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WO2009066080A2 - Réseau - Google Patents

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Publication number
WO2009066080A2
WO2009066080A2 PCT/GB2008/003904 GB2008003904W WO2009066080A2 WO 2009066080 A2 WO2009066080 A2 WO 2009066080A2 GB 2008003904 W GB2008003904 W GB 2008003904W WO 2009066080 A2 WO2009066080 A2 WO 2009066080A2
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Prior art keywords
dithio
disulfide
bis
array
sam
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WO2009066080A3 (fr
Inventor
Gianfranco Gilardi
Andrea Fantuzzi
Vikash Rajnikant Dodhia
Lok Hang Mak
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NanoBioDesign Ltd
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NanoBioDesign Ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D1/00—Processes for applying liquids or other fluent materials
    • B05D1/18—Processes for applying liquids or other fluent materials performed by dipping
    • B05D1/185—Processes for applying liquids or other fluent materials performed by dipping applying monomolecular layers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00635—Introduction of reactive groups to the surface by reactive plasma treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00632—Introduction of reactive groups to the surface
    • B01J2219/00637—Introduction of reactive groups to the surface by coating it with another layer
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00657—One-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides

Definitions

  • Immobilisation of proteins is an important aspect in the production of nano-devices, as sensitivity may depend on both concentration of the protein on the surface and specific orientation of the protein.
  • a common example of a SAM is an alkanethiol on gold.
  • Sulphur has particular affinity for gold, with a binding energy in the range of 20-35 kcal/mol (85-145 kJ/mol).
  • An alkane with a thiol head group will stick to the gold surface and form an ordered assembly with the alkyl chains packing together due to van der Waals forces.
  • the extended alkyl chains typically orient with an angle of ⁇ 30 degrees from the perpendicular of the substrate and are assumed to be in a fully extended linear arrangement.
  • alkane thiol molecules first bind to the gold surface in a 'lying down' position, where the alkyl chain tails of the molecules lie flat on the gold surface.
  • the thiol interaction provides about 20-30 kcal/mol (85-130 kJ/mol) of driving force for the initial binding.
  • These binding events continue until the lying down molecules are dense enough on the surface to interact with each other.
  • the alkyl chains lift off the substrate and point outwards, tethered by the thiol anchor to the surface.
  • cystamine-maleimide as a SAM
  • a two-step process is required to prepare the SAM. Firstly, the cystamine is deposited onto the gold surface and, in a second step, 3-maleimidopropionic acid N-hydroysuccinimide ester is joined to the deposited cystamine to produce a maleimide terminated SAM.
  • 3-maleimidopropionic acid N-hydroysuccinimide ester is joined to the deposited cystamine to produce a maleimide terminated SAM.
  • An even bigger problem is that the reaction between the cystamine and 3- maleimidopropior ⁇ c acid N-hydroxysuccinimide ester is not totally specific.
  • the reaction product shown in Figure IA is the desired product that allows covalent linkage to surface accessible cysteines in a protein, such as a cytochrome P450.
  • the GATRTM (Grazing angle Attenuated Total Reflectance) is a single reflection ATR accessory designed for analysing monolayers and adsorbed species on semiconductor and metallic substrates.
  • the present invention resides in a self-assembly monolayer (S AM) on a metal surface, the monolayer comprising maleimido(C 2 -C 28 )alkanethio groups bound to the metal surface via the sulphur atom.
  • S AM self-assembly monolayer
  • the (C 2 -C 28 )alkane chain may also be cyclic or aromatic, substituted or unsubstituted, saturated or unsaturated, branched or unbranched, and mixtures thereof.
  • C 12 )alkanethio groups are selected from maleimidoethanethio, maleimidopropanethio, maleimidobutanethio, maleimidopentanethio, maleimidohexanethio, maleimidoheptanethio, maleimidooctanethio, maleimidononanethio maleimidodecanethio maleimidoundecanethio maleimidododecanethio and mixtures thereof.
  • the maleimido(C 2 -C 12 )alkanethio group is maleimidohexanethio.
  • maleimido(C 2 -C 28 )alkanethio groups may be maleimidotridecanethio, maleimidotetradecanethio, maleimidopentadecanethio, maleimidohexadecanethio, maleimidoheptadecanethio, maleimidooctadecanethio, maleimidononadecanethio, maleimidoeicosanethio, maleimidoheneicosanethio, maleimidodocosanethio, maleimidotricosanethio, maleimidotetracosanethio, maleimidopentacosanethio, maleimidohexacosanethio, maleimidoheptacosanethio or maleimidooctacosanethio.
