Electroenzymatic cholesterol biosensor, manufacture and use thereof

Abstract

The subject matter of the invention is an enzymatic system comprising carbon nanostructures modified with a cationic polyelectrolyte, wherein cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte. Cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte by means of electrostatic interactions between its negatively charged molecules and positively charged groups of the cationic polyelectrolyte. The subject matter of the invention is also an electrode comprising a conductive substrate on which the above- mentioned enzymatic system is placed, and a method of producing the electrode and its use for determining cholesterol. Furthermore, the subject matter of the invention is a use of the above-mentioned electrode as one of the working electrodes in a biosensor comprising more than one working electrode and adapted for determining more than one analyte. Another subject matter of the invention is a biosensor for determining cholesterol comprising the above-mentioned electrode as a working electrode. Finally, the subject matter of the invention is a method of determining cholesterol, which comprises contacting a sample containing cholesterol with the above-mentioned biosensor in the presence of a cofactor, and electrochemical determination of cholesterol in the sample.

Description

[0001] ELECTROENZYMATIC CHOLESTEROL BIOSENSOR, MANUFACTURE AND USE THEREOF

[0002] The subject matter of the invention is an enzymatic system comprising modified carbon nanostructures on which an enzyme is immobilized. The subject matter of the invention is also an electrode comprising a substrate on which the enzymatic system is deposited, a method of producing the electrode and its use for determining cholesterol. Moreover, the subject matter of the invention is a biosensor comprising the above-mentioned electrode and a method of determining cholesterol using the biosensor.

[0003] Determination of cholesterol is very important due to the fact that high levels of this compound in an organism may lead to many diseases.

[0004] In the state of the art, various enzymatic (electrocatalytic) systems used in biosensors for determining the level of cholesterol are described. The most commonly used enzyme in the systems of this type is cholesterol oxidase (ChOx). Cholesterol oxidase is also known to be used in combination with another enzyme, such as cholesterol esterase (ChEt), diaphorase (DH) or horseradish peroxidase (HRP). Furthermore, it is also known to immobilize an enzyme / enzymes on carbon nanostructures, for example, on carbon nanotubes functionalized with -COO’ groups or modified with different compounds.

[0005] For example, the publication by P.R. Solanki et al., Multi- walled carbon nanotubes / sol-gel- derived silica / chitosan nanobiocomposite for total cholesterol sensor, Sensors and Actuators B, 137 (2009) 727-735 describes an enzymatic system in which cholesterol oxidase (ChOx) combined with cholesterol esterase (ChEt) is immobilized via glutaraldehyde on multiwalled carbon nanotubes modified with chitosan and silica (MWCNT / SiCh-CHIT / ITO).

[0006] The use of glutaraldehyde to immobilize cholesterol oxidase is also described in the publication by M. Eguilaz et al., Designing Electrochemical Interfaces with Functionalized Magnetic Nanoparticles and Wrapped Carbon Nanotubes as Platforms for the Construction of High-Performance Bienzyme Biosensors, Anal. Chem, 83 (2011) 7807-7814, which discloses a system in which cholesterol oxidase (ChOx) combined with horseradish peroxidase (HRP) is immobilized on a composite consisting of multi-walled carbon nanotubes, poly(diallyldimethylammonium chloride) and magnetic nanoparticles functionalized with glutaraldehyde (GA-MNP / PDDA / MWCNT). Modification of the carbon nanotubes with poly(diallyldimethylammonium chloride) was preceded by carboxylation of the nanotubes by treating them with a mixture of sulphuric acid and nitric acid in appropriate conditions.

[0007] Another known system, described in the publication by Ch. Dhand et al., Polyaniline-carbon nanotube composite film for cholesterol biosensor, Analytical Biochemistry, 383 (2008) 194-199, is a system in which cholesterol oxidase is immobilized on an electrophoretically deposited composite film composed of polyaniline and multi-walled carbon nanotubes (PANI-MWCNT). The covalent attachment of the enzyme to the composite film occurred as a result of amide bond formation between the NH2 groups of polyaniline and the -COOH groups of oxidase.

[0008] Another example of a system in which cholesterol oxidase is immobilized on carbon nanotubes as a result of covalent interactions is the system described in the publication by A. Wisitsoraat et al.; Fast cholesterol detection using flow injection microfluidic device with functionalized carbon nanotubes based electrochemical sensor, Biosensors and Bioelectronics, 2010, 1514-1520. In the described system, the carbon nanotubes are modified with hydrolysed polyvinyl alcohol.

[0009] Apart from multi-walled carbon nanotubes, graphene can also be used to immobilize an enzyme. For example, the publication by R. Manjunatha et al., An amperometric bienzymatic cholesterol biosensor based on functionalized graphene modified electrode and its electrocatalytic activity towards total cholesterol determination, Taianta, 99 (2012) 302- 309 describes an enzymatic system in which cholesterol oxidase, alone or in combination with cholesterol esterase, is immobilized via glutaraldehyde and bovine serum albumin on graphene functionalized with -COO’ groups.

[0010] There are also known multilayer enzymatic systems, e.g. the publication by X. Cai et al., A layer-by-layer assembled and carbon nanotubes / gold nanoparticles-based bienzyme biosensor for cholesterol detection, Sensors and Actuators B, 181 (2013) 575 - 583 describes a multilayer system consisting of alternately deposited layers of a composite composed of poly(allylamine hydrochloride), multi-walled carbon nanotubes and gold nanoparticles (PAH-MWCNT-GNP) and layers of horseradish peroxidase (HRP) on which layers of the above-mentioned composite and layers of cholesterol oxidase (ChOx) are also deposited alternately (i.e. (PAH-MWCNT-GNP / HRP)m / (PAH-MWCNT-GNP / ChOx)n). Modification of the carbon nanotubes with poly(allylamine hydrochloride) was preceded by carboxylation of the nanotubes. Another multilayer system, described in the publication by M. Guo et al., Carbon Nanotubes- Based Amperometric Cholesterol Biosensor Fabricated Through Layer-by-Layer Technique, Electroanalysis, 2004, 16, No. 23, 1992-1998, is a system comprising a layer of multi-walled carbon nanotubes on which layers of poly(diallyldimethylammonium chloride) (PDDA) and layers of cholesterol oxidase (ChOx) are deposited alternately. In that case, the carbon nanotubes are not modified with poly(diallyldimethylammonium chloride) but are only functionalized with -COO’ groups by treating them with nitric acid.

