CA2262011A1 - Biosensor with novel passivating layer - Google Patents
Biosensor with novel passivating layer Download PDFInfo
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- CA2262011A1 CA2262011A1 CA 2262011 CA2262011A CA2262011A1 CA 2262011 A1 CA2262011 A1 CA 2262011A1 CA 2262011 CA2262011 CA 2262011 CA 2262011 A CA2262011 A CA 2262011A CA 2262011 A1 CA2262011 A1 CA 2262011A1
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- Prior art keywords
- biosensor according
- vinyl
- transducer
- passivating layer
- ether
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- 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
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
Disclosed is a biosensor having an immobilized biocomponent for specific binding of a biomolecule (analyte) which is present in an aqueous solution, comprising a transducer and a layer of an organic hydrogel as a passivating layer which suppresses nonspecific binding. The passivating layer is preferably formed by graft polymerization of a hydrophilic vinyl monomer onto an activated polymeric intermediate layer formed on the transducer.
Description
BIOSENSOR WITH NOVEL PASSIVATING LAYER
1. Field of the Invention The invention relates to a biosensor having an immobilized biocomponent for detecting a specific biomolecule (or analyte)in an aqueous matrix (or solution) and having a novel, highly effective passivating layer for suppressing nonspecific binding. The invention moreover relates to a process for producing the biosensor.
1. Field of the Invention The invention relates to a biosensor having an immobilized biocomponent for detecting a specific biomolecule (or analyte)in an aqueous matrix (or solution) and having a novel, highly effective passivating layer for suppressing nonspecific binding. The invention moreover relates to a process for producing the biosensor.
2. Prior Art The mode of operation of biosensors is based on combining a biocomponent capable of specifically binding an analyte that is a biomolecule present in an aqueous matrix, with a transducer (or signal converter) which converts a primary chemical or physical signal created by the binding into an optical or electrical secondary signal which can be amplified and evaluated in a conventional manner. There have been disclosures of reversibly operating biosensors (biosensors in the narrower sense) and also irreversible biosensors (probes), which are discarded after a single use.
Examples of biospecific bind pairs composed of the biocomponent of the sensor and the biomolecule as analyte are an antibody and an antigen, a receptor and a ligand, and also O.Z. 5269 an enzyme and a substrate. In modern biosensors, the biocomponent is normally immobilized on a surface of the transducer.
The transducer may, for example, be a piezoelectric crystal which reacts to the change in mass on its surface as a result of the specific binding of the biomolecule with a change in its oscillation behaviour, the change indicating the existence of binding (qualitatively) and, if desired, its extent (quantitatively). Instead of using piezoelectric oscillations for the process of converting the signal, it is also possible to use surface acoustic waves (SAW) of piezoelectric substrates for this purpose. The depth of penetration of the surface acoustic wave is in the range of its wavelength, and therefore biomolecules which are specifically bound at the surface have an effect on the transmission of the waves. Biosensors which operate with transducers based on piezoelectric oscillations or on surface acoustic waves are mass sensors, since they fundamentally give information on the change in mass (i.e., weight) at the transducer surface. Another known biosensor type utilizes transducers which convert the signals received into optical phenomena and determine the analyte by making use of changes in radiation absorption, in emission after excitation of the probe, in reflection, refraction and diffraction, or in polarization.
O.Z. 5269 The signals emitted by the transducer are electronically amplified and subjected to comparative evaluation, the result being a yes/no statement or, in the case of quantitative analysis, a numerical value.
The sensitivity of detection of a biomolecule in a matrix with a biosensor is diminished by the fact that the transducer converts into electrical or optical signals not only the signals deriving from specific binding of the biomolecule to the biocomponent of the sensor but also signals deriving from nonspecific binding of other components of the matrix. This nonspecific binding may be adsorptive, absorptive, covalent or ionic in nature and produces false analysis results, since the signal passed on by the transducer for evaluation is composed of the sum of the signal from specific and from nonspecific binding. The error caused by the nonspecific signal could be tolerated if it were constant for a particular analytical task in the same matrix, or at least a matrix of the same type. This is, however, not the case. For example, the extent of nonspecific binding during determination of antigens in blood differs from donor to donor, and therefore even if the antigen is absent the values obtained have a scatter the extent of which can be signified using the standard deviation of the blank measurements from the mean value. The greater the standard deviation, the lower the detection sensitivity, since a test is usually only judged to be positive if the measured value exceeds three times O.Z. 5269 times the standard deviation.
It is therefore desirable to prevent nonspecific binding, or at least to suppress the same, so that the standard deviation is minimized. In a variant which has been disclosed (see, for example, K.A. Davis, T.R. Leary:
Continuous Liquid Phase Piezoelectric Biosensor for Kinetic Immunoassays, Analy. Chem. 61 (1989), 1227-1230), nonspecific binding of proteins is suppressed with bovine serum albumin (BSA) as passivating layer in the determination of immunoglobulin G (IgG) in phosphate-buffered sodium chloride solution (PBS). However, the effectiveness of this passivating layer is not ideal because, on the one hand, the macromolecules of the BSA do not seal the transducer surface hermetically, so that there are unoccupied positions remaining for nonspecific binding and, on the other hand, BSA in its own right nonspecifically binds hydrophobic groups, e.g. in fatty acid molecules.
3. Summary of the Invention It has now been found that organic hydrogels give passivating layers for biosensors which have excellent effectiveness with respect to nonspecific binding. The invention therefore provides a biosensor with an immobilized biocomponent for specific binding of a biomolecule (analyte) which is present in an aqueous matrix. A significant feature of this biosensor is a layer of an organic hydrogel as passivating layer with respect to nonspecific binding.
O.Z. 5269 The invention further provides a process for producing the biosensor by the use of an organic hydrogel for a passivating layer.
The invention also provides a method for determining a biomolecule or analyte in an aqueous matrix by the use of the novel biosensor.
The novel biosensors are distinguished by a considerably smaller standard deviation of blank measurements from the mean value, and therefore by greater sensitivity than known biosensors with or without a passivating layer. This may be due to a hydrated envelope of the hydrogels, which deters the approach of, and nonspecific bind of, molecules which differ from the analyte. The passivating layer adheres particularly firmly, and therefore the biosensors are particularly suitable as reversible sensors if the hydrogel is applied by well known graft polymerization techniques. A
further advantage is that the cross-sensitivity is reduced (or the specificity for the sought-for analyte is increased).
4. Description of Preferred Embodiment of the Invention 2o The passivating layer of an organic hydrogel is the significant feature of the biosensor of the invention. It is preferably a mass sensor and the biocomponent which it contains is generally an antibody, an enzyme or a receptor, desirably sonically or covalently bound to the transducer, directly or via a polymeric intermediate layer. The transducer may be composed of conventional materials known for O.Z. 5269 this purpose, e.g. silicon nitride (Si3N4), silica or lanthanum niobate (LaNb03). The novel biosensor likewise has no particular features with regard to the processing and evaluation of the secondary signal created in the transducer.
