INTRODUCTION

The progress of modern analytical chemistry is closely associated with the increase of sensitivity and selectivity of the analysis, as well as with the development of new equipment that would allow quick and easy analysis of complex objects outside laboratory. These requirements have led to the emergence of a new trend in analytical chemistry -chemical sensors. One of the most promising types of chemical sensors is biosensors, containing bio-recognition element.

Biosensors can be characterized, above all, by extraordinary selectivity due to the high specificity of bio-recognition element, such as an enzyme, allowing in some cases separate identification of stereoisomers. Secondly, biosensors demonstrate fast response time that allows express analysis. Thirdly, the analysis with biosensors is highly sensitive because, in most cases, it refers to the kinetic methods based on measuring of reaction rate.

Biosensors are particularly promising in the analysis of organics, including biologically active substances. Acute need for such analysis is associated with high demands for quality control of food, medicines. It associated also with the importance of the definition of such substances in biological fluids for the purposes of diagnosis of large number of diseases. However, routine analysis of the above objects is complicated by the presence of large variety of components inside the matrix. That leads to the analysis with reduced sensitivity and selectivity. In fact, nowadays only chromatography allows to analyze such objects with confidence, suffering from such disadvantages as long duration and high cost of analysis, complex procedure of sample preparation and high demands for the purity of the reagents. Considering the absence of these disadvantages, biosensors are real alternative to the chromatographic methods in the analysis of organic substances in complex objects.

The research conducted so far for the development of new biosensors is limited, in most cases, to the glucose oxidase as a bio-recognition element. This enzyme allows to implement different approaches for enzyme immobilization on the electrode surface due to its high activity and stability. At the same time, other available enzymes from the oxidoreductases class, offer more opportunities for the selection of analyte and object of analysis. However, these enzymes can often be characterized by worse stability and activity that prevents the application of traditional immobilization approaches and directs research efforts to finding of new matrices for bio-encapsulation.

One of the promising methods of enzyme immobilization is their encapsulation in thin SiO 2 film on the electrode surface by sol-gel technology. This allows to keep them active and to ensure unhindered access of substrate molecules to the enzyme, increasing the sensitivity of the modified electrodes. The electrodeposition method acquired good reputation for deposition of such films on the electrode surface, allowing to obtain uniform and homogeneous coating. This method was previously proposed in our laboratory for encapsulating of glucose and hemoglobin. Another pathway for increasing of the biosensors sensitivity consists in the use of metallic and carbonaceous nanomaterials, which can be characterized by high surface area and catalytic properties.

CHAPTER 1. LITERATURE SURVEY

General overview of amperometric biosensors

Development of electrochemical sensors is one of the most popular areas of analytical chemistry, widely developed in recent years. The electrode is used as a transducer in such sensors contributing to their cheapness and availability. Owing to easy registration of electrical signal, electrochemical sensors first reached commercialization and are widely used in clinical, industrial, agrochemical and environmental analysis [1].

Electrochemical biosensors combine the analytical power of electrochemical techniques with unprecedented specificity of biological processes of recognition. The general idea of such sensors is appropriate immobilization bio-recognition element in close proximity to the electrode surface and generating electrochemical (more often amperometric or potentiometric) signal, the magnitude of which depends on the concentration of the analyte. The level of development of modern technologies can get tiny, cheap and easy to use biosensors, which are already exploited in many fields of analytical chemistry -analysis of food [2,3], the environmental [4] and clinical analysis [START_REF] Rastislav | Application of Electrochemical Biosensors in Clinical Diagnosis / R. Monošík[END_REF]. Unanimously adopted by the world scientific community as a powerful analytical methods, biosensors own chapters in modern textbooks about analytical chemistry [START_REF] Otto | Modern methods in analytical chemistry[END_REF][START_REF] David | Modern Analytical Chemistry / D. Harvey[END_REF][8].

The term "biosensor" usually refers to the use of any material of biological origin as a recognition element. Microorganisms, organelles, nucleic acids, antibodies, they all find their application in biosensors. However, the most popular are biosensors, containing an enzyme (s). This is because of relative simplicity of their preparation and the fact that enzymes, conceived as natural catalysts, are characterized by high specificity, efficiency and response rate. Therefore, below the term "biosensors" will refer to enzyme-based biosensors.

Concept of amperometric biosensors

Unlike many chemical reactions, electrochemical processes always occur at the interface between the electrode and the solution. Depending on conditions, the electrochemical measurements can be carried out in potentiometric (equilibrium) or potentiostatic (nonequilibrium) modes. In the first case, the experiment is carried out in static mode with no current flowing through the electrochemical cell. The established potential of electrode allows to determine the concentration of analyte in solution. Potentiometry -an important method in analytical chemistry, and new ionselective membranes that have been developed over the past 10-20 years, allow direct monitoring of many ions in complex samples using this method [START_REF] Evans Alun | Potentiometry and Ion Selective Electrodes[END_REF].

Potentiostatic or voltammetric methods of analysis are based on dynamic nonequilibrium situation, when applied to the electrode potential induces electrochemical reactions at the electrode-solution boundary. The electric current passing through the cell is different from zero. This current can be used for the characterization of occurring reaction and for detection any electroactive substances in solution. The advantages of voltammetry are a high sensitivity and selectivity, wide linear range, portable and cheap equipment, and a large number of different available electrodes [START_REF] Joseph | Analytical Electrochemistry[END_REF].

Immobilized on the electrode enzyme catalyzes some reaction, which can be schematically represented as follows:

Substrate + Co-Reactant(coenzyme)

𝑒𝑛𝑧𝑦𝑚𝑒 → Product + Co-Reactant(coenzyme)',
Therefore the choice of transducer depends primarily on the enzymatic system that is used in each case. For example, the enzymatic reaction of urease leads to changes in pH, so the best choice is pH-sensitive electrodes, while decarboxylase initiating carbon dioxide release can be used together with potentiometric gas sensors.

However, the use of voltammetry is advantageous for most enzymes, because of release or consumption of electroactive substances easily detectable with electrodes.

Historically amperometric biosensors can be divided into three generations [START_REF] Brian | Chemical Sensors and Biosensors[END_REF] (Fig. 1.1): 1) In the first-generation biosensors (Fig. 1.1a) the enzymatic oxidation of the substrate involves participation of dissolved oxygen. The first such biosensor was developed on the basis of glucose oxidase and Clark oxygen electrode [START_REF] Clark | Continuous recording of blood oxygen tensions by polarography[END_REF]. It allowed to measure the concentration of glucose by detecting the decrease in the concentration of dissolved oxygen consumed as a result of enzymatic reaction [START_REF] Updike | The enzyme electrode / S[END_REF]. However, these biosensors have significant limitations invoked by the need to maintain a constant concentration of dissolved oxygen and very low potential of its reduction (-0.7 V). Therefore, cathodic reduction of oxygen has been replaced by anodic oxidation of hydrogen peroxide released as a result of the reaction.

2) The second generation biosensors (Fig. 1.1b) uses so-called mediator or electrons-carrier involved in the enzymatic reaction. Mediators are usually molecules that can be easily and reversible oxidized and reduced on the electrode at low potential (e.g., ferrocene and ferrocyanides). Their role is electron transfer from the molecule of the enzyme to the electrode, inducing current, which depends on the substrate concentration. The use of mediators gave a significant boost to the development of new types of biosensors, while the problems remain such as an effective mediator immobilization on the electrode surface and very strict demands to the molecule (low redox potential, pH independence, lack of reaction with other components of biosensors) [START_REF] Dzyadevych | Amperometric enzyme biosensors[END_REF].

3) Third-generation biosensors (Fig. 1.1c) operate on the basis of direct electron transfer between the electrode and the active center of the enzyme.

This approach makes possible getting rid of any intermediary molecules and almost directly converts the concentration of the substrate on the measurable electrochemical signal. However, the design of such biosensors is not easy task, especially because the electrochemically-active group of the enzyme is usually located deep inside of the protein molecule beyond the shield protein groups [START_REF] Freire | Direct Electron Transfer : An Approach for Electrochemical Biosensors with Higher Selectivity and Sensitivity[END_REF].

