EP1379680A2 - Procede pour fixer des biomolecules sur des surfaces chimiquement inertes - Google Patents

Procede pour fixer des biomolecules sur des surfaces chimiquement inertes

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Publication number
EP1379680A2
EP1379680A2 EP02732600A EP02732600A EP1379680A2 EP 1379680 A2 EP1379680 A2 EP 1379680A2 EP 02732600 A EP02732600 A EP 02732600A EP 02732600 A EP02732600 A EP 02732600A EP 1379680 A2 EP1379680 A2 EP 1379680A2
Authority
EP
European Patent Office
Prior art keywords
chemically inert
biomolecule
immobilized
biomolecules
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02732600A
Other languages
German (de)
English (en)
Inventor
Hans-Willi Kling
Jens Seemann
Johannes Georg Schindler
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cognis IP Management GmbH
Original Assignee
Cognis Deutschland GmbH and Co KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE2001118553 external-priority patent/DE10118553A1/de
Priority claimed from DE2001118554 external-priority patent/DE10118554A1/de
Application filed by Cognis Deutschland GmbH and Co KG filed Critical Cognis Deutschland GmbH and Co KG
Publication of EP1379680A2 publication Critical patent/EP1379680A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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

Definitions

  • the present invention relates to a method for binding biomolecules, in particular enzymes or enzymatic systems, to chemically inert carrier surfaces.
  • the present invention relates to a method for immobilizing biomolecules, in particular enzymes or enzymatic systems, by attachment, in particular physical and / or chemical attachment, to chemically inert support surfaces and the use of the immobilized systems produced in this way, preferably for use in bioreactors , Biosensors and chromatographic systems.
  • the binding can be carried out by adsorption, ion binding or covalent binding.
  • the binding to the carrier can also take place within the original microbial cell.
  • the activity of the enzyme is not affected by the fixation; it can be used multiple or continuously in a carrier-bound manner.
  • the enzyme When immobilized by inclusion, the enzyme is mostly enclosed between semipermeable membranes and / or gels, microcapsules or fibers.
  • the encapsulated enzymes are e.g. B. separated by a semipermeable membrane from the surrounding substrate and product solution. Cells can also be encapsulated. The activity of the enzyme is not affected by the fixation in space.
  • Immobilized enzymes, enzyme-producing microorganisms or cells are used in particular in biotechnological processes.
  • the first technical processes with immobilized cells were empirically optimized and are still used today.
  • Another older process is vinegar production using the generator process.
  • the most important process is the use of cells with glucose isomerase to produce syrup containing fructose.
  • Glucose amylase for the production of glucose in the starch process is also used immobilized.
  • the cleavage of lactose using the immobilized ß-galactosidase from yeast to glucose and galactose is also a common procedure.
  • a disadvantage of the methods for immobilizing enzymes known from the prior art is in particular the fact that the immobilization of the enzymes has to be carried out in a relatively complex manner and a combination with systems which are inert to the enzymes is not possible.
  • the problem underlying the present invention now consists in providing a method with which biomolecules, in particular enzymes or enzymatic systems, can also be attached to chemically inert carrier surfaces.
  • a new method is to be provided with which biomolecules, in particular enzymes or enzymatic systems, can be immobilized by binding to chemically inert carrier surfaces.
  • Another problem on which the present invention is based is to provide a method with which biomolecules, in particular enzymes or enzymatic systems, can be immobilized in a simple manner, the reactivity of the biomolecules immobilized in this way being essentially to be retained, i. H. the reactivity of the biomolecules immobilized in this way should essentially not be restricted.
  • the present invention thus relates to a process for immobilizing biomolecules, in particular enzymes or enzymatic systems, by fixing or binding them to a chemically inert support surface, the process comprising the following process steps: (a) activation of the chemically inert support surface by modification of the support surface using plasma chemical methods; subsequently
  • step (b) Linking the biomolecule (s) to be immobilized, if necessary after converting them into an activated or attachable state, to the carrier surface activated in step (a).