[0011] Individual layers in the above-described systems are generally deposited by immersing an appropriate substrate (an electrode or an electrode already modified with another layer) in a solution containing the components of the layer to be deposited or by dropping such a solution onto an appropriate substrate.

[0012] Cholesterol oxidase catalyses cholesterol oxidation using oxygen as the acceptor of electrons, as a result of which hydrogen peroxide is formed. Thus, cholesterol determination with the use of biosensors based on the above-mentioned enzymatic systems is carried out by the quantitative measurement of produced hydrogen peroxide. Such measurements are dependent on the availability of oxygen, which may affect the repeatability of analysis. Therefore, work is underway on biosensors using cholesterol dehydrogenase, which is an enzyme that also catalyses the cholesterol oxidation, but unlike cholesterol oxidase, it does not utilize oxygen as the acceptor of electrons. The acceptor of electrons in the reaction catalysed by cholesterol dehydrogenase is nicotinamide adenine dinucleotide (NAD+), and as a result of this reaction, a reduced form of dinucleotide (NADH) is produced, and not hydrogen peroxide. Determination of cholesterol level is carried out by measuring the signal from NADH oxidation.

[0013] For example, a biosensor based on cholesterol dehydrogenase is described in patent description EP 0794 429 Bl. In one of the embodiments, the biosensor described in that document comprises a reaction solution, which contains cholesterol dehydrogenase, nicotinamide adenine dinucleotide and a compound that facilitates the transfer of electrons. In another embodiment, dehydrogenase together with other reagents (inter alia other enzymes) is contained in a layer deposited on a substrate.

[0014] Moreover, patent description US 7 087 149 Bl describes a biosensor in which an enzyme (for example, cholesterol dehydrogenase) is immobilized on a plasma-polymerized membrane containing appropriate functional groups for binding the enzyme. Another patent description EP 0795 601 Bl describes a biosensor comprising a reaction layer deposited on an electrode system. The layer contains a hydrophilic polymer, an enzyme and an electron acceptor. One of the exemplary enzymes mentioned in that description is cholesterol dehydrogenase.

[0015] In the state of the art, a system comprising cholesterol dehydrogenase and carbon nanostructures, i.e. nanodots is also known. Namely, the publication by M. del Barrio et al., Carbon nanodot, modified-electrode for peroxide-free cholesterol biosensing and biofuel cell design, Sensors & Actuators: B. Chemical, 375 (2023) 132895 describes a biosensor comprising an electrode modified with the carbon nanodots functionalized with a phenothiazine dye. Dehydrogenase is covalently immobilized on the functionalized nanodots using carbodiimide hydrochloride and N-hydroxy succinimide.

[0016] Another example of a system that can be applied in biosensors for determining cholesterol is the system described in the publication by T. Quah et al., Cholesterol as a Promising Alternative Energy Source: Bioelectrocatalytic Oxidation Using NAD-Dependent Cholesterol Dehydrogenase in Human Serum, Journal of The Electrochemical Society, 164 (3) H3024-H3029 (2017). It is composed of two layers. The first layer contains linear poly(ethylenimine) modified with dimethylferrocene (FcMei-LPEI) and diaphorase (DH), whereas the second layer contains linear poly(ethylenimine) modified with octyl (Cs-LPEI) and cholesterol dehydrogenase (ChDH).

[0017] Apart from biosensors comprising one working electrode for determining one analyte, in the state of the art, biosensors comprising several different working electrodes are also known. Such biosensors are adapted for determining more than one analyte.

[0018] For example, the publication by H. Kanso et al., Electroanalysis, 29 (2017) 87 describes a bioelectrocatalytic system for simultaneous determination of glucose and lactates on screen- printed electrodes modified with graphene. On one electrode, cellobiose dehydrogenase is immobilized, and on the other - lactate oxidase.

[0019] The publication by M. Thapa et al., Biosensors, 11 (2021) 507 discloses a biosensor comprising screen-printed electrodes also for simultaneous determination of glucose and lactates in blood. In that case, biocatalytic layers containing respectively glucose oxidase and lactate oxidase are used.

[0020] The publication by J. Pilas et al., Anal. Chem., 91 (2019) 15293 describes a bioelectrocatalytic system for simultaneous determination of ethanol, formates and D- and L-enantiomers of lactate. To construct a biosensor, enzymes from the group of dehydrogenases (alcohol, formate, L-lactate and D-lactate dehydrogenases) are used. Graphene oxide is a matrix for immobilizing the enzymes.

[0021] As it results from the above description of the state of the art, in this field there is still a need for enzymatic systems for use in biosensors for determining cholesterol, which systems would comprise cholesterol dehydrogenase immobilized on a matrix based on carbon nanostructures, in particular on modified carbon nanostructures, which nanostructures are known to have unique physicochemical properties i.e. chemical stability, good mechanical properties and high electron conduction. Furthermore, it is of great importance to develop an appropriate method for immobilizing the enzyme and modifying the matrix, as it affects the obtained measurement results, inter alia the linearity range of an analytical signal.

[0022] Therefore, the purpose of the invention was to develop a simple and stable enzymatic system, which would be applicable in enzymatic electrodes used for the construction of biosensors for determining cholesterol, which biosensors would be characterized by a wide linearity range of an analytical signal and insensitivity to the presence of oxygen. The purpose of the invention was also to develop a simple method of producing the electrodes comprising the enzymatic system.

[0023] These purposes have been achieved by means of the solutions presented in the appended claims.

[0024] The subject matter of the invention is an enzymatic system comprising carbon nanostructures modified with a cationic polyelectrolyte, wherein cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte. Cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte by means of electrostatic interactions between its negatively charged molecules and positively charged groups of the cationic polyelectrolyte.

[0025] Preferably, the carbon nanostructures are selected from the group consisting of carbon nanotubes, carbon nanodots, fullerenes, graphene and their mixture.

[0026] Preferably, the carbon nanotubes are single-walled, double-walled or multi-walled carbon nanotubes.

