WO2011078800A1

WO2011078800A1 - Method for the detection of an analyte

Info

Publication number: WO2011078800A1
Application number: PCT/SG2010/000482
Authority: WO
Inventors: Y. Jackie Ying; Jie Zhang; Zhiqiang Gao; Boon Ping Ting
Original assignee: Agency For Science, Technology And Research
Priority date: 2009-12-23
Filing date: 2010-12-22
Publication date: 2011-06-30
Also published as: SG172498A1

Abstract

The present invention relates to a method for the detection of an analyte in a sample, the method comprising (a) immobilizing the analyte on the surface of an electrode; (b) contacting the electrode comprising the analyte with an analyte detection agent comprising a metal nanoparticle; (c) contacting the electrode obtained in (b) with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent, wherein the metal nanoparticle serves as a nucleation seed to deposit the metal on the complex-modified electrode surface to form a metal catalyst-modified electrode; (d) contacting the metal catalyst-modified electrode with a proton-rich electrolyte,; and (e) detecting a change in electrical current caused by the hydrogen generation and correlating the obtained electrochemical signal to the presence or amount of the analyte in the sample. The invention further relates to the production of an electrode or sensor that can be used in such a detection method, the thus obtainable electrode or sensor and its use for the detection of an analyte.

Description

METHOD FOR THE DETECTION OF AN ANALYTE

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of Singapore Patent Application No. 200908580-4, filed 23 December 2009, the contents of which being hereby incorporated by reference it its entirety for all purposes.

Technical Field

[0002] The invention relates to a method for the detection of an analyte, and in particular, to a method for the electrochemical detection of an analyte via hydrogen generation.

Background

[0003] The following discussion of the background is intended to facilitate an understanding of the invention. However, it should be appreciated that the discussion is not an acknowledgment or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.

[0004] There is an increasing need for the development of simple, rapid and ultrasensitive immunobiosensors for the detection of proteins. These sensors would be immensely beneficial for applications such as early stage disease diagnosis, detection of biological threats, and drug screening. The advantage of using immunobiosensors over other types of sensors in these applications lies in their high selectivity, which originates from the high selectivity and binding affinity of an antibody to its antigen. Consequently, the need for analyte enrichment and purification may be bypassed. [0005] However, since the antibody-antigen binding only causes minimal physicochemical changes, this binding event cannot be detected by most analytical instruments. Typical detection methods employ antibodies that are conjugated with a substance that can be read out easily by spectrophotometric, piezoelectric or electrochemical methods to improve detection sensitivity..

[0006] However, there still remains a need in the art for detection methods that allow analyte detection with increased sensitivity.

Summary

[0007] The present invention meets this object and provides for a method for the detection of analytes with very high sensitivity that is based on the inventors' finding that assay sensitivity can be significantly improved by using a method that involves analyte-dependent deposition of a metal that can catalyze hydrogen generation on the surface of an electrode and then using the hydrogen generation as a readout.

[0008] In a first aspect, the present invention thus relates to a method for the detection of an analyte in a sample, the method comprising:

(a) immobilizing the analyte on the surface of an electrode;

(b) contacting the electrode comprising the analyte with an analyte detection agent comprising a metal nanoparticle to form a complex of analyte and analyte detection agent on the surface of the electrode;

(c) contacting the complex-modified electrode obtained in (b) with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle, wherein the metal nanoparticle serves as a nucleation seed to deposit the metal on the complex- modified electrode surface to form a metal catalyst-modified electrode;

(d) contacting the metal catalyst-modified electrode with a proton-rich electrolyte, wherein the metal deposited on the metal catalyst-modified electrode catalyses the reduction of the protons to hydrogen; and (e) detecting a change in electrical current caused by the hydrogen generation and correlating the obtained electrochemical signal to the presence or amount of the analyte in the sample.

[0009] In another aspect, the present invention relates to a method for the production of an electrochemical sensor, comprising:

(a) immobilizing an analyte on the surface of an electrode;

(b) contacting the electrode comprising the analyte with an analyte detection agent comprising a metal nanoparticle to form a complex of analyte and analyte detection agent on the surface of the electrode; and

(c) contacting the complex-modified electrode obtained in (b) with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle to deposit the metal on the complex-modified electrode surface.

[0010] In a further aspect, the present invention relates to an electrochemical sensor obtainable according to a method of the invention.

[0011] In yet another aspect, the present invention relates to the use of an electrochemical sensor of the invention for the detection of an analyte.

[0012] In one aspect, the present invention relates to a method for the production of an electrode, comprising contacting an electrode comprising a complex of an analyte and an analyte detection agent comprising a metal nanoparticle with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle to deposit the metal on the electrode surface.

[0013] In another aspect, the present invention relates to an electrode obtainable according to a method of the invention. [0014] In a further aspect, the present invention relates to the use of an electrode of the invention in an electrochemical sensor for the detection of an analyte.

Brief Description of the Drawings

[0015] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings.

[0016] Fig. 1 shows a schematic illustration of detecting analyte prostate-specific antigen (PSA) in accordance with one aspect of the invention.

[0017] Fig. 2 shows TEM images of Pt nanoparticles synthesized with (a) 0.25 g/100 ml of capping agent, scale bar 5 nm; (b) 0.10 g/100 ml of capping agent, scale bar 0.5 μπι; (c) 0.10 g/100 ml of Capping agent, scale bar 10 nm; (d) Pt nanoparticle cluster- antibody conjugates, scale bar 0.2 μιη in accordance with one embodiment of the invention.

[0018] Fig. 3 shows cyclic voltammograms of proton redox process obtained in an acidic solution containing 10 mM of HC1 and 1 M of KC1 with different electrodes (2 mm diameter) when the PSA concentration is 10 nM: (i) bare Au electrode, (ii) Au electrode modified with a mixed thiol monolayer (10% MUOH, 90% MHA), (iii) bare Pt electrode, and (o) metal catalyst-modified electrode of in accordance with one embodiment of the invention.

[0019] Fig. 4 shows (a) effect of Pt enhancement time (from the top curve to the bottom curve: 0, 2, 5, 10, 15, 30, 45 and 60 min) on the voltammetric response of the electrochemical sensor of one embodiment of the invention in an aqueous solution containing 10 mM of HC1 and 1 M of KC1; (b) dependence of the steady-state diffusion controlled limiting current and peak current (data obtained from (a)) on the enhancement time for (■) 0.1 pg/ml and (□) 0 pg/ml of PSA.

[0020] Fig. 5 shows calibration curves of the electrochemical sensor of one embodiment of the invention obtained with a Pt enhancement time of (Δ) 10 min and (□) 30 min.

[0021] Fig. 6 shows the formation of disulfide-based polyamido amines (mixed oligomers) 2 used for conjugating Pt nanoparticles to first analyte-binding molecules in one embodiment of the invention.

Description

[0022] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0023] The invention is based on the finding that the sensitivity of the detection of analytes such as, but not limited to, proteins, peptides, lipids, nucleic acids, small organic molecules, organic polymers, carbohydrates and haptens, which may be present in a sample fluid in only trace amounts, can be significantly improved by electrochemical detection via hydrogen generation in the presence of a metal catalyst.

[0024] By use of the present method, in which the metal catalyst catalyzing the hydrogen generation reaction is used for amplification of the analyteranalyte-binding molecule binding event, an analyte concentration as low as 1 fg/ml can be detected. The increased sensitivity of the detection method of the invention can be attributed in part by the catalytic activity of the metal catalyst in accelerating the hydrogen generation reaction in an acidic aqueous medium and the high sensitivity of the electrochemical detection technique in detecting the generation of hydrogen.

[0025] In addition, the sensitivity of the electrochemical detection technique in detecting the generation of hydrogen can be increased further by increasing the concentration of the metal catalyst using a catalyst-growth solution, thereby accelerating the hydrogen generation even further, thus allowing a simple, accurate and affordable detection and quantification of the concentration of the analytes present in the sample.

[0026] In a first aspect, the present invention relates to a method for the detection of an analyte, the method comprising:

(a) immobilizing the analyte on the surface of an electrode;

(d) contacting the metal catalyst-modified electrode with a proton-rich electrolyte, wherein the metal deposited on the metal catalyst-modified electrode catalyses the reduction of the protons to hydrogen; and

(e) detecting a change in electrical current caused by the hydrogen generation and correlating the obtained electrochemical signal to the presence or amount of the analyte in a sample.

[0027] The step of immobilizing the analyte on the surface of an electrode may comprise (i) covalently coupling a first analyte-binding molecule to the surface of the electrode, and (ii) contacting the electrode coupled to the first analyte-binding molecule with the sample containing the analyte to form a first analyte-binding molecule: analyte complex.

[0028] The step of contacting the electrode comprising the analyte with an analyte detection agent may comprise contacting the electrode comprising the first analyte- binding molecule:analyte complex with the analyte detection agent, wherein the analyte detection agent comprises a second analyte-binding molecule conjugated to the metal nanoparticle, to form a first analyte-binding molecule:analyte:detection agent complex.

[0029] The formation of the complex of (i) the analyte and the first analyte-binding molecule, or (ii) the analyte and the first analyte-binding molecule and the analyte detection agent may be carried out prior to coupling the first analyte-binding molecule to the electrode surface.

