WO2016018148A1

WO2016018148A1 - Biosensor comprising a modified metal surface and method for the modification of a metal surface

Info

Publication number: WO2016018148A1
Application number: PCT/NL2015/050550
Authority: WO
Inventors: 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
Original assignee: Biomarque B.V.
Priority date: 2014-07-30
Filing date: 2015-07-28
Publication date: 2016-02-04

Abstract

The present invention 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: (1) the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au; (2) an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety; (3)the alkyloxy or alkenyloxy moiety is covalently bonded to said metal surface via the alkyloxy or alkenyloxy O- atom; and (4) the linker moiety, if present, is covalently bonded to theenzyme and to the alkyloxy or alkenyloxy moiety; (b) a reference electrode; and (c) means for detecting an electricalsignal, the means being operationally coupled to at least working electrode (a) and reference electrode (b). 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 and to a modified metal surface obtainable by the process. Furthermore, the invention relates to an electrode comprising said modified metal surface, and to a biosensor comprising said modified metal surface.

Description

Biosensor comprising a modified metal surface and method for the modification of a metal surface

Field of the invention

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.

Background of the invention

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. Typically, 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. Typically, a biosensor comprises a signal collector that is connected to the working electrode, and that collects, amplifies and displays the signal.

In an electrochemical biosensor the interaction of the biological component with the analyte results in an electrical 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, incorporated by reference, 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). In this biosensor, the glucose oxidases react with a biomolecule to form hydrogen peroxide, and the nano-Ptlr particles carry out an electrochemical oxidation-reduction reaction with the hydrogen peroxide.

Quan et al, Bull. Korean Chem. Soc. 2002, 23, 385 - 390, incorporated by reference herein, discloses a biosensor wherein laccase was assembled on an amine- derivatized platinum electrode by glutaraldehyde coupling. A Pt disc electrode (4 mm diameter) was oxidized by 5% solution of potassium dichromate in 15% nitric acid (2 hours, 80 °C). The enzyme layer formed on the surface did not communicate electrons directly with the electrode, but the enzymatic activity of the surface was followed by electrochemical detection of enzymatically oxidized products.

WO 2013/058879, incorporated by reference, discloses microneedle arrays for biosensing and drug delivery. 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.

Several methods are known in the art for the immobilization of an enzyme to a biosensor surface. When 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. Alternatively, 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.

For example 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.

Putzbach et al., Sensors 2013, 13, 4811 - 4840, incorporated by reference, discloses that for gold nanoparticles there are four basic methods of enzyme immobilisation: physical adsorption, chemical adsorption, self-assembling monolayers (SAMs) and co-modification with electrode component matrix. SAMs provide a simple and well-studied method of immobilizing gold nanoparticles and enzymes onto electrodes, allowing a high degree of control of the composition and thickness of the transducer surface. Colloidal gold-modified electrodes can be prepared by covalently tethering the gold nanoparticles with surface-functional groups (-CN, - H₂ 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.

Parra et al., Analytica Chimica Acta 2006, 555, 308 - 315, incorporated by reference, discloses a lactate biosensor wherein lactate oxidase is covalently bonded to a gold substrate. This approach is based on the covalent attachment of proteins containing lysine residues, through acylation of free primary or secondary aliphatic groups, to gold surfaces modified with a bifunctional reagent containing succinimide functionalities, in particular 3,3'-dithiodipropionic acid di(N-succinimidyl ester (DTSP). Since DTSP adsorbs onto gold surfaces through the disulfide group, the terminal succinimidyl groups are exposed to the solution allowing further covalent immobilization of amine-containing biomolecules.

Gamero et al., Biosensors and Bioelectronics 2010, 25, 2038 - 2044, incorporated by reference, discloses a lactate biosensor using a nanostructured rough gold surface as a transducer. The biosensor is developed by immobilization of lactate oxidase (Lox), on a rough gold electrode modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate (DTSP). This bifunctional reagent preserves the rough gold structure and allows further covalent immobilization of the enzyme through the terminal succinimidyl groups. Biosensors known in the art typically suffer from several disadvantages. Often, their biocompatibility is insufficient. Biocompatibility is important because e.g. 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. Furthermore, not all biosensors are able to measure analytes in a continuous fashion.

Summary of the invention

The present invention 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:

(1) the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au; (2) an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;

(3) the alkyloxy or alkenyloxy moiety is covalently bonded to said metal surface via the alkyloxy or alkenyloxy O-atom; and

(4) the linker moiety, if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety;

(b) a reference electrode; and

(c) means for detecting an electrical signal, the means being operationally coupled to at least working electrode (a) and reference electrode (b).

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:

(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 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;

(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; and

(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; or

(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.

Description of the figures

In Figure 1, a schematic overview of an embodiment of the biosensor according to the invention is shown.

In Figure 2, several examples of a modified metal surface, wherein an enzyme (E) is covalently attached to the metal surface via an alkyloxy or an alkenyloxy group, are depicted.

In Figure 3, additional examples of a modified metal surface, wherein an enzyme (E) is covalently attached to the metal surface via an alkyloxy or an alkenyloxy group, are depicted.

In Figure 4, several examples of a modified metal surface, wherein an enzyme (E) is covalently attached to the metal surface via an alkyloxy or an alkenyloxy group and a linker moiety (G), are depicted.

In Figure 5, additional examples of a modified metal surface, wherein an enzyme (E) is covalently attached to the metal surface via an alkyloxy or an alkenyloxy group and a linker moiety (G), are depicted.

In Figure 6, the process for the detection of lactate is schematically shown.

In Figure 7, a calibration curve for a glucose biosensor according to the invention, wherein glucose oxidase (GOX) is the enzyme, is shown.

Detailed description of the invention

Definitions

The verb "to comprise" as is used in this description and in the claims and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

In addition, reference to an element by the 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. In addition, the description of 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. When 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. When 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.

The term "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_nH_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. Unless stated otherwise, an alkene may optionally be substituted with one or more, independently selected, substituents as defined below. Unless stated otherwise, an alkene may optionally be interrupted by one or more heteroatoms independently selected from the group consisting of O, N and S.

The term "alkyne" herein 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_nH_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. Unless stated otherwise, an alkyne may optionally be substituted with one or more, independently selected, substituents as defined below. Unless stated otherwise, 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_nH_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_nH_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_nH_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_nH_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_nH_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_nH_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_nH_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. Unless stated otherwise, 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 C₂) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of 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 C₃) and may include monocyclic and bicyclic structures. Optionally, 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.