  • step (a) of the method according to the invention the chemically inert carrier surface is activated by modifying the carrier surface using plasma-chemical methods.
  • the plasma-chemical modification of surfaces is known per se to the person skilled in the art. You can refer to the relevant specialist literature here. So far, however, this method has not been used to prepare surfaces for the attachment or immobilization of biomolecules.
  • the - initially - chemically inert carrier surface is functionalized.
  • “chemically inert support surface” is understood to mean a surface that is non-reactive with respect to the respective application. In the present case, it is particularly meant that the support surface is initially, ie originally or per se, not suitable for binding biomolecules, that is, in other words, the support surface initially does not have any reactive functional groups which are associated with the biomolecule to be connected or coupled ( can react. In the sense of the present invention, “chemically inert” means in particular non-reactive towards the respective biomolecules or not suitable for binding biomolecules.
  • Suitable chemically inert surfaces according to the invention are all surfaces which do not or essentially do not impair the catalytic activity of the biomolecule to be connected or coupled in process step (b), in particular enzyme, and which do not or essentially do not interfere with enzymatically catalyzed processes and methods , These can be, for example, chemically inert metal surfaces, in particular surfaces made of precious metal or their alloys (e.g. platinum or stainless steel).
  • Chemically inert plastic surfaces in particular polyhalogenated polymers, preferably polyalkyls or polyalkylenes such as polytetrafluoroethylene ( Teflon® ) or polyvinyl chloride (PVC), are also suitable according to the invention.
  • polyhalogenated polymers preferably polyalkyls or polyalkylenes such as polytetrafluoroethylene ( Teflon® ) or polyvinyl chloride (PVC)
  • Teflon® polytetrafluoroethylene
  • PVC polyvinyl chloride
  • Teflon ® Polytetrafluoroethylene (Teflon ® ) or PVC. It is also possible to combine different materials, for example chemically resistant ones
  • metal surfaces e.g. stainless steel or platinum
  • polytetrafluoroethylene (Teflon ® ) or PVC e.g. B.
  • Teflon ® polytetrafluoroethylene
  • PVC polytetrafluoroethylene
  • Another material suitable for binding biomolecules is, for example, cellulose acetate.
  • the activation of the chemically inert support surface in step (a) of the method according to the invention takes place in particular in that at least one suitable functional group which is reactive towards the biomolecules to be attached is attached or fixed directly to the chemically inert support surface under plasma chemical conditions.
  • This method is known per se to the person skilled in the art.
  • the activation of the chemically inert carrier surface in step (a) of the method according to the invention can take place in reactive plasma, in particular high-frequency plasma. This happens, for example, in a - reactive - plasma, in particular high-frequency plasma, from inert gas (s), such as, for example, B. noble gases, and reactant gas (s), such as. B. ammonia.
  • suitable functional group reactive towards the biomolecule or enzyme to be bound denotes a functional group which is suitable for direct (direct) attachment or coupling or else for indirect (indirect) attachment or coupling of the respective biomolecule, in particular enzyme is, ie is reactive towards the respective biomolecule or enzyme or reacts with it with binding or coupling.
  • the originally chemically inert surface has functional groups which are reactive towards the biomolecules to be attached or coupled.
  • Non-limiting examples of reactive functional groups suitable according to the invention are in particular groups or groupings which comprise or represent a carboxyl group, an amino group, a hydroxyl group and / or a thio group, optionally in protonated or deprotonated form.
  • the plasma chemical conducted amino, hydroxyl and / or carboxyl modification chemically inert surfaces in particular chemically inert surfaces of polytetrafluoroethylene (eg., Teflon ® membrane) or PVC. This method is known per se to the person skilled in the art.
  • the peculiarity of the plasma chemical activation in step (a) of the method according to the invention is in particular that the activation of the chemically inert carrier surface takes place selectively only on the surface.
  • the chemically inert carrier surface is activated by means of plasma chemical methods in step (a) of the method according to the invention, the bulk properties of the originally chemically inert carrier surface are retained, for example.