[0030] The formation of the complex of the analyte and the analyte detection agent may be carried out prior to contacting the analyte with the first analyte-binding molecule coupled to the electrode surface.

[0031] The terms "analyte", "target compound", "target molecule" or "target" as interchangeably used herein, refer to any substance that can be detected in an assay by binding to a binding molecule, and which, in one embodiment, may be present in a sample. Therefore, the analyte can be, without limitation, any substance for which there exists a naturally occurring antibody or for which an antibody can be prepared. The analyte may, for example, be an antigen, a protein, a polypeptide, a nucleic acid, a hapten, a carbohydrate, a lipid, a cell or any other of a wide variety of biological or non- biological molecules, complexes or combinations thereof Generally, the analyte will be a protein, peptide, carbohydrate or lipid derived from a biological source such as bacterial, fungal, viral, plant or animal samples. Additionally, however, the target may also be a small organic compound such as a drug, drug-metabolite, dye or other small molecule present in the sample. [0032] The term "sample", as used herein, refers to an aliquot of material, frequently biological matrices, an aqueous solution or an aqueous suspension derived from biological material. Samples to be assayed for the presence of an analyte by the methods of the present invention include, for example, cells, tissues, homogenates, lysates, extracts, and purified or partially purified proteins and other biological molecules and mixtures thereof.

[0033] Non-limiting examples of samples typically used in the methods of the invention include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates, urine, semen, lymph fluids and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supematants; tissue specimens which may or may not be fixed; and cell specimens which may or may not be fixed. The samples used in the methods of the present invention will vary based on the assay format and the nature of the tissues, cells, extracts or other materials, especially biological materials, to be assayed. Methods for preparing protein extracts from cells or samples are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the methods of the invention. Detection in a body fluid can also be in vivo, i.e. without first collecting a sample.

[0034] "Peptide" generally refers to a short chain of amino acids linked by peptide bonds. Typically peptides comprise amino acid chains of about 2-100, more typically about 4-50, and most commonly about 6-20 amino acids. "Polypeptide" generally refers to individual straight or branched chain sequences of amino acids that are typically longer than peptides. "Polypeptides" usually comprise at least about 20 to 1000 amino acids in length, more typically at least about 100 to 600 amino acids, and frequently at least about 200 to about 500 amino acids. Included are homo-polymers of one specific amino acid, such as for example, poly-lysine. "Proteins" include single polypeptides as well as complexes of multiple polypeptide chains, which may be the same or different. [0035] Multiple chains in a protein may be characterized by secondary, tertiary and quaternary structure as well as the primary amino acid sequence structure, may be held together, for example, by disulfide bonds, and may include post-synthetic modifications such as, without limitation, glycosylation, phosphorylation, truncations or other processing.

[0036] Antibodies such as IgG proteins, for example, are typically comprised of four polypeptide chains (i.e., two heavy and two light chains) that are held together by disulfide bonds. Furthermore, proteins may include additional components such associated metals (e. g., iron, copper and sulfur), or other moieties. The definitions of peptides, polypeptides and proteins includes, without limitation, biologically active and inactive forms; denatured and native forms; as well as variant, modified, truncated, hybrid, and chimeric forms thereof.

[0037] The terms "contacting" or "incubating" as used interchangeably herein refer generally to providing access of one component, reagent, analyte or sample to another. For example, contacting can involve mixing a solution comprising an analyte binding protein or conjugate thereof with a sample. The solution comprising one component, reagent, analyte or sample may also comprise another component or reagent, such as dimethyl sulfoxide (DMSO) or a detergent, which facilitates mixing, interaction, uptake, or other physical or chemical phenomenon advantageous to the contact between components, reagents, analytes and/or samples.

[0038] The term "detecting" and associated term "detection", as used herein, refer to a method of verifying the presence of a given molecule. The technique used to accomplish this is an electrochemical detection method involving a signal amplification strategy based on catalytic hydrogen generation reaction. The term "electrochemical detection" as used herein refers to the utilization of electrochemical means to indicate the presence or absence, either qualitatively or quantitatively, of an analyte, i.e. include correlating the detected signal with the amount of analyte. The detection includes in vitro as well as in vivo detection. [0039] The term "hapten" as used herein, refers to a small proteinaceous or nonprotein antigenic determinant which is capable of being recognized by an antibody. Typically, haptens do not elicit antibody formation in an animal unless part of a larger species. For example, small peptide haptens are frequently coupled to a carrier protein such as keyhole limpet hemocyanin in order to generate an anti-hapten antibody response.

[0040] "Antigens" are macro molecules capable of generating an antibody response in an animal and being recognized by the resulting antibody. Both antigens and haptens comprise at least one antigenic determinant or "epitope", which is the region of the antigen or hapten which binds to the antibody. Typically, the epitope on a hapten is the entire molecule.

[0041] The term "analyte-binding molecule" as used herein refers to any molecule capable of binding to an analyte of choice so as to form a complex consisting of the analyte-binding molecule and the analyte. Preferably, this binding is specific so that a specific complex between analyte and analyte-binding molecule is formed.

[0042] "Specifically binding" and "specific binding" as used herein mean that the analyte-binding molecule binds to the analyte based on recognition of a binding region or epitope on the analyte. The analyte-binding molecule preferably recognizes and binds to the analyte with a higher binding affinity than it binds to other compounds in the sample. In various embodiments of the invention, "specifically binding" may mean that an antibody or other biological molecule, binds to an analyte with at least about a 10⁶- fold greater affinity, preferably at least about a 10⁷-fojd greater affinity, more preferably at least about a 10 -fold greater affinity, and most preferably at least about a 10 -fold greater affinity than it binds molecules unrelated to the analyte. Typically, specific binding refers to affinities in the range of about 10⁶-fold to about 10⁹-fold greater than non-specific binding. In some embodiments, specific binding may be characterized by affinities greater than 10⁹-fold over non-specific binding. The binding affinity may be determined by any suitable method. Such methods are known in the art and include, without limitation, surface plasmon resonance and isothermal titration calorimetry. In a specific embodiment, the analyte-binding molecule uniquely recognizes and binds to the analyte.

[0043] The analyte-binding molecule may be a proteinaceous molecule, such as an antibody, for example a monoclonal or polyclonal antibody, which immunologically binds to the analyte at a specific determinant or epitope. The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies as well as antibody variants or fragments (e.g., Fab, F(ab')₂, scFv, Fv diabodies and linear antibodies), so long as they exhibit the desired binding activity.

[0044] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies can include "chimeric" antibodies and humanized antibodies. A "chimeric" antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

[0045] Monoclonal antibodies may be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Koehler and Milstein (U. S. Patent No. 4,376,110), the human B-cell hybridoma technique, and the EBV-hybridoma technique. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb may be cultivated in vitro or in vivo. Production of high titres of mAbs in vivo makes this a very effective method of production.

[0046] "Polyclonal antibodies" are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, or an antigenic functional derivative thereof. For the production of polyclonal antibodies, host animals such as rabbits, mice and goats, may be immunized by injection with an antigen or hapten-carrier conjugate optionally supplemented with adjuvants.

[0047] Alternatively, techniques described for the production of single chain antibodies (U. S. Patent No. 4,946,778) can be used to produce suitable single chain antibodies. Single chain antibodies are typically formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

[0048] Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

[0049] The analyte-binding molecule may also be any other proteinaceous scaffold that has been adapted or mutated to bind a given ligand with sufficient binding affinity.

[0050] Examples of useful scaffolds include those scaffolds described in US patent application 2005/0089932 or US Patent 6,682,736. Another example of suitable scaffolds are members of the lipocalin protein family as described in the international patent applications WO 99/16873, WO 00/75308, WO 03/029471, WO 03/029462, WO 03/029463, WO 2005/019254, WO 2005/019255 or WO 2005/019256, for instance. [0051] In accordance with the above, scaffolds besides members of the lipocalin family include, but are not limited to, a EGF-like domain, a Kringle-domain, a fibronectin type I domain, a fibronectin type II domain, a fibronectin type III domain, a PAN domain, a Gla domain, a SRCR domain, a Kunitz/Bovine pancreatic trypsin inhibitor domain, tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil (P-type) domain, a von Willebrand factor type C domain, an Anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, LDL-receptor class A domain, a Sushi domain, a Link domain, a Thrombospondin type I domain, an immunoglobulin domain or a an immunoglobulin-like domain (for example, domain antibodies or camel heavy chain antibodies), a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a Somatomedin B domain, a WAP-type four disulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2 domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain, Kappabodies, Minibodies, Janusins, a nanobody, a adnectin, a tetranectin, a microbody, an affilin, an affibody or an ankyrin, a crystallin, a knottin, ubiquitin, a zinc- finger protein, an ankyrin or ankyrin repeat protein or a leucine-rich repeat protein, an avimer; as well as multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains.

