 1 in Table 1) solution
  • FIG. 5 shows scanning electron microscopy (SEM) images of the gold electrodes after hybridization reaction and GOx-catalyzed deposition of copper hexacyano ferrate nanoparticles.
  • Figure 5(a) is a SEM image of the control electrode (Totally Mismatched Target 2 in Table 1).
  • Figure 5(b) is a SEM image of the electrode hybridized with 100 fM target DNA (Complementary Target 1 in Table 1). No copper hexacyano ferrate nanoparticles were observed on the surface of the control electrode, whereas granular copper hexacyano ferrate nanoparticles were observed on the electrode hybridized with 100 fM target DNA.
  • Fig. 6 shows the differential pulse voltammogram (DPV) of the electrodes after hybridization reaction with 1.0 pM of complementary target DNA (Complementary Target 1 in Table 1), 1.0 pM of one-base mismatched target DNA (One-base Mismatched Target 1 in Table 1) and 1.0 pM of totally mismatched (non-complementary) DNAs (Totally Mismatched Target 1 and 2 in Table 1). Difference in DPV peak current values shown can be used in a biosensor to differentiate between fully complementary and mismatched targets.
  • Fig. 6 shows the differential pulse voltammogram (DPV) of the electrodes after hybridization reaction with 1.0 pM of complementary target DNA (Complementary Target 1 in Table 1), 1.0 pM of one-base mismatched target DNA (One-base Mismatched Target 1 in Table 1) and 1.0 pM of totally mismatched (non-complementary) DNAs (Totally Mismatched Target 1 and 2 in Table 1). Difference in DPV peak current values shown can be used in a biosensor to differentiate between fully
  • control 7 is a graph showing DPV peak current performance of control electrode (Totally Mismatched Target 2 in Table 1) (curve denoted as “control”) and electrode hybridized with 10 fM complementary target DNA (Complementary Target 1 in Table 1) (curve denoted as “10 fM complementary target”) as a function of copper hexacyano ferrate nanoparticle deposition time.
  • control totally Mismatched Target 2 in Table 1
  • 10 fM complementary target Complementary Target 1 in Table 1
  • Fig. 8 is a graph showing the differential pulse voltammogram (DPV) of electrode hybridized with complementary target DNA (Complementary Target 1 in Table 1) at various levels of target DNA concentrations.
  • the DPV peak current increased with an increase in target DNA concentration.
  • Insert of Figure 8 is a graph showing DPV peak current performance of electrode hybridized with the complementary target DNA as a function of target DNA concentration.
  • Fig. 9 shows a specific embodiment of the method according to the present invention.
  • a biotinylated target probe was used.
  • Avidin-labelled glucose oxidase (GOx) was used as the molecule capable of transferring electrons to a metallic precursor, and glucose was used as the substrate compound.
  • Potassium ferricyanide in copper chloride solution was used as the metallic nanoparticle precursor material.
  • glucose is oxidised to form glucono lactone which may be further oxidised to form gluconic acid, and ferricyanide ion is reduced in solution to form ferrocyanide.
  • Ferroxyanide formed subsequently reacts with copper (II) ion present to form copper hexacyano ferrate nanoparticles.
  • Fig. 10 shows a specific embodiment of the method according to the present invention.
  • a biotinylated target nucleic acid was used.
  • Avidin- labelled glucose oxidase (GOx) was used as the molecule capable of transferring electrons to a metallic precursor, and glucose was used as the substrate compound.
  • Potassium ferricyanide in copper chloride solution was used as the metallic nanoparticle precursor material.
  • glucose is oxidised to form glucono lactone which may be further oxidised to form gluconic acid, and ferricyanide ion is reduced in solution present to form copper hexacyano ferrate nanoparticles.
  • FIG. 11 shows cyclic voltammograms of a gold electrode which was exposed to totally mismatched (non-complementary) target (Totally Mismatched Target 3 in Table 1) (curve denoted by “Control”) and used as a control electrode, and a gold electrode hybridized with 100 fM target DNA (Complementary Target 2 in Table 1) (curve denoted by "100 fM”), both of which were subjected to GOx-catalyzed deposition of copper hexacyano ferrate nanoparticles.
  • 100 fM target DNA Complementary Target 2 in Table 1
  • Fig. 12 is a graph showing the differential pulse voltammogram (DPV) of electrode hybridized with complementary target DNA (Complementary Target 2 in Table 1) at various levels of target DNA concentrations.
  • the DPV peak current increased with an increase in target DNA concentration.
  • the invention is based on the finding that the sensitivity of the detection of analytes, such as target nucleic acids which may be present in only trace amounts, can be significantly improved by electrochemical detection utilizing an enzyme/substrate reaction in the presence of a metallic nanoparticle precursor material.
  • an enzyme catalyzed reaction is used for amplification of nucleic acid hybridization event
  • a nucleic acid concentration as low as about 1 fM i.e. 10 "15 M
  • the increased sensitivity of the detection method of the invention compared to other procedures known in the art can be attributed in part by the high sensitivity of the electrochemical detection technique and catalytic activity of the enzyme.
  • a polymeric binder which may be used to increase sensitivity of detection electrode for detection of specific analyte is not necessarily required using this process.
  • the present invention refers to a method for electrochemical detection of a target nucleic acid molecule.
  • the term 'electrochemical detection' refers to the utilization of electrochemical means to indicate the presence or absence, either qualitatively or quantitatively, of an analyte.
  • detection refers to both qualitative and quantitative detection of analytes in a sample, as well as qualitative measurements in which for instance different types of analyte molecules in a given sample are identified, or, as a further example, the behaviour of a particular analyte molecule in a given environment is observed.
  • electrochemical detection involves the use of electrodes immersed in an analyte, and connected to an instrument that varies the voltage applied to the electrodes while measuring the current flow between the electrodes. By varying the electrode potential, an electric current that is characteristic of the electrochemical active substances in the electrolyte flows between the electrodes.
  • two or more electrodes are used.
  • One of the electrodes can be a detection electrode, also known as the working electrode, which makes contact with the analyte and facilitate transfer of electrons to and from the analyte.
  • a second electrode can be a counter electrode, which works to balance the electrons added or removed by the working electrode.
  • a third electrode may be used to act as a reference electrode which acts as a reference in measuring and controlling the working electrodes potential.
  • Figure 1 shows a general scheme of the method for electrochemical detection of a target nucleic acid molecule according to the present invention.
  • the present invention includes providing a detection electrode having a capture nucleic acid molecule immobilized within a sensing region of the detection electrode.
  • detection 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.
  • a respective detection electrode may for example be used for the detection of an electric signal in the method of the present invention.
  • the electrode such as a detection electrode is one of at least two terminals of an electrically conducting medium.
  • a detection electrode can be used in any electrode arrangement known in the art that comprises a detecting or working electrode. Such an electrode arrangement can also comprise a counter electrode as well as a reference electrode.
  • An electrode, such as a detecting electrode may be a conventional metal electrode, such as a noble metal electrode, or an electrode made from polymeric material or carbon, the surface of which has been optionally modified in order to facilitate the immobilization of the capture nucleic acid molecule.
