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WO2013039455A1 - Capteur ampérométrique - Google Patents

Capteur ampérométrique Download PDF

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Publication number
WO2013039455A1
WO2013039455A1 PCT/SG2012/000334 SG2012000334W WO2013039455A1 WO 2013039455 A1 WO2013039455 A1 WO 2013039455A1 SG 2012000334 W SG2012000334 W SG 2012000334W WO 2013039455 A1 WO2013039455 A1 WO 2013039455A1
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Prior art keywords
working electrode
electrode
amperometric sensor
molecule
solution
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English (en)
Inventor
Chee Seng Toh
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Nanyang Technological University
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Nanyang Technological University
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Priority to SG2014013353A priority Critical patent/SG2014013353A/en
Publication of WO2013039455A1 publication Critical patent/WO2013039455A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood

Definitions

  • the invention relates to an amperometric sensor, and in particular, to an amperometric sensor for hydrogen peroxide (H 2 0 2 ) operating without application of an external electrical potential.
  • Electrochemical sensors and biosensors detect analytes typically by monitoring electron flow through the sensor under an applied electrical potential which provides an equivalent power magnitude of /. V (current.voltage) to drive the redox reaction between the analyte and electrode, mediator or electrocatalytic species including enzymes.
  • a biofuel cell design which measures the changes in the open-circuit potential in response to the biofuel concentration, utilizes biological catalysts as the biocathode and/or bioanode in which the reduction of oxygen and the oxidation of biofuel occurred.
  • a self-powered amperometric sensor for detecting analyte present in a sample is disclosed herein.
  • the sensor derives current signal from the spontaneous reaction between hydrogen peroxide (H 2 0 2 ) and the sensor in the absence of an externally applied electrical potential.
  • the sensor does not rely on the open-circuit potential as the responsive signal; rather, a complete closed circuit which allows electrons to flow is employed in the present sensor.
  • Additions or depletion of H 2 0 2 directly or indirectly, results in a change in the detected current flow, correlating to the amount of analyte present in the sample.
  • an amperometric sensor for measuring the amount of hydrogen peroxide (H 2 0 2 ) present in a sample.
  • the sensor comprises:
  • a working electrode in contact with a sensing solution, wherein the working electrode comprises a porous membrane structure, wherein the sensing solution comprises the sample containing H 2 0 2 or a precursor of H 2 0 2 , wherein H 2 0 2 is reduced at the working electrode;
  • a counter electrode in contact with a reference solution, wherein the porous membrane structure of the working electrode provides a physical barrier between the sensing solution and the reference solution, wherein a reducer is oxidized at the counter electrode; a connector to electrically connect the working electrode and the counter electrode to allow a current flow therethrough, wherein the current flow is formed by a flow of electrons from the electrode where oxidation has occurred to the electrode where reduction has occurred, with the proviso that the current flow is not induced by an externally applied electrical potential; and
  • a current detector to detect and measure the current flow.
  • a method for measuring the amount of H 2 0 2 present in a sample comprises:
  • an amperometric sensor of the first aspect contacting a sensing solution with a working electrode of the amperometric sensor, wherein the working electrode comprises a porous membrane structure, wherein the sensing solution comprises the sample containing H 2 0 2 or a precursor of H 2 0 2 , wherein H 2 0 2 is reduced at the working electrode;
  • FIG. 1 shows a schematic of (a) the H 2 0 2 -powered sensor using a 2- compartment cell separated by a Prussian blue nanotubes membrane; (b) the H 2 0 2 - powered virus sensor using a 2 -compartment cell separated by a Prussian blue nanotubes membrane, showing the binding of virus to antibody molecules within the nanochannels of the porous membrane.
  • FIG. 2 shows the current-potential curves for solution oxygen reduction under ambient condition (— ⁇ ), oxygen reduction in air-saturated solution (— ), and hydrogen peroxide (8 mM) reduction in nitrogen-saturated solution (— ) at (a) porous PB-nt membrane (i.e. porous Pt membrane coated with PB); (b) porous Pt membrane.
  • Fig. 3 shows (a) the effect of adding H 2 0 2 on the driving force measured from the potential difference between VB-nt and auxiliary electrode during closed-circuit condition when current flows through the cell; (b) the effect of adding H 2 0 2 on the potential difference between ?B-nt and counter electrodes under open-circuit condition when no current flows through the cell; (c) the plot of driving force obtained from (a) versus the logarithm of H 2 0 2 concentration.
  • Sensing solution contains 1M TRIS buffer
  • reference solution contains 0.5M KCl.
  • Fig. 4 shows the dependence of the power density on the potential of the ?B-nt membrane electrode vs auxiliary electrode in sensing solution containing 30mM H 2 0 2 .
  • Sensing solution contains 1M TRIS buffer
  • reference solution contains 0.5M KCl.