[0052] The analyte-binding molecule may be a mutein of the member of the lipocalin protein family. In some of these embodiments, the open end of the β-barrel structure of the lipocalin fold (which encompasses the natural ligand binding site of the lipocalin family) is used to form the analyte binding site. Members of the lipocalin family of proteins include, but are not limited to the bilin binding protein of Pieris brassicae (SWISS-PROT Data Bank Accession Number P09464), human tear lipocalin (SWISS-PROT Data Bank Accession Number M90424), human apo lipoprotein D (SWISS-PROT Data Bank Accession Number P05090), the retinol binding protein (RBP) (for example of human or porcine origin, SWISS-PROT Data Bank Accession Number of the human RBP: P02753, SWISS-PROT Data Bank Accession Number of the porcine RBP P27485), human neutrophil gelatinase-associated lipocalin (hNGAL, SWISS-PROT Data Bank Accession Number P80188), rat G¾-microglobulin-related protein (A2m, (SWISS-PROT Data Bank Accession Number P31052), and mouse 24p3/uterocalin (24p3, (SWISS-PROT Data Bank Accession Number PI 1672), Von Ebners gland protein 2 of Rattus norvegicus (VEG protein 2; SWISS-PROT Data Bank Accession Number P41244), Von Ebners gland protein 2 of Sus scrofra (pig) (LCN1 ; SWISS-PROT Data Bank Accession Number P53715), the Major allergen Can fl precursor of dog (ALL 1, SWISS-PROT Data Bank Accession Number 018873), and insecticyanin A or insecticyanin B of the tobacco hawkmoth Manducta sexta (SWISS- PROT Data Bank Accession Number P00305 and Q00630, respectively).

[0053] The analyte binding molecule may also be a binding protein, receptor or extracellular domain (ECD) thereof capable of forming a binding complex with a ligand, typically a polypeptide or glycopeptide ligand.

[0054] Those skilled in the art will recognized that the non-limiting examples given above describing various forms of antibodies as analyte-binding molecules can also be extended to other proteinaceous receptors such as recombinant, chimeric, hybrid, truncated etc., forms of non-antibody receptors.

[0055] The analyte-binding molecule can also be a non-proteinaceous receptor, such as for example a nucleic acid based molecule, such as an Aptamer or Spiegelmer (Aptamer made of L-ribonucleotides).

[0056] Generally, electrochemical detection involves the use of electrodes immersed in a sample containing an analyte, and connected to an instrument that can detect changes in the electric current. In one embodiment, voltage may be applied to the electrodes and may be varied while measuring the current flow between the electrodes (voltammetry electrochemical detector). By varying the electrode potential, an electric current that is characteristic of electrochemical active; substances in an electrolyte flows between the electrodes. Typically, 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 facilitates 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. Optionally, a third electrode may be used to act as a reference electrode which acts as a reference in measuring and controlling the working electrode's potential.

[0057] Fig. 1 shows a schematic illustration of electro chemically detecting analyte in accordance with one embodiment of a first aspect of the invention. The detecting method comprises providing a detection electrode having first analyte-binding molecules immobilized within a sensing region of the detection electrode. The term "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 electrical signal such as an electrical current in the method of the invention.

[0058] The detection electrode may be part of an electrode arrangement further comprising a counter electrode and/or a reference electrode. The detection electrode may be a conventional metal electrode, such as a noble metal group electrode, the surface of which has been optionally modified in order to facilitate the immobilization of analyte. 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 Pt-Ir, Pd- Pt, Pd-Rh or Pd-Pt-Rh, to name only a few. In another embodiment, the detection electrode comprises gold, or an alloy comprising gold. Alternatively, the detection electrode may be made from suitable conductive materials such as, but not limited to, polymeric material or carbon, common silicon or gallium arsenide substrate, ceramics to which a gold layer and a silicon nitride layer may have been applied.

[0059] The analyte may be selected from the group consisting of proteins, peptides, lipids, nucleic acids, small organic molecules, organic polymers, carbohydrates and haptens. The first analyte-binding molecule may be selected from the group consisting of antibodies and fragments or variants thereof, antibody-like molecules, and binding proteins, receptor proteins, and domains thereof. The complex formed between analyte and analyte-binding molecule may include, without limitation, antigen-antibody, protein-protein, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme- substrate, and IgG-protein A complexes.. The first analyte-binding molecules immobilized within the sensing region of the detection electrode in this illustration are PSA antibodies that bind specifically to analyte prostate-specific antigens (PSA) to form a complex of PSA:PSA antibody, which binding event may be detected via subsequent hydrogen generation reaction.

[0060] The first analyte-binding molecules, irrespective of the analytes for which they have binding activity, 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 first analyte-binding molecule is caused to be located. Generally, the sensing region may be any area on the detection electrode. In some embodiments, the sensing region may be an aperture to which a solution containing the analytes is caused to flow. The first analyte-binding molecules may be immobilized continuously throughout the detection electrode surface, or isolated on certain areas of the detection electrode surface.

[0061] A protection layer, also called a blocking layer, adapted to prevent nonspecific binding of analytes on a surface of the detection electrode may be formed on the detection electrode surface or in the sensing region of the detection electrode. In one embodiment, the protection layer may comprise a thin film comprising a blocking agent. In another embodiment, the protection layer may comprise a thin film comprising a blocking agent and a coupling molecule capable of binding to the first analyte-binding molecule and immobilizing it on the electrode surface. Such a thin film can be a monolayer, a bilayer, or a multilayer of any thickness. In certain embodiments, the protection layer may be partially formed on the detection electrode surface, which layer may be continuous throughout, or isolated on certain areas of the detection electrode surface. In other embodiments, the protection layer may be absent.

[0062] The term "blocking agent" as used herein refers to an agent, e.g. a molecule which can inhibit or block the unspecific binding of other molecules, such as the analytes or any other compounds which can be comprised in a test sample and can interfere with the electrochemical detection, to the detection electrode surface. In principle, any agent that can be immobilized on the detection electrode and that is able to prevent (or at least to significantly reduce) the unspecific interaction between first analyte-binding molecules or analytes or interfering agents and the detection electrode surface is suitable. Examples of blocking agents are thiol molecules, disulfides, thiophene derivatives, and polythiophene derivatives, to name only a few. In some embodiments, 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. In one embodiment, 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-mercapto-l- hexadecanoic acid (16-MHA), 1 1-mercapto-l-undecanic acid, 12-mercaptododecanoic, 1 1 -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 11-mercapto-l-undecanol (11-MUOH), 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 analytes and/or first analyte-binding molecules present and the type of detection electrode used. In various embodiments, the blocking agents comprise a thiol group for binding to the electrode surface and a carboxyl or hydroxy group that can be used to immobilize the analyte or first analyte-binding molecule. In one embodiment, if the first analyte-binding molecule is PSA antibody and the detection electrode is a gold electrode, a suitable blocking agent can be a thiol molecule such as 11-MUOH, which contains a thiol (-SH) group for binding to the gold detection electrode surface through formation of a thiol-gold bond, and an alcohol (-OH) group at the other end which does not interact with the first analyte-binding molecules.

[0063] The blocking agent may be added either separately or together with the first analyte-binding molecules. When added separately, the blocking agent may be added after the first analyte-binding molecules have been immobilized on the detection electrode to block all unoccupied binding sites on the electrode surface or added prior of the first analyte-binding molecules, for example to block unspecific binding and to facilitate the immobilization of the first analyte-binding molecule on the electrode surface.

[0064] When the blocking agent is added after the first analyte-binding molecules have been immobilized on the detection electrode, the blocking agent may be bound on the detection electrode in areas between the immobilized first analyte-binding molecules, i.e. the spaces left unbound by the first analyte-binding molecules. This may stabilize the neighbouring first analyte-binding molecules which can, for example, be bound to the detection electrode via functional groups described herein. The blocking agent can also prevent unspecific binding of the analyte to the electrode surface. The blocking agent can also be used to minimize steric effects caused by immobilization of the first analyte-binding molecules on the detection electrode surface. The bound blocking agent molecules can form a uniform and dense monolayer with the immobilized first analyte-binding molecules on the detection electrode surface.

[0065] In one embodiment, as shown in Fig. 1, the blocking agent can be added prior to the first analyte-binding molecules. When the blocking agent is added prior to the first analyte-binding molecules, it may be added together with a coupling molecule or it may itself be suitable as a coupling molecule. The term "coupling molecule" as used herein refers to a molecule that is adapted to bind to a first analyte-binding molecule and to the detection electrode surface, thus facilitating the immobilization of the first analyte-binding molecule on the electrode surface. In principle, any molecule that can be immobilized on the detection electrode and that is able to interact with the first analyte-binding molecules is suitable. By adding the blocking agent together with the coupling molecules, a thin film comprising the blocking agent and coupling molecules may be formed on the surface of the detection electrode. Therefore, areas on the detection electrode bound by coupling molecules allow for specific binding of first analyte-binding molecules, and areas on the detection electrode onto which a blocking agent is immobilized prevent non-specific binding of first analyte-binding molecules or other molecules, such as analyte molecules. [0066] Choice of the coupling molecule may depend on the type of first analyte- binding molecules present and the type of detection electrode used. The coupling 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 first analyte- binding molecule. Examples of coupling molecule can include modified fatty acids, such as the fatty acids described above in connection with the blocking agent. Fatty acids comprising a thiol group can include, but are not limited to, 16-MHA, 11- mercapto-l-undecanic acid, 12-mercaptododecanoic acid, 11 -mercaptodecanoic acid or 10-mercaptodecanoic acid. In one embodiment, if the first analyte-binding molecule is PSA antibody and the detection electrode is a gold electrode, a suitable coupling molecule can be a thiol molecule such as 16-MHA, which contains a thiol (-SH) group for binding to the gold detection electrode surface through formation of a thiol-gold bond, and an carboxyl (-COOH) group at the other end which interacts with the amino group of the PSA antibody to form a peptide bond, thereby immobilizing the first analyte-binding molecule on the detection electrode surface. This is exemplified in the embodiment illustrated in Fig. 1.

