CA2202043A1 - Biosensor with biomaterial covalently fixed on a signal-active surface - Google Patents
Biosensor with biomaterial covalently fixed on a signal-active surfaceInfo
- Publication number
- CA2202043A1 CA2202043A1 CA002202043A CA2202043A CA2202043A1 CA 2202043 A1 CA2202043 A1 CA 2202043A1 CA 002202043 A CA002202043 A CA 002202043A CA 2202043 A CA2202043 A CA 2202043A CA 2202043 A1 CA2202043 A1 CA 2202043A1
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- Prior art keywords
- group
- crosslinking agent
- signal
- groups
- active surface
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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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Pathology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Holo Graphy (AREA)
- Measurement Of The Respiration, Hearing Ability, Form, And Blood Characteristics Of Living Organisms (AREA)
- Photoreceptors In Electrophotography (AREA)
Abstract
The successful bonding of bioactive material, especially enzymes, to the signal-active surface of a biosensor can be achieved by means of a heterobifunctional crosslinking agent having a group A reactive for the signal-active surface, e.g. a group reacting with -OH, -SH or -NHx, and a photoactivatable, biomaterial-reactive group B. Known groups B are, in particular, azidophenyl and azidosalicylic groups.
Crosslinking agents containing such groups B on the one hand and succinimide ester groups on the other are commercially available and hence particularly attractive. The signal-active surface can be especially a Si3N4 surface with reactive NHx groups, as can be obtained from the gas phase by SiH4/NH3 reaction, especially on a fresh SiO2 surface, and has been freed from oxides especially by treatment with dilute hydrofluoric acid.
Crosslinking agents containing such groups B on the one hand and succinimide ester groups on the other are commercially available and hence particularly attractive. The signal-active surface can be especially a Si3N4 surface with reactive NHx groups, as can be obtained from the gas phase by SiH4/NH3 reaction, especially on a fresh SiO2 surface, and has been freed from oxides especially by treatment with dilute hydrofluoric acid.
Description
BIOSE~SOR WITH BIOMATERIAL COVALENTLY FIXED ON A SIGNAL-ACTIVE SURFACE
The invention relates to biosensors particularly to bio-sensors based on the field effect with, if appropriate, sila-nized signal active surface to which the sensor biomaterial islinked b~ means of a cross linker.
Biosensors which utilize enzymatic reactions to selec-tively determine the presence of certain analytes have been un-der development for some time.
Basically, such sensors comprise a surface layer including a biomaterial such as an enzyme which is exposed to the medium to be tested, particularly a liquid. The information provided by the biomaterial under the influence of the analyte is con-verted by a transducer element and is finally presented by means, for example by integra~ed electronic signal processing means, in a recordable form.
As transducers especially potentiometric and amperometric electrodes such as field effect structures are under considera-tion.
A particular problem encountered with such biomaterial containing sensors is the sufficient, that is particularly per-manent, fixing or immobilizing of the biomaterial on the sensorsurface with a high surface-specific activity in such a way that this type of immobilization does not detrimentally affect the activity of the biomaterial.
As sensor biomaterial, various forms such as enzymes, mi-croorganisms, cells, antioodies, antigens, organelles, or tis-sue sections can be considered. However, in the respective literature, research is mainly concerned with enzyme-containing biosensors so that, for simplification, only enzyme-containing biosensors are considered below.
As immobilization techniques only the purely adsorptive s addition, the binding to polymer layers, the cross-linking of enzyme molecules among each other and the covalent attachment of the enzymes to the sensors are known. For the covalent at-tachment of the enzyme to the mainly oxidic sensor surface, the sensor surface is generally silanized (mostly with an amino-alcylalcoxysilan) and subsequently the enzyme is coupled to thesilanized surface by means of a glutaraldehyde (see Anal. Chim.
Acta 245 (1991) pages 89 - 99). But a silanization of Si3N4 surfaces with subsequent covalent attachment by means of dif-ferent cross-linking means has also already been described (see R.A. Williams et al., Biosensors ~ Bioelectronics 9 (1994) 159 - 67).
Also, the use of thiol-terminated silanes with the subse-quent protein immobilization by means of a heterobifunctional crosslinking means, for example with N-hydroxy-succinimide es-ters such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDS), N-succinimidyl-p-iodine acetyl-amido-benzoate (SIAB), N
succinimidyl-4-(p-male-imido-phenyl)-butyrate (SMPB) or N-succinimidyl-m-maleimido-benzoate-derivates (MBS-derivates), (see S.K. Bathea et al. Anal. Brochem. 178 (1989) 408-13) has been recommended.