Regardless of biosensor's generation, the enzyme must be firmly fixed in close proximity to the electrode, and the unobstructed diffusion of substrate to the enzyme as well as the reaction products to the electrode must exist.

As noted above, the concentration of analyte in solution is determined by the amount of current flowing through the working electrode. There are two methods of its measurement that can achieve the best results. The voltammograms (linear or cyclic) are being obtained by gradual change of the potential at the working electrode at a certain rate. The electrochemical reaction occurs at some point characterized by increased current and peak appearance at the voltammogram. The value of the peak is proportional to the concentration of electrochemically-active substances according to the equation of Randles-Sevcik [START_REF] Brian | Chemical Sensors and Biosensors[END_REF] (see Appendix B)

However, the methods with potential scanning is not very practical for the applications in biosensors due to the impact of charging current of the electrical double layer [START_REF] Budnikov | Fundamentals of modern electrochemical analysis[END_REF] and the considerable time required for one scan. A more convenient method is amperometry, where a constant potential is applied to the working electrode inducing an electrochemical reaction. At such conditions current firstly decreases sharply due to the depletion of near-electrode layer, and then goes to the constant value, which can be calculated from the modified equation Cottrell (see.

Appendix B). The value of quasistationary current under these conditions depends on the concentration of electrochemically-active substances, and the use of stirred solution reduces the thickness of the diffusion layer and leads to increased current.

Enzyme types used in biosensors

Enzymes -substances of protein nature that catalyze chemical reactions in living systems. At present several thousands of individual enzymes existing in living organisms are known [17]. The International Union of Biochemistry and Molecular

Biology has developed a 4-levels system of their classification and nomenclature in accordance with the type of reaction accelerated by corresponding enzyme [18].

According to this classification oxidoreductases are the first, and probably one of the largest classes among 6 types of enzymes. Such enzymes catalyze biological redox reactions that transfer electrons from one molecule to another. These enzymes are ideal for the creation of electrochemical biosensors given that all enzymatic reactions of oxidoreductases involve the electron transfer from one molecule to the other.

Oxidoreductases class can be divided into two subclasses, depending on the type of oxidant used. If molecular oxygen acts as an electron acceptor, thus turning into a molecule of hydrogen peroxide, these enzymes belong to oxidases. If a special molecule (coenzyme) acts as oxidant, these enzymes belong to the subclass of dehydrogenases. Among the variety of coenzymes (PQQ, FMN, TPP, Coenzyme A etc.), the two of them are most widespread in the oxidoreductase class: the nicotinamide adenine dinucleotide (NAD or NADP) or flavin adenine dinucleotide (FAD). These molecules are able to be oxidized and reduced in a reversible redox process.

The wide popularity of oxidases as biosensors sensitive elements is primarily evoked by relative ease of electrochemical detection of hydrogen peroxide at metallic electrodes. The electrochemical detection of dehydrogenases coenzyme NAD + /NADH is complicated (see paragraph 1.5.2), but they are also widely used in the development of biosensors.

The methods of biomolecules immobilization on the electrode surface

Biomolecules such as enzymes may lose their activity quickly in aqueous solutions because of their gradual oxidation or destruction of quaternary structure on the edge of the liquid/air [START_REF] Nikolas | Enzyme stabilization strategies based on electrolytes and polyelectrolytes for biosensor applications[END_REF]. Given the relatively high cost of pure enzyme preparations their cost-beneficial use requires the reusability of enzyme based biosensors. For this reason, the use of enzymes in solution is an exception, and research efforts are aimed at finding new ways of enzyme immobilization, i.e.

attachment to the surface.

When choosing an immobilization method one should pay attention to the retention of enzyme activity and conformation of the active center. In addition, enzyme should be in biocompatible environment protected from microbial attack and pollutants. Substrate molecules should be able to diffuse freely to it from the external solution [START_REF] Iqbal | Bioencapsulation within synthetic polymers (Part 1): sol-gel encapsulated biologicals[END_REF][START_REF] Sassolas | Immobilization strategies to develop enzymatic biosensors[END_REF]. Biosensors characteristics are very dependent on the method of enzyme immobilization. The purpose of this immobilization is to ensure close contact between the enzyme and the transducer, while maintaining (and sometimes even improving) stability of the enzyme. There are physical and chemical methods of immobilization, which consist in the following [START_REF] Wilhelm | Immobilized enzymes: methods and applications[END_REF][START_REF] Krajewska | Application of chitin-and chitosan-based materials for enzyme immobilizations: a review / B. Krajewska // Enzyme and Microbial Technology[END_REF]:

1) Adsorption. A simple and cheap method, but it is often reversible, i.e.

enzyme gradually desorbs from the surface during measurements. This leads to poor stability of biosensors.

2) Micro-encapsulation using solid or liquid membranes. This method is often used in first biosensors, it allows to place the biomolecules inside the semipermeable membrane in close contact with the transducer. The disadvantage of this method is the complexity and high cost of such membranes, and, most importantly, complicated diffusion of reactants through the membrane.

3) Encapsulation. The enzyme is mixed with a solution of polymer and then the polymerization is initiated. The result is a gel containing encapsulated enzyme. The absence of chemical bonding and biocompatible environment makes the enzyme more active. Unfortunately, enzyme can leach from the gel over time, resulting in signal loss.

4)

Crosslinking. The enzyme is bound using so-called bifunctional reagents such as glutaraldehyde, which could form a Schiff base with aminogroups of the protein. The mild binding method almost does not change steric configuration of the enzyme, but such electrodes are characterized by low mechanical properties and diffusion of the substrate through the material is rather slow.

5) Covalent binding. The chemical binding of the enzyme to the carrier by a variety of functional groups is used. This method provides the best stability of the immobilized enzyme, however, it is difficult and tedious, with a lack of results reproducibility. In addition, the chemical binding leads to disruption of steric configuration of the enzyme molecule, which leads to the degradation of its activity and denaturation.

Summing up the above, the choice of method of immobilization depends on the each particular case [START_REF] Linqiu | Immobilised enzymes: science or art? / L. Cao[END_REF]. However, it can be concluded that very tough or very weak binding of the enzyme does not lead to good results because of a lack of the electrode activity or stability respectively. In search of the optimal immobilization method the preference should be given to a "golden mean" -an encapsulation and cross-linking methods, they could provide a strong enzyme fixing together with the preservation of its activity [START_REF] Sassolas | Immobilization strategies to develop enzymatic biosensors[END_REF].

One of these soft methods is the encapsulation of enzyme in a polymer film of silicon oxide (SiO 2 ). Such material is biocompatible, has a high porosity and several other important advantages making promising the development of SiO 2 -based biosensors [START_REF] Gupta | Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects[END_REF].

Amperometric SiO2-based biosensors

Hybrid silica materials obtained by the sol-gel technology are widely used in electroanalytical chemistry [START_REF]intersection of functionalized sol-gel materials with electrochemistry[END_REF]. Interest to these materials is due to ease of synthesis, and their unique properties, including a variety of chemical compositions and structure (in the form of monoliths or thin films). Being solid inorganic substances, they have a high specific surface area (200-1500 m 2 /g) at the same time and threedimensional structure, consisting of a large number of open interconnected pores. It provides a high diffusion rate of analytes inside. Together with lots of available active sites this is a key factor in the development of highly sensitive electrochemical sensors [START_REF] Walcarius | Electroanalysis with Pure, Chemically Modified and Sol-Gel-Derived Silica-Based Materials / A. Walcarius // Electroanalysis[END_REF]. Another advantage of silica-based materials is the ease of modification with various mediators that can alter their characteristics, increasing the selectivity analysis or providing electrocatalytic properties [START_REF] Walcarius | Electroanalysis with Pure, Chemically Modified and Sol-Gel-Derived Silica-Based Materials / A. Walcarius // Electroanalysis[END_REF][START_REF] Walcarius | Electrochemical Applications of Silica-Based Organic-Inorganic Hybrid Materials / A[END_REF].