In a preferred embodiment of the novel biosensor, the passivating layer of the organic hydrogel has been applied to the surface of the transducer directly or, preferably indirectly via an intermediate layer, especially desirably by grafting hydrophilic monomers, and contains on its free surface, covalently or sonically bound or integrated into the hydrogel, the biocomponent for specific binding with the biomolecule in the matrix. Biosensors having such a passivating layer grafted onto an activated polymeric intermediate layer and containing the biocomponents sonically or covalently bound on their free surface are distinguished by particularly low sensitivity.
4.1. Passivatinq Layer/Hydrophillic Vinyl Monomers The organic hydrogel of the passivating layer may be produced by polymerization, preferably by graft polymerization, of hydrophilic vinyl monomers, optionally together with proportions of hydrophobic vinyl monomers. The hydrophilic polymer becomes a hydrogel on contacting an aqueous medium, a condition which is always fulfilled in the use as specified. Preparation of hydrogels or of polymers which give a hydrogel may be conducted as described for example in German Patent Application Nos. 197 00 079.7 (O. Z.
O.Z. 5269 _ 7 _ 5146), 197 15 449.2 (O. Z. 5176) and 197 27 556.7 (O. Z. 5210).
The hydrogel of the passivating layer is, of course, itself hydrophillic. Preferred passivating layers are made of hydrogels which exhibit a contact angle of less than 40°, more preferably less than 30°, when the contact angle of an air bubble is determined by the method of R.J. Good et al., Techniques of Measuring Contact Angles in Surface and Colloid Science (ed. R.J. Good) Vol. 11, Plenum Press, New York, N.Y.
This is done by allowing an air bubble to form below the specimen immersed in water, and this bubble coats the surface to a greater or lesser extent depending on its hydrophilicity.
Suitable hydrophilic vinyl monomers contain at least one olefinic double bond, and also at least one hydrophilic group. The olefinic double bonds may be present in functional groups of various types, for example in alkenyl groups, such as vinyl or allyl radicals, or in radicals derived from unsaturated carboxylic acids or from their derivatives, for example acrylic acid or methacrylic acid, or from the amides of these carboxylic acids or malefic acid. There is also great scope for variation with regard to the hydrophilic groups.
Examples which may be mentioned of suitable hydrophilic groups are: hydroxyl groups, ether groups, acyloxy groups, carboxyl groups, carboxylic ester groups, carboxamide groups, carboalkoxy groups and nitrile groups; 1,2-epoxy groups;
sulfuric esters, sulfonic acid, sulfinic acid groups, phosphoric acid groups, phosphonic acid groups and phosphinic O.Z. 5269 _ g _ acid groups, including the salts and esters corresponding to these; primary, secondary and tertiary amino groups; acylamino groups, which may be in open chains or incorporated into a ring; polyalkylene oxide groups, such as polyethylene oxide groups and polypropylene oxide groups, with or without a terminal hydroxyl group; polyester groups, polyesteramide groups and polyetheresteramide groups; the pyrrolidone ring and similar heterocycles, and also radicals of olefinically functionalized sugars. The hydrophilicity of a monomer is, of course, dependent of the balance between hydrophilic and hydrophobic fractions in its molecule. Monomers suitable for the invention are soluble in water at 20°C to an extent of at least 1% by weight, preferably at least 10% by weight and in particular at least 40% by weight, based in each case on the entire solution.
The hydrophilic vinyl monomers used for the invention preferably contain one olefinic double bond and one hydrophilic group. However, they may also have more than one olefinic double bond and/or hydrophilic group. Open-chain polyalkylene oxides having two terminal vinyl, allyl, acryloxy or methacryloxy groups, for example, are therefore very suitable.
Examples of suitable hydrophilic vinyl monomers which may be mentioned are: acrylic acid and its derivatives, e.g. acrylamide, N,N-diemethylacrylamide, acrylonitrile, methyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl O.Z. 5269 acrylate and 2-methoxyethyl acrylate; 2-ethyoxyethyl acrylate, 4-hydroxybutyl acrylate and 1,4-butanediol diacrylate, and also methacrylic acid and its corresponding derivatives; vinyl derivatives of carboxylic acids, such as vinyl acetate, N-vinylactetamide and N-vinylpyrrolidone; vinylsulfonic acids and their alkali metal salts, such as sodium vinylsulfonate;
alkenylarylsulfonic acids and their alkali metal salts, such as styrenesulfonic acid and sodium styrene sulfonate, vinyl ethers, such as vinyl methyl ether, vinyl ethyl ether, vinyl glycidyl ether, diethylene glycol divinyl ether and vinyl n-butyl ether; vinyl ketones, such as vinyl methyl ketone, vinyl ethyl ketone and vinyl n-propyl ketone; vinylamines, such as N-vinylpyrrolidine; polyalkyleneoxy compounds with terminal allyl, vinyl, acrylyl or methacylyl groups, such as ethoxytetraethoxyethyl acrylate or methacrylate, n-propoxydodecaethyleneoxyethyl vinyl ether, polyethylene glycol mono- or diacrylates with molecular weights of 600 or 1200, poly(ethylene/propylene) glycol mono- or dimethacrylates with molecular weights of 400 and 800, and also allyloxyoctapropyl-eneoxyethanol; sugar derivatives, such as vinyl-substituted arbinose or acryloylated hyroxypropylcellulose; and functionalized polyalkylene glycols, such as triethylene glycol diacrylate or tetraethylene glycol diallyl ether.
In cases where trifunctional or higher-functional vinyl monomers are used concomitantly, the result is a crosslinked copolymer and this, in particular in the graft O.Z. 5269 polymerization described subsequently, achieves a particularly good seal of the surface of the transducer and therefore a greater accuracy of measurement of the biosensor. Examples of suitable trifunctional or higher-functional vinyl monomers are polyacrylates, such as trimethylolpropane triacylate, trimethylolpropane trimethacrylate and pentaerythritol tetraacrylate; polyvinyl ethers, such as the trivinyl ether of glycerol-12E0 and the tetraallyl ether of pentaerythritol.
The crosslinking vinyl monomers may be used in amounts of preferably from 0.01 to 1 mol%, based on all of the monomers.
4.2. Hydrophobic Vinyl Monomers as Comonomers Besides the hydrophilic monomers, concomitant use may be made, in preparing the hydrogels or the polymers which are hydrogels on contact with an aqueous medium, of certain amounts, e.g. up to 60 mol%, based on all of the monomers, of hydrophobic vinyl monomers, e.g. a-olefins, such as propene, 1-butene and 1-octene; vinyl chloride; and vinyl aromatics, such as styrene, a-methylstyrene and vinyl toluene.
Instead of the crosslinking hydrophilic vinyl monomers mentioned which may optionally be used concomitantly, it is also possible to make concomitant use of trifunctional or higher-functional hydrophobic vinyl monomers in the amounts mentioned, the effect achieved being essentially the same as that described.
The molar proportion of the hydrophobic vinyl monomers should be judged in such a way that the hydrogel O.Z. 5269 shows a contact angle of less than 40°, preferably less than 30°C, in the test of R.J. Good et al. described above.
4.3. Preparation of the Organic Hydro~els in Bulk The monomers, in each case individually or else as a mixture appropriate for the respective application, may be polymerized in a conventional manner, preferably by free-radical-initiated solution or emulsion polymerization. The monomers are generally used as solutions of concentrations of from 1 to 40% by weight, preferably from 5 to 20% by weight.