  • the monolayer further comprises (C 2 -C 28 )alkanethio groups of Formula II:
  • the mixed monolayer comprises (C 2 -C 12 )alkanethio groups bound to the metal surface via the sulphur atom.
  • the (C 2 -C 28 )alkanethio groups may be selected from ethanethio, propanethio, butanethio, pentanethio, hexanethio, heptanethio, octanethio, nonanethio, decanethio, undecanethio, dodecanethio, tridecanethio, tetradecanethio, dipentadecanethio, hexadecanethio, heptadecanethio, octadecanethio, nonadecanethio, eicosanethio, heneicosanethio, docosanethio, tricosanethio, tetracosanethio, pentacosanethio, he
  • the (C 2 -C 28 )alkane chain may also be cyclic or aromatic, substituted or unsubstituted, saturated or unsaturated, branched or unbranched, and mixtures thereof.
  • Dithio-bismaleimido(C 2 -C28)alkane may also be represented by the general formula of Formula III:
  • n 1 to 28.
  • dithio-bismaleimidoalkane is dithio-bismaleimidoethane, which is commercially available from PIERCE (Rockford, Illinois) as a protein cross-linking agent (Chen et al (1991) J. Biol. Chem. 266, 18237-43).
  • the dithio-bismaleimido(C 2 -C 2 g)alkane may be selected from dithio-bismaleimidoethane, dithio-bis-maleimidopropane, dithio-bis-maleimidobutane, dithio- bis-maleimidopentane, dithio-bis-maleimidohexane, dithio-bis-maleimidoheptane, dithio-bis-maleimidooctane, dithio-bis-maleimidononane, dithio-bis- maleimidodecane, dithio-bis-maleimidoundecane, dithio-bis-maleimidododecane and mixtures thereof.
  • the dithio-bismaleimido(C 2 -C 28 )alkane is dithio-bis-maleimidohexane.
  • dithio-bismaleimido(C 2 -C 28 )alkane groups may be dithio- bismaleimidotridecane, dithio-bismaleimidotetradecane, dithio- bismaleimidopentadecane, dithio-bismaleimidohexadecane, dithio- bismaleimidoheptadecane, dithio-bismaleimidooctadecane, dithio- bismaleimidononadecane, dithio-bismaleimidoeicosane, dithio- bismaleimidoheneicosane, dithio-bismaleimidodocosane, dithio- bismaleimidotricosane, dithio-bismaleimidotetracosane, dithio- bismaleimidopentacosane, dithio-bismaleimidohexacosane, dithio- bismaleimidohept
  • the invention also encompasses a process for preparing a self assembly monolayer comprising contacting dithio-bismaleimido(C 2 -C 28 )alkane with a metal surface.
  • the comprises contacting dithio-bismaleimido(C 2 -C 12 )alkane with a metal surface.
  • the dithio-bismaleimido(C 2 -C 12 )alkane may be selected from dithio- bis-maleimidoethane, dithio-bis-maleimidopropane, dithio-bis-maleimidobutane, dithio-bis-maleimidopentane, dithio-bis-maleimidohexane, dithio-bis- maleimidoheptane, dithio-bis-maleimidooctane, dithio-bis-maleimidononane, dithio- bis-maleimidodecane, dithio-bis-maleimidoubdeane, dithio-bis-maleimidododecane and mixtures thereof.
  • a particularly preferred dithio-bismaleimido(C 2 -C 12 )alkane group is dithio
  • dithio-bismaleimido(C 2 -C 28 )alkane groups may be dithio- bismaleimidotridecane, dithio-bismaleimidotetradecane, dithio- bismaleimidopentadecane, dithio-bismaleimidohexadecane, dithio- bismaleimidoheptadecane, dithio-bismaleimidooctadecane, dithio- bismaleimidononadecane, dithio-bismaleimidoeicosane, dithio- bismaleimidoheneicosane, dithio-bismaleimidodocosane, dithio- bismaleimidotricosane, dithio-bismaleimidotetracosane, dithio- bismaleimidopentacosane, dithio-bismaleimidohexacosane, dithio- bismaleimidohept
  • the SAM is a mixed monolayer and so the process further comprises contacting an alkanethiol, an alkenethiol or dialkyl disulfide of Formula IV, or mixtures thereof with the metal surface.