Unless stated otherwise, 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 - C₁₂ alkyl groups, C₂ - C₁₂ alkenyl groups, C₂ - C₁₂ alkynyl groups, C₃ - C₁₂ cycloalkyl groups, C₅ - C₁₂ cycloalkenyl groups, C₈ - C₁₂ cycloalkynyl groups, Ci - C₁₂ alkyloxy groups, C₂ - C₁₂ alkenyloxy groups, C₂ - C₁₂ alkynyloxy groups, C₃ - C₁₂ cycloalkyloxy groups, halogens, amino groups, oxo and silyl groups, wherein the silyl groups can be represented by the formula (R¹⁰)₃Si-, wherein R¹⁰ is independently selected from the group consisting of Ci - C₁₂ alkyl groups, C₂ - C₁₂ alkenyl groups, C₂ - C₁₂ alkynyl groups, C₃ - C₁₂ cycloalkyl groups, Ci - C₁₂ alkoxy groups, C₂ - C₁₂ alkenyloxy groups, C₂ - C₁₂ alkynyloxy groups and C₃ - C₁₂ cycloalkyloxy groups, wherein the alkyl groups, alkenyl groups, alkynyl groups, cycloalkyl groups, alkoxy groups, alkenyloxy groups, alkynyloxy groups and cycloalkyloxy groups are optionally substituted, the alkyl groups, the alkoxy groups, the cycloalkyl groups and the cycloalkoxy groups being optionally interrupted by one of more hetero-atoms selected from the group consisting of O, N and S. Biosensor

(a) a working electrode comprising a modified metal surface, wherein:

(1) the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au;

(2) an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety;

(b) a reference electrode; and

In a preferred embodiment, the device according to the invention comprises:

(a) a working electrode comprising a modified metal surface, wherein:

(2) an enzyme is covalently attached to the metal surface via an alkyloxy moiety and, optionally, a linker moiety;

(3) the alkyloxy moiety is covalently bonded to said metal surface via the alkyloxy O-atom; and

(4) the linker moiety, if present, is covalently bonded to the enzyme and to the alkyloxy moiety;

(b) a reference electrode; and

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:

(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;

(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; and

(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.

(b) a reference electrode; and

(c) means for detecting an electrical signal, the means being operationally coupled to at least working electrode (a) and reference electrode (b). preferred embodiment, the device according to invention comprises:

(a) 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;

(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;

(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; and

(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.

a reference electrode; and

means for detecting an electrical signal, the means being operationally coupled to at least working electrode (a) and reference electrode (b).

The process for the modification of a metal surface, and preferred embodiments thereof, are described in more detail below. In a particularly preferred embodiment, in 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. The term "biosensor" herein relates to an analytical device that is used for the detection of an analyte, wherein the device comprises a biological component.

The term "analyte" herein refers to a substance that is of interest in an analytical procedure. The analyte is discussed in more detail below. The term "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. In a preferred embodiment, 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. In a further preferred embodiment, 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.

Similarly, 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. In a preferred embodiment, said means detect an electrical signal quantitatively, and more preferably said means detect an electrical signal quantitatively and continuously.

The term "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.

The term "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 (H₂O₂). In this embodiment, the electro- active species as described above is thus hydrogen peroxide.

In Figure 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.

Working electrode

As described above, 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:

(4) the linker moiety, if present, is covalently bonded to the enzyme and to the alkyloxy or alkenyloxy moiety.

In a preferred embodiment, the modified metal surface comprises the following features:

(4) the 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. In other words, the working electrode comprises a metal, and the surface of said metal, or a part thereof, is modified. In particular, (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.

The term "modified metal surface" herein 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.

Optionally, 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. In a preferred embodiment, 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. For example Charnley et al., Reactive and Functional Polymers , 2011, 71, 329 - 334, Banerjee et al., Advanced Materials 2011, 23, 690 - 718 and Dalsin et al , Materials Today 2005, 8, 38 - 46, all incorporated by reference, review several classes of compounds having anti-fouling properties. Examples of suitable 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. Zhao et al., Biomaterials 2013, 34, 4714 - 4724, incorporated by reference), 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), and zwitterionic linkers as sulfobetaines (e.g. Nguyen et al., Langmuir 2012, 28, 604 - 610, incorporated by reference), carboxybetaines (e.g. Braul et al., Analytical Chemistry 2013, 85, 1447 - 1453, incorporated by reference), sulfopyridinium betaines (e.g. Meng et al., Journal of Membrane Science 2014, 461, 123 - 129, incorporated by reference), phosphoryl choline (e.g. Su et. al., Journal of Membrane Science 2008, 322 111 - 111, incorporated by reference) and cysteine derivatives (e.g. Rosen et al., Langmuir 2011, 27, 10507 - 10513, incorporated by reference).

In a preferred embodiment of the working electrode (a), a linker moiety is present in between the enzyme and the alkyloxy or alkenyloxy moiety. In a further preferred embodiment, the linker moiety is a compound having anti-fouling properties. In a further preferred embodiment, 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. In Figures 2, 3, 4 and 5, 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. In Figures 2 and 3, an enzyme E (5) is covalently bonded to the alkyloxy or alkenyloxy group. In Figures 4 and 5, a linker moiety G (7) is present in between the alkyloxy or alkenyloxy moiety and the enzyme E (5). In Figures 2, 3, 4 and 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. In other words, 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. For example a carboxyl group, carboxylate group, an aldehyde group or an amine 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. In addition, other functional groups may be introduced into the enzyme prior to attachment to the alkyloxy or alkenyloxy moiety. Examples of functional groups that may be present in the alkyloxy or alkenyloxy moiety, as well as the process to covalently attach the enzyme to the alkyloxy or alkenyloxy moiety, are described in more detail below.

In Figures 4 and 5 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). In a preferred embodiment, 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. Preferably 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.

In a preferred embodiment, 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.

More preferably, 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-quinone, L-galactonolactone, albonoursin, aclacinomycin-A, D-aspartate, L-amino-acids, D-amino-acids, amines, pyridoxal 5'- phosphate, D-glutamate, ethanolamine, putrescine, L-glutamate, L-lysine, L-aspartate, glycine, primary-amines, diamines, pseudooxynicotine, sarcosine, N-methyl-L-amino- acids, N6-methyl-lysine, (S)-6-hydroxynicotine, (R)-6-hydroxynicotine, L-pipecolate, dimethylglycine, dihydrobenzophenanthridine, Nl-acetylpolyamine, Nl- acetylspermidine, Nl-acetylspermine, N8-acetylspermidine, spermine, polyamine, L- saccharopine, 4-methylaminobutanoate, N-alkylglycine, 4-methylaminobutanoate, nitroalkanes, N-acetylindoxyl, urate, thiols, glutathione, methanethiol, S-prenyl-L- cysteine, farnesylcysteine, 3-hydroxyanthranilate, rifamycin B, 2-amino-4- hydroxypteridine, heterocyclic compounds, xanthine, heterocyclic compounds, 6- hydroxynicotinate, reticuline, cannabigerolate and cannabidiolic acid.

Even more preferably, 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 amine, polyamine, N-alkylglycines, thiols, glutathione, heterocyclic compounds, xanthine

Yet even more preferably, the analyte is selected from the group consisting of glucose, lactate, cholesterol, histamine, heterocyclic compounds, L-phenylalanine and D-aspartate.

Most preferably, 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. In a preferred embodiment, 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. In a further preferred embodiment, 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.