  • Process step (a) is then followed in process step (b) by the connection or coupling of the biomolecule (s) to be immobilized to the plasma-chemically modified support surface, this being done by connection or coupling of the biomolecules via the functional groups reactive in step (a).
  • the biomolecules can be attached directly to the reactive functional groups applied to the support surface or indirectly, e.g. B. via suitable linkers or linker molecules. Methods for this are known per se to the person skilled in the art.
  • a "linker” - also synonymously referred to as a “linker molecule” - is understood to mean a molecule or a part of a molecule which serves to link fragments and / or other molecules (here: linkage or connection of plasma-chemically activated or modified, chemically inert carrier surfaces with biomolecules, especially enzymes).
  • the biomolecule in particular the enzyme or the enzymatic enzyme
  • the plasma-chemically activated or modified carrier surface it may be advisable to convert the biomolecule to an activated or connectable state. Methods for this are well known to the person skilled in the art.
  • Such methods are known per se to the person skilled in the art.
  • the biomolecules can be directly or indirectly attached to a plasma chemical by means of covalent and / or ionic bonding, preferably covalent bonding, via the reactive functional group (s) attached in step (a) activated, chemically inert carrier surface.
  • This causes the biomolecule, in particular the enzyme, to be immobilized.
  • biomolecules in particular all types of enzymes, can be immobilized using the method according to the invention.
  • the biomolecule to be immobilized is an enzyme, this can for example be selected from the group of oxidoreductases, transferases, hydrolases (e.g. esterases such as lipases), lyases, isomerases and ligases (synthetases) and their mixtures and combinations with one another.
  • Process step (b) can optionally be followed by a process step (c) which comprises crosslinking the enzymes coupled in process step (b).
  • cross-linking reagents eg glutard ialdehyde
  • Process step (c) which comprises crosslinking the enzymes coupled in process step (b).
  • FIG. 1 schematically shows the sequence of the method according to the invention with method steps (a) and (b).
  • the activation of the chemically inert carrier surface 1 in method step (a) is carried out by modifying the carrier surface 1 with plasma chemical methods, FIG. 1 showing an embodiment according to which - starting from a starting molecule 2 - a suitable functional one which is reactive towards the biomolecule to be bound Group 2 '(z. B. an amino group) is attached directly to the chemically inert support surface 1.
  • process step (b) the direct coupling or connection of the biomolecule 3 to be immobilized to the carrier surface 1 activated in step (a) then follows, thereby immobilizing the biomolecule 3 is effected.
  • the method according to the invention makes it possible to bind biomolecules, in particular enzymes, to chemically inert surfaces and to immobilize them in this way, whereby the reactivity of the biomolecule, in particular the enzyme, is not or essentially not limited in its reactivity.
  • the immobilization makes the biomolecules, in particular enzymes, reusable. After use, the enzymes can be easily separated. On in this way they can be used, in particular, in a high local concentration and, for example, in a continuous flow.
  • the substrate specificity and the specificity of the reaction and the reactivity of the biomolecules, in particular enzymes are retained in the immobilization according to the invention.
  • the present invention also relates to the biomolecules which can be produced, immobilized and, if appropriate, crosslinked by the process according to the invention, in particular enzymes or enzymatic systems.
  • the immobilized biomolecules, in particular enzymes or enzymatic systems are fixed directly or indirectly to a chemically inert support surface, in particular attached or coupled, the attachment or coupling of the biomolecule being effected via suitable reactive functional groups attached to the chemically inert support surface, for example directly via ionic and / or covalent bonds or indirectly via a suitable linker with biomolecule- or enzyme-reactive functional groups.
  • the immobilized biomolecules which can be produced by the process according to the invention, in particular enzymes or enzymatic systems, can be used, for example, in biosensors or bioreactors. Furthermore, their use in chromatographic systems, in particular chromatographic columns, is also possible, either for preparative purposes or synthesis purposes or else for analytical purposes.
