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WO2008139375A2 - Chemical sensor comprising a gel and a fluorescer - Google Patents

Chemical sensor comprising a gel and a fluorescer Download PDF

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Publication number
WO2008139375A2
WO2008139375A2 PCT/IB2008/051786 IB2008051786W WO2008139375A2 WO 2008139375 A2 WO2008139375 A2 WO 2008139375A2 IB 2008051786 W IB2008051786 W IB 2008051786W WO 2008139375 A2 WO2008139375 A2 WO 2008139375A2
Authority
WO
WIPO (PCT)
Prior art keywords
sensor
present
change
analyte
sensor according
Prior art date
Application number
PCT/IB2008/051786
Other languages
French (fr)
Other versions
WO2008139375A3 (en
Inventor
Ralph Kurt
Derk Jan Wilfred Klunder
Emiel Peeters
Roel Penterman
Dirk Jan Broer
Maarten Marinus Johannes Wilhelmus Van Herpen
Original Assignee
Koninklijke Philips Electronics N. V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Koninklijke Philips Electronics N. V. filed Critical Koninklijke Philips Electronics N. V.
Publication of WO2008139375A2 publication Critical patent/WO2008139375A2/en
Publication of WO2008139375A3 publication Critical patent/WO2008139375A3/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G01N2021/7706—Reagent provision
    • G01N2021/7723—Swelling part, also for adsorption sensor, i.e. without chemical reaction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence

Definitions

  • the present invention is directed to the field of devices for the detection of one or more analytes in a sample, especially to the field of devices for the detection of biomolecules in aqueous solution.
  • the present invention is directed to the detection of analytes in fluids, especially to the detection of biomolecules in aqueous solution.
  • a chemical sensor is disclosed in the US 5,821,129 which is hereby incorporated by reference.
  • the device layout is such that the sensor and the sensor read- out are spatially separated, enabling remote sensing.
  • the sensor in the US 5,821,129 suffers drawbacks when employing it for continuous monitoring of physiological parameters inside the body of a human.
  • the sensor read-out is done by subjecting an alternating magnetic field to the stacked sensor structure and measuring a voltage from a detection unit comprising a coiled structure. This methodology hampers miniaturization of the device.
  • the device requires a sensor unit consisting of at least three layers, two being magnetic and one being responsive to a certain stimulus.
  • the invention considers remote sensing which is not desirable in case of long-term implantation where one would desire to have the complete device implanted.
  • a chemical sensor especially for determining the presence, identity, amount and/or concentration of at least one analyte in a fluid sample is provided, comprising
  • At least one second material adapted to interact with the at least one sensor whereby the second material comprises at least one fluorescent material. and whereby the first, at least one second material and the at least one sensor are so provided towards each other that upon a change of the physical properties of the first material a change in at least one of the signals detected by the at least one sensor occurs.
  • a sensor according to the present invention shows for most applications at least one of the following advantages a suppressed biofouling due to a high water content in said first elastic material a high biocompatibility due to a high water content in said first elastic material - a fast response due to small thickness of said first elastic material a high accuracy a small form factor a low-cost system design a very miniaturized design, especially in cases where an incident evanescent field is used (as will be described later on).
  • the term “elastic” especially means, includes and/or describes a property of a material, that can be at least partially elastic deformed, i.e. the deformation is at least partially (or completely) reversible (gets its old shape, size, dimension back).
  • “elastic” especially means includes and/or describes a fully reversible process of shape recovery.
  • the term "interact" in section (c) especially means and/or includes that the at least one second material is able to emit light which is detected by the at least one sensor.
  • fluorescence especially means and/or includes that the second material comprises a material which is capable of emitting light upon incident radiation.
  • the term “fluorescence” especially means and/or includes that the second material comprises a material which is capable of emitting light upon incident radiation.
  • fluorescence includes fluorescence as well as phosphorescence.
  • the at least one changing direction essentially in which the change of the first material in response to the at least one analyte occurs.
  • the at least one sensor and/or the at least one second material are provided in the changing direction at opposite ends of the first material.
  • the at least one second material is provided next to the first material.
  • the term “next" includes that the second material is provided in contact with the first material as well as they are separated by further material(s) which may e.g. be provided in form of a thin layer(s) or other provisions.
  • the term “next” especially includes that said third matrix material is provided in contact with the first material as well as they are separated by further material(s) which may e.g. be provided in form of a thin layer(s) or other provisions.
  • the at least one second material is embedded in said at least one first material.
  • embedded in the context of this invention expressly especially includes and/or means that the second material is connected and/or bonded to the first material e.g. via covalent bonds (which may be formed e.g. by co -polymerization) or via non-covalent bonds such as hydrogen bonds or van-der Waals-forces.
  • covalent bonds which may be formed e.g. by co -polymerization
  • non-covalent bonds such as hydrogen bonds or van-der Waals-forces.
  • the term “embedded” in the context of this invention is meant also to especially include embodiments where the second material is provided as a separate material.
  • the at least one second material is embedded in said at least one first material in form of and/or comprises molecules, dyes, labels, small beads, moieties, preferably with a diameter of >0.5nm and ⁇ lOOnm, more preferably between ⁇ lnm and ⁇ lOnm.
  • the average particle size of the at least one second material is >0.5nm and ⁇ lOOnm.
  • the average particle size of the at least one second material is ⁇ lnm and ⁇ lOnm.
  • the at least one second material has an anisotropic shape, preferably a disk or rod like shape, (typical sizes are 0.5nm x lnm x 3nm).
  • the at least one second material has an anisotropic shape, and is substantially aligned with it's longest axis parallel to the surface of the sensor.
  • the at least one second material is randomly oriented. This has especially for many applications the advantage of that it is easy to manufacture.
  • the at least one second material has an anisotropic dipole moment, which is substantially parallel to the polarization direction of the incident radiation.
  • the at least one second material comprises a fluorescent material with an extinction coefficient > 10000 L mo 1-1 cm-1 and ⁇ 300000 L mol-1 cm-1, preferably >20000 L mol-1 cm-1 and ⁇ 200000 L mol-1 cm-1. Said extinction coefficient is measured at the wavelength ⁇ max and/or +/- 50nm, at which the maximum extinction is obtained.
  • the at least one second material comprises a fluorescent material with a fluorescence efficiency in the range of >5% and ⁇ 100%, preferably >10% and ⁇ 60% at the excitation wavelength used in the device.
  • the at least one second material is excited by an incident electromagnetic radiation characterized by a wavelength in the range >280nm and ⁇ 1500nm, preferably >390 and ⁇ 850nm.
  • the at least one second material is excited by an incident electromagnetic radiation characterized sharp spectral distribution FWHM of ⁇ 100 nm, preferably ⁇ 50nm, most preferably ⁇ 30nm.
  • the second material comprises at least one fluorescent material selected out of the group comprising triphenylmethane dyes, azo dyes, cyanine dyes, phtalocyanin dyes, acridine dyes, anthrachinone dyes, anthocyane dyes, phenazin dyes, phenoxazin dyes, phenothiazine dyes, furanone dyes, coumarine dyes, oxazines dyes, rhodomine dyes, carbocyanine dyes, stilbene dyes, oligophenyl dyes, perylene dyes, styryl dyes, benzophenoxazonium dyes, oligophenylenevinylene or mixtures thereof.
  • fluorescent material selected out of the group comprising triphenylmethane dyes, azo dyes, cyanine dyes, phtalocyanin dyes, acridine dyes, anthrachinone dyes, anthocyane dyes,
  • the senor comprises a light emitting means which emits polarized light towards the second material.
  • the senor comprises a light emitting means which emits polarized light and which is capable to change the polarization direction, especially rotating and/or switching the polarization direction. By doing so it is possible to "tune" the excitation depth for a wide range of applications within the present invention.
  • the excitation of light of the second material is caused by means of an incident evanescent field. It has been shown for many applications within the present invention that by doing so, the efficacy and accuracy of the sensor can be greatly increased while it is possible miniature the sensor.
  • the first and second material are provided with and/or in between a wire grid as described in WO 2006/136991 and/or a 2 dimensional array of absorbing and/or reflecting elements.
  • said evanescent field can be build up very easily and efficiently for a wide range of applications within the present invention.
  • a wire grid especially means and/or includes an array of at least one aperture defined in at least one layer (layer-x) that is essentially non-transparent for the incident light.