[0027] Preferably, the cationic polyelectrolyte is a polybase or its salt having functional groups with a positively charged nitrogen atom, and more preferably it is poly(diallyldimethylammonium chloride). Preferably, the enzymatic system is in the solid form consisting of a first layer and a second layer, wherein the first layer contains the carbon nanostructures modified with the cationic polyelectrolyte and the second layer placed on the first layer contains cholesterol dehydrogenase.

[0028] The subject matter of the invention is also an electrode comprising a conductive substrate on which the above-defined enzymatic system is placed.

[0029] Preferably, the conductive substrate is a metallic substrate, and more preferably, the metallic substrate is selected from gold, platinum, silver or palladium.

[0030] Also preferably, the conductive substrate is a carbon substrate, and more preferably the carbon substrate is selected from graphite, glassy carbon, graphene, carbon nanotubes or carbon paste, wherein the carbon paste contains carbon nanotubes or graphene.

[0031] The subject matter of the invention is a use of the above-defined electrode for determining cholesterol.

[0032] Furthermore, the subject matter of the invention is a use of the above-defined electrode as one of the working electrodes in a biosensor comprising more than one working electrode and adapted for determining more than one analyte.

[0033] The subject matter of the invention is also a biosensor for determining cholesterol, wherein the biosensor comprises a system of electrodes including a working electrode, which is the electrode defined above.

[0034] The subject matter of the invention is a method of determining cholesterol, wherein the method comprises contacting a sample containing cholesterol with the above-defined biosensor in the presence of a cofactor and electrochemical determination of cholesterol in the sample.

[0035] Preferably, nicotinamide adenine dinucleotide or its analogue is used as the cofactor.

[0036] Preferably, acetyl pyridine adenine dinucleotide, thio-nicotinamide adenine dinucleotide or its potassium salt, acetyl pyridine hypoxanthine dinucleotide, nicotinic acid adenine dinucleotide, nicotinamide hypoxanthine dinucleotide or nicotinamide guanine dinucleotide is used as the analogue of nicotinamide adenine dinucleotide.

[0037] Preferably, the cofactor is used at a concentration falling within the range of 2 to 10 mM. Preferably, the electrochemical determination of cholesterol in the sample is carried out by applying a potential to a system of electrodes and recording an analytical signal from a cofactor oxidation reaction.

[0038] Another subject matter of the invention is a method of producing the above-defined electrode, wherein the method comprises the following steps: a) modifying carbon nanostructures with a cationic polyelectrolyte by: i) adding a solution of the cationic polyelectrolyte in an organic or inorganic solvent to the carbon nanostructures in a weight ratio of the carbon nanostructures to the cationic polyelectrolyte from 3:1 to 5:1, to produce a mixture; ii) placing the mixture produced in step i) in an ultrasonic bath for a period of 2 to 3 hours; iii) stirring the mixture produced in step ii) for a period of 12 to 48 hours; and iv) centrifuging the mixture produced in step iii) and decanting the solvent to obtain a precipitate of the carbon nanostructures modified with the cationic polyelectrolyte; b) pouring an organic or inorganic solvent over the precipitate of the carbon nanostructures modified with the cationic polyelectrolyte obtained in step iv) to produce a colloidal solution of the carbon nanostructures modified with the cationic poly electrolyte; c) immobilizing the carbon nanostructures modified with the cationic polyelectrolyte on a conductive substrate; d) producing a solution of cholesterol dehydrogenase; and e) immobilizing cholesterol dehydrogenase on the carbon nanostructures modified with the cationic polyelectrolyte.

[0039] Preferably, steps i) to iv) are repeated for the second time.

[0040] Preferably, a polybase or its salt having functional groups with a positively charged nitrogen atom, and more preferably poly(diallyldimethylammonium chloride), is used as the cationic polyelectrolyte.

[0041] Preferably, nanostructures selected from the group consisting of carbon nanotubes, carbon nanodots, fullerenes, graphene and their mixture are used as the carbon nanostructures.

[0042] Preferably, single-walled, double-walled or multi-walled carbon nanotubes are used as the carbon nanotubes. Preferably, in step i) water, toluene, chloroform, dimethylformamide or tetrahydrofuran is used as the solvent.

[0043] Preferably, in step b) water, toluene, chloroform, dimethylformamide or tetrahydrofuran is used as the solvent.

[0044] Preferably, step c) is carried out by depositing the colloidal solution of the carbon nanostructures modified with the cationic polyelectrolyte produced in step b) onto the conductive substrate and allowing it to dry to form a layer containing the carbon nanostructures modified with the cationic polyelectrolyte.

[0045] Preferably, a metallic substrate, and more preferably gold, platinum, silver or palladium, is used as the conductive substrate.

[0046] Also preferably, a carbon substrate, and more preferably graphite, glassy carbon, graphene, carbon nanotubes or carbon paste, wherein the carbon paste contains carbon nanotubes or graphene, is used as the conductive substrate.

[0047] Preferably, step d) is carried out by dissolving cholesterol dehydrogenase in a buffer or an aqueous salt solution having a pH value higher than 4.5.

[0048] Preferably, in step d) the solution of cholesterol dehydrogenase, which contains cholesterol dehydrogenase in an amount of 3 to 15 U is produced.

[0049] Preferably, step e) is carried out by depositing the solution of cholesterol dehydrogenase produced in step d) onto the layer containing the carbon nanostructures modified with the cationic polyelectrolyte formed in step c) and allowing it to dry to form a layer containing cholesterol dehydrogenase.

[0050] Preferably, the method of producing the electrode additionally comprises depositing protective layers after steps c) and e).

[0051] The inventors of the present invention have unexpectedly found out that, due to the modification of carbon nanostructures with an appropriate cationic polyelectrolyte, preferably a polymer composed of macromolecules containing positively charged nitrogen atoms, it is possible to obtain a matrix for stable immobilization of cholesterol dehydrogenase. The developed method of modifying the carbon nanostructures is characterized by great simplicity as it has significantly reduced the number of steps. This definitely translates into greater comfort and shorter working time for the researcher and also reduces the possibility of making an error to a minimum. Furthermore, the obtained solutions of modified nanostructures are characterized by stability for several weeks. Immobilization of cholesterol dehydrogenase on the modified carbon nanostructures takes place as a result of electrostatic interactions. The enzymatic system according to the invention obtained in this way is very stable and simple to obtain. Depositing the system according to the invention on a conductive substrate makes it possible to obtain an enzymatic electrode for determining cholesterol. Measurements carried out using a biosensor comprising the above-mentioned electrode as a working electrode are characterized by high sensitivity, a wide linearity range of readings, as well as independence of oxygen presence in a sample. Moreover, no significant decrease is observed in the activity of cholesterol dehydrogenase immobilized on the modified nanostructures towards cholesterol oxidation. These and other advantages of the present invention will be obvious in the light of the following description of the invention, and in particular in the light of its embodiments.

