HK1249766A1 - Enzyme stabilization in cm sensors - Google Patents

Description

Enzyme stabilization in CM sensors

The present application is a divisional application of an invention patent application entitled "enzyme stabilization in CM sensor" filed on 5/14/2010, and having a national application number of 201080019693.9.

The present invention relates to a composition for forming an electrode, an electrochemical sensor comprising the same, and a method for determining an analyte using the electrochemical sensor.

Measurement systems for biochemical analysis are important components of clinically relevant analytical methods. This mainly relates to the measurement of analytes which can be determined directly or indirectly with the aid of enzymes. The concentration of a clinically useful parameter is typically determined using an in vitro assay system. However, in vitro assays are inadequate when determining analytes whose concentration exhibits significant variation over the course of a day due to the limited temporal resolution and difficulties encountered in sampling.

In this case, biosensors (i.e. measuring systems equipped with biological components which allow a continuous or discontinuous repeated measurement of the analyte and which can be used ex vivo as well as in vivo) have proven particularly suitable for measuring analytes. Ex vivo biosensors are typically used in flow cells, while in vivo biosensors may be implanted, for example, into subcutaneous adipose tissue. In this connection, it is possible to distinguish between percutaneous implants which are introduced into the tissue only for a short time and are in direct contact with a measuring device located on the skin, and full implants which are inserted into the tissue together with the measuring device by means of a surgical operation.

Electrochemical biosensors comprising an enzyme as the biological component contain the enzyme in or on the working electrode, in which case, for example, the analyte can serve as a substrate for the enzyme and can be changed physicochemically (e.g. oxidized) with the aid of the enzyme. An electrical measurement signal caused by the flow of electrons released during analyte conversion at the working electrode is correlated with the measured concentration of the analyte such that the electrical measurement signal can be used to determine the presence and/or amount of the analyte in the sample.

In practice, the working electrode must meet a number of requirements in order to be suitable for use in an electrochemical sensor:

the working electrode should have a low contact resistance and should therefore be highly conductive.

The working electrode should not contain any components that will electrochemically convert at the selected polarizing voltage. This can be achieved by appropriate selection of binders and fillers.

The electrochemically active surface area of the working electrode must remain constant throughout its operation. For this purpose, a reduction in the surface area due to absorption of the component by the surrounding liquid must be avoided. This is usually achieved by applying one or several highly biocompatible polymer coatings.

In order to minimize the decomposition voltage and thus to achieve a specific conversion of the parameters, the electrochemical reaction of the conversion products of the enzymatic reaction should be achieved at a low overpotential. For this purpose, a rapid transfer of electrons from the prosthetic group of the enzyme to the diverting electrode should be provided.

The working electrode should contain a sufficient amount of analyte-specific enzyme with sufficient and constant activity to ensure that the enzymatic reaction that overlaps with the electrochemical conversion is not limited by the available enzyme activity, but by the amount of analyte available. In other words, sensitivity must be maintained throughout the run. Diffusion of the enzyme from the working electrode into the surrounding tissue must be avoided, also due to possible enzyme toxicity. Finally, it must be ensured that the enzyme activity does not fall below a predetermined limit during storage.

A number of electrode compositions are known to be useful in minimizing overpotentials. With respect to H₂O₂By using rhodium-and glucose oxidase-coated carbon fibers, the oxidation potential can be reduced by, for example, 450 mV compared to carbon fibers coated with glucose oxidase alone [ Wang et al, Analytical Chemistry (1992), 64, 456-]. A more easily achieved process is described in EP 0603154 a2, which provides an electrode composite prepared as follows: thoroughly mixing the oxides and/or hydroxides of the elements of period 4 of the periodic table with graphite and a binder, resulting in H of the anode₂O₂Overvoltage reduction for oxidation>200 mV。

Electrically conductive electrocatalysts, such as carbon nanotubes, are known, in addition to the electrically non-conductive and/or semi-conductive metal oxides usually introduced into electrode complexes, which, due to their small volume, can be arranged in the vicinity of the repair center (prototypical center) of the enzyme, have a high electrical conductivity and achieve efficient transfer of electrons [ Wang et al, analysis (2003), 128, 1382-. Due to their high surface area, small amounts of nanotubes are sufficient to achieve a reduction in decomposition potential US 2006/0021881 a 1.

In order to provide a constant enzymatic activity, different measures are known in the art. One possibility to avoid diffusion of the enzyme from the surface of the working electrode into the environment is to provide the working electrode with a suitable coating, for example a cover film. However, the use of such coatings in electrochemical sensors is associated with certain problems, such as the necessity of depositing pinhole-free films. Second, for mass transfer limited systems, the capping film must be deposited in a highly reproducible layer thickness. This requirement represents a broad limitation of possible coatings, since it is difficult to achieve very thin layers that exhibit a sufficiently high barrier to mass transfer.

In addition, electrochemical sensors for the determination of different analytes must usually also contain different cover membranes in order to provide different mass transfer rates of the substrate and the co-substrate to the electrodes. At the same time, it must be ensured that the mulch is highly biocompatible for in vivo applications. Since even the smallest defects in the membrane are sufficient to cause leakage of the enzyme from the electrode into the environment, a huge amount of quality control is necessary, especially in the case of in vivo biosensors, leading to considerable technical requirements and increased production costs.

Alternatively, by immobilizing the enzyme in the electrode matrix of the working electrode, the extent of enzyme leakage can be reduced, which has led to a focused search for suitable methods of enzyme immobilization in electrochemical biosensors. In practice, the enzyme may be coupled to one or more of the slurry components chemically covalently, or physically inserted into the complex such that the enzyme is adsorptively bound to, and/or incorporated into, one or more of the slurry components.