Recently it was shown that these materials can also be used to encapsulate biomolecules preserving their activity [START_REF]Biochemically active sol-gel glasses: the trapping of enzymes[END_REF][START_REF] Dave | Sol-gel encapsulation methods for biosensors[END_REF][START_REF]Organically modified sol-gel sensors / O[END_REF]. Silica-materials possess several key characteristics that make them promising for bioencapsulation. Simple lowtemperature synthesis method avoids protein denaturation and unique approach of polymer chains formation around the enzyme molecule, does not lead to a violation of its steric configuration [START_REF] Gupta | Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects[END_REF]. These materials may contain a high amount of water in the structure that improves the long-term stability of immobilized biorecognition elements [START_REF]Enzymes and Other Proteins Entrapped in Sol-Gel Materials / D[END_REF]. In addition, silica-materials have excellent biocompatibility and ability to protect against microbial attack [START_REF] Vivek | Immobilization of Biomolecules in Sol-Gels: Biological and Analytical Applications / V. Kandimalla[END_REF].

However, the SiO 2 -based materials have several shortcomings that need to be removed. First, it is a gradual leaching of modifier molecules from the film and the destruction of the film. This problem can be solved by the introduction of the structuring and stabilizing agents (e.g., surfactants and polyelectrolytes) [START_REF] Nadzhafova | Heme proteins sequestered in silica sol-gels using surfactants feature direct electron transfer and peroxidase activity[END_REF][START_REF]Surfactant-Induced Modification of Dopants Reactivity in Sol-Gel Matrixes / C. Rottman[END_REF][START_REF]silica-nanocomposite films electrogenerated on pyrolitic graphite electrode[END_REF].

Secondly, it is a need for a uniform distribution of enzymes in the film (without the formation of conglomerates), that can be achieved by selecting the appropriate method of SiO2-film obtaining.

Biomolecules encapsulation into the silica-matrix on the electrode surface

In general, the sol-gel method involves hydrolysis of the precursor (alkoxide) in acidic or alkaline medium with subsequent condensation and polycondensation of monomers leading to the formation of porous gel [START_REF] Brinker | Sol-Gel Science: the physics and chemistry of sol-gel processing[END_REF]: The properties of the formed gel, such as porosity, surface area, polarity, hardness, largely depend on the rate of hydrolysis and condensation reactions (Fig. 1.2), as well as on the choice of the precursor, molar ratio, choice of solvent, temperature, processes drying and aging [START_REF] Joseph | Sol-gel materials for electrochemical biosensors[END_REF]. Moreover, the process of aging may occur long time after the formation of the gel, forming additional bonds inside sol-gel matrix. During the aging process the solvent can be removed from the pores, which leads to a change in polarity and viscosity and to reduction of the pores diameter [START_REF] Brinker | Sol-Gel Science: the physics and chemistry of sol-gel processing[END_REF][START_REF] Kulwinder | Characterization of the Microenvironments of PRODAN Entrapped in Tetraethyl Orthosilicate Derived Glasses / K. K. Flora[END_REF].

For analytical purposes, sol-gel materials can be obtained either as monoliths or thin films [START_REF] Gupta | Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects[END_REF]. Monoliths can be from hundreds of micrometers to several centimeters of thickness and can effectively immobilize large number of (a)

(b) (c)
biomolecules that are kept inside because of its size and molecular weight. However, the main drawback of monoliths is very large response time due to slow diffusion. In addition, they usually do not find their use in electrochemistry due to lack of conductivity of thick layers of silica. One way to solve this problem can be the creation of composite bioelectrode [START_REF] Joseph | Sol-gel materials for electrochemical biosensors[END_REF] by mixing the sol-gel precursor with enzyme and conductive materials -carbon paste [START_REF] Pankratov | Sol-gel derived renewable-surface biosensors / I. Pankratov[END_REF], graphite [START_REF] Gun | Sol-gel derived, ferrocenyl-modified silicate-graphite electrode: Wiring of glucose oxidase / J. Gun[END_REF] or metal particles [START_REF] Bai | Gold nanoparticles-mesoporous silica composite used as an enzyme immobilization matrix for amperometric glucose biosensor construction[END_REF].

At the same time, thin sol-gel films with thickness less than one micrometer offer significantly faster diffusion of analyte to biorecognition centers guarantying fast response. Therefore they are considered more promising for application in electrochemical sensors [START_REF]Organically modified sol-gel sensors / O[END_REF].

There are several ways of enzyme immobilization within the sol-gel film on the electrode surface:

Covalent binding of the enzyme to the SiO 2 -matrix (Fig. 1.3) by carbodiimide coupling reaction was used for glucose oxidase [START_REF]Covalent immobilization of an enzyme (glucose oxidase) onto a carbon sol-gel silicate composite surface as a biosensing platform / X[END_REF] and lactate dehydrogenase [START_REF] Cheng-Li | Amperometric L-lactate sensor based on sol-gel processing of an enzyme-linked silicon alkoxide[END_REF],

but this method has not gained widespread use due to the loss of enzyme activity as result of binding to hard matrix. The so-called «sandwich»-configuration (Fig. 1.3) imply enzyme placing between two layers of sol-gel film. Such configuration have been used for the first time for glucose oxidase in work [START_REF]Glucose Biosensor Based on a Sol-Gel-Derived Platform / U. Narang[END_REF] and shown higher activity and fast response compared to conventional methods. Later it was also used for lactate dehydrogenase [START_REF] Ramanathan | Immobilization and Characterization of Lactate Dehydrogenase on TEOS Derived Sol-Gel Films[END_REF][START_REF]Immobilization of lactate dehydrogenase on tetraethylorthosilicate-derived sol-gel films for application to lactate biosensor[END_REF]. Unfortunately, the result of this configuration is uneven distribution of the enzyme throughout the film-modifier reducing the reproducibility of the analytical response.

Double-layer SiO 2 -film (Fig. 1.3) was used for immobilization of lactate oxidase [START_REF]Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate[END_REF] and peroxidase [START_REF] Stephen | Development of a sol-gel based amperometric biosensor for the determination of phenolics[END_REF]50], together with the osmium redox mediator. But the enzyme and the mediator in this configuration may contact only at the boundary between two layers, making efficiency of such biosensors significantly lower.

The encapsulation of enzyme in whole thin SiO 2 -film (with or without a mediator) is one of the most popular methods. Thus, a uniform distribution of modifier-molecules in the film and close contact with the electrode can be achieved.

The important key factors of thin film formation are homogeneity and thickness of the film, adhesion to the electrode, resistance to cracking and minimization of possible enzyme leaching. The film thickness is a major parameter for the obtained modified electrodes, as its increase slows diffusion of the analyte to the active centers inside the film, reducing the response [START_REF] Vivek | Immobilization of Biomolecules in Sol-Gels: Biological and Analytical Applications / V. Kandimalla[END_REF]. However, the amount of immobilized enzyme is low in very thin films, which also leads to the signal drop.

Methods of thin sol-gel films obtaining on the electrode surface

The main methods for the obtaining of thin sol-gel coatings on the surface of the electrodes is dip-coating and spin-coating [START_REF]Review of sol-gel thin film formation[END_REF], less common methods are dropcoating and spray-coating (Table 1.1). Despite the variety of methods for the obtaining of thin sol-gel films several difficulties need to be got round on the road to a successful application of such films in biosensors [START_REF] Gupta | Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects[END_REF]. Firstly, most of the techniques do not allow to reproducibly modify surfaces with complex morphology, such as fibers. Second, for achievement of measurable signal level, thin films require large biomolecules content, and this can create problems for the enzymes that are insoluble or precipitate in the sol. Thirdly, homogeneous films often require the presence of significant amounts of alcohol as a viscosity modifier, that can lead to denaturation of immobilized biomolecules.

Finally, unlike the monoliths with slow processes of drying and aging, these processes for thin films occur simultaneously and very fast. This could potentially lead to the film cracking and dehydration of immobilized biomolecules.