  • Fig. 5 shows the effect of increasing K + concentration in the reference or sensing solution during the addition of H 2 0 2 to the sensing solution.
  • the following three arrangements of cell solutions are used: (a) Pt
  • FIG. 6 shows the chemical reaction driven amperometric responses of (a) H 2 0 2 sensor during successive additions of H 2 0 2 (indicated by arrows); (b) glucose sensor during successive additions of glucose (indicated by arrows).
  • Fig. 7 shows the chemical reaction driven amperometric responses of virus biosensor during successive additions of stock solution of virus (indicated by arrows).
  • Fig. 8 illustrates the construction of a porous PB-nanotube membrane electrode.
  • the PB-nt membrane is fabricated by sputtering a -50 run thick platinum layer on one side of a 60 ⁇ thick nanoporous alumina membrane, followed by electrodeposition of PB onto the porous Pt membrane electrode.
  • Fig. 9 shows (A) typical closed-circuit steady-state current response of a H 2 0 2 - powered sensor toward DENV-2 virus; (B) its calibration plot of normalized closed circuit steady-state current versus virus concentration; line is non-linear best fitted data; error bars are standard deviation of experiment data (points); (C) selective and nonselective responses of the H 2 0 2 -powered virus sensor toward DENV-2and DENV-3, added sequentially to the sensing solution. (A)-(C) use 200 nm pore size membranes as templates; (D) unstable closed-circuit current response of a H 2 0 2 -powered sensor toward DENV-2 virus, using a 20 nm pore size membrane. [0017] Fig. 10 shows a photograph and schematic of a standalone Nafion-filled membrane probe using the nanometer thick metal layer of the membrane at the air- electrolyte interface as the reference and counter electrode.
  • Fig. 11 shows a closed-circuit current response of a dry membrane probe of Fig. 10 immobilized with anti-dengue type II monoclonal antibody toward (A) DENV-2 and (B) DENV-3.
  • H 2 0 2 The spontaneous redox reactions between hydrogen peroxide (H 2 0 2 ) and the present sensor is selected of interest because of the significance of H 2 0 2 in treating waste in groundwater, in cell metabolism, signal transduction, as stress indicators of living cells, as clinical markers for diseases and is the product of several hundreds of oxidases enzymes, commonly used in many enzyme based biosensors.
  • an amperometric sensor for measuring the amount of H 2 0 2 present in a sample.
  • the sensor comprises:
  • a working electrode in contact with a sensing solution, wherein the working electrode comprises a porous membrane structure, wherein the sensing solution comprises the sample containing H 2 0 2 or a precursor of H 2 0 2 , wherein H 2 0 2 is reduced at the working electrode;
  • a counter electrode in contact with a reference solution, wherein the porous membrane structure of the working electrode provides a physical barrier between the sensing solution and the reference solution, wherein a reducer is oxidized at the counter electrode; a connector to electrically connect the working electrode and the counter electrode to allow a current flow therethrough, wherein the current flow is formed by a flow of electrons from the electrode where oxidation has occurred to the electrode where reduction has occurred, with the proviso that the current flow is not induced by an externally applied electrical potential; and
  • a current detector to detect and measure the current flow.
  • a method for measuring the amount of H 2 0 2 present in a sample comprises:
  • the working electrode comprises a porous membrane structure
  • the sensing solution comprises the sample containing H 2 0 2 or a precursor of H 2 0 2 , wherein H 2 0 2 is reduced at the working electrode;
  • an amperometric sensor refers to a galvanic
  • H 2 0 2 for the detection of ions in a solution based on electric current or changes in electric current.
  • changes in the concentrations of the H 2 0 2 and of a reducer (or an oxidizer) at a respective electrode of the sensor enable a flow of electrons in a connector electrically connecting the respective electrodes of the sensor, thereby allowing an electric current or a change thereof to be detected.
  • the detected current or a change thereof is then correlated to the amount of H 2 0 2 present in the sample or the amount of H 2 0 2 produced or consumed in the reaction.
  • the H 2 0 2 is described as being reduced at the working electrode. However, it is to be understood and appreciated that in certain embodiments, H 2 0 2 is oxidzed at the working electrode and an oxidizer is reduced at the counter electrode.
  • the sample contains H 2 0 2 as the analyte to be detected.
  • changes in the concentration of H 2 0 2 are correlated to the amount of an analyte present in the sample.
  • analyte glucose is added to a solution comprising glucose oxidase (Gox)
  • glucose is oxidized by Gox to gluconic acid and H 2 0 2 is produced in the reaction.
  • the amount of H 2 0 2 being produced during the reaction correlates to the amount of glucose being converted into gluconic acid.
  • H 2 0 2 may be consumed during a reaction and likewise, the change in the concentration of H 2 0 2 may be correlated to the analyte of interest.
  • amperometric sensor may also be termed as an amperometric biosensor if the analyte to be detected is a biomolecule.