  • the present invention thus also relates to biosensors, bioreactors and chromatographic systems which comprise the immobilized biomolecules obtainable according to the invention, in particular enzymes or enzymatic systems.
  • biosensors are sensors with a bioactive component, based on the coupling of biomolecules that specifically recognize analytes as receptors in the broadest sense, with physicochemical transducers that generate a biologically generated signal (e.g. oxygen concentration, pH value) , Dye, etc.) into electrical measurement signals.
  • a biologically generated signal e.g. oxygen concentration, pH value
  • FIG. 2 shows the typical structure of a biosensor for the specific detection of an analyte 1, the biosensor comprising a receptor 2 and a transducer 3 which converts the biological signal generated by the receptor 2 into an electronic signal 4 which is forwarded to electronics 5 ,
  • biomolecules can be used for specific recognition, in particular enzymes.
  • Potentiometric sensors, amperometric electrodes, piezoelectric sensors, thermistors or optoelectronic sensors can be used as transducers. Due to the reaction or interaction of the analyte with the receptor, a distinction is made between two basic types of biosensors, namely, on the one hand, bioaffinity sensors, which take advantage of changes in the electron density that occur during complex formation, and, on the other hand, metabolism sensors, which are based on the specific detection and conversion of substrates.
  • Biosensors are used - especially in the form of enzyme electrodes - in healthcare, for the control of biotechnological processes, in the food industry or in environmental protection. A wide variety of systems can be analyzed with biosensors.
  • the enzyme molecules are either introduced into polymer matrices (such as PVC, gels, graphites or zeolites) or between foils (e.g. cellulose acetate).
  • polymer matrices such as PVC, gels, graphites or zeolites
  • foils e.g. cellulose acetate
  • Oxygen based and have a chemically inert membrane eg a Teflon membrane
  • a chemically inert membrane eg a Teflon membrane
  • suitable biomolecule- or enzyme-reactive functional groups must be bound to the chemically inert membrane surface.
  • B. amino or carboxyl groups, and these groups can be attached in a plasma coating process. If necessary, cross-linking reagents can then be used to bind the immobilized enzymes.
  • suitable biosensor systems according to the invention are e.g. B.
  • Further exemplary embodiments provide for the binding of 1,000 to 100,000 U (units) of catalase or 10 to 100 U of glucose oxidase with the bifunctional glutardialdehyde on aminated PTFE membranes with a membrane diameter of 1 to 10 mm, preferably 8 mm, but represent the ranges given are not restrictions, but rather have to be based on the types of application, taking into account the desired concentration range of the analyte to be measured.
  • the service life of such 0 2 -sensitive-enzymatic sensors is approximately 2 months.
  • biosensors according to the invention enable, for example, the production of microelectrodes (arrays) for small volumes and high sample throughput (e.g. for combinatorial use).
  • the solution to the problem lies in the simultaneous introduction of chemically bound oxygen, which then has to be released within the enzyme membrane for the substrate conversion of the ⁇ -D-glucose by GOD.
  • This can be done in a particularly elegant manner on the basis of a bienzyme membrane, for example by B.
  • catalase with glutardialdehyde is additionally covalently bound and crosslinked according to the invention on an aminized PTFE membrane.
  • the two enzymes can be immobilized in mixed form, but also in layers.
  • the immobilization in layers can in turn be carried out in two or more layers.
  • the order of the enzyme layers can, but need not, be application-oriented.
  • oxygen from hydrogen peroxide for the substrate conversion of glucose oxidase. Since on the one hand there are 0 2 -sensitive-enzymatic membrane electrodes and on the other hand oxygen is one of the reactants of glucose oxidase, the hydrogen peroxide should be offered in a constant concentration. In the case of an oxygen-containing measuring medium, it should also be provided that the proportion of the physically dissolved is also Oxygen in constant concentration reaches the sensor.
  • the present invention thus relates to a biosensor, in particular for measuring glucose, preferably ⁇ -D-glucose, the biosensor having at least one on an activated, plasma-chemically modified and / or functionalized carrier surface of a chemically inert carrier material, preferably polytetrafluoroethylene.
