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WO2016018148A1 - Biocapteur comprenant une surface métallique modifiée et procédé pour la modification d'une surface métallique - Google Patents

Biocapteur comprenant une surface métallique modifiée et procédé pour la modification d'une surface métallique Download PDF

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Publication number
WO2016018148A1
WO2016018148A1 PCT/NL2015/050550 NL2015050550W WO2016018148A1 WO 2016018148 A1 WO2016018148 A1 WO 2016018148A1 NL 2015050550 W NL2015050550 W NL 2015050550W WO 2016018148 A1 WO2016018148 A1 WO 2016018148A1
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WO
WIPO (PCT)
Prior art keywords
metal surface
alkyloxy
enzyme
alkenyloxy
oxidase
Prior art date
Application number
PCT/NL2015/050550
Other languages
English (en)
Inventor
Jose Maria ALONSO CARNICERO
Maurice Charles René Franssen
Abraham Antonius Maria BIELEN
Luc Maria Wilhelmus SCHERES
Anke Kristin SCHÜTZ-TRILLING
Wouter Bastiaan Zeper
Johannes Teunis Zuilhof
Petrus Adrianus Maria VAN PAASSEN
Wouter Olthuis
Liza RASSAEI
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Biomarque B.V.
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Publication date
Application filed by Biomarque B.V. filed Critical Biomarque B.V.
Publication of WO2016018148A1 publication Critical patent/WO2016018148A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction

Definitions

  • Biosensor comprising a modified metal surface and method for the modification of a metal surface
  • the present invention is in the field of biosensors.
  • the invention relates to a biosensor comprising a working electrode, said working electrode comprising a modified metal surface wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, and wherein an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety.
  • the invention is therefore also in the field of modified metal surfaces.
  • a biosensor is generally defined as an analytical device that is used for the detection of an analyte, wherein the device comprises a biological component.
  • a biosensor comprises a biological component and a working electrode (also referred to as a transducer).
  • the biological component of the biosensor interacts with the analyte, resulting in a signal that may be detected.
  • the biological component may e.g. be an enzyme, an antibody, a nucleic acid, a microorganism, etc.
  • the signal resulting from the interaction of the analyte with the biological element may be transformed (transduced) into a signal that is more easily measured and/or quantified.
  • a biosensor comprises a signal collector that is connected to the working electrode, and that collects, amplifies and displays the signal.
  • Electrochemical biosensors wherein the biological component is an enzyme are known in the art. This type of biosensor is based on the detection of an electrical signal produced by an electro-active species, wherein the electro-active species is produced or depleted by an enzymatic reaction.
  • the electrical signal may be detected and quantified in several ways. For example in an amperometric biosensor, a voltage is applied to the working electrode, inducing a redox reaction of the electro-active species. In a potentiometric sensor, the electrical signal is a change in electrode potential. Typically, the signal that is generated and measured is proportional to the concentration of the analyte that is detected. For example Ronkainen et al, Chem. Soc. Rev. 2010, 39, 1747 - 1763, incorporated by reference, describes various methods suitable for the electrochemical detection in a biosensor.
  • a device for the continuous monitoring of subcutaneous lactate is for example disclosed in Poscia et al., Biosensors and Bioelectronics 2005, 20, 2244 - 2250, incorporated by reference.
  • This device was developed by modifying the GlucoDay ® portable medical device (A. Menarini Diagnostics), and is based on a biosensor comprising lactate oxidase.
  • the enzyme is immobilized on a nylon net and placed on a Pt electrode.
  • This biosensor was connected to a portable device provided with a micropump and coupled to a microdialysis system. The device is able to record subcutaneous lactate concentration every 3 minutes.
  • US 2011/0155576 discloses a homogeneously- structured catalyst/enzyme composite, which may be applied in e.g. a biosensor.
  • a glucose biosensor is disclosed, the biosensor comprising a working electrode and an electrochemical transducer in which nano-Ptlr catalyst particles are used as the catalyst particles, and glucose oxidases are used as the enzymes.
  • the nano-Ptlr catalyst particles and the glucose oxidases are simultaneously deposited on the working electrode, e.g. by electrophoresis deposition (EPD).
  • EPD electrophoresis deposition
  • the glucose oxidases react with a biomolecule to form hydrogen peroxide
  • the nano-Ptlr particles carry out an electrochemical oxidation-reduction reaction with the hydrogen peroxide.
  • a device may include e.g. an array of hollowed microneedles, in which each needle includes a protruded needle structure including an exterior wall forming a hollow interior and an opening at a terminal end of the protruded needle structure exposing the hollow interior, and a probe inside the exterior wall to interact with one or more chemical or biological substances that come into contact with the probe via the opening to produce a probe sensing signal, and an array of wires that are coupled to the probes of the array of hollowed needles, respectively, each wire being electrically conductive to transmit the probe sensing signal produced by a respective probe.
  • One or more of the probes may include a functionalized coating to interact with an analyte within a fluid, and an electrochemical interaction between the analyte and the coating can be detected e.g. by using amperometry, voltammetry or potentiometry.
  • the device can be integrated into an adhesive patch for placement on skin to detect the analyte residing in transdermal fluid, and biosensing can then be implemented directly at the microneedle-transdermal interface without the uptake and subsequent processing of biological fluids.
  • the device may e.g. be used for the biosensing of glucose or lactate. Glutamate oxidase, lactate oxidase and glucose oxidase may be immobilized on a platinum working electrode by entrapment within a conducting poly(o-phenylenediamine) (PPD) thin film.
  • PPD conducting poly(o-phenylenediamine)
  • the surface is e.g. silicon or silica glass
  • the surface may for example be functionalised with polylysine, aminosilane, epoxysilane or nitrocellulose, followed by reaction with the enzyme.
  • the enzyme may be for example be immobilised by deposition techniques, e.g. electrophoresis deposition (EPD) or Layer-by-Layer deposition of polyelectrolytes.
  • the enzyme may also be immobilized in a conductive linker film present on a biosensor surface.
  • Ronkainen et al Chem. Soc. Rev. 2010, 39, 1747 - 1763, incorporated by reference, discloses an overview of immobilization techniques in the fabrication of nanomaterial-based electrochemical biosensors.
  • SAMs self-assembling monolayers
  • Colloidal gold-modified electrodes can be prepared by covalently tethering the gold nanoparticles with surface-functional groups (-CN, - H 2 or -SH) of SAMs-modified electrode surface, and alkanethiols are the most intensely studied. Short-chain molecules such as 3-mercaptopropionic acid and cystamine can be self- assembled on the modified electrode for further nanoparticle binding.
  • blood and other biological fluids generally comprise components that may rapidly foul the working electrode, resulting in a decreased effectivity.
  • Another important issue is that the enzyme may leach from the biosensor into the fluid to be analysed or denature on the electrode surface.
  • biosensors are able to measure analytes in a continuous fashion.
  • the present invention relates to a device for the detection of an analyte in a fluid, the device comprising:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety;
  • the device according to the invention is also referred to as a biosensor.
  • the invention also relates to a process for the modification of a metal surface, the process comprising the steps of:
  • step (iii) optionally, reacting the alkyloxy- or alkenyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy- or linker- alkenyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy- or an enzyme-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy- or an enzyme-linker-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy or linker-alkenyloxy moiety.
  • the invention further relates to a modified metal surface obtainable by the process according to the invention, to an electrode comprising said modified metal surface and to a biosensor comprising said modified metal surface.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • the compounds disclosed in this description and in the claims may comprise one or more asymmetric centres, and different diastereomers and/or enantiomers may exist of the compounds.
  • the description of any compound in this description and in the claims is meant to include all diastereomers, and mixtures thereof, unless stated otherwise.
  • any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer.
  • the compounds may occur in different tautomeric forms.
  • the compounds according to the invention are meant to include all tautomeric forms, unless stated otherwise.
  • the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer.
  • alkene herein refers to an unsaturated chemical compound comprising one or more carbon-carbon double bonds.
  • An alkene may be linear or branched.
  • An alkene may comprise a terminal carbon-carbon double bond and/or an internal carbon- carbon double bond.
  • a terminal alkene is an alkene wherein a carbon-carbon double bond is located at a terminal position of a carbon chain.
  • An unsubstituted alkene comprising one carbon-carbon double bond has the general formula C n H 2n .
  • An alkene may also comprise two or more carbon-carbon double bonds. When an alkene comprises two or more carbon-carbon double bonds, the alkene may comprise two or more terminal carbon-carbon double bonds.
  • An alkene may also comprise a combination of two or more terminal carbon-carbon double bonds and one or more internal carbon-carbon double bonds.
  • Examples of an unsubstituted terminal alkene comprising two or more carbon-carbon double bonds include 3-vinylhex-l-ene, 3- ethyl-penta-l,4-diene, 4,4-diallyldec-l-ene and 3,3-divinyldec-l-ene.
  • an alkene may optionally be substituted with one or more, independently selected, substituents as defined below.
  • an alkene may optionally be interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.
  • alkyne refers to an unsaturated chemical compound comprising one or more carbon-carbon triple bonds.
  • An alkyne may be linear or branched.
  • An alkyne may comprise a terminal carbon-carbon triple bond and/or an internal carbon-carbon triple bond.
  • a terminal alkyne is an alkyne wherein a carbon- carbon triple bond is located at a terminal position of a carbon chain.
  • An unsubstituted alkyne comprising one carbon-carbon triple bond has the general formula C n H 2n-2 .
  • An alkyne may also comprise two or more carbon-carbon triple bonds. When an alkyne comprises two or more carbon-carbon triple bonds, the alkyne may comprise two or more terminal carbon-carbon triple bonds.
  • An alkyne may also comprise a combination of two or more terminal carbon-carbon triple bonds and one or more internal carbon- carbon triple bonds.
  • Examples of an unsubstituted terminal alkyne comprising two or more carbon-carbon triple bonds include 3-ethynylhex-l-yne, 3-ethyl-penta-l,4-diyne, 4,4-di(prop-2-yn-l-yl)dec-l-yne and 3,3-diethynyldec-l-yne.
  • an alkyne may optionally be substituted with one or more, independently selected, substituents as defined below.
  • an alkyne may optionally be interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.
  • Unsubstituted alkyl groups have the general formula C n H 2n +i and may be linear or branched. Unsubstituted alkyl groups may also contain a cyclic moiety, and thus have the concomitant general formula C n H 2n- i . Unless stated otherwise, the alkyl groups are optionally substituted by one or more, independently selected, substituents as defined below, and/or optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S. Examples of alkyl groups include methyl, ethyl, propyl, 2-propyl, t-butyl, 1-hexyl, 1-dodecyl, etc.
  • Unsubstituted alkyloxy groups have the general formula OC n H 2n +i and may be linear or branched. Unsubstituted alkyloxy groups may also contain a cyclic moiety, and thus have the concomitant general formula OC n H 2n- i . Unless stated otherwise, the alkyloxy groups are optionally substituted by one or more, independently selected, substituents as defined below, and/or optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S. Examples of alkyloxy groups include methoxy, ethoxy, propoxy, 2-propyloxy, t-butyloxy, 1- hexyloxy, 1-dodecyloxy, etc.
  • An alkenyl group comprises one or more carbon-carbon double bonds and may be linear or branched.
  • An unsubstituted alkenyl group comprising one carbon-carbon double bond has the general formula C n H 2n- i .
  • a terminal alkenyl is an alkenyl group wherein the carbon-carbon double bond is located at a terminal position of a carbon chain. Unless stated otherwise, the alkenyl group is optionally substituted by one or more, independently selected, substituents further specified in this document, and/or optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S. Examples of alkenyl groups include ethenyl, propenyl, butenyl, octenyl, etc.
  • An alkenyloxy group comprises one or more carbon-carbon double bonds and may be linear or branched.
  • An unsubstituted alkenyloxy group comprising one carbon- carbon double bond has the general formula OC n H 2n -i.