Regarding adsorptive immobilization, Rege et al [ Nano Letters (2003), 3, 829-832] disclose an electrode composite comprising single-walled carbon nanotubes (SWCNTs) and/or graphite (as conductive fillers) and PMMA (as binders), wherein chymotrypsin is physically retained. In this context, it was found that composites containing SWCNTs showed less enzyme leakage than composites containing graphite, which seems to be due to the higher surface energy of SWCNTs than graphite.

Tang et al [ Analytical Biochemistry (2004), 331, 89-97] demonstrated that by using a CNT-electrode on which Pt particles are electrochemically deposited and which is sorptively modified with glucose oxidase, the long-term stability of glucose oxidase can be significantly increased compared to conventional graphite electrodes.

Tsai et al Langmuir (2005), 21, 3653-. Immobilization of the matrix refers to the electrostatic interaction between negatively charged MWCNT and Nafion (on the one hand) and positively charged glucose oxidase (on the other hand).

Guan et al [ Biosensors and Bioelectronics (2005), 21, 508-. Increased linearity of the response curve is observed and is due to the increased electron transfer rate of MWCNTs compared to conventional carbon electrodes.

Finally, Kurusu et al [ Analytical Letters (2006), 39, 903-911-]It is disclosed that the use of an electrode comprising a mixture of MWCNT, GOD and mineral oil results in H₂O₂A significant reduction in oxidative decomposition voltage.

However, the adsorption immobilization operation has many problems. One major drawback of physical immobilization is the dependence of the binding constant on the composition of the medium surrounding the electrode, which requires a barrier membrane to prevent enzyme leakage.

In particular, however, physical coupling has a great need for reproducibility of the active surface of the working electrode (and thus for its production). As mentioned above, adsorptive immobilization requires either application of a solution and/or suspension containing the enzyme to the surface of a pre-fabricated working electrode or introduction of a solution and/or suspension containing the enzyme into an electrode complex. However, the dispensing application of solutions and/or suspensions containing enzymes on the surface of the working electrode has the following disadvantages: the addition of small volumes (e.g.in the nanoliter range) of enzyme solution places high demands on the accuracy of the automated metering device. In addition, the distribution of the enzyme on the surface of the working electrode and the transfer of the enzyme into the pores of the working electrode depend on the topography (topograph) and energy of the electrode surface, which are difficult to reproduce.

In view of the above, it is preferable to mix an enzyme into the electrode complex. However, loss of effective enzyme activity by cleavage, solvent effects and thermal effects cannot be avoided due to the need for uniform distribution of the enzyme in the predominantly hydrophobic complex. Furthermore, since the deposition of the electrode slurry requires specific rheological characteristics, limitations with respect to overall slurry conductivity and adhesion on the substrate must be considered, while a constant slurry consistency must be provided after the dry enzyme is homogeneously distributed in the composite.

As an alternative, the enzyme may be introduced in a complex in aqueous solution or suspension in order to minimize denaturation. However, the necessity arises for later removal of the solvent or suspending agent, so that the complex does not presumably have constant rheological characteristics during production.

Therefore, in view of the drawbacks of adsorptive immobilization, there is thus a particular need for immobilizing enzymes in electrochemical biosensors by covalently bonding at least one component of an electrode matrix.

EP 0247850 a1 discloses a biosensor for amperometric detection of an analyte. These sensors contain an electrode with an immobilized enzyme immobilized or adsorbed on the surface of an electrically conductive support, wherein the support consists of or contains a platinum-plated porous layer of resin-bound carbon or graphite particles. For this purpose, electrodes made of platinum-plated graphite and polymeric binders are first prepared and subsequently exposed to enzymes. In this case, the enzyme is immobilized by adsorption on the electrode surface, or by coupling it to a polymeric binder using a suitable reagent.

Also in EP 0603154 a2, amperometric biosensors with electrodes comprising an enzyme immobilized or adsorbed on or in an electrically conductive, porous electrode material are described. To produce the enzyme electrode, the catalyst-acting oxide or oxide hydrate of the 4 th-cycle transition metal (e.g. manganese dioxide) is brought together with graphite and a non-conductive polymeric binder into a slurry and, in a second step, the porous electrode material obtained after drying the slurry is contacted with the enzyme. The enzyme may be immobilized on or within the porous electrode material by using a cross-linking agent such as glutaraldehyde.

One major disadvantage of the electrochemical biosensors described in EP 0247850 a1 and EP 0603154 a2 is that the enzyme is first immobilized on an already prefabricated, enzyme-free electrode. As a result, there is a problem in that the enzyme cannot be coupled to the electrode component in a controlled manner. Thus, when glutaraldehyde is used as a cross-linking agent, the enzyme not only binds any reactive components of the electrode material in an uncontrolled manner, but also cross-links with each other. Furthermore, this operation can contaminate the electrodes with the reagents used, and therefore the electrodes must be thoroughly cleaned again, particularly before use in an in vivo biosensor, which increases production complexity and thus costs.

Cho et al (Biotechnology and Bioengineering (1977), 19, 769-]Immobilization of enzymes on activated carbon by covalent coupling is described. More specifically, immobilization of glucose oxidase on petroleum-based activated carbon is described as follows: (a) adsorbing the enzyme and then cross-linking with glutaraldehyde, or (b) activating the carbon surface with diimide followed by reaction with the enzyme. In this way, it was confirmed that H is present in comparison with the soluble enzyme₂O₂A significantly slower inactivation of the enzyme in the presence.

Li et al [Analytical and Bioanalytical Chemistry (2005), 383, 918-922]A glucose biosensor is disclosed which comprises a modified glassy carbon electrode as the working electrode. The improved glassy carbon electrode was prepared as follows: the surface of a commercial electrode was coated with functionalized multi-walled carbon nanotubes (MWCNTs, having oxidized glucose oxidase covalently attached thereto) in a solution containing Nafion^®And ferrocene monocarboxylic acid in PBS buffer. The catalytic action of the functionalized nanotubes on glucose oxidation is particularly emphasized.