Thus, the researchers face with the task of developing new, simple and effective ways of electrodes modification with bio-composite films SiO 2 -enzyme that would allow to solve the above problems. One of the promising methods that can be used for this purpose is electrochemically-assisted deposition.

Electrochemically-assisted deposition method

The method of electrochemically-assisted sol-gel deposition (EAD) is a relatively new way for getting thin coatings, it was first described in 1999 [START_REF] Shacham | Electrodeposition of Methylated Sol-Gel Films on Conducting Surfaces[END_REF]. This method now can be applied only to the conductive surfaces, but can solve the basic problem of the traditional methods of sol-gel processing -impossibility of modification of surfaces with complex morphology and small size [START_REF] Shacham | Pattern recognition in oxides thin-film electrodeposition: Printed circuits[END_REF].

This method consists in immersion of the electrode in a solution of prehydrolyzed sol-gel precursor and application of sufficient negative potential (EAD at positive potential is also possible [START_REF] Collinson | Electrodeposition of Porous Silicate Films from Ludox Colloidal Silica[END_REF]) over some time (Fig. 1.4): When applying a negative potential to the electrode the reactions of water (and oxygen) reduction are occurring according to the scheme [START_REF]Electrochemically deposited sol-gel-derived silicate films as a viable alternative in thin-film design[END_REF]:

2H 2 O + 2ē → 2OH -+ H 2 O 2 + 2H 2 O + 4ē → 4OH - O 2 + 2H 2 O + 2 ē → H 2 O 2 + 2OH - (1.1)
Hydroxyl-ions generated by these reactions, enhance pH in the near-electrode area, significantly accelerating the polycondensation reaction of SiO 2 -precursor (Fig.

1 .2b, c) and the formation of SiO 2 -film on the electrode surface. Since the time of potential application is typically less than a few minutes and even seconds, it does not lead to significant changes in the overall pH of the whole solution. Thickness of the formed film can be easily changed by controlling the value [START_REF] Shacham | Electrodeposition of Methylated Sol-Gel Films on Conducting Surfaces[END_REF] and time [START_REF]the functionalization of macroporous electrodes / F. Qu[END_REF] of negative potential applying. This affects the amount of the formed catalyst (OH --ions)

and thus -the rate of the precursor polycondensation. In particular, the EAD method was used to obtain ultra-thin SiO 2 -films with ordered vertically-oriented pores [START_REF]Electrochemically assisted self-assembly of mesoporous silica thin films[END_REF].

Since the gelation and drying processes during EAD occur independently of each other, coatings obtained by this method are much more porous than ones obtained by classical methods of sol-gel deposition [START_REF]intersection of functionalized sol-gel materials with electrochemistry[END_REF][START_REF] Collinson | Electrodeposition of Porous Silicate Films from Ludox Colloidal Silica[END_REF]. The higher number of pores facilitates diffusion of reactants through the film increasing the sensitivity and response time, which is especially important for electrochemical sensors. The addition to the sol-gel solution during EAD can be used for the formation of films containing encapsulated mediators such ferrocenedimethanol and ruthenium bipyridyl [START_REF]Electrochemically deposited sol-gel-derived silicate films as a viable alternative in thin-film design[END_REF].

The EAD method is also suitable for the encapsulation of biomolecules, particularly due to lower amount of produced alcohol [START_REF] Collinson | Electrodeposition of Porous Silicate Films from Ludox Colloidal Silica[END_REF][START_REF]Electrochemically deposited sol-gel-derived silicate films as a viable alternative in thin-film design[END_REF], which is formed during deposition and can lead to denaturation of proteins. It was used for the immobilization of biomolecules such as glucose oxidase [START_REF]A glucose biosensor based on chitosan-glucose oxidase-gold nanoparticles biocomposite formed by one-step electrodeposition[END_REF][START_REF] Jia | One-Step Immobilization of Glucose Oxidase in a Silica Matrix on a Pt Electrode by an Electrochemically Induced Sol-Gel Process[END_REF][START_REF] Oksana | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon / O. Nadzhafova[END_REF], hemoglobin [START_REF]silica-nanocomposite films electrogenerated on pyrolitic graphite electrode[END_REF][START_REF] Oksana | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon / O. Nadzhafova[END_REF], peroxidase [START_REF] Yang | Simple approach for efficient encapsulation of enzyme in silica matrix with retained bioactivity[END_REF]. These biomolecules are among the most stable and can withstand relatively harsh chemical conditions, so widely used in the development of new types of biosensors [START_REF] Wilson | Glucose oxidase: an ideal enzyme / R. Wilson[END_REF]. However, the application of EAD method for the immobilization of other types of enzymes is still unresolved and poorly researched issue. In addition, owing to the vast possibilities of the thickness control of the formed film this method is of interest for the modification of nanostructured electrodes [START_REF] Shacham | Pattern recognition in oxides thin-film electrodeposition: Printed circuits[END_REF][START_REF]the functionalization of macroporous electrodes / F. Qu[END_REF][START_REF] Mandakini | Quantitative Control over Electrodeposition of Silica Films onto Single-Walled Carbon Nanotube Surfaces[END_REF][START_REF] Stefanie | Local electrocatalytic induction of sol-gel deposition at Pt nanoparticles[END_REF].

Application of nanomaterials in biosensors development

Nanomaterials having the size less than 100 nm in at least one dimension, are widely used in modern analytical chemistry, particularly in the field of chemical sensors. Their uniqueness is associated with significant differences in the properties of nanosized particles from those of macrosize made from the same material [START_REF] Pratibha | Prospects of Nanomaterials in Biosensors[END_REF].

This creates the possibility of modification of the nanomaterials properties by varying their size [START_REF] Tewodros | Recent advances in nanostructured chemosensors and biosensors[END_REF]. They have exceptional thermal and electrical properties, high activity and surface area, thus can be used to improve the sensitivity and response time of (electrochemical) sensors [START_REF] Joseph | Nanomaterial-based electrochemical biosensors[END_REF]. Nanosize materials were used for the achievement of direct electron transfer between the electrode and the enzyme, for acceleration of electrochemical reaction, for amplification of the biorecognition element signal etc. [68]. 1.3.1. General properties of nanomaterials: nanoparticles, nanotubes, nanofibers.

Nanomaterials that have found application in the development of sensors can be classified by the dimensionality [START_REF] Valentini | Nanomaterials and Analytical Chemistry[END_REF][START_REF]Functional One-Dimensional Nanomaterials: Applications in Nanoscale Biosensors[END_REF]:

zero-dimensional (0D) nanomaterials. These include particles with the size in all three dimensions less than 100 nm, nanoparticles and quantum dots;

one-dimensional (1D) nanomaterials. These particles have a size larger than 100 nm in only one dimension, the two others do not exceed 100 nm. These include a variety of nanofibers, nanotubes, nanowires; two-dimensional (2D) nanomaterials. Such materials have a size greater than 100 nm in two dimensions, representing a variety of nanosheets and nanofilms with thickness less than 100 nm. In recent years, rapid development of sensors based on this type of material is associated with the discovery of graphene [71,72].

Although nanomaterials may be made of any material, only metal or carbon nanomaterials and conductive polymers are mainly used in electrochemistry, because of their high electrical conductivity.