  • a first in-plane dimensions (that is parallel to layer-x) of the wire grid is below the diffraction limit of the incident light in the medium that fills the aperture (diffraction limit is defined as wavelength/(2*refractive index of medium that fills aperture), while a second dimensions is above the diffraction limit.
  • An evanescent field in the space between the wires of the wire grid may be generated by illumination of the wire grid with light having R-polarized light, which is actually one embodiment of the present invention. It has been shown that for a wide range of applications within the present invention R-polarized light is essentially reflected or absorbed by the wire grid and has an electric field such that the projection of the electric field on a plane parallel to the wires of the wire grid is along the second dimension of the wire grid.
  • the term "with and/or in between” especially means that the wire grid is so provided in the vicinity of the first and/or second material to allow an evanescent field to function as an incident radiation for inducing emittance of light from the at least one second material.
  • the at least one second material has an anisotropic shape, and is substantially aligned with its longest axis parallel to the direction of the wire grid.
  • the senor is and/or comprises a waveguide biosensor.
  • waveguide biosensor in the sense of the present invention especially means that the first material layer is used as cladding material for the waveguide.
  • the evanescent excitation of fluorescence can be performed by exciting a waveguide mode.
  • the first and/or second material is within the evanescent field of the waveguide mode, and results in a fluorescent signal that depends on thickness of hydrogel layer.
  • the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and ⁇ 200 nm.
  • the senor uses total internal reflection.
  • This especially includes and/or means that the first material is provided on a substrate with an index of refraction higher than the first material.
  • the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and ⁇ 200 nm.
  • the senor is and/or comprises a surface plasmon biosensor.
  • Preferred materials for metal film are gold and/or silver, preferably with a thickness ⁇ 100 nm, more preferred 60 nm ⁇ lOnm.
  • surface plasmon waves can propagate. These surface plasmons waves have an evanescent field in both the metal film and the hydrogel. By excitation of surface plasmon waves, it is possible to locally excite the fluorophores and this way perform a surface specific fluorescence measurement.
  • the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and ⁇ 200 nm.
  • the sensor includes confocal excitation.
  • the first material is provided on top of a transparent substrate.
  • a focused excitation spot preferably with a NA of the lens of >0.5
  • the surface specificity of the fluorescence measurement is obtained by focusing the collected fluorescence on a small pinhole.
  • the fluorescence generated away from the hydrogel-substrate interface may be focused in front or behind the pinhole and is usually substantially blocked by the pinhole.
  • the depth of the measurement volume is >1 and ⁇ 2 micrometers.
  • the change of the first material includes shrinking and/or swelling.
  • the device comprises a sensor having a sensor direction and the changing direction is essentially perpendicular to the sensor direction.
  • sensor direction especially means and/or includes that in case the sensor extends itself in one or two dimensions larger than in the other two (or one), so that the sensor is either somewhat flat or forms a needle, the “sensor direction” would then be the direction where the sensor has its longest extension.
  • the uniformity of distribution of the at least one second material in the first material is homogenous in a plane parallel to the sensor direction. This has been shown to be advantageous for many applications within the present invention.
  • the uniformity of distribution of the at least one second material in the first material in the direction perpendicular to the senor surface is non-uniform, preferably having a gradient, which may be either linear or stepwise. This has been shown to be advantageous for many applications within the present invention.
  • the first material changes its size and/or thickness when interacting with the at least one analyte.
  • the first material is provided in form of a gel.
  • said second material is substantially forming a layer.
  • said second material is embedded in a third material substantially forming a layer.
  • the thickness of the at least one second material and or said third material is >0.5 nm and ⁇ IO nm, preferably >1 nm and ⁇ 8 nm, most preferably >1.5 nm and ⁇ 5 nm.
  • the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.1% and ⁇ 40%.
  • the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.2% and ⁇ 10%.
  • the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.5% and ⁇ 5%.
  • the first material is provided in form of a layer.
  • the term "layer” means and/or includes especially that the thickness of the first material in one dimension is >0% and ⁇ 50% than in either one of the other dimensions.
  • the layer thickness is bound to a range in order to obtain optimum device properties: On the one hand a too thin layer will yield a low signal and insufficient sensitivity whereas a too thick layer will yield a slow response due to long diffusion times.
  • the first material is provided in form of a layer with a thickness of >5 nm and ⁇ IOOO nm.
  • the first material is provided in form of a layer with a thickness of >20 nm and ⁇ 500 nm.
  • the first material is provided in form of a layer with a thickness of >50 nm and ⁇ 200 nm.
  • the maximum thickness change of the first material upon interaction with said at least one analyte in the sensing direction is between >0.2% and ⁇ 90%, preferably between >1% and ⁇ 30%.
  • the maximum thickness change of the first material upon interaction with said at least one analyte in the sensing direction is between >0.5nm and ⁇ 50nm preferably between ⁇ lnm and ⁇ 20 nm.
  • the first material comprises a hydrogelic material.
  • hydrogelic material means and/or includes especially that this material comprises polymers that in water form a water-swollen network.
  • hydrogel material in the sense of the present invention furthermore especially means that at least a part of the hydrogel material comprises polymers that in water form a water-swollen network and/or a network of polymer chains that are water-soluble.
  • the hydrogel material comprises in swollen state >50% water and/or solvent, more preferably >70% and most preferred >90%, whereby preferred solvents include organic solvents, preferably organic polar solvents and most preferred alkanols such as Ethanol, Methanol and/or (Iso-) Propanol.
  • hydrogelic material means and/or includes especially that the hydrogel is responsive which means that it displays a change of shape and total volume upon a change of a specific parameter.
  • a specific parameter can be a physical (temperature, pressure) or chemical property (ionic concentration, pH, analyte concentration) or biochemical property (enzymatic activity).
  • the hydrogel material comprises a material selected out of the group comprising poly(meth)acrylic materials, silicagel materials, substituted vinyl materials or mixture thereof.
  • the hydrogel material comprises a substituted vinyl material, preferably vinylcaprolactam and/or substituted vinylcaprolactam.
  • the hydrogel material comprises a poly(meth)acrylic material made out of the polymerization of at least one (meth)acrylic monomer and at least one polyfunctional (meth)acrylic monomer.
  • the (meth)acrylic monomer is chosen out of the group comprising (meth)acrylamide, acrylic esters, hydroxyethyl(meth)acrylate, ethoxyethoxyethyl(meth)acrylate or mixtures thereof.
  • (meth)acrylic monomer is a bis-(meth)acryl and/or a tri-(meth)acryl and/or a tetra- (meth)acryl and/or a penta-(meth)acryl monomer.
  • the polyfunctional (meth)acrylic monomer is chosen out of the group comprising bis(meth)acrylamide, tetraethylene glycol di(meth)acrylate, Methylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, tripropyleneglycol di(meth)acrylates, pentaerythritol tri(meth)acrylate polyethyleneglycoldi(meth)acrylate, ethoxylated bisphenol-A- di(meth)acrylate , hexanedioldi(meth)acrylate or mixtures thereof.
  • the hydrogel material comprises an anionic poly(meth)acrylic material, , preferably selected out of the group comprising (meth)acrylic acids, arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid, sulfonamides or mixtures thereof, and/or a cationic poly(meth)acrylic material , preferably selected out of the group comprising vinyl pyridine, vinyl imidazole, aminoethyl (meth)acrylates or mixtures thereof, co- polymerized with at least one monomer selected out of the group neutral monomers, preferably selected out of the group vinyl acetate, hydroxy ethyl (meth)acrylate (meth)acrylamide, ethoxyethoxyethyl(meth)acrylate or mixture thereof, or mixtures thereof.
  • These co-polymers change their shape as a function of pH and can respond to an applied electrical field and/or current by as well. Therefore these co-polymers change
  • the first material comprises a hydrogelic material comprising thermo-sensitive polymers.
  • the first material comprises a hydrogelic material comprising monomers selected out of the group comprising poly-N-isopropylamide (PNIPAAm) and copolymers thereof with monomers selected out of the group comprising polyoxyethylene, trimethylol-propane distearate, poly- ⁇ -caprolactone or mixtures thereof.
  • PNIPAAm poly-N-isopropylamide
  • the hydrogelic material is based on thermo -responsive monomers selected out of the group comprising N-isopropylacrylamide, di(m)ethylacrylamide, carboxyisopropylacrylamide, hydroxymethylpropylmethacrylamide, acryloylalkylpiperazine, N-vinylcaprolactam.