[0067] The coupling molecule may additionally be activated using an activating agent. The term "activating agent" as used herein refers to an agent e.g. a molecule used to catalyze formation of bonds between the coupling molecule and the first analyte- binding molecule. Choice of the activating agent may depend on the type of bond that exists between the coupling molecule and the first analyte-binding 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 coupling molecule and/or the first analyte-binding molecule. This may be carried out by activating the carboxyl or phosphate groups present to form an O- urea derivative, which reacts readily with nucleophiles. Examples of carbodiimide includes, but are not limited to, carbodiimide reagents include Ν,Ν'- dicyclohexylcarbodiimide (DCC), N,Ndiisopropylcarbodiimide, or N-efhyl-N'-(3- dimethylarninopropyl)carbodiirnide such as 1 -Ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC). Carbodiimides are usually used together with succinimide type compounds to assist carbodiimide coupling. Examples of succinimide include, but are not limited to, N-hydroxysuccinimide (NHS), succinimide, N-methylsuccinimide, N-2- dimethylsuccinimide, 2-methylsuccinimide, or 2,3-dimethylsuccinimde. In one embodiment, NHS can be used with EDC to catalyse formation of amide bonds between the carboxyl group (-COOH) of 16-MHA, which acts as the coupling molecule, and the amino group (-NH₂) of the first analyte-binding molecule.

[0068] As discussed earlier, one way of immobilizing the first analyte-binding molecules on the detection electrode is via interaction with coupling molecules bound to the surface of the detection electrode. In other embodiments, immobilization of the first analyte-binding molecules can be carried out on the detection 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 first analyte-binding 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. In some embodiments, the first analyte-binding molecules may carry at least one functional group, such as an amine, a hydroxyl, an epoxide or thiol group, which allow direct immobilization on the surface of the first analyte-binding electrode through chemical interaction, for example formation of a covalent bond.

[0069] After immobilizing the first analyte-binding molecules on the detection electrode, any remaining first analyte-binding molecules that were not immobilized may be removed from the detection electrode. Removing an unbound first analyte-binding molecule may be desired to avoid subsequent binding of such unbound first analyte- binding molecule with the analyte. Removing an unbound first analyte-binding molecule may also be desired to avoid a non-specific binding of such first analyte- binding 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 first analyte-binding molecule may for instance be removed by exchanging the medium, e.g. a solution that contacts the detection electrode. In one embodiment, the detection electrode may be washed with a washing buffer, for example a glycine-containing washing buffer, to remove the unbound first analyte-binding molecules.

[0070] The detection electrode comprising immobilized first analyte-binding molecules is then contacted with a solution comprising analytes. The analytes are allowed to bind specifically with the first analyte-binding molecules. As illustrated in Fig. 1, the analyte may comprise a first binding site for binding to the immobilised first analyte-binding molecule. The first binding site may be located at any position on the analyte. The analyte may additionally comprise a second binding site located at a different part of the analyte. In one embodiment, the first and second binding sites are not overlapping. The analyte may also comprise more than one first binding sites and/or more than one second binding sites. This may, for example, be the case, if the analyte exists in form of a dimer, trimer or or multimer.

[0071] The complex formed between the analyte and the first analyte-binding molecule may then be contacted with a second analyte binding molecule to form a complex, for example a ternary complex. Alternatively, the analyte may first be contacted with the second analyte-binding molecule and the resulting complex contacted with the first analyte-binding molecule. The second analyte-binding molecule may bind to the second binding site of the analyte. The second analyte-binding molecule has a binding affinity for the analyte. The second analyte-binding molecule may have a lower, equal, or higher binding affinity for the analyte than the first analyte-binding molecule. In certain embodiments such as in Fig. 1, the first analyte-binding molecule that is immobilised on the detection electrode surface and binds to the first region of the analyte is an antibody (primary antibody) and the second analyte-binding molecule that binds to the second binding site of the analyte is also an antibody (secondary antibody). The second analyte-binding molecule may thus be defined similar to the first analyte- binding molecule and may, for example, be also selected from the group consisting of antibodies and fragments or variants thereof, antibody-like molecules, binding proteins, protein receptors, and extracellular domains thereof. The second analyte-binding molecule may also be specific for the analyte. Although in certain embodiments, the first and second analyte-binding molecules are different, in other embodiments, the first and second analyte-binding molecules are the same or bind to the same binding site. The binding sites, i.e. the first and/or second binding sites, may be epitopes. A single given analyte may comprise one or more similar or identical epitopes. In such a case, the first and second analyte binding molecules may have the same binding specificity or may even be the same antibody.

[0072] The second analyte-binding molecule is allowed to contact the analyte, such that it can specifically bind to the second binding site of the analyte. The second analyte-binding molecule may be added either separately or together with the analyte. In one embodiment, the second analyte-binding molecule may be added after the analyte has been bound with the immobilized first analyte-binding molecules on the detection electrode surface. In another embodiment, the second analyte-binding molecule may be added to the sample solution containing the analyte. In this case, the solution containing both the second analyte-binding molecule and the analyte, optionally also containing preformed complexes of analyte and second analyte-binding molecule, can then be contacted with the sensing region of the detection electrode comprising the immobilized first analyte-binding molecule, for example, by immersing the sensing region in a solution comprising the analyte and the second analyte-binding molecule. The second analyte-binding molecule can bind specifically to the second binding site of the analyte. After the second analyte-binding molecules have been bound to the analytes, excess unbound second analyte-binding molecules may be separated from the solution by centrifugation, which is but one example of the separation technique.

[0073] In various embodiments of the claimed invention, the second analyte- binding molecules comprise a metal nanoparticle that is bound to the second analyte- binding molecule. The second analyte-binding molecule having a bound or otherwise conjugated metal nanoparticle is herein also referred to as "analyte detection agent". The metal nanoparticle may be bound to the second analyte-binding molecule via a coupling reagent. The term "coupling reagent" as used herein refers to a reagent, e.g. a molecule or compound that binds or conjugates the metal nanoparticle to the second analyte-binding molecule. In certain embodiments, the coupling reagent may comprise a compound having both thiol groups and primary amine groups. The thiol groups of the coupling reagent may be used to bind the metal nanoparticle, whereas the amine groups may be used to bind the second analyte-binding molecule. The primary amine groups of the coupling reagent may also bind to a linker which in turn binds to the second analyte- binding molecule. In such an embodiment, a first region of the linker interacts with the primary amine groups of the coupling reagent while a second region interacts with the second analyte-binding molecule. In one embodiment, the coupling reagent is a polyamidoamine, for example a disulfide-based polyamidoamine. In one specific embodiment, the coupling reagent is a mixture of oligomers of disulfide-based polyamido amines (2), as illustrated in Fig. 6 (the synthesis of which is provided in the Example section). In certain embodiments where the coupling reagent comprises primary amine groups, the linker that helps to bind to the second analyte-binding molecule is a N-succinimidyl compound. In one embodiment, the linker is 0,0'-bis[2- ( -succinimidyl-succinylamino)ethyl] polyethylene glycol 3,000 (NHS-PEG-NHS).

[0074] In one embodiment, the metal nanoparticle may be a noble metal including platinum group metals. Noble metal includes silver, palladium, gold, platinum, iridium, osmium, rhodium and ruthenium. In one embodiment, the metal nanoparticle comprises a metal that catalyses the formation and/or growth of a metal catalyst for hydrogen generation reaction. In one embodiment, the metal nanoparticle is a platinum nanoparticle.

[0075] The resultant detection electrode comprising the analyte:analyte-detection agent complex including the bound metal nanoparticle is thus formed, and the detection electrode so-formed is hereinafter termed "complex-modified electrode".

[0076] A metal catalyst for catalyzing hydrogen generation reaction is formed and deposited on the complex-modified electrode. The complex-modified electrode is contacted with a catalyst-growth solution to form and grow the metal catalyst. The term "catalyst-growth solution" as used herein refers to a solution providing suitable conditions under which the formation of a metal by reducing the metal salt is initiated, and that allows the formed metal to grow over time. The formation and/or growth of the metal may be thermodynamically favoured and occurs spontaneously. In certain embodiments, the rate of the formation and/or growth of the metal may be accelerated by application of external conditions such as heat or pressure.

