EP1379680A2

EP1379680A2 - Method for fixing biomolecules onto chemically inert surfaces

Info

Publication number: EP1379680A2
Application number: EP02732600A
Authority: EP
Inventors: Hans-Willi Kling; Jens Seemann; Johannes Georg Schindler
Original assignee: Cognis Deutschland GmbH and Co KG
Current assignee: Cognis IP Management GmbH
Priority date: 2001-04-14
Filing date: 2002-04-11
Publication date: 2004-01-14
Also published as: EP1385985A2; JP2004533238A; US20040209340A1; WO2002083932A3; WO2002083931A3; US20040175811A1; WO2002083931A2; WO2002083932A2

Abstract

The invention relates to a method for fixing biomolecules, particularly enzymes or enzymatic systems onto chemically inert support surfaces which are activated or are functionalised by plasma-chemicals. The invention also relates to a method for immobilising biomolecules, preferably enzymes or enzymatic systems, by direct or indirect connection, particularly chemical and/or physical fixing onto chemically inert support surfaces which are activated or are functionalised by plasma-chemicals. The immobilised biomolecules can be used, for e.g., in bioreactors, biosensors and chromatographic systems.

Description

Process for connecting biomolecules to chemically inert surfaces

The present invention relates to a method for binding biomolecules, in particular enzymes or enzymatic systems, to chemically inert carrier surfaces. In particular, 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.

In applied microbiology, especially in biotechnology, it is known to fix enzymes, enzyme-producing microorganisms or cells on certain carriers, especially when they are used as biocatalysts. This process is commonly referred to as immobilization.

Since native enzymes are reduced in their activity during storage or in a single batch approach by biological, chemical or physical effects, there is a need to stabilize these enzymes because of the high production costs of enzymes. Immobilization makes them reusable. After use, the enzymes can be easily separated. In this way they can be used in high local concentration and in continuous flow. The substrate specificity and the specificity of the reaction as well as the reactivity of the enzyme must not be lost by the immobilization.

When immobilizing enzymes, three basic methods are generally distinguished, namely firstly the immobilization by crosslinking, secondly the immobilization by binding to a support and thirdly the immobilization by inclusion. When immobilized by crosslinking, crosslinked enzymes are obtained which are fixed to one another without their activity being influenced. However, the enzymes are no longer soluble. The crosslinking takes place, for example, with glutardialdehyde.

If the immobilization is carried out by binding to a carrier, 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.

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. B. wastewater treatment in the trickling filter. Another older process is vinegar production using the generator process. In the food sector, 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. There are further technical processes with immobilized systems in the production of amino acids, in the cleavage of penicillin G to 6-aminopenicillinic acid and in the production of ethanol with growing, immobilized cells from Saccharomyces sp. Immobilized enzyme and cell systems are used not only in biotechnological production processes, but also in analytics. B. in so-called biosensors. The principle of the analysis with the aid of immobilized systems is based on the fact that a substrate to be determined is implemented by an immobilized system, whereby the change in the product, substrate or cosubstrate concentration can be tracked, for example with several coupled methods (e.g. Enzyme electrodes).

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. In particular, 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

(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).

In 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. In other words, in step (a) of the method according to the invention, the - initially - chemically inert carrier surface is functionalized.

For the purposes of the present invention, “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. Only through the plasma chemical treatment in step (a) of the process according to the invention is the surface of the chemically inert carrier material prepared for the fixation, ie coupling or binding of the biomolecule or enzymes in the subsequent process step (b). 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. Furthermore, chemically inert surfaces for binding the biomolecules or enzymes are all common polymers that are used for reactor manufacture, for example also the already mentioned polyhalogenated polymers such as

Polytetrafluoroethylene (Teflon ^® ) or PVC. It is also possible to combine different materials, for example chemically resistant ones

To coat metal surfaces (e.g. stainless steel or platinum) with polytetrafluoroethylene (Teflon ^® ) or PVC, e.g. B. by vapor deposition. 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. In particular, 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. The phrase "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.

After the plasma chemical activation or modification according to step (a) of the method according to the invention, 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.

Particularly preferred is 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. In other words, when 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. B. hydrophobicity or hydrophilicity properties, permeabilities, microporosities, mechanical properties such as hardness, brittleness etc. 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.

According to the invention, 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).

Before the biomolecule, in particular the enzyme or the enzymatic enzyme, is connected or coupled to 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. As an alternative to this, or simultaneously with this, it may also be advisable to activate the reactive functional group (s) fixed to the support surface in step (a) of the process according to the invention, for example by protonation, deprotonation etc., depending on the chemical nature of the functional group (n). Such methods are known per se to the person skilled in the art.