It is expedient for the solvent to be water. The resultant polymer is then immediately a hydrogel. If an organic solvent is used for the operation, the polymer becomes a hydrogel at the latest during use of the biosensor in contact with the aqueous matrix. Use may be made of conventional polymerization initiators, e.g. organic peroxy compounds or hydroperoxides, azo compounds, peracids, persulfates or percarboneates, in the conventional amounts, the polymerization being initiated thermally or by irradiation.
The solutions or emulsions are immediately suitable for coating the transducers, preferably by spincoating, by which means passivating layers with thicknesses of from 0.1 to 100 ~.m can be created from the polymer.
Alternatively, the passivating layer may be created by grafting the hydrogel polymer depicted above onto the surface of the transducer. In this case, it is preferred to apply an intermediate layer of a (co)polymer before grafting;
O.Z. 5269 the intermediate layer is, or may be, activated so as to permit grafting. Grafting is preferred, since it gives a firmly adhering, particularly thin but nevertheless coherent coating on the surface of the transducer.
4.4. Polymeric Intermediate Layer and its Activation Suitable polymers for the intermediate layer are commodity polymers with or without radiation-sensitive groups, for example polyurethanes, polyamides, polyesters, polyethers, polyether-block-amides, polyester-block-amides, polysiloxanes, polystyrene, polyvinyl chloride, polycarbonates, polyolefins, polysulfones, polyisoprene, polychlorprene, polytetra-fluoroethylene, polyacrylates, polymethacrylates, and also the corresponding copolymers and blends. The intermediate layer may be applied preferably by spincoating from organic solutions and generally likewise has a thickness of from 0.01 to 100 Vim.
The surface of the intermediate layer may be activated by any of a large number of methods in preparation for grafting, and mention is made of the most important of these.
(1) Commodity polymers without W-radiation-sensitive groups may advantageously be activated by W radiation, e.g.
in the wavelength range from 100 to 400 nm, preferably from 125 to 310 nm. Particularly good results are achieved with continuous and largely monochromatic radiation, as produced, for example, by Excimer W sources (Heraeus, Kleinostheim, O.Z. 5269 Germany), for example using F2, Xe2, ArF, XeCl, KrCl or KrF as the medium for the lamp. However, other radiation sources, such as mercury vapor lamps with a broad radiation spectrum and a proportion of visible radiation are suitable as long as they emit considerable proportions of radiation in the wavelength ranges mentioned. It has been found that the presence of small amounts of oxygen is advantageous. The preferred partial pressures of oxygen are from 2x10-5 to 2x10-2 bar. The operation may, for example, be carried out at a reduced pressure of from l0-4 to 10-1 bar, or using an inert gas, such as helium, nitrogen or argon, with an oxygen content of from 0.02 to 20 parts per thousand. The ideal irradiation time depends on the polymeric substrate, on the composition of the surrounding gaseous medium, on the wavelength of the radiation, and also on the power of the radiation source, and may readily be determined by exploratory trials. The substrates are generally irradiated for from 0.1 seconds to 20 minutes, in particular from 1 second to 10 minutes. These very short irradiation times give only very little heating of the polymeric substrate and, even with radiation whose wavelength is at the hard end of the wider range mentioned, there is no occurrence of undesirable side reactions which could cause damage to the exposed surfaces.
(2) According to the invention, it is also possible to achieve activation by using a high-frequency plasma or microwave plasma (Hexagon, Technics Plasma, 85551 Kirchheim, O.Z. 5269 Germany) in air or in a nitrogen or argon atmosphere. The exposure times are generally from 30 seconds to 30 minutes, preferably from 2 to 10 minutes. The energy input, for laboratory apparatus, is from 100 to 500 W, preferably from 200 to 300 W.
(3) It is also possible to use corona apparatus (SOFTAL, Hamburg, Germany) for activation. The exposure times in this case are generally from 1 second to 10 minutes, preferably from 1 to 60 seconds.
(4) Activation by electron beams or gamma rays (e. g.
from a cobalt 60 source) permits short exposure times, generally from 0.1 to 60 seconds.
(5) Flame treatment of surfaces likewise leads to their activation. Suitable apparatus, in particular that which has a barrier flame front, may readily be constructed or, for example, purchased from ARCOTEC, 71297 Monsheim, Germany.
Such apparatus may be operated using hydrocarbons or hydrogen as the gas for combustion. In all cases, overheating which damages the intermediate layer has to be avoided, and this is readily achieved by intimate contact between a cooled metal surface and the surface opposite to that being flame-treated.
The exposure times generally amount to from 0.1 second to 1 minute, preferably from 0.5 to 2 seconds, the flames in all cases being nonluminous and the distance of the surface of the intermediate layer to the outer flame front being from 0.2 to 5 cm, preferably from o.5 to 2 cm.
O.Z. 5269 (6) The surfaces of the intermediate layers may moreover be activated by treatment with strong acids or bases. Among the strong acids which are suitable, mention may be made of sulfuric acid, nitric acid and hydrochloric acid. For example, polyamides may be treated for from 5 seconds to 1 minute with concentrated sulfuric acid at room temperature.
Particularly suitable strong bases are alkali metal hydroxides in water or in an organic solvent. For example, the substrate may be exposed to dilute sodium hydroxide solution for from 1 l0 to 60 minutes at from 20 to 80°C. Alternatively, polyamides, for example, may be activated by exposing the substrate surface to 2% strength KOH in tetrahydrofuran for from 1 minute to 30 minutes.
Examples of biospecific bind pairs composed of the biocomponent of the sensor and the biomolecule as analyte are an antibody and an antigen, a receptor and a ligand, and also O.Z. 5269 an enzyme and a substrate. In modern biosensors, the biocomponent is normally immobilized on a surface of the transducer.
The transducer may, for example, be a piezoelectric crystal which reacts to the change in mass on its surface as a result of the specific binding of the biomolecule with a change in its oscillation behaviour, the change indicating the existence of binding (qualitatively) and, if desired, its extent (quantitatively). Instead of using piezoelectric oscillations for the process of converting the signal, it is also possible to use surface acoustic waves (SAW) of piezoelectric substrates for this purpose. The depth of penetration of the surface acoustic wave is in the range of its wavelength, and therefore biomolecules which are specifically bound at the surface have an effect on the transmission of the waves. Biosensors which operate with transducers based on piezoelectric oscillations or on surface acoustic waves are mass sensors, since they fundamentally give information on the change in mass (i.e., weight) at the transducer surface. Another known biosensor type utilizes transducers which convert the signals received into optical phenomena and determine the analyte by making use of changes in radiation absorption, in emission after excitation of the probe, in reflection, refraction and diffraction, or in polarization.
O.Z. 5269 The signals emitted by the transducer are electronically amplified and subjected to comparative evaluation, the result being a yes/no statement or, in the case of quantitative analysis, a numerical value.