  • Noble metal includes silver, palladium, gold, platinum, iridium, osmium, rhodium and ruthenium. In one embodiment, silver, gold, platinum, mixtures thereof or alloys thereof can be used. Examples of noble metal alloys include alloys of platinum and iridium, Pd-Pt, Pd-Rh or Pd-Pt-Rh, to name only a few. In one embodiment, the noble metal is gold or an alloy comprising gold.
  • the electrodes of the electrode arrangement such as the detecting electrode may also be 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), such as the detection electrode of the electrode arrangement.
  • the distance between the detecting and the counter electrodes may vary, depending on the kind of structuring technique used and the type of target nucleic acid to be detected.
  • the distance between the electrodes can be between about 50 ⁇ m to 1000 ⁇ m or at least 50 ⁇ m or at least 1000 ⁇ m.
  • the capture nucleic acid molecules immobilized within a sensing region of the detection electrode according to the present invention may refer to a single type of nucleic acid molecule.
  • the term "nucleic acid molecule" as used herein 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), protein nucleic acids molecules (PNA) and tecto-RNA molecules (e.g. Liu, B., et al., J. Am. Chem. Soc.
  • LNA has a modified RNA backbone with a methylene bridge between C4' and 02', providing the respective molecule with a higher duplex stability and nuclease resistance.
  • DNA or RNA may be of genomic or synthetic origin.
  • a respective nucleic acid may furthermore contain non-natural nucleotide analogues and/or be linked to a functional group.
  • the capture nucleic acid molecules may also comprise different types of nucleic acids, for example, nucleic acid molecules having different nucleic acid sequences (which therefore also exhibit different binding specificities).
  • capture nucleic acid molecules allows the simultaneous or consecutive detection of different target nucleic acid molecules such as two or more genomic DNAs, each of them having binding specificity for one particular type of capture nucleic acid molecule, and also the detection of the same target nucleic acid molecule via different recognition sequences, e.g., the 5'- and 3'- termini of a nucleic acid molecule or two ligand binding sites of a receptor molecule, which enhances the likelihood to detect even a few copies of a target nucleic acid molecule in a sample.
  • different capture nucleic acid molecules capable of hybridizing to the same target nucleic acid can be immobilized within the sensing region.
  • the capture nucleic acid molecule may also be a synthetic oligonucleotide or longer nucleic acid sequences, as long as the latter do not fold in any structure preventing hybridization of the capture nucleic acid molecule with the target nucleic acid to be detected.
  • the capture nucleic acid molecules can comprise at least one or at least two modified nucleotides such as nucleotides carrying functional group such as a biotin, an avidin, a streptavidin, a digoxigenin, and anti-digoxigenin, an amine, a hydroxyl, an epoxide or thiol group.
  • the capture nucleic acid molecule may additionally comprise a spacer.
  • spacer refers to a chemical structure which links two components to each other. The spacer can be used to modify the length of the capture nucleic acid molecule. The spacer can also be used to minimize steric effects caused by immobilization of the capture nucleic acid molecules on the detection electrode surface. The spacer may be located between the capture nucleic acid molecule and the surface of the detection electrode.
  • the spacer can be located between a chemical linker which is bound to the surface of the detection electrode and the capture nucleic acid molecule.
  • spacer can include, but are not limited to, a nucleic acid spacer, an alkane spacer, a polypeptide spacer, a nucleotide spacer, such as an adenosine spacer or a thymine spacer; a polyethylene glycol (PEG) spacer, phosphoramidite, or l',2'-dideoxyribose, and derivatives thereof. Length of the spacer can be varied for specific applications.
  • thymine spacer can be in the form of thymine T5, which is a 5 thymine base-long oligonucleotide or in its longer form, thymine T9, which is a 9 thymine base-long oligonucleotide.
  • Suitable capture nucleic acid molecules for the detection of target nucleic acids may be provided in a suitable buffer to stabilize it.
  • a capture nucleic acid molecule having a sequence that is partially or fully complementary to the single-stranded region of the respective target nucleic acid is preferably used.
  • a single- stranded nucleic acid molecule may be selected as the capture nucleic acid molecule.
  • Such a single- stranded nucleic acid molecule may have a nucleic acid sequence that is at least partially complementary to at least a portion of a strand of the nucleic acid molecule that is the target nucleic acid.
  • the single-stranded nucleic acid molecule may be specified such that it can hybridize to the first region of the target nucleic acid.
  • the respective nucleotide sequence of the nucleic acid capture molecule may for example be 70, for example 80 or 85, including 100 % complementary to the target 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.
  • complementary refers to two nucleotides that can form multiple favourable interactions with one another. It has been demonstrated herein that the method of the present invention is suitable to differentiate a single-mismatched target nucleic acid molecule from a fully complementary target nucleic acid molecule.
  • the capture nucleic acid molecule used in a method according to the present invention may be of any suitable length so that it is capable of hybridizing with the target nucleic acid molecule.
  • the capture nucleic acid molecule can have a length of about 10 nucleotides to about 30 nucleotides, for example a length of about 10 nucleotides to about 25 nucleotides, such as a length of about 10 nucleotides to about 20 nucleotides.
  • the capture nucleic acid molecule can have a length of at least 10 nucleotides.
  • the capture nucleic acid molecules can be immobilized within a sensing region of the detection electrode.
  • the sensing region is usually a zone or aperture on the detection electrode into which the capture nucleic acid molecule is caused to be located.
  • the sensing region may be any area on the detection electrode.
  • the sensing region may be an aperture to which the target nucleic acid molecule is caused to flow.
  • a protection layer adapted to prevent non-specific binding of nucleic acid molecules on the electrode surface may be formed on the surface of the detection electrode or in the sensing region of the detection electrode.
  • the protection layer may comprise a thin film comprising a blocking agent and a capture nucleic acid binding molecule capable of binding to the capture nucleic acid molecule.
  • Such a thin film can be a monolayer, a bilayer or a multilayer of any thickness.
  • blocking agent refers to an agent, e.g. a molecule which can inhibit or block nucleic acid molecules, such as the target nucleic acid molecule or non- target nucleic acid molecules or any other compounds which can be comprised in a test sample and can interfere with the electrochemical detection, from interacting with the electrode surface.
  • an agent e.g. a molecule which can inhibit or block nucleic acid molecules, such as the target nucleic acid molecule or non- target nucleic acid molecules or any other compounds which can be comprised in a test sample and can interfere with the electrochemical detection, from interacting with the electrode surface.
  • any agent that can be immobilized on the electrode and that is able to prevent (or at least to significantly reduce) interaction between nucleic acid molecules and the electrode surface is suitable.
  • blocking agents are thiol molecules, disulfides, thiophene derivatives, and polythiophene derivatives, to name only a few.
  • thiol molecules comprising a terminal hydroxyl group (-OH) or carboxyl group (-COOH) can be used.
  • One particular useful class of blocking reagents can be fatty acids or fatty alcohols. Both can comprise a functional group, such as a thiol group, for binding to the electrode surface.
  • the fatty acids comprise between about 1 to about 20, or between about 1 to about 6 carbon atoms.