  • the alkanethiol, alkenethiol or dialkyl disulfide may include cyclic, saturated, unsaturated, straight, branched, substituted or unsubstituted chains and mixtures thereof.
  • the dialkyl disulfide may be selected from diethyl disulfide, dipropyl disulfide, dibutyl disulfide, dipentyl disulfide, dihexyl disulfide, diheptyl disulfide, dioctyl disulfide, dinonyl disulfide, didecyl disulfide, diundecyl disulfide, didodecyl disulfide, ditridecyl disulfide, ditetradecyl disulfide, dipentadecyl disulfide, dihexadecyl disulfide, diheptadecyl disulfide, dioctadecyl disulfide, dinonadecyl disulfide, dieicosyl disulfide, diheneicosyl disulfide, didocosyl disulfide, ditricosyl disulfide, ditetracosyl disulfide,
  • any suitable metal surface may be used but a noble metal such as gold, silver or platinum are particularly preferred.
  • the array may also incorporate a reference electrode.
  • the reference electrode is separate from the working and counter electrodes, thereby forming a three-electrode array.
  • the reference electrode is short circuited to the counter electrode, thereby providing a two-electrode array.
  • the reference electrode provides a baseline potential and is ideally a silver/silver chloride electrode, but may also be a silver/silver nitrate electrode.
  • the materials for each of the electrodes are deposited on a plastic film and the shapes of each of the electrodes may be formed, for example, by laser ablation or screen printing.
  • the array may further comprise a protein, the protein being immobilised to the metal surface via the SAM.
  • immobilised is used in the context that the protein is confined on the surface of the SAM and encompasses physiosorption or chemisorption. Expressed in another way, the surface of the SAM is functionalised by the protein.
  • the gold electrode is a working electrode, to which the P450 haem domain is attached, via a SAM. While a glassy carbon electrode working could be used, gold has been specifically selected because of properties of the metal. In particular, chemicals can be covalently, as well as non-covalently, bonded to a gold surface via a thiol or mercaptan group. Such binding is more stable than non-covalent binding achieved with a glassy carbon electrode, and provides a more stable array. A further advantage of using gold is that it allows molecules bound to its surface to be specifically orientated.
  • Attachment of the P450 to the electrode by a SAM as described above provides distance between the electrode and the haem domain, thus protecting the protein from denaturing, as well as providing a layer of insulation between the electrode and the reaction solution.
  • the electrodes are sized to fit in the well of a standard microtitre plate.
  • a microtitre plate or microplate is a flat plate with multiple "wells" used as small test tubes.
  • the microplate has become a standard tool in analytical research and clinical diagnostic testing laboratories and typically has 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. It will be appreciated that any number of wells, from one upwards, may be used. Indeed, a preferred embodiment has as array of 8 wells. It will also be appreciated that the wells may be arranged in any format. While a typical microtitre plate has the wells arranged in a square or rectangular format, the wells may be in any suitable format such as a linear or circular arrangement.
  • wells may be defined by open-ended tubes of a suitable capacity that may be overlaid onto a base that carries an array of the electrodes suitably arranged to correspond with an arrangement of the tubes acting as wells.
  • a potentiostat is a control and measuring device that, in an electrolytic cell, keeps the potential of the working electrode at a constant level respect to the reference electrode. It consists of an electric circuit which controls the potential across the cell by sensing changes in its resistance, varying accordingly the current supplied to the system: a higher resistance will result in a decreased current, while a lower resistance will result in an increased current, to keep the voltage constant.
  • the electrical contacts may extend from the or each electrode at any suitable angle or orientation.
  • the contacts may be arranged in the same horizontal plane as the electrodes.
  • the contacts may extend from the array in an orientation that is perpendicular to the plane of the electrodes.
  • a cytochrome P450 isoform is immobilised on a working electrode in a well. Buffer is then added to the well followed by a test compound, such as a drug candidate. The working and counter electrodes are connected via an interface to a potentiostat and the current produced upon substrate turnover is recorded. By using different concentrations of test compound, a Michaelis Menten curve can be generated, providing an indication of the efficacy of the compound as a substrate, effector or inhibitor for the particular cytochrome P450 isoform in the well.