[0052] The results of tests concerning the electrochemical characterization of layers of modified nanostructures and determination of cholesterol using a biosensor comprising a working electrode with the enzymatic system according to the invention are presented in the drawing in which:

[0053] Fig. 1 shows cyclic voltammetric curves for glassy carbon electrodes with MWCNT / PDDA (a) and SWCNT / PDDA (b) layers and for a glassy carbon electrode without these layers (c);

[0054] Fig. 2 shows cyclic voltammetric curves recorded for glassy carbon electrodes (c) covered with MWCNT / PDDA (a) and SWCNT / PDDA (b) layers and after adding 5 mM NADH solution (a’, b’, c’);

[0055] Fig. 3 shows cyclic voltammetric curves for oxidation of different cholesterol concentrations: 1.0 mM (a’, b’), 2.0 mM (a”, b”), 3.0 mM (a’”) in MWCNT / PDDA-ChDH (a) and SWCNT / PDDA-ChDH (b) layers deposited on glassy carbon electrodes;

[0056] Fig. 4 shows amperometric curves recorded for MWCNT / PDDA-ChDH (a) and SWCNT / PDDA-ChDH (b) layers for different cholesterol concentrations (0; 0.25; 0.5; 0.75; 1.0 mM);

[0057] Fig. 5 shows the amperometric dependence of current intensity on time, recorded for MWCNT / PDDA-ChDH (a) and SWCNT / PDDA-ChDH (b) layers when adding subsequent portions of cholesterol (every 0.5 mM); Fig. 6 shows a calibration curve of the dependence of stationary current intensity on changes in cholesterol concentration for MWCNT / PDDA-ChDH (a) and SWCNT / PDDA-ChDH (b) layers; and

[0058] Fig. 7 shows a graph of the dependence of the activity of cholesterol dehydrogenase immobilized on MWCNT / PDDA-ChDH (a) and SWCNT / PDDA-ChDH (b) layers, measured by a decrease in current intensity over time, in the presence of cholesterol (1.0 mM).

[0059] The abbreviations used in the description and in the figures have the meanings commonly known and used by those skilled in the field of the present invention. For the sake of clarity, however, the following abbreviations should be understood as follows:

[0060] - MWCNT - multi-walled carbon nanotubes;

[0061] - SWCNT - single-walled carbon nanotubes;

[0062] - PDDA - poly(diallyldimethylammonium chloride);

[0063] - ChDH - cholesterol dehydrogenase;

[0064] - NAD+- nicotinamide adenine dinucleotide, oxidized form;

[0065] - NADH - nicotinamide adenine dinucleotide, reduced form;

[0066] - Triton X-100 - 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol;

[0067] - GCE - glassy carbon electrode.

[0068] Below is presented a detailed description of the invention.

[0069] The cationic polyelectrolyte used in the invention is any prior-art cationic polyelectrolyte (i.e. a polybase or its salt) composed of structural units having positively charged functional groups. According to the invention, a polyelectrolyte having functional groups with a positively charged nitrogen atom (for example, -N+H3X“, -N+RH2X“, -N+R2HX“ or -N+R3X ) is preferably used, and more preferably it is poly(diallyldimethylammonium chloride). Such polyelectrolyte dissociates in a solution into polycations having the positively charged nitrogen atom and negative counterions. Therefore, as a result of modifying carbon nanostructures with the solution of the above-mentioned polyelectrolyte, a positive electric charge present on the nitrogen atom is obtained on their surface. On the other hand, by dissolving cholesterol dehydrogenase in a solution having an appropriate pH value (higher than the isoelectric point of the enzyme), enzyme molecules in anionic form (with a negative electric charge) are obtained. This makes it possible to immobilize the enzyme on the modified nanostructures by adsorption of the negatively charged enzyme molecules on the positively charged modified carbon nanostructures as a result of electrostatic interactions.

[0070] According to the invention, any carbon nanostructures selected from the group consisting of carbon nanotubes (single-walled, double-walled and multi- walled), carbon nanodots, fullerenes, graphene and their mixture can be used as the carbon nanostructures. These structures increase the rate of electron transport between an electrode and the active centre of enzyme molecule and additionally reduce the overpotential of NADH oxidation.

[0071] In one embodiment, the enzymatic system according to the invention is in the solid form consisting of two layers. The first layer contains the carbon nanostructures modified with the cationic polyelectrolyte, whereas the second layer placed on the first layer contains cholesterol dehydrogenase. The layers are formed by using two separate solutions, the first solution containing the carbon nanostructures modified with the cationic polyelectrolyte and the second solution containing cholesterol dehydrogenase.

[0072] In another embodiment, the enzymatic system is in the form of one layer containing both components. In this embodiment, dehydrogenase is immobilized directly on the modified carbon nanostructures, and not on an earlier formed layer of the nanostructures. This one layer is formed by using one solution containing both the nanostructures and the enzyme.

[0073] In both cases, the key is the presence of appropriate electric charges on individual components, which is easier to achieve and control when forming two separate layers by using two separate solutions. Furthermore, in the case of the two-layer system, the risk of enzyme denaturation is eliminated. Therefore, the two-layer system is preferable.

[0074] By placing the enzymatic system according to the invention on a conductive substrate, an enzymatic electrode for determining cholesterol is obtained. The conductive substrate is not specifically limited and, according to the invention, any conductive substrate known in the field of electrochemistry can be used. Namely, both a metallic substrate and a carbon substrate can be used. Among the metallic substrates, gold, platinum, silver or palladium is preferably used, whereas among the carbon substrates, graphite, glassy carbon, graphene, carbon nanotubes or carbon paste is preferably used, wherein the carbon paste contains carbon nanotubes or graphene. In one aspect of the invention, the enzymatic electrode according to the invention is used as a working electrode in a biosensor for determining cholesterol.