A direct attachment of enzymes and microorganisms to a Si3N4 surface produced by plasma-polymerization is described in JP 61 087 699 published in 1986 wherefor glutaraldehyde or dia-socyanate are to be used.
The reactivity of the functional groups of the homo as well as the bifunctional cross linking means and of the respec-tive bonding partners permits the formation of oligomers and the diversification of the bonds so that the accurate covalent attachment of a biomolecule, by way of a cross-linking mole-cule, to the signal-action surface is not guaranteed.
Consequently, a new way of immobilizing biomaterial on sensor surfaces has been developed which provides for well-defined products with high sensitivity and a long service life.
The biosensors according to the invention which are all of the type referred to initially are characterized in that the cross-linking means is provided by a heterobifunctional cross-linking structure with a reactive group (A) specific for thesignal-active surface on one hand and a photo-activatable bio-material reactive group (B) on the other hand.
With such a heterobifunctional crosslinking structure whose biomaterial-reactive group can be caused to react only by photoactivation, a well defined bonding of the biomaterial at the surface can be achieved by first providing for a reaction of the cross-linking means with the surface by way of a corre-spondingly reactive group while avoiding photoactivations.
After removal of the (unreacted) excess cross-linking means the biomaterial is converted by appropriate exposure to light.
As surfaces, particularly semiconductor material such as Germanium or Silicon as well as oxidic materials such as sili-con oxide, aluminum oxide, tantalum oxide, etc., which may be in reactive form or silanized may be used. Particularly mer-capto- or amino group-containing silanization layers are ap-plied, for example, to silicon dioxide. Suitable is also a Si3N4-surface layer with reactive groups, particularly NHX-groups which provide for a coupling particularly with aldehyde groups, ester groups or imido-ester groups.
As photoactivatable groups which react with biomaterial, alicyclically or aromatically bonded nitro or azido groups which, upon exposure to light, react with protein-type material are particularly suitable. In this respect, azidophenyl rests or azidosalicyl rests are known as group B containing crosslinking means with a N-hydroxy-succinimido ester- group as group A. Such cross-linking agents are commercially available ~see for example "Immuno Technology Catalog & Handbook" of the firm Pierce of 1992/3; E34 - 46).
The photoactivated reaction of a azidophenyl compound with protein under exposure to light of a wave length of 265 - 275 nm takes place for example in accordance with the equation:
R ~ N3 + protein ---> R- ~ -NH-protein Suitable for R are, because of their availability, par-ticularly rests with an N-hydroxy succinimido ester group as an lS end group a which can react wit NHx groups in the following manner:
O O
R'-C~O-N~ + ~ NH2 --> R'-C-NH- ~ + HO-N~
O
As function A of the crosslinking agent reacting with SH
groups, disulfide groups are known.
Silicon nitride surfaces can be formed particularly well, using precipitation by a CVD technique, from a SiH4/NH3 mixture (see A. Garde et al., in ESSDERC 1994 - 11 - 15 Sept. 1994, Ed.
C. Hill & P. Ashburn). When exposed to air, they take oxygen and when exposed to humidity, they tend to hydrolyze whereby they form Si-OH, Si-NH and Si-NH2 groups. These groups react with the group A of the crosslinking agent and can be used for the bonding of biomaterial to the nitride surface.
For the realization of the sensors according to the inven-tion suitably an Si3N4 surface is used from which oxides have _.
been removed by a treatment with diluted hydrofluoric acid. A
crosslinking agent reacts with the NH2 groups or with the NH
groups of the surface and its photoactivatable functions B then react with the protein.
As NH2 reactive groups A of the crosslinking agents, halo-genide, epoxide, imide or isocyanate-functions may be used in addition to those already mentioned. A plurality of reaction possibilities with amino groups are given for example in US
patent 5,234,820.
The particular amino-specific reactions given below occur at room temperature with neutral to slightly alkaline pH val-ues. An increase in the temperatures or of the pH value in-creases the reaction speed but also the hydrolysis rate of the crosslinking agents. The buffer used should contain neither amines nor other compounds with which the functional groups of the crosslinking agents could resist.
N-hydroxysuccinimide ester reacts particularly specific with primary amines. Under splitting off of N-hydroxy-succinimide an amide compound is formed between the primary amine and rest group of the ester used. If no water soluble analogon is used, a cross-linking agent which includes this functional group must first be dissolved in a small amount of an organic solvent (for example, DMSO); only then can it be di-luted in an aqueous buffer to the final concentration. The ion strength of the buffer should not be too high to prevent crys-tallization effects. A slightly alkaline pH value (7-9) guar-antees that the primary amines are in an improportioned state.
Aldehydes have a highly reduced carbonyl group. This group re-acts with primary amines while forming water.