Gold and platinum nanoparticles are the most widely used type of nanomaterials for the development of amperometric biosensors [START_REF] Eugenii | Electroanalytical and Bioelectroanalytical Systems Based on Metal and Semiconductor Nanoparticles[END_REF]. Several layers of nanoparticles deposited on the electrode lead to the formation of porous layer with large surface area, which can adsorb and concentrate a large number of substances [START_REF] Fang | Electrochemical sensors based on metal and semiconductor nanoparticles[END_REF]. They can also be considered as an array of nanoelectrodes with own advantages [START_REF] Wenlong | Colloid chemical approach to nanoelectrode ensembles with highly controllable active area fraction[END_REF]. Gold nanoparticles are able to provide stable immobilization of enzymes while preserving their activity, to achieve the direct electron transfer between the electrode and the enzyme without addition of mediators [START_REF] José | Gold nanoparticle-based electrochemical biosensors[END_REF][START_REF] Shaojun | Synthesis and electrochemical applications of gold nanoparticles[END_REF]. Platinum nanoparticles are mainly used in amperometric biosensors based on oxidases, due to their ability to greatly facilitate oxidation and reduction of hydrogen peroxide and oxygen [START_REF]Amperometric glucose biosensor based on integration of glucose oxidase with platinum nanoparticles/ordered mesoporous carbon nanocomposite / X[END_REF][START_REF] Hrapovic | Electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes[END_REF][START_REF] Minhua | Electrocatalysis on platinum nanoparticles: particle size effect on oxygen reduction reaction activity[END_REF].

However, of the greatest interest in the development of electrochemical biosensors are zero-dimensional materials [START_REF] Joseph | Nanomaterial-based electrochemical biosensors[END_REF][START_REF]Functional One-Dimensional Nanomaterials: Applications in Nanoscale Biosensors[END_REF]. Due to the considerable length-todiameter ratio, they can act as nanowires, increasing conductivity of film-modifier and connecting the electrode surface with molecules encapsulated in the film [START_REF] Joseph | Nanomaterial-based electrochemical biosensors[END_REF].

Metal nanofibers can be used for direct detection of biological and chemical substances [START_REF] Fernando | Nanowire-Based Biosensors / F[END_REF], but their use in the design of enzymatic biosensors, except several examples [START_REF]Platinum nanowire nanoelectrode array for the fabrication of biosensors[END_REF][START_REF] Qu | Electrochemical biosensing utilizing synergic action of carbon nanotubes and platinum nanowires prepared by template synthesis[END_REF], is not enough explored.

Among carbon nanomaterials carbon nanotubes (CNT) attract great interest since their discovery [START_REF] Sumio | Helical microtubules of graphitic carbon / S. Iijima[END_REF]. Single-walled CNT consist of a monatomic layer of graphite rolled into a cylinder, which has a large length-to-diameter ratio. Multiwalled CNT consist of several cylinders of different diameters, placed one inside another (Fig. 1.5). CNT have unique electrical, mechanical and structural properties that make them very attractive for use in electrochemical sensors [START_REF]New materials for electrochemical sensing VI: Carbon nanotubes / A[END_REF][START_REF] Lourdes | Role of carbon nanotubes in electroanalytical chemistry: a review[END_REF]. High sensitivity of CNT conductivity to the adsorbed molecules allows their use as nanoscale DNAsensors, and the ability to accelerate the electron transfer of many important biomarkers can significantly improve the characteristics of CNT-based enzyme electrodes [START_REF] Joseph | Carbon-Nanotube Based Electrochemical Biosensors[END_REF]. Also CNT can accumulate important biomolecules (e.g., nucleic acids [START_REF] Joseph | Carbon-nanotube-modified glassy carbon electrodes for amplified label-free electrochemical detection of DNA hybridization[END_REF]), and neutralize the process of the electrode surface poisoning by reaction products [START_REF] Musameh | Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes[END_REF].

Possibility of H 2 O 2 and NADH detection at low potential and limited surface passivation during the oxidation of NADH makes CNT an ideal material for use in amperometric biosensors based on oxidases and dehydrogenases [START_REF] Joseph | Carbon-Nanotube Based Electrochemical Biosensors[END_REF] . Moreover, studies have shown that vertically-oriented CNT can be used as a direct conductor between the electrode and the active center of the enzyme (which is usually located deep inside the molecule and isolated by protein chains) that covalently bound to the end of nanotube [START_REF]Protein electrochemistry using aligned carbon nanotube arrays[END_REF][START_REF] Fernando | Long-range electrical contacting of redox enzymes by SWCNT connectors[END_REF]. a b

Methods of the electrodes modification with carbon nanotubes. Method of electrophoretic deposition

Successful implementation of CNT in amperometric biosensors requires adequate control of their chemical and physical properties, their functionalization and immobilization on the surface [START_REF] Joseph | Carbon-Nanotube Based Electrochemical Biosensors[END_REF]. Simple mixing CNT with a solution of the enzyme in most cases leads to very low final concentration of nanotubes and often to enzyme denaturation or aggregation of CNT.

There are two basic approaches for surface modification with CNT: direct synthesis of CNT from metal catalysts located on the electrode surface, or surface modification with pre-synthesized CNT obtained by various techniques. Direct methods of synthesis allow to obtain a big number of firmly fixed on the electrode surface CNT and sometimes even to get vertically-oriented CNT [START_REF]Vertically Aligned Carbon Nanotube Electrodes Directly Grown on a Glassy Carbon Electrode[END_REF]. However, the most synthesis methods require high temperatures, pressures and sophisticated equipment, which limit their use in ordinary laboratories. Therefore most of modified electrodes are being obtained by deposition of CNT dispersion and subsequent drying or preparation of composite electrodes based on CNT mixed with carbon paste [START_REF]Carbon nanotube purification: preparation and characterization of carbon nanotube paste electrodes[END_REF][START_REF] María | Enzymatic Biosensors Based on Carbon Nanotubes Paste Electrodes[END_REF], teflon [START_REF] Joseph | Carbon nanotube/teflon composite electrochemical sensors and biosensors[END_REF], polymers [START_REF]Carbon nanotube-polymer composites: Chemistry, processing, mechanical and electrical properties[END_REF], ceramics [START_REF] Biuck | Simultaneous determination of acetaminophen and dopamine using SWCNT modified carbon-ceramic electrode by differential pulse voltammetry[END_REF].

Because the synthesized CNT usually contain many impurities of other allotropic modifications of carbon their purification before use by treating with acidoxidants is often necessary [98]. Besides elimination of impurities, this treatment also aims to create carboxyl functional groups on the places of defects in the structure of CNT. The presence of these groups enables covalent immobilization of biorecognition molecules or integration of CNT in polymer structure [99].

The limitation on the way of broad application of CNT in the development of biosensors is their negligible solubility in most solvents (including complete insolubility in inorganic solvents) [100], which makes the preparation of composite electrodes with high content of CNT quite challenging. Moreover, the difficulties of CNT manipulating are related to their small size and tendency to aggregation that prevents the formation of homogeneous and reproducible coatings on the electrode surface [101]. Therefore, the additives are often used to improve the dispersion of CNT in solvents such as surfactants, nafion, chitosan, DNA and others [102].

Two conditions are desirable for fabrication of enzyme electrodes based on CNT: a) a sufficiently high content of CNT in the final film-modifier in order to take full advantage of their properties, and b) the biocompatibility and mild conditions of bio-composite electrode fabrication to ensure the retention of the enzymatic activity (hence the undesirability of organic solvents and a large quantity of surfactants).

Given this, it would be optimal simultaneous deposition and concentration of CNT from the aqueous solution. To date, CNT-coatings, obtained by this method are mainly used for the creation of new composite materials [108], field emission devices [109,110],

supercapacitors [111,112], fuel cells [113][114][115], and for biomedical applications [116][117][118]. However, the EPD method, due to inherent advantages such as the possibility of CNT deposition from low-concentrated aqueous solutions can be used for the fabrication of CNT-matrix for amperometric biosensors. Currently, in the literature there are only a few examples of electrophoretically-deposited CNT application (as a part of CNT-polyaniline composite) for construction of biosensors based on cholesterol oxidase [119] and glycerol dehydrogenase [120], but the influence of the characteristics of CNT layer in the above works is not discussed.