[0075] It is also possible to use the enzymatic electrode according to the invention as one of the working electrodes in a biosensor comprising more than one working electrode and adapted for determining more than one analyte, including cholesterol. Such biosensors for determining more than one analyte are known in the field, as described in the section relating to the state of the art in the present description. Selection of the number and types of working electrodes and the appropriate design of the biosensor comprising more than one working electrode is within the abilities of a person skilled in the art.

[0076] A method of determining cholesterol using the biosensor according to the invention consists in contacting a sample containing cholesterol with the said biosensor in the presence of a cofactor.

[0077] The enzymatic reaction catalysed by cholesterol dehydrogenase consists in oxidizing cholesterol to cholest-4-en-3-one. This reaction requires the presence of a cofactor being the acceptor of electrons from cholesterol oxidation. The reduced cofactor is subsequently oxidized at an electrode.

[0078] ChDH cholesterol + NAD+- * cholest — 4 — en — 3 — on + NADH

[0079] The cofactor in the above reaction is nicotinamide adenine dinucleotide. According to the invention, its analogues can also be used; namely, acetyl pyridine adenine dinucleotide (APAD+), thio-nicotinamide adenine dinucleotide (TNAD+) or its potassium salt (TNADK+), acetyl pyridine hypoxanthine dinucleotide (ADH+), nicotinic acid adenine dinucleotide (NAAD+), nicotinamide hypoxanthine dinucleotide (NHD+) or nicotinamide guanine dinucleotide (NGD+). Preferably, the cofactor is used in a concentration falling within the range of 2 to 10 mM. Electrochemical determination of cholesterol is carried out by applying a potential to the system of biosensor electrodes and recording the analytical signal from the reaction of cofactor oxidation (NADH).

[0080] Before starting the electrochemical determination of cholesterol in a sample, the sample containing cholesterol must be properly prepared. To this end, a surfactant (a Triton X-100 solution) is added to the said sample in an amount that makes it possible to obtain a homogenous mixture, and then a buffer or an aqueous salt solution having a pH value higher than 4.5 is added to obtain the assumed volume. Preferably, the buffer or the aqueous salt solution is added after being heated to a temperature in the range of 50 to 70°C. Then, the produced homogenous mixture is heated at a temperature in the range of 50 to 70°C, preferably in an ultrasonic bath, for a period of 0.5 to 2 hours. Triton X-100 is added in such an amount that it constitutes from 1% to 10% by volume relative to the total volume of the sample.

[0081] Additionally, the sample to be determined is deoxidized before the electrochemical measurement.

[0082] A method of producing an electrode comprising the enzymatic system according to the invention comprises, firstly, the step of modifying carbon nanostructures with a cationic polyelectrolyte. To this end, a solution of the cationic polyelectrolyte is poured over the carbon nanostructures. The solvent used in this step is not specifically limited and both an organic solvent, such as toluene, chloroform, dimethylformamide and tetrahydrofuran, and an inorganic solvent, such as water, can be used. However, the ratio of the carbon nanostructures to the cationic polyelectrolyte is essential; namely, it falls within the range of 3: 1 to 5: 1, and preferably is 4: 1. The mixture containing the nanostructures and the polyelectrolyte produced in this way is placed in an ultrasonic bath for a period of 2 to 3 hours and then it is stirred for a period of 12 to 48 hours. Then the mixture is centrifuged and a solution from above a precipitate containing modified nanostructures is decanted. All the above-mentioned steps are preferably repeated once again and then an organic or inorganic solvent is poured over the obtained precipitate in order to produce a colloidal solution of the carbon nanostructures modified with the cationic polyelectrolyte. In this case, the same solvents as mentioned above can be used. Immobilization of the obtained modified carbon nanostructures on a conductive substrate can be carried out by dropping the produced colloidal solution onto the conductive substrate or by immersing the conductive substrate in this solution; preferably this is carried out by dropping the solution onto the substrate. Then, the solution is allowed to dry as a result of which a layer containing the carbon nanostructures modified with the cationic polyelectrolyte is formed. In the subsequent (or parallel) step, a solution of cholesterol dehydrogenase is prepared. The pH of this solution must be higher than the isoelectric point of the enzyme so that it can be transformed into anionic form. The appropriate solution is any buffer or any aqueous salt solution having a pH value higher than 4.5. Preferably, phosphate buffer having pH 7.0 is used. According to the invention, the solution containing cholesterol dehydrogenase in an amount of 3-15 U is used. The dehydrogenase solution produced in this way is subsequently deposited onto the layer of the modified carbon nanostructures and is allowed to dry in order to form a layer containing dehydrogenase. This can be carried out in a similar way as in the case of forming the layer of the nanostructures by dropping the produced enzyme solution onto the conductive substrate with the layer of the nanostructures or by immersing the conductive substrate with the layer of the nanostructures in the solution, preferably this is carried out by dropping the solution.

[0083] Preferably, protective layers are deposited onto the individual layers containing the carbon nanostructures and cholesterol dehydrogenase. These protective layers are usually formed by using sulfonated polytetrafluoroethylene with the registered brand name Nafion® (by DuPont). It is a copolymer of tetrafluoroethylene (TFE) and perfluoro-3,6-dioxa-4-methyl- 7-octenesulfonic acid. The structure and properties of the copolymer are regulated by the production method and appropriate selection of monomer ratio during copolymerization, i.e. of tetrafluoroethylene (TFE) and perfluoro sulfonic acid. As a result of these modifications, commercially available analogues of Nafion are produced: Aciplex® (Asahi Chemical), Flemion® (Asahi Glass), 3M™, fumion® F (FuMA-Tech) and Aquivion™ (Solvay- Solexis), which also can be used in the present invention to form the protective layers.

[0084] The types of cationic polyelectrolyte, carbon nanostructures and conductive substrate used in the method of producing the electrode according to the invention are specified above in the detailed description of the enzymatic system and the electrode according to the invention.

[0085] The invention is illustrated by the following examples, which however do not limit the invention.