  • an amperometric sensor or sometimes simply termed as a sensor, is generally described herein and it is to be understood that it may also refer to an amperometric biosensor, when appropriate.
  • analyte and "target molecule” as interchangeably used herein, refer to any substance that can be detected in an assay by binding to a binding or capture 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
  • sample refers to an aliquot of material, frequently but not necessarily always, 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.
  • 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;
  • 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 supernatants; 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.
  • 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.
  • 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.
  • Antibodies such as IgG proteins are typically comprised of four polypeptide chains (i.e., two heavy and two light chains) that are held together by disulfide bonds.
  • 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.
  • contact refer generally to providing access of one component, reagent, analyte or sample to another.
  • 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, which facilitates mixing, interaction, uptake, or other physical or chemical phenomenon advantageous to the contact between components, reagents, analytes and/or samples.
  • 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 generating an electric current signal based on changes in the concentration of H 2 0 2 as described above.
  • 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.
  • hapten refers to a small proteinaceous or non-protein 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.
  • carrier protein such as keyhole limpet hemocyanin
  • Antigens are macromolecules 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.
  • capture molecule refers to any molecule capable of binding to an analyte or target molecule of choice so as to form a complex consisting of the capture molecule and the target molecule. Preferably, this binding is specific so that a specific complex is formed.
  • Specifically binding and “specific binding” as used herein mean that the capture molecule binds to the target molecule based on recognition of a binding region or epitope on the target molecule.
  • the capture molecule preferably recognizes and binds to the target molecule with a higher binding affinity than it binds to other compounds in the sample.
  • “specifically binding” may mean that an antibody or other biological molecule, binds to a target molecule with at least about a 10 6 -fold greater affinity, preferably at least about a 10 7 -fold greater affinity, more preferably at least about a 10 8 -fold greater affinity, and most preferably at least about a 10 9 -fold greater affinity than it binds molecules unrelated to the target molecule.
  • specific binding refers to affinities in the range of about 10 6 -fold to about 10 9 -fold greater than non-specific binding. In some embodiments, specific binding may be characterized by affinities greater than 10 9 -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 capture molecule uniquely recognizes and binds to the target molecule.
  • the capture 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.
  • 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.
  • 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.
  • 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.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen, or an antigenic functional derivative thereof.
  • host animals such as rabbits, mice and goats, may be immunized by injection with an antigen or hapten- carrier conjugate optionally supplemented with adjuvants.
  • 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.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • 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.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • the capture molecule may also be any other proteinaceous scaffold that has been adapted or mutated to bind a given ligand with sufficient binding affinity.
  • useful scaffolds include those scaffolds described in US patent application
  • 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.
  • 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
  • the capture molecule may be a mutein of the member of the lipocalin protein family.
  • the open end of the ⁇ -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.
  • 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 apolipoprotein 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 a2-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).
  • the capture 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.
  • ECD extracellular domain
  • the capture 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) .
  • a nucleic acid based molecule such as an Aptamer or Spiegelmer (Aptamer made of L-ribonucleotides) .
  • the working electrode comprises a porous membrane structure.
  • the working electrode comprises a porous alumina membrane coated with a conducting element provided on a surface thereon and the working electrode is in contact with a sensing solution.
  • the conducting element facilitates the flow of electrons generated from or received at the working electrode.
  • a separate conducting element may not be necessary.
  • the sensing solution comprises the sample containing H 2 0 2 or a precursor of H 2 0 2 . H 2 0 2 is reduced or oxidized at the working electrode, depending the electrochemistry of the sensing solution and the working electrode, which will be elaborated in greater details in later paragraphs below.
  • the conducting element may be, but is not limited to, a platinum or gold layer.
  • the thickness of the conducting element may be less than 100 nm such as ⁇ 90 ran, ⁇ 80 nm, -70 nm, ⁇ 60 nm, ⁇ 50 nm, ⁇ 40 nm, or less.
  • the working electrode comprises an electrocatalyst for the reduction of H 2 0 2 at the working electrode. The electrocatalyst speeds up the rate of electro-reduction of H 2 0 2 at the working electrode, thereby reducing the response time for current generation and detection.
  • the electrocatalyst is comprised in the pores of the porous membrane structure.
  • the electrocatalyst may also be comprised, or further comprised in addition to the pores of the porous membrane structure, at a surface of the working electrode, including a portion of the surface having a conducting element thereon.
  • the electrocatalyst for the reduction of H 2 0 2 at a surface of the working electrode and/or in the pores of the porous membrane structure of the working electrode may be electro-deposited prior to contacting the sensing solution with the working electrode.
  • the electrocatalyst comprises a hexacyanoferrate- derived material or Prussian blue (PB).
  • PB is known to possess high activity for H 2 0 2 electrocatalysis and may thus be used to enhance the electrochemical activity of the porous membrane towards H 2 0 2 .
  • PB may be deposited onto the porous membrane using potential cycling method which forms nanotubes (nt) structures embedded within the channels or pores of the porous membrane.