  • test compound is ideally in solution, diluted in a suitable solvent such as acetonitrile, buffer, water, ethanol or dimethyl sulfoxide (DMSO).
  • suitable solvent such as acetonitrile, buffer, water, ethanol or dimethyl sulfoxide (DMSO).
  • the present invention also encompasses a method for the preparation of a self-assembly monolayer (SAM) on a gold surface.
  • the method comprises cleaning the gold surface, combining the cleaned surface with one or more alkanethiol or arylthiol groups or mixtures thereof to form a self-assembled monolayer (S AM) on the surface and washing and agitating the resulting SAM- modified surface.
  • SAM self-assembly monolayer
  • the gold surface may be cleaned electrochemically or using an oxygen or argon plasma.
  • oxides are formed on the gold surface. These oxides may be removed by washing the cleaned surface.
  • Alcohols such as methanol or ethanol, have been found to be particularly effective. Oxides form an unstable bond with thiol groups, hence removal of the oxide layer is necessary when forming a thio-bound SAM. Alternatively, the oxides may be retained on the surface.
  • washing and agitating the electrode after deposition of the SAM is essential as this substantially disperses any aggregates that may have formed. Agitation may be by shaking, sonication and/or repeated aspiration.
  • a protein of interest may be contacted with the modified surface and the resulting assembly stored with a suitable buffer.
  • the protein may be a redox protein, such as a drug metabolising enzyme.
  • a drug metabolising enzyme may be any suitable enzyme such as a flavin-containing mono-oxygenase (FMO), a monoamine oxidase (MAO), an alcohol dehydrogenase (ADH) or a cytochrome P450 enzyme.
  • FMO flavin-containing mono-oxygenase
  • MAO monoamine oxidase
  • ADH alcohol dehydrogenase
  • the protein may be derived from a mammal, plant, bacteria or fungus.
  • the protein is a cytochrome P450 haem domain.
  • haem domain in the context of the present invention refers to the active site of P450 containing a haem iron centre.
  • the protein may include a scaffold or structure around the haem that may be derived from the wild-type cytochrome P450 from which the haem domain is derived.
  • the scaffold may be derived from a different cytochrome P450 and indeed a cytochrome P450 derived from a different organism or species.
  • the protein may also include domains having a different function to the haem domain.
  • the haem domain may be linked, attached or integral to a flavodoxin.
  • the assembly may then be washed with an appropriate buffer.
  • the SAM may be a bilayer, monolayer or a mixed monolayer.
  • the SAM may comprise a thiolipid.
  • a thiolipid SAM may be derived from 1,2- dipalmitoyl-sn-glycero-S-phosphothioethanol.
  • a protein may be carried in micelles or liposomes so that when the micelles or liposomes are added to a SAM-modified surface, the micelles/liposomes open up and release the protein onto and/or into the SAM, with the lipid of the micelles/liposomes providing a second layer on the SAM, thereby resulting in a bilayer SAM. The protein is then non- covalently attached to the SAM.
  • the protein may be attached to the SAM via cysteine or in a non-cysteine specific manner.
  • the SAM may be a mixture of two or more alkanethio or arylthio groups having differing chain lengths.
  • the proteins may be bound to the SAM via amine groups, which covalently bond, for example via a peptide bond, to carboxy groups on the surface of the SAM.
  • the mixed monolayer may consist of 10-carboxy- decanethiol and 8-hydroxy-l-octanethiol.
  • the SAM may comprise a maleimido(C 2 -C 28 )alkanethio SAM as described above.
  • Figure 1 which illustrates the product formed as a result of the reaction between cystamine and 3-maleimidopropionic acid N-hydroxysuccinimide ester (A) and the product of the reaction between the amine of cystamine and the maleimide of 3-maleimidopropionic acid N-hydroxysuccinimide ester (B).
  • Figure 2 shows GATR FT-IR spectra of bare gold (black line), cystamine monolayer on gold (red line) and cystamine-maleimide monolayer on gold. The arrow indicates the peak resulting from the presence of succinimidyl ester.
  • Figure 3 shows the effect of stripping of gold oxide in 0.1 M H 2 SO 4 without (dashed line) and with (continuous line) incubation in ethanol.