  • a chemically inert carrier material preferably polytetrafluoroethylene.
  • fixed peroxide-sensitive biomolecule in particular enzyme, preferably glucose oxidase, optionally in combination with catalase.
  • the biosensor is a peroxide sensor or a biochemical peroxide sensor.
  • this is understood to mean a biosensor or a measuring element which is qualitatively or quantitatively sensitive or sensitive to the presence or concentration of inorganic or organic peroxides (e.g. hydrogen peroxide, dissolved peroxides from inorganic or organic salts, organic peracids etc.)
  • inorganic or organic peroxides e.g. hydrogen peroxide, dissolved peroxides from inorganic or organic salts, organic peracids etc.
  • Such a (biochemical) peroxide sensor contains - quasi as an active component - molecules which indicate the presence of peroxides or react with peroxides.
  • These can be, for example, molecules, in particular hydrogen peroxide-sensitive enzymes, which are bound to an activated, chemically inert carrier surface by binding, without this fundamentally changing their reactivity.
  • the biochemical peroxide sensor according to the invention is a biosensor for the qualitative and / or quantitative determination of organic or inorganic peroxides, in particular hydrogen peroxide, of dissolved peroxides from inorganic or organic salts and / or of organic peracids, wherein the biochemical peroxide sensor has at least one, in particular on an activated, plasma-chemically modified or functionalized carrier surface of a chemically inert carrier material
  • polytetrafluoroethylene z. B. Tefion ® membrane
  • peroxide-sensitive biomolecule in particular enzyme, preferably catalase.
  • one or more can be on the carrier Biomolecules or enzymes - either directly or indirectly via linkers - that are mutually complementary in their effect: the enzyme that actually reacts with hydrogen peroxide can be a catalase, for example.
  • Such enzymes are commercially available, for example from Merck KGaA in Germany.
  • the biomolecules or enzymes bound to the chemically inert support surface can then be cross-linked, for example with glutardialdehyde.
  • the biomolecules or enzymes are fixed or bound to the surface by means of suitable, reactive functional groups which have previously been applied to the support surface by known plasma-chemical methods.
  • the carrier surface consists of surface chemistry modified by plasma chemistry
  • Polytetrafluoroethylene Teflon ®
  • a coating thereof e.g. a metal vapor-coated with polytetrafluoroethylene, such as platinum
  • Teflon ® layer is modified as described above and the enzyme or enzymes are fixed thereon.
  • the superficial modification of the Teflon preferably consists in generating or attaching plasma-chemically reactive groups, such as amino or carboxyl groups, to the support surface and then attaching the enzyme or enzymes and then possibly crosslinking them.
  • the unit consisting of the biomolecule (s), in particular enzyme (s), and the carrier surface provides a preferably electrical signal, on the basis of which the presence and / or concentration of peroxides can be deduced (e.g. using a previously created calibration curve ).
  • the biochemical peroxide sensor according to the invention thus enables the qualitative and / or quantitative determination of peroxides in free or in bound form, for example in the form of free hydrogen peroxide, in the form of peracids, in the form of perborates or in the form of soluble peroxides.
  • a certain concentration of the per-compound is in equilibrium with a certain concentration of hydrogen peroxide, the can be determined by the peroxide sensor according to the invention.
  • the level of the electrical signal of the peroxide sensor according to the invention can be correlated with the concentration of the per compound present in each case by means of corresponding calibration curves. If one wishes to determine the concentration of hydrogen peroxide in free or bound form, the measurement solution is preferably buffered to a pH in the range from 4.5 to 8.
  • the presence or concentrations of molecules which react with the peroxides or which cause peroxide consumption can also be determined indirectly (for example nitrite or hydroxylamine).
  • the reaction of the peroxide-consuming molecules with the peroxides (e.g. hydrogen peroxide) or the competitive reaction of the peroxide sensor is used for this. Accordingly, one can, for example, add a known amount of hydrogen peroxide to the solution of the peroxide-consuming molecules, measure the level of the electrical signal of the biochemical peroxide sensor, compare it with the theoretically expected level (e.g. based on calibration) and, from the difference, the concentration of the peroxide-consuming level Derive molecules.