US 2007/0029195 a1 discloses the immobilization of biomolecules, such as proteins, by covalent coupling to a conductive polymeric matrix reinforced with nanoparticles to improve mechanical stability, electrical conductivity and immobilization of biomolecules. The matrix is a nanocomposite comprising polypyrrole coated gold nanoparticles formed from pyrrole and pyrrole propylic acid, wherein the pyrrole propylic acid provides covalent attachment of biomolecules.

US 2008/0014471 a1 discloses an electrochemical sensor comprising an electrode employing a cluster of cross-linked enzymes immobilized on Carbon Nanotubes (CNTs). In detail, the immobilization includes: functionalization of the nanotube surface, followed by covalent attachment of an enzyme such as glucose oxidase with the aid of a linking reagent to produce a CNT-enzyme conjugate, precipitation of the free enzyme with a precipitating agent, and finally treatment with a cross-linking agent to form a cross-linked enzyme cluster, which is covalently attached to the CNTs by the CNT-enzyme conjugate.

US 2008/044911 a1 discloses a glucose sensor comprising nanowires (nanowires) having glucose oxidase covalently attached to their surface, the functionalized nanowires being prepared by: by contacting conventional nanowires with a linking agent (e.g., silane) and an enzyme. The conversion of glucose in the sample to be examined by glucose oxidase immobilized on the surface of the nanowires results in a change in the pH of the test solution, wherein the pH change generates a signal in the nanowires, which signal can be detected by suitable means.

It is known in the art that covalent coupling of enzymes to supports (e.g.MANAE-agarose, activated glyoxyl-agarose and glutaraldehyde agarose) leads to enzyme stabilization against thermal decomposition [ Betancor et al, Journal of Biotechnology (2006), 121, 284-289)]. However, in addition to thermal decomposition, biosensors employing such immobilized enzymes are also confronted with problems caused by organic solvents during storage of the biosensors or by, for example, H during handling₂O₂And the like, and the enzyme decomposition caused by the oxidizing agent.

In practice, biosensors must meet a number of requirements to allow accurate measurement of immediate or time-delayed therapeutic measures. In particular, it is of utmost importance that the target analyte can be determined with high specificity and sensitivity in order to determine low amounts of clinically relevant parameters. As a result, the significant loss of enzyme activity typically observed in commercially available biosensors is unacceptable for diagnostic and/or clinical purposes when stored for more than 1 month.

It is therefore an object of the present invention to provide a composition for forming an electrode, wherein the disadvantages of the prior art are at least partially eliminated. In particular, the composition will ensure specific and durable immobilization of the enzyme, prevent or at least reduce loss of enzyme activity after production of the electrode complex, preserve for a period of more than 1 month as well as biosensor function, and ensure high sensitivity throughout the working period.

This object is achieved according to the invention by means of a composition for forming an electrode, comprising an electrically conductive component having an analyte-specific enzyme covalently linked thereto, wherein the composition additionally comprises at least one electrically non-conductive or semi-conductive enzyme stabilizing component.

According to the present invention, the composition must comprise an electrically conductive component having covalently attached thereto an analyte-specific enzyme. The term "electrically conductive component" as used within this application denotes any such arbitrary componentSubstance (b): it contains freely movable charges and has a rho<10^-4Ω cm, thus allowing the transport of current. The electrically conductive substance may be an electronic conductor (primary conductor) or an ionic conductor (secondary conductor), with an electronic conductor being preferred.

Preferably, the electrically conductive component is H₂O₂-a decomposition (decomposition) catalyst. This means that the electrically conductive component not only transports electrical current, but is additionally capable of catalyzing the decomposition of hydrogen peroxide that is present or formed in the sample that contacts the electrically conductive component. The decomposition of hydrogen peroxide in biochemical test elements is particularly important since hydrogen peroxide often causes the destruction of most enzymes used in the detection of clinically relevant analytes and may additionally act as an inhibitor of the analyte or of a synergistic substrate, such as oxygen. To this end, the electrically conductive component may, for example, catalyze the chemical conversion of hydrogen peroxide to a chemically less active or inert substance, including the oxidation of hydrogen peroxide to water.

In another preferred embodiment of the present invention, the electrically conductive component is selected from the group consisting of: activated carbon, carbon black, graphite, carbon-containing nanotubes, palladium, platinum and hydroxides of iron oxides such as feo (oh), with graphite and carbon-containing nanotubes being particularly preferred.

The term "carbon-containing nanotubes" as used herein denotes all kinds of nanotubes, i.e. tubes with an average inner diameter <1 μm, which contain carbon as one of their components. In particular, carbon-containing nanotubes within the meaning of the present invention include Carbon Nanotubes (CNTs), which may be in the form of, for example, single-walled carbon nanotubes (SWCNTs), double-walled carbon nanotubes (DWCNTs), multi-walled carbon nanotubes (MWCNTs), and the like. Since nanotubes generally provide a highly efficient surface, they can be sufficiently modified with appropriate substances to be immobilized on their surface (e.g. enzymes employed in the detection of analytes).

In another embodiment, the composition according to the invention may comprise at least one additional electrically conductive component in addition to the electrically conductive component having the analyte-specific enzyme covalently linked thereto. The additional electrically conductive component may be any substance capable of transporting an electric current and is preferably selected from: activated carbon, carbon black, graphite, carbon-containing nanotubes, palladium, platinum and iron oxide hydroxides. In principle, the component can also form a conjugate with the analyte-specific enzyme by means of a covalent bond, but preferably is not covalently linked to the analyte-specific enzyme.