More preferably, 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-alcohol oxidase (EC 1.1.3.18), a 4-hydroxymandelate oxidase (EC 1.1.3.19), a long-chain-alcohol oxidase (EC 1.1.3.20), a glycerol-3 -phosphate oxidase (EC 1.1.3.21), a thiamin oxidase (EC 1.1.3.23), a hydroxyphytanate oxidase (EC 1.1.3.27), an N-acylhexosamine oxidase (EC 1.1.3.29), a vanillyl-alcohol oxidase (EC 1.1.3.38), a nucleoside oxidase (H₂O₂- forming) (EC 1.1.3.39), a D-mannitol oxidase (EC 1.1.3.40), an alditol oxidase (EC 1.1.3.41), an aclacinomycin-N oxidase (EC 1.1.3.45), an aldehyde oxidase (EC 1.2.3.1), a pyruvate oxidase (EC 1.2.3.3), an oxalate oxidase (EC 1.2.3.4), a glyoxylate oxidase (EC 1.2.3.5), an indole-3-acetaldehyde oxidase (EC 1.2.3.7), an aryl-aldehyde oxidase (EC 1.2.3.9), an acyl-CoA oxidase (EC 1.3.3.6), a dihydrouracil oxidase (EC 1.3.3.7), a tryptophan a,B-oxidase (EC 1.3.3.10), a pyrroloquinoline-quinone synthase (EC 1.3.3.11), an L-galactonolactone oxidase (EC 1.3.3.12), an albonoursin synthase (EC 1.3.3.13), an aclacinomycin-A oxidase (EC 1.3.3.14), a D-aspartate oxidase (EC 1.4.3.1), an L-amino-acid oxidase (EC 1.4.3.2), a D-amino-acid oxidase (EC 1.4.3.3), an amine oxidase (EC 1.4.3.4), a pyridoxal 5'-phosphate synthase (EC 1.4.3.5), a D- glutamate oxidase (EC 1.4.3.7), an ethanolamine oxidase (EC 1.4.3.8), a putrescine oxidase (EC 1.4.3.10), an L-glutamate oxidase (EC 1.4.3.11), an L-lysine oxidase (EC 1.4.3.14), a D-glutamate(D-aspartate) oxidase (EC 1.4.3.15), an L-aspartate oxidase (EC 1.4.3.16), a glycine oxidase (EC 1.4.3.19), an L-lysine 6-oxidase (EC 1.4.3.20), a primary-amine oxidase (EC 1.4.3.21), a diamine oxidase (EC 1.4.3.22), a pseudooxynicotine oxidase (EC 1.4.3.24), a sarcosine oxidase (EC 1.5.3.1), an N- methyl-L-amino-acid oxidase (EC 1.5.3.2), an N6-methyl-lysine oxidase (EC 1.5.3.4), an (S)-6-hydroxynicotine oxidase (EC 1.5.3.5), an (R)-6-hydroxynicotine oxidase (EC 1.5.3.6), an L-pipecolate oxidase (EC 1.5.3.7), a dimethylglycine oxidase (EC 1.5.3.10), a dihydrobenzophenanthridine oxidase (EC 1.5.3.12), an Nl-acetylpoly amine oxidase (EC 1.5.3.13), a polyamine oxidase (propane- 1, 3 -diamine-forming) (EC

1.5.3.14) , an N8-acetylspermidine oxidase (propane- 1,3 -diamine-forming) (EC

1.5.3.15) , 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 1.8.3.4), a prenylcysteine oxidase (EC 1.8.3.5), a farnesylcysteine lyase (EC 1.8.3.6), a 3-hydroxyanthranilate oxidase (EC 1.10.3.5), a rifamycin-B oxidase (EC 1.10.3.6), a pteridine oxidase (EC 1.17.3.1), a xanthine oxidase (EC 1.17.3.2), a 6-hydroxynicotinate dehydrogenase (EC 1.17.3.3), a reticuline oxidase (EC 1.21.3.3), a tetrahydrocannabinolic acid synthase (EC 1.21.3.7) and a cannabidiolic acid synthase (EC 1.21.3.8).

Even more preferably, 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 1.1.3.29), a nucleoside oxidase (H₂0₂-forming) (EC 1.1.3.39), an D-mannitol oxidase (EC 1.1.3.40), an alditol oxidase (EC 1.1.3.41), an aldehyde oxidase (EC 1.2.3.1), a pyruvate oxidase (EC 1.2.3.3), an oxalate oxidase, (EC 1.2.3.4), a glyoxylate oxidase (EC 1.2.3.5), an aryl-aldehyde oxidase (EC 1.2.3.9), an acyl-CoA oxidase (EC 1.3.3.6), a tryptophan a,B-oxidase (EC 1.3.3.10), 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 amine oxidase (EC 1.4.3.4), an D-glutamate oxidase (EC 1.4.3.7), a putrescine oxidase (EC 1.4.3.10), an L-glutamate oxidase (EC 1.4.3.11), an L-lysine oxidase (EC 1.4.3.14), an D-glutamate(D-aspartate) oxidase (EC 1.4.3.15), an L-aspartate oxidase (EC 1.4.3.16), a glycine oxidase (EC 1.4.3.19), an L-lysine 6-oxidase (EC 1.4.3.20), a primary-amine oxidase (EC 1.4.3.21), a diamine oxidase (EC 1.4.3.22), an N-methyl-L-amino-acid oxidase (EC 1.5.3.2), an Nl-acetylpoly amine oxidase (EC 1.5.3.13), a non-specific polyamine oxidase (EC 1.5.3.17), an N-alkylglycine oxidase (EC 1.5.3.20), a thiol oxidase (EC 1.8.3.2), a glutathione oxidase (EC 1.8.3.3), and a xanthine oxidase (EC 1.17.3.2).

Yet even more preferably, 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).

Most preferably, 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).

It should be noted that in the prior art the term "lactate oxidase" is often used to refer to (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).

As described above, 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. For example when the analyte to be detected is lactate, it is preferred that the enzyme is (S)-2-hydroxy-acid oxidase. When the analyte to be detected is glucose, it is preferred that the enzyme is a glucose oxidase, a hexose oxidase, or a pyranose oxidase. When the analyte to be detected is cholesterol, it is preferred that the enzyme is cholesterol oxidase. When the analyte to be detected is histamine, it is preferred that the enzyme is diamine oxidase. When the analyte to be detected is D- aspartate, it is preferred that the enzyme is a D-amino-acid oxidase, a D-glutamate (D- aspartate) oxidase or a D-aspartate oxidase.

As described above, 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 (H₂O₂). 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.

As an example, the process for the detection of lactate is schematically shown in Figure 6. 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. When the analyte to be detected is lactate, 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 (0₂) are converted into pyruvate and hydrogen peroxide (H₂O₂) by the enzyme. Hydrogen peroxide migrates through layer (8) to the surface of the metal, where it is oxidized, resulting in an electrical signal.

Similary, when the analyte to be detected is glucose, the enzyme that is attached to the working electrode is preferably a glucose oxidase, a hexose oxidase or a pyranose oxidase. When the enzyme is a glucose oxidase or a hexose oxidase, β-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. When the enzyme is a pyranose oxidase, β-D-glucose is converted into 2- dehydro-D-glucose by the enzyme, under formation of hydrogen peroxide.

As described in more detail above, 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.

In a preferred embodiment, the working electrode comprises a film of the metal, wherein an outer surface of the metal, or a part thereof, is modified. The term "outer surface" herein 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.