  • Figure 5 shows A) GATR FT-IR spectra of the bare gold electrode (black line), DTME monolayer on a gold electrode (dark grey line) and CYP2D6 bound to the DTME monolayer on a gold electrode (light grey line); B) GATR FT-IR spectra of DTME monolayer (solid line) and CYP2E1 bound to the DTME monolayer (dashed line).
  • Figure 6 shows AFM imaging topography and cross-sections of A) annealed gold; B) DTME on gold; C) DTME modified gold electrode with 0.5 ⁇ M CYP2E1 ; D) DTME modified gold electrode with 2 ⁇ M CYP2E1; and E) DTME modified gold electrode with 5 ⁇ M CYP2E1.
  • Figure 8 is a cyclic voltammogram of 0.1 M potassium hexacyanoferrate(III) (K 3 [Fe(CN) 6 ]) in IM potassium chloride (KCl) at a scan rate of 5OmVs '1 .
  • the black line shows the bare gold electrode and the grey line shows the DTME modified gold surface.
  • Figure 9 illustrates a series of dithio-bismaleimidoalkanes.
  • Figure 11 is a diagram of a mixed monolayer of dithio-bismaleimidohexane and dihexyl disulphide.
  • Figure 12 shows GATR FT-IR spectra of bare gold (dashed line), dihexyl disulphide monolayer on gold (light grey line), dithio-bismaleimidohexane (DTMH) monolayer on gold (dark grey line) and dihexyl disulphide + dithio- bismaleimidohexane mixed monolayer on gold (black line).
  • Figure 13 is a schematic illustrating the formation of the thiolipid bilayer SAM and subsequent binding of P450. The tail illustrated as part of the P450 represents an expression tag and plays no part in the assembly of the array or function of the P450.
  • Figure 16 shows GATR-FITR spectra of a 1 ,2-dipalmitoyl-5n-glycero-3- phosphothioethanol monolayer on a gold surface.
  • Right hand panel change in current at -57OmV measured upon titration of metoprolol for the protein (squares) and control (circles) electrodes.
  • Figure 18 illustrates the repeatability of the current response upon titration of metoprolol on to three different electrodes.
  • Figure 19 shows AFM imaging (topography) of A) bare annealed gold; B) 10- carboxy-decanethiol and 8-hydroxy-l-octanethiol on gold; and C) CYP2C9/BMR immobilised on 10-carboxy-decanethiol and 8-hydroxy-l-octanethiol on gold.
  • Figure 20 is a cyclic voltammogram showing the insulation properties of a mixed monolayer.
  • Figure 21 is a Diclofenac titration by CYP2C9/BMR immobilised onto gold using a mixed monolayer.
  • Figure 22: Left hand panel cysteine residues in CYP2D6; Right hand table
  • Figure 23 illustrates the position of the cysteine residues in the structure of CYP2C9 shown as ribbon. The most exposed one, CYS 372, is indicated.
  • Figure 25 is A) perspective photograph showing one embodiment of an array;
  • Evaporated gold electrodes were prepared in clean room conditions using a V2000 evaporator with chamber conditioned at 300 degrees centigrade.
  • the electrodes consist of 150 nm of gold evaporated on 50 nm of Chromium that was evaporated on a Silicon wafer with 300 nm of thermally generated silicon oxide.
  • the electrodes were cleaned in an oxygen plasma (RF power 9OW; Oxygen zero grade at 12.5 ml/min; time depends on the surface from 3 to 60 min). This cleaning process generates a hydrophilic surface due to the formation of gold oxides. However, gold oxides form an unstable bond with thiol groups and so the gold oxides are reduced to gold by immersing the electrode, after cleaning, into ethanol.
  • RF power 9OW oxygen plasma
  • Oxygen zero grade at 12.5 ml/min time depends on the surface from 3 to 60 min.
  • the effect of the ethanol treatment after plasma cleaning can be checked using cyclic voltammetry.
  • the stripping of the oxides can be illustrated in a 0.1 M sulphuric acid solution scanning between +1.0V and OV against SCE (saturated calomel electrode). As shown in Figure 3, within 5 minutes reduction of the oxides in ethanol is complete. In comparison, no ethanol treatment results in an oxide peak at around +0.75V.