The sensitivity of detection of a biomolecule in a matrix with a biosensor is diminished by the fact that the transducer converts into electrical or optical signals not only the signals deriving from specific binding of the biomolecule to the biocomponent of the sensor but also signals deriving from nonspecific binding of other components of the matrix. This nonspecific binding may be adsorptive, absorptive, covalent or ionic in nature and produces false analysis results, since the signal passed on by the transducer for evaluation is composed of the sum of the signal from specific and from nonspecific binding. The error caused by the nonspecific signal could be tolerated if it were constant for a particular analytical task in the same matrix, or at least a matrix of the same type. This is, however, not the case. For example, the extent of nonspecific binding during determination of antigens in blood differs from donor to donor, and therefore even if the antigen is absent the values obtained have a scatter the extent of which can be signified using the standard deviation of the blank measurements from the mean value. The greater the standard deviation, the lower the detection sensitivity, since a test is usually only judged to be positive if the measured value exceeds three times O.Z. 5269 times the standard deviation.
It is therefore desirable to prevent nonspecific binding, or at least to suppress the same, so that the standard deviation is minimized. In a variant which has been disclosed (see, for example, K.A. Davis, T.R. Leary:
Continuous Liquid Phase Piezoelectric Biosensor for Kinetic Immunoassays, Analy. Chem. 61 (1989), 1227-1230), nonspecific binding of proteins is suppressed with bovine serum albumin (BSA) as passivating layer in the determination of immunoglobulin G (IgG) in phosphate-buffered sodium chloride solution (PBS). However, the effectiveness of this passivating layer is not ideal because, on the one hand, the macromolecules of the BSA do not seal the transducer surface hermetically, so that there are unoccupied positions remaining for nonspecific binding and, on the other hand, BSA in its own right nonspecifically binds hydrophobic groups, e.g. in fatty acid molecules.
3. Summary of the Invention It has now been found that organic hydrogels give passivating layers for biosensors which have excellent effectiveness with respect to nonspecific binding. The invention therefore provides a biosensor with an immobilized biocomponent for specific binding of a biomolecule (analyte) which is present in an aqueous matrix. A significant feature of this biosensor is a layer of an organic hydrogel as passivating layer with respect to nonspecific binding.
O.Z. 5269 The invention further provides a process for producing the biosensor by the use of an organic hydrogel for a passivating layer.
The invention also provides a method for determining a biomolecule or analyte in an aqueous matrix by the use of the novel biosensor.
The novel biosensors are distinguished by a considerably smaller standard deviation of blank measurements from the mean value, and therefore by greater sensitivity than known biosensors with or without a passivating layer. This may be due to a hydrated envelope of the hydrogels, which deters the approach of, and nonspecific bind of, molecules which differ from the analyte. The passivating layer adheres particularly firmly, and therefore the biosensors are particularly suitable as reversible sensors if the hydrogel is applied by well known graft polymerization techniques. A
further advantage is that the cross-sensitivity is reduced (or the specificity for the sought-for analyte is increased).
4. Description of Preferred Embodiment of the Invention 2o The passivating layer of an organic hydrogel is the significant feature of the biosensor of the invention. It is preferably a mass sensor and the biocomponent which it contains is generally an antibody, an enzyme or a receptor, desirably sonically or covalently bound to the transducer, directly or via a polymeric intermediate layer. The transducer may be composed of conventional materials known for O.Z. 5269 this purpose, e.g. silicon nitride (Si3N4), silica or lanthanum niobate (LaNb03). The novel biosensor likewise has no particular features with regard to the processing and evaluation of the secondary signal created in the transducer.
In a preferred embodiment of the novel biosensor, the passivating layer of the organic hydrogel has been applied to the surface of the transducer directly or, preferably indirectly via an intermediate layer, especially desirably by grafting hydrophilic monomers, and contains on its free surface, covalently or sonically bound or integrated into the hydrogel, the biocomponent for specific binding with the biomolecule in the matrix. Biosensors having such a passivating layer grafted onto an activated polymeric intermediate layer and containing the biocomponents sonically or covalently bound on their free surface are distinguished by particularly low sensitivity.
4.1. Passivatinq Layer/Hydrophillic Vinyl Monomers The organic hydrogel of the passivating layer may be produced by polymerization, preferably by graft polymerization, of hydrophilic vinyl monomers, optionally together with proportions of hydrophobic vinyl monomers. The hydrophilic polymer becomes a hydrogel on contacting an aqueous medium, a condition which is always fulfilled in the use as specified. Preparation of hydrogels or of polymers which give a hydrogel may be conducted as described for example in German Patent Application Nos. 197 00 079.7 (O. Z.
O.Z. 5269 _ 7 _ 5146), 197 15 449.2 (O. Z. 5176) and 197 27 556.7 (O. Z. 5210).
The hydrogel of the passivating layer is, of course, itself hydrophillic. Preferred passivating layers are made of hydrogels which exhibit a contact angle of less than 40°, more preferably less than 30°, when the contact angle of an air bubble is determined by the method of R.J. Good et al., Techniques of Measuring Contact Angles in Surface and Colloid Science (ed. R.J. Good) Vol. 11, Plenum Press, New York, N.Y.
This is done by allowing an air bubble to form below the specimen immersed in water, and this bubble coats the surface to a greater or lesser extent depending on its hydrophilicity.
Suitable hydrophilic vinyl monomers contain at least one olefinic double bond, and also at least one hydrophilic group. The olefinic double bonds may be present in functional groups of various types, for example in alkenyl groups, such as vinyl or allyl radicals, or in radicals derived from unsaturated carboxylic acids or from their derivatives, for example acrylic acid or methacrylic acid, or from the amides of these carboxylic acids or malefic acid. There is also great scope for variation with regard to the hydrophilic groups.
Examples which may be mentioned of suitable hydrophilic groups are: hydroxyl groups, ether groups, acyloxy groups, carboxyl groups, carboxylic ester groups, carboxamide groups, carboalkoxy groups and nitrile groups; 1,2-epoxy groups;
sulfuric esters, sulfonic acid, sulfinic acid groups, phosphoric acid groups, phosphonic acid groups and phosphinic O.Z. 5269 _ g _ acid groups, including the salts and esters corresponding to these; primary, secondary and tertiary amino groups; acylamino groups, which may be in open chains or incorporated into a ring; polyalkylene oxide groups, such as polyethylene oxide groups and polypropylene oxide groups, with or without a terminal hydroxyl group; polyester groups, polyesteramide groups and polyetheresteramide groups; the pyrrolidone ring and similar heterocycles, and also radicals of olefinically functionalized sugars. The hydrophilicity of a monomer is, of course, dependent of the balance between hydrophilic and hydrophobic fractions in its molecule. Monomers suitable for the invention are soluble in water at 20°C to an extent of at least 1% by weight, preferably at least 10% by weight and in particular at least 40% by weight, based in each case on the entire solution.
The hydrophilic vinyl monomers used for the invention preferably contain one olefinic double bond and one hydrophilic group. However, they may also have more than one olefinic double bond and/or hydrophilic group. Open-chain polyalkylene oxides having two terminal vinyl, allyl, acryloxy or methacryloxy groups, for example, are therefore very suitable.