Preferably the working electrode comprises at least one skin-piercing means. In a further preferred embodiment 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. In a further preferred embodiment, 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 μπι and about 1 mm, preferably between about 10 μπι and about 500 μπι, and more preferably between about 30 μπι and about 200 μπι. The outer diameter is typically between about 10 nm and about 1 mm, preferably between about 1 μπι and about 500 μπι, and more preferably between about 10 μπι and about 100 μπι. 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. Alternatively, 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. polycarbonate, polymethacrylic acid, ethylenevinyl acetate, polytetrafluoroethylene (TEFLON™), polyesters), etc. Methods to apply a layer of a metal as a coating are also known in the art. For example WO 2009/097660, US 2009/0297913 and US 2013/216694, all incorporated by reference, disclose methods for the coating of microneedles with a metal. Deposition of a metal on a surface via a sputtering process is e.g. described in Li et al., Surface Science 2003, 529, 410 - 418, incorporated by reference.

In a preferred embodiment, 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.

When the working electrode comprises an array of microneedles, 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.

When the device according to the invention is to detect an analyte in interstitial fluid, it is preferred that 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

The term "reference electrode" herein 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).

Means for detecting an electrical signal

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.

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. 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. In 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.

In a preferred embodiment of the device according to the invention, 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. When 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 presence of a linker moiety having anti-fouling properties in between the alkyloxy or alkenyloxy moiety and the enzyme prevents fouling of the working electrode.

In addition, 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

As described above, 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;

(2) 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);

(2) 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);

Said modified metal surface is described in more detail above.

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.

In a first embodiment, the invention relates to a process for the modification of a metal surface, the 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; and

(ii) reacting the oxidized metal surface 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. In this embodiment, the metal surface is modified, i.e. functionalized, with alkyloxy moieties or alkenyloxy moieties, the alkyloxy moieties or alkenyloxy moieties being covalently bonded to the metal surface. In a preferred embodiment of the process for the modification of a metal surface, 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.

In a preferred embodiment of the process according to the invention, 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. In a further preferred embodiment of the process, the metal surface is comprised in an array of microneedles.

In this embodiment, 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.

In step (i), an oxidized metal surface is provided. The metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au. Preferably, the metal is selected from the group consisting of Pt, Ag and Au, more preferably Pt and Au. Most preferably, the metal is Pt.

The term "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. As will be clear to a person skilled in the art, 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.

In an oxidized metal surface, typically 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. As an example, when the metal is platinum (Pt), the oxidized Pt surface may e.g. comprise -OH groups, platinum(II) oxide (PtO), platinum(IV) oxide (Pt0₂), and/or a combination thereof. In a preferred embodiment, the oxidized Pt surface comprises a mixture of platinum(II) oxide (PtO) and platinum(IV) oxide (Pt0₂).

As another example, when the metal is gold (Au), the oxidized Au surface may e.g. comprise -OH groups, gold(I) oxide (Au₂0), gold(III) oxide (Au₂0₃), and/or a combination thereof. In a preferred embodiment, the oxidized Au surface comprises gold(I) oxide (Au₂0). 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.

In a wet etching process, 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 HN0₃ (commercially available solution at 70%) or in a mixture, e.g. a 3 : 1 to 5: 1 mixture (preferably a 4: 1 mixture) of H₂S0₄/H₂0₂, 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 0₂ 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.

In a plasma activation process, 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). In a preferred embodiment, in step (i) the oxidized metal surface is provided by exposing a metal surface to an oxygen plasma.

In 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.

In this step of the process, 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)). In a preferred embodiment, the attachment of the alkene occurs via a Markovnikov-type addition. In another preferred embodiment, 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.

When the reaction in step (ii) is a thermal reaction, it is preferred that the reaction is performed at an elevated temperature. Preferably, 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. As will be clear to a person skilled in the art, the temperature range wherein the reaction is performed depends, amongst others, on the nature of the alkene or alkyne.

When the reaction in step (ii) is a photochemical reaction, it is preferred that the reaction is performed under the action of radiation. In a preferred embodiment, 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.

In a preferred embodiment, the reaction in step (ii) is a thermal reaction.

In another preferred embodiment, in step (ii) the oxidized metal surface is reacted with an alkene.

In a preferred embodiment, in step (ii) the oxidized metal surface is reacted with an alkene according to Formula (1) or an alkyne according to Formula (2):

A- Q B- Q

J n (i) (2)

wherein:

n is an integer in the range of 1 to 5.

A is a linear, branched or cyclic C₂ - C50 alkenyl group, the alkenyl group being a 1-alkenyl group or an internal alkenyl group;

B is a linear, branched or cyclic C₂ - 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 -XR²,

- R²R³, -CH=CR²R³, -C_≡CR², - R²-C(0)-N(R²)₂, -0-[(C(R⁴)₂)_pO]_q-R²

-C(X)XR , -C(X)R , -C(X) R²R³, -S(0)OR¹, -S(0)₂OR¹, -S(0) R²R³,

-OP(0)(OR¹)₂, wherein:

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; R¹ is a linear, branched or cyclic Ci - C₁₂ alkyl group; a phenyl group; a C₇ - Ci2 alkaryl group; or a C₇ - C₁₂ arylalkyl group; wherein the alkyl, phenyl, alkaryl and arylalkyl groups are optionally substituted with one or more of F or CI;

R² and R³ are independently selected from the group consisting of hydrogen and R¹;

R⁴ is independently selected from hydrogen or Ci - C₄ alkyl; and R⁵ is a monofunctional hydroxy or thiohydroxy protecting group. In a further preferred embodiment, in step (ii) the oxidized metal surface is reacted with an alkene according to Formula (1).

R⁵ is a monofunctional hydroxy or thiohydroxy protecting group. Such protecting groups are well known in the art. Also 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). However, it is preferred that 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(R¹)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.

A is a linear, branched or cyclic C₂ - C₅o, preferably C₂ - C40, more preferably C₂ - C30, even more preferably C₄ - C₂₀, yet even more preferably C₄ - C₁₂, most preferably C₆ - Cn alkenyl group, the alkenyl group comprising a 1-alkenyl group and/or an internal alkenyl group. In a preferred embodiment, 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. In another preferred embodiment, 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.

B is a linear, branched or cyclic C₂ - C50, preferably C₂ - C₄₀, more preferably C₂ - C30, even more preferably C₄ - C₂₀, yet even more preferably C₄ - C₁₂, most preferably C₆ - Cn alkynyl group, the alkynyl group comprising a 1-alkynyl group and/oror an internal alkynyl group. In a preferred embodiment, 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. In another preferred embodiment, 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 sp³ 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 sp³ C-atom of the alkynyl group B in (2).

In one embodiment, 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 -XR², - R²R³, -C(X)XR², -C(X)R², -C(X) R²R³, -CI, -Br, -I, -XC(X)R¹, - R²C(X)R¹ and -XR⁵, wherein X, R¹, R², R³ and R⁵ are as defined above. It is further preferred that X is O. Even more preferably, Q is selected from the group consisting of -OR², - R²R³, -C(0)OR², -C(0)R², -C(0) R²R³, -CI, -Br, -I, -OC(0)R¹, - R²C(0)R¹ and -OR⁵.

As defined above, R¹ is a linear, branched or cyclic Ci - C12 alkyl group, a phenyl group, a C₇ - C12 alkaryl group or a C₇ - C12 arylalkyl group, wherein the alkyl, phenyl, alkaryl and arylalkyl groups are optionally substituted with one or more of F atoms or CI atoms. In a preferred embodiment, R¹ comprises one or more F atoms. More preferably, R¹ comprises a -CF₃ group.