  • Examples for fatty acids comprising a thiol group include, but are not limited to 16-mercaptohexadecanoic acid (also known as MHA), 11-mercapto-l-undecanic acid (also known as MUA), 12-mercaptododecanoic, 11- mercaptodecanoic acid or 10-mercaptodecanoic acid.
  • Examples of fatty alcohols can include, but are not limited to saturated alcohols or unsaturated alcohols. Further examples of fatty alcohols can include, but are not limited to capric alcohol, lauryl alcohol, undecanol, myristil alcohol, or cetyl alcohol, to name only a few.
  • Choice of blocking agent may depend on the type of nucleic acid molecules present and the type of detection electrode used.
  • a suitable blocking agent can be a thiol molecule such as 11-Mercapto-l- undecanol, which contains a thiol (-SH) group for binding to the gold surface through formation of a thiol-gold bond, and an alcohol (-OH) group at the other end which does not interact with the nucleic acid molecule.
  • the blocking agent may be added either individually or together with the capture nucleic acid molecules. When added individually, the blocking agent may be added after the capture nucleic acid molecules have been immobilized on the detection electrode or added in advance of the capture nucleic acid molecules.
  • the blocking agent When the blocking agent is added after the capture nucleic acid molecules have been immobilized on the detection electrode, the blocking agent may be bound on the detection electrode in areas between the immobilized capture nucleic acid molecules.
  • the blocking agent can provide support for neighbouring capture nucleic acid molecules which can, for example, be bound to the detection electrode via functional groups described herein. In this way, the capture nucleic acid molecules can maintain in an upright position, thereby facilitating coupling efficiencies for hybridization with target nucleic acid molecules.
  • the blocking agent 11-mercapto-l-undecanic acid can be used.
  • the blocking agent can be added after the capture nucleic acid molecules are immobilized on the detection electrode, in which the blocking agent molecules can take up the space left unbound by the capture nucleic acid molecules, and bind on the surface of the detection electrode.
  • the bound blocking agent molecules can form a uniform and dense monolayer with the immobilized capture nucleic acid molecules on the detection electrode surface.
  • the blocking agent can be added in advance of the capture nucleic acid molecules.
  • the blocking agent When the blocking agent is added in advance of the capture nucleic acid molecules, it may be added together with a capture nucleic acid binding molecule.
  • capture nucleic acid binding molecule refers to a molecule that is adapted to bind with a capture nucleic acid. In principle, any molecule that can be immobilized on the electrode and that is able to interact with the capture nucleic acid molecules is suitable.
  • a thin film comprising the blocking agent and capture nucleic acid binding molecules may be formed on the surface of the electrode.
  • the capture nucleic acid binding molecule may depend on the type of capture nucleic acid molecules present and the type of detection electrode used.
  • the capture nucleic acid binding molecule may comprise one or two functional groups, such as a thiol group, an amino group, a carboxyl group, a hydroxyl group, or an epoxide group, which may be the same or different.
  • One functional group may be used for binding to the detection electrode surface, and another functional group may be used for binding to the capture nucleic acid.
  • Examples of capture nucleic acid binding molecule can include fatty acids, such as fatty acids described above in connection with the blocking agent.
  • Fatty acids comprising a thiol group can include, but are not limited to, 16-mercaptohexadecanoic acid (also known as MHA), 11-mercapto-l-undecanic acid (also known as MUA), 12-mercaptododecanoic acid, 11 -mercaptodecanoic acid or 10-mercaptodecanoic acid.
  • a suitable capture nucleic acid binding molecule can be a thiol molecule such as 16- mercaptohexadecanic acid, which contains a thiol (-SH) group for binding to the gold surface through formation of a thiol-gold bond, and an carboxyl (-COOH) group at the other end which interacts with the amino group on the capture nucleic acid molecule to form a peptide bond, thereby immobilizing the capture nucleic acid on the detection electrode surface.
  • thiol molecule such as 16- mercaptohexadecanic acid, which contains a thiol (-SH) group for binding to the gold surface through formation of a thiol-gold bond, and an carboxyl (-COOH) group at the other end which interacts with the amino group on the capture nucleic acid molecule to form a peptide bond, thereby immobilizing the capture nucleic acid on the detection electrode surface.
  • the capture nucleic acid binding molecule may additionally be activated using an activating agent.
  • activating agent refers to an agent e.g. a molecule used to catalyze formation of bonds between the capture nucleic acid binding molecule and the capture nucleic acid molecule. Choice of the activating agent may depend on the type of bond that exists between the capture nucleic acid binding molecule and the capture nucleic acid molecule.
  • An example of activating agent can be carbodiimides. Carbodiimides can catalyze formation of amide bonds between carboxylic acids or phosphates and amines, which may be present on the capture nucleic acid binding molecule and/or the nucleic acid molecules.
  • carbodiimide includes, but are not limited to, carbodiimide reagents include N 5 N'- dicyclohexylcarbodiimide (also known as DCC), N,Ndiisopropylcarbodiimide, or N-ethyl-N'- (3-dimethylaminopropyl)carbodiimide such as l-Ethyl-3-(3-dimethylaminopropyl)- carbodiimide (also known as EDC).
  • Carbodiimides are usually used together with succinimide type compounds to assist carbodiimide coupling.
  • succinimide include, but are not limited to, N-hydroxysuccinimide (also known as NHS), succinimide, N- methylsuccinimide, N-2-dimethylsuccinimide, 2-methylsuccinimide, or 2,3- dimethylsuccinimde.
  • NHS can be used with EDC to catalyse formation of amide bonds between the carboxyl group (-COOH) of 16-mercaptohexadecanoic acid, which acts as the capture nucleic acid binding molecule, and the amino group (-NH 2 ) of a modified capture nucleic acid molecule.
  • one way of immobilizing the capture nucleic acid molecules on the detection electrode is via interaction with capture nucleic acid binding molecules bound to the surface of the detection electrode.
  • immobilization of the capture nucleic acid molecules can be carried out on the electrode surface by any suitable physical or chemical interaction. These interactions include, for example, hydrophobic interactions, van der Waals interactions, or ionic (electrostatic) interactions as well as covalent bonds. This further means that a capture nucleic acid molecule can directly be immobilized on the surface of the electrode by hydrophobic interaction, van der Waals interactions or electrostatic interaction or through covalent coupling using a linker molecule should the surface of the electrode not be suitable for direct immobilization.
  • the capture nucleic acid molecules may comprise modified nucleotides carrying at least one functional group, such as a biotin, an avidin, a streptavidin, a digoxigenin, and anti-digoxigenin, an amine, a hydro xyl, an epoxide or thiol group, which can therefore immobilize directly on the surface of the electrode through chemical interaction.
  • a functional group such as a biotin, an avidin, a streptavidin, a digoxigenin, and anti-digoxigenin, an amine, a hydro xyl, an epoxide or thiol group, which can therefore immobilize directly on the surface of the electrode through chemical interaction.
  • any remaining nucleic acid capture molecule, or molecules, that were not immobilized may be removed from the detection electrode.
  • Removing an unbound nucleic acid capture molecule may be desired to avoid subsequent hybridization of such capture molecule with the target nucleic acid molecule.
  • Removing an unbound nucleic acid capture molecule may also be desired to avoid a non-specific binding of such capture 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 electrochemical measurement.