  • a terminal alkenyloxy group is an alkenyloxy group wherein the carbon-carbon double bond is located at a terminal position of a carbon chain. Unless stated otherwise, the alkenyloxy group is optionally substituted by one or more, independently selected, substituents further specified in this document, and/or optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S. Examples of alkenyloxy groups include ethenyloxy, propenyloxy, butenyloxy, octenyloxy, etc.
  • An alkynyl group comprises one or more carbon-carbon triple bonds and may be linear or branched.
  • An unsubstituted alkynyl group comprising one carbon-carbon triple bond has the general formula C n H 2n -3 -
  • a terminal alkynyl group is an alkynyl group wherein the carbon-carbon triple bond is located at a terminal position of a carbon chain.
  • the alkynyl group is optionally substituted by one or more, independently selected, substituents further specified in this document, and/or optionally interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.
  • Examples of alkynyl groups include ethynyl, propynyl, butynyl, octynyl, etc.
  • An aryl group comprises six to twelve carbon atoms and may include monocyclic and bicyclic structures. Unless stated otherwise, the aryl group may optionally be substituted by one or more, independently selected, substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl.
  • Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures. Unless stated otherwise, the arylalkyl groups and alkylaryl groups may optionally be substituted by one or more, independently selected, substituents further specified in this document.
  • An arylalkyl group is for example benzyl.
  • An alkylaryl group is for example 4-t-butylphenyl.
  • Heteroaryl groups comprise at least two carbon atoms (i.e. at least C 2 ) and one or more heteroatoms N, O, P or S.
  • a heteroaryl group may have a monocyclic or a bicyclic structure.
  • heteroaryl group may be substituted by one or more substituents further specified in this document.
  • suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.
  • Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C 3 ) and may include monocyclic and bicyclic structures.
  • the heteroaryl groups may be substituted by one or more substituents further specified in this document.
  • a (hetero)aryl group comprises an aryl group and a heteroaryl group.
  • An alkyl(hetero)aryl group comprises an alkylaryl group and an alkylheteroaryl group.
  • a (hetero)arylalkyl group comprises a arylalkyl group and a heteroarylalkyl group.
  • alkenes, alkynes, alkyl groups, alkyloxy groups, alkenyl groups, alkenyloxy groups, alkynyl groups, (hetero)aryl groups, (hetero)arylalkyl groups and alkyl(hetero)aryl groups may be substituted with one or more substituents independently selected from the group consisting of Ci - C 12 alkyl groups, C 2 - C 12 alkenyl groups, C 2 - C 12 alkynyl groups, C 3 - C 12 cycloalkyl groups, C 5 - C 12 cycloalkenyl groups, C 8 - C 12 cycloalkynyl groups, Ci - C 12 alkyloxy groups, C 2 - C 12 alkenyloxy groups, C 2 - C 12 alkynyloxy groups, C 3 - C 12 cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl groups can
  • the present invention relates to a device for the detection of an analyte in a fluid, the device comprising:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety;
  • the device according to the invention comprises:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy moiety and, optionally, a linker moiety;
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy moiety;
  • the present invention also relates to a device for the detection of an analyte in a fluid, the device comprising: (a) a working electrode comprising a modified metal surface, wherein the modified metal surface is obtainable by a process comprising the steps of:
  • step (ii) reacting the oxidized metal surface of step (i) with an alkene or an alkyne, the alkene or alkyne being optionally substituted and/or being optionally interrupted by one or more heteroatoms, to form an alkyloxy- or alkenyloxy-modified metal surface, wherein the alkyloxy or alkenyloxy moiety is covalently bonded to the metal surface via the alkyloxy or alkenyloxy O-atom;
  • step (iii) optionally, reacting the alkyloxy- or alkenyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy- or linker-alkenyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy- or an enzyme-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy- or an enzyme-linker-alkenyloxy- modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy or linker-alkenyloxy moiety.
  • the device according to invention comprises:
  • a working electrode comprising a modified metal surface, wherein the modified metal surface is obtainable by a process comprising the steps of: (i) providing an oxidized metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • step (ii) reacting the oxidized metal surface of step (i) with an alkene, the alkene being optionally substituted and/or being optionally interrupted by one or more heteroatoms, to form an alkyloxy- modified metal surface, wherein the alkyloxy moiety is covalently bonded to the metal surface via the alkyloxy O-atom;
  • step (iii) optionally, reacting the alkyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker- alkyloxy moiety.
  • means for detecting an electrical signal the means being operationally coupled to at least working electrode (a) and reference electrode (b).
  • step (ii) of said process the oxidized metal surface is reacted with an alkene according to Formula (1) or an alkyne according to Formula (2), more preferably with an alkene according to Formula (1), as described in more detail below.
  • the device according to the invention is herein also referred to as a biosensor.
  • biosensor herein relates to an analytical device that is used for the detection of an analyte, wherein the device comprises a biological component.
  • analyte herein refers to a substance that is of interest in an analytical procedure.
  • the analyte is discussed in more detail below.
  • detection herein not only refers to the qualitative detection of an analyte in a fluid, but also to the quantitative detection of an analyte in a fluid.
  • the device according to the invention is a device for the quantitative detection of an analyte in a fluid. Consequently, in a preferred embodiment the concentration of an analyte in a fluid is determined by the device according to the invention.
  • the device detects an analyte in a fluid continuously, and more preferably the device detects an analyte in a fluid quantitatively and continuously. Therefore, in a further preferred embodiment, the device according to the invention is a device for the continuous measuring of the concentration of an anlyte in a fluid.
  • the term "means for detecting an electrical signal” refers to means for qualitatively detecting an electrical signal as well as to means for quantitatively detecting an electrical signal.
  • said means detect an electrical signal quantitatively, and more preferably said means detect an electrical signal quantitatively and continuously.
  • working electrode is herein used in its normal scientific meaning and refers to the electrode on which the reaction of interest is occurring in the device according to the invention. As is known in the art, the working electrode may be used in conjunction with an auxiliary electrode (also referred to as "counter electrode”), and together with a reference electrode form a three electrode system.
  • auxiliary electrode also referred to as "counter electrode”
  • reference electrode is herein used in its normal scientific meaning and refers to an electrode which has a stable and well-known electrode potential.
  • the biosensor according to the invention is an electrochemical biosensor, comprising an enzyme as the biological component.
  • An electrical signal produced by an electro-active species is detected by the device, and the electro-active species is produced by an enzymatic reaction.
  • the biosensor according to the invention is particularly suitable for the detection of an analyte that is oxidizable by an enzyme under the formation of hydrogen peroxide (H 2 O 2 ).
  • the electro- active species as described above is thus hydrogen peroxide.
  • FIG. 1 a schematic overview of an embodiment of the biosensor according to the invention is shown.
  • a working electrode (1) and a reference electrode (10) are operationally coupled via conducting means (3), to means for detecting an electrical signal (2).
  • Means for detecting an electrical signal (2) are known to the person skilled in the art.
  • Means (3) for operationally coupling working electrode (1) and reference electrode (10) to said means for detecting an electrical signal (2) are also known to the person skilled in the art, and comprise e.g. a conducting wire.
  • Working electrode (1) comprises a modified metal surface (4).
  • An enzyme (5) is covalently attached to modified metal surface (4), via an alkyloxy or alkenyloxy moiety and, optionally, a linker moiety.
  • the layer of alkyloxy or alkenyloxy moieties and optional linker moieties is depicted as (8) in Figure 1.
  • the device according to the invention comprises: (a) a working electrode comprising a modified metal surface, wherein the modified metal surface comprises the following features:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety.
  • the modified metal surface comprises the following features:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy moiety and, optionally, a linker moiety;
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy moiety.
  • the working electrode is conductively connected to the means for detecting an electrical signal (c).
  • the working electrode comprises a modified metal surface.
  • the working electrode comprises a metal, and the surface of said metal, or a part thereof, is modified.
  • (part of) the outer surface of the metal is modified, i.e. (part of) the metal surface that is in contact with the fluid comprising the analyte to be detected.
  • modified metal surface refers to a metal surface that is modified with an enzyme.
  • the enzyme is covalently attached to the metal surface, in other words, the bonds that are present in between the enzyme and the metal surface are covalent bonds.
  • the enzyme is covalently attached to the metal surface via the O-atom of an alkyloxy or an alkenyloxy moiety.
  • the alkyloxy or alkenyloxy moiety may be a branched alkyloxy or alkenyloxy moiety wherein more than one alkyloxy or alkenyloxy O-atom is covalently bonded to the metal surface.
  • the modified metal surface therefore comprises covalent M-O-C bonds, wherein M represents the metal.
  • a linker moiety is present in between the enzyme and the alkyloxy or alkenyloxy moiety. If present, the linker moiety is covalently bonded both to the enzyme and to the alkyloxy or alkenyloxy moiety.
  • the presence of a linker moiety preferably prevents fouling of the modified metal surface by e.g. adsorption of proteins that may be present in the fluid wherein the analyte to be detected is present. Consequently, the linker moiety preferably has anti-fouling properties.
  • the linker moiety is therefore a compound having anti-fouling properties. Compounds having anti-fouling properties are known to the person skilled in the art.
  • linker moieties having anti-fouling properties include polyethylene glycol (PEG) linkers (e.g. Lundberg et al., Applied Materials and Interfaces 2010, 2, 903 - 912, incorporated by reference), polyacrylamide and polyacrylate linkers (e.g.
  • oligosaccharides e.g. Fyrner et al., Langmuir 2011, 27, 15034 - 15047, incorporated by reference
  • linker mimics of phospholipids e.g. Huang et al., Polymer 2006, 47, 3141 - 3149, incorporated by reference
  • peptoids or poly-N-substituted glycines e.g. Lau et al., Langmuir 2Q ⁇ 2, 28, 16099 - 16107, incorporated by reference
  • zwitterionic linkers as sulfobetaines (e.g.
  • carboxybetaines e.g. Braul et al., Analytical Chemistry 2013, 85, 1447 - 1453, incorporated by reference
  • sulfopyridinium betaines e.g. Meng e
  • a linker moiety is present in between the enzyme and the alkyloxy or alkenyloxy moiety.
  • the linker moiety is a compound having anti-fouling properties.
  • the linker moiety is selected from the group consisting of polyethylene glycol (PEG), polyacrylamides, polyacrylates, oligosaccharides, phospholipids, peptoids, sulfobetaines, carboxybetaines, sulfopyridinium betaines, phosphoryl cholines and cysteine derivatives.
  • PEG polyethylene glycol
  • polyacrylamides polyacrylates
  • oligosaccharides phospholipids
  • peptoids sulfobetaines
  • carboxybetaines sulfopyridinium betaines
  • cysteine derivatives examples of a modified metal surface are shown in more detail.
  • Figures 2, 3, 4 and 5 show that the surface (4) of the metal (6) is modified.
  • Alkyloxy groups (Figure 2(a), (3a), (4a) and 5(a)) or alkenyloxy groups ( Figure 2(b), 3(b), 4(b) and 5(b)) are bonded covalently to metal surface (4), via the alkyloxy or alkenyloxy O-atom.
  • an enzyme E (5) is covalently bonded to the alkyloxy or alkenyloxy group.
  • a linker moiety G (7) is present in between the alkyloxy or alkenyloxy moiety and the enzyme E (5).
  • s and t are integers, preferably ranging from 0 to 50, more preferably ranging from 0 to 30, and most preferably ranging from 0 to 20. It is further preferred that s is an integer ranging from 0 to 10 and t is an integer ranging from 1 to 30, more preferably s is 0, 1, 2, 3, 4, 5 or 6 and t is an integer ranging from 1 to 20, even more preferably s is 0, 1, 2, 3 or 4 and t is an integer ranging from 1 to 25, and most preferably s is 0 or 1 and t is an integer ranging from 1 to 20.
  • the enzyme is covalently bonded to the alkyloxy or the alkenyloxy moiety.