Particularly preferred examples of 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.

Particularly preferred examples of an alkyne (2) include trifluoroethyl undec-10- ynoate, succinimidyl undec-10-ynoate, 5-hexyn-l-ol and 10-undecyn-l-ol.

In another embodiment, the invention relates to a process for the modification of a metal surface, the process comprising the steps of:

(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.

In this embodiment, it is preferred that the process comprises the steps of:

(ii) reacting the oxidized metal surface 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;

(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). In this embodiment, when the process comprises steps (i), (ii) and (iv-a), the metal surface is modified, i.e. functionalized, with enzyme-alkyloxy moieties or enzyme-alkenyloxy moieties. When the process comprises steps (i), (ii), (iii) and (iv-b), 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. In a preferred embodiment, 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.

In step (iii) of the process, the alkyloxy- or alkenyloxy-modified metal surface of step (ii) is reacted with a linker moiety. Preferably, 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.

As was described 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, F¹ and F², wherein F¹ is a functional group that is able to react with a functional group on the alkyloxy or alkenyloxy moiety and F² 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):

F^G-F²

(3)

wherein:

G is the linker backbone;

F¹ 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 F² is a functional group able to react with a functional group on the enzyme in step (iv- a) or (iv-b) of the process. F¹ 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, and F² is a functional group that is complementary to a functional group on the enzyme in step (iv-a) or (iv-b). The term "complementary functional groups" herein 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.

F¹ and F² may for example be independently selected from the group consisting of hydrogen, halogen, XR², C₄ - C₁₀ (hetero)cycloalkyne groups, -CH=C(R²)₂, -C≡CR², -[C(R²)₂C(R²)₂0]_q-R², wherein q is in the range of 1 to 200, -CN, -N₃, -NCX, -XCN, -XR⁵, -N(R²)₂, -⁺N(R²)_3, -C(X)N(R²)₂, -C(R²)₂XR², -C(X)R², -C(X)XR², -S(0)R², -S(0)₂R², -S(0)OR², -S(0)₂OR², -S(0)N(R²)₂, -S(0)₂N(R²)_2, -OS(0)R², -OS(0)₂R², -OS(0)OR², -OS(0)₂OR², -P(0)(R²)(OR²), -P(0)(OR²)₂, -OP(0)(OR²)₂, -Si(R¹)₃, -XC(X)R², -XC(X)XR², -XC(X)N(R²)₂, -N(R²)C(X)R², -N(R²)C(X)XR² and -N(R²)C(X)N(R²)₂, wherein X is oxygen or sulphur and wherein R¹, R² and R⁵ are as defined above. Linker backbone G may for example be selected from the group consisting of linear or branched Ci-C₂oo alkylene groups, C₂-C₂oo alkenylene groups, C₂-C₂oo alkynylene groups, C₃-C₂oo cycloalkylene groups, C₅-C₂oo cycloalkenylene groups, C₈- C₂oo cycloalkynylene groups, C₇-C₂oo alkylarylene groups, C₇-C₂oo arylalkylene groups, C₈-C₂oo arylalkenylene groups, C9-C₂oo arylalkynylene groups. Optionally the 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 NR⁵, wherein R⁵ is independently selected from the group consisting of hydrogen, halogen, Ci - C₂₄ alkyl groups, C₆ - C₂₄ (hetero)aryl groups, C₇ - C₂₄ alkyl(hetero)aryl groups and C₇ - C₂₄ (hetero)arylalkyl groups. Most preferably, the heteroatom is O. Preferably, G has anti-fouling properties.

Examples of suitable 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. In step (iv) of the process, the modified metal surface is reacted with an enzyme.

The process may comprise step (iv-a) or step (iv-b). 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). 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).

In a preferred embodiment, 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. In a further preferred embodiment, 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.

1.5.3.14) , an N8-acetylspermidine oxidase (propane- 1,3 -diamine-forming) (EC

Yet even more preferably, 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). Most preferably, 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).

As already described above, it should be noted that in the prior art the term "lactate oxidase" is occasionally used to refer to (S)-2-hydroxy-acid oxidase (EC 1.1.3.15).

Modified metal surface

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:

(ii) reacting the oxidized metal surface 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.

In a preferred embodiment, the invention relates to a modified metal surface, obtainable by a process comprising the steps of:

(ii) reacting the oxidized metal surface 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. 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.

Steps (i) and (ii) of said process are described in more detail above. In a preferred embodiment, 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):

-O— Y^Q -O— Z^Q (3) (4)

wherein:

Y is a linear, branched or cyclic C₂ - C50 alkylene group;

Z is a linear, branched or cyclic C₂ - C50 alkenylene group, the alkenylene group being a 1 -alkenylene group or an internal alkenylene group; and

Q and n are as defined above. In a particular embodiment, the alkyloxy or alkenyloxy moieties are according to Formula (5) or (6):

0 -Y- -Q -o -Q

' J X

(5) (6)

wherein:

Q and n are as defined above;

Y is a branched C₂ - C₅o alkylene group;

Z is a branched C₂ - C50 alkenylene group, the alkenylene group being an internal alkenylene group; and

x is an integer in the range of 2 to 10.

In this embodiment, 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. In this embodiment it is preferred that x is 2, 3, 4, 5 or 6, more preferably x is 2, 3, or 4 and most preferably x is 2 or 3.

In a further preferred embodiment, 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:

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).

In a preferred embodiment, the modified metal surface is obtainable by a process comprising the steps of:

(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. Steps (i), (ii), (iii), (iv-a) and (iv-b) of said process are described in more detail above. In a preferred embodiment, 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. In a further preferred embodiment, in step (iii) 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. In an embodiment, the invention therefore relates to an electrode, comprising a modified metal surface, wherein:

(4) the 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:

(3) 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:

(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;

(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; and

In a preferred embodiment, 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:

(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;

Steps (i), (ii), (iii), (iv-a) and (iv-b) of said process are described in more detail above.

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.

Examples

General

Chemicals

1-Hexadecene (standard for GC, >99.5%), 1-octadecene (standard for GC,

>99.5%), ethylene diamine (99.5%), tris(2-aminoethyl)amine (96%), 2- bromoisobutyryl bromide (98%), triethylamine (NEt₃, >99%), [2-(methacryloyloxy)- ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (97%), [3-(methacryloylamino)- propyl]dimethyl(3-sulfopropyl)ammonium hydroxide inner salt (96%), copper-(I) chloride (99.995%) (CuCl), copper-(II) chloride (99.995%) (CuCl₂), 2,2^'-bipyridine (99%), sodium citrate tribasic dehydrate (>98%), D(+)-glucose (>99.5%), and Na₂HP0₄ (>99%) were purchased from Sigma- Aldrich. 1-octadecyne (>95%) was obtained from TCI Europe N. V. and distilled twice under reduce pressure before use. KC1 (p.a.) and KH₂P0₄ (p.a.) were provided by Merck KGaA. NaCl (p.a., 99.5%) was obtained from Acros Organics. Glutaraldehyde (50% aqueous solution) was provided by Alfa Aesar. Acetone, isopropanol, ethanol and CH₂C1₂ were purchased from Sigma- Aldrich (HPLC grade). For surface modification CH₂C1₂ 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), Na₂HP0₄ (1.41 g/L), KH₂P0₄ (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.