[0077] In one embodiment, the catalyst-growth solution comprises a metal salt of the metal catalyst to be formed and a reducing agent. In one embodiment, the catalyst- growth solution comprises about 0,01 mM to about 1 M of the metal salt. In one embodiment, the catalyst-growth solution comprises about 0.1 mM to about 1 M of the reducing agent. The metal catalyst is one that catalyzes hydrogen generation reaction and is formed by reducing the metal salt by the reducing agent. The metal catalyst may be a platinum group metal including palladium, platinum, iridium, osmium, rhodium and ruthenium. The metal catalyst may comprise a metal that is the same as the metal nanoparticle bound to the second analyte-binding molecule. Alternatively, the metal catalyst may comprise a metal that is different from the metal nanoparticle bound to the second analyte-binding molecule. In one embodiment, the metal catalyst is platinum. In yet another embodiment, the metal nanoparticle bound to the second analyte-binding molecule and the metal catalyst are both platinum.

[0078] In one embodiment, the metal salt is a hexachloroplatinate salt. In the embodiments where the metal catalyst is platinum, the metal hexachloroplatinate salt includes, but is not limited to, hexachloroplatinate (IV) hydrate (H₂PtCl₆), potassium hexachloroplatinate (IV) (K₂PtC¼), sodium hexachloroplatinate (IV) (Na₂PtCl₆), and ammonium hexachloroplatinate (IV)

In one embodiment, the metal salt is H₂PtCl6. In certain embodiments, the metal salt may include a combination of hexachloroplatinate salts.

[0079] In another embodiment, the metal salt is a tetrachloroplatinate salt. In the embodiments where the metal catalyst is platinum, the metal tetrachloroplatinate salt includes, but is not limited to, tetrachloroplatinate (II) hydrate (H₂PtC ), potassium tetrachloroplatinate (II) (K₂PtC ), sodium tetrachloroplatinate (II) (Na₂PtC ), and ammonium tetrachloroplatinate (II) ((NH₄)₂PtC ). In one embodiment, the metal salt is K₂PtCl₄. In certain embodiments, the metal salt may include a combination of tetrachloroplatinate salts. In yet certain embodiments, the metal salt may include a combination of hexachloroplatinate salts and tetrachloroplatinate salts. In one embodiment, the metal salt includes a mixture of H₂PtCl<5 and K₂PtC .

[0080] The reducing agent comprised in the catalyst-growth solution reduces the metal salt to metal catalyst in the presence of the metal nanoparticle bound to the second analyte-binding molecule. In one embodiment, the metal nanoparticle bound to the second analyte-binding molecule catalyzes the reduction process of the metal salt by the reducing agent. Alternatively, the metal salt may be reduced by the reducing agent in the presence of a catalyst other than or in addition to the metal nanoparticle bound to the second analyte-binding molecule. The reducing agent may be an organic acid or an inorganic acid. In one embodiment, the reducing agent is an organic acid. The organic acid suitable for reducing the metal salt includes a carboxylic acid, and the carboxylic acid includes formic acid, ascorbic acid, and acetic acid, which are but few examples of the reducing agent. In one embodiment, the reducing agent is formic acid. In certain embodiments, the reducing agent may include a mixture of reducing acids, such as a mixture of formic acid, ascorbic acid, citratic acid, oxalatic acid and acetic acid. In the embodiment where formic acid is used, the reduction of metal salt to metal catalyst may be performed at room temperature under physiological pH. In embodiments where ascorbic acid or acetic acid is used, the reduction of metal salt to metal catalyst may be performed at 100 °C or 200 °C, respectively, under physiological pH.

[0081] The catalyst-growth solution may further comprise a stabilizing agent for stabilising the catalyst-growth solution over a period of time. In one embodiment, the catalyst-growth solution comprises about 0.05 to about 2 % by weight of the stabilizing agent. When a stabilizing agent is said to stabilise the catalyst-growth solution, it is to be understood and appreciated that it means that the rate of formation and/or deposition of the metal catalyst is not adversely affected or reduced over a period of time. The stabilizing agent may stabilise the catalyst-growth solution for more than 30 minutes, or more than 1 hour, or more than 1 day. The stablizing agent may comprise a polysorbate detergent. The polysorbate detergent includes Tween 80 and Tween 20, which are but few examples of a stabilizing agent. In one embodiment, the stabilizing agent is Tween 80. The pH of the catalyst-growth solution may need to be adjusted to avoid the detachment of the analyte, or the first or second analyte-binding molecule from the detection electrode surface in an acidic environment due to the presence of the acidic reducing agent. In one embodiment, the pH is adjusted to 6.5.

[0082] In one embodiment where the catalyst-growth solution comprises PtC ^2" salt and formic acid reducing agent in the presence of platinum nanoparticle bound to the second analyte-binding molecule, the stabilizing agent may comprise Tween 80. In one embodiment, the catalyst-growth solution comprises 1 mM PtCl ^" and 0.1 M formic acid, and the reduction process is performed at room temperature under physiological pH to form the platinum catalyst. In yet another embodiment, the catalyst- growth solution comprises 1 mM PtCL^2", 0.1 M formic acid, and 0.5 % Tween 80, and the reduction process is performed at room temperature with an adjustment of the pH to 6.5 to form the platinum catalyst.

[0083] In addition to catalyzing the reduction of metal salt to metal catalyst in the catalyst-growth solution by the metal nanoparticle bound to the second analyte-binding molecule, the metal nanoparticle advantageously acts as a nucleation seed for the deposition and/or growth of the metal catalyst formed by the reduction of the metal salt. The catalyst-growth solution may therefore be seen as facilitating a seed-mediated nucleation and growth mechanism for the metal catalyst. Due to the presence of the metal nanoparticle bound to the second analyte-binding molecule, the metal catalyst formed by the reduction of the metal salt is thermodynamically favoured to cluster together and deposit on the seeding metal nanoparticle. The amount of seeded and deposited metal catalyst on the metal nanoparticle increases over time. The so-formed metal catalyst may comprise nanocrystals.

[0084] In this context, when the metal catalyst is said to be deposited on the complex-modified electrode, it is to be understood and appreciated that the metal catalyst may not be physically deposited or bound to the complex-modified electrode surface, since the surface may already have bound blocking agent, for example. Rather, the metal catalyst is bound, and therefore 'deposited' to the complex-modified electrode surface via the seeding metal nanoparticle. Hereinafter, the deposition of the metal catalyst on the complex-modified electrode forms a metal catalyst-modified electrode.

[0085] The catalyst-modified electrode is contacted with a proton-rich electrolyte to generate hydrogen. The hydrogen generation causes a change in electrical current, which is one example of a electrochemical signal, to be detected. The obtained electrochemical signal may then be correlated to the presence of amount of the analyte in the sample. As mentioned earlier, the deposited metal catalyst favourably catalyzes the hydrogen generation reaction, i.e. the reduction of protons to hydrogen. The proton- rich electrolyte contains proton (i.e. H⁺ ions) concentration rich enough to enable detection of the electrical current upon reduction of the protons to hydrogen gas.

[0086] In one embodiment, the proton-rich electrolyte comprises a solution of an acid in an aqueous medium. In one embodiment, the concentration of the acid in the proton-rich electrolyte is about 0.1 mM to about 1 M. The acid comprised in the proton- rich electrolyte may be an inorganic acid. In one embodiment, the aqueous medium of the proton-rich electrolyte comprises a solution of a salt. In one embodiment, the concentration of salt in the proton-rich electrolyte is about 1 mM to about 10 M. The salt may dissociate into its constituent ions, e.g. cations and anions that flow to respective electrodes in the electrochemical electrode arrangement, thereby causing a change in electrical current to be detected. In one embodiment, the proton-rich electrolyte comprises an aqueous solution of hydrochloric acid (HCl) and a salt selected from the group consisting of potassium chloride (KCl), sodium chloride (NaCl), ammonium chloride (NH₄C1), and calcium chloride (CaCl₂). In one embodiment, the proton-rich electrolyte comprises an aqueous solution of HCl and KCl. In another embodiment, the proton-rich electrolyte comprises an aqueous solution of sulfuric acid (H₂S0₄) and a salt selected from the group consisting of sodium sulfate (Na₂S0₄), and aluminum sulfate (A1₂(S0₄)₃). In further embodiment, the proton-rich electrolyte comprises an aqueous solution of nitric acid (HN0₃) and a salt selected from the group consisting of sodium nitrate (NaNQ₃) and potassium nitrate (KN0₃). [0087] Subsequently, 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. As an illustrative example, 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.

[0088] In another aspect, the present invention relates to a method for the production of an electrochemical sensor, comprising:

(a) immobilizing an analyte on the surface of an electrode;

[0089] In a further aspect, the present invention relates to an electrochemical sensor obtainable according to a method of the invention.

[0090] In yet another aspect, the present invention relates to the use of an electrochemical sensor of the invention for the detection of an analyte. 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. Another application is the use of such 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.

[0091] In one aspect, the present invention relates to a method for the production of an electrode, comprising contacting an electrode comprising a complex of an analyte and an analyte detection agent comprising a metal nanoparticle with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle to deposit the metal on the electrode surface.

[0092] In another aspect, the present invention relates to an electrode obtainable according to a method of the invention.

[0093] In a further aspect, the present invention relates to the use of an electrode of the invention in an electrochemical sensor for the detection of an analyte.

[0094] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non- limiting examples.