  • the enzyme may for example be bonded to the alkyloxy or the alkenyloxy moiety via a functional group on the side chain of an amino acid in the enzyme, or via the C- terminal amino acid carboxyl group or the N-terminal amino acid amine group of the enzyme. Bonding of the enzyme functional group to the alkyloxy or alkenyloxy moiety occurs via a functional group that is present on the alkyloxy or alkenyloxy moiety, preferably on the co-position of the alkyloxy or alkenyloxy moiety.
  • the enzyme may be attached to the alkyloxy or alkenyloxy moiety via reaction of a functional group present on the alkyloxy or alkenyloxy moiety with a functional group present on the enzyme.
  • a functional group present on the alkyloxy or alkenyloxy moiety may react with a functional group that is present on the enzyme in order to covalently attach the enzyme to the alkyloxy or alkenyloxy moiety.
  • Functional groups that may be present on the enzyme include the C-terminal carboxyl group, the N-terminal amine group, and functional groups that may be present in amino acid side chains, which are known to a person skilled in the art.
  • a linker moiety G (7) is present in between the alkyloxy or alkenyloxy moiety and the enzyme E (5).
  • the linker moiety G is covalently bonded to the alkyloxy or the alkenyloxy moiety and to the enzyme E.
  • the linker moiety G is bonded to the enzyme E via a functional group that is present in the enzyme, as discussed above, and a functional group present in the linker moiety.
  • the linker moiety G is bonded to the alkyloxy or alkenyloxy moiety via a functional group present in the linker moiety and a functional group present in the alkyloxy or alkenyloxy moiety.
  • the metal is selected from the group consisting of ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), iridium (Ir), platinum (Pt) and gold (Au).
  • the metal is selected from the group consisting of Ag, Pt and Au, more preferably from the group consisting of Pt and Au. Most preferably, the metal is Pt.
  • the modified metal surface is thus a modified Ru surface, a modified Rh surface, a modified Pd surface, a modified Ag surface, a modified Ir surface, a modified Pt surface or a modified Au surface.
  • the modified metal surface is a modified Ag surface, a modified Pt surface or a modified Au surface, more preferably a modified Pt surface or a modified Au surface. Most preferably the modified metal surface is a modified Pt surface.
  • the analyte in a fluid that is detected by the device according to the invention is preferably an analyte in a biological fluid. As described below in more detail, it is particularly preferred that the analyte that is detected by the device is an analyte in interstitial fluid.
  • the analyte that is detected by the device according to the invention is an analyte that is oxidizable by an enzyme under the formation of hydrogen peroxide.
  • the analyte is selected from the group consisting of malate, glucose, cholesterol, aromatic primary alcohols, L-gulono-l,4-lactone, galactose, L- sorbose, pyridoxine 4, alcohols, (S)-2-hydroxy-acids, lactate, glycolate, choline, secondary-alcohols, 4-hydroxymandelate, long-chain-alcohols, glycerol-3 -phosphate, thiamin, hydroxyphytanate, N-acylhexosamine, vanillyl-alcohol, nucleosides, D- mannitol, alditols, aclacinomycin-N, aldehydes, pyruvate, oxalate glyoxylate, aldehydes with an indole-ring structure, aryl-aldehydes, acyl-CoA, dihydrouracil, tryptophan, pyrroloquinoline-quinon
  • the analyte is selected from the group consisting of malate, glucose, cholesterol, aromatic primary alcohols, galactose, alcohols, (S)-2-hydroxy- acids, lactate, glycolate, secondary-alcohols, long-chain-alcohols, glycerol-3 - phosphate, N-acylhexosamine, nucleoside, D-mannitol, alditols, aldehydes, pyruvate, oxalate, glyoxylate, aryl-aldehydes, acyl-CoA, tryptophan, D-aspartate, L-amino-acids, D-amino-acids, amines, D-glutamate, putrescine, L-glutamate, L-lysine, L-aspartate, glycine, primary-amines, diamine, N-methyl-L-amino-acid, Nl-acetylpoly
  • the analyte is selected from the group consisting of glucose, lactate, cholesterol, histamine, heterocyclic compounds, L-phenylalanine and D-aspartate.
  • the analyte is selected from the group consisting of glucose and lactate.
  • the type of enzyme that is covalently attached to the modified metal surface depends on the analyte that is to be detected by the device according to the invention.
  • the enzyme is selected from the group consisting of hydrogen peroxide forming oxidases, i.e. oxidoreductases that catalyse oxidation- reduction reactions involving molecular oxygen as electron acceptor and that form hydrogen peroxide during the reduction of the molecular oxygen.
  • the enzyme is a hydrogen peroxide forming oxidase selected from the Enzyme Classes (EC) 1.1.3, 1.2.3, 1.3.3, 1.4.3, 1.5.3, 1.6.3, 1.7.3, 1.8.3, 1.10.3, 1.17.3 or 1.21.3.
  • the enzyme is selected from the group consisting of a malate oxidase (EC 1.1.3.3), a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), an aryl-alcohol oxidase (EC 1.1.3.7), an L- gulonolactone oxidase (EC 1.1.3.8), a galactose oxidase (EC 1.1.3.9), a pyranose oxidase (EC 1.1.3.10), an L-sorbose oxidase (EC 1.1.3.11), a pyridoxine 4-oxidase (EC 1.1.3.12), an alcohol oxidase EC 1.1.3.13, an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), a choline oxidase (EC 1.1.3.17), a secondary-alco
  • a spermine oxidase EC 1.5.3.16), a non-specific polyamine oxidase (EC 1.5.3.17), an L-saccharopine oxidase (EC 1.5.3.18), a 4-methylaminobutanoate oxidase (formaldehyde-forming) (EC 1.5.3.19), an N-alkylglycine oxidase (EC 1.5.3.20), a 4- methylaminobutanoate oxidase (methylamine-forming) (EC 1.5.3.21), a nitroalkane oxidase (EC 1.7.3.1), an acetylindoxyl oxidase (EC 1.7.3.2), a factor-independent urate hydroxylase (EC 1.7.3.3), a thiol oxidase (EC 1.8.3.2), a glutathione oxidase (EC 1.8.3.3), a methanethiol oxidase (EC EC 1.5.3.1
  • the enzyme is selected from the group consisting of a malate oxidase (EC 1.1.3.3), a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), an aryl-alcohol oxidase (EC 1.1.3.7), a galactose oxidase (EC 1.1.3.9), a pyranose oxidase (EC 1.1.3.10), an alcohol oxidase (EC 1.1.3.13), an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), a secondary-alcohol oxidase (EC 1.1.3.18), a long-chain-alcohol oxidase (EC 1.1.3.20), a glycerol-3- phosphate oxidase (EC 1.1.3.21), an N-acylhexosamine oxidase (EC EC 1.1
  • the enzyme is selected from the group consisting of a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), a pyranose oxidase (EC 1.1.3.10), an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), an aldehyde oxidase (EC 1.2.3.1) an D-aspartate oxidase (EC 1.4.3.1), an L- amino-acid oxidase (EC 1.4.3.2), an D-amino-acid oxidase (EC 1.4.3.3), an D- glutamate(D-aspartate) oxidase (EC 1.4.3.15), a diamine oxidase (EC 1.4.3.22), and a xanthine oxidase (EC 1.17.3.2).
  • a glucose oxidase EC 1.1.
  • the enzyme is selected from the group consisting of a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), pyranose oxidase (EC 1.1.3.10) and an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).
  • lactate oxidase is often used to refer to (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).
  • the type of enzyme that is covalently attached to the modified metal surface depends on the analyte that is to be detected by the device.
  • the person skilled in the art knows which enzyme(s) is (are) suitable for the detection of a specific analyte.
  • the enzyme is (S)-2-hydroxy-acid oxidase.
  • the enzyme is a glucose oxidase, a hexose oxidase, or a pyranose oxidase.
  • the enzyme is cholesterol oxidase.
  • the enzyme is diamine oxidase.
  • the enzyme is a D-amino-acid oxidase, a D-glutamate (D- aspartate) oxidase or a D-aspartate oxidase.
  • the analyte that is detected by the device according to the invention is preferably an analyte that is oxidizable by an enzyme under the formation of hydrogen peroxide.
  • the enzyme catalyzes the oxidation of the analyte.
  • Oxygen acts as electron acceptor, and is reduced to hydrogen peroxide (H 2 O 2 ).
  • Subsequent oxidation or reduction of hydrogen peroxide at the modified metal surface results in an electrical signal that is detected by the device.
  • the electrical signal that is detected is proportional to the concentration of the analyte in the fluid.
  • Hydrogen peroxide is an electrochemically active species, which can be oxidized or reduced at certain potentials at the electrode. For example, this can be done amperometrically, in which a potential is applied and current is monitored, or voltammetrically, in which the potential is changed and current change is monitored. For example, as the hydrogen peroxide concentration changes at the electrode in which a potential is applied, the corresponding current change is monitored as an electrical signal that can be further processed using signal processing techniques.
  • the process for the detection of lactate is schematically shown in Figure 6.
  • a metal (6) comprising a modified metal surface (4) is shown.
  • An enzyme (5) is covalently attached to the metal surface via an alkyloxy or alkenyloxy moiety and, optionally, a linker moiety.
  • the layer of alkyloxy or alkenyloxy moieties and optional linker moieties is depicted as (8) in Figure 6.
  • the enzyme E (5) that is attached to the working electrode is preferably an (S)-2-hydroxy-acid oxidase.
  • the modified metal surface is in contact with the fluid comprising the analyte to be detected (9).
  • Lactate and oxygen (0 2 ) are converted into pyruvate and hydrogen peroxide (H 2 O 2 ) by the enzyme. Hydrogen peroxide migrates through layer (8) to the surface of the metal, where it is oxidized, resulting in an electrical signal.
  • the enzyme that is attached to the working electrode is preferably a glucose oxidase, a hexose oxidase or a pyranose oxidase.
  • the enzyme is a glucose oxidase or a hexose oxidase
  • ⁇ -D- glucose is converted into D-glucono-l,5-lactone by the enzyme, under formation of hydrogen peroxide.
  • D-Glucono-l,5-lactone spontaneously hydrolyses into gluconic acid.
  • the enzyme is a pyranose oxidase
  • ⁇ -D-glucose is converted into 2- dehydro-D-glucose by the enzyme, under formation of hydrogen peroxide.
  • the working electrode of the device according to the invention comprises a modified metal surface.
  • the electrode may e.g. be composed essentially of the metal, or the electrode may e.g. comprise a film of the metal at a surface of the electrode.
  • the working electrode comprises a film of the metal, wherein an outer surface of the metal, or a part thereof, is modified.
  • outer surface refers to a surface of the metal that, during operation of the device according to the invention, will be in contact with the fluid comprising the analyte to be detected.
  • the working electrode comprises at least one skin-piercing means.
  • the skin-piercing means comprises a needle, preferably one or more microneedles, more preferably an array of microneedles.
  • the microneedles may be hollow or solid microneedles.
  • the microneedles are solid microneedles. Microneedles are known in the art, and described in more detail in e.g. WO 99/64580, WO 2009/097660 and Ji et al, Journal of Physics: Conference Series 2006, 1127 - 1131, all incorporated by reference.
  • the length of a microneedle is typically between about 1 ⁇ and about 1 mm, preferably between about 10 ⁇ and about 500 ⁇ , and more preferably between about 30 ⁇ and about 200 ⁇ .
  • the outer diameter is typically between about 10 nm and about 1 mm, preferably between about 1 ⁇ and about 500 ⁇ , and more preferably between about 10 ⁇ and about 100 ⁇ .
  • the cross-section of a microneedle may be circular or non- circular, e.g. square, oblong, triangle, polygonal, and the shaft may be straight or tapered.
  • the one or more microneedles may for example be composed essentially of the metal, wherein an outer surface of said metal, or a part thereof, is modified.
  • the one or more microneedles may be composed essentially of a certain material, the outer surface of said material, or a part thereof, being coated with a layer of a metal, wherein the outer surface of said metal, or a part thereof, is modified.