The reaction of primary amines with imidoesters occurs in the pH range between 8 and 9. The ester is formed thereby and the primary amine forms, with the imido group, a guanidino com-pound.
CA 02202043 l997-04-07 If the crosslinking agent includes as the functional end A
a thiol specific group such as maleimide, activated halogenide or pyridyldisulfide, the surface to be bonded thereon must have a free sulfhydryle group (generally a mercaptoalkyle group of an alkoxysilane). In order to prevent an oxidation of this group, the buffer used must be degasified.
Maleimide reacts in lightly sour to neutral medium (pH 6.5 - 7.5) whereas, for halogenide and pyridylsulfide, pH-values of 7 or greater are to be recommended.
The invention is usable for the bonding to the biosensor of biomaterial, especially protein-type biomaterial comprising specifically various enzymes such as hydrolases oxidoreducta-ses, transferases, especially penicillinase, urease glucoseoxi-dase, urokinase, acetylcholinesterase, butyrylcholinesterase or lactatoxidase. Tests have been performed especially for the example of pencillin sensors for which reason the following de-scription refers to penicillin sensors.
In the description reference is made to the attached Fig-ures.
They show schematically:
Fig. l a sensor principle (measurement arrangement) Fig. 2 a typical measurement curve for a concentration range of 10-4 to 10~1 Mol/l penicillin, and Fig. 3 the calibration curve of a sensor according to the in vention.
Example:
On p~ or respectively, pt_ doped silicon wafers (lm Ohm-cm to 30 Ohm-cm) an electrically non-conductive layer of silicon dioxide with a thickness of 5 - l00nm was first generated by thermal dry oxidation between 700 and 1200~C (here at 1000~C) in a diffusion furnace. On this layer, an also non-conductive layer of silicon nitride with a thickness of l0 - l00nm was de-posited by chemical precipitation in the gas phase (PCED). The ratio SiH4/NH3 in the reaction gas was 2/1, the substrate tem-perature was 200 - 500~C (here 300~C) and the pressure during the precipitation was 1 - 3 Torr (here 1.5 Torr). An annealing step under N2 gas followed (5-60 minutes at 700 - 100~C). Fi-nally, the unpolished side of the substrate was provided with an Ohm-type contact structure (for example, 10 - 1000 nm Al, Au). The material used was applied in vacuum, by thermal vapor deposition, under a basis pressure <10~3mbar. The deposition rate was between 0.1 and 10 nm/s. Subsequently, the wafer was annealed in an RTA furnace at 150-500~C (here 400~C) under an N2 atmosphere.
Immediately before the start of the enzyme immobilization process, the wafer was cleaned with acetone, 2-propanol and distilled water in an ultrasonic bath and was etched for 10-60 seconds (here 30 seconds) in diluted fluoridic acid (1-10%HF).
When using the heterobifunctional cross-linking agent ANB-NOS (N-5- Azido-2-nitrobenzoyloxysuccinimide), this crosslink-ing agent was first dissolved in a small amount of DMSO and then diluted with 0.2M triethanolamine buffer (pH5-9) to a fi-nal concentration of 0.5-10mM. This solution was applied to the Si3N4 surface and remained there at room temperature for 5 to 40 minutes. At a lower temperature, it needs to remain for a longer period. Molecules which were not bonded to the sili-con nitride surface were removed by flashing with triethanolamine buffer (TEA). Subsequently, the enzyme (penicillinase, type I from the Bacillus Cereus, Sigma P 0389) was dissolved (1000-5000 units/ml) in a buffer which does not contain any amino groups (for example, TEA, particularly not TRIS- or gly-cin buffer) and was applied to the silicon nitride surfacewhich had been treated with cross-linking agents. After an in-cubation time of 1-240 min.(here 15min) at temperatures of be-tween 4 and 60~C, especially at room temperature, the bonding of the enzyme molecules to the still free functional group of the cross-linking agent was induced by light of a wavelength range of 320 - 350nm. After completion of the immobilization process, the finished penicillin sensors were washed with 0.l M
TRIS buffer (pH 7-8) and distilled water and then dried in air, or respectively under an N2- or inert gas atmosphere for at least l0 minutes.
With the filed effect sensors made in this way measure-ments for determining the penicillin concentration in aqueous solutions were performed.
Fig. l shows schematically the measuring arrangement. The field effect sensors made in accordance with the invention and consisting of a silicon substrate l, the insulating layer 2 (silicon dioxide and silicon nitride), the crosslinking agent layer 3 and the enzyme layer (penicillin layer) 4 were inte-grated into a measuring cell. The measuring cell was filled with an aqueous measuring solution which contained penicillin G
in a concentration of between l0-5 and l mol/l. A reference electrode 7 (for example Ag/AgCl) is immersed in the measuring solution. The potentials are measured at the silicon substrate by way of the reference electrode 7 and a contact electrode 8.