Examples

[0095] Example 1: Materials and Reagents

[0096] Prostate-specific antigen (PSA) from human serum (P-3338) was purchased from Sigma- Aldrich. Monoclonal antibodies to PSA were obtained from Meridian Life Science Inc., Biodesign International (M86433M as capture antibody and M86111M as detection antibody). 0,0'-bis[2-(N-succinimidyl-succinylamino)ethyl] polyethylene glycol 3,000 ( HS-PEG-NHS), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-succinimidyl ester (NHS), 16-mercapto-l-hexadecanoic acid (16-MHA), 11- mercapto-l-undecanol (11-MUOH), ethylamine, NaBH₄, H₂PtCl , K₂PtCLt, formic acid, Tween 80, NaOH, boric acid, sodium tetraborate, Tris hydroxymethyl (aminomethane) (TRIS), Na₂HP0₄, NaH₂P0₄, KC1 and HC1 were obtained from Sigma- Aldrich. Nanopure water (resistivity > 18 ΚΩ cm) was used in the studies.

[0097] Example 2: Apparatus

[0098] Cyclic voltammetry was performed with a CHI 760C electrochemical workstation (CH Instruments, Texas). A conventional 3 -electrode system was employed. A 2 mm-diameter gold electrode (CH Instruments), a platinum wire and an Ag/AgCl (3M KC1) electrode (CH Instruments) were employed as the working electrode, counter electrode, and reference electrode, respectively. TEM experiments were performed on a JEOL JEM-3010 electron microscope (200 kV). Centrifugation was conducted with an Allegra 64R Centrifuge (Beckman Coulter, California).

[0099] Example 3: Synthesis and Characterization of Disulfide-based Polvamidoamines

[00100] Materials

[00101] Methyl-3-mercaptopropionate, tris(2-aminoethyl)amine (96%), pentaethylene-hexamine, dimethylsulfoxide (DMSO) and tetrahydroiuran (THF) were all purchased from Sigma- Aldrich and used as received.

[00102] Methods

[00103] Ή and ¹³ C NMR spectra. Spectra were recorded on a Bruker AVANCE 400 at 400 MHz and 100 MHz, respectively, using the deuterated solvent indicated. CS ChemNMR Pro version 6.0 (Upstream Solutions GbmH Scientific Software Engineering CH-6052 Hergiswil, Switzerland) was employed to analyze the spectra.

[00104] Gel permeation chromatography (GPC). The molecular weight (M_w) of polyamidoamine was analyzed by GPC (Waters 2690, MA, USA) with a differential refractometer detector (Waters 410, MA, USA). The mobile phase consisted of 0.5 M of sodium acetate and 0.5 M of acetic acid solution with a flow rate of 1 mL/min. A Shodex OHpak SB-803 HQ (8.0 mm x 300 mm) column was used. Number and weight average molecular weights (M_n and M_w) as well as polydispersity indices were calculated from a calibration curve using a series of dextran standards (Aldrich, Missouri, USA) with molecular weights ranging from 667 to 778000.

[00105] Elemental analysis. The nitrogen content of the polyamido amine was determined by elemental analysis using Perkin-Elmer Instruments Analyzer 2400 CHN/CHNS and Eurovector EA3000 Elemental Analyzers.

[00106] Synthesis and characterization of disulfide-based polyamidoamines (mixed oligomers)

[00107] Formation of amine monomer (1). 10 ml of tris(2-arninoethyl)amine (0.067 mol), 60 ml of methanol and 17 ml of epichlorohydrin (0.22 mol) were placed in a flask under nitrogen. The mixture was left to stir in an ice bath for 24 h. Next, 250 ml of pentaethylenehexamine (1.08 mol) were added to the contents of the flask in conjunction with 100 ml of DMSO. The mixture was left to stir at 90°C for 24 h. The contents of the flask were then dialyzed against flowing water using dialysis tubing with a molecular weight cut-off of 500 Da (Spectrum Laboratories, USA) for 1 day, followed by freeze drying. δ_Η(400 MHz, D₂0) 3.70 (lH, m, -CH₂-CH(OH)CH₂-); 3.10-2.20 (m, - NH-GZ-VCi^-NH-, -CH₂-CH(OH)CH₂-, and -NH-CH₂-CH₂-N(-CH₂-CH₂-NH-)₂). 5c(100 MHz, D₂0) 67, 58, 56, 55, 53, 52, 49, 48, 47, 45, 44, 39, 38 and 37.

[00108] Formation of disulfide-based polyamidoamines (mixed oligomers) (2). 6.5 g of the amine monomer (1) (0.0081 mol) were added to a flask with 150 ml of methanol and 0.70 ml of methyl-3-mercaptopropionate (0.0064 mol). The mixture was then left to stir at 60°C for 24 h. The excess methanol was removed under vacuum, and then the mixture was precipitated in THF and dried using a vacuum oven. 5H(400 MHZ, D₂0) 3.70 (1H, m, -CH₂-CH^"(OH)CH₂-); 3.30-3.20 (2H, t, -NH-CH₂-C^₂-NH-C(=0)-CH₂- CH₂-S-); 3.10-2.20 (m, -NH-C ¼-CH₂-NH-, -NH-CH₂-CH₂-NH-C(=0)-CH₂-C//₂-S-S- , CH₂-CH₂-NH-C(=0)-C^₂-CH₂-SH, -CH₂-CH(OH)CH₂-, and -NH-CH₂-CH₂-N(-CH₂- CH₂-NH-)₂). 6c(100 MHz, D₂0) 165, 67, 58, 56, 55, 53, 52, 49, 48, 47, 45, 44, 39, 38, 37, 27 and 21.

[00109] Synthesis and characterization of disulfide-based polyamidoamines (mixed oligomers). The oligomeric polyamidoarnine was prepared via a two-step procedure. The first step involved a ring-opening mechanism in conjunction with nucleophilic substitution. For this simple procedure, pentaethylenehexamine was employed as the main amine as shown in Scheme 1. Briefly, we initially formed the amine monomer (1) compound by reacting the core amine, tris(2-aminoethylene)amine with epichlorohydrin in methanol via ring opening in an ice bath for 24 h. This was followed by reacting the core with excess pentaethylenehexamine via nucleophilic substitution in an oil bath at 90°C for 24 h. Subsequently, the amine monomer (1) compound was purified via dialysis in water. We then reacted the purified amine monomer (1) with the thiol ester, methyl-3-mercaptopropionate, via nucleophilic substitution at 60°C for 24 h in methanol. Lastly, the excess methanol was removed under vacuum, and the disulfide- based polyamidoamines (mixed oligomers) were obtained as a crude mixture via precipitation in THF. The oligomeric mixture was then dried in a vacuum oven.

[00110] The chemical structure of both the amine monomer (1) and the disulfide- based polyamidoamines (mixed oligomers) (2) were confirmed using Ή and ¹³C NMR spectroscopy. In addition, with the aid of GPC analysis, the weight average molecular weight of the disulfide-based polyamidoamines (mixed oligomers) was determined to be 2.8 kDa with a polydispersity index of 1.4.

[00111] Example 4; Synthesis of Pt Nanoparticles and Pt Nanoparticle- Antibody Conjugates

[00112] 1 mM of H₂PtCl₆ and 2.5 mg/ml of disulfide-based polyamidoamines were dissolved in 200 ml of nanopure water, and stirred for 10 min. Disulfide-based polyamidoamines contained both thiol groups for strong Pt nanoparticle stabilization, and primary amine groups for further bioconjugation (see Example 3). The solution turned brown upon the addition of 48 mg of NaBH₄ dissolved in 2 ml water, suggesting the formation of Pt nanoparticles. The Pt nanoparticles were then concentrated to 10 ml via water evaporation. 30 ml of acetone were added to the solution to precipitate the Pt nanoparticles via centrifugation. The precipitate was collected and re-dissolved in 10 ml of nanopure water. This procedure was repeated twice to obtain pure Pt nanoparticles. TEM image (Fig. 2a) showed that the size of the Pt nanocrystals was approximately 3 nm. However, sedimentation of these Pt nanocrystals in a pure aqueous solution via centrifugation was impossible even at 21,000 rpm. For ease of centrifugation and separation of the Pt nanoparticle-antibody conjugates from the excess antibodies after conjugation, less capping reagent (1 mg/ml) was used while keeping the amount of the other reagents unchanged. The resulting Pt nanoparticles were purified using the same protocol as described above. Low-resolution transmission electron microscopy (TEM) showed that the Pt particles have a diameter of 40 nm (Fig. 2b). However, high- resolution TEM image (Fig. 2c) illustrated that these particles were aggregates of 3 nm Pt nanocrystals.

[00113] The Pt nanoparticle clusters were conjugated with detection antibodies using NHS-PEG-NHS linker, which reacted with the primary amines from both Pt nanoparticles and detection antibodies. Pt nanoparticle clusters (2.5 mg) were diluted in 1 ml of borate buffer (pH 7.5), and mixed with an excess amount of NHS-PEG-NHS 3000 (10 mg dissolved in 100 μ\ of dimethyl sulfoxide (DMSO)). After 15 min of incubation, the NHS-PEG-NHS-conjugated nanoparticles were sedimented by centrifugation at 13,000 rpm to remove excess NHS-PEG-NHS. The recovered activated particles were immediately dissolved in 1 ml of borate buffer (pH 7.5) containing 0.1 mg of M86111M detection antibodies, and incubated for 2 h under shaking. 100 μ\ of TRIS buffer (1 M, pH 7.4) were added to block any free NHS groups. The final Pt nanoparticle-antibody conjugates were separated by centrifugation at 13,000 rpm to remove excess detection antibodies. TEM image indicated that the Pt nanoparticles were stable upon conjugation (Fig. 2d). The conjugated nanoparticles were kept at 4°C.