  • Materials suitable for the manufacturing of microneedles are known in the art, and include e.g. various metals (e.g. stainless steel, gold, titanium, nickel), silicon, silicon dioxide, ceramics, various polymers (e.g.
  • the working electrode comprises an array of microneedles, wherein the outer surface of the microneedles, or a part thereof, is coated with a metal film, and wherein the outer surface of the metal film, or a part thereof, is modified.
  • the metal film is preferably a Pt film or a Au film, more preferably a Pt film.
  • the device according to the invention is particularly well suited for transdermal analyte sensing, i.e. the qualitative or quantitative detection of an analyte in the interstitial fluid (interstitial fluid may also be referred to as "transdermal fluid").
  • the microneedles penetrate into the skin at a depth less than the subcutaneous layer, and are in contact with the interstitial fluid comprising the analyte to be detected. Detection of the analyte takes place at the microneedle-interstitial fluid interface.
  • the device according to the invention is particularly suitable for the qualitative or quantitative detection of lactate or glucose in interstitial fluid.
  • the array of microneedles is integrated into a patch that may be applied to the skin.
  • Said patch is preferably an adhesive patch.
  • Adhesive patches that may be applied to the skin are known to the person skilled in the art. For example adhesive patches suitable for incorporation of microneedles are disclosed in e.g. US 2013/0216694, incorporated by reference.
  • reference electrode refers to an electrode having a stable and well-known electrode potential. Reference electrodes are known to the person skilled in the art. Examples of a reference electrode include a standard hydrogen electrode (SHE), a normal hydrogen electrode (HE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper/copper(II) sulfate electrode (CSE) , a silver / silver chloride electrode (Ag/AgCl), a palladium-hydrogen electrode and a dynamic hydrogen electrode. In a preferred embodiment, the reference electrode is a silver/silver chloride (Ag/AgCl) electrode.
  • the reference electrode is conductively connected to the means for detecting an electrical signal (c).
  • the device according to the invention further comprises means for detecting an electrical signal, the means being operationally coupled to at least working electrode (a) and reference electrode (b).
  • Working electrode (a) is conductively connected to the means for detecting an electrical signal.
  • Reference electrode (b) is conductively connected to the means for detecting an electrical signal.
  • the means for detecting an electrical signal are arranged to detect, during use, an electrical signal generated between the working electrode and the reference electrode, based on the redox reaction induced on the working electrode by the analyte in a fluid.
  • Means for detecting an electrical signal are known to the person skilled in the art.
  • Ronkainen et al Chem. Soc. Rev. 2010, 39, 1747 - 1763, incorporated by reference, describes various methods suitable for the electrochemical detection in a biosensor.
  • Ronkainen et al. further describe that voltammetric and amperometric techniques are characterized by applying a potential to a working electrode versus a reference electrode and measuring the current.
  • the term voltammetry is used for those techniques in which the potential is scanned over a set potential range.
  • amperometry changes in current are monitored in time while a constant potential is maintained at the working electrode with respect to a reference electrode.
  • the electrical signal may e.g. be determined by a so-called potentiostat, where a potential is applied to the working electrode with respect to the reference electrode.
  • the resulting flow of electrical current is a measure for the concentration of the analyte.
  • the means for detecting an electrical signal are integrated with the metal of which the surface is modified.
  • the device according to the invention has several advantages.
  • the working electrode comprises a modified metal surface, wherein the enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety. Due to said covalent attachment, the enzyme is strongly attached to the metal surface, and leaching of the enzyme into the fluid to be analysed is reduced to a minimum.
  • the alkyloxy or alkenyloxy moiety is a branched alkyloxy or alkenyloxy moiety wherein more than one alkyloxy or alkenyloxy O-atom is covalently bonded to the metal surface, the attachment of the enzyme to the metal surface is particularly strong.
  • the device when the working electrode comprises an array of microneedles and is integrated into a patch that may applied to the skin, the device enables the in situ detection of analytes in interstitial fluid in a minimally invasive manner, and without pain or harm to the user of the device. Detection of the analyte takes places directly at the microneedle-interstitial fluid (also referred to as transdermal fluid) interface, and the uptake and processing of interstitial liquid is not necessary. Process for the modification of a metal surface
  • the device according to the invention comprises a working electrode, the working electrode comprising a modified metal surface.
  • the modified metal surface comprises the following features: (1) the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety (preferably, a linker moiety comprising anti-fouling properties);
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety.
  • the modified metal surface comprises the following features:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy moiety and, optionally, a linker moiety (preferably, a linker moiety comprising anti-fouling properties);
  • linker moiety if present, is covalently bonded to the enzyme and to the alkyloxy moiety.
  • the present invention also relates to a process for the preparation of a modified metal surface, in other words, to a process for the modification of a metal surface.
  • the invention relates to a process for the modification of a metal surface, the process comprising the steps of:
  • the oxidized metal surface is reacted with an alkene in step (ii). Consequently, in this preferred embodiment, the metal surface is modified with alkyloxy moieties, the alkyloxy moieties being covalently bonded to the metal surface.
  • the metal surface to be modified is a metal surface, or a part thereof, of an electrode.
  • the electrode may e.g. be composed essentially of the metal, or the electrode may e.g. comprise a film of the metal at (part of) a surface of the electrode.
  • the metal surface is comprised in an array of microneedles.
  • the outer surface, or a part thereof, of an array of microneedles is coated with a metal film, and the outer surface, or a part thereof, of the metal film is modified by the process according to the invention.
  • the invention therefore also relates to a method for the manufacturing of an electrode, said method comprising the process for the modification of a metal surface according to the invention.
  • an oxidized metal surface is provided.
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au.
  • the metal is selected from the group consisting of Pt, Ag and Au, more preferably Pt and Au.
  • the metal is Pt.
  • oxidized metal surface herein refers to a metal surface comprising a metal oxide (MO x , wherein M is metal) and/or surface-bonded hydroxy groups (OH groups). Said hydroxy groups are covalently bonded to the metal surface, via an M-0 bond (M is metal).
  • MO x refers to a metal oxide, wherein M is Ru, Rh, Pd, Ag, Ir, Pt or Au, and x is 1, 2, 3 or 4.
  • the value of x depends on the type of metal. When the metal is e.g. Pt or Pd, x is 1 or 2. When the metal is e.g. Au or Ag, x is 1 or 3. When the metal is e.g. Ru, x is 2 or 4.
  • the bulk of the metal is essentially oxygen- free, whereas a surface of the metal, or a part thereof, comprises -OH groups, covalently bonded to the metal surface, and/or a metal oxide MO x .
  • the oxidized Pt surface may e.g. comprise -OH groups, platinum(II) oxide (PtO), platinum(IV) oxide (Pt0 2 ), and/or a combination thereof.
  • the oxidized Pt surface comprises a mixture of platinum(II) oxide (PtO) and platinum(IV) oxide (Pt0 2 ).
  • the oxidized Au surface may e.g. comprise -OH groups, gold(I) oxide (Au 2 0), gold(III) oxide (Au 2 0 3 ), and/or a combination thereof.
  • the oxidized Au surface comprises gold(I) oxide (Au 2 0).
  • An oxidized metal surface may e.g. be comprised of a metal film, wherein the metal bulk is essentially oxygen- free, and wherein the surface of the metal film comprises -OH groups and/or a metal oxide MO x .
  • the oxidized metal surface may be provided in several ways. Oxidation (i.e. the formation of MO x on the surface of the metal, the formation of surface-bonded OH- groups and/or the activation of surface-bonded OH-groups) may occur e.g. in the air, or upon some sort of activation reaction. Preferably, the oxidized metal surface is provided via wet etching, dry etching or plasma activation.
  • the metal surface is e.g. contacted with an acid, or with a mixture of an acid and an organic solvent.
  • a wet etching process may e.g. comprise immersing the metal surface in HN0 3 (commercially available solution at 70%) or in a mixture, e.g. a 3 : 1 to 5: 1 mixture (preferably a 4: 1 mixture) of H 2 S0 4 /H 2 0 2 , e.g. for about 30 minutes or more (preferably for 60 minutes or more, and more preferably for 120 minutes or more).
  • Dry etching may for example be performed using 0 2 gas in an inductively coupled plasma etching (ICP) and/or in a reactive ion etching (RIE etching) equipment, as is known to a person skilled in the art.
  • ICP inductively coupled plasma etching
  • RIE etching reactive ion etching
  • a metal surface is exposed to an oxygen plasma.
  • the oxidation of a metal surface by an oxygen plasma is known in the art, see for example Li et al., Surface Science 2003, 529, 410-418, incorporated by reference herein, wherein the oxidation of Pt surfaces by an oxygen plasma is described.
  • An oxidized Pt surface may for example be provided by exposing Pt films (e.g. of 200 nm thickness) to oxygen plasma (e.g. 0.1 mbar, 15 seem, 50 W) for a certain amount of time (e.g. about 30 minutes or more).
  • the oxidized metal surface is provided by exposing a metal surface to an oxygen plasma.
  • step (ii) of the process according to the invention the oxidized metal surface of step (i) is reacted with an alkene or an alkyne, preferably an alkene, the alkene or alkyne optionally being substituted and/or optionally being interrupted by one or more heteroatoms.
  • an alkene or alkyne reacts with MO x or with an OH- group present on the metal surface, to form a covalently bonded alkyloxy moiety or a covalently bonded alkenyloxy moiety.
  • An alkyloxy- or alkenyloxy-modified metal surface is thus formed, wherein the alkyloxy or alkenyloxy moiety is covalently bonded to the metal surface via the alkyloxy or alkenyloxy O-atom.
  • the alkyloxy or alkenyloxy moiety may also be a branched alkyloxy or alkenyloxy moiety wherein more than one alkyloxy or alkenyloxy O-atom is covalently bonded to the metal surface.
  • the attachment of an alkene or alkyne to the oxidized metal surface may occur via a Markovnikov-type addition, or via an anti-Markovnikov-type addition.
  • the modified metal surfaces shown in Figures 2 and 4 may be obtained via Markovnikov- type addition of the alkene ( Figures 2(a) and 4(a)) or alkyne ( Figures 2(b) and 4(b)).
  • the modified metal surfaces shown in Figures 3 and 5 may be obtained via an anti- Markovnikov-type addition of the alkene ( Figures 3(a) and 5(a)) or alkyne ( Figures 3(b) and 5(b)).
  • the attachment of the alkene occurs via a Markovnikov-type addition.
  • the attachment of the alkyne occurs via a Markovnikov-type addition.
  • the reaction of the oxidized metal surface with the alkene or alkyne may be a thermal reaction or a photochemical reaction.
  • the reaction in step (ii) is a thermal reaction
  • the reaction is performed at an elevated temperature.
  • the reaction is performed at a temperature in the range of about 40°C to about 180°C, preferably in the range of about 50°C to about 170°C, more preferably in the range of about 60°C to about 160°C, even more preferably in the range of about 70°C to about 150°C, and most preferably in the range of about 80°C to about 140°C.
  • the temperature range wherein the reaction is performed depends, amongst others, on the nature of the alkene or alkyne.
  • the reaction in step (ii) is a photochemical reaction
  • the reaction is performed under the action of radiation.
  • the reaction is performed under ultraviolet radiation, preferably having a wavelength in the range of about 200 nm to about 300 nm, more preferably having a wave length in the range of about 200 nm to 295 nm, even more preferably in the range of about 220 nm to about 285 nm and most preferably in the range of about 245 nm to about 275 nm.
  • the reaction in step (ii) is a thermal reaction.
  • step (ii) the oxidized metal surface is reacted with an alkene.
  • step (ii) the oxidized metal surface is reacted with an alkene according to Formula (1) or an alkyne according to Formula (2):
  • n is an integer in the range of 1 to 5.