  • An unbound capture molecule may for instance be removed by exchanging the medium, e.g. a solution that contacts the detection electrode.
  • the present method includes contacting the detection electrode comprising capture nucleic acid molecules with a solution comprising a target nucleic acid molecule.
  • the target nucleic acid molecules are allowed to hybridize with the capture nucleic acid molecules, thereby forming a first complex between the target nucleic molecule and the capture nucleic acid molecule.
  • the target nucleic acid molecule that can be detected (including quantified) by the method of the present invention can originate from a large variety of sources.
  • Samples that include or are suspected or expected to include the respective target nucleic acid include biological samples derived from microorganism, plant material and animal tissue (e.g. insects, fish, birds, cats, livestock, domesticated animals and human beings), as well as blood, urine, sperm, stool samples obtained from such animals.
  • Biological tissue of not only living animals, but also of animal carcasses or human cadavers can be analysed, for example, to carry out post mortem tissue biopsy or for identification purposes, for instance.
  • samples may be water that is obtained from non-living sources such as from the sea, lakes, reservoirs, or industrial water to determine the presence of a target nucleic acid from bacteria.
  • Microorganism can include prokaryotes, eukaryotes or archaea. Further embodiments include a target nucleic acid, dissolved in liquids or comprised in suspensions of solids.
  • the sample comprising the target nucleic acid may have been prepared in form of a fluid, such as a solution.
  • a fluid such as a solution.
  • examples include, but are not limited to, a solution or a slurry of a nucleotide, a polynucleotide, or a nucleic acid.
  • a sample comprising a target nucleic acid 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 target nucleic acid molecule may also be present in the form of a modified target nucleic acid molecule (also called composite target nucleic acid molecule).
  • the target nucleic acid molecule may comprise one or two functional groups as already mentioned herein, such as a thiol group, an amino group, a carboxyl group, a hydro xyl group, or an epoxide group, which may be the same or different.
  • an aqueous solution may include one or more buffer compounds. Numerous buffer compounds are used in the art and may be used to carry out the various processes described herein.
  • buffers 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-amino-ethanesulfonate (also called ACES), N-(2-hydroxyethyl)-piperazine-N'-2-ethanesul- fonic acid (also called HEPES), 4-(2-hydroxyethyl)-l-piperazine-propanesulfonic acid (also called HEPPS), piperazine-l,4-bis(2-ethanesulfonic acid) (also called PIPES), (2-[Tris(hydro- xymethyl)-methylamino]-l-ethansulfonic acid (also called TES), 2-cyclohexylamino-ethane- sulfonic acid (also called CHES)
  • buffers include, but are not limited to, triethanol- amine, diethanolamine, ethylamine, triethylamine, glycine, glycylglycine, histidine, tris(hy- droxymethyl)aminomethane (also called TRIS), bis-(2-hydroxyethyl)-imino-tris(hydroxy- methyl)methane (also called BIS-TRIS), and N-[Tris(hydroxymethyl)-methyl]-glycine (also called TRICINE), to name a few.
  • TRIS tris(hy- droxymethyl)aminomethane
  • BIS-TRIS bis-(2-hydroxyethyl)-imino-tris(hydroxy- methyl)methane
  • TRICINE N-[Tris(hydroxymethyl)-methyl]-glycine
  • TE buffer comprising sodium chloride and ethylenediaminetetraacetic acid (also called EDTA) was used.
  • phosphate buffered saline also called PBS
  • the buffers may be or be included in aqueous solutions of such buffer compounds or solutions in a suitable polar organic solvent.
  • One or more respective solutions may be used to accommodate the suspected biological target nucleic acid as well as other matter used, throughout an entire method of the present invention.
  • the target nucleic acid molecule used in a method according to the present invention may comprise a first region for hybridization to a capture nucleic acid molecule.
  • the first region may be located at any position on the target nucleic acid. In one embodiment, the first region is located at or near to one end of the target nucleic acid.
  • the target nucleic acid may additionally comprise a second region. Also the second region can be located at or near to one end of the target nucleic acid. In one embodiment, the first region and the second region of the target nucleic acid are located at different parts of the target nucleic acid and are not overlapping.
  • the second region can be located at or near to the other end of the target nucleic acid.
  • the target nucleic acid molecule can be of any suitable length for hybridization, including a short oligonucleotide. In some embodiments it has a length of about 10 nucleotides to about 200 nucleotides or 10 to about 80 nucleotides, for example a length of about 15 nucleotides to about 40 nucleotides, such as a length of about 25 to about 60 nucleotides. In some embodiments, the target nucleic acid molecule can have a length of at least 10 nucleotides.
  • a solution suspected to contain the target nucleic acid molecule to be detected is contacted with the detection electrode.
  • the detection electrode may for example be immersed in a solution, to which the solution suspected to include the target nucleic acid molecule is added.
  • the method further includes allowing the target nucleic acid molecule to form a first complex with the capture nucleic acid molecule in the sensing region of the detection electrode. This occurs by allowing the first region of the target nucleic acid molecule to hybridize to the capture nucleic acid molecule. If the solution contains a plurality of different target nucleic acid molecules to be detected, the conditions may be chosen so that the target nucleic acid molecules can either bind simultaneously or consecutively to their respective capture molecules.
  • the method of the present invention may further include adding a target probe.
  • target probe refers herein to a nucleic acid molecule capable of hybridizing to a second region on the target nucleic acid molecule. Characteristics of the capture nucleic acid molecule also apply to the target probe.
  • the target probe is allowed to contact with the target nucleic acid molecule, such that it can hybridize with the second region of the target nucleic acid molecule to form a second complex.
  • the target probe may be added either individually or together with the target nucleic acid molecules. When added individually, the target probe may be added after the target nucleic acid molecules have been hybridized with the capture nucleic acid molecules.
  • the target probe may be added to a solution containing the target nucleic acid molecule.
  • the solution containing both the target probe and the target nucleic acid molecule can then be contacted with the sensing region of the detection electrode comprising a capture nucleic acid molecule, for example, by immersing the sensing region in a solution comprising the target nucleic acid molecule and the target probe.
  • the target probe can contain a linking moiety such as a functional group already described herein.
  • Examples of a functional group can include, but are not limited to a biotin, an avidin, a streptavidin, a digoxigenin, and anti- digoxigenin, an amine, a carboxyl, a hydroxyl, an epoxide or thiol group.
  • the target probe can hybridize with a second region of the target nucleic acid molecule.
  • the target probe can be of any suitable length for hybridization, including a short oligonucleotide. In some embodiments it has a length of about 10 nucleotides to about 50 nucleotides, for example a length of about 10 to about 35 nucleotides, such as a length of about 10 nucleotides to about 25 nucleotides .
  • the target probe can have a length of at least 10 nucleotides.
  • the present invention further includes adding an agent (alternatively called composition), denoted for example as Ml in Figure 1, comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target nucleic acid molecule, as shown by Figure l(c).
  • an agent alternatively called composition
  • the agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material can bind to the target probe of the second complex so that the agent binds to the target nucleic acid molecule via the target probe.