Fig. 2 shows a typical measurement curve which was taken in the CONCAP (CONstant CAPacitance) mode in the concentration range of between l0 4 and l0 mol/l penicillin. For this pur-pose, pencillin G sodium salt (Sigma P 3032) was dissolved in10 mM TRIS-HCl buffer, pH7. With increasing penicillin concen-tration, the concentration of the penicilloacid formed in-creases and, consequently, also the concentration of the hydro-gen ions in close proximity of the silicon nitride surface which acts as pH transducer. This results in a shift of the potential at the interface silicon nitride/electrolyte to more positive or, respectively, negative voltage values. The plot-ting of the values over time permits an observation of the con-centration-dependent potential curve of the enzyme reaction.
The changing of the measurement solution occurred at the indi-cated times.
Fig. 3 shows the characteristic chemical transmission curve determined from Fig. l. It represents the calibration curve of the field effect sensor made in accordance with the invention. Particulars concerning the sensitivity of a poten-tiometric chemo- or bio-sensor are given with regard to the ba-sic Nernst relationship in the linear range of this curve, that is, in the range in which there is a logarithmic relation be-tween the penicillin concentration and the applied potential.
This range is, for the sensor made in accordance with the in-vention, between p-Penicillin G 2, 3 and 3, which corresponds to 0.5 and 5mM. The sensitivity is 50mV per decade.
The exact location of the linear measurement range and the absolute sensitivity depend to a large extent on the selection of the buffer composition, its concentration, that is the buffer capacity and the pH value. By a suitable selection of these parameters, the measurement range required for a particu-lar measurement can be accurately adjusted. For example, using a IMIDAZOL buffer (pH7), the linear measurement range is be-tween 2 and 20 in M penicillini with a HEPES buffer, it is about between l and l0 mM. Increasing the pH value shifts the location toward higher penicillin concentrations, reducing the pH value shifts it correspondingly to lower concentrations.
The sensors made in accordance with the invention have a high time-stability of more than 250 days. The sensitivity is in the area of 50mV per penicillin decade.
The manufacture of a field effect transistor which has in the gate area the same makeup as the capacitive layer arrange-ment described by the invention is possible.
The invention relates to biosensors particularly to bio-sensors based on the field effect with, if appropriate, sila-nized signal active surface to which the sensor biomaterial islinked b~ means of a cross linker.
Biosensors which utilize enzymatic reactions to selec-tively determine the presence of certain analytes have been un-der development for some time.
Basically, such sensors comprise a surface layer including a biomaterial such as an enzyme which is exposed to the medium to be tested, particularly a liquid. The information provided by the biomaterial under the influence of the analyte is con-verted by a transducer element and is finally presented by means, for example by integra~ed electronic signal processing means, in a recordable form.
As transducers especially potentiometric and amperometric electrodes such as field effect structures are under considera-tion.
A particular problem encountered with such biomaterial containing sensors is the sufficient, that is particularly per-manent, fixing or immobilizing of the biomaterial on the sensorsurface with a high surface-specific activity in such a way that this type of immobilization does not detrimentally affect the activity of the biomaterial.
As sensor biomaterial, various forms such as enzymes, mi-croorganisms, cells, antioodies, antigens, organelles, or tis-sue sections can be considered. However, in the respective literature, research is mainly concerned with enzyme-containing biosensors so that, for simplification, only enzyme-containing biosensors are considered below.
As immobilization techniques only the purely adsorptive s addition, the binding to polymer layers, the cross-linking of enzyme molecules among each other and the covalent attachment of the enzymes to the sensors are known. For the covalent at-tachment of the enzyme to the mainly oxidic sensor surface, the sensor surface is generally silanized (mostly with an amino-alcylalcoxysilan) and subsequently the enzyme is coupled to thesilanized surface by means of a glutaraldehyde (see Anal. Chim.
Acta 245 (1991) pages 89 - 99). But a silanization of Si3N4 surfaces with subsequent covalent attachment by means of dif-ferent cross-linking means has also already been described (see R.A. Williams et al., Biosensors ~ Bioelectronics 9 (1994) 159 - 67).
Also, the use of thiol-terminated silanes with the subse-quent protein immobilization by means of a heterobifunctional crosslinking means, for example with N-hydroxy-succinimide es-ters such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDS), N-succinimidyl-p-iodine acetyl-amido-benzoate (SIAB), N
succinimidyl-4-(p-male-imido-phenyl)-butyrate (SMPB) or N-succinimidyl-m-maleimido-benzoate-derivates (MBS-derivates), (see S.K. Bathea et al. Anal. Brochem. 178 (1989) 408-13) has been recommended.