According to the method according to the invention, 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. In principle, all types of biomolecules, in particular all types of enzymes, can be immobilized using the method according to the invention. If 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). In general, cross-linking reagents (eg glutard ialdehyde) which are known per se to the person skilled in the art are used here to bind the enzymes.

1 schematically shows the sequence of the method according to the invention with method steps (a) and (b). First of all, 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. As shown in the embodiment according to FIG. 1, in 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. Here, 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.

As described above, the immobilized biomolecules obtainable by the method according to the invention, in particular enzymes or enzymatic systems, can also be used in biosensors. According to the invention, “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.

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 ,

Various 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. B. glucose, galactose, lactose, ethanol, lactic acid or uric acid.

When using the immobilized enzymes in conventional biosensors, the enzyme molecules are either introduced into polymer matrices (such as PVC, gels, graphites or zeolites) or between foils (e.g. cellulose acetate). The concept according to the invention, on the other hand, is that with sensors that for example on the enzymatic production or consumption of

Oxygen based and have a chemically inert membrane (eg a Teflon membrane), which is used to bind enzymes by the method according to the invention; As described above, 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. Examples of suitable biosensor systems according to the invention are e.g. B. catalase and / or glucose oxidase, in which, after coupling the respective enzyme to a suitable functional group which is bound to a chemically inert surface, crosslinking with glutardialdehyde can be carried out (e.g. 5% in buffer, pH Value = 7 and 20 mg catalase (Merck) or glucose oxidase (Merck) in 500 μl solution). 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.

The 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 combined immobilization of the two enzymes catalase and glucose oxidase has already been pointed out. This is of analytically relevant importance if e.g. B. is about the continuous monitoring of measured values of glucose in low-oxygen or even oxygen-free media. In addition, the method described below allows the measuring range to be expanded. Since oxygen is an important reactant in substrate conversion of glucose oxidase (GOD),

β-D-glucose + 0 ₂ + H ₂ 0 ^GQP H ₂ 0 ₂ + glucose ^acid

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 β-D-glucose by GOD. This can be done in a particularly elegant manner on the basis of a bienzyme membrane, for example by B. In addition to glucose oxidase, 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.

If now in a 0 ₂ -sensitive-enzymatic flow sensor with the bienzyme membrane described in its embodiment according to the invention besides ß-D-glucose, which is in equilibrium with the - form when the mutarotation equilibrium is set, simultaneously floods chemically bound oxygen in the form of H ₂ O ₂ , catalase becomes according to the reaction equation

2 H ₂ 0 ₂ ^Catalysis 0 ₂ + 2 H ₂ 0

Provide 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. According to a particular embodiment, the present invention thus relates to a biosensor, in particular for measuring glucose, preferably β-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. fixed peroxide-sensitive biomolecule, in particular enzyme, preferably glucose oxidase, optionally in combination with catalase.

According to a further special embodiment of the present invention, the biosensor is a peroxide sensor or a biochemical peroxide sensor. According to the invention, 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.) , 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.

According to a preferred embodiment of the present invention, 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), fixed or attached peroxide-sensitive biomolecule, in particular enzyme, preferably catalase. In other words, 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.

In a particularly preferred embodiment thereof, the carrier surface consists of surface chemistry modified by plasma chemistry

Polytetrafluoroethylene (Teflon ^® ) or has a coating thereof (e.g. a metal vapor-coated with polytetrafluoroethylene, such as platinum), the

Surface of the 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.

In the biochemical peroxide sensor according to the invention, 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. In all of these cases, 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. For example, 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.

With the peroxide sensor according to the invention, 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).

When using the 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). To this For example, several substances can be determined in parallel by several enzymes (or enzyme chains) in a measuring cell or in measuring systems. In this regard, reference can be made to the patent application with the official file number DE 101 18 554.5, the entire content of which is hereby incorporated by reference.

As described above, the immobilized biomolecules obtainable by the process according to the invention, in particular enzymes or enzymatic systems, can also be used in bioreactors.

According to the invention, “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. B. reactors with planar surfaces as well as tubular reactors. Examples of this are enzyme reactors coated with polytetrafluoroethylene, to the walls of which the enzyme is bound after the pretreatment according to the invention described above, so that a more efficient generation of reactors can be achieved in which it is not necessary to separate the enzymes from the reaction solution. 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.

According to another embodiment of the present invention, when the biomolecules immobilized according to the invention, in particular enzymes or enzymatic systems, are used in 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.

When using the 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, there is the possibility of at least two different types of biomolecules, in particular enzymes or enzymatic Systems to combine with each other, d. H. use so-called biomolecule or enzyme chains or biomolecule or enzyme degradation chains. The various biomolecules, in particular enzymes or enzymatic systems, 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.