[00114] Example 5: Preparation of Sandwich Immunosensor [00115] Au electrode of 2 mm-diameter for the immunosensor was first polished carefully using 0.3-μηι alumina slurry, and electrochemically cleansed in a H₂S0₄ solution (0.5 M) by cycling the potential between -0.2 V and 0.8 V vs. a Pt wire quasi- reference electrode for 5 min. The electrode was then washed with nanopure water, and dipped into 100 μΐ of an ethanolic solution containing 0.1 mM of 16-MHA and 0.9 mM of 11-MUOH overnight. 11-MUOH was used as a spacer to obtain an optimal density of the COOH groups on the electrode surface and to prevent non-specific adsorption. The electrode was then soaked for 2 h in 100 μΐ of phosphate buffered saline (PBS) (0.05 M, pH 7.5) containing 5 mM of NHS, 20 mM of EDC and 100 jig ml of M86433M capture antibodies. The antibodies were covalently bound to the electrode through the conjugation of amine groups from the antibodies with the activated carboxyl groups on the electrode surface. Next, 10 μΐ of ethanolamine solution (1 M) were added to block any free activated carboxyl groups. The resulting antibody-modified electrode was washed with nanopure water, and then with 10 mM of glycine (pH 2.2) to remove any non-covalently bound antibodies. The electrode prepared was exposed to PSA analyte concentrations of 0-10 ng/ml for 30 min. It was washed with PBS before dipping into the Pt nanoparticle-labeled detection antibodies. After 15 min, the electrode was thoroughly washed with 0.01 M of TRIS buffer (pH 7.4) containing 0.15 M of NaCl to remove non-specifically bound Pt nanoparticle-labeled detection antibodies. The electrode was then carefully washed with nanopure water. It was next dipped into the Pt catalyst-growth solution containing 1 mM of PtC ^2", 0.1 M of formic acid and 0.5% of Tween 80 (pH 6.5) for a certain amount of time. The electrode was washed again with nanopure water before conducting cyclic voltammetric measurements in an aqueous solution containing 01 M of KC1 and 0.01 M of HC1.

[00116] Example 6; Formulation of the Pt Catalyst-Growth Solution

[00117] An important step in this immunosensing strategy was the rapid seed- mediated generation and deposition of bare Pt particles on the electrode surface in the aqueous phase at room temperature under physiological pH using a Pt catalyst-growth solution. It was found that a Pt catalyst -growth solution containing 1 mM of PtCU^2" and 0.1 M of formic acid reductant was stable for approximately 30 min, and that the Pt nanoparticle seeds could efficiently catalyze the reduction of PtC ^2" by formic acid to produce Pt. To increase the stability of the Pt catalyst-growth solution from approximately 30 min to more than 1 day without significantly decreasing the rate of seed-mediated deposition of Pt, 0.5% of Tween 80 was added to the catalyst-growth solution. The pH of the catalyst-growth solution was adjusted to 6.5 to avoid the detachment of PSA or detection antibody from the electrode surface in an acidic environment (due to the presence of 0.1 M of formic acid in the Pt enhancement step).

[00118] Fig. 3 shows the cyclic voltammograms of proton redox processes obtained at different electrodes in an acidic solution containing 10 mM of HCl and 1 M of KC1. Since the reduction of H⁺ occurred at a potential close to that of 0₂ reduction, 10 mM of HCl was used so that the signal due to 0₂ reduction was negligible compared to that of H⁺ reduction. Consequently, removal of 0₂ was not required during the experiments. The results showed that the bare Pt electrode was much more active for facilitating the hydrogen evolution process. A well-defined voltammogram was obtained with a formal potential of -0.417 V vs. Ag/AgCl (taken as the average of the anodic peak potential, -0.384 V vs. Ag/AgCl, and the cathodic peak potential, -0.449 V vs. Ag/AgCl). In contrast, proton reduction at the bare Au electrode surface was much slower with a very large overpotential. The peak potential of -0.741 V vs. Ag/AgCl for proton reduction at a bare Au electrode was almost 0.3 V more negative than that at the Pt electrode. The overpotential of proton reduction was even higher at the Au electrode modified with a mixed thiol monolayer. In contrast, the voltammogram obtained using the PSA immunosensor when the analyte PSA concentration was 10 nM, was essentially identical to that obtained at a bare Pt electrode. This suggested that the Pt particles generated with the catalyst-growth solution have good activity for proton reduction.

[00119] Example 7: Effect of Pt enhancement time

[00120] The amount of Pt deposited onto the electrode surface during the Pt enhancement step, and hence the electrochemical response of the immunosensor was time-dependent. Experiments were therefore conducted to investigate the effect of the Pt enhancement time on the voltammetric response of the PSA immunosensor. The transition from a steady-state sigmoidal-shaped voltammogram to a transient peak- shaped voltammogram is clearly illustrated in Fig. 4a. When the enhancement time was short enough (2-5 min) and the Pt particle density and/or size was sufficiently small, (i.e. the distance between the particles was 10 times their diameter), every individual Pt particle behaved like an independent micro electrode. Subsequently, the mass transport was governed by radial diffusion, and sigmoidally shaped steady-state voltammograms were observed. When the enhancement time was longer than 1 h, the electrode surface would be almost entirely covered with Pt particles. The mass transport to the electrode was then governed by planar diffusion. The voltammogram obtained after Pt enhancement treatment resembled that obtained at a Pt disc electrode (see Fig. 3). When the enhancement time increased from 10 min to 45 min, the Pt particle density and/or size increased, causing the diffusion layer of each Pt particle microelectrode to overlap. Therefore, the transition of steady- state voltammogram to an ideal transient voltammogram of a macro-disc electrode was observed. Controlled experiments conducted in the absence of PSA showed that background currents were small. This suggested that thiol- modified Au electrodes were almost inert towards the catalytic growth of Pt, and that the non-specific adsorption of PSA or PSA antibody was minimal. Values of the steady-state diffusion-controlled limiting currents or peak currents obtained from Fig. 4a are summarized in Fig. 4b.

[00121] Example 8: Calibration curves

[00122] The results in Fig. 4 implied that a higher sensitivity would be expected if a longer Pt enhancement time was applied. However, longer enhancement time might also decrease the dynamic concentration response range of the immunosensor. To examine this, two calibration curves were obtained using different Pt enhancement times (Fig. 5). In both cases, the PSA immunosensors have a good response to PSA over a wide concentration range. When a Pt enhancement time of 10 min was used, the steady-state diffusion limiting current or peak current increased when the PSA concentration increased from 10 fg/ml to 10 pg/ml. The peak current approached a plateau when the PSA concentration was higher than 0.1 ng/ml. The results also showed that the sensitivity of the PSA immunosensor increased and its dynamic response range shifted towards a lower concentration range (1 fg/ml to 1 pg/ml) when a longer Pt enhancement time of 30 min was used.

[00123] It has been shown that a new Pt catalyst-growth solution containing 1 mM of PtC , 0.1 M of formic acid and 0.5% of Tween 80 (pH 6.5) was very effective for the seed-mediated growth of Pt particles. The Pt particles generated using the method of the present invention were active towards electrochemical hydrogen evolution. The advantage of using this Pt enhancement strategy and the Pt-catalyzed hydrogen evolution for the development of ultrasensitive immunosensors has been successfully demonstrated in the example of PSA detection. A detection limit of 1 fg/ml has been achieved.

[00124] The inventions illustratively described herein may suitably be practised in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by specific embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00125] The content of all documents cited herein is incorporated by reference in their entirety.

[00126] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00127] Other embodiments are in the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method for the detection of an analyte, the method comprising:

(a) immobilizing the analyte on the surface of an electrode;

(c) contacting the complex-modified electrode obtained in (b) with a catalyst- growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle, wherein the metal nanoparticle serves as a nucleation seed to deposit the metal on the complex-modified electrode surface to form a metal catalyst-modified electrode;

2. The method of claim 1, wherein (a) comprises

(i) covalently coupling a first analyte-binding molecule to the surface of the electrode; and

(ii) contacting the electrode coupled to the first analyte-binding molecule with the sample containing the analyte to form a first analyte-binding molecule: analyte complex.

3. The method of claim 2, wherein (b) comprises contacting the electrode comprising the first analyte-binding molecule: analyte complex with the analyte detection agent, wherein the analyte detection agent comprises a second analyte-binding molecule conjugated to the metal nanoparticle, to form a first analyte-binding molecule:analyte:detection agent complex.

4. The method of claim 2 or 3, wherein the formation of the complex of (i) the analyte and the first analyte-binding molecule, or (ii) the analyte and the first analyte- binding molecule and the analyte detection agent is carried out prior to coupling the first analyte-binding molecule to the electrode surface.

5. The method of any one of claims 2-4, wherein the formation of the complex of the analyte and the analyte detection agent is carried out prior to contacting the analyte with the first analyte-binding molecule coupled to the electrode surface.