  • the molecule capable of transferring electrons to a metallic nanoparticle precursor material can be an enzyme or an enzyme-conjugate. Usually, any enzyme may be used that leads to the generation of a detectable electric current.
  • the enzyme may be selected from the group of oxidoreductases.
  • oxidoreductases are, but not limited to, glucose oxidase (also called GOx), catalase, lactate oxidase, alcohol dehydrogenase, hydroxybutyrate dehydrogenase, lactic dehydrogenase, glycerol dehydrogenase, sorbitol dehydrogenase, glucose dehydrogenase, malate dehydrogenase, galactose dehydrogenase, malate oxidase, galactose oxidase, xanthine dehydrogenase, alcohol oxidase, choline oxidase, xanthine oxidase, choline dehydrohenase, pyruvate dehydrogenase, pyruvate oxidase, oxalate oxidase, bilirubin oxidase, glutamate dehydrogenase, glutamate oxidase,
  • glucose oxidase is used as the molecule.
  • Glucose oxidase is a flavin containing glycoprotein which catalyzes the oxidation of glucose to gluconic acid.
  • the glycoprotein contains two moles of flavinadenine dinucleotide (FAD) per mole of enzyme, and has a molecular weight of 160,000 consisting of two identical subunits.
  • the molecule capable of transferring electrons to a metallic nanoparticle precursor material can bind with the target probe of the second complex such that the molecule binds to the target nucleic acid molecule via the target probe.
  • the molecule may be bound to the target probe via physical interactions such as van der Waals forces or chemical interactions such as covalent bonding or ionic bonding. It may comprise a functional group adapted to interact with the target probe or the target nucleic acid molecule. Examples of such functional groups have already been described herein.
  • avidin labeled glucose oxidase is used as the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the avidin labelled glucose oxidase can bind to a biotin labeled target nucleic acid or a biotin labeled target probe.
  • the agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material further comprises the target probe.
  • the target probe is bound to the agent so that the agent binds indirectly to the target nucleic acid molecule via the target probe.
  • the agent comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material would bind to the target nucleic acid molecule via the target probe. Therefore, referring to Figure 2(c), the agent may comprise the target probe. Therefore, the agent can be bound to the target probe, and can subsequently bind to the target nucleic acid molecule via the target probe after contacting it.
  • the agent comprising the molecule capable of transferring electrons to a metallic nanoparticle precursor material may bind directly to the target nucleic acid molecules without the use of a target probe.
  • agent denoted by Ml may comprise a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target probe of the second complex, and without a target probe.
  • the said molecule may contain a functional group adapted to interact with the target nucleic acid molecule. Examples of such functional groups have already been described herein
  • the method of the present invention further comprises contacting the sensing region of the detection electrode with a metallic nanoparticle precursor material and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • a metallic nanoparticle precursor material denoted as NPM
  • a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material is illustrated in Figure l(d), in which the detection electrode is contacted with a metallic nanoparticle precursor material (denoted as NPM) and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material and substrate compound can react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material, in which the substrate compound is oxidized (denoted as o- substrate compound), and due to reduction of the metallic nanoparticle precursor material, metallic nanoparticles (denoted as NPM red.) are formed which can deposit on the detection electrode.
  • the sensing region of the detection electrode may be immersed in a solution, to which a reactive solution comprising the metallic nanoparticle precursor material and a substrate compound for the molecule capable of transferring electrons to the metallic nanoparticle precursor material is added.
  • the reactive solution may be substantially oxygen free.
  • substantially oxygen free means having an oxygen concentration less than 100 parts per million (ppm).
  • the reactive solution may be made substantially oxygen free by purging the solution with an inert gas.
  • Inert gas includes nitrogen, argon, helium, to name only a few.
  • Purging may be carried out through passing the inert gas through the solution for a period of time. A suitable amount of time which is dependent on the type of gas used, the gas flow rate and amount of reactive solution. A person skilled in the art would be able to determine the appropriate conditions so as to achieve a substantially oxygen free reactive solution.
  • the metallic nanoparticle precursor material comprises material that is adapted to form metallic nanoparticles, the metallic nanoparticles selected from the group comprising noble metal nanoparticles, transition metal nanoparticles, and metal hexacyano ferrate nanoparticles.
  • the metallic nanoparticle precursor material can be a mixture of two or more chemical compounds.
  • the metallic nanoparticle precursor material can be a mixture of two or more salts.
  • the term "salt" refers to any water soluble salts including organic salts and inorganic salts such as nitrate, phosphate, and sulfate salts.
  • the salt may dissociate into its constituent ions, e.g. cation and anion, for example, by electrolytic dissociation in a suitable solvent.
  • metallic nanoparticle precursor material suitable for the method of the invention include, but are not limited to, a molybdenum salt, a copper salt, a germanium salt, a tin salt, a rhenium salt, an antimony salt, a platinum salt (e.g. Pt 2+ ), a palladium salt (e.g. Pd 2+ , Pd 4+ ), a silver salt (e.g. Ag + ), a nickel salt, a cobalt salt (e.g. Co 3+ ), an iron salt (e.g. Fe 2+ ), a bismuth salt (e.g. Bi 2+ ) and a mercury salt (e.g. Hg 2+ ).
  • a molybdenum salt e.g. Pt 2+
  • a palladium salt e.g. Pd 2+ , Pd 4+
  • a silver salt e.g. Ag +
  • nickel salt e.g. Ag +
  • a cobalt salt e
  • At least one of the metallic nanoparticle precursor materials used in the method of the invention is capable of acting as an oxidant.
  • Oxidation is a reaction in which an atom loses electrons.
  • an oxidant also termed oxidizing agent or oxidiser, is capable of oxidizing another substance by accepting electrons, while it is itself reduced in the process.
  • the metallic nanoparticle precursor material may oxidize another substance in a substantially oxygen free environment, for example, in a solution that has been purged of oxygen.
  • the metallic nanoparticle precursor material may be a salt.
  • one of the constituent ions usually the anion, may accept one electron.
  • the chemically reduced ion may react with another ion and precipitate out from the solution to form a metallic nanoparticle.
  • Noble metal nanoparticle precursors and metal oxide nanoparticle precursors include, but are not limited to silver nitrate (AgNO 3 ), [Ag(NH 3 ) 2 ] + (aq), gold chloride (AuCl 3 ), hydrogen tetrachloroaureate(III) hydrate (HAuCU*3H 2 O), chloroplatinic acid hexahydrate (H 2 PtCl 6 » 6H 2 O), chloroplatinic acid hexahydrate (H 2 PdCl 6 -OH 2 O), manganese nitrate (Mn(NOs) 2 ), or potassium permanganate (KMnO 4 ).
  • precursor for titanium dioxide (TiO 2 ) nanoparticles can include titanium alkoxides or titanium organometallic precursors.
  • titanium alkoxides can include, but are not limited to titanium methoxide, titanium ethoxide, titanium isopropoxide, titanium propoxide and titanium butoxide.
  • Metal hexacyano ferrate nanoparticle precursor materials can include, for example, a mixture of a copper salt and a ferricyanide salt.
  • a copper salt include, but are not limited to, copper chloride, copper acetate, copper sulphate, copper halides such as copper bromide, copper fluoride or copper iodide.