  • hydrophilic monomers comprising hydroxyethyl(meth)acrylate, (meth)acrylic acid, acrylamide, polyethyleneglycol(meth)acrylate, N-vinyl pyrolidone, dimethylaminopropylmethacrylamide, dimethylaminoethylacrylate, N- hydroxymethylacrylamide or mixtures thereof, and/or co -polymerized with monomers selected out of the group hydrophobic monomers, comprising (iso)butyl(meth)acrylate, methylmethacrylate, isobornyl(meth)acrylate, glycidyl methacrylate or mixtures thereof.
  • group hydrophilic monomers comprising hydroxyethyl(meth)acrylate, (meth)acrylic acid, acrylamide, polyethyleneglycol(meth)acrylate, N-vinyl pyrolidone, dimethylaminopropylmethacrylamide, dimethylaminoethylacrylate, N- hydroxymethyl
  • the first material comprises a hydrogelic material with a swelling ratio of >1% and ⁇ 500% at 20 0 C.
  • the swelling ratio especially includes, means or refers to a measurement according to the following procedure:
  • the first material was dried to form a film in an oven under the temperature of 50 0 C.
  • the film was immersed in an excess of deionized water to remove the residual unreacted compounds.
  • the swollen polymer film was then cut into disk forms with 8mm in diameter and dried at 50 0 C until the weight no longer changed.
  • a preweighed dried sample (Wo) was immersed in an excess of deionized water in a thermostatic water bath until the swelling equilibrium was attained.
  • the weight of the wet sample (Wi) was determined after the removal of the surface water via blotting with filter paper.
  • the first material comprises a hydrogelic material with a swelling ratio of >3% and ⁇ 200% at 20 0 C.
  • the first material comprises a hydrogelic material with a swelling ratio of >5% and ⁇ 100% at 20 0 C.
  • the first material comprises a hydrogelic material with a swelling ratio of >1% and ⁇ 30% at 20 0 C.
  • the first material comprises a hydrogelic material with a swelling ratio of >1% and ⁇ 25% at 20 0 C.
  • the first material comprises a hydrogelic material with a swelling ratio of >1% and ⁇ 20% at 20 0 C.
  • the first material comprises a receptor for the analyte to be detected.
  • the term "receptor” means and/or includes especially that some chemical moieties are present in the first material which are capable to interact with a selected analyte e.g. by hydrostatic interactions, hydrogen bonding, chemical reception, molecular recognition and the like.
  • calmodulin which binds to both calcium as well as to a range of anti-psychotic molecules, referred to as the phenothiazines, [J.D. Ehrick (Nature Materials p. 298-302, Vol. 4, April 2005)], which is hereby fully incorporated by reference.
  • the first material is a polymeric material. According to an embodiment of the present invention, the first material is a polymeric material with a conversion of >50% and ⁇ 100%.
  • the conversion especially includes, means or refers to a measurement according to the following procedure:
  • the first material is a polymeric material with a conversion of >70% and ⁇ 95%.
  • the term "essentially” means and/or includes especially a wt-% content of >90 %, according to an embodiment >95 %, according to an embodiment >99 %.
  • the crosslink density in the first material is >0.002 and ⁇ 1, preferably >0.05 and ⁇ 1.
  • crosslink density means or includes especially the following definition:
  • the crosslink density ⁇ x is here defined as
  • the second material is embedded and/or incorporated in a third matrix material.
  • the interaction of the first material includes a phase transition in said first material, preferably an phase separation such a an LCST (lower critical solution temperature).
  • LCST lower critical solution temperature
  • the matrix material is a polymeric organic material, which is preferably and according to a further embodiment of the present invention permeable to the sample to be analyzed by the first material.
  • Suitable and insofar preferred materials for the matrix material include hydrogels, brush polymers and/or cross-linked polymer brush layers, porous polymeric networks obtained e.g. by polymerization induced phases separation. Typical pore size range from a few nanometer to several micrometer.
  • the first material is functionalized in order to bind to the at least one second material and/or to the matrix material described above.
  • the first material is partially made out of a material compolymeried with glycidylmethacrylate and/or acrylic acid N-hydroxy succinimide ester.
  • the second material may bind itself to the epoxy groups (in case of the glycidylmethacrylate) or the ester groups (in case of the succinimide ester)s for many applications within the present invention.
  • the first material comprises biotinylated and/or streptavidin functionalized end-groups.
  • the biotin /streptavidin link may be used to bind the second material to the first material, which has been shown to be advantageous for a wide range of applications within the present invention.
  • the adhesion between the first and second material(s) is enhanced by applying an adhesion promoter.
  • the first material is provided with primary amine side groups, which may covalently bond to the second material provided with epoxy or NHS functionalities.
  • the responsive hydrogel monomers can be copolymerized with glycidylmethacrylate (to obtain epoxy side groups) or acrylic acid N-hydroxy succinimide ester (NHS).
  • the magnetite particles can be covalently linked to the hydrogel surface.
  • biotinylated beads can be coupled to a strepavidin- functionalized hydrogel.
  • the first material is at least partly surrounded by a well coated with a non-sticking material, which has a surface tension of ⁇ 30 mN/m, preferably ⁇ 25 mN/m.
  • the first material is surrounded by the non- sticking material which has a surface tension of ⁇ 30 mN/m, preferably ⁇ 25 mN/m in substantially all directions which are perpendicular to the changing direction.
  • the non- sticking material is a fluor-containing material, preferably a fluorinated monolayer material, which was preferably made using plasma treatment, e.g. CF 4 plasma treatment or by vapour deposition of a fluorsilane e.g. perfluoroalkylchlorosilane.
  • the device comprises a substrate and/or matrix material in the vicinity of the first material, whereby the device comprises at least one adhesion promoting layer between the first material and the substrate and/or matrix material.
  • the adhesion promoting layer is a monolayer.
  • the at least one adhesion promoting layer is chosen from the group silane-containing layers, thiol-containing layers, amine-containing layers or mixtures thereof.
  • silane-containing layer especially means and/or includes a layer which comprises a material of the form
  • Ri is selected out of the group comprising acrylate, methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol;
  • R-2 is selected out of the group alkylene, arylene, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or poly disulfides,
  • R3 and R4 are independently selected out of the group halogen
  • R6-R7 (whereby R5 is selected out of the group comprising acrylate, methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol and R7 is selected out of the group alkyl, aryl, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or polydisulf ⁇ des)
  • 0-R 8 (whereby R 8 is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, heteroaryl, halogen)
  • R5 represents the group 0-R 9 , where R 9 is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, halogen and/or R 5 is a hydrolyzable moiety.
  • alkyl linear and branched Cl-C8-alkyl
  • alkylene selected from the group consisting of: methylene; 1,1 -ethylene; 1,2-ethylene; 1,1-propylidene; 1,2-propylene; 1,3- propylene; 2,2-propylidene; butan-2-ol- 1 ,4-diyl; propan-2-ol- 1 ,3-diyl; 1 , 4-butylene; cyclohexane-l,l-diyl; cyclohexan-l,2-diyl; cyclohexan-1,3- diyl; cyclohexan-l,4-diyl; cyclopentane-l,l-diyl; cyclopentan-l,l-diyl, cyclopentan-l,
  • alkyl linear and branched Cl-C6-alkyl
  • long-chain alkyl linear and branched C5-C10 alkyl
  • cycloalkyl C6-C8-cycloalkyl
  • alkoxy Cl-C4-alkoxy
  • long-chain alkoxy linear and branched C5-C10 alkoxy, preferably linear C6-C8 alkoxy aryl: selected from group consisting of: phenyl; biphenyl; naphthalenyl; anthracenyl; and phenanthrenyl, heteroaryl: selected from the group consisting of: pyridinyl; pyrimidinyl; quinolinyl; pyrazolyl; triazolyl; isoquinolinyl; imidazolyl; and oxazolidinyl, wherein the heteroaryl may be connected to the compound via any atom in the ring of the selected heteroaryl, heteroarylene: selected from the group consisting of: pyridin 2,3-diyl; pyridin-2,4-diyl; pyridin-2,6-diyl; pyridin-3,5-diyl; quinolin-2,3-diyl; quinolin-2,4-diy
  • thiol-containing layer especially means and/or includes a layer which comprises a material of the form R-SH with R chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl.
  • this thiol- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If a thiol-containing layer is used, the surface of the matrix material is chosen out of a thiol-binding material, especially the surface of the matrix material is an Au-surface.