X-ray photoelectron spectroscopy (XPS) measurements

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).

Static water contact angle measurements

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°).

Infrared reflection absorption spectroscopy (IRRAS)

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.

Estimation of the thickness of the oxidized Pt layer

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.

ox

In this equation, IoJIp_t is the ratio of the Pt 4f peak area in the oxidized layer and the metallic layer, Θ is the takeoff angle (angle between the sample and the detector: 80°), do_x is the thickness of the oxide layer, λ 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/cm³ (density of the hydrated Pt0₂)' as described in A. Cotton, G. Wilkinson, C. A. Murillo, M. Bochmann, "Advanced Inorganic Chemistry", 6^th ed., Wiley, New York 1999, incorporated by reference. The escape depth of Pt 4f photoelectrons through the oxide overlayer is assumed to be about the same as in Pt metal, which can be approximated using the empirical equation (2), as described in Briggs et al. (see above). λ=0Λ\α^{1 5}Ε ^{0 5} (2)

In this equation, 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. Based on Eq. (2), λ 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. 1991, 95, 7017-7021; Geissler et al, "Comparative Study of Monolayers Self-Assembled from Alkylisocyanides and Alkanethiols on Polycrystalline Pt Substrates", Langmuir 2004, 20, 6993-6997, both incorporated by reference): d= - pt₄f cosy ln(Pt₄f^Grafted/Pt₄f^0x) (3) where Pt_4f ^Grafted is the intensity of the Pt4f signal for the oxidized Pt film grafted with the alkyl layer, Pt_4f ^0x is the intensity of the Pt4f signal from the oxidized Pt substrate, λ is the attenuation length of the Pt_4f electrons in the hydrocarbon layer, and γ is the electron take-off angle relative to the sample surface normal (10°). The value of λ was determined from the following empirical expression, if monochromatic Al Ka irradiation is used as the X-ray source (Wallart et al, "Quantitative XPS Characterization of Organic Monolayers on Silicon: Study of Alkyl and Alkoxy Monolayers on H-Si(l l l)", J. Am. Chem. Soc. 2005, 127, 7871-7878, incorporated by reference): λ(Α) = 9.0 + 0.022(1487 - E) (4)

For Pt_4f photoelectrons with bonding energy of 73 eV, equation 4 results in a value of 4.01 nm for λ

Preparation of oxidized platinum substrates

Platinum films (200 nm thickness) were sputtered over 6 μπι thermal silicon oxide on silicon wafers, using lOnm Ta as adhesion layer. Pt pieces (l x l or 1 x2.5 cm² for IRRAS) were cleaned by sonication using a solvent series with increasing polarity: hexane, CH₂CI₂ 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

30 min and stored in an Ar-glovebox until further utilization.

In 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.

Table 1. XPS binding energy and curve fittings in % for a freshly oxidized Pt film.

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 (Pt₄f7/₂) and at 74.5 eV (Pt₄f₅/₂) 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). The Pt4n/₂ peak at 72.0 eV and Pt₄f₅/₂ peak at 75.3 eV correspond to Pt(OH)₂ chemical species while the Pt₄f signals of Pt0₂ appear at 73.3 eV (PUnn) and at 77.2 eV (Pt₄f₅/₂). Integration of Pt₄f components permits to assess the composition of the oxidized Pt films as a mixture of Pt/Pt(OH)₂/Pt0₂ in a ratio 2.3/1.1/1.

Table 2: XPS Pt 4f binding energies and curve fittings for a freshly oxidized Pt film

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 Pt0₂ while the peak of oxygen of Pt(OH)₂ species appears at 531.1 eV. The ratio 01s(Pt(OH)₂)/01s(Pt0₂) is 1.1. This value would correspond to Pt(OH)₂/Pt0₂ ~ 1 and corroborates the composition of the Pt oxide thin layer we determined from the Pt₄f 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.

Table 3: XPS O Is binding energies and curve fittings for a freshly oxidized Pt film

Covalent attachment of alkene- or alkyne-derived organic layers

Method A

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). After reaction, the substrate was rinsed with CH₂CI₂ and sonicated for 3 min in CH₂CI₂. The substrate was dried with Ar and stored in an Ar-glovebox.

Method B

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 CH₂CI₂, and sonicated for 3 min in CH₂CI₂ before being dried with a stream of dry argon.

Example 1: Modified metal surfaces

Several examples 1 A - 1L of modified metal surfaces were prepared as described below. These examples are shown in Table 4.

Table 4: Examples of modified metal surfaces.

Example Alkene / alkyne Linking unit (G) Enzyme (E)

1A 1-hexadecene

IB 1- octadecyne

1C 2,2,2-Trifluoroethyl undec- 10-enoate (TFEE)

ID 2,2,2-Trifluoroethyl undec- tris(2-aminoethyl)- 10-enoate (TFEE) amine

IE 2,2,2-Trifluoroethyl undec- tris(2-aminoethyl)- 10-enoate (TFEE) amine and

glutaraldehyde

IF 2,2,2-Trifluoroethyl undec- tris(2-aminoethyl)- human HAOX- 10-enoate (TFEE) amine and enzyme

glutaraldehyde

1G 2,2,2-Trifluoroethyl undec- ethylenediamine

10-enoate (TFEE)

1H Ethylendiamine + TFEE Isobutyryl

11 2,2,2-Trifluoroethyl undec- Ethylenediamine, Example Alkene / alkyne Linking unit (G) Enzyme (E)

10-enoate (TFEE) isobutyryl and

poly(sulfobetaine)-N- methy 1 aery 1 ami de

1J tris(2-aminoethyl)-amine Isobutyryl

and TFEE

IK 2,2,2-Trifluoroethyl undec- tris(2-aminoethyl)- 10-enoate (TFEE) amine, isobutyryl and

poly(sulfobetaine)-N- methylacrylate

1L 2,2,2-Trifluoroethyl undec- tris(2-aminoethyl)- glucose oxidase

10-enoate (TFEE) amine and

glutaraldehyde

Example I A

An oxidized Pt surface was reacted with 1-hexadecene according to Method B as described above, at 120°C for 16 h.

In Table 5 the surface composition and organic layer thickness (determined by

XPS) and static water contact angle for Example 1 A are shown.

Table 5: Surface composition and organic layer thickness (XPS) and static water contact angle for Example 1A.

In Table 6 the XPS C Is binding energies and curve fittings for Example 1A are shown.

Table 6: XPS C Is binding energies and curve fittings for Example 1A.

Species Curve fitting

[binding energy, eV] (%)

C-C [285 eV] 93.5 Species Curve fitting

[binding energy, eV] (%)

C-0 [286.9 eV] 6.5

Deconvolution of the XPS high resolution Cls spectrum shows two contributions. The binding peak at 286.9 eV is assigned to a C-0 bond (cf. J. ter Maat et al, Langmuir 2009, 25, 11592-11597, incorporated by reference), and the binding peak at 285.0 eV to the CHx alkyl chain. Analysis of the Cls peak areas in Table 3 yields a C-O/C-C area ratio around 0.07. This value is very close to the stoichiometrical ratio obtained (0.066), considering that one carbon atom from 1- hexadecene is oxidized to a C-O-Pt linkage upon binding to the surface.