According to the invention, the electrically conductive component has an analyte-specific enzyme covalently linked thereto. The enzyme may be any enzyme specific for the analyte to be detected and suitable for achieving the desired effect by the person skilled in the art. The enzyme immobilized on the electrically conductive component is preferably an oxidase, in particular an alcohol oxidase (1.1.3.13), an aryl alcohol oxidase (EC 1.1.3.7), a catechol oxidase (EC 1.1.3.14), a cholesterol oxidase (EC1.1.3.6), a choline oxidase (1.1.3.17), a galactose oxidase (EC 1.1.3.9), a glucose oxidase (EC1.1.3.4), a glycerol-3-phosphate oxidase (EC 1.1.3.21), a hexose oxidase (EC 1.1.3.5), a malate oxidase (EC 1.1.3.3), a pyranose oxidase (EC 1.1.3.10), a pyridoxine-4-oxidase (EC 1.1.3.12), or a thiamine oxidase (EC 1.1.3.23). More preferably, the enzyme is glucose oxidase.

Preferably, the analyte-specific enzyme is selectively covalently bound to the electrically conductive component. Covalent binding of the enzyme to the electrically conductive component ensures a constancy of the function of the electrode comprising the composition of the invention, since detachment of the enzyme can be excluded under typical measurement conditions (physiological electrolyte concentration, physiological pH, body temperature). Thus, an electrochemical sensor comprising such electrodes can operate over a long period of time and operate substantially without drift.

In order to covalently bind the analyte-specific enzyme to the electrically conductive component, in a preferred embodiment, the invention foresees that the electrically conductive component has a surface comprising a functional group to which the enzyme is bound. The surface may, for example, exhibit hydroxyl, carboxyl or amino functionality. Alternatively, the surface may be functionalized as follows: coating the electrically conductive component with a reagent adapted to form functional groups, whereby the enzyme can be covalently bound to the surface of the electrically conductive component.

Coating agents used within the scope of the invention are substances which: on the one hand, it is covalently bound to the electrically conductive component and, on the other hand, it contains at least one functional group for covalently binding an enzyme. This means that the coating agent is at least bifunctional, i.e. comprises at least 2 functional groups, which may be the same or different.

In a preferred embodiment, the enzyme is covalently bound directly to the surface of the electrically conductive component, which means that at least one covalent bond is formed between a functional group of the enzyme and a functional group on the surface of the electrically conductive component. The enzyme may be coupled to the surface in any manner and may comprise functional groups and/or enzymes that are pre-activated on the surface of the electrically conductive component.

The functional groups may be activated by, for example, reacting the functionalized electrically conductive component and/or enzyme with a suitable activator. Preferred activators include carbodiimides such as Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and combinations of carbodiimide and succinimide. Activators particularly suitable for the purposes of the present invention include combinations of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS).

Other Methods suitable for covalently binding, particularly selectively covalently binding, analyte-specific enzymes to the electrically conductive component are known to those skilled in the art and are described, for example, in H.Weetall, Methods of Enzymology (1976), 33, 134-.

According to the invention, the composition additionally comprises: an electrically non-conductive or semi-conductive enzyme stabilizing component. The term "electrically non-conductive component" as used within this application denotes a component having ρ>10⁹Ω cm and is not capable of transporting any substance of electric current. In contrast, the term "electrically semi-conductive component" as used herein means having a conductivity of 10^-4<ρ<10⁹Ω cm resistivity. The term "enzyme-stabilized" means that the non-conductive or semi-conductive component is capable of stabilizing an analyte-specific enzyme, which is achieved by: the enzyme is broken down by reaction with an analyte-specific enzyme, formation of a complex or conjugate with the enzyme, or by conversion of a chemical.

In a preferred embodiment, the electrically non-conductive or semi-conductive enzyme stabilizing component is H₂O₂A decomposition catalyst, which means that the electrically non-conductive or semi-conductive enzyme stabilizing component decomposes hydrogen peroxide, e.g. by chemical conversion, thereby preventing damage to the analyte-specific enzyme employed in the composition according to the invention.

The electrically non-conductive or semi-conductive enzyme stabilizing component is preferably selected from the group consisting of: h₂O₂-degrading metal oxide and H₂O₂-a degrading enzyme. For the purposes of the present invention, said H₂O₂The degrading metal oxides may be any metal oxides capable of catalyzing the decomposition of hydrogen peroxide, for example by oxidizing them, where Ag₂O、Al₂O₃Or the metal oxide of period 4 of the periodic table has proven particularly advantageous. The oxide of the metal of the 4 th period of the periodic Table is preferably an oxide of Mn, CuO or ZnO, wherein MnO is₂、Mn₃O₄Or Mn₅O₈Is particularly preferred. H₂O₂The degrading enzyme may be one known to decomposeAny enzyme that oxidizes hydrogen specifically includes peroxidases and catalases.

In another preferred embodiment, the composition according to the invention additionally comprises an analyte-specific enzyme covalently linked to an electrically non-conductive or semi-conductive enzyme stabilizing component. This means that the analyte-specific enzyme employed in the composition of the invention may be covalently linked not only to the electrically conductive component, but additionally to an electrically non-conductive or semi-conductive enzyme-stabilizing component by means of covalent bonds formed between the enzyme and, for example, functional groups located on the surface of the electrically non-conductive or semi-conductive enzyme-stabilizing component.

According to the invention, the electrically conductive component and/or the electrically non-conductive or semi-conductive enzyme-stabilizing component can be provided in particulate form, wherein the particle size can be varied according to the respective requirements. In a preferred embodiment, the composition according to the invention comprises an electrically conductive component and/or an electrically non-conductive or semi-conductive enzyme stabilizing component in the form of nanoparticles (i.e. particles having an average diameter <1 μm). Within the scope of the present invention, 90% of the nanoparticles generally have a diameter of from 10 nm to 100 nm, diameters of from 15 nm to 30 nm having proven particularly preferred.