  • the working electrode thus-formed may be termed a PB-nt membrane electrode.
  • Fig. 8 illustrates the construction of the porous PB- «/ membrane electrode by sputter-coating a 50 nm thick platinum layer on one side of a nanoporous 60 ⁇ thick alumina membrane (step (A)), followed by
  • step (B) electrodeposition of PB onto the porous Pt membrane electrode.
  • step (B) electrodeposition of PB onto the porous Pt membrane electrode.
  • the thus- formed ?B-nt is then employed as the working electrode in this embodiment.
  • PB has been widely used as an important pigment for dyes, paints, inks and others because of its intense and durable blue colour.
  • Prussian blue is a face-centered cubic crystal lattice with a unit cell constant of about 10.2 A and is a three dimensional polymer which consisted of alternating high-spin Fe + and low-spin Fe" ions in which Fe 11 ions are surrounded octahedrically by the carbon atoms of cyano ligands while Fe 3+ ions are linked to the nitrogen atoms of cyano ligands.
  • analogues of Prussian blue may also be suitable for use as the material for the working electrode.
  • suitable analogues of Prussian blue include, but are not limited to, Prussian blue analogues incorporating Fe and Cu or Fe and Ni.
  • FIG. 1 shows various embodiments of a schematic of (a) the H 2 0 2 -powered sensor using a 2-compartment cell separated by a Prussian blue nanotubes membrane; and (b) the H 2 0 2 -powered virus sensor using a 2-compartment cell separated by a Prussian blue nanotubes membrane, showing the binding of virus to antibody molecules within the nanochannels of the porous membrane.
  • the porous membrane separates the sensing solution from the reference solution which contains the reference and/or counter electrodes.
  • the working electrode is in contact with the sensing solution and facilitates transfer of electrons.
  • a counter electrode is in contact with the reference solution, wherein the porous membrane structure of the working electrode provides a physical barrier between the sensing solution and the reference solution.
  • the sensing solution is H 2 0 2 , KC1, a buffer, or a mixture thereof.
  • the KC1 solution may have a concentration of about 1 mM to 1 M.
  • the buffer may be, but is not limited to Tris or phosphate buffer.
  • the sensing solution may be a mixture of H 2 0 2 and Tris.
  • the counter electrode works to balance the electrons added or removed by the working electrode.
  • a third electrode may be used to act as a reference electrode which acts as a reference in measuring and controlling the working electrode's potential.
  • glucose is added to the sensing solution comprising glucose oxidase and is oxidized by oxygen to gluconic acid, and produces H 2 0 2 during the reaction at the working electrode.
  • H 2 0 2 undergoes reduction (O " in peroxide to O " in water) to form water while at the counter electrode, water undergoes oxidation (OH " or H 2 0 to 0 2 ) to form oxygen gas and electrons are released.
  • Electrons released from the oxidation reaction at the counter electrode flows via an electrically connecting connector to working electrodes where the electrons are received to form the reduction reaction at the working electrode, thereby generating an electrical current flow between the working and counter electrodes.
  • the sensor design comprises the nanoporous PB-nt membrane to separate the two solutions in a 2-compartment sensor cell.
  • H 2 0 2 oxidizes PB-nt in the sensing solution, followed by Galvanic current flow between the porous PB-nt membrane electrode in the sensing solution and counter electrode in the reference solution, resulting in the sensing signal detected due to changes in the H 2 0 2 concentration.
  • a current detector is provided to detect and measure the current flow. The present sensor design thus allows a current flow to be not induced by an externally applied electrical potential.
  • PB crystal The channels of PB crystal were found to be crucial for diffusion of small hydrated molecules such as oxygen and hydrogen peroxide through the crystal so that PB could act as a three dimensional electrocatalyst.
  • the main advantage of PB -modified electrodes when compared to conventional platinum electrodes is the availability of low operating potential which could be as low as 0.0V vs Ag/AgCl when used for H 2 0 2 detection and for biosensor applications.
  • the self-powered amperometric sensor since nanoporous membrane is used as template for the fabrication of PB-nt membrane electrode, the self-powered amperometric sensor exhibits a better electrochemical and mechanical stability because PB-nt is retained from leaking and at the same time, the porous layers and channels can load a large amount of PB-nt compared to conventional solid electrodes.
  • PB acts as a selective electron transfer mediator which catalyzes the electro-reduction or electro-oxidation of H 2 0 2 because the channels of PB crystal lattice only selectively allowed low molecular weight molecules to penetrate through while blocking the molecules with higher molecular weight.
  • PB can be reduced to the Everitt's salt (ES) at E 0 ' ⁇ +0.24V (vs. Ag/AgCl, 1M KC1) or oxidize to Berlin green (BG) at more positive potential of E° ⁇ +0.90V (vs Ag/AgCl, 1 M KC1).