In Table 7 the static water contact angle and organic layer thickness as determined by XPS for Example 1 A, after various modification times, are shown.

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.

Infrared reflection-absorption spectroscopy (IRRAS) spectra of hexadecyl functionalized layers exhibit the symmetric and antisymmetric -CH₂- stretching peaks at 2855 cm^-1 and at 2928 cm^-1 respectively. These data indicate that the adsorbed alkyl layer is not ordered (see M. D. Porter et al, J. Am. Chem. Soc. 1987, 109, 3559-3568, incorporated by reference).

Example IB

An oxidized Pt surface was reacted with 1-octadecyne according to Method B as described above, at 120°C for 16 h.

In Table 8 the surface composition, organic layer thickness (determined by XPS) and static water contact angle for Example IB are shown. In 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.

Table 9. XPS C Is binding energies and curve fittings in % for Example

Species Curve fittings

[binding energy, eV] (%)

C-C [285 eV] 97.3

C-0 [287 eV] 2.7 Deconvolution of the XPS high resolution Cls spectrum shows two contributions. The binding peak at 287.0 eV is assigned to a C-0 bond (cf. J. ter Maat et al, Langmuir 2009, 25, 11592-11597, incorporated by reference), and the binding peak at 285.0 eV to the CHx alkyl chain. Analysis of the Cls peak areas in Table 3 yields a C-O/C-C area ratio around 0.028. 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. The 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.

Example 1C

An oxidized Pt surface was reacted with 2,2,2-trifluoroethyl undec-10-enoate (TFEE) according to Method B as described above, at 120°C for 16 h.

In Table 10 the surface composition, organic layer thickness (determined by XPS) and static water contact angle for Example 1C are shown.

Table 10: Surface composition, organic layer thickness (XPS) and static water contact angle of Example 1C.

XPS analysis on the TFEE grafted substrates showed that elements Pt, O, C, and F were present at the surface. For a 1.5 nm thick layer the experimental Cls/Fls ratio was calculated to be 4.2 which is in good agreement with the theoretical value of 4.3. The C/O ratio of 4.0 is near to the theoretical value 4.3 for a monolayer of oxygen atoms bound to a TFEE monolayer. Besides, the estimated O/F ratio was 1.1, which is closed to the theoretical rate 0/F=l that would correspond to a perfect monolayer of oxygen atoms connected to a monolayer of TFEE.

In Table 11 the XPS C I s binding energies and curve fittings for Example 1C are shown.

Table 11: XPS C Is binding energies, curve fittings in % and surface ratios for Example 1C.

The values in Table 11 indicate that the TFE group remains intact after attachment onto the surface.

In Table 12 the static water contact angle and organic layer thickness as determined by XPS for Example 1C, after various modification times, are shown.

Table 12: Organic layer thickness as determined by XPS for Example 1C after various modification times.

Based on XPS measurements a thickness of 0.4 nm after 6h of reaction was calculated, while grafting for 16h produced a 1.5 nm thick layer. Further extension of the grafting time to 20h and 48h resulted in layers having a thickness of 2.5 nm and 3.0nm thick layers respectively. These results show that the thermal grafting of unsubstituted alkenes on oxidized platinum substrates is not self-limited and may lead to the formation of multilayers.

Further characterization was performed by IRRAS. The C-H stretching area shows a -CH₃ band at 2976 cm^"1. This signal confirms that the attachment of the alkenes to oxidized Pt surfaces occurs via Markovnikov addition, as observed for the modification of silica and silicon carbide (SiC) with alkenes. The antisymmetric and symmetric stretching bands of -CH₂- appear at 2931 cm^"1 and 2858 cm^"1 respectively. These wave number values are characteristic of disordered monolayers. Markovnikov addition may explain why the monolayers remain disordered, since the presence of a methyl group close to the surface does not allow the hydrocarbon chains to closely pack together. Example ID

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.

Table 14: XPS C Is binding energies and curve fittings in % for Example

Species Curve fittings

[binding energy, eV] (%)

C-C [285.1 eV] 52.2

C-O&C-N [286.2 eV] 36.6

-C=0 [288.2 eV] 11.2 Example IE

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.

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

Example IE.

Table 16: XPS C Is binding energies and curve fittings in % for Example

Example IF

Human alpha-hydroxyacid oxidase enzyme (HAOX enzyme) was immobilised on the aldehyde terminated Pt surface of Example IE by depositing 150 microliters of a 0.5 mg/mL solution of HAOX 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, Na₂HP0₄. (sodium phosphate dibasic) 1.41 g/L, KH₂PO₄ (potassium dihydrogen phosphate) 0.27 g/L, KC1 (potassium chloride) 0.20 g/L. Enzyme modified metal surface IF was obtained. Table 17 shows the surface composition and organic layer thickness as determined by XPS of Example IF, and Table 18 shows XPS C Is binding energies and curve fittings in %. Table 17: Surface composition and organic layer thickness as determined by XPS of

Example IF.

Table 18: XPS C Is binding energies and curve fittings in % for Example

Example 1G

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 19: Surface composition and organic layer thickness as determined by XPS of

Example 1G.

Example 1H

An amine terminated surface 1G is reacted with isobutyryl 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 20 shows the surface composition and organic layer thickness as determined by XPS of bromo terminated surface Example 1H.

Table 20: Surface composition and organic layer thickness as determined by XPS of Example 1H.

Example II

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 CuCl₂ (4.8 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum. Subsequently, the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl₂ 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 CuCl₂. Afterward, the mixture was transferred to a reaction flask containing the bromo- terminated surface Example 1H by means of a syringe (argon flushed). Polymerization was carried out under argon pressure with stirring at room temperature for a period of time. The samples were removed and rinsed with warm water (60°C) for 5 min, cleaned by sonication in water, and dried under a stream of argon. As a result a metal surface coated with poly(sulfobetaine)-N-methylacrylamide was obtained.

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.

Table 22: XPS C Is binding energies and curve fittings in % for Example

Example 1J

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 IK

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 CuCl₂ (4.8 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum. Subsequently, the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl₂ 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 CuCl₂. Afterward, the mixture was transferred to a reaction flask containing the bromo-terminated surface Example 1 J by means of a syringe (argon flushed). Polymerization was carried out under argon pressure with stirring at room temperature for a period of time. The samples were removed and rinsed with warm water (60°C) for 5 min, cleaned by sonication in water, and dried under a stream of argon. As a result a metal surface coated with poly(sulfobetaine)-N-methylacrylate was obtained.

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.

Table 25: XPS C Is binding energies and curve fittings in % for Example

Example 1L

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, Na₂HP0₄. (sodium phosphate dibasic) 1.41 g/L, KH₂PO₄ (potassium dihydrogen phosphate) 0.27 g/L, KC1 (potassium chloride) 0.20 g/L.

Enzyme modified metal surface 1L was obtained.

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.