The ability to control the electrically conductive component and/or the electrically non-conductive or semi-conductive enzyme stabilizing effective surface of the component by means of particle size is particularly important, for example, in connection with functionalization with chemicals. More specifically, a more efficient surface may increase the loading of the enzyme and, thus, may result in higher enzyme activity, expressed as: units per mg enzyme-loaded fraction. The electrically conductive component for the purposes of the present invention generally has an enzymatic activity of from about 10 mU/mg to about 5U/mg, an enzymatic activity of from about 100 mU/mg to about 1U/mg having proven particularly advantageous. The term "unit" as used within the scope of the present application denotes the amount of enzyme required to convert 1 μmol of substrate per minute under standard conditions.

In addition to the electrically conductive component and the electrically non-conductive or semi-conductive enzyme stabilizing component, the composition according to the present invention may include other components conventionally used to form electrodes, for example, binders, fillers, and the like. With respect to the binder, the composition preferably comprises at least one binder selected from the group consisting of: fluorinated hydrocarbons such as Teflon^®Polycarbonate, polyisoprene, polyurethane, acrylic, polyvinyl and silicone, with polyurethane, acrylic and polyvinyl being more preferred.

To form the electrode, the electrically conductive component having the analyte-specific enzyme covalently attached thereto, the electrically non-conductive or semi-conductive enzyme-stabilizing component, and all other components required to form the electrode matrix are thoroughly mixed and then dried such that the electrically conductive component and/or the electrically non-conductive or semi-conductive enzyme-stabilizing component are preferably uniformly dispersed in the composition.

Depending on the specific requirements of the electrode to be formed, the skilled person is able to determine without any difficulty the amounts of the different components required to provide the composition according to the invention. However, with respect to the electrically conductive component, it has proved advantageous when the composition contains a conjugate formed by the electrically conductive component and the analyte-specific enzyme in an amount of 0.5-10% (by weight, based on the dry weight of the composition). On the other hand, the composition preferably contains an electrically non-conductive or semi-conductive enzyme stabilizing component in an amount of 5 to 50% (by weight, based on the dry weight of the composition).

The compositions described herein will significantly reduce the loss of activity of the analyte-specific enzyme required for detection of the target analyte by means of a biosensor, such as an electrochemical sensor, comprising an electrode using a composition according to the invention. In particular, after storage of the composition at a temperature of 4 ℃ for at least 4 weeks, preferably at least 12 weeks, more preferably at least 28 weeks, the enzyme has a residual activity of at least 90% in the composition according to the invention, based on the total enzyme activity prior to storage, i.e. based on the total enzyme activity in the composition immediately after preparation is complete.

In another aspect, the present invention thus relates to an electrochemical sensor for the determination of an analyte, said sensor comprising at least one working electrode and at least one reference electrode, wherein the working electrode comprises a composition according to the present invention.

Although the working electrode of the electrochemical sensor according to the invention is used for converting the analyte to be determined, the reference electrode allows the polarization potential of the working electrode to be adjusted and may consist of any substance suitable for the purpose of the invention. Metal/metal ion electrodes, in particular silver/silver chloride electrodes, are preferably used as reference electrodes. In addition to the at least one working electrode and the at least one reference electrode, the electrochemical sensor may also comprise at least one counter electrode, which is preferably in the form of a noble metal electrode, in particular a gold electrode or a graphite electrode.

According to the present invention, the electrochemical sensor preferably comprises a biocompatible coating covering at least one working electrode, at least one reference electrode and optionally a counter electrode. The biocompatible coating allows analyte to permeate the electrode matrix, but prevents the electrode components from entering the surrounding medium containing the target analyte.

In view of the fact that the enzyme does not flow out of the working electrode or electrochemical sensor due to its covalent binding to the electrically conductive component, a biocompatible coating is not absolutely necessary for many applications. Thus, the electrochemical sensor according to the invention can also be used for in vivo applications when the biocompatible coating is not an enzyme barrier. In such cases, a biocompatible coating may be selected that provides optimal interaction with surrounding tissue and/or blood or serum.

Biocompatible coatings can be prepared in different ways. One preferred method is to use a pre-processed membrane, which is applied to the electrochemical sensor. The membrane can be fixed on the sensor by different techniques, while adhesive or laser welding are considered to be preferred. Alternatively, the biocompatible coating can be prepared in situ, wherein a solution of a suitable polymer is applied to the electrochemical sensor, followed by drying it. Preferably, the polymer is applied to the biosensor by spraying, dip coating or dispersing a dilute solution of the polymer in a low boiling point organic solvent, but is not limited to these methods.

Polymers suitable for these purposes include in particular: polymers with zwitterionic structure and mimicking cell surface, such as 2-methacryloyloxyethyl-phosphorylcholine-co-n-butyl-methacrylate (MPC-co-BMA). The resulting biocompatible coating generally has a thickness of from about 1 μm to about 100 μm, preferably from about 3 μm to about 25 μm.

The electrochemical sensor according to the invention is preferably designed for multiple measurements, i.e. the sensor is capable of repeatedly measuring the analyte to be determined. This is particularly desirable in applications where it is desirable to control the presence and/or amount of analyte constantly (i.e. continuously or discontinuously) over a long period of time (e.g. 1 day or more, especially 1 week or more). In a particularly preferred embodiment, the electrochemical sensor according to the invention can thus be implanted completely or partially, and can be implanted, for example, in adipose tissue or in blood vessels. Alternatively, the present invention allows the electrochemical sensor to be designed as a flow cell through which a fluid sample containing the analyte passes.