6. The method of any one of claims 2-5, wherein the first and/or the second analyte- binding molecules are specific for the analyte.

7. The method of claim 6, wherein the first and/or the second analyte-binding molecules are selected from the group consisting of antibodies and fragments or variants thereof, antibody-like molecules, binding proteins, protein receptors, and extracellular domains (ECD) thereof.

8. The method of any one of claims 3-5, wherein the first and second analyte- binding molecules are the same or different.

9. The method of any one of claims 1-8, wherein the metal nanoparticle and the metal catalyst are each formed of the same or a different metal.

10. The method of any one of claims 1-9, wherein the metal nanoparticle comprises or consists of a metal selected from the group consisting of gold, silver, ruthenium, rhodium, palladium, osmium, iridium, and platinum.

11. The method of claim 10, wherein the metal nanoparticle consists of platinum.

12. The method of any one of claims 1-1 1 , wherein the metal catalyst comprises or consists of a metal selected from the group consisting of ruthenium, rhodium, palladium, osmium, iridium, and platinum.

13. The method of claim 12, wherein the metal catalyst consists of platinum.

14. The method of claim 13, wherein the metal salt is a hexachloroplatinate or tetrachloroplatinate salt.

15. The method of claim 14, wherein the metal salt is selected from the group consisting of dihydrogen hexachloroplatinate (IV) hydrate (H₂PtCl₆- xH₂0), potassium hexachloroplatinate (IV) (KjPtCk), sodium hexachloroplatinate (IV) (Na₂PtCl ), ammonium hexachloroplatinate (IV) ((NH₄)₂PtCl₆), dihydrogen tetrachloroplatinate (II) hydrate (H₂PtCl₄' xH₂0), potassium tetrachloroplatinate (II) (K₂PtC ), sodium tetrachloroplatinate (II) (Na₂PtC ), ammonium tetrachloroplatinate (II) (( H₄)₂PtCLt), and mixtures thereof.

16. The method of claim 15, wherein the metal salt is H₂PtCl - xH₂0 or K₂PtC .

17. The method of any one of claims 1 -16, wherein the catalyst-growth solution comprises about 0.01 mM to about 1 M of the metal salt.

18. The method of any one of claims 1 -17, wherein the reducing agent is a carboxylic acid.

19. The method of claim 18, wherein the reducing agent is selected from the group consisting of formic acid, ascorbic acid, and acetic acid.

20. The method of claim 19, wherein the reducing agent is formic acid.

21. The method of any one of claims 1 -20, wherein the catalyst-growth solution comprises about 0.1 mM to about 1 M of the reducing agent.

22. The method of any one of claims 1-21 , wherein the catalyst-growth solution further comprises a stabilizing agent to stabilize the catalyst-growth solution.

23. The method of claim 22, wherei the stabilizing agent is selected from the group consisting of a polysorbate detergent, Tween 80, and Tween 20.

24. The method of claim 22 or 23, wherein the catalyst-growth solution comprises about 0.05 to about 2 % by weight of the stabilizing agent.

25. The method of any one of claims 1-24, wherein the proton-rich electrolyte comprises a solution of an acid in an aqueous medium.

26. The method of claim 25, wherein the proton-rich electrolyte comprises an aqueous solution of hydrochloric acid (HCl) and a salt selected from the group consisting of potassium chloride (KC1), sodium chloride (NaCl), ammonium chloride (NH₄C1), and calcium chloride (CaCl₂).

27. The method of claim 25, wherein the proton-rich electrolyte comprises an aqueous solution of sulfuric acid (H₂S0₄) and a salt selected from the group consisting of sodium sulfate (Na₂S0₄), and aluminum sulfate (A1₂(S0₄)₃).

28. The method of claim 25, wherein the proton-rich electrolyte comprises an aqueous solution of nitric acid (HNO₃) and a salt selected from the group consisting of sodium nitrate (NaN0₃) and potassium nitrate (K 0₃).

29. The method of any one of claims 25 to 28, wherein the concentration of the acid in the proton-rich electrolyte is about 0.1 mM to about 1 M.

30. The method of any one of claims 25 to 29, wherein the concentration of salt in the proton-rich electrolyte is about 1 mM to about 10 M.

31. The method of any one of claims 1-30, wherein the analyte is selected from the group consisting of proteins, peptides, lipids, nucleic acids, small organic molecules, organic polymers, carbohydrates and haptens.

32. The method of any one of claims 1 -31 , wherein the method comprises one or more washing steps prior to (c).

33. A method for the production of an electrochemical sensor, comprising:

(a) immobilizing an analyte on the surface of an electrode;

(c) contacting the complex-modified electrode obtained in (b) with a catalyst- growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle to deposit the metal on the complex-modified electrode surface.

34. The method of claim 33, wherein (a) comprises

(ii) contacting the electrode coupled to the first analyte-binding molecule with a sample containing the analyte to form a first analyte-binding molecule:analyte complex.

35. The method of claim 34, wherein (b) comprises contacting the electrode comprising the first analyte-binding molecule:analyte complex with the analyte detection agent, wherein the analyte detection agent comprises a second analyte-binding molecule conjugated to the metal nanoparticle, to form a first analyte-binding molecule:analyte:detection agent complex.

36. The method of claim 34 or 35, wherein the formation of the complex of (i) the analyte and the first analyte-binding molecule, or (ii) the analyte and the first analyte- binding molecule and the analyte detection agent is carried out prior to coupling the first analyte-binding molecule to the electrode surface.

37. The method of any one of claims 34-36, wherein the formation of the complex of the analyte and the analyte detection agent is carried out prior to contacting the analyte with the first analyte-binding molecule coupled to the electrode surface.

38. The method of any one of claims 34-37, wherein the first and/or the second analyte-binding molecules are specific for the analyte.

39. The method of claim 38, wherein the first and/or the second analyte binding molecules are selected from the group consisting of antibodies and fragments or variants thereof, antibody-like molecules, and receptor proteins.

40. The method of any one of claims 35-39, wherein the first and second analyte- binding molecules are the same or different.

41. The method of any one of claims 33-40, wherein the metal nanoparticle and the metal catalyst are each formed of the same or a different metal.

42. The method of any one of claims 33-41 , wherein the metal nanoparticle comprises or consists of a metal selected from the group consisting of gold, silver, ruthenium, rhodium, palladium, osmium, iridium, and platinum.

43. The method of claim 42, wherein the metal nanoparticle consists of platinum.

44. The method of any one of claims 33-43, wherein the metal catalyst comprises or consists of a metal selected from the group consisting of ruthenium, rhodium, palladium, osmium, iridium, and platinum.

45. The method of claim 44, wherein the metal catalyst consists of platinum.

46. The method of claim 45, wherein the metal salt is a hexachloroplatinate or tetrachloroplatinate salt.

47. The method of, claim 46, wherein the metal salt is selected from the group consisting of dihydrogen hexachloroplatinate (IV) hydrate (Η₂Ρί¾· xH₂0), potassium hexachloroplatinate (IV) (K₂PtCle), sodium hexachloroplatinate (IV) (Na₂PtCl ), ammonium hexachloroplatinate (IV) ((NH₄)2PtCl ), dihydrogen tetrachloroplatinate (II) hydrate (H₂PtCLt'xH₂0), potassium tetrachloroplatinate (II) (K₂PtCl₄), sodium tetrachloroplatinate (II) (Na₂PtCl4), ammonium tetrachloroplatinate (II) ((NKL^PtCL*), and mixtures thereof.

48. The method of claim 47, wherein the metal salt is Η₂Ρί¾· xH₂0 or K₂PtCl₄.

49. The method of any one of claims 33-48, wherein the catalyst-growth solution comprises about 0.01 mM to about 1 M of the metal salt.

50. The method of any one of claims 33-49, wherein the reducing agent is a carboxylic acid.

51. The method of claim 50, wherein the reducing agent is selected from the group consisting of formic acid, ascorbic acid, and acetic acid.

52. The method of claim 51, wherein the reducing agent is formic acid.

53. The method of any one of claims 33-52, wherein the catalyst-growth solution comprises about 0.1 mM to about 1 M of the reducing agent.

54. The method of any one of claims 33-53, wherein the catalyst-growth solution further comprises a stabilizing agent to stabilize the catalyst-growth solution.

55. The method of claim 54, wherein the stabilizing agent is selected from the group consisting of a polysorbate detergent, Tween 80, and Tween 20.

56. The method of claim 54 or 55, wherein the catalyst-growth solution comprises about 0.05 to about 2 % by weight of the stabilizing agent.

57. Electrochemical sensor obtainable according to the method of any one of claims 33-56.

58. Use of the electrochemical sensor of claim 57 for the detection of an analyte.

59. A method for the production of an electrode, comprising contacting an electrode comprising a complex of an analyte and an analyte detection agent comprising a metal nanoparticle with a catalyst-growth solution, wherein the catalyst-growth solution comprises a metal salt and a reducing agent for reducing the metal salt in the presence of the metal nanoparticle to deposit the metal on the electrode surface.

60. An electrode obtainable according to the method of claim 59.

61. Use of the electrode of claim 60 in an electrochemical sensor for the detection of an analyte.