  • the peroxide sensor according to the invention is suitable, for example, for process monitoring, control or control (for example during phosphating).
  • biomolecules immobilized according to the invention in particular enzymes or enzymatic systems, in biosensors, it is therefore possible to combine at least two different types of biomolecules, in particular enzymes or enzymatic systems, with one another, ie in particular to use so-called enzyme chains or enzyme degradation chains.
  • the various enzymes can either be present in a reaction system (for example in a measuring cell) or can be sequentially connected in series (for example in successive measuring cells). If necessary, however, a parallel multi-electrode structure can also be advantageous (e.g. for differential measurements).
  • several substances can be determined in parallel by several enzymes (or enzyme chains) in a measuring cell or in measuring systems.
  • the immobilized biomolecules obtainable by the process according to the invention in particular enzymes or enzymatic systems, can also be used in bioreactors.
  • bioreactors are understood to mean the physical container in which biological substance conversions are carried out, in particular with biomolecules such as enzymes.
  • the method according to the invention can be used to achieve, for example, a modification of the wall surfaces of bioreactors.
  • It can be any type of bioreactor, such.
  • Reactor surfaces suitable according to the invention can, for example, consist of metal or can be coated (for example Teflon® or PVC) or have a combination of these materials with all the common polymers used for reactor production.
  • biomolecules immobilized according to the invention in particular enzymes or enzymatic systems
  • bioreactors there is also the possibility, particularly in the case of fixed bed reactors, of the immobilized biomolecule, in particular to bind enzyme or enzymatic system to the stationary carrier material or bulk material.
  • biomolecules immobilized according to the invention in particular enzymes or enzymatic systems, in bioreactors, in particular for modifying the wall surface or when binding the enzyme to the carrier material or bulk material
  • biomolecules in particular enzymes or enzymatic Systems
  • bioreactors in particular for modifying the wall surface or when binding the enzyme to the carrier material or bulk material
  • biomolecules in particular enzymes or enzymatic Systems
  • biomolecule or enzyme chains in particular enzymes or enzymatic Systems
  • biomolecule or enzyme chains or biomolecule or enzyme degradation chains can be connected either in a single reaction zone or sequentially (e.g. in successive reaction zones). This enables, for example, multi-stage, enzymatically catalyzed syntheses and processes.
  • FIG. 3A shows a stirred tank reactor in which the energy is introduced by mechanically moving units.
  • G denotes the gas flow and M the mechanical drive device (e.g. motor). Because of their versatility, stirred tank reactors are used most frequently.
  • 3B shows a bubble column reactor in which the mixing is carried out by supplying air or another gas.
  • G denotes the gas flow.
  • 3C shows a so-called airlift fermenter with an internal passage, a liquid circulation and mixing being generally generated by the introduction of air or another gas.
  • G denotes the gas flow.
  • 3D shows a so-called airlift fermenter with an external passage, a liquid circulation and mixing being generally generated by the introduction of air or another gas.
  • G denotes the gas flow.
  • bioreactor types of the prior art can be modified according to the invention, for example by modifying the wall surface (for example by coupling biomolecules, in particular enzymes or enzymatic systems according to the invention, to the chemically inert reactor walls) or in the case of fixed bed Bioreactors by connecting the biomolecules, in particular enzymes or enzymatic systems, to the carrier material or bulk material.
  • FIG. 4 shows an example and schematically some embodiments of bioreactors according to the present invention:
  • FIG. 4A shows a bioreactor, the chemically inert walls of which are modified by coupling an immobilized biomolecule, in particular type A enzyme.
  • G denotes the gas flow.
  • FIG. 4B shows a bioreactor, the chemically inert walls of which are modified by coupling an immobilized biomolecule, in particular enzyme, of type A and an immobilized biomolecule, in particular enzyme, of type B, which are arranged in different, successive reaction zones.