Examples of suitable hydrophilic vinyl monomers which may be mentioned are: acrylic acid and its derivatives, e.g. acrylamide, N,N-diemethylacrylamide, acrylonitrile, methyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl O.Z. 5269 acrylate and 2-methoxyethyl acrylate; 2-ethyoxyethyl acrylate, 4-hydroxybutyl acrylate and 1,4-butanediol diacrylate, and also methacrylic acid and its corresponding derivatives; vinyl derivatives of carboxylic acids, such as vinyl acetate, N-vinylactetamide and N-vinylpyrrolidone; vinylsulfonic acids and their alkali metal salts, such as sodium vinylsulfonate;
alkenylarylsulfonic acids and their alkali metal salts, such as styrenesulfonic acid and sodium styrene sulfonate, vinyl ethers, such as vinyl methyl ether, vinyl ethyl ether, vinyl glycidyl ether, diethylene glycol divinyl ether and vinyl n-butyl ether; vinyl ketones, such as vinyl methyl ketone, vinyl ethyl ketone and vinyl n-propyl ketone; vinylamines, such as N-vinylpyrrolidine; polyalkyleneoxy compounds with terminal allyl, vinyl, acrylyl or methacylyl groups, such as ethoxytetraethoxyethyl acrylate or methacrylate, n-propoxydodecaethyleneoxyethyl vinyl ether, polyethylene glycol mono- or diacrylates with molecular weights of 600 or 1200, poly(ethylene/propylene) glycol mono- or dimethacrylates with molecular weights of 400 and 800, and also allyloxyoctapropyl-eneoxyethanol; sugar derivatives, such as vinyl-substituted arbinose or acryloylated hyroxypropylcellulose; and functionalized polyalkylene glycols, such as triethylene glycol diacrylate or tetraethylene glycol diallyl ether.
In cases where trifunctional or higher-functional vinyl monomers are used concomitantly, the result is a crosslinked copolymer and this, in particular in the graft O.Z. 5269 polymerization described subsequently, achieves a particularly good seal of the surface of the transducer and therefore a greater accuracy of measurement of the biosensor. Examples of suitable trifunctional or higher-functional vinyl monomers are polyacrylates, such as trimethylolpropane triacylate, trimethylolpropane trimethacrylate and pentaerythritol tetraacrylate; polyvinyl ethers, such as the trivinyl ether of glycerol-12E0 and the tetraallyl ether of pentaerythritol.
The crosslinking vinyl monomers may be used in amounts of preferably from 0.01 to 1 mol%, based on all of the monomers.
4.2. Hydrophobic Vinyl Monomers as Comonomers Besides the hydrophilic monomers, concomitant use may be made, in preparing the hydrogels or the polymers which are hydrogels on contact with an aqueous medium, of certain amounts, e.g. up to 60 mol%, based on all of the monomers, of hydrophobic vinyl monomers, e.g. a-olefins, such as propene, 1-butene and 1-octene; vinyl chloride; and vinyl aromatics, such as styrene, a-methylstyrene and vinyl toluene.
Instead of the crosslinking hydrophilic vinyl monomers mentioned which may optionally be used concomitantly, it is also possible to make concomitant use of trifunctional or higher-functional hydrophobic vinyl monomers in the amounts mentioned, the effect achieved being essentially the same as that described.
The molar proportion of the hydrophobic vinyl monomers should be judged in such a way that the hydrogel O.Z. 5269 shows a contact angle of less than 40°, preferably less than 30°C, in the test of R.J. Good et al. described above.
4.3. Preparation of the Organic Hydro~els in Bulk The monomers, in each case individually or else as a mixture appropriate for the respective application, may be polymerized in a conventional manner, preferably by free-radical-initiated solution or emulsion polymerization. The monomers are generally used as solutions of concentrations of from 1 to 40% by weight, preferably from 5 to 20% by weight.
It is expedient for the solvent to be water. The resultant polymer is then immediately a hydrogel. If an organic solvent is used for the operation, the polymer becomes a hydrogel at the latest during use of the biosensor in contact with the aqueous matrix. Use may be made of conventional polymerization initiators, e.g. organic peroxy compounds or hydroperoxides, azo compounds, peracids, persulfates or percarboneates, in the conventional amounts, the polymerization being initiated thermally or by irradiation.
The solutions or emulsions are immediately suitable for coating the transducers, preferably by spincoating, by which means passivating layers with thicknesses of from 0.1 to 100 ~.m can be created from the polymer.
Alternatively, the passivating layer may be created by grafting the hydrogel polymer depicted above onto the surface of the transducer. In this case, it is preferred to apply an intermediate layer of a (co)polymer before grafting;
O.Z. 5269 the intermediate layer is, or may be, activated so as to permit grafting. Grafting is preferred, since it gives a firmly adhering, particularly thin but nevertheless coherent coating on the surface of the transducer.
4.4. Polymeric Intermediate Layer and its Activation Suitable polymers for the intermediate layer are commodity polymers with or without radiation-sensitive groups, for example polyurethanes, polyamides, polyesters, polyethers, polyether-block-amides, polyester-block-amides, polysiloxanes, polystyrene, polyvinyl chloride, polycarbonates, polyolefins, polysulfones, polyisoprene, polychlorprene, polytetra-fluoroethylene, polyacrylates, polymethacrylates, and also the corresponding copolymers and blends. The intermediate layer may be applied preferably by spincoating from organic solutions and generally likewise has a thickness of from 0.01 to 100 Vim.
The surface of the intermediate layer may be activated by any of a large number of methods in preparation for grafting, and mention is made of the most important of these.
(1) Commodity polymers without W-radiation-sensitive groups may advantageously be activated by W radiation, e.g.
in the wavelength range from 100 to 400 nm, preferably from 125 to 310 nm. Particularly good results are achieved with continuous and largely monochromatic radiation, as produced, for example, by Excimer W sources (Heraeus, Kleinostheim, O.Z. 5269 Germany), for example using F2, Xe2, ArF, XeCl, KrCl or KrF as the medium for the lamp. However, other radiation sources, such as mercury vapor lamps with a broad radiation spectrum and a proportion of visible radiation are suitable as long as they emit considerable proportions of radiation in the wavelength ranges mentioned. It has been found that the presence of small amounts of oxygen is advantageous. The preferred partial pressures of oxygen are from 2x10-5 to 2x10-2 bar. The operation may, for example, be carried out at a reduced pressure of from l0-4 to 10-1 bar, or using an inert gas, such as helium, nitrogen or argon, with an oxygen content of from 0.02 to 20 parts per thousand. The ideal irradiation time depends on the polymeric substrate, on the composition of the surrounding gaseous medium, on the wavelength of the radiation, and also on the power of the radiation source, and may readily be determined by exploratory trials. The substrates are generally irradiated for from 0.1 seconds to 20 minutes, in particular from 1 second to 10 minutes. These very short irradiation times give only very little heating of the polymeric substrate and, even with radiation whose wavelength is at the hard end of the wider range mentioned, there is no occurrence of undesirable side reactions which could cause damage to the exposed surfaces.
(2) According to the invention, it is also possible to achieve activation by using a high-frequency plasma or microwave plasma (Hexagon, Technics Plasma, 85551 Kirchheim, O.Z. 5269 Germany) in air or in a nitrogen or argon atmosphere. The exposure times are generally from 30 seconds to 30 minutes, preferably from 2 to 10 minutes. The energy input, for laboratory apparatus, is from 100 to 500 W, preferably from 200 to 300 W.