  • a ferricyanide salt comprises at least one cation which can include, but is not limited to, ions of lithium, sodium, potassium, caesium, ammonium, magnesium, calcium, barium and aluminium, and at least one ferricyanide anion.
  • a ferricyanide salt can include, but are not limited to an alkali metal ferrycyanide salt, such as potassium ferricyanide, sodium ferricyanide or lithium ferricyanide.
  • copper chloride and potassium ferricyanide salt are used as the metallic nanoparticle precursor materials.
  • Suitable replacements for ferricyanide salts can include ferrocene/ferrocenium derivatives which have similar electrochemical properties to ferricyanide salts.
  • metal hexacyano ferrate nanoparticles include, but are not limited to hexacyano ferrate nanoparticles of iron, cobalt, nickel, cupric, neodymium, manganese, molybdenum, iridium, zinc, lead, platinum and palladium.
  • the substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material refers to a reactant that becomes at least partially oxidized when acted upon by an enzyme.
  • the enzyme can be a molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • substrate compounds include, but are not limited to alcohol, glucose, hydrogen peroxide, bicarbonate, amino acid, sodium perborate, sodium percarbonate, and mixtures thereof.
  • suitable enzyme/substrate compound pairs may be glucose oxidase/glucose, catalase/hydrogen peroxide, lactate oxidase/lactic acid, to name only a few.
  • the molecule capable of transferring electrons to a metallic nanoparticle precursor material is glucose oxidase and the substrate compound is glucose.
  • a metallic nanoparticle may be formed after allowing the substrate compound to react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material and the metallic nanoparticle precursor material.
  • the substrate compound may be oxidized in the presence of the molecule capable of transferring electrons to a metallic nanoparticle precursor material, of which the molecule can act as the catalyst.
  • the catalyst can accept the electron from the substrate compound to form an intermediate compound, and can be regenerated when the electron is transferred to the metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material is reduced as a result of the electron transfer, and may react with another ion present to form a metallic nanoparticle.
  • the metallic nanoparticle may precipitate out of the solution and may be deposited on the surface of the detection electrode. Therefore, it is demonstrated herein that no nanoparticle seeds are required for precipitation of the metallic nanoparticles from solution and deposition on the surface of the detection electrodes.
  • an electrical measurement is performed at the detection electrode.
  • Electrical measurements according to the invention can include measurements of current as well as of voltage. Any detection technique for electric signals may be used in the method of the present invention.
  • a detection technique according to the invention may for instance include a measurement of a conductance, a voltage, a current, a capacitance or a resistance.
  • conductance may be measured by linear cyclic voltammetry (also called CV), square wave voltammetry (also called SWV), normal pulse voltammetry, differential pulse voltammetry (also called DPV) and alternating current voltammetry.
  • CV linear cyclic voltammetry
  • SWV square wave voltammetry
  • DPV differential pulse voltammetry
  • alternating current voltammetry alternating current voltammetry.
  • the kit can include an electrode arrangement comprising a detection electrode.
  • the kit can further include a capture nucleic acid molecule.
  • the capture nucleic acid comprises a nucleotide sequence that is at least partially complementary to at least a first portion of the target nucleic acid molecule.
  • the kit can optionally further include a target probe.
  • the target probe comprises a nucleotide sequence that is at least partially complementary to at least a second portion of the target nucleic acid molecule.
  • the kit can further include an agent.
  • the agent comprises a molecule capable of transferring electrons to a metallic nanoparticle precursor material and being capable of binding to the target nucleic acid molecule, wherein the molecule capable of transferring electrons to a metallic nanoparticle precursor material is capable of reducing the metallic nanoparticle precursor material.
  • the kit can further include a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material is capable of accepting an electron to form a metallic nanoparticle.
  • the kit can further include a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor solution.
  • the detection electrode of the invention may be used as biosensor. Such sensors are needed in many fields such as analytical chemistry, biochemistry, pharmacology, microbiology, food technology, medicine, forensic investigation, or environmental monitoring in order to analyze the presence and concentration of certain analytes in a given sample. For example, biosensors may be used for the diagnosis of infectious diseases.
  • biosensors in genome projects, for example, for detecting genes or gene mutations such as single nucleotide polymorphisms (SNPs) that are causative or indicative for a genetic disease.
  • SNPs single nucleotide polymorphisms
  • a detection electrode comprising capture nucleic acid molecules immobilized within a sensing region of the detection electrode is shown in Figure 2(a) (see also Example 2 and 3). It is contacted with a solution comprising target nucleic acid molecules and target probes, in which the target nucleic acid molecules are allowed to hybridize with the capture nucleic acid molecules, thereby forming a first complex between the target nucleic molecule and the capture nucleic acid molecule.
  • the target probe can hybridize to a second region on the target nucleic acid to form a second complex. This is shown in Figure 2(b) (see also Example 4).
  • the detection electrode is then contacted with an agent (denoted as Ml) comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target probe of the second complex.
  • the agent can bind with the target probe of the second complex so that the agent binds to the target nucleic acid molecule via the target probe, as shown in Figure 2(c) (see also Example 5).
  • the detection electrode is contacted with a metallic nanoparticle precursor material (denoted as NPM) and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material and substrate compound can react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material to form metallic nanoparticles capable of depositing in the sensing region of the detection electrode (see also Example 6).
  • Example 1 Reagents and apparatus
  • Nucleic acids used in the experiments i.e. thiolated oligonucleotide capture probes, biotinylated detection probes, target DNA with its sequence associated with influenza A virus RNA, and totally mismatched target DNA used as controls were custom-made by 1st base Pte Ltd. (Singapore), and summarized in Table 1.
  • Phosphate-buffered saline PBS, pH 7.4
  • TE buffer was used to prepare target DNA solutions. Double distilled water was used throughout the experiments.
  • Gold electrode was polished with a 0.3 ⁇ m alumina slurry, followed by 5 min sonication in water to remove any alumina particles.
  • the purpose of the polishing step is to remove any contaminants that may be on the electrode.
  • the electrode was soaked in Nanostrip ® reagent (Cyantek Corporation, Fremont, CA), which is a stabilized formulation of sulphuric acid and hydrogen peroxide compounds, for 10 min and rinsed with a copious amount of water to remove excess Nanostrip ® reagent.
  • the electrode was then dried with a stream of nitrogen. Nanostrip ® reagent is used to remove any organic impurities that may be present on the electrode.
  • Example 3 Addition of MUA blocking agent [0078] After thorough rinses with water and ethanol to remove unbound thiolated DNA capture probes, the capture probe modified gold electrode was soaked in 1 mM ethanoic 11- mercapto-1-undecanic acid (MUA) solution for 4 hours to form a MUA-DNA monolayer on the electrode.
  • MUA mM ethanoic 11- mercapto-1-undecanic acid
  • Example 4 Hybridization of target DNA and target probe on capture probe
  • Target DNA Complementary Target 1 in Table 1
  • biotinylated target probe Detection Probe 1 in Table 1
  • Each sample comprises a fixed concentration of 100 nM target probes and different target concentrations ranging from 1.0 fM to 1.0 nM.