  • ES Everitt's salt
  • BG Berlin green
  • E° ⁇ +0.90V vs Ag/AgCl, 1 M KC1
  • the working electrode comprises a capture molecule for capturing a target molecule, wherein the binding reaction between the capture molecule and the target molecule produces or consumes H 2 0 2 .
  • H 2 0 2 is added to the sensing solution prior to the addition of the target molecule.
  • the response to the H 2 0 2 in the sensing solution is measured before and after the capture of the target molecule and changes in the concentration of H 2 0 2 during the binding reaction is thus determined.
  • the capture molecule may be comprised in the pores of the porous membrane structure, such as immobilized onto the pore or channel surface.
  • Immobilization of the capture molecule onto the pore or channel surface may include, but is not limited to, interaction with coupling molecules bound to the surface of the pores of channels.
  • immobilization of the capture molecules can be carried out on the surface by any suitable physical or chemical interaction. These interactions include, for example, hydrophobic interactions, van der Waals interactions, or ionic (electrostatic) interactions as well as covalent bonds. This further means that a capture molecule can directly be immobilized on the surface of the pores or channels by hydrophobic interaction, van der Waals interactions or electrostatic interaction or through covalent coupling.
  • the capture 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 through chemical interaction, for example formation of a covalent bond.
  • the working electrode comprises a target molecule bound to the capture molecule.
  • the capture molecule may be immobilized on a surface of the working electrode.
  • the capture molecule, the target molecule and the binding relationship between the capture molecule and the target molecule have been described in earlier paragraphs above.
  • the capture molecule may be, but is not limited to, an antibody and fragment or variant thereof, antibody-like molecule, binding protein, protein receptor, extracellular domain (ECD) thereof, or a mixture thereof.
  • the target molecule may be, but is not limited to, a protein, peptide, lipid, nucleic acid, small organic molecule, organic polymer, carbohydrate, hapten, or a mixture thereof.
  • this self-powered amperometric sensor design is extended to a virus biosensor in which labeling of the target is not required for detection (i.e. enzyme- labelling is not required).
  • Dengue virus is chosen as the target in this example because dengue is one of the infectious diseases that is widely spread over global regions with yearly occurrence of epidemics and could be deadly in some cases.
  • Nanoporous alumina membranes (AnodiscTM, 13mm diameter, 0.02 ⁇ pore size) from Whatman (Maidstone, Kent, UK). 37% HC1 from Analar Normapur, 35% ⁇ 2 0 2 and KCl from Scharlau, potassium hexacyanoferrate (III) and D(+)-Glucose anhydrous from Merck, anhydrous ferric chloride from GCE Laboratories, 1M Tris buffer pH 7.0 from 1st Base. Enzyme glucose oxidase (GOx) from Aspergillus niger (EC 1.1.3.4, -200 units/mg), was purchased as lyophilized powder from Sigma and stored at -20°C. Stock solutions of glucose in 1M Tris buffer were left overnight and then stored at 4°C before used. All solutions were prepared in Ultrapure water (Sartorius Ultrapure Water System).
  • JEOL JFC-1600 Auto Fine Coater was used to prepare platinum-coated porous membrane electrode. Electrochemical experiments were studied with a CHI750D electrochemical workstation, an e-corder 401 (eDAQ) and a potentiostat (eDAQ EA161) and performed in a 2-compartment cell with 3 -electrode system with porous PB-nanotubes (nt) membrane electrode as the working electrode, a Ag/AgCl (1M KCl) reference electrode and a platinum mesh as the counter electrode were used.
  • eDAQ e-corder 401
  • eDAQ EA161 potentiostat
  • the Prussian blue nanotubes sensor was fabricated as briefly described as follows. Conductive platinum layer sputtered on one side of the membrane was subsequently electrodeposited with a Prussian blue layer to give the porous PB-nt membrane. The electrodeposition of PB-nt was achieved by a potential sweep from -0.5 to +0.6V at 50mV s "1 for 30 cycles in a solution containing 5.0mM K 3 Fe HI (CN) 6 , 5.0mM FeCl 3 , 0.1M KC1 and 0.01M HC1. The porous PB-nt membrane was
  • H 2 0 2 solution was used to prepare the stock hydrogen peroxide solutions.
  • a sensing solution refers to the solution that was in direct contact with the porous PB-nt membrane while a reference solution refers to the solution where both the reference and the counter electrodes were placed in.
  • Stock solution of glucose in 1M Tris was added successively into a 0.1M KC1 in 1M Tris solution containing 4.0mg/mL glucose Oxidase for glucose sensing (Fig. 1(a)). For virus determination. 20 ⁇ . of 0.2mg/mL
  • Immunoglobulin G (IgG) dengue II was adsorbed on the electrode surface for an hour.
  • Stock solution of heat-inactivated dengue virus type II was added successively to the sensing solution containing 1M Tris and H 2 0 2 .