(3) It is also possible to use corona apparatus (SOFTAL, Hamburg, Germany) for activation. The exposure times in this case are generally from 1 second to 10 minutes, preferably from 1 to 60 seconds.
(4) Activation by electron beams or gamma rays (e. g.
from a cobalt 60 source) permits short exposure times, generally from 0.1 to 60 seconds.
(5) Flame treatment of surfaces likewise leads to their activation. Suitable apparatus, in particular that which has a barrier flame front, may readily be constructed or, for example, purchased from ARCOTEC, 71297 Monsheim, Germany.
Such apparatus may be operated using hydrocarbons or hydrogen as the gas for combustion. In all cases, overheating which damages the intermediate layer has to be avoided, and this is readily achieved by intimate contact between a cooled metal surface and the surface opposite to that being flame-treated.
The exposure times generally amount to from 0.1 second to 1 minute, preferably from 0.5 to 2 seconds, the flames in all cases being nonluminous and the distance of the surface of the intermediate layer to the outer flame front being from 0.2 to 5 cm, preferably from o.5 to 2 cm.
O.Z. 5269 (6) The surfaces of the intermediate layers may moreover be activated by treatment with strong acids or bases. Among the strong acids which are suitable, mention may be made of sulfuric acid, nitric acid and hydrochloric acid. For example, polyamides may be treated for from 5 seconds to 1 minute with concentrated sulfuric acid at room temperature.
Particularly suitable strong bases are alkali metal hydroxides in water or in an organic solvent. For example, the substrate may be exposed to dilute sodium hydroxide solution for from 1 l0 to 60 minutes at from 20 to 80°C. Alternatively, polyamides, for example, may be activated by exposing the substrate surface to 2% strength KOH in tetrahydrofuran for from 1 minute to 30 minutes.
(7) Finally, monomers with W-radiation-sensitive groups may be incorporated at an early stage, during preparation of the polymers for the intermediate layer. Examples of suitable monomers of this type are furyl and cinnamoyl derivatives, which may, for example, be used in amounts of from 3 to 15 mol%. Monomers of this type which have good suitability are 20 cinnamoylethyl acrylate and methacrylate.
In many cases, e.g. in the case of highly hydrophobic polymers, it may be advisable to activate the surfaces of the intermediate layer by a combination of two or more of the activation methods mentioned. The preferred activation method is W-radiation as in number (1).
O.Z. 5269 4.5. Preparation of the Passivating Layer by Graft (co)polymerization of monomers The passivating layer may be created by graft (co)polymerization, by grating the monomers onto the activated intermediate layer. If the intermediate layers have been activated by one of the methods described under (1) to (6), it is expedient to expose the activated surfaces to the action of oxygen, e.g. in the form of air, for from 1 to 20 minutes, preferably from 1 to 5 minutes.
l0 The surfaces of the intermediate layers which have been activated (also, if desired, as in (7)) are then coated by known methods, such as dipping, spraying or brushing, with solutions of the vinyl monomers) to be used according to the invention. Solvents which have proven successful are water-ethanol mixtures, but other solvents may also be used as long as they have sufficient ability to dissolve the monomers) and give good wetting of the surfaces. Depending on the solubility of the monomers and on the desired thickness of the finished coating, the concentrations of the monomers in the 2o solution may be from 1 to 40% by weight. Solutions with monomer contents of from 5 to 20% by weight, for example of about 10% by weight, have proven successful in practice and generally in a single operation give cohesive coatings with thicknesses which may be more than 0.1 ~m and which cover the surface of the intermediate layers.
After the solvent has evaporated, or during its O.Z. 5269 evaporation, the polymerization or copolymerization of the monomers) applied to the activated surface is expediently induced by means of radiation in the short-wavelength segment of the visible ranged or in the long-wavelength UV range of electromagnetic radiation. An example of radiation which has good suitability is that with wavelengths of from 250 to 500 nm, preferably from 290 to 320 nm. Radiation in the wavelength range mentioned is relatively soft and is selective in polymerization, and does not attack the polymeric intermediate layer. As in the activation of the surfaces of the intermediate layer, it is again advantageous here to operate with a radiation source which emits continuous and largely monochromatic radiation. A particularly suitable source is again the Excimer UV source with continuous radiation, e.g. with XeCl or XeF as the medium for the radiation. The required intensity of radiation and the exposure time depend on the particular hydrophilic monomers and may readily be determined by exploratory experiments. In principle, mercury vapor lamps may also be used here, as long as they emit considerable proportions of radiation in the wavelength ranges mentioned. The exposure times in all cases are generally from 10 seconds to 30 minutes, preferably from 2 to 15 minutes.
Alternatively, the passivating layer may also be created by irradiation of the transducer which has been provided with an activated polymeric intermediate layer and O.Z. 5269 dipped into the monomer solution.
Depending on the concentration of the monomer solution, on the coating conditions (application method, dip-irradiation), on the intensity of the radiation and on the exposure time, grafting gives a firmly adhering barrier layer of the hydrogel, generally with a thickness of from 10 nm to 500 ~,m.
4.6. Incorporation of the Biocomponents Since the biocomponents are generally sensitive l0 molecules, it is preferred to incorporate them on the surface of the barrier layer of the organic hydrogel after this layer has been applied. The binding which links the biocomponent with the barrier layer is preferably of covalent or ionic type. The details depend on the nature of the biocomponent, where the biocomponent contains, for example, free primary or secondary amino groups, and this is frequently the case, it may be attached ionically with an ammonium structure or covalently via a carboxamide bridge, an example of the latter being the known method using N-hydroxysuccinimide and a 20 carboxyl group in the hydrogel. The coat of the biocomponent achieved in this way on the surface of the barrier layer has good density, increases measurement sensitivity, and at the same time is stable and resistant to abrasion.
4.7. Use of the Biosensor The novel biosensor is suitable, as are corresponding biosensors of the prior art, for determining a O.Z. 5269 biomolecule (or analyte) in an aqueous matrix, the biomolecule (or analyte) being specifically bound by the biocomponent of the sensor. It is therefore possible by this means, for example, to detect HIV antibodies in human blood, in order to identify infection of donated blood.
O.Z. 5269
In many cases, e.g. in the case of highly hydrophobic polymers, it may be advisable to activate the surfaces of the intermediate layer by a combination of two or more of the activation methods mentioned. The preferred activation method is W-radiation as in number (1).
O.Z. 5269 4.5. Preparation of the Passivating Layer by Graft (co)polymerization of monomers The passivating layer may be created by graft (co)polymerization, by grating the monomers onto the activated intermediate layer. If the intermediate layers have been activated by one of the methods described under (1) to (6), it is expedient to expose the activated surfaces to the action of oxygen, e.g. in the form of air, for from 1 to 20 minutes, preferably from 1 to 5 minutes.
l0 The surfaces of the intermediate layers which have been activated (also, if desired, as in (7)) are then coated by known methods, such as dipping, spraying or brushing, with solutions of the vinyl monomers) to be used according to the invention. Solvents which have proven successful are water-ethanol mixtures, but other solvents may also be used as long as they have sufficient ability to dissolve the monomers) and give good wetting of the surfaces. Depending on the solubility of the monomers and on the desired thickness of the finished coating, the concentrations of the monomers in the 2o solution may be from 1 to 40% by weight. Solutions with monomer contents of from 5 to 20% by weight, for example of about 10% by weight, have proven successful in practice and generally in a single operation give cohesive coatings with thicknesses which may be more than 0.1 ~m and which cover the surface of the intermediate layers.