  • A is a linear, branched or cyclic C 2 - C50 alkenyl group, the alkenyl group being a 1-alkenyl group or an internal alkenyl group;
  • B is a linear, branched or cyclic C 2 - C50 alkynyl group, the alkynyl group being a 1-alkynyl group or an internal alkynyl group;
  • Q is hydrogen or a functional group selected from the group consisting of -XR 2 ,
  • p is an integer in the range of 2 to 4.
  • q is an integer in the range of 1 to 500;
  • X is independently O or S;
  • R 1 is a linear, branched or cyclic Ci - C 12 alkyl group; a phenyl group; a C 7 - Ci2 alkaryl group; or a C 7 - C 12 arylalkyl group; wherein the alkyl, phenyl, alkaryl and arylalkyl groups are optionally substituted with one or more of F or CI;
  • R 2 and R 3 are independently selected from the group consisting of hydrogen and R 1 ;
  • R 4 is independently selected from hydrogen or Ci - C 4 alkyl; and R 5 is a monofunctional hydroxy or thiohydroxy protecting group.
  • step (ii) the oxidized metal surface is reacted with an alkene according to Formula (1).
  • R 5 is a monofunctional hydroxy or thiohydroxy protecting group.
  • protecting groups are well known in the art.
  • methods for adding such groups to -XH groups and methods for removing such protecting groups, under conditions that do not affect the molecular structure of the modified metal surface obtained, are known in the art, see for example McOmie, "Protective Groups in Organic Chemistry", Plenum Press, 1973, Greene, “Protective Groups in Organic Synthesis", 3 Edition, John Wiley & Sons, 1999 and F.A Carey and R.J Sundberg, "Advanced Organic Chemistry Part B: Reactions and Synthesis", 3 Ed., Plenum Press 1990, p. 678 - 686, all incorporated by reference.
  • Suitable examples of monofunctional hydroxy and thiohydroxy protecting groups include methoxymethyl, methylthiomethyl, 2-methoxyethoxymethyl, bis(2-chloro- ethoxy)methyl, tetrahydropyranyl, tetrahydrothiopyranyl, 4-methoxytetrahydropyranyl, 4-methoxytetrahydrothiopyranyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1- ethoxyethyl, 1-methoxy ethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, benzyl and optionally substituted triphenylmethyl (trityl).
  • the monofunctional hydroxyl or thiohydroxy protecting group is selected from the group consisting of allyl, benzyl, optionally substituted trityl, and tetrahydropyranyl. It is even more preferred that the monofunctional hydroxyl or thiohydroxy protecting group is selected from benzyl and tetrahydropyranyl.
  • Suitable examples of the -Si(R 1 ) 3 group include trimethylsilyl, triethylsilyl, triisopropylsilyl, isopropyldimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and tribenzylsilyl. Methods of the introduction and removal of such groups are known in the art, see for example Lalonde et al., Synthesis 1985, 9, 817 - 908, incorporated by reference.
  • alkenyl group A is a linear, branched or cyclic C 2 - C 5 o, preferably C 2 - C40, more preferably C 2 - C30, even more preferably C 4 - C 20 , yet even more preferably C 4 - C 12 , most preferably C 6 - Cn alkenyl group, the alkenyl group comprising a 1-alkenyl group and/or an internal alkenyl group.
  • alkenyl group A is a 1- alkenyl group, i.e. in a preferred embodiment the alkene according to Formula (1) is a terminal alkene. In this embodiment it is further preferred that alkene (1) is a linear alkene.
  • alkenyl group A is a branched alkenyl group comprising more than one terminal carbon-carbon double bond, i.e. in another preferred embodiment the alkene according to Formula (1) is a branched alkene comprising two or more terminal carbon-carbon double bond.
  • alkynyl group B is a linear, branched or cyclic C 2 - C50, preferably C 2 - C 40 , more preferably C 2 - C30, even more preferably C 4 - C 20 , yet even more preferably C 4 - C 12 , most preferably C 6 - Cn alkynyl group, the alkynyl group comprising a 1-alkynyl group and/oror an internal alkynyl group.
  • alkynyl group B is a 1- alkynyl group, i.e. in a preferred embodiment the alkyne according to Formula (2) is a terminal alkyne. In this embodiment it is further preferred that alkyne (2) is a linear alkyne.
  • alkynyl group B is a branched alkynyl group comprising more than one terminal carbon-carbon triple bond, i.e. in another preferred embodiment the alkyne according to Formula (2) is a branched alkyne comprising two or more terminal carbon-carbon triple bonds.
  • Alkene (1) and alkyne (2) may comprise more than one functional group Q (n is 1, 2, 3, 4 or 5). Preferably, n is 1, 2 or 3, more preferably, n is 1 or 2 and most preferably, n is 1. It is preferred that Q is in the co-position of alkene (1) or alkyne (2). It is further preferred that alkene (1) is a 1 -alkene, n is 1 or 2 (preferably 1) and Q is present at the co-position, i.e. at the terminal sp 3 C-atom of the alkenyl group A in (1). It is also further preferred that alkyne (2) is a 1 -alkyne, n is 1 or 2 (preferably 1) and Q is present at the co-position, i.e. at the terminal sp 3 C-atom of the alkynyl group B in (2).
  • Q is hydrogen. In another embodiment, Q is a functional group, said functional group optionally being masked or protected. In a preferred embodiment, Q is a functional group, said functional group optionally being masked or protected. In a further preferred embodiment, Q is selected from the group consisting of -XR 2 , - R 2 R 3 , -C(X)XR 2 , -C(X)R 2 , -C(X) R 2 R 3 , -CI, -Br, -I, -XC(X)R 1 , - R 2 C(X)R 1 and -XR 5 , wherein X, R 1 , R 2 , R 3 and R 5 are as defined above.
  • X is O.
  • Q is selected from the group consisting of -OR 2 , - R 2 R 3 , -C(0)OR 2 , -C(0)R 2 , -C(0) R 2 R 3 , -CI, -Br, -I, -OC(0)R 1 , - R 2 C(0)R 1 and -OR 5 .
  • R 1 is a linear, branched or cyclic Ci - C12 alkyl group, a phenyl group, a C 7 - C12 alkaryl group or a C 7 - C12 arylalkyl group, wherein the alkyl, phenyl, alkaryl and arylalkyl groups are optionally substituted with one or more of F atoms or CI atoms.
  • R 1 comprises one or more F atoms. More preferably, R 1 comprises a -CF 3 group.
  • an alkene (1) include 2,2,2-trifluoroethyl undec-10-enoate (TFEE), succinimidyl undec-10-enoate), 5-hexen-l-ol and 10- undecen-l-ol.
  • an alkyne (2) include trifluoroethyl undec-10- ynoate, succinimidyl undec-10-ynoate, 5-hexyn-l-ol and 10-undecyn-l-ol.
  • the invention relates to a process for the modification of a metal surface, the process comprising the steps of:
  • step (iii) optionally, reacting the alkyloxy- or alkenyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy- or linker- alkenyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy- or an enzyme-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy- or an enzyme-linker-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy or linker-alkenyloxy moiety.
  • the process comprises the steps of:
  • step (iii) optionally, reacting the alkyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy moiety.
  • This embodiment of the process for the modification of a metal surface thus comprises steps (i), (ii) and (iv-a), or steps (i), (ii), (iii) and (iv-b).
  • the metal surface is modified, i.e. functionalized, with enzyme-alkyloxy moieties or enzyme-alkenyloxy moieties.
  • the metal surface is modified, i.e. functionalized, with enzyme-linker-alkyloxy moieties or enzyme-linker-alkenyloxy moieties.
  • Steps (i) and (ii), and preferred embodiments thereof, are described in more detail above.
  • the oxidized metal surface is reacted in step (ii) with an alkene according to Formula (1) or an alkyne according to Formula (2), preferably with an alkene according to Formula (1).
  • Alkene (1) and alkyne (2), and preferred embodiments thereof, are described in more detail above.
  • step (iii) of the process the alkyloxy- or alkenyloxy-modified metal surface of step (ii) is reacted with a linker moiety.
  • the linker moiety has anti-fouling properties. Therefore, in a preferred embodiment, the linker moiety is a compound having anti-fouling properties. Compounds having anti-fouling properties are known in the art, and are described in more detail above.
  • the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety of the alkyloxy- or alkenyloxy-modified metal surface of step (i). Bonding occurs via reaction of a functional group present on the alkyloxy or alkenyloxy moiety with a functional group present on the linker moiety.
  • the linker moiety preferably comprises two or more functional groups F, wherein one or more functional group F is able to react with a functional group on the alkyloxy or alkenyloxy moiety, and one or more functional group F is able to react with a functional group on the enzyme. More preferably, the linker moiety comprises two functional groups, F 1 and F 2 , wherein F 1 is a functional group that is able to react with a functional group on the alkyloxy or alkenyloxy moiety and F 2 is a functional group that is able to react with a functional group on the enzyme in step (iv-a) or step (iv-b) of the process (see below).
  • the linker moiety therefore preferably is according to Formula (3):
  • G is the linker backbone
  • F 1 is a functional group able to react with a functional group on the alkyloxy or alkenyloxy moiety of the modified surface obtained by step (ii) of the process; and F 2 is a functional group able to react with a functional group on the enzyme in step (iv- a) or (iv-b) of the process.
  • F 1 is a functional group that is complementary to, i.e. able to react with, a functional group on the alkyloxy or alkenyloxy moiety of the modified surface obtained by step (ii) of the process
  • F 2 is a functional group that is complementary to a functional group on the enzyme in step (iv-a) or (iv-b).
  • complementary functional groups refers to functional groups that are able to react with one another.
  • Complementary functional groups are known to the person skilled in the art.
  • Methods for introducing a functional group into a molecule, e.g. into a linker moiety, are also known to the person skilled in the art.
  • Linker backbone G may for example be selected from the group consisting of linear or branched Ci-C 2 oo alkylene groups, C 2 -C 2 oo alkenylene groups, C 2 -C 2 oo alkynylene groups, C 3 -C 2 oo cycloalkylene groups, C 5 -C 2 oo cycloalkenylene groups, C 8 - C 2 oo cycloalkynylene groups, C 7 -C 2 oo alkylarylene groups, C 7 -C 2 oo arylalkylene groups, C 8 -C 2 oo arylalkenylene groups, C9-C 2 oo arylalkynylene groups.
  • alkylene groups, alkenylene groups, alkynylene groups, cycloalkylene groups, cycloalkenylene groups, cycloalkynylene groups, alkylarylene groups, arylalkylene groups, arylalkenylene groups and arylalkynylene groups may be substituted, and optionally said groups may be interrupted by one or more heteroatoms, preferably 1 to 100 heteroatoms, said heteroatoms preferably being selected from the group consisting of O, S and NR 5 , wherein R 5 is independently selected from the group consisting of hydrogen, halogen, Ci - C 24 alkyl groups, C 6 - C 24 (hetero)aryl groups, C 7 - C 24 alkyl(hetero)aryl groups and C 7 - C 24 (hetero)arylalkyl groups.
  • the heteroatom is O.
  • G has anti-fouling properties.
  • linker moieties include (poly)ethylene glycol diamines (e.g. l,8-diamino-3,6-dioxaoctane or equivalents comprising longer ethylene glycol chains), polyethylene glycol or polyethylene oxide chains, polypropylene glycol or polypropylene oxide chains and l,x-diaminoalkanes wherein x is the number of carbon atoms in the alkane, polyacrylamides, polyacrylates, oligosaccharides, phospholipids, peptoids, sulfobetaines, carboxybetaines, sulfopyridinium betaines, phosphoryl cholines, and cysteine derivatives.
  • the modified metal surface is reacted with an enzyme.
  • the process may comprise step (iv-a) or step (iv-b).
  • step (iii) When the process according to the invention comprises optional step (iii), then the modified metal surface of step (iii) is reacted with an enzyme in step (iv-b).
  • step (iii) When the process does not comprise optional step (iii), then the modified metal surface of step (ii) is reacted with an enzyme in step (iv-a).