  • H 2 0 2 solution is added prior to the virus detection. No external electrical potential was applied during the amperometric detection of H 2 0 2 , glucose and the dengue virus type II was not labelled.
  • Fig. 2(a) and 2(b) show that hydrogen peroxide reduction at the PB-nt commences at -+0.05V (vs. Ag/AgCl, 1M KC1), with an overpotential of ⁇ 250mV compared to a control voltammetric experiment carried out using porous platinum (Pt) membrane electrode.
  • Pt membrane gives lower reduction currents towards H 2 0 2 compared to the PB-nt membrane (Fig. 2(b)).
  • Ag/AgCl at the VB-nt membrane electrode indicates significantly reduced Pt activity when coated with PB (Fig. 2(a) and 2(b)).
  • a reduction current towards oxygen occurs at ⁇ 0V vs. Ag/AgCl is observed for the Pt membrane electrode (Fig. 2(b)).
  • the oxygen reduction current is negligible when compared to H 2 0 2 reduction (Fig. 2(a)).
  • the VB-nt do not suffer significant interferences from the underlying Pt layer nor reduction of solution oxygen under ambient condition.
  • PB can be reduced to the Everitt's salt (ES) as follows:
  • PB ES E°- 3 ⁇ 4 +0.24V vs. Ag/AgCl, lM KCl
  • BG Berlin green
  • H 2 0 2 is known to undergo electrocatalytic reduction or oxidation at a PB modified electrode when the polymer exists in the ES or BG form, respectively at potentials less than 0.3V or larger than 0.85V (vs. Ag/AgCl).
  • the overall sensing mechanism comprises the following H 2 0 2 reduction:
  • PB is subsequently converted back to ES via the Galvanic cell reaction which comprises a cathodic reaction at VB-nt:
  • the sensor reaction comprises the H 2 0 2 reduction by PB-nt membrane in the sensing solution, coupled to the Galvanic cell reaction between PB-nt and Pt counter electrode.
  • K selective and dual -ion PB-nt sensors show that the nanotubes potential is influenced by the ratio of PB to ES according to the Nernst relation. That is, the relation between the electrochemical potential of the PB-nt membrane and H 2 0 2 concentration under steady-state condition can be related.
  • Fig. 3(a) shows the driving force between the PB-nt membrane and auxiliary electrode under closed-circuit condition, measured from the potential difference between the nanotubes and auxiliary electrode during closed-circuit condition and when aliquots of H 2 0 2 are added to the sensing solution.
  • the incremental change in the driving force is evident of a second reaction that can regenerate the reduced form of PB even in excess amount of H 2 0 2 .
  • a control using the same sensor under open- circuit condition when no current flows between the working and auxiliary electrodes shows a typical sigmoidal titration curve expected for exhaustive depletion of ES in increasing H 2 0 2 concentration (Fig. 3(b)).
  • the driving force of the sensor reaches steady-state values in ⁇ 30 to 60s, upon addition of each aliquot of H 2 0 2 as noted from the time needed to achieve plateau values of the potential-time curve (Fig. 3(a), inset). Best fitted linear line for the change in driving force versus the logarithm of H 2 0 2 concentration gives a slope of 29.7mV (Fig. 3(c)). This confirms the steady-state condition can be achieved when the sensor is challenged with concentrations of H 2 0 2 ranging from ⁇ to 5mM (Fig. 3(c)).
  • Fig. 4 shows the power density of the cell potential in 1M TRIS buffer solution containing 30mM H 2 0 2 .
  • FIG. 5 A typical sensor response (Fig. 5) shows the increase in steady-state current towards increasing concentration of H 2 0 2 . In the absence of K + , the sensor seems to give least sensitive response towards H 2 0 2 due to the low activity of PB. At increasing K + concentration, the sensor sensitivity (response slope) improves significantly (Fig. 5). This sensitivity attenuation by K + ion can be explained by the K + dependent conversion reaction between PB and ES (Eq. 1) which influences its electrochemical reduction and oxidation rate (Eq. 1 1). [0097] In general, the loading amount of PB nanotubes is determined to be ⁇ lxl0 "7 mol cm "2 , which is 10 to 100 times higher than thin films of PB.
  • the sensor gives similar increasing analytical signal responses when K + is added in either the reference or sensing solution (Fig. 5). Placement of high K + concentration in the reference solution is the more favourable cell arrangement to avoid the additional procedure of adding K + ion to the sample solution and to reduce interference from unknown amount of K + ion when present in some samples.
  • This demonstration of a high sensitivity H 2 0 2 sensor without the use of an applied electrical potential opens up the development of analytical sensors with low energy needs by a simple change from a single compartment cell to a two-compartment cell comprising a porous electrocatalytic electrode as a sensor and a counter anodic (or cathodic) element of a Galvanic cell.
  • this self-powered amperometeric sensor in a one- compartment cell by selecting the anodic and cathodic reactions with suitable thermodynamic potentials with consideration of reactions at the sensing and auxiliary electrodes modified with appropriate mediators or catalysts.