  • amine-containing layer especially means and/or includes a layer which comprises a material of the form Ri-NH-R 2 with Ri chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether and R 2 chosen out of the group hydrogen, alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether. It has been shown for a wide range of applications that this amine- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If a amine- containing layer is used, the surface of the matrix material is preferably equipped with amine-binding groups, preferably epoxy groups and/ or reactive esters, halogenides and/or amines.
  • the present invention furthermore relates to a method of measuring the presence, identity, amount and/or concentration of at least one analyte in a sample using a sensor as described above, comprising the steps of
  • the first material changes its size and/or thickness when interacting with the at least one analyte.
  • a sensor and/or a method according to the present invention may be of use in a broad variety of systems and/or applications, amongst them one or more of the following: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures and body fluids such as e.g. blood, urine or saliva - high throughput screening devices for chemistry, pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research - tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics tools for combinatorial chemistry analysis devices
  • biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures and body fluids such as e.g. blood, urine or saliva - high throughput screening devices for chemistry
  • pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e
  • Fig. 1 shows a very schematic cross-sectional view of a sensor according to a first embodiment of the present invention
  • Fig. 2 shows a very schematic cross-sectional view of a sensor according to Fig. 1 with the first material in a swollen state.
  • Fig. 3 shows a very schematic cross-sectional view of a sensor according to a second embodiment of the present invention
  • Fig. 4 shows a diagram of the fluorescence of a sensor according to
  • Example I vs. the swelling of the first material in the sensor.
  • Fig. 1 shows a very schematic cross-sectional view of a sensor 1 according to a first embodiment of the present invention.
  • the sensor comprises a first material 10 with a second material 20 embedded therein.
  • the second material and the first material are covalently bonded together via co -polymerization.
  • the first material 10 is provided on a sensor substrate 60, which is transparent for the light emitted by the second material. Between the sensor substrate 60 and the first material 10 there is a silane monolayer 40 as described above (it should be added that Fig. 1 is highly schematic and in actual embodiments the thickness of the layer 40 will be of course much smaller). Left and right of the first material 10 there is a non- sticking material 30.
  • the first material 10 is placed between a wire grid 50, which functions in order to create an evanescent field. It should be noted that in Fig. 1 the height of the grid 50 is higher than the first material 10; however, in most actual applications, the height of the first material will be larger, as described above.
  • Fig. 2 shows a very schematic cross-sectional view of a sensor according to Fig. 1 with the first material in a swollen state.
  • the components which are identical with the embodiment of Fig. 1 are not discussed to avoid repetitions.
  • an evanescent field (as indicated by the area "F") will be created, whose strength decreases exponentially with the distance from the substrate material 60.
  • the second material 20 Upon stimulation with the evanescent field, the second material 20 will emit light via fluorescence.
  • Fig. 3 shows a very schematic cross-sectional view of a sensor according to a second embodiment of the present invention.
  • the components, which are identical with the embodiment of Fig. 1 are not discussed to avoid repetitions.
  • Fig. 3 differs from the embodiment of Fig. 1 in the form of the wire grid (i.e. a trapezoid cross-section) but also on the provision of the first and second material.
  • the second material 20 is placed next (i.e. on top) of the first material 10.
  • a sensor according to Fig. 1 was used, whereby the initial thickness of the first material was 150 nm.
  • the used evanescent decay length corresponded with the 1/e intensity of 20 nm.
  • the duty cycle of wire grid i.e. fraction of wire grid not covered with wires
  • the height of the wire grid i.e. the numeral 50 in Fig. 1 is 150 nm, too.

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Abstract

The invention relates to a Chemical sensor having a gel-like first material and a fluorescent second material. The detection occurs via the change of the first material upon stimulation with an analyte.

Description

CHEMICAL SENSOR
The present invention is directed to the field of devices for the detection of one or more analytes in a sample, especially to the field of devices for the detection of biomolecules in aqueous solution.
The present invention is directed to the detection of analytes in fluids, especially to the detection of biomolecules in aqueous solution.
A chemical sensor is disclosed in the US 5,821,129 which is hereby incorporated by reference. The device layout is such that the sensor and the sensor read- out are spatially separated, enabling remote sensing. However, the sensor in the US 5,821,129 suffers drawbacks when employing it for continuous monitoring of physiological parameters inside the body of a human. For example the sensor read-out is done by subjecting an alternating magnetic field to the stacked sensor structure and measuring a voltage from a detection unit comprising a coiled structure. This methodology hampers miniaturization of the device. Furthermore the device requires a sensor unit consisting of at least three layers, two being magnetic and one being responsive to a certain stimulus. The invention considers remote sensing which is not desirable in case of long-term implantation where one would desire to have the complete device implanted.
It is therefore an object of the present invention to provide a chemical sensor which allows a quicker detection and can for most applications be miniaturized.
This object is solved by a sensor according to claim 1 of the present invention.
Accordingly, a chemical sensor, especially for determining the presence, identity, amount and/or concentration of at least one analyte in a fluid sample is provided, comprising
(a) a first material, whereby the first material is an elastic material adapted to change its physical properties according to interaction with said at least one analyte;
(b) at least one sensor;
(c) at least one second material adapted to interact with the at least one sensor whereby the second material comprises at least one fluorescent material. and whereby the first, at least one second material and the at least one sensor are so provided towards each other that upon a change of the physical properties of the first material a change in at least one of the signals detected by the at least one sensor occurs.
A sensor according to the present invention shows for most applications at least one of the following advantages a suppressed biofouling due to a high water content in said first elastic material a high biocompatibility due to a high water content in said first elastic material - a fast response due to small thickness of said first elastic material a high accuracy a small form factor a low-cost system design a very miniaturized design, especially in cases where an incident evanescent field is used (as will be described later on).
In the sense of the present invention, the term "elastic" especially means, includes and/or describes a property of a material, that can be at least partially elastic deformed, i.e. the deformation is at least partially (or completely) reversible (gets its old shape, size, dimension back). In the sense of the present invention "elastic" especially means includes and/or describes a fully reversible process of shape recovery.
In the sense of the present invention, the term "interact" in section (c) especially means and/or includes that the at least one second material is able to emit light which is detected by the at least one sensor.
In the sense of the present invention, the term "fluorescence" especially means and/or includes that the second material comprises a material which is capable of emitting light upon incident radiation. In the sense of the present invention the term
"fluorescence" includes fluorescence as well as phosphorescence.
According to a preferred embodiment of the present invention, there is at least one changing direction, essentially in which the change of the first material in response to the at least one analyte occurs. According to a preferred embodiment of the present invention, the at least one sensor and/or the at least one second material are provided in the changing direction at opposite ends of the first material.
According to a preferred embodiment of the present invention, the at least one second material is provided next to the first material. The term "next" includes that the second material is provided in contact with the first material as well as they are separated by further material(s) which may e.g. be provided in form of a thin layer(s) or other provisions.
In case the second material is embedded and/or incorporated into a third matrix material (as will be described later on), the term "next" especially includes that said third matrix material is provided in contact with the first material as well as they are separated by further material(s) which may e.g. be provided in form of a thin layer(s) or other provisions.
According to a preferred embodiment of the present invention, the at least one second material is embedded in said at least one first material. It should be noted that the term "embedded" in the context of this invention expressly especially includes and/or means that the second material is connected and/or bonded to the first material e.g. via covalent bonds (which may be formed e.g. by co -polymerization) or via non-covalent bonds such as hydrogen bonds or van-der Waals-forces. However, the term "embedded" in the context of this invention is meant also to especially include embodiments where the second material is provided as a separate material. According to a preferred embodiment of the present invention, the at least one second material is embedded in said at least one first material in form of and/or comprises molecules, dyes, labels, small beads, moieties, preferably with a diameter of >0.5nm and ≤lOOnm, more preferably between ≥lnm and ≤lOnm. According to an embodiment of the present invention, the average particle size of the at least one second material is >0.5nm and ≤lOOnm.
According to an embodiment of the present invention, the average particle size of the at least one second material is ≥lnm and ≤lOnm.
According to an embodiment of the present invention, the at least one second material has an anisotropic shape, preferably a disk or rod like shape, (typical sizes are 0.5nm x lnm x 3nm).
According to an embodiment of the present invention, the at least one second material has an anisotropic shape, and is substantially aligned with it's longest axis parallel to the surface of the sensor. However, according to another embodiment of the present invention, the at least one second material is randomly oriented. This has especially for many applications the advantage of that it is easy to manufacture.