After the solvent has evaporated, or during its O.Z. 5269 evaporation, the polymerization or copolymerization of the monomers) applied to the activated surface is expediently induced by means of radiation in the short-wavelength segment of the visible ranged or in the long-wavelength UV range of electromagnetic radiation. An example of radiation which has good suitability is that with wavelengths of from 250 to 500 nm, preferably from 290 to 320 nm. Radiation in the wavelength range mentioned is relatively soft and is selective in polymerization, and does not attack the polymeric intermediate layer. As in the activation of the surfaces of the intermediate layer, it is again advantageous here to operate with a radiation source which emits continuous and largely monochromatic radiation. A particularly suitable source is again the Excimer UV source with continuous radiation, e.g. with XeCl or XeF as the medium for the radiation. The required intensity of radiation and the exposure time depend on the particular hydrophilic monomers and may readily be determined by exploratory experiments. In principle, mercury vapor lamps may also be used here, as long as they emit considerable proportions of radiation in the wavelength ranges mentioned. The exposure times in all cases are generally from 10 seconds to 30 minutes, preferably from 2 to 15 minutes.
Alternatively, the passivating layer may also be created by irradiation of the transducer which has been provided with an activated polymeric intermediate layer and O.Z. 5269 dipped into the monomer solution.
Depending on the concentration of the monomer solution, on the coating conditions (application method, dip-irradiation), on the intensity of the radiation and on the exposure time, grafting gives a firmly adhering barrier layer of the hydrogel, generally with a thickness of from 10 nm to 500 ~,m.
4.6. Incorporation of the Biocomponents Since the biocomponents are generally sensitive l0 molecules, it is preferred to incorporate them on the surface of the barrier layer of the organic hydrogel after this layer has been applied. The binding which links the biocomponent with the barrier layer is preferably of covalent or ionic type. The details depend on the nature of the biocomponent, where the biocomponent contains, for example, free primary or secondary amino groups, and this is frequently the case, it may be attached ionically with an ammonium structure or covalently via a carboxamide bridge, an example of the latter being the known method using N-hydroxysuccinimide and a 20 carboxyl group in the hydrogel. The coat of the biocomponent achieved in this way on the surface of the barrier layer has good density, increases measurement sensitivity, and at the same time is stable and resistant to abrasion.
4.7. Use of the Biosensor The novel biosensor is suitable, as are corresponding biosensors of the prior art, for determining a O.Z. 5269 biomolecule (or analyte) in an aqueous matrix, the biomolecule (or analyte) being specifically bound by the biocomponent of the sensor. It is therefore possible by this means, for example, to detect HIV antibodies in human blood, in order to identify infection of donated blood.
O.Z. 5269
Claims (20)
1. A biosensor for detecting a biomolecule analyte present in an aqueous solution, which comprises:
a transducer;
a passivating layer formed on a surface of the transducer either directly or via a polymeric intermediate layer, wherein the passivating layer is made of an organic hydrogel; and a biocomponent which is capable of specifically binding the biomolecule analyte and is immobilized on a free surface of the passivating layer by being covalently or sonically bound to or being integrated into the hydrogel;
wherein when the biocomponent binds the biomolecule analyte, a primary chemical or physical signal created by the binding is converted by the transducer into an optical or electrical second signal which can be amplified and evaluated;
and wherein the passivating layer suppresses non-specific binding of proteins with the biocomponent.
a transducer;
a passivating layer formed on a surface of the transducer either directly or via a polymeric intermediate layer, wherein the passivating layer is made of an organic hydrogel; and a biocomponent which is capable of specifically binding the biomolecule analyte and is immobilized on a free surface of the passivating layer by being covalently or sonically bound to or being integrated into the hydrogel;
wherein when the biocomponent binds the biomolecule analyte, a primary chemical or physical signal created by the binding is converted by the transducer into an optical or electrical second signal which can be amplified and evaluated;
and wherein the passivating layer suppresses non-specific binding of proteins with the biocomponent.
2. A biosensor according to claim 1, wherein the organic hydrogel exhibits a contact angle of less than 40°.
3. A biosensor according to claim 2, wherein the organic hydrogel exhibits a contact angle of less than 30°.
4. A biosensor according to claim 1, 2 or 3, wherein the passivating layer has a thickness of 0.1 to 100 µm.
5. A biosensor according to any one of claims 1 to 4, wherein the hydrogel is made of a polymer or copolymer of a hydrophilic vinyl monomer which is soluble in water to an extent of at least 1% by weight at 20°C.
6. A biosensor according to claim 5, wherein the hydrophilic vinyl monomer is soluble in water to an extent of at least 10% by weight at 20°C.
7. A biosensor according to claim 5, wherein the hydrophilic vinyl monomer is soluble in water to an extent of at least 40% by weight at 20°C.
8. The biosensor according to any one of claims 1 to 7, wherein the hydrophilic vinyl monomer comprises at least one olefinic double bond and at least one hydrophilic group.
9. The biosensor according to claim 8, wherein the hydrophilic vinyl monomer is selected from at least one of the group consisting of acrylic acid and its derivatives acrylamide, N,N-dimethylacrylamide, acrylonitrile, methyl acrylate, 2-hydroxyethyl acrylate, 2-hydroxypropyl acrylate, 2-methoxyethyl acrylate, 2-ethoxyethylacrylate, 4-hydroxybutyl acrylate, 1,4-butanediol diacrylate; methacrylic acid and its corresponding derivatives; vinyl acetate, N-vinyl acetamide;
N-vinyl pyrrolidone, vinylsulfonic acid, sodium vinylsulfonate, styrenesulfonic acid, sodium styrene sulfonate, vinyl methyl ether, vinyl ethyl ether, vinyl glycidyl ether, diethylene glycol divinyl ether, vinyl n-butyl ether, vinyl methyl ketone, vinyl ethyl ketone, vinyl n-propyl ketone, N-vinylpyrrolidine, ethoxytetraethyoxyethyl acrylate or methacrylate, n-propoxydodecaethyleneoxyethyl vinyl ether, polyethylene glycol mono- or diacrylates (molecular weight about 600 or about 1200), poly(ethylene/propylene) glycol mono- or dimethacrylate (molecular weight about 400 or about 800), vinyl substituted arabinose, acryloylated hydroxypropyl-cellulose, triethylene glycol diacrylate and tetraethylene glycol diallyl ether.
N-vinyl pyrrolidone, vinylsulfonic acid, sodium vinylsulfonate, styrenesulfonic acid, sodium styrene sulfonate, vinyl methyl ether, vinyl ethyl ether, vinyl glycidyl ether, diethylene glycol divinyl ether, vinyl n-butyl ether, vinyl methyl ketone, vinyl ethyl ketone, vinyl n-propyl ketone, N-vinylpyrrolidine, ethoxytetraethyoxyethyl acrylate or methacrylate, n-propoxydodecaethyleneoxyethyl vinyl ether, polyethylene glycol mono- or diacrylates (molecular weight about 600 or about 1200), poly(ethylene/propylene) glycol mono- or dimethacrylate (molecular weight about 400 or about 800), vinyl substituted arabinose, acryloylated hydroxypropyl-cellulose, triethylene glycol diacrylate and tetraethylene glycol diallyl ether.