  • 2 ⁇ L aliquots of the mixture solutions were applied on the electrode and incubated in a moisture-saturated environmental chamber at 50 °C for 1 hour. After hybridization, the electrode was thoroughly rinsed with a blank hybridization buffer solution to remove excess solution.
  • the electrode was incubated in a 2 ⁇ L aliquot of 25 ⁇ g/mL avidin- labeled glucose oxidase (GOx-A) conjugates in TE buffer at room temperature for 1 hour. During incubation, the avidin-labeled glucose oxidase molecules were attached to the biotinylated target probes on the electrode via biotion-avidin interaction. After attachment of the glucose oxidase molecules (referred herein as GOx), the electrode was rinsed with copious amounts of phosphate-buffered saline and soaked in phosphate-buffered saline under stirring for 10 min to wash away any non-specifically adsorbed GOx.
  • GOx-A avidin-labeled glucose oxidase
  • Example 6 Formation of copper hexacyano ferrate nanoparticles
  • Enzyme-catalyzed deposition of copper hexacyano ferrate nanoparticles was carried out by incubating the hybridized GOx-tagged electrode in a nitrogen saturated citrate buffer (0.1 M, pH 5.0) containing 1.OmM copper (II) chloride, 50 mM potassium ferricyanide and 50 mM glucose for about 20 min at room temperature.
  • ferricyanide Fe(CN) 6 3"
  • oxygen acts as the electron acceptor for the oxidation of glucose catalyzed by GOx.
  • the reactions can be represented using the following equations: [0087] Glucose + GOx-FAD ⁇ Glucono lactone + GOx-FADH 2 Eqn.(l) [0088] GOx-FADH 2 + 2Fe(CN) 6 3" +2OH " ⁇ GOx-FAD + 2Fe(CN) 6 4" + 2H 2 O Eqn. (2) [0089] Fe(CN) 6 4" + 2Cu 2+ ⁇ Cu 2 [Fe(CN) 6 ]
  • Equation (1) glucose reacts with enzyme glucose oxidase (GOx-FAD), and is itself oxidized to form glucono lactone.
  • the reduced enzyme GOx-FADH 2
  • ferricyanide Fe(CN) 6 3"
  • ferrocyanide Fe(CN) 6 4"
  • Ferrocyanide reacts with copper (II) ion in the solution and precipitates out as copper hexacyano ferrate [Cu 2 [Fe(CN) 6 ] nanoparticles as shown in Equation (3).
  • Example 7 Characterization using differential pulse voltammetry (DPV)
  • DPV differential pulse voltammetry
  • DPV differential pulse voltammetry
  • Settings of the differential pulse voltammetry are: initial potential 0 V; final potential 1.0 V; step potential 4 mV; amplitude 50 mV; pulse 0.05 s; and pulse period 0.2 s.
  • Example 8 Characterization using fluorescence imaging
  • Example 9 Characterization using cyclic voltammetry experiments
  • Figure 4(b) shows the cyclic voltammograms corresponding to a control electrode which was exposed to non-complementary target (Totally Mismatched Target 2) (curve denoted by “Control”) and to an electrode hybridized with 100 fM target DNA (Complementary Target 1) (curve denoted by "100 fM”), both of which were subjected to GOx-catalyzed deposition of copper hexacyano ferrate nanoparticles.
  • ⁇ E P The peak-to-peak potential separation value, of about 0.12 V in Figure 4(b), is significantly larger than that of thin copper hexacyano ferrate films on conductive electrodes reported by other research groups (eg. Siperko et al, Journal of the Electrochemical Society
  • Insert of Figure 4(b) shows a differential pulse voltammogram (DPV) of the electrode hybridized with 100 fM target DNA solution.
  • DUV differential pulse voltammogram
  • Differential pulse voltammogram was carried out with settings of an initial potential of 4mV and a final potential of 1.0V, step potential at 4mV, amplitude of 5OmV, pulse at 0.05 s, pulse period of 0.2s, and with an electrolyte solution of 0.10 M potassium nitrate (KNO 3 ).
  • FIG. 5(a) is a SEM image of the control electrode (Totally Mismatched Target 2 in Table 1)
  • Figure 5(b) is a SEM image of the electrode hybridized with 100 fM target DNA (Complementary Target 1 in Table 1).
  • Differential pulse voltammogram was carried out with settings of an initial potential of 4 mV and a final potential of 1.0 V, step potential at 4mV, amplitude of 50 mV, pulse at 0.05 s, pulse period of 0.2 s, and with an electrolyte solution of 0.10 M potassium nitrate (KNO 3 ).
  • FIG. 6 is a graph showing the differential pulse voltammogram (DPV) of the electrodes.
  • the DPV peak current of the electrode hybridized with fully complementary target DNA (Complementary Target 1 in Table 1) was 5.98 ⁇ 0.35 ⁇ A.
  • the DPV peak current was 1.18 ⁇ 0.21 ⁇ A.
  • This difference in DPV peak current can be used in a biosensor according to an embodiment of the present invention to differentiate between fully complementary and mismatched targets.
  • no detectable DPV peak current was observed for the electrode hybridized with totally mismatched target DNAs.
  • FIG. 7 is a graph showing DPV peak current performance of control electrode (curve denoted as “control”) and electrode hybridized with 10 fM complementary target DNA (curve denoted as “10 fM complementary target”) as a function of copper hexacyano ferrate nanoparticle deposition time.
  • control control electrode
  • 10 fM complementary target 10 fM complementary target DNA
  • the background current increased significantly from 0.09 ⁇ A after a deposition time of 20 minutes to 0.91 ⁇ A after a deposition time of 40 minutes. Therefore, a deposition time of 20 minutes was chosen under the present experimental conditions to ensure good sensitivity and a good signal to noise ratio.
  • Figure 8 is a graph showing the differential pulse voltammogram (DPV) of electrode hybridized with complementary target DNA (Complementary Target 1 in Table 1) at various levels of target DNA concentrations.
  • the DPV peak current increased with an increase in target DNA concentration. This implies that a higher level of hybridization of target DNA would subsequently lead to a higher level of GOx attachment and increased amount of copper hexacyanoferrate nanoparticles deposition on the electrode surface.
  • Insert of Figure 8 is a graph showing DPV peak current performance of electrode hybridized with complementary target DNA as a function of target DNA concentration.
  • the dynamic range was found to be between 1.0 fM and 10 pM with a regression coefficient of 0.98.
  • Detection of synthetic DNA with its sequence associated with influenza A virus RNA was successfully demonstrated with a detection limit of about 10 3 copies (1 fM in a sample volume size of 2 ⁇ L). Given that the reported mean viral load of patients is around 10 5 to 10 7 copies/mL, the proposed method of the present invention can be applied to detection of influenza virus A RNA without the need for reverse transcription process and PCR amplification.
  • Total assay time of actual samples which comprises processes of (i) extraction of viral RNA, (ii) direct hybridization of target RNA with biosensor, (iii) attachment of GOx via the detection probes, and (iv) enzyme-catalyzed deposition of hexacyno ferrate and their electrochemical detection, is about 2 hours, which is comparable to the assay time using state of the art PCR methods.