According to an embodiment of the present invention, the at least one second material has an anisotropic dipole moment, which is substantially parallel to the polarization direction of the incident radiation.
According to an embodiment of the present invention, the at least one second material comprises a fluorescent material with an extinction coefficient > 10000 L mo 1-1 cm-1 and <300000 L mol-1 cm-1, preferably >20000 L mol-1 cm-1 and <200000 L mol-1 cm-1. Said extinction coefficient is measured at the wavelength λmax and/or +/- 50nm, at which the maximum extinction is obtained.
According to an embodiment of the present invention, the at least one second material comprises a fluorescent material with a fluorescence efficiency in the range of >5% and <100%, preferably >10% and <60% at the excitation wavelength used in the device. According to an embodiment of the present invention, the at least one second material is excited by an incident electromagnetic radiation characterized by a wavelength in the range >280nm and <1500nm, preferably >390 and <850nm.
According to an embodiment of the present invention, the at least one second material is excited by an incident electromagnetic radiation characterized sharp spectral distribution FWHM of <100 nm, preferably <50nm, most preferably < 30nm.
According to an embodiment of the present invention, the second material comprises at least one fluorescent material selected out of the group comprising triphenylmethane dyes, azo dyes, cyanine dyes, phtalocyanin dyes, acridine dyes, anthrachinone dyes, anthocyane dyes, phenazin dyes, phenoxazin dyes, phenothiazine dyes, furanone dyes, coumarine dyes, oxazines dyes, rhodomine dyes, carbocyanine dyes, stilbene dyes, oligophenyl dyes, perylene dyes, styryl dyes, benzophenoxazonium dyes, oligophenylenevinylene or mixtures thereof.
According to an embodiment of the present invention, the sensor comprises a light emitting means which emits polarized light towards the second material. By doing so it has been shown that the excitation depth of the light into the first and/or second material may be optimized for a wide range of applications within the present invention..
Preferably the sensor comprises a light emitting means which emits polarized light and which is capable to change the polarization direction, especially rotating and/or switching the polarization direction. By doing so it is possible to "tune" the excitation depth for a wide range of applications within the present invention.
According to an embodiment of the present invention, the excitation of light of the second material is caused by means of an incident evanescent field. It has been shown for many applications within the present invention that by doing so, the efficacy and accuracy of the sensor can be greatly increased while it is possible miniature the sensor.
According to an embodiment of the present invention, the first and second material are provided with and/or in between a wire grid as described in WO 2006/136991 and/or a 2 dimensional array of absorbing and/or reflecting elements. By doing so, said evanescent field can be build up very easily and efficiently for a wide range of applications within the present invention.
In the context of the present invention, a wire grid especially means and/or includes an array of at least one aperture defined in at least one layer (layer-x) that is essentially non-transparent for the incident light. A first in-plane dimensions (that is parallel to layer-x) of the wire grid is below the diffraction limit of the incident light in the medium that fills the aperture (diffraction limit is defined as wavelength/(2*refractive index of medium that fills aperture), while a second dimensions is above the diffraction limit.
An evanescent field in the space between the wires of the wire grid may be generated by illumination of the wire grid with light having R-polarized light, which is actually one embodiment of the present invention. It has been shown that for a wide range of applications within the present invention R-polarized light is essentially reflected or absorbed by the wire grid and has an electric field such that the projection of the electric field on a plane parallel to the wires of the wire grid is along the second dimension of the wire grid.
The term "with and/or in between" especially means that the wire grid is so provided in the vicinity of the first and/or second material to allow an evanescent field to function as an incident radiation for inducing emittance of light from the at least one second material.
According to an embodiment of the present invention, the at least one second material has an anisotropic shape, and is substantially aligned with its longest axis parallel to the direction of the wire grid.
According to an embodiment of the present invention, the sensor is and/or comprises a waveguide biosensor.
The term "waveguide biosensor" in the sense of the present invention especially means that the first material layer is used as cladding material for the waveguide. By doing so, the evanescent excitation of fluorescence can be performed by exciting a waveguide mode. The first and/or second material is within the evanescent field of the waveguide mode, and results in a fluorescent signal that depends on thickness of hydrogel layer. In this embodiment it is especially preferred that the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and <200 nm.
According to a further embodiment of the present invention, the sensor uses total internal reflection. This especially includes and/or means that the first material is provided on a substrate with an index of refraction higher than the first material. By illumination via the glass under angles of incidence above the critical angle for the substrate- hydrogel interface, it is possible to excite evanescent waves and similar to the waveguide biosensor use this for surface specific excitation of the fluorophores in the hydrogel.
In this embodiment it is especially preferred that the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and <200 nm.
According to a further embodiment of the present invention, the sensor is and/or comprises a surface plasmon biosensor. This especially includes and/or means that the first material is provided on top of a metal film. Preferred materials for metal film are gold and/or silver, preferably with a thickness <100 nm, more preferred 60 nm ± lOnm. Along the interface between the metal film and the hydrogel layer, surface plasmon waves can propagate. These surface plasmons waves have an evanescent field in both the metal film and the hydrogel. By excitation of surface plasmon waves, it is possible to locally excite the fluorophores and this way perform a surface specific fluorescence measurement.
In this embodiment it is especially preferred that the penetration depth of evanescent field (1/e intensity decay) is > 100 nm and <200 nm. According to a further embodiment of the present invention, the sensor includes confocal excitation.
This especially means and/or includes that the first material is provided on top of a transparent substrate. By illuminating the interface between hydrogel and substrate with a focused excitation spot (preferably with a NA of the lens of >0.5) it is possible to detect the generated fluorescence through the same lens. In a further embodiment, the surface specificity of the fluorescence measurement is obtained by focusing the collected fluorescence on a small pinhole. In this case, the fluorescence generated away from the hydrogel-substrate interface may be focused in front or behind the pinhole and is usually substantially blocked by the pinhole.
According to a preferred embodiment, the depth of the measurement volume is >1 and <2 micrometers.
Preferably the change of the first material includes shrinking and/or swelling. According to a further embodiment of the present invention, the device comprises a sensor having a sensor direction and the changing direction is essentially perpendicular to the sensor direction.
The term "sensor direction" especially means and/or includes that in case the sensor extends itself in one or two dimensions larger than in the other two (or one), so that the sensor is either somewhat flat or forms a needle, the "sensor direction" would then be the direction where the sensor has its longest extension.
According to an embodiment of the present invention, the uniformity of distribution of the at least one second material in the first material is homogenous in a plane parallel to the sensor direction. This has been shown to be advantageous for many applications within the present invention.
According to an embodiment of the present invention, the uniformity of distribution of the at least one second material in the first material in the direction perpendicular to the senor surface is non-uniform, preferably having a gradient, which may be either linear or stepwise. This has been shown to be advantageous for many applications within the present invention.
According to a preferred embodiment of the present invention, the first material changes its size and/or thickness when interacting with the at least one analyte.
According to an embodiment of the present invention, the first material is provided in form of a gel. According to an embodiment of the present invention said second material is substantially forming a layer. According to an embodiment of the present invention said second material is embedded in a third material substantially forming a layer.
According to a preferred embodiment of the present invention, the thickness of the at least one second material and or said third material is >0.5 nm and ≤IO nm, preferably >1 nm and <8 nm, most preferably >1.5 nm and <5 nm.
According to an embodiment of the present invention, the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.1% and <40%.
According to an embodiment of the present invention, the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.2% and <10%.
According to an embodiment of the present invention, the concentration (expressed as percent of the total volume) of the at least one second material in said third material is >0.5% and <5%. According to an embodiment of the present invention, the first material is provided in form of a layer.
The term "layer" means and/or includes especially that the thickness of the first material in one dimension is >0% and <50% than in either one of the other dimensions. For some applications within the present invention, the layer thickness is bound to a range in order to obtain optimum device properties: On the one hand a too thin layer will yield a low signal and insufficient sensitivity whereas a too thick layer will yield a slow response due to long diffusion times.
According to an embodiment of the present invention, the first material is provided in form of a layer with a thickness of >5 nm and ≤IOOO nm.
According to an embodiment of the present invention, the first material is provided in form of a layer with a thickness of >20 nm and <500 nm.
According to an embodiment of the present invention, the first material is provided in form of a layer with a thickness of >50 nm and <200 nm. According to an embodiment of the present invention, the maximum thickness change of the first material upon interaction with said at least one analyte in the sensing direction is between >0.2% and <90%, preferably between >1% and <30%.