  • a two-compartment cell design is selected at this stage because of the side reaction of H 2 0 2 and Pt auxiliary electrode, leading to a lower responsive signal.
  • the amperometric signal towards H 2 0 2 gives rapid response time of 30s, with excellent linearity at low H 2 0 2 concentration which has a low detection limit of 0.1 ⁇ and sensitivity of 48mA M " 1 cm “2 (Fig. 6(a)). Its detection limit is comparable to reported potential controlled PB sensors. Reasonably reproducible responses with standard deviation of -20% can be obtained from three different sensors by normalizing the sensors' signals against the amount of charges passed during initial charging process of PB-ni upon closing the circuit in the absence of H 2 0 2 . For individual sensors, the typical standard errors are in the range of 1 to 3% and can be used up to four sets of 15 measurements each.
  • a chemical energy driven sensor for the detection of hydrogen peroxide and glucose based on a porous PB-nt membrane electrode is described herein.
  • the unique Galvanic cell sensor configuration does not utilize any external electrical potential, thus requiring minimal electrical energy input.
  • Potential- controlled voltammetric experiments and measurements of the sensor electrochemical potentials indicate that the FB-nt are of similar characteristics as the usual PB films and that the sensing reaction proceeds via reduction of hydrogen peroxide.
  • Excellent curve fits of the potential and current data to theory indicates the spontaneous reduction of hydrogen peroxide at the FB-nt, coupled to the Galvanic cell regeneration of the reduced form of PB, proceeds under steady-state condition.
  • Outstanding analytical performance of the chemical energy driven sensor towards hydrogen peroxide is comparable to potential-controlled PB sensors.
  • MAC-ELISA immunoglobulin M-antibody capture ELISA
  • MAC-ELISA the most common and widely used serological test.
  • the detection of the immune-complexes formation is carried out by enzyme conjugated-monoclonal or polyclonal antibodies that will transform the colorless substrate into a colored product, measured with a spectrophotometer.
  • Fig. 7 shows the preliminary result of using the present self-powered amperometric biosensor in virus detection. It is observed that the current has changed after stock solutions of virus are added into the sensing solution containing H 2 0 2 .
  • Prussian blue nanotubes membrane was prepared as follows: First, a -50 nm thick platinum layer was sputtered on one side of the membrane (either 20 or 200 nm pore size). Then the conductive membrane was subjected to potential cycling from - 0.5 V to +0.6 V at 50 mV s "1 for 30 cycles in a solution containing 5.0 mM
  • PB-wi membranes were immobilized with antibody before use as virus sensor as follows: Mouse anti-dengue type II or III virus monoclonal antibody stock was diluted to 0.2 ⁇ g mL "1 in 1 M Tris buffer, pH 7 and was stored in 4 °C before used. 50 ⁇ ,, of the 0.2 ⁇ g mL "1 antibody was applied onto the V -nt side of the membrane and incubated at 4 °C for 45 min and subsequently at room temperature for further 15 min before use.
  • the electrolyte used was Nafion ® perfluorinated resin, incorporated within the 60 ⁇ thick membrane structure and the counter electrode consists of the -50 nm thick platinum coating on the other side of the ⁇ -nt membrane side exposed to surrounding air, in a two-electrode cell arrangement.
  • 80 ⁇ . of 0.5 mM H 2 0 2 was first added onto the PB- nt coated side of the dry membrane probe under closed-circuit condition and allowed to generate steady-state current values after ⁇ 7 min. After that, aliquots (2 iL) of 1000 pfu mL "1 virus stock solution were successively added into the H 2 0 2 solution.
  • Fig. 9 shows (A) typical closed-circuit steady-state current response of a H 2 02-powered sensor toward DENV-2 virus; (B) its calibration plot of normalized closed circuit steady-state current versus virus concentration; line is non-linear best fitted data; error bars are standard deviation of experiment data (points); (C) selective and non-selective responses of the H 2 0 2 -powered virus sensor toward DENV-2and DENV-3, added sequentially to the sensing solution. (A)-(C) use 200 nm pore size membranes as templates; (D) unstable closed-circuit current response of a H 2 0 2 - powered sensor toward DENV-2 virus, using a 20 nm pore size membrane.
  • Fig. 10 shows a photograph and schematic of a standalone Nafion- filled membrane probe using the nanometer thick metal layer of the membrane at the air- electrolyte interface as the reference and counter electrode.
  • a standalone membrane probe, filled with Nafion® perfluorinated resin that allows it to be kept under dry ambient condition and activated in the presence of small amount of solution or solution droplets is provided.
  • the Nafion® perfluorinated resin-incorporated dry membrane probe is designed with the air-platinum as the reference/counter and the P -nt membrane as the working electrode. Nafion® provides the ionic conduction between the two electrodes.