  • a contact angle is the angle between the electrode surface and a tangent drawn at the point where the outer surface of a bead of water meets the electrode surface. Because the oxidised surface after plasma treatment is hydrophilic, if a droplet of 1- 2 ⁇ l water is dropped onto the cleaned surface, the droplet will spread due to the hydrophilicity of the surface. Therefore, the angle between the electrode surface and the outer surface of the droplet will be small (acute). After removal of the oxides by ethanol, the droplet will retain a beaded shape on the surface, due to the hydrophobicity and so the contact angle becomes less acute. This is illustrated in Table 1.
  • Dithio-bismaleimidoethane is a cleavable sulfhydryl-to-sulfhydryl crosslinking agent that is used to cross-link proteins (Chen et al (1991) J. Biol. Chem. 266, 18237-43).
  • a SAM of DTME on a gold surface was prepared by incubating a cleaned (as per Example 1) gold surface in a 2mM solution of DTME in acetonitrile. The resulting SAM electrode was then washed with acetonitrile, then with acetonitrile and sonication (or vigorous agitation) for 5 minutes, and finally with water.
  • the wash and sonication (and/or agitation) steps after deposition of the SAM are important as this substantially removes any aggregates.
  • DTME binds to the gold surface
  • the short length of the molecule may mean that the SAM is not sufficiently insulating.
  • Scanning the DTME modified electrode in 0.01M potassium hexacyanoferrate(III) (K 3 [Fe(CN) 6 ]) in IM potassium chloride (KCl) between -0.1 V and +0.65 V (versus SCE) a method that is frequently used to study the uniformity of SAMs (Ganesh and Lakshminarayana (2006) Langmuir, 22, 1561-70), results in a cyclic voltammetry similar to that of the bare gold electrode (see Figure 8).
  • the first spacer molecule to be tested was dithio-bismaleimidohexane (DTMH).
  • DTMH dithio-bismaleimidohexane
  • a mixed monolayer was prepared by incubating a cleaned gold surface in acetonitrile containing 2mM dithio-bismaleimidohexane and 2mM dihexyl disulphide for 48hours at room temperature.
  • Figure 11 is a schematic depiction of the dithio- bismaleimidohexane and dihexyl disulphide mixed monolayer.
  • the modified SAM electrode was washed vigorously in acetonitrile and Milli-Q water .
  • the presence of the mixed monolayer was then confirmed using contact angle studies and GATR FT-IR.
  • Table 2 contact angles of bare gold, dihexyl disulphide on gold, dithio- bismaleimidohexane on gold and dihexyl disulphide + dithio-bismaleimidohexane mixed monolayers on gold.
  • a protein concentration of 30 ⁇ M on a C8/C10 mixed monolayer showed a distribution having a higher uniformity and compactness compared to 5 ⁇ M, as observed by the higher reproducibility of the electrochemical behaviour of the 30 ⁇ M protein concentration (results not shown).

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Abstract

La présente invention porte sur des groupes maléimido-(alcane en C2-C20)thio comme monocouches auto-assemblées (SAM) et sur des procédés de construction de ceux-ci. L'invention porte également sur un réseau comprenant une telle SAM, sur un procédé de construction du réseau et sur un procédé de préparation d'une électrode en or.
PCT/GB2008/003904 2007-11-23 2008-11-21 Réseau Ceased WO2009066080A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013160749A3 (fr) * 2012-04-27 2014-01-23 Fondazione Bruno Kessler Surface polymère hydrophile, son procédé de production, applications dudit procédé et articles comprenant ladite surface
KR101841362B1 (ko) 2016-10-24 2018-03-22 부경대학교 산학협력단 암세포 표적 기능을 갖는 신규 가교 마이셀, 이를 포함하는 약물 전달체 및 이의 제조방법

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US6489102B2 (en) * 1999-08-05 2002-12-03 Wisconsin Alumni Research Foundation Biomolecule and/or cellular arrays on metal surfaces and product produced thereby

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013160749A3 (fr) * 2012-04-27 2014-01-23 Fondazione Bruno Kessler Surface polymère hydrophile, son procédé de production, applications dudit procédé et articles comprenant ladite surface
KR101841362B1 (ko) 2016-10-24 2018-03-22 부경대학교 산학협력단 암세포 표적 기능을 갖는 신규 가교 마이셀, 이를 포함하는 약물 전달체 및 이의 제조방법

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