Amperometric biosensors based on SiO2 and oxidases

Overview of oxidases used in amperometric biosensors

Oxidases subclass contains more than 100 enzymes that catalyze specific oxidation of many important biologically-active organic compounds by molecular oxygen [START_REF] Joseph | Carbon-Nanotube Based Electrochemical Biosensors[END_REF]. In most cases, enzymatic reactions catalyzed by oxidases are following:

𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 + О 2 𝑜𝑥𝑖𝑑𝑎𝑠𝑒 → 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 + Н 2 О 2 (1.2)
The enzyme glucose oxidase (GOx) -"pioneer" in the field of amperometric biosensors, the rapid development of this industry began from a message about the first GOx-based biosensor [121]. The reason of its popularity is in the spread of diabetes disease, which affects the people around the globe. The regular screening of glucose concentration in the blood becomes for them a daily routine task and amperometric biosensors may be the solution of this problem [122]. However, the popularity of GOx is also linked to its unique properties -a high specificity, activity and stability [START_REF] Wilson | Glucose oxidase: an ideal enzyme / R. Wilson[END_REF]. That is the reason for its applicability for testing new methods of In addition to the above, there are existing biosensors based on xanthine oxidase, ascorbate oxidase, bilirubin oxidase, lysine oxidase and other enzymes [1].

There is a considerable number of works about the immobilization of mentioned enzymes into SiO 2 -film by sol-gel method for the development of amperometric biosensors. This particularly concerns cholesterol oxidase [139,141,[155][156][157], lactate oxidase [START_REF]Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate[END_REF]126,158,159], tyrosinase [160][161][162][163], glucose oxidase [START_REF]Glucose Biosensor Based on a Sol-Gel-Derived Platform / U. Narang[END_REF][START_REF] Oksana | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon / O. Nadzhafova[END_REF][164][165][166][167]. Among other types of enzymes choline oxidase has an important substrate -choline, the determination of which is necessary in biomedical practice as well as in the analysis of food. In addition, this enzyme can be used to analyze important inhibitors -pesticides. However, in the literature we found only a few reports of the ChOx immobilization in SiO 2 -film [168,169]. Taking into account the mentioned above properties of SiO 2 -materials, the development of ChOx-and SiO 2based biosensors is a promising possibility.

Problems of electrochemical detection of H 2 O 2

As noted above (see section 1.1.1) the indicator reaction of the first-generation oxidases-based biosensors may be electrochemical detection of consumed oxygen by its reduction on the electrode at potential -0.7 V. Although the first known biosensors used this principle, this approach has some significant drawbacks leading to its renunciation. First, this is significant variation of oxygen content depending on pH, temperature and composition of the solution, and secondly, it is the simultaneous reduction of hydrogen peroxide at the same potential. Therefore the electrochemical detection of hydrogen peroxide, which is one of the products of enzymatic reactions of almost all oxidases, is considered more promising [170]. The detection can be performed by its oxidation at potential about 0.6 V -the dissolved oxygen does not interfere with the reaction under these conditions.

However, the use of such biosensors in the analysis of real objects faces the problems associated with side reactions that may occur at the electrode at this potential. For example the analysis of biological fluids is complicated due to a lot of components may be oxidized at the electrode at potential 0.6 V, such as ascorbic acid, dopamine, bilirubin and some others [START_REF] Brian | Chemical Sensors and Biosensors[END_REF].

Amperometric biosensors based on SiO2 and dehydrogenases

Overview of dehydrogenases used in amperometric biosensors

Dehydrogenases subclass includes more than 300 enzymes that use coenzyme NAD + /NADH (or its modification NAD(P) + /NAD(P)H) as an electron acceptor.

Dehydrogenases can (due to reversibility of NAD + /NADH) also catalyze the reverse reaction in most cases [187]. In general, the enzymatic reaction of dehydrogenases can be represented as:

𝑆𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 + 𝑁𝐴𝐷 + 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒 ↔ 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 + 𝑁𝐴𝐷𝐻 (1.3)
The number and diversity of dehydrogenases provide more choices for the construction of various amperometric biosensors. The absence of oxygen in the enzymatic reaction scheme can eliminate most of the disadvantages associated with it (difficulty of detecting. interfering influence, etc.) [188]. The reversibility of enzymatic reactions allows to expand analytical applications with the ability to determine the reaction product instead of the substrate. Nevertheless, biosensors based on dehydrogenases are much less common than ones on the basis of oxidases. This is associated with the difficulty of electrochemical detection of NAD + /NADH (see paragraph 1.5.

2)

The most widespread enzymes from the dehydrogenases subclass used in the development of biosensors (alcohol dehydrogenase, glucose dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase) more or less duplicate the functions of the corresponding enzymes of oxidases subclass (see paragraph 1.4.1) and their applications. However, alcohol dehydrogenase is used more than analog from oxidases due to its higher activity and stability in the sensors and much higher specificity towards ethanol [145]. Lactate dehydrogenase is also characterized by higher selectivity than lactate oxidase [189].

(Form)aldehyde dehydrogenase -an enzyme that catalyzes the oxidation of formaldehyde to formic acid. Besides the coenzyme NAD + , the reaction also requires the presence of coenzyme glutathione. Biosensors based on this enzyme can be used to determine the formaldehyde having allergic, mutagenic and toxic effects in the food, pharmaceutical and cosmetic industries [190][191][192][193]. The disadvantage of this enzyme is low activity, high cost and difficulty of application due to the need of two coenzymes [193].

Glycerol dehydrogenase provides oxidation of glycerol by coenzyme NAD + .

Biosensors based on this enzyme may be useful for wine quality control during fermentation [194,195] as well as in clinical analysis of blood [196]. At the same time, the lack of selectivity of this enzyme and reversibility of enzymatic reaction are reported [197].

Sorbitol dehydrogenase (DSDH) -an enzyme that catalyzes the conversion of polyhydric alcohol sorbitol to fructose. Measurement of sorbitol content is important in the analysis of diabetic food and clinical analysis to prevent the development of diabetes. To date there are only several reports on the biosensors development based on this enzyme [198][199][200][201], among them only in work [198] it was used for the real objects analysis. In addition, immobilized DSDH can be used for the design of the bioreactor for electro-enzymatic synthesis [202,203].

Malate dehydrogenase catalyzes the oxidation of malic acid and its salts.

Biosensors based on this enzyme can be used to determine the malic acid in food:

fruits, juices and wines, where it affects the organoleptic [187,204,205].

3-hydroxybutyrate dehydrogenase can be used in the biosensors for clinical analysis of blood, where the determination of 3-hydroxybutyrate is important to avoid life-threatening diabetic ketoacidosis in diabetic patients [206][207][208].

As mentioned above, the data mainly is missing in the literature about the immobilization of dehydrogenases in SiO 2 -film by sol-gel method and the development of amperometric biosensors based on such modified electrodes. Among all the above enzymes such procedure has been described only for lactate dehydrogenase [209][210][211] and malate dehydrogenase [212]. Given the small number of publications and the importance of substrate the point of interest is the development of biosensor based on sorbitol dehydrogenase immobilized in SiO 2 -film.

Problems of electrochemical detection of NAD + /NADH

The coenzyme NAD + serves as an electron acceptor in most enzymatic A particular interest is the presence of well-developed aromatic system in phenothiazine dyes that determines their ability to be easily and firmly adsorbed on carbon surfaces, including carbon nanotubes [233,234]. Such combination of nanostructured electrode and mediator leads to a synergistic effect -the potential decrease and the increase of the sensitivity and stability NADH detection [235,236].

This allows to apply this approach in the development of dehydrogenase-based biosensors [189,[237][238][239].

Conclusions from the literature survey

Analysis of the literature data has shown that electrochemical biosensors is a promising branch of the sensor development. An approach that deserves attention for the immobilization of biomolecules on the electrode surface is their encapsulation into a thin polymer film of silica, due to the large porosity and biocompatibility of the latter. However, there are unresolved issues about the use of such bio-composite films in the biosensors development, e.g., the achievement of strong enzyme fixing in the film, as well as maintaining its sufficient activity. In addition, the fabrication of SiO 2 -films by traditional methods does not always lead to reproducible results.

The method of electrochemically-assisted deposition is an alternative to this methods allowing to obtain reproducible porous films with controlled thickness. On the example of glucose oxidase and hemoglobin this method was successfully applied also for bioencapsulation, but information about its application for other types of enzymes, particularly dehydrogenases, is absent in the literature.