  • the enzyme is selected from the group consisting of hydrogen peroxide forming oxidases, i.e. oxidoreductases that catalyse oxidation- reduction reactions involving molecular oxygen as electron acceptor and that form hydrogen peroxide during the reduction of the molecular oxygen.
  • the enzyme is a hydrogen peroxide forming oxidase selected from the Enzyme Classes (EC) 1.1.3, 1.2.3, 1.3.3, 1.4.3, 1.5.3, 1.6.3, 1.7.3, 1.8.3, 1.10.3, 1.17.3 or 1.21.3.
  • the enzyme is selected from the group consisting of a malate oxidase (EC 1.1.3.3), a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), an aryl-alcohol oxidase (EC 1.1.3.7), an L- gulonolactone oxidase (EC 1.1.3.8), a galactose oxidase (EC 1.1.3.9), a pyranose oxidase (EC 1.1.3.10), an L-sorbose oxidase (EC 1.1.3.11), a pyridoxine 4-oxidase (EC 1.1.3.12), an alcohol oxidase EC 1.1.3.13, an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), a choline oxidase (EC 1.1.3.17), a secondary-alco
  • a spermine oxidase EC 1.5.3.16), a non-specific polyamine oxidase (EC 1.5.3.17), an L-saccharopine oxidase (EC 1.5.3.18), a 4-methylaminobutanoate oxidase (formaldehyde-forming) (EC 1.5.3.19), an N-alkylglycine oxidase (EC 1.5.3.20), a 4- methylaminobutanoate oxidase (methylamine-forming) (EC 1.5.3.21), a nitroalkane oxidase (EC 1.7.3.1), an acetylindoxyl oxidase (EC 1.7.3.2), a factor-independent urate hydroxylase (EC 1.7.3.3), a thiol oxidase (EC 1.8.3.2), a glutathione oxidase (EC 1.8.3.3), a methanethiol oxidase (EC EC 1.5.3.1
  • the enzyme is selected from the group consisting of a malate oxidase (EC 1.1.3.3), a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), an aryl-alcohol oxidase (EC 1.1.3.7), a galactose oxidase (EC 1.1.3.9), a pyranose oxidase (EC 1.1.3.10), an alcohol oxidase (EC 1.1.3.13), an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), a secondary-alcohol oxidase (EC 1.1.3.18), a long-chain-alcohol oxidase (EC 1.1.3.20), a glycerol-3- phosphate oxidase (EC 1.1.3.21), an N-acylhexosamine oxidase (EC EC 1.1
  • the enzyme is selected from the group consisting of a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), a cholesterol oxidase (EC 1.1.3.6), a pyranose oxidase (EC 1.1.3.10), an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15), an aldehyde oxidase (EC 1.2.3.1) an D-aspartate oxidase (EC 1.4.3.1), an L- amino-acid oxidase (EC 1.4.3.2), an D-amino-acid oxidase (EC 1.4.3.3), an D- glutamate(D-aspartate) oxidase (EC 1.4.3.15), a diamine oxidase (EC 1.4.3.22), and a xanthine oxidase (EC 1.17.3.2).
  • a glucose oxidase EC 1.1.
  • the enzyme is selected from the group consisting of a glucose oxidase (EC 1.1.3.4), a hexose oxidase (EC 1.1.3.5), pyranose oxidase (EC 1.1.3.10) and an (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).
  • lactate oxidase is occasionally used to refer to (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).
  • the present invention also relates to a modified metal surface obtainable by the process for the modification of a metal surface according to the invention.
  • the process for the modification of a metal surface, and preferred embodiments thereof, are described in more detail above.
  • the invention therefore relates to a modified metal surface, obtainable by a process comprising the steps of:
  • the invention relates to a modified metal surface, obtainable by a process comprising the steps of:
  • the invention further relates to a modified metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, wherein alkyloxy moieties are covalently bonded to the metal surface via the alkyloxy O-atom, the alkyloxy moieties optionally being substituted and/or optionally being interrupted by one or more heteroatoms.
  • the invention also relates to a modified metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, wherein alkenyloxy moieties are covalently bonded to the metal surface via the alkenyloxy O- atom, the alkenyloxy moieties optionally being substituted and/or optionally being interrupted by one or more heteroatoms.
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, wherein alkenyloxy moieties are covalently bonded to the metal surface via the alkenyloxy O- atom, the alkenyloxy moieties optionally being substituted and/or optionally being interrupted by one or more heteroatoms.
  • Steps (i) and (ii) of said process are described in more detail above.
  • the oxidized metal surface is reacted in step (ii) with an alkene according to Formula (1) or an alkyne according to Formula (2), preferably an alkene according to Formula (1).
  • Alkene (1) and alkyne (2), and preferred embodiments thereof, are described in more detail above.
  • the invention therefore relates, in a preferred embodiment, to a modified metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, wherein alkyloxy or alkenyloxy moieties are covalently bonded to the metal surface via the alkyloxy or alkenyloxy O-atom, wherein the alkyloxy or alkenyloxy moieties are according to Formula (3) or (4):
  • Y is a linear, branched or cyclic C 2 - C50 alkylene group
  • Z is a linear, branched or cyclic C 2 - C50 alkenylene group, the alkenylene group being a 1 -alkenylene group or an internal alkenylene group;
  • alkyloxy or alkenyloxy moieties are according to Formula (5) or (6):
  • Y is a branched C 2 - C 5 o alkylene group
  • Z is a branched C 2 - C50 alkenylene group, the alkenylene group being an internal alkenylene group;
  • x is an integer in the range of 2 to 10.
  • the alkyloxy or alkenyloxy moiety is a branched alkyloxy or alkenyloxy moiety wherein more than one alkyloxy or alkenyloxy O-atom is covalently bonded to the metal surface, resulting in a particularly stable attachment to the metal surface.
  • x is 2, 3, 4, 5 or 6, more preferably x is 2, 3, or 4 and most preferably x is 2 or 3.
  • the invention relates to a modified metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au, wherein alkyloxy moieties are covalently bonded to the metal surface via the alkyloxy O-atom, wherein the alkyloxy moieties are according to Formula (3), (5) or (6) as defined above, more preferably according to Formula (3).
  • the invention further relates to a modified metal surface, obtainable by a process comprising the steps of:
  • step (iii) optionally, reacting the alkyloxy- or alkenyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy- or linker- alkenyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy- or an enzyme-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy- or an enzyme-linker-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy or linker-alkenyloxy moiety.
  • the modified metal surface is thus obtainable by a process comprising steps (i), (ii) and (iv-a), or by a process comprising steps (i), (ii), (iii) and (iv-b).
  • the modified metal surface is obtainable by a process comprising the steps of:
  • step (iii) optionally, reacting the alkyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy moiety; or (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy moiety.
  • the oxidized metal surface is reacted in step (ii) with an alkene according to Formula (1) or an alkyne according to Formula (2), preferably with an alkene according to Formula (1).
  • Alkene (1) and alkyne (2), and preferred embodiments thereof, are described in more detail above.
  • the linker moiety is according to Formula (3). Linker moiety (3), and preferred embodiments thereof, are described in more detail above.
  • the invention further relates to the use of a modified metal surface according to the invention in an electrode.
  • the invention therefore also relates to an electrode, comprising a modified metal surface according to the invention.
  • the invention therefore relates to an electrode, comprising a modified metal surface, wherein:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;
  • linker if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety.
  • Said electrode preferably comprises a modified metal surface, wherein:
  • the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;
  • an enzyme is covalently attached to the metal surface via an alkyloxy moiety and, optionally, a linker moiety;
  • the alkyloxy moiety is covalently bonded to said metal surface via the alkyloxy O-atom; and the linker, if present, is covalently bonded to the enzyme and to the alkyloxy moiety.
  • the electrode according to the invention and preferred embodiments thereof, are described in more detail above. Also the modified metal surface, and preferred embodiment thereof, are described in more detail above.
  • the invention therefore also relates to an electrode, comprising a modified metal surface, wherein the modified metal surface is obtainable by a process comprising the steps of:
  • step (ii) reacting the oxidized metal surface of step (i) with an alkene or an alkyne, the alkene or alkyne being optionally substituted and/or being optionally interrupted by one or more heteroatoms, to form an alkyloxy- or alkenyloxy- modified metal surface, wherein the alkyloxy or alkenyloxy moiety is covalently bonded to the metal surface via the alkyloxy or alkenyloxy O-atom;
  • step (iii) optionally, reacting the alkyloxy- or alkenyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy- or linker-alkenyloxy- modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy- or an enzyme-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy or alkenyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy- or an enzyme-linker-alkenyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy or linker-alkenyloxy moiety.
  • the electrode according to the invention comprises a modified metal surface, wherein the modified metal surface is obtainable by a process comprising the steps of:
  • step (i) providing an oxidized metal surface, wherein the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au; (ii) reacting the oxidized metal surface of step (i) with an alkene, the alkene being optionally substituted and/or being optionally interrupted by one or more heteroatoms, to form an alkyloxy-modified metal surface, wherein the alkyloxy moiety is covalently bonded to the metal surface via the alkyloxy O- atom;
  • step (iii) optionally, reacting the alkyloxy-modified metal surface of step (ii) with a linker moiety, to form a linker-alkyloxy-modified metal surface, wherein the linker moiety is covalently bonded to the alkyloxy moiety;
  • step (iv-a) reacting the modified metal surface of step (ii) with an enzyme to form an enzyme-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the alkyloxy moiety;
  • step (iv-b) reacting the modified metal surface of step (iii) with an enzyme to form an enzyme-linker-alkyloxy-modified metal surface, wherein the enzyme is covalently bonded to the linker moiety of the linker-alkyloxy moiety.
  • the invention further relates to the use of an electrode according to the invention in a device for the detection of an analyte in a fluid, and to the use of an electrode according to the invention in a biosensor.
  • the invention further relates to the use of the modified metal surface according to the invention in a device for the detection of an analyte in a fluid, and to the use of a modified metal surface according to the invention in a biosensor.
  • KC1 p.a.
  • KH 2 P0 4 p.a.
  • NaCl p.a., 99.5%
  • Glutaraldehyde 50% aqueous solution
  • Acetone, isopropanol, ethanol and CH 2 C1 2 were purchased from Sigma- Aldrich (HPLC grade).
  • CH 2 C1 2 was dried in a PureSolv EN solvent purification system (Innovative Technology, USA).
  • Deionized (DI) water was obtained from a Milli-Q Integral water purification system (Merck-Millipore, USA).
  • Phosphate buffered saline (10 mM, pH 7.4) was prepared from a solution of NaCl (8.01 g/L), Na 2 HP0 4 (1.41 g/L), KH 2 P0 4 (0.27 g/L) and KC1 (0.20 g/L) in DI water.
  • SSC buffer (pH 7.0) was prepared from a 150 mM of sodium chloride (8.77 g/L) and 15 mM of sodium citrate tribasic dehydrate (4.41 g/L).
  • 2,2,2-Trifluoroethyl undec-10-enoate (TFEE) was synthesized as described in M. Rosso et al, Langmuir 2009, 25, 2172-2180, incorporated by reference.
  • XPS X-ray photoelectron spectroscopy
  • the XPS analysis of surfaces was performed using a JPS-9200 Photoelectron Spectrometer (JEOL, Japan).
  • the high-resolution spectra were obtained under UHV conditions using monochromatic Al Ka X-ray radiation at 12 kV and 20 mA, using an analyzer pass energy of 50 eV for wide scan and 10 eV for narrow scan.
  • the emitted electrons were collected at 10° from the surface normal (take-off angle relative to the surface normal 10°). All XPS spectra were evaluated by Casa XPS software (version 2.3.15). High-resolution spectra were corrected with linear or Shirley background before fitting.
  • Atomic area ratios were determined after a baseline correction and normalizing the peak area ratios by the corresponding atomic sensitivity factors (1.00 for Cl s, 1.80 for Nl s, 2.93 for Ol s, 4.43 for F I s, 15.5 for Pt4f).