A direct attachment of enzymes and microorganisms to a Si3N4 surface produced by plasma-polymerization is described in JP 61 087 699 published in 1986 wherefor glutaraldehyde or dia-socyanate are to be used.
The reactivity of the functional groups of the homo as well as the bifunctional cross linking means and of the respec-tive bonding partners permits the formation of oligomers and the diversification of the bonds so that the accurate covalent attachment of a biomolecule, by way of a cross-linking mole-cule, to the signal-action surface is not guaranteed.
Consequently, a new way of immobilizing biomaterial on sensor surfaces has been developed which provides for well-defined products with high sensitivity and a long service life.
The biosensors according to the invention which are all of the type referred to initially are characterized in that the cross-linking means is provided by a heterobifunctional cross-linking structure with a reactive group (A) specific for thesignal-active surface on one hand and a photo-activatable bio-material reactive group (B) on the other hand.
With such a heterobifunctional crosslinking structure whose biomaterial-reactive group can be caused to react only by photoactivation, a well defined bonding of the biomaterial at the surface can be achieved by first providing for a reaction of the cross-linking means with the surface by way of a corre-spondingly reactive group while avoiding photoactivations.
After removal of the (unreacted) excess cross-linking means the biomaterial is converted by appropriate exposure to light.
As surfaces, particularly semiconductor material such as Germanium or Silicon as well as oxidic materials such as sili-con oxide, aluminum oxide, tantalum oxide, etc., which may be in reactive form or silanized may be used. Particularly mer-capto- or amino group-containing silanization layers are ap-plied, for example, to silicon dioxide. Suitable is also a Si3N4-surface layer with reactive groups, particularly NHX-groups which provide for a coupling particularly with aldehyde groups, ester groups or imido-ester groups.
As photoactivatable groups which react with biomaterial, alicyclically or aromatically bonded nitro or azido groups which, upon exposure to light, react with protein-type material are particularly suitable. In this respect, azidophenyl rests or azidosalicyl rests are known as group B containing crosslinking means with a N-hydroxy-succinimido ester- group as group A. Such cross-linking agents are commercially available ~see for example "Immuno Technology Catalog & Handbook" of the firm Pierce of 1992/3; E34 - 46).
The photoactivated reaction of a azidophenyl compound with protein under exposure to light of a wave length of 265 - 275 nm takes place for example in accordance with the equation:
R ~ N3 + protein ---> R- ~ -NH-protein Suitable for R are, because of their availability, par-ticularly rests with an N-hydroxy succinimido ester group as an lS end group a which can react wit NHx groups in the following manner:
O O
R'-C~O-N~ + ~ NH2 --> R'-C-NH- ~ + HO-N~
O
As function A of the crosslinking agent reacting with SH
groups, disulfide groups are known.
Silicon nitride surfaces can be formed particularly well, using precipitation by a CVD technique, from a SiH4/NH3 mixture (see A. Garde et al., in ESSDERC 1994 - 11 - 15 Sept. 1994, Ed.
C. Hill & P. Ashburn). When exposed to air, they take oxygen and when exposed to humidity, they tend to hydrolyze whereby they form Si-OH, Si-NH and Si-NH2 groups. These groups react with the group A of the crosslinking agent and can be used for the bonding of biomaterial to the nitride surface.
For the realization of the sensors according to the inven-tion suitably an Si3N4 surface is used from which oxides have _.
been removed by a treatment with diluted hydrofluoric acid. A
crosslinking agent reacts with the NH2 groups or with the NH
groups of the surface and its photoactivatable functions B then react with the protein.
As NH2 reactive groups A of the crosslinking agents, halo-genide, epoxide, imide or isocyanate-functions may be used in addition to those already mentioned. A plurality of reaction possibilities with amino groups are given for example in US
patent 5,234,820.
The particular amino-specific reactions given below occur at room temperature with neutral to slightly alkaline pH val-ues. An increase in the temperatures or of the pH value in-creases the reaction speed but also the hydrolysis rate of the crosslinking agents. The buffer used should contain neither amines nor other compounds with which the functional groups of the crosslinking agents could resist.
N-hydroxysuccinimide ester reacts particularly specific with primary amines. Under splitting off of N-hydroxy-succinimide an amide compound is formed between the primary amine and rest group of the ester used. If no water soluble analogon is used, a cross-linking agent which includes this functional group must first be dissolved in a small amount of an organic solvent (for example, DMSO); only then can it be di-luted in an aqueous buffer to the final concentration. The ion strength of the buffer should not be too high to prevent crys-tallization effects. A slightly alkaline pH value (7-9) guar-antees that the primary amines are in an improportioned state.