3 shows, by way of example and schematically, different types of prior art bioreactors:

3A shows a stirred tank reactor in which the energy is introduced by mechanically moving units. Here 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. Here 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. Here 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. Here G denotes the gas flow.

The previously described 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.

4 shows an example and schematically some embodiments of bioreactors according to the present invention:

4A shows a bioreactor, the chemically inert walls of which are modified by coupling an immobilized biomolecule, in particular type A enzyme. Here G denotes the gas flow.

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. Here 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.

Numerous other variants for the modification of bioreactors according to the invention, in particular 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.

There is also the possibility of using the 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).

The use of the biomolecules immobilized according to the invention, in particular enzymes or enzymatic systems, in biosensors, bioreactors and chromatographic systems has the advantage that, on the one hand, the biomolecules, in particular enzymes or enzymatic systems, can be reused by the immobilization and, on the other hand, they are easy to use after use Separation is possible (e.g. after synthesis in the bioreactor, for example by draining the reaction mixture). In this way, the biomolecules, in particular enzymes or enzymatic systems, can be used efficiently and inexpensively in a high local concentration and in a continuous flow. However, 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.

Further refinements and variations of the present invention are readily recognizable and realizable for the person skilled in the art when reading the patent specification, without thereby leaving the scope of the present invention.

The present invention is illustrated using the following exemplary embodiments, which, however, are in no way intended to limit the invention.

embodiments

Production of bioelectrochemical peroxide and glucose sensors according to the invention

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.

After punching out aminized PTFE membranes with a diameter of 13 mm, they are clamped onto an acrylic glass ring using an O-ring in such a way that a flat surface of 8 mm in diameter is available so that the enzymes can be covalently bound and cross-linked on the side of the measurement solution. z. B. application-oriented (measuring range):

for different H ₂ 0 ₂ sensors: 1,000 to 100,000 U catalase /

membrane

for various glucose sensors: 10 to 100 U glucose oxidase /

Membrane.

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.

After pushing the acrylic glass ring with membrane system onto a 0 ₂ electrode and locking it in a flow chamber, bioelectrochemical sensors according to the invention result, as previously defined in the general description. The service life of such sensors is approximately two months. Strictly speaking, the glucose sensors according to the invention measure β-D-glucose. However, since the β- and α-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.

For example, the following biosensors according to the invention were produced:

1. Glucose sensor with laboratory code 2.) SBC-1320-ß-D-glucose-HDKS-No. 1 :

40 U glucose oxidase covalently bound with glutardialdehyde to the carboxylated PTFE membrane of a 0 ₂ detector.

2. Glucose sensor with laboratory code 3.) SBC-1321-ß-D-glucose-HDKS-No. 1 :

40 U glucose oxidase covalently bound with glutardialdehyde to aminized PTFE membrane of a 0 ₂ detector.

3. Hydrogen peroxide sensor with laboratory code 5.) SBC-1323-H ₂ 0 ₂ -HDKS1-No. 1: 26,000 U catalase covalently bound with glutardialdehyde to aminized PTFE membrane of a 0 ₂ detector.

For the glucose sensor with laboratory code 3.) 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. For this purpose, the sensor 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.

Furthermore, 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. The 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:

Catalase glucose oxidase

2.6 x 10 U 40 U

5.2 x 10 ⁴ U 80 U

7.8 x 10 ⁴ U 120 U

In the bioelectrochemical flow sensors according to the invention, the measuring medium can be sucked in with a roller pump connected downstream.

Claims

claims:

1. A method for immobilizing biomolecules, in particular enzymes or enzymatic systems, by fixing and / or binding them to a chemically inert support surface, the method comprising the following process steps:

(a) activation of the chemically inert support surface by modification of the support surface using plasma chemical methods; subsequently

(b) Linking at least one biomolecule to be immobilized, if necessary after converting it into an activated, bindable state, to the carrier surface activated in step (a).

2. The method according to claim 1, characterized in that the activation of the chemically inert carrier surface in step (a) comprises functionalizing the chemically inert carrier surface.

3. The method according to claim 1 or 2, characterized in that the activation of the chemically inert support surface in step (a) takes place in that at least one suitable functional group which is reactive toward the biomolecules to be attached is attached directly to the chemically inert support surface under plasma-chemical conditions ,

4. The method according to any one of the preceding claims, characterized in that the reactive functional group comprises a carboxyl, amino, hydroxyl and / or thio group, optionally in activated, in particular protonated or deprotonated form.