The use of nanomaterials can significantly improve the analytical characteristics of biosensors based on oxidases and dehydrogenases, including sensitivity and selectivity. The platinum nanoparticles are promising for the choline oxidase immobilization, since they can increase the sensitivity of detection of hydrogen peroxide. Carbon nanotubes are suitable for the stable and low potential detection of coenzyme NADH, so they can be used for the immobilization of sorbitol dehydrogenase. However, there is not much information in the literature about the selection of nanomaterials and the methods of their immobilization.

CHAPTER 2. EXPERIMENTAL PART.

Chemicals and reagents

Enzymes

Three enzymes from the oxidoreductases class were used in this work:

-Glucose oxidase (GOx, EC 1.1. Solution with concentration 10 mg/mL (activity 100 units/mg). Isoelectric point 4,3.

Enzyme solutions (except DSDH) were prepared by dissolving an appropriate amount (final concentration 10 mg/mL) in 0.067 M PBS (pH 6.0) and stored at 4С

when not used.

Reagents for sol-gel synthesis.

For the synthesis of SiO 2 -based sol the tetraethoxysilane (TEOS, 98%, «Alfa Aesar»), concentrated hydrochloric acid (HCl, 36%, «Prolabo») and deionized water from a «Purelab Option» water purification system were used.

The polyelectrolytes and surfactants were added to the sol as additives: poly- Addition of positively-charged polyelectrolytes and surfactants was intended for improvement of the enzyme interaction with silica-groups of the sol-gel film.

Silaca-groups are negatively charged at the pH used during the sol-gel synthesis (5 -7) due to their deprotonation [240]. This results in electrostatic repulsion taking into account the negative charge of the enzyme molecules at such pH. Therefore, the use of positively-charged polyelectrolyte, that plays a role of stabilizing agent between SiO 2 and protein, leads to significant improvements of the enzyme encapsulation [241]. In addition, cationic surfactants, such as CTAB, improve structure of the solgel film and can enhance the response stability of the encapsulated in SiO 2 -film biomolecules due to their inclusion into micelles [START_REF] Nadzhafova | Heme proteins sequestered in silica sol-gels using surfactants feature direct electron transfer and peroxidase activity[END_REF][START_REF]silica-nanocomposite films electrogenerated on pyrolitic graphite electrode[END_REF].

While immobilizing coenzyme NAD + into SiO 2 -film 3-glycidoxypropyltrimethoxysilane (GPS, 98%, «Sigma») was used as bonding agent. It is able to bind the adenine residue in the coenzyme molecule and react with silanol-groups in the condensation reaction [203].

Reagents for voltammetric measurements.

All solutions were prepared using deionized water by dissolving of accurate amounts and/or appropriate dilution. Standardization of solutions was performed using titrimetry.

1,1'-Ferrocenedimethanol (98%, «Aldrich»), hydrogen peroxide (H 2 O 2 , 35%, «Acros»), oxidized and reduced forms of β-nicotinamide adenine dinucleotide (NAD + /NADH, 98%, «Sigma») were used as electrochemical probes.

Glucose («Acros»), D-sorbitol (98%, «Sigma») and choline chloride (97%, «Fluka») were used as substrates of the corresponding enzymes. Solutions of glucose were left to mutorotate at least for 24 h before using.

The voltammetric measurements were conducted using phosphate and Tris-HCl buffer solutions as background electrolytes unless otherwise specified. Acidity and composition of buffer solutions were chosen taking into consideration optimal pH of enzymatic activity and the absence of interfering influence of solution components for the substrate determination.

The adsorption of dye methylene green (MG, >80%, «Sigma») was used for the determination of electroactive surface area of electrodes.

Substances studied for interfering influence.

To investigate the interfering effect on the determination of sorbitol the number of carbohydrates that can be found in the food was chosen as well as representatives of the homologous series of polyhydric alcohols, including sorbitol stereoisomer (mannitol). The components that can be part of cosmetic products (glycerol, sodium lauryl sulfate) and biological fluids (ascorbic acid, urea) were tested as well. All solutions were prepared by dissolving accurate amounts of substances or by dilution of initial solutions. Iron(III) nitrate, acidified to prevent its hydrolysis, was used for ascorbic acid masking.

To investigate the interfering effect on the determination of choline we studied mono-and disaccharides that can be part of the food (glucose, sucrose, lactose), substances that are part of the biological fluids (uric and ascorbic acid, urea), ethanol and some metal ions (Pb (II), Zn (II), Cu (II), Fe (III)), which are classical inhibitors of enzymes. All solutions were prepared by dissolving accurate amounts of substances or by dilution of initial solutions.

Apparatus

All -For the purpose of comparison platinum and gold macroelectrodes were useds.

For Pt-Nfb and GCE a specially designed Teflon cell were used in which the working electrode is located on the bottom side, and its required surface area is limited by rubber ring (ø = 6 and 9,5 mm). The electrode was connected to the potentiostat using copper wire and silver glue (див. Scheme 2.1 для Pt-Nfb).

Scheme 2.1. The connection way of Pt-Nfb to electrochemical cell.

For electrophoretic deposition of CNT a specially designed device was used consisting of a DC source of alternating voltage and two steel plates-electrodes that were placed strictly parallel to each other at a distance of 6 mm (Fig. 1.6). Plate, which acted as an anode, was shorter providing possibility of GCE connection.

The study of electrodes surface was carried out by method of scanning electron microscopy (using commercial microscope Hitachi FEG S4800, SCMEM, University of Nancy) and by atomic-force microscopy (using a microscope Asylum Research MFP-3D-Bio).

To ensure continuous mixing of solutions magnetic stirrers with adjustable speed was applied. Acidity of the solution was checked using pH-meter Mettler Toledo S220 and Piccolo HI 1290.

Argumentation of the choice of objects and methods

Modification of electrodes by electrochemically-assisted deposition method

The modification of electrodes with SiO 2 -film was performed by electrochemically-assisted deposition method using alcohol-free sol composition, which does not inhibit the enzyme activity [START_REF] Oksana | Direct electrochemistry of hemoglobin and glucose oxidase in electrodeposited sol-gel silica thin films on glassy carbon / O. Nadzhafova[END_REF].

-For the preparation of the sol for GOx immobilization 2.28 mL of TEOS, 2.0 mL of H 2 O, and 2.5 mL of 0.01 M HCl were mixed with a magnetic stirrer for 16 h. Then, prior to introduction of the enzyme in the medium, 1.66 mL of 0.1 M NaOH was added to neutralize the sol (to avoid possible enzyme denaturation in acidic medium). The enzyme solution (50 μL of PBS (0.067 M, pH 6.0) and 100 μL of 10 mg/mL GOx solution) was added to 0.5 mL of the hydrolyzed sol and left to stay for 1 h.

-For the preparation of the sol for ChOx immobilization 0,21 mL of TEOS, 0,15 ml of H 2 O and 0,26 mL of 0,01 М HCl were mixed with a magnetic stirrer for 12 h. Prior to EAD 0,4 mg of CTAB, 0,03 mL of 0,067 М PBS (рН 6,0) and 0,01 mL of ChOx solution (10 mg/mL) were added to 0.5 mL of the hydrolyzed sol.

-For the preparation of the sol for DSDH immobilization 2.28 mL of TEOS, 2.0 mL of H 2 O, and 2.5 mL of 0.01 M HCl were mixed with a magnetic stirrer for 16 h. The final sol was diluted three times with pure water and, then, a 100 μL aliquot of this solution was mixed with 100 μL of PDDA (20 wt% in water) and 100 μL of DSDH solution.

-For the preparation of the sol for co-immobilization of DSDH and NAD+ [203] 2.28 mL of TEOS, 2.0 mL of H 2 O, and 2.5 mL of 0.01 M HCl were mixed with a magnetic stirrer for 16 h. The sol was diluted five times with water and its 20 μL aliquot was mixed with 10 μL of 20% PEI-solution, 10 μL ofNAD + -GPS solution (prepared by mixing 25 mg of NAD + and 37.6 mg of GPS in 400 μL of Tris-HCl buffer solution pH 7.5 for 14 h at room temperature) and 20 μL of DSDH-solution.