Aldehydes have a highly reduced carbonyl group. This group re-acts with primary amines while forming water.
The reaction of primary amines with imidoesters occurs in the pH range between 8 and 9. The ester is formed thereby and the primary amine forms, with the imido group, a guanidino com-pound.
CA 02202043 l997-04-07 If the crosslinking agent includes as the functional end A
a thiol specific group such as maleimide, activated halogenide or pyridyldisulfide, the surface to be bonded thereon must have a free sulfhydryle group (generally a mercaptoalkyle group of an alkoxysilane). In order to prevent an oxidation of this group, the buffer used must be degasified.
Maleimide reacts in lightly sour to neutral medium (pH 6.5 - 7.5) whereas, for halogenide and pyridylsulfide, pH-values of 7 or greater are to be recommended.
The invention is usable for the bonding to the biosensor of biomaterial, especially protein-type biomaterial comprising specifically various enzymes such as hydrolases oxidoreducta-ses, transferases, especially penicillinase, urease glucoseoxi-dase, urokinase, acetylcholinesterase, butyrylcholinesterase or lactatoxidase. Tests have been performed especially for the example of pencillin sensors for which reason the following de-scription refers to penicillin sensors.
In the description reference is made to the attached Fig-ures.
They show schematically:
Fig. l a sensor principle (measurement arrangement) Fig. 2 a typical measurement curve for a concentration range of 10-4 to 10~1 Mol/l penicillin, and Fig. 3 the calibration curve of a sensor according to the in vention.
Example:
On p~ or respectively, pt_ doped silicon wafers (lm Ohm-cm to 30 Ohm-cm) an electrically non-conductive layer of silicon dioxide with a thickness of 5 - l00nm was first generated by thermal dry oxidation between 700 and 1200~C (here at 1000~C) in a diffusion furnace. On this layer, an also non-conductive layer of silicon nitride with a thickness of l0 - l00nm was de-posited by chemical precipitation in the gas phase (PCED). The ratio SiH4/NH3 in the reaction gas was 2/1, the substrate tem-perature was 200 - 500~C (here 300~C) and the pressure during the precipitation was 1 - 3 Torr (here 1.5 Torr). An annealing step under N2 gas followed (5-60 minutes at 700 - 100~C). Fi-nally, the unpolished side of the substrate was provided with an Ohm-type contact structure (for example, 10 - 1000 nm Al, Au). The material used was applied in vacuum, by thermal vapor deposition, under a basis pressure <10~3mbar. The deposition rate was between 0.1 and 10 nm/s. Subsequently, the wafer was annealed in an RTA furnace at 150-500~C (here 400~C) under an N2 atmosphere.
Immediately before the start of the enzyme immobilization process, the wafer was cleaned with acetone, 2-propanol and distilled water in an ultrasonic bath and was etched for 10-60 seconds (here 30 seconds) in diluted fluoridic acid (1-10%HF).
When using the heterobifunctional cross-linking agent ANB-NOS (N-5- Azido-2-nitrobenzoyloxysuccinimide), this crosslink-ing agent was first dissolved in a small amount of DMSO and then diluted with 0.2M triethanolamine buffer (pH5-9) to a fi-nal concentration of 0.5-10mM. This solution was applied to the Si3N4 surface and remained there at room temperature for 5 to 40 minutes. At a lower temperature, it needs to remain for a longer period. Molecules which were not bonded to the sili-con nitride surface were removed by flashing with triethanolamine buffer (TEA). Subsequently, the enzyme (penicillinase, type I from the Bacillus Cereus, Sigma P 0389) was dissolved (1000-5000 units/ml) in a buffer which does not contain any amino groups (for example, TEA, particularly not TRIS- or gly-cin buffer) and was applied to the silicon nitride surfacewhich had been treated with cross-linking agents. After an in-cubation time of 1-240 min.(here 15min) at temperatures of be-tween 4 and 60~C, especially at room temperature, the bonding of the enzyme molecules to the still free functional group of the cross-linking agent was induced by light of a wavelength range of 320 - 350nm. After completion of the immobilization process, the finished penicillin sensors were washed with 0.l M
TRIS buffer (pH 7-8) and distilled water and then dried in air, or respectively under an N2- or inert gas atmosphere for at least l0 minutes.
With the filed effect sensors made in this way measure-ments for determining the penicillin concentration in aqueous solutions were performed.