[0086] In the examples presented below the following reagents and apparatus have been used:

[0087] Reagents:

[0088] - multi-walled carbon nanotubes: outer diameter x length 6-13 nm x 2.5-20 pm > 99%, Aldrich, USA,

[0089] - single-walled carbon nanotubes: carbon >80 %, diameter 1.2-2.0 nm, Aldrich, USA,

[0090] - poly(diallyldimethylammonium chloride), Sigma- Aldrich, USA,

[0091] - Nafion®, 5% solution in a mixture of aliphatic alcohols and water, 15-20% H2O, Aldrich, USA,

[0092] - cholesterol dehydrogenase (Nocardia bacterial strain) (ChDH), Sigma- Aldrich, USA, - cholesterol, Sigma- Aldrich, USA,

[0093] - nicotinamide adenine dinucleotide, oxidized form (NAD+), Sigma- Aldrich, USA,

[0094] - nicotinamide adenine dinucleotide, reduced form (NADH), Sigma- Aldrich, USA,

[0095] - Triton TM X-100, Sigma- Aldrich, USA,

[0096] - high-purity argon (99.999%) for solution deoxidation.

[0097] Apparatus

[0098] - electrochemical analyser CHI 1030B, CH Instruments, Inc. Austin, Texas, USA;

[0099] - ultrasonic bath SONIC-0.5 (POLSONIC Sp. J.), frequency 40kHz, power 80W.

[0100] The tests were carried out with a three-electrode system consisting of the following electrodes:

[0101] - working electrode - a glassy carbon disc electrode having the diameter of 3 mm (surface area of 0.07 cm2), CH Instruments Inc., Austin, Texas, USA, cleaned before each measurement on a polishing felt on which there was a suspension of aluminium oxide (AI2O3) having various grain size;

[0102] - counter electrode - a platinum wire having the diameter of about 0.08 mm, CH Instruments Inc., Austin, Texas, USA; and

[0103] - reference electrode - Ag / AgCl / lM KC1, CH Instruments Inc., Austin, Texas, USA

[0104] Example 1: Producing an electrode comprising a conductive substrate on which the enzymatic system according to the invention is deposited. a) Producing a colloidal solution of carbon nanotubes modified with poly (diallyldimethylammonium chloride) .

[0105] In the first step, 5 mg of each single-walled carbon nanotubes (SWCNT) and multi-walled carbon nanotubes (MWCNT) was weighed into separate vessels and each of them was poured with 5 ml of 5% aqueous solution of poly(diallyldimethylammonium chloride) (PDDA). Then, the obtained suspensions were placed in an ultrasonic bath for two hours, and subsequently were stirred with a magnetic stirrer for 24 hours. The next step was centrifugation of the samples (at the speed of 10000 rpm for 10 minutes and then at the speed of 12000 rpm for 10 minutes), followed by decantation of solutions from above precipitates. 5 ml of 5% PDDA solution was again poured over the precipitates of the carbon nanotubes. The previous actions were repeated and finally, distilled water was poured over the precipitates of modified carbon nanotubes. As a result, colloidal solutions of the modified carbon nanotubes (MWCNT / PDDA and SWCNT / PDDA) were obtained, which were characterized by stability for several weeks. b) Immobilizing the carbon nanotubes modified with poly(diallyldimethylammonium chloride) on a conductive substrate - forming MWCNT / PDDA and SWCNT / PDDA layers.

[0106] 3 pl of each of the above colloidal solutions (MWCNT / PDDA and SWCNT / PDDA) was dropped onto glassy carbon electrodes and then was allowed to dry in order to form layers containing the modified carbon nanotubes. Then, 5 pl of Nafion® was dropped onto each of the two layers and allowed to dry in order to protect the layers from desorption. c) Preparing a solution of cholesterol dehydrogenase.

[0107] 0.72 mg of cholesterol dehydrogenase (ChDH) was weighed on an analytical balance, and then 15 pl of 0.1 mol dm-3phosphate buffer having pH=7.0 was added. d) Immobilizing cholesterol dehydrogenase on the layer of the nanotubes modified with poly(diallyldimethylammonium chloride) - producing MWCNT / PDDA-ChDH and SWCNT / PDDA-ChDH systems.

[0108] 7 pl of the above-prepared enzyme solution was deposited onto each MWCNT / PDDA layer and SWCNT / PDDA layer formed earlier and was allowed to dry in order to form layers containing cholesterol dehydrogenase. After about 15-20 minutes, when the layers were already dry, 5 pl of 0.5% Nafion solution was dropped onto each of them and was again allowed to dry.

[0109] Example 2: Electrochemical characterization of SWCNT / PDDA and MWCNT / PDDA layers deposited on glassy carbon electrodes.

[0110] To examine the electrochemical properties of the MWCNT / PDDA and SWCNT / PDDA layers, measurements were performed in deoxidized 0.1 M phosphate buffer having pH=7.0, using the technique of cyclic voltammetry, at the polarization rate of 5 mV / s.

[0111] On the recorded voltammetric curves (Fig. 1), a higher capacity current intensity can be observed in the case of electrodes modified with the MWCNT / PDDA (curve a) and SWCNT / PDDA (curve b) layers compared with the intensity obtained for a pure glassy carbon electrode (curves c). This is related to the presence of the carbon nanostructures and increased active surface of the electrode. Higher values of capacity currents were observed for the electrode with the SWCNT / PDDA layer. It was further examined what was the influence of the individual layers of modified carbon nanotubes on the process of oxidizing NADH to NAD+. To this end, 5 mM NADH was oxidized in the MWCNT / PDDA and SWCNT / PDDA layers using cyclic voltammetry in deoxidized 0.1 M phosphate buffer having pH=7.0.

[0112] For the redox couple NAD+ / NADH, the redox potential is relatively low and amounts to -0.56V, whereas NADH oxidation at a pure glassy carbon electrode occurs at a high overpotential and may lead to the formation of nicotinamide dimers, which contaminate the surface of the electrode.

[0113] To avoid the above-mentioned problem, the overpotential of NADH oxidation should be reduced.