According to an embodiment of the present invention, the maximum thickness change of the first material upon interaction with said at least one analyte in the sensing direction is between >0.5nm and <50nm preferably between ≥lnm and <20 nm.
According to an embodiment of the present invention, the first material comprises a hydrogelic material.
In the sense of the present invention, the term "hydrogelic material" means and/or includes especially that this material comprises polymers that in water form a water-swollen network.
The term "hydrogel material" in the sense of the present invention furthermore especially means that at least a part of the hydrogel material comprises polymers that in water form a water-swollen network and/or a network of polymer chains that are water-soluble. Preferably the hydrogel material comprises in swollen state >50% water and/or solvent, more preferably >70% and most preferred >90%, whereby preferred solvents include organic solvents, preferably organic polar solvents and most preferred alkanols such as Ethanol, Methanol and/or (Iso-) Propanol.
In the sense of the present invention, the term "hydrogelic material" means and/or includes especially that the hydrogel is responsive which means that it displays a change of shape and total volume upon a change of a specific parameter. Such parameter can be a physical (temperature, pressure) or chemical property (ionic concentration, pH, analyte concentration) or biochemical property (enzymatic activity).
According to an embodiment of the present invention, the hydrogel material comprises a material selected out of the group comprising poly(meth)acrylic materials, silicagel materials, substituted vinyl materials or mixture thereof.
According to an embodiment of the present invention, the hydrogel material comprises a substituted vinyl material, preferably vinylcaprolactam and/or substituted vinylcaprolactam.
According to an embodiment of the present invention, the hydrogel material comprises a poly(meth)acrylic material made out of the polymerization of at least one (meth)acrylic monomer and at least one polyfunctional (meth)acrylic monomer.
According to an embodiment of the present invention, the (meth)acrylic monomer is chosen out of the group comprising (meth)acrylamide, acrylic esters, hydroxyethyl(meth)acrylate, ethoxyethoxyethyl(meth)acrylate or mixtures thereof. According to an embodiment of the present invention, the polyfunctional
(meth)acrylic monomer is a bis-(meth)acryl and/or a tri-(meth)acryl and/or a tetra- (meth)acryl and/or a penta-(meth)acryl monomer.
According to an embodiment of the present invention, the polyfunctional (meth)acrylic monomer is chosen out of the group comprising bis(meth)acrylamide, tetraethylene glycol di(meth)acrylate, Methylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, tripropyleneglycol di(meth)acrylates, pentaerythritol tri(meth)acrylate polyethyleneglycoldi(meth)acrylate, ethoxylated bisphenol-A- di(meth)acrylate , hexanedioldi(meth)acrylate or mixtures thereof.
According to an embodiment of the present invention, the hydrogel material comprises an anionic poly(meth)acrylic material, , preferably selected out of the group comprising (meth)acrylic acids, arylsulfonic acids, especially styrenesulfonic acid, itaconic acid, crotonic acid, sulfonamides or mixtures thereof, and/or a cationic poly(meth)acrylic material , preferably selected out of the group comprising vinyl pyridine, vinyl imidazole, aminoethyl (meth)acrylates or mixtures thereof, co- polymerized with at least one monomer selected out of the group neutral monomers, preferably selected out of the group vinyl acetate, hydroxy ethyl (meth)acrylate (meth)acrylamide, ethoxyethoxyethyl(meth)acrylate or mixture thereof, or mixtures thereof. These co-polymers change their shape as a function of pH and can respond to an applied electrical field and/or current by as well. Therefore these materials may be of use for a wide range of applications within the present invention.
According to an embodiment of the present invention, the first material comprises a hydrogelic material comprising thermo-sensitive polymers.
According to an embodiment of the present invention, the first material comprises a hydrogelic material comprising monomers selected out of the group comprising poly-N-isopropylamide (PNIPAAm) and copolymers thereof with monomers selected out of the group comprising polyoxyethylene, trimethylol-propane distearate, poly-ε-caprolactone or mixtures thereof.
According to an embodiment of the present invention, the hydrogelic material is based on thermo -responsive monomers selected out of the group comprising N-isopropylacrylamide, di(m)ethylacrylamide, carboxyisopropylacrylamide, hydroxymethylpropylmethacrylamide, acryloylalkylpiperazine, N-vinylcaprolactam. and copolymers thereof with monomers selected out of the group hydrophilic monomers, comprising hydroxyethyl(meth)acrylate, (meth)acrylic acid, acrylamide, polyethyleneglycol(meth)acrylate, N-vinyl pyrolidone, dimethylaminopropylmethacrylamide, dimethylaminoethylacrylate, N- hydroxymethylacrylamide or mixtures thereof, and/or co -polymerized with monomers selected out of the group hydrophobic monomers, comprising (iso)butyl(meth)acrylate, methylmethacrylate, isobornyl(meth)acrylate, glycidyl methacrylate or mixtures thereof. These co-polymers are known to be thermo -responsive and therefore may be of use for a wide range of applications within the present invention. According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >1% and <500% at 200C.
In the sense of the present invention, the swelling ratio especially includes, means or refers to a measurement according to the following procedure: The first material was dried to form a film in an oven under the temperature of 500C. The film was immersed in an excess of deionized water to remove the residual unreacted compounds. The swollen polymer film was then cut into disk forms with 8mm in diameter and dried at 500C until the weight no longer changed. A preweighed dried sample (Wo) was immersed in an excess of deionized water in a thermostatic water bath until the swelling equilibrium was attained. The weight of the wet sample (Wi) was determined after the removal of the surface water via blotting with filter paper. The equilibrium swelling ration was calculated with the following formula swelling ratio = (Wi - Wo) / Wo
According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >3% and <200% at 200C. According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >5% and <100% at 200C.
According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >1% and <30% at 200C.
According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >1% and <25% at 200C.
According to an embodiment of the present invention, the first material comprises a hydrogelic material with a swelling ratio of >1% and <20% at 200C.
According to an embodiment of the present invention, the first material comprises a receptor for the analyte to be detected. In the sense of the present invention, the term "receptor" means and/or includes especially that some chemical moieties are present in the first material which are capable to interact with a selected analyte e.g. by hydrostatic interactions, hydrogen bonding, chemical reception, molecular recognition and the like.
An example of such a receptor system is "calmodulin" which binds to both calcium as well as to a range of anti-psychotic molecules, referred to as the phenothiazines, [J.D. Ehrick (Nature Materials p. 298-302, Vol. 4, April 2005)], which is hereby fully incorporated by reference.
According to an embodiment of the present invention, the first material is a polymeric material. According to an embodiment of the present invention, the first material is a polymeric material with a conversion of >50% and <100%.
In the sense of the present invention, the conversion especially includes, means or refers to a measurement according to the following procedure:
After the polymerization of the first material and the embedding of the at least one second material, a quantitative amount of inhibitor was introduced into a sample of the first material and the sample was quickly quenched in an ice bath. For the removal of remaining monomers and initiators, the sample as washed with deionized water several times. After that, the sample was dried in vacuum oven at 700C until there was no change in weight anymore. The conversion was calculated as follows: conversion = (P-F)/Mo * 100 % where P is the weight of the dry copolymer composite network obtained from the sample, F is the theoretical weight of the at least one second material incorporated in the first material (if present) and M0 is the weight of the monomers in the feed. According to an embodiment of the present invention, the first material is a polymeric material with a conversion of >70% and <95%.
The term "essentially" means and/or includes especially a wt-% content of >90 %, according to an embodiment >95 %, according to an embodiment >99 %.
According to an embodiment of the present invention, the crosslink density in the first material is >0.002 and <1, preferably >0.05 and <1.
In the sense of the present invention, the term "crosslink density" means or includes especially the following definition: The crosslink density δx is here defined as
δ „ = where X is the mole fraction of polyfunctional monomers and L the mole x L + X fraction of linear chain (= non polyfunctional) forming monomers. In a linear polymer δx = 0 , in a fully crosslinked system δx = 1 .
According to a further embodiment of the present invention, the second material is embedded and/or incorporated in a third matrix material. By doing so, it can for a wide range of applications within the present invention be ensured that the arrangement of the second material does not change during the lifetime of the sensor. According to a further embodiment of the present invention, the interaction of the first material includes a phase transition in said first material, preferably an phase separation such a an LCST (lower critical solution temperature). Such a phase separation in the first material (as a cause of the volume change) has for many applications within the present invention the advantage that non-linear effects may be obtained. By doing so it has been shown that the change in shape for a certain change of "trigger" (e.g. concentration) may even be increased for many applications within the present invention.