  • G denotes the gas flow.
  • 4C shows a bioreactor whose chemically inert walls are modified by coupling an immobilized biomolecule, in particular enzyme, of type A and an immobilized biomolecule, in particular enzyme, of type B, which are arranged within a single reaction zone.
  • 4D shows a bioreactor in the form of a fixed bed reactor, on the carrier material or bulk material immobilized biomolecules, in particular enzymes, of type A and type B are coupled, which are arranged within a reaction zone.
  • bioreactor wall surfaces and / or bulk bioreactors and the like are possible, which the person skilled in the art will readily consider when reading the present description without departing from the scope of the present invention.
  • biomolecules immobilized according to the invention in particular enzymes or enzymatic systems, in chromatographic systems, in particular in chromatographic columns. This can be done for preparative or synthetic purposes (e.g. carrying out enzymatically catalyzed reactions on a chromatographic column) or else for analytical purposes (e.g. for analytical column chromatography).
  • biomolecules immobilized according to the invention in particular enzymes or enzymatic systems
  • biosensors bioreactors and chromatographic systems
  • biomolecules in particular enzymes or enzymatic systems
  • they are easy to use after use Separation is possible (e.g. after synthesis in the bioreactor, for example by draining the reaction mixture).
  • the biomolecules, in particular enzymes or enzymatic systems can be used efficiently and inexpensively in a high local concentration and in a continuous flow.
  • the substrate specificity and the specificity of the reaction and the reactivity of the enzymes are not lost by the immobilization according to the invention.
  • the aim of the process of the invention is (for example Teflon ®.), Among other things, the chemically inert surface material as a diffusion barrier - in the plasma - sputter, and then the functional groups for sole or Reactor applications directly e.g. B. to apply the amino or carboxyl groups to which the biomolecules, in particular enzymes or enzymatic systems, can then be coupled.
  • the chemically inert surface material as a diffusion barrier - in the plasma - sputter, and then the functional groups for sole or Reactor applications directly e.g. B. to apply the amino or carboxyl groups to which the biomolecules, in particular enzymes or enzymatic systems, can then be coupled.
  • a chemically inert carrier surface in this case a PTFE membrane
  • Teflon ® membrane is first functionalized in reactive high-frequency plasma under conditions known per se. Suitable, enzyme-reactive functional groups are bound to the chemically inert membrane surface, in particular amino and / or carboxyl groups.
  • the cavity of the acrylic glass ring is charged with an internal electrolyte on the detector side for the purpose of connecting the measuring cathode made of Pt and Ag / AgCI reference anode.
  • bioelectrochemical sensors according to the invention result, as previously defined in the general description.
  • the service life of such sensors is approximately two months.
  • the glucose sensors according to the invention measure ⁇ -D-glucose.
  • the ⁇ - and ⁇ -forms of glucose are present in a constant ratio after the mutarotation equilibrium has been set, one can speak of glucose sensors in the present case, since the calibration process also takes these circumstances into account.
  • biosensors according to the invention were produced:
  • SBC-1321-ß-D-glucose-HDKS-No. 1 is a membrane system based on an aminized PTFE membrane with GOD covalently bound by glutardialdehyde, the amination of the PTFE membrane and the subsequent immobilization of the GOD being carried out by the method according to the invention.
  • This membrane system was integrated in an amperometric flow cell with Pt measuring cathode and Ag / AgCI reference anode: A 0 2 -sensitive-enzymatic ß-D-glucose sensor for flow measurements was created.
  • the sensor system according to the invention was tested in an endurance test.
  • the senor according to the invention was continuously perfused with a phosphate buffer (pH value of 7.04 at 25 ° C.). During this period, about 100 liters of this buffer were pumped through the measuring system. Finally, glucose measurements with the phosphate buffer as Solvent for the analyte to demonstrate the functionality of the membrane system carried out (pump: Permax 12/6 at 20 digits with silicone tubing 1.9 x 4.5 mm). Despite continuous perfusion of the sensor according to the invention with phosphate buffer (HPL) at a temperature of 20 to 25 ° C. for one year, the enzymatic activity of the membrane system can still be described as sufficient even after one year.