10. The biosensor according to claim 9, wherein the hydrophilic vinyl monomer further comprises at least one of the group consisting of trimethylol propane triacrylate, trimethylolpropane trimethacrylate, pentacrythritol tetraacrylate, trivinyl ether of glycol-12F0 and tetraallyl ether of pentaerythritol.
11. A biosensor according to any one of claims 5 to 10, wherein the passivating layer is formed directly on the surface of the transducer.
12. A biosensor according any one of claims 5 to 10, wherein the passivating layer is formed via a polymeric intermediate layer.
13. The biosensor according to claim 12, wherein the polymeric intermediate layer comprises at least one polymer selected from the group consisting of polyurethane, polyamide, polyester, polyether, polyether-block-amide, polyester-block-amide, polysiloxane, polystyrene, polyvinyl chloride, polycarbonate, polyolefin, polysulfone, polyisoprene, polychloroprene, polytetrafluoroethylene, polyacrylate and polymethacrylate.
14. A biosensor according to claim 12 or 13, wherein the passivating layer is formed by graft (co)polymerization of the hydrophilic vinyl monomer onto an activated surface of the polymeric intermediate layer.
15. A biosensor according to any one of claims 1 to 14, wherein the biocomponent is an antibody, a receptor or an enzyme designed to specifically bind with an antigen, a ligand or a substrate, respectively, present in the aqueous solution.
16. A biosensor according to any one of claims 1 to 15, wherein the transducer is made of silicon nitride Si3N4 silica or lanthanum niobate LaNbO3.
17. A biosensor according to any one of claims 1 to 16, wherein the biocomponent is immobilized using a free primary or secondary amino group thereof attached ironically or covalently to a carboxyl group in the hydrogel.
18. A process for producing the biosensor according to claim 11, which comprises:
coating the organic hydrogel directly onto a surface of the transducer; and immobilizing the biocomponent to a free surface of the organic hydrogel.
coating the organic hydrogel directly onto a surface of the transducer; and immobilizing the biocomponent to a free surface of the organic hydrogel.
19. A process for producing the biosensor according to claim 14, which comprises:
coating the polymeric intermediate layer onto a surface of the transducer;
activating a surface of the polymeric intermediate layer;
(co)polymerizing the hydrophilic vinyl monomer on the activated surfaces of the polymeric intermediate layer to form the passivating layer; and immobilizing the biocomponent to a free surface of the passivating layer.
coating the polymeric intermediate layer onto a surface of the transducer;
activating a surface of the polymeric intermediate layer;
(co)polymerizing the hydrophilic vinyl monomer on the activated surfaces of the polymeric intermediate layer to form the passivating layer; and immobilizing the biocomponent to a free surface of the passivating layer.
20. The process according to claim 19 wherein the activation of the polymeric intermediate layer is carried out by an activation source selected from the group consisting of UV radiation, high-frequency plasma, microwave plasma, corona, electron beam, gamma rays, flame, strong acid and strong base.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE1998106642 DE19806642A1 (en) | 1998-02-18 | 1998-02-18 | Biosensor for specific binding assays, having organic hydrogel passivation layer |
| DE19806642.2 | 1998-02-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2262011A1 true CA2262011A1 (en) | 1999-08-18 |
Family
ID=7858080
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2262011 Abandoned CA2262011A1 (en) | 1998-02-18 | 1999-02-16 | Biosensor with novel passivating layer |
Country Status (5)
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| EP (1) | EP0940674A3 (en) |
| JP (1) | JPH11337552A (en) |
| CA (1) | CA2262011A1 (en) |
| DE (1) | DE19806642A1 (en) |
| NO (1) | NO990737L (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7964414B2 (en) | 2005-03-22 | 2011-06-21 | Fujifilm Corporation | Biosensor with suppressed non-specific adsorption |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10032042A1 (en) * | 2000-07-05 | 2002-01-24 | Inventus Biotec Gesellschaft Fuer Innovative Bioanalytik, Biosensoren Und Diagnostika Mbh & Co. Kg | Disposable electrochemical biosensor for the quantitative determination of analyte concentrations in liquids |
| FI118061B (en) | 2001-09-24 | 2007-06-15 | Beanor Oy | Procedure and bio donor for analysis |
| WO2003038131A1 (en) * | 2001-11-01 | 2003-05-08 | Rensselaer Polytechnic Institute | Biocatalytic solgel microarrays |
| FI115166B (en) | 2001-12-31 | 2005-03-15 | Biofons Oy | Diagnostic procedures |
| US8224414B2 (en) * | 2004-10-28 | 2012-07-17 | Echo Therapeutics, Inc. | System and method for analyte sampling and analysis with hydrogel |
| JP4514675B2 (en) * | 2005-01-14 | 2010-07-28 | 株式会社イノアックコーポレーション | Diagnostic container |
| JP4877509B2 (en) * | 2007-02-08 | 2012-02-15 | Ｊｓｒ株式会社 | Blocking agent, probe binding particle and method for producing the same |
| JP5003902B2 (en) * | 2007-11-09 | 2012-08-22 | Ｊｓｒ株式会社 | Non-specific adsorption inhibitor for biological substances and method for coating articles |
| EP2058660B1 (en) | 2007-11-09 | 2016-05-25 | JSR Corporation | Nonspecific adsorption inhibitor of substance relating to living body and method for coating article |
| CN106066400B (en) * | 2016-06-14 | 2017-11-17 | 焦作百奥泰科生物科技有限公司 | A kind of fluorescence immune chromatography test paper for detecting zearalenone |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0539625A1 (en) * | 1991-10-28 | 1993-05-05 | Dräger Medical Electronics B.V. | Electrochemical sensor for measuring the glucose content of glucose containing fluids |
| SE9203118D0 (en) * | 1992-10-26 | 1992-10-26 | Pharmacia Biosensor Ab | A METHOD OF PREVENTING UNDESIRED BINDING IN SOLID PHASE ASSAYS |
| US5543326A (en) * | 1994-03-04 | 1996-08-06 | Heller; Adam | Biosensor including chemically modified enzymes |
-
1998
- 1998-02-18 DE DE1998106642 patent/DE19806642A1/en not_active Withdrawn
- 1998-12-23 EP EP98124626A patent/EP0940674A3/en not_active Withdrawn
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1999
- 1999-02-16 CA CA 2262011 patent/CA2262011A1/en not_active Abandoned
- 1999-02-17 JP JP11039041A patent/JPH11337552A/en active Pending
- 1999-02-17 NO NO990737A patent/NO990737L/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7964414B2 (en) | 2005-03-22 | 2011-06-21 | Fujifilm Corporation | Biosensor with suppressed non-specific adsorption |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH11337552A (en) | 1999-12-10 |
| EP0940674A2 (en) | 1999-09-08 |
| DE19806642A1 (en) | 1999-08-19 |
| NO990737D0 (en) | 1999-02-17 |
| NO990737L (en) | 1999-08-19 |
| EP0940674A3 (en) | 2000-06-21 |
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