The electrochemical sensors described herein may be used to determine an analyte in a fluid medium, which may be derived from any source. In a preferred embodiment, the electrochemical sensor is used to determine an analyte in a bodily fluid including, but not limited to, whole blood, plasma, serum, lymph, bile, cerebrospinal fluid, extracellular tissue fluid, urine, and glandular secretions such as saliva or sweat, with whole blood, plasma, serum, and extracellular tissue fluids being considered particularly preferred. The amount of sample required to perform the assay is generally from about 0.01. mu.l to about 100. mu.l, preferably from about 0.1. mu.l to about 2. mu.l.

The analyte to be determined qualitatively and/or quantitatively may be any biological or chemical substance to be detected by means of a redox reaction. The analyte is preferably selected from: malic acid, alcohol, ammonium, ascorbic acid, cholesterol, cysteine, glucose, glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid, 5' -nucleotidase, peptides, pyruvate, salicylate, and triglycerides. In a particularly preferred embodiment, the analyte to be determined by means of the electrochemical sensor according to the invention is glucose.

In another aspect, the present invention relates to a method for determining an analyte, the method comprising the steps of:

(a) contacting a sample containing an analyte with an electrochemical sensor according to the invention, and

(b) determining the presence and/or amount of the analyte.

For the determination of an analyte, the electrochemical sensor may be designed in any way that allows contact between the electrochemical sensor and a sample containing the analyte. Thus, the sensor may be designed as a flow cell through which the medium containing the analyte flows. On the other hand, the sensor can also be designed as a diffusion sensor, wherein the contact between the sensor and the medium takes place by diffusion. Likewise, the electrochemical sensor may be designed as a device intended to be implanted wholly or partially in the body of a patient, in which case it is implanted in a blood vessel or in tissue, in particular subcutaneous adipose tissue.

A measurable signal is generated by the sensor based on the presence and/or amount of the analyte. The signal is preferably an electrical signal, such as a current, voltage, resistance, etc., which is evaluated or read using a suitable device. The electrochemical sensor is preferably an amperometric sensor.

The figures and examples below are intended to further illustrate the invention.

Drawings

FIG. 1: with respect to time [ s]Plotted measurement signals [ nA ] of the electrochemical sensor according to the invention]Dependent on the glucose concentration [0, 2, 4, 6, 8, 12, 17, 23, 26, 20, 15, 10, 7, 5, 3, 0.8mM ]]The sensor employs a working electrode comprising a conjugate formed of carbon nanotubes and glucose oxidase (CNT-GOD; 1.75% by weight) and MnO₂(20% by weight) as H of semiconductivity₂O₂-a decomposition catalyst. The polarization voltage is 350 mV.

FIG. 2: measurement Signal [ nA/mM ] of an electrochemical sensor according to the invention stored at a temperature of 4 ℃ for a period of 28 weeks]The sensor employs a working electrode comprising a conjugate formed of carbon nanotubes and glucose oxidase (CNT-GOD; 2.4% by weight) and MnO₂(19.5% by weight) as H which is semiconductive₂O₂-a decomposition catalyst.

FIG. 3: in MES buffer (pH 7.4) in the presence of H₂O₂(5% solution) in MES buffer, and in MES buffer containing H₂O₂(5% solution) and 500U/ml Catalase in MES buffer, the enzymatic Activity of the conjugate formed by carbon nanotubes and glucose oxidase (CNT-GOD) [ mU/mg lyophilized conjugate]。

FIG. 4: h caused by different electrically conductive and electrically non-conductive or semi-conductive substances₂O₂Decomposition Rate [ mM/mg/h]。

4A H from different commercially available carbons₂O₂Decomposition Rate [ mM/mg/h]。

4B H from different commercially available metal oxides₂O₂Decomposition Rate [ mM/mg/h]。

4C H resulting from 2 different manganese oxides₂O₂Decomposition Rate [ mM/mg/h]。

FIG. 5: the measurement signals [ nA ] of the different electrochemical sensors plotted against increasing glucose concentrations [ mg/dl ]. The polarization voltage is 350 mV.

5A: measurement signals [ nA ] of 8 electrochemical sensors employing working electrodes comprising conjugates formed of carbon nanotubes and glucose oxidase (CNT-GOD; 2% by weight).

5B: measurement signals [ nA ] of 8 electrochemical sensors according to the invention]The sensor employs a working electrode comprising a conjugate formed of carbon nanotubes and glucose oxidase (CNT-GOD; 2% by weight) and MnO₂(20% by weight) as H of semiconductivity₂O₂-a decomposition catalyst.

FIG. 6: schematic representation of an electrochemical sensor according to the invention.

Examples

Example 1: preparation of conjugates formed from carbon nanotubes and glucose oxidase (CNT-GOD)

To prepare an electrically conductive component with an enzyme covalently attached thereto, 2.5 g of carbon nanotubes (CNT, Nanolab) were added to a solution of 480 mg of glucose oxidase (GOD, Roche), 9.6 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC, Sigma) and 7.2 g N-hydroxysuccinimide (NHS, Sigma) in 480 ml of Millipore water. After incubating the mixture at room temperature for 6 hours on a laboratory shaker, the conjugate was separated from the reaction solution by passing the mixture through a membrane filter. The conjugate was washed with PBS buffer (3X 120 ml), Millipore water (1X 120 ml) and dried under vacuum overnight to give 3.0 g of CNT-GOD conjugate.