  • Example 14 Another embodiment of the present invention [00124] Referring to Figure 1, a detection electrode comprising capture nucleic acid molecules immobilized within a sensing region of the detection electrode is shown in Figure l(a) (see also Example 15).
  • the detection electrode is then contacted with an agent (denoted as Ml) comprising a molecule capable of transferring electrons to a metallic nanoparticle precursor material and capable of binding to the target nucleic acid molecule.
  • agent can bind with the target nucleic acid, as shown in Figure l(c) (see also Example 17).
  • the detection electrode is contacted with a metallic nanoparticle precursor material (denoted as NPM) and a substrate compound for the molecule capable of transferring electrons to a metallic nanoparticle precursor material.
  • the metallic nanoparticle precursor material and substrate compound can react with the molecule capable of transferring electrons to a metallic nanoparticle precursor material to form metallic nanoparticles capable of depositing in the sensing region of the detection electrode (see also Example 18).
  • Example 15 Formation of protection layer and immobilization of capture probe
  • the inventors have also carried out experiments regarding formation of a protection layer prior to immobilisation of capture nucleic acid molecules (corresponding to Figure l(a)).
  • the gold electrode was cleaned following the cleaning procedure outlined in Example 2. Subsequently, the gold electrode is soaked in an ethanolic solution of 5 mM 11- mercapto-1-undecanol (MU) and ImM 16-mercaptohexadecanic acid (MHA) for 24 hours to allow self-assembly of a mixed monolayer of MU and MHA on the gold electrode.
  • MU 11- mercapto-1-undecanol
  • MHA ImM 16-mercaptohexadecanic acid
  • the quality of the mixed monolayer of MU-MHA was evaluated in 0.1 M potassium chloride containing 1 mM potassium ferricyanide. After ascertaining the quality of the mixed monolayer using cyclic voltammetry as outlined in Example 3, the carboxyl groups in the mixed monolayer were activated by soaking in 5 mM EDC and 10 mM NHS in PBS for 30 minute. The capture nucleic acid molecules were coupled to the monolayer through peptide bonding by applying a 2 ⁇ L aliquot of 1 ⁇ M of the capture nucleic acid molecules solution (Capture Probe 2 in Table 1) onto the activated monolayer surface, followed by incubation at room temperature for 1 hour.
  • Example 16 Target DNA Detection
  • Hybridization of the target DNA was carried out by applying a 2 ⁇ L aliquot of the target DNA solution with different concentrations ranging from 1.0 fM to 1.0 nM onto the electrodes and incubated in a moisture-saturated environment chamber at 50 °C for 1 hour. After hybridization, the electrode was thoroughly rinsed with a blank hybridization buffer solution to remove excess solution.
  • Example 17 Attachment of glucose oxidase molecules
  • Attachment of glucose oxidase molecules follows the same procedure as outlined in Example 5, except that a 5 ⁇ L aliquot of 25 ⁇ g/mL avidin-labeled glucose oxidase (GOx- A) conjugates was used.
  • Example 18 Attachment of glucose oxidase molecules and characterization using DPV
  • Enzyme-catalyzed deposition of copper hexacyano ferrate nanoparticles was carried out by incubating the hybridized GOx-tagged electrode in a nitrogen saturated citrate buffer (0.1 M, pH 5.0) containing 1.0 mM copper (II) nitrate, 30 mM potassium ferricyanide and 10 mM glucose for 20 min at room temperature.
  • a nitrogen saturated citrate buffer 0.1 M, pH 5.0
  • Example 19 Characterization using cyclic voltammetry and DPV
  • Figure 11 shows cyclic voltammograms of a gold electrode which was exposed to totally mismatched (non-complementary) target (Totally Mismatched Target 3 in Table 1) (curve denoted by “Control”) and used as a control electrode, and a gold electrode hybridized with 100 fM target DNA (Complementary Target 2 in Table 1) (curve denoted by "100 fM”), both of which were subjected to GOx-catalyzed deposition of copper hexacyano ferrate nanoparticles.
  • 100 fM target DNA Complementary Target 2 in Table 1
  • Figure 12 is a graph showing the differential pulse voltammogram (DPV) of electrode hybridized with complementary target DNA (Complementary Target 2 in Table 1) at various levels of target DNA concentrations.
  • the DPV peak current increased with an increase in target DNA concentration.

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Abstract

La présente invention concerne le domaine des capteurs. En particulier, l'invention concerne un procédé pour la détection d'un acide nucléique cible dans un échantillon par un procédé électrochimique. Le procédé comprend la mise en contact d'une solution suspectée de contenir une molécule d'acide nucléique cible avec une électrode de détection comprenant une molécule d'acide nucléique de capture. La molécule d'acide nucléique cible s'hybride avec la molécule d'acide nucléique de capture. Une sonde cible peut facultativement être ajoutée. Un agent, qui a une molécule capable de transférer des électrons à un matériau précurseur de nanoparticule métallique et étant capable de se lier à la molécule d'acide nucléique cible, s'associe à cette dernière. Un matériau précurseur de nanoparticules métalliques et un composé de substrat sont ajoutés. Des nanoparticules métalliques sont formées. Le procédé comprend la détection d'une modification du courant électrique en cas de formation des nanoparticules métalliques pour déterminer la présence ou l'absence de la molécule d'acide nucléique cible.
PCT/SG2010/000139 2009-04-08 2010-04-08 Biocapteur d'acide nucléique Ceased WO2010117341A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2016190727A1 (fr) * 2015-05-22 2016-12-01 Universiti Kebangsaan Malaysia Biocapteur électrochimique d'adn pour l'identification du sexe et de la variété
CN107922978A (zh) * 2015-05-22 2018-04-17 马来西亚国立大学 用于性别和类别识别的电化学dna生物传感器
CN106442649A (zh) * 2016-09-23 2017-02-22 桂林电子科技大学 一种基于eis结构电化学生物传感器检测1,5‑脱水葡萄糖醇的方法
US11834697B2 (en) 2017-09-15 2023-12-05 Oxford University Innovation Limited Electrochemical recognition and quantification of cytochrome c oxidase expression in bacteria
CN110684652A (zh) * 2019-10-30 2020-01-14 德州学院 一种石墨烯核酸生物传感器、其制备方法及应用
CN113624815A (zh) * 2021-06-30 2021-11-09 江西师范大学 一种基于三维DNA Walker和滕氏蓝的双信号miRNA-21检测方法
CN113624815B (zh) * 2021-06-30 2023-04-11 江西师范大学 一种基于三维DNA Walker和滕氏蓝的双信号miRNA-21检测方法
CN115275164A (zh) * 2022-08-30 2022-11-01 首都师范大学 一种金属离子掺杂的羟基氧化镍电极材料及其修饰的电极以及再生方法
CN115950933A (zh) * 2022-11-29 2023-04-11 常州先趋医疗科技有限公司 自组装单分子膜修饰电极及其制备方法和用途
CN115980159A (zh) * 2022-12-29 2023-04-18 清华大学 电化学核酸传感器
CN116297761A (zh) * 2023-02-28 2023-06-23 南京理工大学 基于肽核酸探针与钯纳米颗粒电化学检测microRNA的方法

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