[0114] Curves a and a’ presented in Fig. 2 were recorded at the glassy carbon electrode covered with the MWCNT / PDDA layer, respectively before and after adding 5 mM NADH, curves b and b’ were recorded at the glassy carbon electrode covered with the SWCNT / PDDA layer, respectively before and after adding 5 mM NADH, whereas curves c and c’ were recorded at a pure glassy carbon electrode, respectively before and after adding 5 mM NADH. When analysing Fig. 2, an increase in capacity current density can be noticed for the measurement at the electrodes covered with the MWCNT / PDDA and SWCNT / PDDA layers relative to the measurement at the pure electrode. As described above, this is due to the fact that the carbon nanostructures enlarged the active surface of the electrode. This also resulted in an increase in current density of the catalytic oxidation of NADH in the MWCNT / PDDA and SWCNT / PDDA layers. Moreover, a shift of the oxidation potential of NADH towards negative values was observed for the MWCNT / PDDA and SWCNT / PDDA layers relative to the values obtained for the unmodified electrode. For the MWCNT / PDDA layer, the oxidation process of NADH begins at E=0.3 V, and in the case of the SWCNT / PDDA layer - at the value E=0 V. For the unmodified electrode the oxidation process occurs at about 0.5 V. The reason for the shift of the potential may be, among other things, the reduced probability of the occurrence of the above-mentioned radical reaction, which leads to the formation of NADH dimers contaminating the electrode surface.

[0115] Example 3: Examining bioelectrocatalytic properties and stability of MWCNT / PDDA- ChDH and SWCNT / PDDA-ChDH layers. First, 10 mM cholesterol solution was prepared. To this end, a cholesterol sample was weighed and Triton X-100 was added in such an amount that it constituted 10% of the cholesterol solution volume. Then, heated 0.1 M phosphate buffer having pH=7.0 was added to the assumed volume and the solution was placed in an ultrasonic bath and was simultaneously heated at the temperature of about 60°C. As a result, a milky suspension was obtained, which showed a tendency to foam.

[0116] To examine the bioelectrocatalytic properties of the MWCNT / PDDA-ChDH and SWCNT / PDDA-ChDH layers, cholesterol oxidation was carried out in the tested layers at different cholesterol concentrations using cyclic voltammetry. The measurements were performed in deoxidized 0.1 M phosphate buffer having pH=7.0, in the presence of 5 mM NAD+, within the potential range of -0.2 V to 0.9 V, at the polarization rate of 5 mV s’1. The first curves were recorded in the absence of cholesterol (curve a for the MWCNT / PDDA-ChDH layer and curve b for the SWCNT / PDDA-ChDH layer), whereas the following curves - for cholesterol concentrations being 1.0 mM, 2.0 mM and 3.0 mM for the MWCNT / PDDA-ChDH layer (curves a’, a” and a’”) and for cholesterol concentrations being 1.0 mM and 2.0 mM for the SWCNT / PDDA-ChDH layer (curves b’ and b”).

[0117] It was noticed that after each addition of cholesterol, the value of anodic current intensity increased. It can be concluded from the analysis of the recorded curves, obviously bearing in mind the oxidation potential of NADH at the electrodes with the MWCNT / PDDA and SWCNT / PDDA layers, which was shifted towards more negative values, that the reaction catalysed by ChDH proceeds towards the formation of cholest-4-en-3-one and NADH, and the recorded analytical signal comes from the regeneration of the cofactor that was used in the enzymatic reaction.

[0118] Using the amperometry technique, current intensity over time at a constant potential was also recorded for both layers. For MWCNT / PDDA-ChDH, the potential was E= 0.7 V (Fig. 4 a), and for SWCNT / PDDA-ChDH it was E= 0.6 V (Fig. 4 b). The measurement was performed in deoxidized 0.1 M phosphate buffer having pH=7.0, containing 5 mM NAD+. Each subsequent curve was recorded for increasing cholesterol concentration (0 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM). It can be seen on the obtained amperometric curves that the increasing concentration of cholesterol causes an increase in the values of current intensity. Higher current intensity values were obtained for the SWCNT / PDDA-ChDH system. The kinetics of both systems was tested in an amperometric experiment by recording catalytic currents over time, after adding subsequent portions of a cholesterol solution, at a constant value of potential (0.7 V for MWCNT / PDDA-ChDH and 0.6 V for SWCNT / PDDA-ChDH). Subsequent portions of 0.5 mM cholesterol solution were added to 0.1 M phosphate buffer having pH=7.0 in the presence of 5 mM NAD+, at the time intervals of 100 seconds (Fig. 5).

[0119] Based on the obtained amperometric curves, calibration curves were plotted for both layers (Fig. 6). Using the calibration curves, the sensitivity and linearity range characterizing both systems were determined (Table 1). The determined parameters of the MWCNT / PDDA- ChDH and SWCNT / PDDA-ChDH systems indicate that both systems are quite similar to each other. The system based on the modified single-walled carbon nanotubes is however characterized by a higher sensitivity and a lower detection limit.

[0120] Table 1. Parameters of the MWCNT / PDDA-ChDH and SWCNT / PDDA-ChDH systems.

[0121] Furthermore, a slight decrease in current intensity over time was observed during the measurement performed in the presence of 1 mM cholesterol in 0.1 M phosphate buffer having pH 7.0 (Fig. 7). This demonstrates a slight decrease in the activity of biosensors towards cholesterol oxidation which is about 10.6 % for the MWCNT / PDDA-ChDH system and about 12.1 % for the SWCNT / PDDA-ChDH system in the period of 33 days (Fig. 7). This means that the obtained systems are very stable.

Claims

Claims1. An enzymatic system, characterized in that the system comprises carbon nanostructures modified with a cationic polyelectrolyte, wherein cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte, and wherein cholesterol dehydrogenase is immobilized on the carbon nanostructures modified with a cationic polyelectrolyte by means of electrostatic interactions between its negatively charged molecules and positively charged groups of the cationic polyelectrolyte.

2. The enzymatic system according to claim 1, characterized in that the carbon nanostructures are selected from the group consisting of carbon nanotubes, carbon nanodots, fullerenes, graphene and their mixture.

3. The enzymatic system according to claim 1 or claim 2, characterized in that the carbon nanotubes are single-walled, double-walled or multi-walled carbon nanotubes.

4. The enzymatic system according to any one of claims 1 to 3, characterized in that the cationic polyelectrolyte is a polybase or its salt having functional groups with a positively charged nitrogen atom.

5. The enzymatic system according to claim 4, characterized in that the cationic polyelectrolyte is poly(diallyldimethylammonium chloride).

6. The enzymatic system according to any one of claims 1 to 5, characterized in that the system is in the solid form consisting of a first layer and a second layer, wherein the first layer contains the carbon nanostructures modified with the cationic polyelectrolyte and the second layer placed on the first layer contains cholesterol dehydrogenase.