Example 2: determination of enzymatic Activity of CNT-GOD conjugates

To determine the enzymatic activity of the CNT-GOD conjugate prepared in example 1, 5 mg of lyophilized conjugate were suspended in 1 ml of MES buffer and stirred at room temperature for 1 hour. In parallel, a Toyobo solution (GLO-201; 30 ml MES buffer (79 mM); 6 ml glucose solution (131 mM); 0.3 ml 4-aminoantipyrine solution (0.2 mM); 0.3 ml N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine solution (0.3 mM); 0.3 ml peroxidase solution (about 4U/ml)) was pipetted into a cuvette and buffered at 37 ℃ for 10 minutes. After the conjugate was added to the buffered Toyobo solution, the absorption of the mixture thus obtained was measured using a wavelength of 555 nm. By this means, the enzyme activity was found to be 870 mU/mg conjugate.

Example 3: using a composition comprising a CNT-GOD conjugate and H₂O₂Stability of electrochemical sensors of working electrodes of decomposition catalysts

To determine the stability of the electrochemical sensor according to the invention, a working electrode was prepared comprising 78.25% (by weight) of electrode slurry (Acheson), 1.75% (by weight) of the CNT-GOD conjugate of example 1 and 20% (by weight) of MnO₂(Merck) electrode matrix.

Subsequently, the electrochemical sensor was contacted with a solution containing glucose at concentrations of 0, 2, 4, 6, 8, 12, 17, 23, 26, 20, 15, 10, 7, 5, 3, and 0.8mM, which were periodically varied over a 5 day period. As can be seen from FIG. 1, the electrochemical sensor did not show any loss of sensitivity at all stages, which is prevention of enzyme leakage due to covalent binding to carbon nanotubes and by MnO₂Catalytic H₂O₂The result of the decomposition.

Example 4: by the inclusion of CNT-GOD conjugates and H₂O₂Long term stability of electrochemical sensors of working electrodes of decomposition catalysts

To determine the long-term stability of the electrochemical sensor according to the invention, a working electrode was prepared comprising a mixture of 78% (by weight) of an electrode slurry (Acheson), 2.4% (by weight) of the CNT-GOD conjugate of example 1 and 19.5% (by weight) of MnO₂(Merck) electrode matrix.

Subsequently, the electrochemical sensor was stored at a temperature of 4 ℃ for a period of 28 weeks. As can be seen from fig. 2, the measurement signal of the electrochemical sensor remained unchanged between the 4 th week and the 28 th week after its preparation. The loss of sensitivity between weeks 0 and 4 is due to the modulating effect of the electrode matrix.

Example 5: comprising CNT-GOD conjugates and H₂O₂Determination of the enzymatic Activity of the catalyst decomposing composition

To determine the enzymatic activity of the compositions according to the invention, 10 mg of lyophilized CNT-GOD-conjugate were suspended in 1 ml of MES buffer (pH 7.4) and incubated for 1 hour at room temperature.

Subsequently, a commercially available H was used₂O₂Assay (Toyobo), measuring the residual enzymatic activity of the CNT-GOD conjugate, determined to be about 600 mU/mg of lyophilized conjugate (see figure 2). In parallel, it is determined that₂O₂Residual enzymatic activity of CNT-GOD conjugate in MES buffer (5% solution), and in MES buffer containing H₂O₂(5% solution) and residual enzyme activity of CNT-GOD in 500U/ml catalase MES buffer.

As can be seen from FIG. 3, when H is present in the sample₂O₂Time (CNT-GOD + H)₂O₂) The residual enzymatic activity of the CNT-GOD conjugate was reduced to less than 100 mU/mg of lyophilized conjugate. On the other hand, H₂O₂Solutions ofAnd H₂O₂Addition of the decomposition catalyst catalase, resulting in a residual enzymatic activity (CNT-GOD + H) of about 650 mU/mg of lyophilized conjugate₂O₂+ catalase), indicating H₂O₂Complete inhibition of GOD denaturation.

Example 6：H₂O₂Determination of the decomposition catalyst

To measure H₂O₂Compounds acting as decomposition catalysts, already in relation to their effective catalysis H₂O₂The ability to decompose, a number of carbons and metal oxides were evaluated.

In detail, Carbon Black Acetylene (Strem Chemicals), Spheron 6400(Cabot Corporation), Mogul E (Cabot Corporation), Carbon Black Acetylene (Alfaaesar), Mogul L (Cabot Corporation), Nanofibres (electrovac), Vulcan Black-605 (Cabot Corporation), Black Pearls2000 (Cabot Corporation; ground for 60min), Black Pearls2000 (Cabot Corporation; ground for 120 min) and nanotubes (Nanolab) were tested as electrically conductive Carbon substances. In this context, carbon nanotubes (Nanolab) and Black Pearls2000 (Cabot Corporation; ground for 120 min) proved to be particularly suitable, providing H at 0.00126 and 0.00098 mM/mg catalyst/H, respectively₂O₂Decomposition rate (see fig. 4A).

With respect to the metal oxides, it is apparent from fig. 4B and 4C that the different compounds employed in the experiments provided very different decomposition rates. However, commercially available Al₂O₃(Sigma-Aldrich, product number 229423), Al₂O₃(Aldrich, product No. 551643) and FeO (OH) (Fluka, product No. 71063) are rather poor catalyzers, Ag₂O (Riedel-de Ha ë n, product No. 10228) and Mn₃O₄(Strem Chemicals, product No. 93-2513) proved to be a very effective H₂O₂Decomposition catalyst (see fig. 4B).

Another very effective catalyst for H₂O₂The decomposed compound was Mn obtained as follows₅O₈: heating commercially available Mn at 25 deg.C-430 deg.C₃O₄(Strem Chemicals, product No. 93-2513) for 6 hours, the temperature was maintained for 12 hours, followed by cooling to 25 ℃. As can be seen from FIG. 4C, in the presence of Mn obtained by the above-mentioned method₅O₈In the presence of H observed₂O₂The decomposition rate is obviously better than that of Mn₃O₄The catalytic action of (1).