  • HPL phosphate buffer
  • biosensors were produced which contain glucose oxidase and catalase. Such biosensors can be used, for example, for the continuous monitoring of measured values of glucose in low-oxygen or even oxygen-free media. In addition, this allows the measuring range to be expanded.
  • concentrations of units (U) of catalase or glucose oxidase to be immobilized for such biosensors are given below.
  • the two aforementioned enzymes can also be present in the membrane system in an immobilized manner without restriction. This is substantiated on the basis of three examples, specifying the membrane composition, with the two enzymes in turn being covalently bound and crosslinked on an aminized PTFE membrane using glutardialdehyde:
  • the measuring medium can be sucked in with a roller pump connected downstream.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé pour fixer des biomolécules, notamment des enzymes ou des systèmes enzymatiques, sur des surfaces de support chimiquement inertes, activées ou fonctionnalisées par voie plasmachimique. L'invention concerne notamment un procédé pour fixer des biomolécules, de préférence des enzymes ou des systèmes enzymatiques, par liaison directe ou indirecte, notamment par liaison chimique et/ou physique, sur des surfaces de support chimiquement inertes, activées ou fonctionnalisées par voie plasmachimique. Les biomolécules ainsi immobilisées sont appropriées par exemple pour être utilisées dans des bioréacteurs, biodétecteurs et systèmes chromatographiques.
EP02732600A 2001-04-14 2002-04-11 Procede pour fixer des biomolecules sur des surfaces chimiquement inertes Withdrawn EP1379680A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10118553 2001-04-14
DE10118554 2001-04-14
DE2001118553 DE10118553A1 (de) 2001-04-14 2001-04-14 Verfahren zur Anbindung von Biomolekülen an chemisch inerte Oberflächen
DE2001118554 DE10118554A1 (de) 2001-04-14 2001-04-14 Enzymabbauketten
PCT/EP2002/004042 WO2002083931A2 (fr) 2001-04-14 2002-04-11 Procede pour fixer des biomolecules sur des surfaces chimiquement inertes

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EP1379680A2 true EP1379680A2 (fr) 2004-01-14

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EP02732601A Withdrawn EP1385985A2 (fr) 2001-04-14 2002-04-11 Chaines de decomposition enzymatiques
EP02732600A Withdrawn EP1379680A2 (fr) 2001-04-14 2002-04-11 Procede pour fixer des biomolecules sur des surfaces chimiquement inertes

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EP02732601A Withdrawn EP1385985A2 (fr) 2001-04-14 2002-04-11 Chaines de decomposition enzymatiques

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US (2) US20040175811A1 (fr)
EP (2) EP1385985A2 (fr)
JP (1) JP2004533238A (fr)
WO (2) WO2002083932A2 (fr)

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JP2005289931A (ja) * 2004-04-02 2005-10-20 Tokai Univ 中空状物品
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JP4649680B2 (ja) * 2005-03-02 2011-03-16 独立行政法人産業技術総合研究所 酵素固定化マイクロリアクター、及びその製造方法
CN100363482C (zh) * 2005-12-09 2008-01-23 清华大学 利用亲水/疏水复合膜中的微结构固定化脂肪酶的方法
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CN101935669B (zh) * 2009-12-16 2013-01-02 中国科学院动物研究所 一种解毒工程菌及其制备方法和应用
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Also Published As

Publication number Publication date
WO2002083931A2 (fr) 2002-10-24
WO2002083932A2 (fr) 2002-10-24
US20040175811A1 (en) 2004-09-09
US20040209340A1 (en) 2004-10-21
EP1385985A2 (fr) 2004-02-04
JP2004533238A (ja) 2004-11-04
WO2002083931A3 (fr) 2003-07-31
WO2002083932A3 (fr) 2003-11-27

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