Preferably the matrix material is a polymeric organic material, which is preferably and according to a further embodiment of the present invention permeable to the sample to be analyzed by the first material. Suitable and insofar preferred materials for the matrix material include hydrogels, brush polymers and/or cross-linked polymer brush layers, porous polymeric networks obtained e.g. by polymerization induced phases separation. Typical pore size range from a few nanometer to several micrometer. According to a further embodiment of the present invention, the first material is functionalized in order to bind to the at least one second material and/or to the matrix material described above.
According to a further embodiment of the present invention, the first material is partially made out of a material compolymeried with glycidylmethacrylate and/or acrylic acid N-hydroxy succinimide ester. By doing so it has been found that the second material may bind itself to the epoxy groups (in case of the glycidylmethacrylate) or the ester groups (in case of the succinimide ester)s for many applications within the present invention.
According to a further embodiment the first material comprises biotinylated and/or streptavidin functionalized end-groups. By doing so, the biotin /streptavidin link may be used to bind the second material to the first material, which has been shown to be advantageous for a wide range of applications within the present invention.
According to a further embodiment the adhesion between the first and second material(s) is enhanced by applying an adhesion promoter. Alternatively or additionally, according to a further embodiment the first material is provided with primary amine side groups, which may covalently bond to the second material provided with epoxy or NHS functionalities. This has been shown to be advantageous for a wide range of applications within the present invention. For example the responsive hydrogel monomers can be copolymerized with glycidylmethacrylate (to obtain epoxy side groups) or acrylic acid N-hydroxy succinimide ester (NHS). When functionalized with primary amine groups the magnetite particles can be covalently linked to the hydrogel surface. Alternatively, biotinylated beads can be coupled to a strepavidin- functionalized hydrogel. According to a further embodiment of the present invention, the first material is at least partly surrounded by a well coated with a non-sticking material, which has a surface tension of <30 mN/m, preferably <25 mN/m.
According to a further embodiment of the present invention the first material is surrounded by the non- sticking material which has a surface tension of <30 mN/m, preferably <25 mN/m in substantially all directions which are perpendicular to the changing direction.
According to a further embodiment of the present invention, the non- sticking material is a fluor-containing material, preferably a fluorinated monolayer material, which was preferably made using plasma treatment, e.g. CF4 plasma treatment or by vapour deposition of a fluorsilane e.g. perfluoroalkylchlorosilane. According to a further embodiment, the device comprises a substrate and/or matrix material in the vicinity of the first material, whereby the device comprises at least one adhesion promoting layer between the first material and the substrate and/or matrix material.
According to a preferred embodiment of the present invention, the adhesion promoting layer is a monolayer.
Preferably the at least one adhesion promoting layer is chosen from the group silane-containing layers, thiol-containing layers, amine-containing layers or mixtures thereof.
The term "silane-containing layer" especially means and/or includes a layer which comprises a material of the form
Figure imgf000018_0001
whereby Ri is selected out of the group comprising acrylate, methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol;
R-2 is selected out of the group alkylene, arylene, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or poly disulfides,
R3 and R4 are independently selected out of the group halogen, R6-R7 (whereby R5 is selected out of the group comprising acrylate, methacrylate, acrylamide, methacrylamide, allyl, vinyl, acetyl, amine, epoxy or thiol and R7 is selected out of the group alkyl, aryl, mono- or polyalkoxy, mono- or polyalkylamine, mono- or polyamide, thioether, mono- or polydisulfϊdes), 0-R8 (whereby R8 is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, heteroaryl, halogen) R5 represents the group 0-R9, where R9 is selected out of the group hydrogen, alkyl, long-chain alkyl, aryl, halogen and/or R5 is a hydrolyzable moiety.
Generic group definition: Throughout the description and claims generic groups have been used, for example alkyl, alkoxy, aryl. Unless otherwise specified the following are preferred groups that may be applied to generic groups found within compounds disclosed herein: alkyl: linear and branched Cl-C8-alkyl, alkylene: selected from the group consisting of: methylene; 1,1 -ethylene; 1,2-ethylene; 1,1-propylidene; 1,2-propylene; 1,3- propylene; 2,2-propylidene; butan-2-ol- 1 ,4-diyl; propan-2-ol- 1 ,3-diyl; 1 , 4-butylene; cyclohexane-l,l-diyl; cyclohexan-l,2-diyl; cyclohexan-1,3- diyl; cyclohexan-l,4-diyl; cyclopentane-l,l-diyl; cyclopentan-l,2-diyl; and cyclopentan-l,3-diyl, long-chain alkyl: linear and branched C5-C20 alkyl alkenyl: C2-C6-alkenyl, cycloalkyl: C3-C8-cycloalkyl, alkoxy: Cl-C6-alkoxy, long-chain alkoxy: linear and branched C5-C20 alkoxy aryl: selected from homoaromatic compounds having a molecular weight under 300, arylene: selected from the group consisting of: 1 ,2-phenylene; 1,3- phenylene; 1,4-phenylene; 1,2-naphtalenylene; 1,3-naphtalenylene; 1,4- naphtalenylene; 2,3-naphtalenylene; l-hydroxy-2,3-phenylene; l-hydroxy-2,4- phenylene; l-hydroxy-2,5- phenylene; and l-hydroxy-2,6-phenylene, heteroaryl: selected from the group consisting of: pyridinyl; pyrimidinyl; pyrazinyl; triazolyl; pyridazinyl; 1,3,5-triazinyl; quinolinyl; isoquinolinyl; quinoxalinyl; imidazolyl; pyrazolyl; benzimidazolyl; thiazolyl; oxazolidinyl; pyrrolyl; carbazolyl; indolyl; and isoindolyl, wherein the heteroaryl may be connected to the compound via any atom in the ring of the selected heteroaryl, amine: the group -N(R)2 wherein each R is independently selected from: hydrogen; Cl-C6-alkyl; Cl-C6-alkyl-C6H5; and phenyl, wherein when both R are Cl- C6-alkyl both R together may form an - NC3 to an -NC5 heterocyclic ring with any remaining alkyl chain forming an alkyl substituent to the heterocyclic ring, halogen: selected from the group consisting of: F; Cl; Br and I, polyether: chosen from the group comprising-(O-CH2-CH(R))n-OH and - (0-CH2-CH(R))n-H whereby R is independently selected from: hydrogen, alkyl, aryl, halogen and n is from 1 to 250
Unless otherwise specified the following are more preferred group restrictions that may be applied to groups found within compounds disclosed herein: alkyl: linear and branched Cl-C6-alkyl, long-chain alkyl: linear and branched C5-C10 alkyl, preferably linear C6- C8 alkyl alkenyl: C3-C6-alkenyl, cycloalkyl: C6-C8-cycloalkyl, alkoxy: Cl-C4-alkoxy,
long-chain alkoxy: linear and branched C5-C10 alkoxy, preferably linear C6-C8 alkoxy aryl: selected from group consisting of: phenyl; biphenyl; naphthalenyl; anthracenyl; and phenanthrenyl, heteroaryl: selected from the group consisting of: pyridinyl; pyrimidinyl; quinolinyl; pyrazolyl; triazolyl; isoquinolinyl; imidazolyl; and oxazolidinyl, wherein the heteroaryl may be connected to the compound via any atom in the ring of the selected heteroaryl, heteroarylene: selected from the group consisting of: pyridin 2,3-diyl; pyridin-2,4-diyl; pyridin-2,6-diyl; pyridin-3,5-diyl; quinolin-2,3-diyl; quinolin-2,4-diyl; isoquinolin-l,3-diyl; isoquinolin-l,4-diyl; pyrazol- 3,5-diyl; and imidazole-2,4-diyl, amine: the group -N (R) 2, wherein each R is independently selected from: hydrogen; Cl-C6-alkyl; and benzyl, halogen: selected from the group consisting of: F and Cl, polyether: chosen from the group comprising-(O-CH2-CH(R))n-OH and - (O-CH2-CH(R))n-H whereby R is independently selected from: hydrogen, methyl, halogen and n is from 5 to 50, preferably 10 to 25. It has been shown for a wide range of applications that this silane- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device.
The term "thiol-containing layer" especially means and/or includes a layer which comprises a material of the form R-SH with R chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl.