  • the wettability of the modified surfaces was determined by automated static water contact angle measurements with the use of a Kriiss DSA 100 goniometer (volume of the drop of demineralized water is 3.0 pL). The reported value is the average of at least 3 droplets with the error of less than ⁇ 2° (and typically ⁇ 1° for any value >90°).
  • IRRAS analysis was performed using a Bruker Tensor 27 FT-IR spectrometer, using a commercial variable-angle reflection unit (Auto Seagull, Harrick Scientific).
  • a Harrick grid polarizer was installed in front of the detector and was used for measuring spectra with p-polarized radiation with respect to the plane of incidence at the sample surface.
  • Single channel transmittance spectra were collected at 80° using 2048 scans in each measurement. The raw data were subtracted by the data recorded on a freshly cleaned reference Pt oxide surface, after which a baseline correction was applied to give the reported spectra.
  • the thickness of the oxidized Pt layer was estimated from high resolution Pt 4f spectrum XPS spectra by using the substrate-overlayer model of Eq. (1), as described in D. Briggs, M. P. Seah, "Practical Surface Analysis: Auger and X-ray Photoelectron Spectroscopy ", vol. 1, 2 nd ed., Wiley, New York, 1990, incorporated by reference.
  • IoJIp t is the ratio of the Pt 4f peak area in the oxidized layer and the metallic layer
  • ⁇ is the takeoff angle (angle between the sample and the detector: 80°)
  • do x is the thickness of the oxide layer
  • ⁇ is the escape depth of Pt 4f photoelectrons
  • po x and pp t are the density of oxidized Pt and Pt metal respectively.
  • the density of bulk Pt and of Pt oxide layer were estimated to be 21.1, and 10.2 g/cm 3 (density of the hydrated Pt0 2 )' as described in A. Cotton, G. Wilkinson, C. A. Murillo, M.
  • E is the electron kinetic energy of the monochromatic Al Ka irradiation source (1486.6 eV)
  • a is the diameter of the atoms (0.27 nm for Pt derived from the lattice constant of 0.39 nm in Pt crystals), as described in G. A. Somorjai, "Introduction to Surface Chemistry and Catalysis", Wiley, New York 1993, incorporated by reference.
  • ⁇ was calculated to be 2.2 nm. Therefore we determined a thickness do x of 3 nm for the oxidized Pt film.
  • Literature reports similar values for oxidized Pt layers obtained by oxygen plasma treatment of Pt, as described in J. J. Blackstock et al, Appl. Phys. A 2005, 80, 1343-13, incorporated by reference. Estimation of the thickness of the grafted organic layer.
  • the thickness d of the alkyl layer was calculated using the equation (Laibnis et al, "Attenuation of Photoelectrons in Monolayers of n-Alkanethiols Adsorbed on Copper, Silver, and Gold", J. Phys. Chem.
  • ⁇ 9.0 + 0.022(1487 - E) (4)
  • equation 4 results in a value of 4.01 nm for ⁇
  • Platinum films were sputtered over 6 ⁇ thermal silicon oxide on silicon wafers, using lOnm Ta as adhesion layer.
  • Pt pieces (l x l or 1 x2.5 cm 2 for IRRAS) were cleaned by sonication using a solvent series with increasing polarity: hexane, CH 2 CI 2 and acetone. Subsequently, Pt surfaces were oxidized by exposure to oxygen plasma (0.1 mbar, 15 seem, 50 W, Plasma System ATTO Diener, Germany) for
  • Table 1 the XPS analysis of oxidized Pt films is summarized, and shows a composition of Pt (54.10%), O (32.05%) and C (13.85%). This composition varied slightly when increasing the oxygen plasma treatment.
  • XPS narrow scan measurements of the Pt 4f and of the Oi s regions provided a more detailed description of the surface chemistry.
  • Deconvoluted signals at 71.2 eV (Pt 4 f7/ 2 ) and at 74.5 eV (Pt 4 f 5 / 2 ) were assigned to Pt metal according to literature data (J. J. Blackstock et al, Appl. Phys. A 2005, 80, 1343-1353, Z. Li et al, Surf. Sci. 2003, 529, 410-418 and M. Peuckert et al., Surf Sci. 1984, 145, 239-259, all incorporated by reference).
  • Pt4n/ 2 peak at 72.0 eV and Pt 4 f 5 / 2 peak at 75.3 eV correspond to Pt(OH) 2 chemical species while the Pt 4 f signals of Pt0 2 appear at 73.3 eV (PUnn) and at 77.2 eV (Pt 4 f 5 / 2 ). Integration of Pt 4 f components permits to assess the composition of the oxidized Pt films as a mixture of Pt/Pt(OH) 2 /Pt0 2 in a ratio 2.3/1.1/1.
  • XPS data analysis of the Ols region displays two peaks, as shown in Table 3.
  • the signal at 529.9 eV corresponds to the oxygen atoms of Pt0 2 while the peak of oxygen of Pt(OH) 2 species appears at 531.1 eV.
  • the ratio 01s(Pt(OH) 2 )/01s(Pt0 2 ) is 1.1. This value would correspond to Pt(OH) 2 /Pt0 2 ⁇ 1 and corroborates the composition of the Pt oxide thin layer we determined from the Pt 4 f region.
  • the thickness of the oxide layer was estimated to be 3 nm according to the model proposed in Z. Li et al, Surf. Sci. 2003, 529, 410-418, incorporated by reference.
  • the reaction flask was filled with neat alkene or alkyne (1.5-2 mL), degassed by three consecutive freeze-pump-thaw cycles and heated at 125°C under argon for 30 min.
  • An oxidized metal substrate was then placed into the alkene or alkyne and left to react for a certain time (preferably for at least 8 h).
  • the substrate was rinsed with CH 2 CI 2 and sonicated for 3 min in CH 2 CI 2 .
  • the substrate was dried with Ar and stored in an Ar-glovebox.
  • Neat alkene or alkyne (1.5-2 mL) was added to a three neck flask equipped with a condenser and degassed for 30 min by bubbling argon through it at 80°C.
  • An oxidized metal surface was transferred to the reaction flask and heated at 125°C under a low argon flow for a certain time (preferably for at least 8 hours, e.g. for 16 h).
  • the metal surface was removed from the solution, rinsed with CH 2 CI 2 , and sonicated for 3 min in CH 2 CI 2 before being dried with a stream of dry argon.
  • Table 5 Surface composition and organic layer thickness (XPS) and static water contact angle for Example 1A.
  • Table 6 XPS C Is binding energies and curve fittings for Example 1A.
  • Table 7 Static water contact angle and organic layer thickness as determined by XPS or Example 1A after various modification times.
  • the degree of modification was initially evaluated by measuring the static water contact angle (CA) of the grafted surfaces.
  • the CA value on oxidized Pt films was ⁇ 15° and after 6 hours of reaction the CA increased to ⁇ 100°, which indicates the formation of a hydrophobic layer (Table 4). Extension of the reaction time up to 67h gave a CA ⁇ 97° and did not improve the hydrophobicity of the layer.
  • This CA value is lower than that of the thermally grafted 1-hexadecene on -OH terminated SiC (106°, see M. Rosso et al, Langmuir 2008, 24, 4007-4012, incorporated by reference), and suggests that the alkyl layer is disordered and less dense.
  • Table 7 further shows the thickness of the alkyl layer as a function of the grafting time. It reached 0.78 nm after 6h of modification. Increasing of the grafting time to 16h and 24h resulted in 1.5 nm, and 2.0 nm thick layers respectively. A reaction time of 48h and 67h produced layers with thicknesses of 2.6 nm and 2.7 nm correspondingly. These values are 1.5 times the length of 1-hexadecene molecule (1.9 nm, as determined with Chem3D) and reveal that multilayer formation occurs under these experimental conditions.
  • Example IB the surface composition, organic layer thickness (determined by XPS) and static water contact angle for Example IB are shown.
  • Table 9 the XPS C I s binding energies and curve fittings for Example IB are shown.
  • Table 8 Surface composition, organic layer thickness (XPS) and static water contact angle of Example IB.
  • This value does not match the stoichiometrical ratio obtained (0.056), considering that one carbon atom from 1- octadecyne is oxidized to a C-O-Pt linkage upon binding to the surface, and may suggest that there may be a carbon contamination in the sample.
  • the degree of modification was also estimated by measuring the static water contact angle (CA) of the grafted surface.
  • CA value on oxidized Pt films ( ⁇ 15°) increased to 97° after 16h reaction with octadecyne, which shows the formation of a hydrophobic layer.
  • This CA value is lower than that of the thermally grafted 1- octadecyne layers on -OH terminated SiC (111°, S. P. Pujari et al, Langmuir 2013, 29, 4019-4031, incorporated by reference), and suggests that the alkyl layer is disordered and less dense.
  • Table 10 Surface composition, organic layer thickness (XPS) and static water contact angle of Example 1C.
  • Table 11 XPS C Is binding energies, curve fittings in % and surface ratios for Example 1C.
  • Table 12 Organic layer thickness as determined by XPS for Example 1C after various modification times.
  • Example 1C The modified metal surface of Example 1C was reacted with neat tris(2- aminoethyl)-amine for 8h at 85°C. An amino terminated surface was obtained.
  • Table 13 shows the surface composition and organic layer thickness as determined by XPS of amino terminated surface Example ID, and Table 14 shows XPS C Is binding energies and curve fittings in %.
  • Table 13 Surface composition and organic layer thickness as determined by XPS of amino terminated surface Example ID.
  • Example ID The modified metal surface of Example ID was reacted with a solution of glutaraldehyde 2% in saline-sodium citrate (SSC) buffer for 3h at room temperature (SSC buffer: 150 mM of sodium chloride and 15 mM of sodium citrate tribasic at pH 7.0). An aldehyde terminated surface was obtained.
  • SSC buffer 150 mM of sodium chloride and 15 mM of sodium citrate tribasic at pH 7.0.
  • Table 15 shows the surface composition and organic layer thickness as determined by XPS of aldehyde terminated surface Example IE, and Table 16 shows XPS C Is binding energies and curve fittings in %.
  • Table 15 Surface composition and organic layer thickness as determined by XPS of
  • HAOX enzyme Human alpha-hydroxyacid oxidase enzyme
  • PBS phosphate buffer saline
  • Table 17 shows the surface composition and organic layer thickness as determined by XPS of Example IF
  • Table 18 shows XPS C Is binding energies and curve fittings in %.
  • Example 1C The modified metal surface of Example 1C was reacted with neat ethylenediamine for 8h at 60°C. An amino terminated surface was obtained.
  • Table 19 shows the surface composition and organic layer thickness as determined by XPS of amino terminated surface Example 1G.
  • Table 20 Surface composition and organic layer thickness as determined by XPS of Example 1H.
  • Example 1H The modified metal surface of Example 1H was reacted with monomer [3- (methacryloylamino)propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt according to literature procedure (A. T. Nguyen et al, Langmuir 2012, 28, 12509-12517, incorporated by reference): [3-(methacryloylamino)propyl]dimethyl(3- sulfopropyl)ammonium hydroxide inner salt (5.12 g) and 2,2 '-bipyri dine (0.14 g) were dissolved in a mixture of isopropanol (4.0 mL) and water (16.0 mL) in a round- bottomed flask by stirring.
  • the solution was degassed for 30 min by purging with argon.
  • a mixture of CuCl (36.0 mg) and CuCl 2 (4.8 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum.
  • the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl 2 by means of a syringe (flushed with argon in advance). The mixture was stirred for an additional 30 min under argon to dissolve all CuCl and CuCl 2 .
  • Table 21 shows the surface composition and organic layer thickness as determined by XPS of Example II, and Table 22 shows XPS C Is binding energies and curve fittings in %.
  • Table 21 Surface composition and organic layer thickness as determined by XPS and static water contact angle of Example II.