Table 27: XPS C Is binding energies and curve fittings in % for Example

Example 1M: carboxybetaine based zwitterionic polymer

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. The solution was degassed for 30 min by purging with argon. A mixture of CuCl (26.0 mg) and CuCl₂ (3.6 mg) was added to a separate round-bottomed flask under argon (in a glovebox), which was closed with a rubber septum. Subsequently, the degassed solution was transferred to a flask containing a mixture of CuCl and CuCl₂ 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 CuCl₂. Afterward, the mixture was transferred to a reaction flask containing the bromo-terminated surface Example 1J by means of a syringe (argon flushed). Polymerization was carried out under argon pressure with stirring at room temperature for a period of time. The samples were removed and rinsed with warm water (60°C) for 5 min, cleaned by sonication in water, and dried under a stream of argon. As a result a metal surface coated with poly(carboxybetaine)-N-methylacrylamide was obtained.

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.

Example IN

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 composition: 0.15 M NaCl and 0.015 M sodium citrate tribasic solution).

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.

Example 10

Glucose oxidase (GOX enzyme) enzyme was immobilised on the poly(carboxybetaine)-N-methylacrylamide coated Pt surface of Example IM. For that, 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. Finally 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.

Example Immobilization time (h) M C O N Thickness (nm) (%) (%) (%) (%)

10 3 1.3 73.1 11.7 13.9 14.5

Table 32: XPS C Is binding energies and curve fittings in % for Example 10.

Example IP

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, Na₂HP0₄. (sodium phosphate dibasic) 1.41 g/L, KH₂PO₄ (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.

Example 10

Lactate oxidase (LOX enzyme) enzyme was immobilised on the poly(carboxybetaine)-N-methylacrylamide coated Pt surface of Example IM. For that, 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. Finally 100 microliters of a 3 mg/mL solution of LOX enzyme in phosphate buffer saline (PBS) were deposited on the NHS-activated surface of Example 1L for 1 hour at room temperature. (PBS composition: NaCl (sodium chloride) 8.01 g/L, Na₂HP0₄ (sodium phosphate dibasic) 1.41 g/L, KH₂PO₄ (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.

Example 1R

Human alpha-hydroxyacid oxidase enzyme (HAOX enzyme) was immobilised on the aldehyde terminated Pt surface of Example IE by depositing 100 microliters of a 1 mg/mL solution of HAOX enzyme in phosphate buffer saline (PBS) on the aldehyde terminated surface for 1 hour at room temperature. Following, the surface was rinsed thoroughly with phosphate buffer saline (PBS) and covered with 100 microliters of 3% glutaraldehyde in saline sodium citrate buffer (SSC) for lh. After rinsing with saline sodium citrate buffer (SSC), the HAOX enzyme and glutaraldehyde coupling steps were repeated once. HAOX enzyme immobilization protocol was completed with a final HAOX coupling step.

Enzyme modified metal surface 1R was obtained.

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 cm²) 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.

The results are shown in Figure 7. 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). The activity of the immobilized enzyme follows a Michaelis-Menten type kinetics.

Example 2b: Chronoamperometry experiments for a glucose biosensor

Chronoamperometry experiments were performed in a PGstatlOO Autolab potentiostat (Metrohm Autolab, U.K.) 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 IN and 10 surfaces (see above, geometric area 1 cm²) 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 8 mL of the blank phosphate buffer in such way that the final volume (8 mL) remained constant.

The results are shown in Figure 8. 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.

Example 3: Chronoamperometry experiments for a lactate biosensor

Chronoamperometry experiments were performed in a PGstatlOO Autolab potentiostat (Metrohm Autolab, U.K.) 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 IP, 1Q and 1R surfaces (see above, geometric area 1 cm²) were used as a working electrode. The electrode response to variation in lactate concentrations was quantify by adding aliquots of 0.02M lactate solution to 8 mL of the blank phosphate buffer in such way that the final volume (8 mL) remained constant.

The results are shown in Figure 9. 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%. Similarly when immersed in deionized (DI) water, 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%.

These results indicate that the stability of alkyloxy layers grafted on oxidized Pt is well comparable to that of alkyl thiols on Au (N. S. Bhairamadgi et al., Langmuir 2014, 30, 5829-5839, incorporated by reference).

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.

Atomic concentration and C/Pt ratio

C% 0% Pt% C/Pt C/PtNorm

As prepared 65.5 10.2 24.3 2.70 1

After 24h in DI water 59.5 12.5 28.0 2.12 0.79

Claims

1. Device for the detection of an analyte in a fluid, the device comprising:

(a) a working electrode comprising a modified metal surface, wherein:

(b) a reference electrode; and

2. Device according to claim 1, wherein said modified metal surface is obtainable by a process comprising the steps of:

Device according to claim 1 or claim 2, wherein the working electrode comprises a plurality of microneedles.

Device according to claim 3, wherein the microneedles are solid microneedles.

Device according to any one of the previous claims, wherein the modified metal surface is a modified Pt surface or a modified Au surface.

Device according to any one of the previous claims, wherein the enzyme is selected from the group consisting of hydrogen peroxide forming oxidases, wherein a hydrogen peroxide forming oxidase is defined as an oxidoreductase that catalyses an oxidation-reduction reaction involving molecular oxygen as electron acceptor and wherein hydrogen peroxide is formed during the reduction of the molecular oxygen.

Device according to any one of the previous claims, wherein the device is incorporated in an adhesive patch.

Process for the modification of a metal surface, the process comprising the steps of:

Process according to claim 8, wherein in step (ii) the oxidized metal surface is reacted with an alkene according to Formula (1) or an alkyne according to Formula (2):

A- -Q B- -Q

J n

(1) (2)

wherein:

n is an integer in the range of 1 to 5.

Q is hydrogen or a functional group selected from the group consisting of -XR², - R²R³, -CH=CR²R³, -C≡CR², - R²-C(0)-N(R²)₂, -0-[(C(R⁴)₂)_pO]_q-R², -C(X)XR², -C(X)R² -C(X) R²R³, -S(0)OR¹, -S(0)₂OR¹, -S(0) R²R³,

-OP(0)(OR¹)₂, wherein: 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;

R¹ is a linear, branched or cyclic Ci - C₁₂ alkyl group; a phenyl group; a C₇ - Ci₂ alkaryl group; or a C₇ - C₁₂ arylalkyl group; wherein the alkyl, phenyl, alkaryl and arylalkyl groups are optionally substituted with one or more of F or CI;

R⁴ is independently selected from hydrogen or Ci - C₄ alkyl; and R⁵ is a monofunctional hydroxy or thiohydroxy protecting group.

10. Process according to claim 9, wherein Q is in the co-position of the alkene or alkyne.

11. Process according to any one of claims 8 - 10, wherein the metal is selected from the group consisting of Pt and Au.

12. Process according to any one of claims 8 - 11, wherein the enzyme is selected from the group consisting of hydrogen peroxide forming oxidases, wherein a hydrogen peroxide forming oxidase is defined as an oxidoreductase that catalyses an oxidation-reduction reaction involving molecular oxygen as electron acceptor and wherein hydrogen peroxide is formed during the reduction of the molecular oxygen.

13. Modified metal surface obtainable by the process according to any one of claims 8 - 12.

14. Electrode comprising the modified metal surface according to claim 13.

15. Biosensor comprising the modified metal surface according to claim 13.