It has been shown for a wide range of applications that this thiol- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If a thiol-containing layer is used, the surface of the matrix material is chosen out of a thiol-binding material, especially the surface of the matrix material is an Au-surface.
The term "amine-containing layer" especially means and/or includes a layer which comprises a material of the form Ri-NH-R2 with Ri chosen out of the group alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether and R2 chosen out of the group hydrogen, alkyl, long-chain alkyl, alkenyl, cycloalkyl, polyether. It has been shown for a wide range of applications that this amine- containing layer helps to link the first material to the substrate and/or matrix material essentially without influencing the performance of the sensor device. If a amine- containing layer is used, the surface of the matrix material is preferably equipped with amine-binding groups, preferably epoxy groups and/ or reactive esters, halogenides and/or amines.
The present invention furthermore relates to a method of measuring the presence, identity, amount and/or concentration of at least one analyte in a sample using a sensor as described above, comprising the steps of
(a) Allowing the first material to interact with the at least one analyte to cause a change of physical properties in the first material
(b) Measuring the change of at least one signal detected by the sensor upon interaction with the at least one second material.
According to an embodiment of the present invention, the first material changes its size and/or thickness when interacting with the at least one analyte.
A sensor and/or a method according to the present invention may be of use in a broad variety of systems and/or applications, amongst them one or more of the following: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures and body fluids such as e.g. blood, urine or saliva - high throughput screening devices for chemistry, pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research - tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics tools for combinatorial chemistry analysis devices
The aforementioned components, as well as the claimed components and the components to be used in accordance with the invention in the described embodiments, are not subject to any special exceptions with respect to their size, shape, material selection and technical concept such that the selection criteria known in the pertinent field can be applied without limitations.
Additional details, features, characteristics and advantages of the object of the invention are disclosed in the subclaims, the figures and the following description of the respective figures and examples, which — in an exemplary fashion — show preferred embodiments of a sensor according to the invention.
Fig. 1 shows a very schematic cross-sectional view of a sensor according to a first embodiment of the present invention; Fig. 2 shows a very schematic cross-sectional view of a sensor according to Fig. 1 with the first material in a swollen state.
Fig. 3 shows a very schematic cross-sectional view of a sensor according to a second embodiment of the present invention; Fig. 4 shows a diagram of the fluorescence of a sensor according to
Example I vs. the swelling of the first material in the sensor.
Fig. 1 shows a very schematic cross-sectional view of a sensor 1 according to a first embodiment of the present invention. The sensor comprises a first material 10 with a second material 20 embedded therein. The second material and the first material are covalently bonded together via co -polymerization.
The first material 10 is provided on a sensor substrate 60, which is transparent for the light emitted by the second material. Between the sensor substrate 60 and the first material 10 there is a silane monolayer 40 as described above (it should be added that Fig. 1 is highly schematic and in actual embodiments the thickness of the layer 40 will be of course much smaller). Left and right of the first material 10 there is a non- sticking material 30.
The first material 10 is placed between a wire grid 50, which functions in order to create an evanescent field. It should be noted that in Fig. 1 the height of the grid 50 is higher than the first material 10; however, in most actual applications, the height of the first material will be larger, as described above.
Fig. 2 shows a very schematic cross-sectional view of a sensor according to Fig. 1 with the first material in a swollen state. In Fig. 2, the components, which are identical with the embodiment of Fig. 1 are not discussed to avoid repetitions. As can be seen in Figs. 1 and 2, upon radiation onto the wire grid, an evanescent field (as indicated by the area "F") will be created, whose strength decreases exponentially with the distance from the substrate material 60. Upon stimulation with the evanescent field, the second material 20 will emit light via fluorescence.
Since the strength of the field varies along the first material, it is clear that only a part of the second material, i.e. this material which is located not too far from the substrate material 60 will be able to emit light via fluorescence, since only then the field F is strong enough. The minimum strength required to induce fluorescence has been marked by a black line in Fig. 1 and 2.
As is immediately apparent from Figs. 1 and 2, in Fig. 2 the second material which is (due to the swelling of the first material) located "over the black line" is not able to fluorescence any more. Therefore a swelling of the first material 10 goes along with a decrease in fluorescence; thus so the swelling state of the first material 10 can be measured easily, effectively and accurate.
Fig. 3 shows a very schematic cross-sectional view of a sensor according to a second embodiment of the present invention; In Fig. 3, the components, which are identical with the embodiment of Fig. 1 are not discussed to avoid repetitions.
Fig. 3 differs from the embodiment of Fig. 1 in the form of the wire grid (i.e. a trapezoid cross-section) but also on the provision of the first and second material. In this embodiment, the second material 20 is placed next (i.e. on top) of the first material 10.
EXAMPLES:
The invention will furthermore be better understood by the following Example I which is presented for illustrative purposes only.
In the example, a sensor according to Fig. 1 was used, whereby the initial thickness of the first material was 150 nm. The first material comprised a second material (= fluorophores) in the hydrogel, as described above, and comprises a glucose-sensitive receptor (as described above). The used evanescent decay length corresponded with the 1/e intensity of 20 nm. The duty cycle of wire grid (i.e. fraction of wire grid not covered with wires) was 50 %. The height of the wire grid (i.e. the numeral 50 in Fig. 1) is 150 nm, too.
A (fitted) curve of the fluorescence signal normalized by initial signal is shown in Fig. 4. In Fig. 4 it can be clearly seen that fluorescence signal depends strongly on change in thickness of gel layer.
The particular combinations of elements and features in the above detailed embodiments are exemplary only; the interchanging and substitution of these teachings with other teachings in this and the patents/applications incorporated by reference are also expressly contemplated. As those skilled in the art will recognize, variations, modifications, and other implementations of what is described herein can occur to those of ordinary skill in the art without departing from the spirit and the scope of the invention as claimed. Accordingly, the foregoing description is by way of example only and is not intended as limiting. The invention's scope is defined in the following claims and the equivalents thereto. Furthermore, reference signs used in the description and claims do not limit the scope of the invention as claimed.

Claims

CLAIMS:
1. A chemical sensor, especially for determining the presence, identity, amount and/or concentration of at least one analyte in a fluid sample comprising
(a) a first material, whereby the first material is an elastic material adapted to change its physical properties according to interaction with said at least one analyte;
(b) at least one sensor;
(c) at least one second material adapted to interact with the at least one sensor whereby the second material comprises at least one fluorescent material, whereby the first, at least one second material and the at least one sensor are so provided towards each other that upon a change of the physical properties of the first material a change in at least one of the signals detected by the at least one sensor occurs.
2. The sensor according to claim 1, comprising a changing direction, essentially in which the change of the first material in response to the at least one analyte occurs.
3. The sensor according to any of the claims 1 to 2, whereby the at least one sensor and/or the at least one second material are provided in the changing direction at opposite ends of the first material.
4. The sensor according to any of the claims 1 to 3, whereby the at least one second material is embedded in said at least one first material.
5. The sensor according to any of the claims 1 to 5 whereby the at least one second material comprises a fluorescent material with an extinction coefficient > 10000 L mol-1 cm-1 and <300000 L mol-1 cm-1.
6. The sensor according to any of the claims 1 to 5 whereby the at least one second material comprises a fluorescent material with a fluorescence efficiency in the range of >5% and <100%, preferably >10% and <60% at the excitation wavelength used in the device.
7. The sensor according to any of the claims 1 to 6 whereby the excitation of light of the second material is caused by means of an incident evanescent field.
8. The sensor according to any of the claims 1 to 7 whereby the first and second material are provided with and/or in between a wire grid.
9. A method of measuring the presence, identity, amount and/or concentration of at least one analyte in a sample using a sensor as described above, comprising the steps of
(a) Allowing the first material to interact with the at least one analyte to cause a change of physical properties in the first material
(b) Measuring the change of at least one signal detected by the sensor upon interaction with the at least one second material.
10. A system incorporating a sensor according to any of the Claims 1 to 8, and/or adapted to carry out the method of the claim 9 and being used in one or more of the following applications: biosensors used for molecular diagnostics rapid and sensitive detection of proteins and nucleic acids in complex biological mixtures such as e.g. blood or saliva - high throughput screening devices for chemistry, pharmaceuticals or molecular biology testing devices e.g. for DNA or proteins e.g. in criminology, for on-site testing (in a hospital), for diagnostics in centralized laboratories or in scientific research tools for DNA or protein diagnostics for cardiology, infectious disease and oncology, food, and environmental diagnostics tools for combinatorial chemistry analysis devices
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