  • An amine terminated surface ID is reacted with isobutiryl bromide (0.15 mL) in dichlorom ethane (2 mL) and in the presence of triethyl amine (0.2 mL) at room temperature for 16 hours. A bromo terminated surface is obtained.
  • Table 23 shows the surface composition and organic layer thickness as determined by XPS of bromo terminated surface Example 1 J.
  • Table 23 Surface composition and organic layer thickness as determined by XPS of Example 1J.
  • Example 1J The modified metal surface of Example 1J was reacted with monomer [2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide according to literature procedure (A. T. Nguyen et al, Langmuir 2012, 28, 12509-12517, incorporated by reference): [2-(methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)- ammonium hydroxide inner salt (4.90 g) and 2,2 ' -bipyridine (0.14 g) were dissolved in a mixture of isopropanol (4.0 mL) and water (16.0 mL) in a round-bottomed flask by stirring.
  • the solution was degassed for 30 min by purging with argon.
  • a mixture of CuCl (36.0 mg) and CuCl 2 (4.8 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum.
  • the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl 2 by means of a syringe (flushed with argon in advance). The mixture was stirred for an additional 30 min under argon to dissolve all CuCl and CuCl 2 .
  • Table 24 shows the surface composition and organic layer thickness as determined by XPS of Example IK, and Table 25 shows XPS C Is binding energies and curve fittings in %.
  • Table 24 Surface composition and organic layer thickness as determined by XPS, and static water contact angle of Example IK.
  • Glucose oxidase (GOX) enzyme was immobilised on the aldehyde terminated Pt surface of Example IE by depositing 150 microliters of a 2.5 mg/mL solution of GOX enzyme in phosphate buffer saline (PBS) on the aldehyde terminated surface for 2.5 hours at room temperature.
  • PBS composition NaCl (sodium chloride) 8.01 g/L, Na 2 HP0 4 . (sodium phosphate dibasic) 1.41 g/L, KH 2 PO 4 (potassium dihydrogen phosphate) 0.27 g/L, KC1 (potassium chloride) 0.20 g/L.
  • Table 26 shows the surface composition and organic layer thickness as determined by XPS of Example 1L, and Table 27 shows XPS C Is binding energies and curve fittings in %.
  • Table 26 Surface composition and organic layer thickness as determined by XPS of Example 1L.
  • Example 1M carboxybetaine based zwitterionic polymer
  • Example 1J The modified metal surface of Example 1J was reacted with monomer 2-carboxy-N,N- dimethyl-N-(2'-(methacryloylamino)propyl)ethanaminium inner salt (A. T. Nguyen et al, Langmuir 2012, 28, 12509-12517, incorporated by reference): 2-carboxy-N,N- dimethyl-N-(2'-(methacryloylamino)propyl)ethanaminium inner salt (3.04 g) and 2,2'- bipyridine (0.11 g) were dissolved in a mixture of isopropanol (3.0 mL) and water (12.0 mL) in a round-bottomed flask by stirring.
  • 2-carboxy-N,N- dimethyl-N-(2'-(methacryloylamino)propyl)ethanaminium inner salt A. T. Nguyen et al, Langmuir 2012
  • the solution was degassed for 30 min by purging with argon.
  • a mixture of CuCl (26.0 mg) and CuCl 2 (3.6 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum.
  • the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl 2 by means of a syringe (flushed with argon in advance). The mixture was stirred for an additional 30 min under argon to dissolve all CuCl and CuCl 2 .
  • Table 28 shows the surface composition and organic layer thickness as determined by XPS of Example 1M, and Table 29 shows XPS C Is binding energies and curve fittings in %. Table 28: Surface composition and organic layer thickness as determined by XPS, and static water contact angle of Example 1M.
  • Table 29 XPS C Is binding energies and curve fittings in % for Example 1M.
  • Glucose oxidase (GOX enzyme) enzyme was immobilised on the aldehyde terminated Pt surface of Example IE by depositing 100 microliters of freshly prepared solution of 20 mg/mL solution of GOX enzyme in 25% glutaraldehyde in saline sodium citrate buffer (SSC) for 3 hours at room temperature.
  • SSC saline sodium citrate buffer
  • Enzyme modified metal surface IN was obtained.
  • Table 30 shows the surface composition and organic layer thickness determined by XPS of Example IN.
  • Table 30 Surface composition and organic layer thickness as determined by XPS of Example IN.
  • Glucose oxidase (GOX enzyme) enzyme was immobilised on the poly(carboxybetaine)-N-methylacrylamide coated Pt surface of Example IM.
  • the surface of Example IM was activated with N-hydroxysuccinimide (NHS) groups by depositing on it 100 microliters of a solution 0.1 M N-hydroxysuccinimide(NHS)/0.4 M ⁇ -(S-dimethylaminopropy ⁇ -jV-ethylcarbodiimide hydrochloride for 15 minutes. Afterwards the modified surface was rinsed with deionized water and dry with a stream of argon.
  • NHS N-hydroxysuccinimide
  • Example IM 100 microliters of 50 mg/mL solution of GOX enzyme in 25% glutaraldehyde in saline sodium citrate buffer (SSC) were deposited on the NHS- activated surface of Example IM for 3 hours at room temperature.
  • SSC composition 0.15 M NaCl and 0.015 M sodium citrate tribasic solution.
  • Enzyme modified metal surface 10 was obtained.
  • Table 31 shows the surface composition and organic layer thickness as determined by XPS of Example 10, and Table 32 shows XPS C Is binding energies and curve fittings in %.
  • Table 31 Surface composition and organic layer thickness as determined by XPS of Example 10.
  • Table 32 XPS C Is binding energies and curve fittings in % for Example 10.
  • Lactate oxidase (LOX enzyme) enzyme was immobilised on the aldehyde terminated Pt surface of Example IE by depositing 150 microliters of a 3 mg/mL solution of LOX enzyme in phosphate buffer saline (PBS) on the aldehyde terminated surface for 2.5 hours at room temperature.
  • PBS composition NaCl (sodium chloride) 8.01 g/L, Na 2 HP0 4 . (sodium phosphate dibasic) 1.41 g/L, KH 2 PO 4 (potassium dihydrogen phosphate) 0.27 g/L, KC1 (potassium chloride) 0.20 g/L.
  • Enzyme modified metal surface IP was obtained.
  • Table 33 shows the surface composition and organic layer thickness as determined by XPS of Example IP.
  • Table 33 Surface composition and organic layer thickness as determined by XPS of Example IP.
  • Lactate oxidase (LOX enzyme) enzyme was immobilised on the poly(carboxybetaine)-N-methylacrylamide coated Pt surface of Example IM.
  • the surface of Example IM was activated with N-hydroxysuccinimide (NHS) groups by depositing on it 100 microliters of a solution 0.1 M N-hydroxysuccinimide(NHS)/0.4 M ⁇ -(S-dimethylaminopropy ⁇ -N'-ethylcarbodiimide hydrochloride for 15 minutes. Afterwards the modified surface was rinsed with deionized water and dry with a stream of argon.
  • NHS N-hydroxysuccinimide
  • PBS composition NaCl (sodium chloride) 8.01 g/L, Na 2 HP0 4 (sodium phosphate dibasic) 1.41 g/L, KH 2 PO 4 (potassium dihydrogen phosphate) 0.27 g/L, KC1 (potassium chloride) 0.20 g/L.
  • Enzyme modified metal surface 1Q was obtained.
  • Table 34 shows the surface composition and organic layer thickness as determined by XPS of Example 1Q.
  • Table 34 Surface composition and organic layer thickness as determined by XPS of Example 1Q.
  • HAOX enzyme Human alpha-hydroxyacid oxidase enzyme
  • Example IE Human alpha-hydroxyacid oxidase enzyme
  • PBS phosphate buffer saline
  • SSC saline sodium citrate buffer
  • HAOX enzyme immobilization protocol was completed with a final HAOX coupling step.
  • Table 35 shows the surface composition and organic layer thickness as determined by XPS of Example 1R.
  • Table 35 Surface composition and organic layer thickness as determined by XPS of Example 1R.
  • Example 2a Chronoamperometry experiments for a glucose biosensor
  • Chronoamperometry experiments were performed in a CHI potentiostat (CH Instruments Inc.) interfaced to a personal computer. All measurements were carried out in phosphate buffer 0.2 M (pH 7.1) at room temperature, without deaeration and without stirring, at 500 mV using the CH Instruments software option of plotting current vs. time.
  • the auxiliary/counter electrode was a Pt wire (Model MW 1033, BASi Inc.).
  • An Ag/AgCl electrode (Model MF 2052, BASi Inc.) was employed as a reference electrode.
  • Example 1L surfaces (see above, geometric area 1 cm 2 ) were used as a working electrode.
  • the electrode response to variation in glucose concentrations was quantify by adding aliquots of 0.1M glucose solution to 10 mL of the blank phosphate buffer in such way that the final volume (10 mL) remained constant.
  • Figure 7 shows a glucose calibration curve for a glucose biosensor according to the invention, wherein a modified surface according to Example IE is coupled to glucose oxidase (GOX).
  • GOX glucose oxidase
  • Example 2b Chronoamperometry experiments for a glucose biosensor
  • Figure 8 shows a glucose calibration curve for a glucose biosensor according to the invention, wherein modified surfaces correspond to Examples IN and 10.
  • the activity of the immobilized enzyme follows a Michaelis- Menten type kinetics.
  • Figure 9 shows a lactate calibration curve for a lactate biosensor according to the invention, wherein modified surfaces correspond to Examples IP, 1Q and 1R.
  • the activity of the immobilized enzyme follows a Michaelis- Menten type kinetics.
  • Example 4 Stability of covalently attached alkyloxy-layer on Pt
  • the hydrolytic stability of alkyl layers was assessed by comparison with CI 8 alkyl thiol monolayers on Au. Therefore CI 8 alkyloxy layers were grafted on oxidized Pt surfaces and subsequently immersed in PBS buffer and deionized water.
  • the initial static water CA value (-100°) stays invariable after 4h immersion in PBS buffer (pH 7.4), while decreased to -98° after 8h and dropped to -95° after 24h.
  • the C/Pt ratio obtained from the survey XPS spectrum decreased ⁇ 10%.
  • DI deionized
  • the initial CA value (-100°) does not change significantly after 8h (-102°), whereas it decreases to -92° after 24h. In this case the C/Pt ratio obtained from the survey XPS spectrum dropped -20%.
  • Table 36 Surface composition as determined by XPS of PtOx grafted with 1- octadecene before and after immersion in PBS for 24h.
  • Table 37 Surface composition as determined by XPS of PtOx grafted with 1- octadecene before and after immersion in deionized water for 24h.

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Abstract

La présente invention concerne un dispositif pour la détection d'un analyte dans un fluide, le dispositif comprenant : (a) une électrode de travail comprenant une surface métallique modifiée : (1) le métal étant choisi dans le groupe constitué par Ru, Rh, Pd, Ag, Ir, Pt et Au ; (2) une enzyme étant liée de manière covalente à la surface métallique par l'intermédiaire d'un fragment alkyloxy ou alcényloxy et, éventuellement, un fragment lieur ; (3) le fragment alkyloxy ou alcényloxy étant lié de manière covalente à ladite surface métallique par l'intermédiaire de l'atome d'O d'alkyloxy ou d'alcényloxy ; et (4) le fragment lieur, s'il est présent, étant lié de manière covalente à l'enzyme et au fragment alkyloxy ou alcényloxy ; (b) une électrode de référence ; et (c) un moyen pour détecter un signal électrique, le moyen étant accouplé de manière fonctionnelle à au moins l'électrode de travail (a) et l'électrode de référence (b). Le dispositif selon l'invention est également appelé biocapteur. L'invention concerne également un procédé pour la modification d'une surface métallique et une surface métallique modifiée pouvant être obtenue par le procédé. En outre, l'invention concerne une électrode comprenant ladite surface métallique modifiée et un biocapteur comprenant ladite surface métallique modifiée.
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