5. The method according to any one of the preceding claims, characterized in that the activation of the chemically inert carrier surface by means of plasma chemical methods in step (a) selectively only on the surface.

6. The method according to any one of the preceding claims, characterized in that when the chemically inert carrier surface is activated by means of plasma chemical methods in step (a) the bulk properties of the chemically inert carrier surface are retained in the rest.

7. The method according to any one of the preceding claims, characterized in that the activation of the chemically inert carrier surface in step (a) in the reactive plasma, in particular high-frequency plasma, takes place.

8. The method according to any one of the preceding claims, characterized in that the chemically inert support surface includes noble metals such as platinum in particular and their alloys, stainless steel or polyhalogenated polymers, in particular polyhalogenated polymeric hydrocarbons such as in particular polytetrafluoroethylene or polyvinyl chloride, or cellulose acetate or combinations of these materials.

9. The method according to any one of the preceding claims, characterized in that in step (b) the connection or coupling of the biomolecule or molecules to be immobilized to the plasma-chemically modified carrier surface is carried out by connecting or coupling the biomolecules via the functional groups reactive in step (a) ,

10. The method according to claim 9, characterized in that in step (b) the biomolecules are attached directly to the reactive functional groups applied to the support surface or indirectly via a suitable linker.

11. The method according to any one of the preceding claims, characterized in that the biomolecules are bound directly or indirectly to the support surface by means of covalent and / or ionic bonding, preferably covalent bonding, via the reactive (s) functional group (s).

12. The method according to any one of the preceding claims, characterized in that the biomolecule to be immobilized is an enzyme and is in particular selected from the group of oxidoreductases, transferases, hydrolases such as in particular esterases such as lipases, lyases, isomerases and ligases (synthetases) and mixtures thereof or combinations with each other.

13. The method according to any one of the preceding claims, characterized in that the method step (b) can be followed by a method step (c) which comprises a crosslinking of the biomolecules connected or coupled in method step (b), the method steps (b) and ( c) if necessary also summarized, in particular can be carried out simultaneously.

14. Immobilized biomolecule, in particular enzyme or enzymatic system, obtainable by the method according to claims 1 to 13.

15. Immobilized biomolecule, in particular enzyme or enzymatic system, characterized in that the biomolecule is fixed directly or indirectly to a chemically inert support surface, in particular is attached or coupled, the attachment or coupling of the biomolecule via attached to the chemically inert support surface, suitable, reactive functional groups has occurred.

16. Use of an immobilized biomolecule, in particular enzyme or enzymatic system, according to claim 14 or 15 in bioreactors or biosensors.

17. Use according to claim 16 for modifying the wall surfaces of bioreactors.

18. Use according to claim 16, wherein the immobilized biomolecule, in particular enzyme or enzymatic system, is bound to the carrier material or bulk material of a bioreactor.

19. Use of an immobilized biomolecule, in particular enzyme or enzymatic system, according to claim 14 or 15 in chromatographic systems, in particular chromatographic columns.

20. Use according to claim 19 for analytical or preparative-synthetic purposes.

21. Biosensors, bioreactors or chromatographic systems containing an immobilized biomolecule, in particular enzyme or enzymatic system, according to claim 14 or 15.

22. Biosensor, in particular for the qualitative and / or quantitative determination of peroxides, in particular inorganic and / or organic peroxides such as hydrogen peroxide, dissolved peroxides from inorganic or organic salts and / or organic peracids, the biosensor being modified and modified by at least one on an activated, plasma-chemical / or functionalized carrier surface of a chemically inert carrier material, preferably polytetrafluoroethylene, fixed peroxide-sensitive biomolecule, in particular enzyme, preferably catalase.

23. biosensor, in particular for the qualitative and / or quantitative determination of glucose, in particular β-D-glucose, the biosensor having at least one peroxide-sensitive biomolecule fixed on an activated, plasma-chemically modified and / or functionalized carrier surface of a chemically inert carrier material, preferably polytetrafluoroethylene, in particular enzyme, preferably glucose oxidase, optionally in combination with catalase.

24. Biosensor according to claim 22 or 23, characterized in that the biomolecule (s) bound to the chemically inert support surface, in particular enzymes, are additionally cross-linked, in particular with glutardialdehyde.

25. Biosensor according to one of claims 22 to 24, characterized in that in the biosensor the unit of biomolecule (s), in particular enzyme (s), and carrier surface provides a preferably electrical signal, in particular being based on the signal for the presence and / or concentration of peroxides or glucose, in particular β-D-glucose, can be concluded.

26. Biosensors, in particular according to one of claims 21 to 25, containing glucose oxidase and / or catalase in each case in immobilized form.