Fig. l shows schematically the measuring arrangement. The field effect sensors made in accordance with the invention and consisting of a silicon substrate l, the insulating layer 2 (silicon dioxide and silicon nitride), the crosslinking agent layer 3 and the enzyme layer (penicillin layer) 4 were inte-grated into a measuring cell. The measuring cell was filled with an aqueous measuring solution which contained penicillin G
in a concentration of between l0-5 and l mol/l. A reference electrode 7 (for example Ag/AgCl) is immersed in the measuring solution. The potentials are measured at the silicon substrate by way of the reference electrode 7 and a contact electrode 8.
Fig. 2 shows a typical measurement curve which was taken in the CONCAP (CONstant CAPacitance) mode in the concentration range of between l0 4 and l0 mol/l penicillin. For this pur-pose, pencillin G sodium salt (Sigma P 3032) was dissolved in10 mM TRIS-HCl buffer, pH7. With increasing penicillin concen-tration, the concentration of the penicilloacid formed in-creases and, consequently, also the concentration of the hydro-gen ions in close proximity of the silicon nitride surface which acts as pH transducer. This results in a shift of the potential at the interface silicon nitride/electrolyte to more positive or, respectively, negative voltage values. The plot-ting of the values over time permits an observation of the con-centration-dependent potential curve of the enzyme reaction.
The changing of the measurement solution occurred at the indi-cated times.
Fig. 3 shows the characteristic chemical transmission curve determined from Fig. l. It represents the calibration curve of the field effect sensor made in accordance with the invention. Particulars concerning the sensitivity of a poten-tiometric chemo- or bio-sensor are given with regard to the ba-sic Nernst relationship in the linear range of this curve, that is, in the range in which there is a logarithmic relation be-tween the penicillin concentration and the applied potential.
This range is, for the sensor made in accordance with the in-vention, between p-Penicillin G 2, 3 and 3, which corresponds to 0.5 and 5mM. The sensitivity is 50mV per decade.
The exact location of the linear measurement range and the absolute sensitivity depend to a large extent on the selection of the buffer composition, its concentration, that is the buffer capacity and the pH value. By a suitable selection of these parameters, the measurement range required for a particu-lar measurement can be accurately adjusted. For example, using a IMIDAZOL buffer (pH7), the linear measurement range is be-tween 2 and 20 in M penicillini with a HEPES buffer, it is about between l and l0 mM. Increasing the pH value shifts the location toward higher penicillin concentrations, reducing the pH value shifts it correspondingly to lower concentrations.
The sensors made in accordance with the invention have a high time-stability of more than 250 days. The sensitivity is in the area of 50mV per penicillin decade.
The manufacture of a field effect transistor which has in the gate area the same makeup as the capacitive layer arrange-ment described by the invention is possible.
Claims (9)
1. Biosensor, particularly a biosensor based on the field effect with, if necessary, a silanized signal-active surface to which the sensoric biomaterial is bonded by a crosslinking agent, characterized in that the crosslinking agent is formed by a hetero bifunctional crosslinking agent with a reactive group (A) which is specific for the signal-active surface on one hand, and a photo-activatable biomaterial-reactive group (B) on the other hand.
2. Biosensor according to claim 1, characterized in that the crosslinking agent is bonded to the signal-active surface by means of a coupling reaction of its group (A) with -OH, -SH, or NHx- groups of the signal-active surface.
3. Biosensor according to claim 2, characterized in that the crosslinker is bonded to a silanized surface or to a Si3N4 surface with reactive NHx- groups by means of a coupling reaction of its group (A).
4. Biosensor according to claims 1 to 3, characterized in that the crosslinking agent is bonded to the protein of the biomaterial by way of a photo activated coupling reaction of an acido- or nitro group (B) of the crosslinking agent.
5. Biosensor according to claim 4, characterized in that the photoactivated coupling reaction occurs by means of an aromatically bonded nitro- or azido-group.
6. Biosensor according to claim 5, characterized in that the coupling occurs by means of a crosslinking agent with an azidophenyl group.
7. Biosensor according to claim 2, characterized in that the crosslinking agent is bonded to the surface by means of an aldehyde ester or imidoester group (A).
8. Biosensor according to claim 7, characterized in that the crosslinking agent is bonded to the surface by means of a coupling reaction of a N-hydroxy succinimid-ester group(A).