  • Fig. 11 shows a closed-circuit current response of a dry membrane probe of Fig. 10 immobilized with anti-dengue type II monoclonal antibody toward (A) DENV-2 and (B) DENV-3.

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Abstract

Cette invention concerne un capteur ampérométrique pour mesurer la quantité de peroxyde d'hydrogène (Η2O2) présente dans un échantillon, ledit capteur ampérométrique comprenant : une électrode de travail en contact avec une solution de détection, l'électrode de travail comprenant une structure de type membrane poreuse et la solution de détection comprenant l'échantillon contenant le Η2O2 ou un précurseur de Η2O2, le Η2O2 étant réduit à l'électrode de travail ; une contre-électrode en contact avec une solution de référence, la structure de type membrane poreuse de l'électrode de travail formant une barrière physique entre la solution de détection et la solution de référence, et un réducteur étant oxydé à la contre-électrode ; un connecteur pour relier électriquement l'électrode de travail et la contre-électrode et permettre la circulation d'un courant entre les deux, le courant électrique étant formé par un flux d'électrons depuis l'électrode où l'oxydation s'est produite vers l'électrode où la réduction s'est produite, à condition que le courant électrique ne soit pas induit par un potentiel électrique appliqué de l'extérieur ; et un détecteur de courant pour détecter et mesurer le courant électrique. Un procédé d'utilisation du capteur ampérométrique selon l'invention pour mesurer la quantité de Η2O2 présente dans un échantillon est également décrit.
PCT/SG2012/000334 2011-09-13 2012-09-13 Capteur ampérométrique Ceased WO2013039455A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112638A1 (fr) * 2014-01-21 2015-07-30 The Regents Of The University Of California Biocapteurs salivaires et cellules de biocarburant
EP4134666A1 (fr) 2021-08-09 2023-02-15 Universitat Rovira I Virgili Capteur électrochimique
GB2622185A (en) * 2022-05-25 2024-03-13 John William Dilleen Electrochemical energy diagnostics device for sample analysis
EP4414700A1 (fr) 2023-02-07 2024-08-14 Universitat Rovira I Virgili (URV) Procédé de détermination de peroxyde d'hydrogène avec un capteur électrochimique multiplex

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US20040245101A1 (en) * 2001-08-29 2004-12-09 Itamar Willner Self-powered biosensor
US20060096859A1 (en) * 1999-06-29 2006-05-11 Drew Scientific Limited Amperometric sensor
US20100006431A1 (en) * 2006-02-06 2010-01-14 Gordon George Wallace Self-Powered Sensing Devices
US20100213057A1 (en) * 2009-02-26 2010-08-26 Benjamin Feldman Self-Powered Analyte Sensor

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Publication number Priority date Publication date Assignee Title
US20060096859A1 (en) * 1999-06-29 2006-05-11 Drew Scientific Limited Amperometric sensor
US20040245101A1 (en) * 2001-08-29 2004-12-09 Itamar Willner Self-powered biosensor
US20100006431A1 (en) * 2006-02-06 2010-01-14 Gordon George Wallace Self-Powered Sensing Devices
US20100213057A1 (en) * 2009-02-26 2010-08-26 Benjamin Feldman Self-Powered Analyte Sensor

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015112638A1 (fr) * 2014-01-21 2015-07-30 The Regents Of The University Of California Biocapteurs salivaires et cellules de biocarburant
CN106104264A (zh) * 2014-01-21 2016-11-09 加利福尼亚大学董事会 唾液生物传感器以及生物燃料电池
JP2017512604A (ja) * 2014-01-21 2017-05-25 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 唾液バイオセンサ及びバイオ燃料セル
US10595759B2 (en) 2014-01-21 2020-03-24 The Regents Of The University Of California Salivary biosensors and biofuel cells
CN106104264B (zh) * 2014-01-21 2021-01-26 加利福尼亚大学董事会 唾液生物传感器以及生物燃料电池
US11980462B2 (en) 2014-01-21 2024-05-14 The Regents Of The University Of California Salivary biosensors and biofuel cells
EP4134666A1 (fr) 2021-08-09 2023-02-15 Universitat Rovira I Virgili Capteur électrochimique
WO2023016954A1 (fr) 2021-08-09 2023-02-16 Universidad Rovira I Virgili (Urv) Capteur électrochimique dans la configuration de celui-ci
GB2622185A (en) * 2022-05-25 2024-03-13 John William Dilleen Electrochemical energy diagnostics device for sample analysis
EP4414700A1 (fr) 2023-02-07 2024-08-14 Universitat Rovira I Virgili (URV) Procédé de détermination de peroxyde d'hydrogène avec un capteur électrochimique multiplex
WO2024165646A1 (fr) 2023-02-07 2024-08-15 Universitat Rovira I Virgili (Urv) Procédé de détermination de peroxyde d'hydrogène avec un capteur électrochimique multiplex

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