7. An electrode, characterized in that the electrode comprises a conductive substrate on which the enzymatic system defined in any one of claims 1 to 6 is placed.

8. The electrode according to claim 7, characterized in that the conductive substrate is a metallic substrate.

9. The electrode according to claim 8, characterized in that the metallic substrate is selected from gold, platinum, silver or palladium.

10. The electrode according to claim 7, characterized in that the conductive substrate is a carbon substrate.

11. The electrode according to claim 10, characterized in that the carbon substrate is selected from graphite, glassy carbon, graphene, carbon nanotubes or carbon paste.

12. The electrode according to claim 11, characterized in that the carbon paste contains carbon nanotubes or graphene.

13. A use of the electrode defined in any one of claims 7 to 12 for determining cholesterol.

14. A use of the electrode defined in any one of claims 7 to 12 as one of the working electrodes in a biosensor comprising more than one working electrode and adapted for determining more than one analyte.

15. A biosensor for determining cholesterol comprising a system of electrodes, characterized in that the system of electrodes includes a working electrode, which is the electrode defined in any one of claims 7 to 12.

16. A method of determining cholesterol, characterized in that the method comprises contacting a sample containing cholesterol with the biosensor defined in claim 15 in the presence of a cofactor, and electrochemical determination of cholesterol in the sample.

17. The method of determining cholesterol according to claim 16, characterized in that nicotinamide adenine dinucleotide or its analogue is used as the cofactor.

18. The method of determining cholesterol according to claim 17, characterized in that acetyl pyridine adenine dinucleotide, thio -nicotinamide adenine dinucleotide or its potassium salt, acetyl pyridine hypoxanthine dinucleotide, nicotinic acid adenine dinucleotide, nicotinamide hypoxanthine dinucleotide or nicotinamide guanine dinucleotide is used as the analogue of nicotinamide adenine dinucleotide.

19. The method of determining cholesterol according to any one of claims 16 to 18, characterized in that the cofactor is used at a concentration falling within the range of 2 to 10 mM.

20. The method of determining cholesterol according to any one of claims 16 to 19, characterized in that the electrochemical determination of cholesterol in the sample is carried out by applying a potential to a system of electrodes and recording an analytical signal from a cofactor oxidation reaction.

21. A method of producing the electrode defined in any one of claims 7 to 12, wherein the method comprises the following steps:a) modifying carbon nanostructures with a cationic polyelectrolyte by: i) adding a solution of the cationic polyelectrolyte in an organic or inorganic solvent to the carbon nanostructures in a weight ratio of the carbon nanostructures to the cationic polyelectrolyte from 3:1 to 5:1 to produce a mixture; ii) placing the mixture produced in step i) in an ultrasonic bath for a period of 2 to 3 hours; iii) stirring the mixture produced in step ii) for a period of 12 to 48 hours; and iv) centrifuging the mixture produced in step iii) and decanting the solvent to obtain a precipitate of the carbon nanostructures modified with the cationic polyelectrolyte; b) pouring an organic or inorganic solvent over the precipitate of the carbon nanostructures modified with the cationic polyelectrolyte obtained in step iv) to produce a colloidal solution of the carbon nanostructures modified with the cationic polyelectrolyte; c) immobilizing the carbon nanostructures modified with the cationic polyelectrolyte on a conductive substrate; d) producing a solution of cholesterol dehydrogenase; and e) immobilizing cholesterol dehydrogenase on the carbon nanostructures modified with the cationic polyelectrolyte.

22. The method of producing the electrode according to claim 21, wherein steps from i) to iv) are repeated for the second time.

23. The method of producing the electrode according to claim 21 or claim 22, wherein a polybase or its salt having functional groups with a positively charged nitrogen atom is used as the cationic polyelectrolyte.

24. The method of producing the electrode according to claim 23, wherein poly(diallyldimethylammonium chloride) is used as the cationic polyelectrolyte.

25. The method of producing the electrode according to any one of claims 21 to 24, wherein nanostructures selected from the group consisting of carbon nanotubes, carbon nanodots, fullerenes, graphene and their mixture are used as the carbon nanostructures.

26. The method of producing the electrode according to claim 25, wherein single-walled, double-walled or multi-walled carbon nanotubes are used as the carbon nanotubes.

27. The method of producing the electrode according to any one of claims 21 to 26, wherein, in step i), water, toluene, chloroform, dimethylformamide or tetrahydrofuran is used as the solvent.

28. The method of producing the electrode according to any one of claims 21 to 27, wherein, in step b), water, toluene, chloroform, dimethylformamide or tetrahydrofuran is used as the solvent.

29. The method of producing the electrode according to any one of claims 21 to 28, wherein, step c) is carried out by depositing the colloidal solution of the carbon nanostructures modified with the cationic polyelectrolyte produced in step b) onto the conductive substrate and allowing it to dry to form a layer containing the carbon nanostructures modified with the cationic polyelectrolyte.

30. The method of producing the electrode according to any one of claims 21 to 29, wherein a metallic substrate or a carbon substrate is used as the conductive substrate.

31. The method of producing the electrode according to claim 30, wherein gold, platinum, silver or palladium is used as the metallic substrate.

32. The method of producing the electrode according to claim 30, wherein graphite, glassy carbon, graphene, carbon nanotubes or carbon paste is used as the carbon substrate.

33. The method of producing the electrode according to claim 32, wherein paste containing carbon nanotubes or graphene is used as the carbon paste.

34. The method of producing the electrode according to any one of claims 21 to 33, wherein, step d) is carried out by dissolving cholesterol dehydrogenase in a buffer or an aqueous salt solution having a pH value higher than 4.5.

35. The method of producing the electrode according to any one of claims 21 to 34, wherein, in step d), the solution of cholesterol dehydrogenase which contains cholesterol dehydrogenase in an amount of 3 to 15 U is produced.

36. The method of producing the electrode according to any one of claims 21 to 35, wherein step e) is carried out by depositing the solution of cholesterol dehydrogenase produced in step d) onto the layer containing the carbon nanostructures modified with the cationic polyelectrolyte formed in step c) and allowing it to dry to form a layer containing cholesterol dehydrogenase.

37. The method of producing the electrode according to any one of claims 21 to 36, wherein the method additionally comprises depositing protective layers after steps c) and e).

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