9. Biosensor according to any of the preceding claims, characterized by a signal active surface of a 10-1000 nm Si3N4 surface layer deposited by CVD from a SiH4/NH3 mixture on a SiO2 surface area.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4436001A DE4436001C2 (en) | 1994-10-08 | 1994-10-08 | Biosensor with enzyme fixed on a Si¶3¶N¶4¶ surface |
| DEP4436001.0 | 1994-10-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2202043A1 true CA2202043A1 (en) | 1996-04-18 |
Family
ID=6530280
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002202043A Abandoned CA2202043A1 (en) | 1994-10-08 | 1995-09-30 | Biosensor with biomaterial covalently fixed on a signal-active surface |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0784791B1 (en) |
| JP (1) | JPH10506994A (en) |
| KR (1) | KR970706495A (en) |
| CN (1) | CN1166874A (en) |
| AT (1) | ATE209357T1 (en) |
| BR (1) | BR9509127A (en) |
| CA (1) | CA2202043A1 (en) |
| DE (2) | DE4436001C2 (en) |
| MX (1) | MX9702515A (en) |
| WO (1) | WO1996011402A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19621165C1 (en) * | 1996-05-24 | 1997-10-02 | Karlsruhe Forschzent | Immobilisation of macromolecules, especially enzymes |
| DE19749216C1 (en) * | 1997-11-06 | 1999-04-15 | Forschungszentrum Juelich Gmbh | Assay device containing electrolyte and sensor |
| DE10312296B4 (en) * | 2003-03-20 | 2007-02-15 | Forschungszentrum Jülich GmbH | Reagent biosensor for detection of nitriles and cyanides |
| DE10320898A1 (en) * | 2003-05-09 | 2004-12-02 | Iongate Biosciences Gmbh | Biocompatible sensor electrode arrangement and method for its production |
| DE10320899A1 (en) * | 2003-05-09 | 2004-12-02 | Iongate Biosciences Gmbh | Apparatus with sensors for amperometric- or potentiometric testing of pharmaceutical sites of action and active substances, includes membrane biosensor region |
| FR2945124B1 (en) | 2009-04-29 | 2011-07-08 | Burkert Werke Gmbh & Co Kg | METHOD AND DEVICE FOR MEASURING THE CONCENTRATION OF AN ANALYTE IN A SAMPLE LIQUID |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB8314523D0 (en) * | 1983-05-25 | 1983-06-29 | Lowe C R | Diagnostic device |
| WO1988008875A1 (en) * | 1987-05-05 | 1988-11-17 | The Washington Technology Center | Cycling biochip sensor |
| US5234820A (en) * | 1989-06-30 | 1993-08-10 | Board Of Regents Of The University Of Nebraska | Exopeptidase catalyzed site-specific bonding of supports, labels and bioactive agents to proteins |
| ATE160634T1 (en) * | 1992-03-23 | 1997-12-15 | Siemens Ag | BIOSENSOR |
| CA2064683A1 (en) * | 1992-03-26 | 1993-09-27 | Krishna Mohan Rao Kallury | Formation of thermostable enzymes with extra-ordinary heat tolerance by immobilization on phospholipid matrices |
| US5412083A (en) * | 1992-04-16 | 1995-05-02 | Northeastern University | Carbohydrate heterobifunctional cross-linking reagent |
-
1994
- 1994-10-08 DE DE4436001A patent/DE4436001C2/en not_active Expired - Fee Related
-
1995
- 1995-09-30 WO PCT/DE1995/001371 patent/WO1996011402A1/en active IP Right Grant
- 1995-09-30 DE DE59509868T patent/DE59509868D1/en not_active Expired - Fee Related
- 1995-09-30 KR KR1019970702298A patent/KR970706495A/en not_active Application Discontinuation
- 1995-09-30 CN CN95196188A patent/CN1166874A/en active Pending
- 1995-09-30 AT AT95933316T patent/ATE209357T1/en not_active IP Right Cessation
- 1995-09-30 CA CA002202043A patent/CA2202043A1/en not_active Abandoned
- 1995-09-30 BR BR9509127A patent/BR9509127A/en unknown
- 1995-09-30 MX MX9702515A patent/MX9702515A/en unknown
- 1995-09-30 EP EP95933316A patent/EP0784791B1/en not_active Expired - Lifetime
- 1995-09-30 JP JP8512256A patent/JPH10506994A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| KR970706495A (en) | 1997-11-03 |
| EP0784791A1 (en) | 1997-07-23 |
| DE4436001C2 (en) | 1996-07-25 |
| MX9702515A (en) | 1998-02-28 |
| EP0784791B1 (en) | 2001-11-21 |
| WO1996011402A1 (en) | 1996-04-18 |
| DE59509868D1 (en) | 2002-01-03 |
| BR9509127A (en) | 1997-09-02 |
| DE4436001A1 (en) | 1996-04-11 |
| ATE209357T1 (en) | 2001-12-15 |
| CN1166874A (en) | 1997-12-03 |
| JPH10506994A (en) | 1998-07-07 |
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