Example 7: using a composition comprising a CNT-GOD conjugate and H₂O₂Reduction of overpotential in electrochemical sensors with working electrodes for decomposition of catalysts

To determine the effect of an electrically non-conductive or semi-conductive enzyme-stabilizing component on the overvoltage observed at the working electrode of an electrochemical sensor, 8 electrochemical sensors employing working electrodes comprising 2% (by weight) of the CNT-GOD conjugate of example 1, and 8 electrochemical sensors employing working electrodes comprising 2% (by weight) of the CNT-GOD conjugate of example 1 and 20% (by weight) of MnO were prepared₂(Merck) electrochemical sensor with a working electrode.

As can be seen from FIG. 5A, the use of a working electrode comprising only the CNT-GOD conjugate resulted in a low sensitivity of 0.01 + -0.03 nA/mM glucose solution, where a polarization voltage of 350 mV (vs. Ag/AgCl reference electrode) was applied and a current dependent on the glucose concentration was measured.

In contrast, under the same reaction conditions, with MnO₂The application of the working electrode in combination with the CNT-GOD conjugate resulted in a significantly increased sensitivity of the 0.54 ± 0.11 nA/mM glucose solution, indicating a significant reduction of the overvoltage at the working electrode (see fig. 5B).

Claims (17)

1. A composition for forming an electrode, the composition comprising an electrically conductive component having an analyte-specific enzyme covalently attached thereto,

it is characterized in that the preparation method is characterized in that,

the composition additionally comprises at least one electrically non-conductive or semi-conductive component that stabilizes the analyte-specific enzyme by converting chemical species that cause enzymatic breakdown, the analyte-specific enzyme being selectively covalently bound to the electrically conductive componentThe electrically conductive component is of ρ<10^-4Omega cm, a substance that allows the transport of electric current, and is provided in the form of nanoparticles,

the electrically non-conductive or semi-conductive enzyme stabilizing component is H₂O₂-a decomposition catalyst.

2. The composition according to claim 1, wherein the composition,

it is characterized in that the preparation method is characterized in that,

the electrically conductive component is H₂O₂-a decomposition catalyst.

3. The composition according to claim 1 or 2,

it is characterized in that the preparation method is characterized in that,

the electrically conductive component is selected from: activated carbon, carbon black, graphite, carbon-containing nanotubes, palladium, platinum, and hydroxides of iron oxide, in particular selected from: graphite and carbon-containing nanotubes.

4. The composition according to any one of claims 1 to 3,

it is characterized in that the preparation method is characterized in that,

the enzyme is an oxidase, in particular glucose oxidase.

5. The composition according to any one of claims 1 to 4,

it is characterized in that the preparation method is characterized in that,

the electrically non-conductive or semi-conductive enzyme stabilizing component is selected from the group consisting of: h₂O₂Degradation of metal oxides, in particular Ag₂O、Al₂O₃Or oxides of metals of period 4 of the periodic Table and H₂O₂Degrading enzymes, in particular peroxidases or catalases.

6. The composition of claim 5, wherein the composition is,

it is characterized in that the preparation method is characterized in that,

the oxide of the metal of period 4 of the periodic Table is an oxide of Mn, especially MnO₂、Mn₃O₄Or Mn₅O₈CuO or ZnO.

7. The composition according to any one of claims 1 to 6,

it is characterized in that the preparation method is characterized in that,

it additionally comprises: an analyte-specific enzyme covalently linked to an electrically non-conductive or semi-conductive enzyme stabilizing component.

8. The composition according to any one of claims 1 to 7,

it is characterized in that the preparation method is characterized in that,

the electrically conductive component and/or the electrically non-conductive or semi-conductive enzyme-stabilizing component is provided in the form of nanoparticles.

9. The composition according to any one of claims 1 to 8,

it is characterized in that the preparation method is characterized in that,

it additionally comprises at least one binder selected from: fluorinated hydrocarbons, polycarbonates, polyisoprenes, polyurethanes, acrylics, polyvinyls, and siloxanes.

10. The composition according to any one of claims 1 to 9,

it is characterized in that the preparation method is characterized in that,

the at least one binder is selected from: polyurethane, acrylic and polyethylene resins.

11. The composition according to any one of claims 1 to 10,

it is characterized in that the preparation method is characterized in that,

the electrically conductive component and/or the electrically non-conductive or semi-conductive enzyme stabilizing component are homogeneously dispersed in the composition.

12. The composition according to any one of claims 1 to 11,

it is characterized in that the preparation method is characterized in that,

after storage of the composition at a temperature of 4 ℃ for at least 4 weeks, preferably at least 12 weeks, more preferably at least 28 weeks, the enzyme has a residual activity of at least 90%, based on the total enzyme activity prior to storage.

13. An electrochemical sensor for the determination of an analyte, said sensor comprising at least one working electrode and at least one reference electrode,

it is characterized in that the preparation method is characterized in that,

the working electrode comprises the composition of any one of claims 1-12.

14. The electrochemical sensor according to claim 13,

it is characterized in that the preparation method is characterized in that,

it comprises a biocompatible coating covering the working and reference electrodes.

15. Electrochemical sensor according to claim 13 or 14 for the determination of an analyte in a body fluid, in particular in whole blood, plasma, serum or extracellular tissue fluid.

16. An electrochemical sensor according to claim 13 or 14 for the determination of an analyte selected from the group consisting of: malic acid, alcohols, ammonium, ascorbic acid, cholesterol, cysteine, glucose, glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid, 5' -nucleotidase, peptides, pyruvate, salicylate and triglycerides, especially glucose.

17. Use of an electrochemical sensor according to claim 13 or 14 in the manufacture of a device for use in a method of determining an analyte, the method comprising the steps of:

a contacting a sample containing an analyte with an electrochemical sensor according to claim 13 or 14, and

b determining the presence and/or amount of the analyte.