Prepared sol was introduced in the electrochemical cell where some negative potential was applied to the working electrode in order to initiate the generation of OH-ions (typically from -1.1 to -1.3 V). Potential and/or duration of its application were optimized for each individual case.

After electrodeposition the working electrode was kept in the sol for 5 minutes, then gently washed with water and dried at room temperature for 1 hour before use.

Modification of electrodes with carbon nanotubes by the electrophoretic deposition method

This work used single-walled carbon nanotubes functionalized carboxylic groups (CNT, > 90%, 4-5 nm × 0,5-1,5 microns, 1-3 atom.% COOH-groups, «Sigma»). The presence of carboxyl groups improves dispersancy of CNT in water and gives them a negative charge required for their movement in a constant electric field. For the suspension preparation the appropriate amount of CNT was weighed on an analytical balance (considering concentration 0.1 mg/mL), then deionized water was added, and the suspension was treated in ultrasonic bath for 12 hours.

Glassy carbon plates were polished before the modification by wet emery paper 4000 with Al 2 O 3 powder (0.05 micron, «Buehler»). The same procedure was used to renew the electrode surface after suc-cessful modification and measurements.

The two parallel electrodes were introduced in a 4-mL aliquot of the dispersion and a constant potential difference of 60 V (corresponding to the magnitude of the electric field between the electrodes 100 V/cm) was applied for required time usually from 5 to 120 seconds. The optimal applied voltage of 60 V was used throughout because it constitutes a good compromise between a high speed of deposition and limited decomposition of ultra-pure water (which would generate oxygen bubbles that may affect CNT assembly). The level of electrodes dipping was kept constant in order to ensure the same area (1 cm 2 ) of their contact with dispersion and reproducibility of deposition. After deposition, the glassy carbon plate was carefully removed from the remaining dispersion, gently washed in water, first dried horizontally at room temperature and then put in an oven at 450•C for 1 h. The For the preparation of macroporous CNT-layers the mixture of CNT (0,1 mg/mL) and polystyrene beads (0.05 mg/mL to 0.5 mg/mL) was used. It was obtained by adding to the suspension of CNT certain aliquots of concentrated (5%) suspension of polystyrene beads (PS-beads, 500 nm), obtained by the method [243].

Polystyrene was chosen because of facility of homogeneous beads synthesis and their removal by heating. This mixture was stirred, brought into the cell and applied to EPD as was described above. After the deposition electrode was carefully removed from the suspension and left at room temperature until dry. The template removal was carried out in an oven at 450 C for 1 h with 15 C/min ramp.

For the purpose of comparison, GCE was also modified with CNT-layer by drop-coating method. The CNTs were deposited from aqueous solution, by dropping 10 μL of the same water suspension as for EPD and left to dry completely.

For electrochemical generation of platinum nanoparticles on the surface of CNT we used technique described in [START_REF] Stefanie | Local electrocatalytic induction of sol-gel deposition at Pt nanoparticles[END_REF]. Electrode was dipped in the solution of 1 mM Pt(NO 3 ) 2 , that also contained 0.1 M NaNO 3 , and was exposed to a series of pulses. The sequence of pulses in each series was as follows: 0.035 V for 1 s, -0.7 V for 0.2 s, 0.035 V for 1 s. The electrochemical reduction of platinum and the formation of nanoparticles occurs during the application of negative potential.

Application of the positive potential was used for dumping, it does not happen any electrochemical reaction at such potential. The choice of the number of series of pulses is justified in section 3.2.1.1.

Voltammetric measurements

To study the properties of modified electrodes we used methods of cyclic and linear voltammetry, amperometry and hydrodynamic voltammetry.

All voltammogramms were obtained using saturated silver chloride electrode The sensitivity of the modified electrode to the substrate was calculated as the slope of a graph current-substrate concentration [244].

Surface observations

Investigation of the surface of modified electrodes by scanning electron microscopy (SEM) was carried out by connecting the modified electrode with substrate using a special conductive tape. Since the conductivity of the electrodes was sufficient spraying metal plating wasn't used. Energy of scanning electron beam ranged from 1 to 15 kV, magnification -from 1,000 to 400,000 times. If necessary, the electrode was placed at a certain angle to the electron beam.

Investigation of the surface by atomic force microscopy (AFM) was conducted in the temperature-controlled room using V-shaped silicon nitride probe (MLCT-EXMT-BF, «Veeco Instruments», USA) with a radius of curvature 50 nm and a constant of elasticity 0.1 N/m. Scanning was carried out in contact mode, the speed ranged from 0.5 to 2 Hz, the data was captured from laser optical detector. A thin scratch was made with a needle for profile and film thickness measurements. AFM images were captured on the border of scratches, allowing to estimate the difference in film thickness. Processing of AFM images was carried out using software WSxM 5.0 («Nanotec Electronica SL», Spain) [245].

Scanning electrochemical microscopy (SECM) with a special device developed on the basis of commercial appliance Sensolytics (Germany) was used to study the conductivity of bio-composite films. The measurements were performed using a platinum microelectrode (ø = 25 microns) in a solution containing 0.1 M KCl, 1 mM ferrocenedimethanol. Profilometry with SECM was also applied to study the morphology of the layers of CNT with a thickness greater than 500 nm, in this case a glass needle was used, measuring the distance from it to the surface using piezoelectric sensors [246].

For electrochemical studies of electroactive surface area the dye methylene green was adsorbed on the electrode surface by immersion of the electrode in a 1 mM solution of the dye and stirring on a magnetic stirrer for 12 hours. Then the electrode was washed thoroughly with water and dried at room temperature.

Calculations based on electrochemical measurements

Calculation of apparent Michaelis constant

The apparent Michaelis constant was calculated in order to determine the degree of the relationship of the corresponding enzyme to substrates, as well as to determine the upper limit of the linear range of modified electrodes. The assumption was made that the enzymatic reaction is one-substrate or the concentration of the second substrate (in the case of coenzyme) is saturating, that allows to apply the Michaelis-Menten kinetics to it [247]:

𝑉 = 𝑉 𝑚𝑎𝑥 × 𝑆 𝐾 𝑀 + 𝑆 (2.1)
wh. V -the rate of enzymatic reaction;

V max -the maximum rate of enzymatic reaction at such conditions; S -equilibrium concentration of substrate, mM; K M -Michaelis constant, mM.

The current flowing through the electrochemical cell with a known concentration of the substrate was taken as rate of enzymatic reaction. In this case, the graph of V-S relation for equation (2.1) has the form of hyperbole that approaches to a straight line V = V max . The processing of such curves is difficult (although possible with modern software) so the linearization of this equation was performed by the method of Lineweaver-Burk [248]:

1 𝑉 = 1 𝑉 𝑚𝑎𝑥 + 𝐾 𝑀 𝑉 𝑚𝑎𝑥 × 𝑆 (2.2)
In the coordinates 1/V -1/S the graph of the equation ( 2.2) has the form of a straight line that intersects the abscissa at the point -1/K M making possible finding Michaelis constant.

Further, in all cases apparent Michaelis constant was found out from the equation of the graph 1/V against 1/S, calculated by the method of least squares, and the point of intersection of this line with the abscissa. As an alternative method direct processing of the V-S graph with filter «Hill» in software «Origin 8.5» was used.

Calculation of the electroactive surface area using dye adsorption

The adsorption of the dye methylene green (MG) was used for the calculation of approximate electroactive surface area of electrodes modified with CNT. It was assumed that the dye molecules form a monolayer on the surface of nanotubes [234].

The area of anodic or cathodic peak of adsorbed dye on voltammogram of modified electrode was calculated using computer program «Origin 8.5» (e.g. Fig. 4.5a).

Electroactive surface area calculation was performed using the formula:

𝑆 𝑒𝑙.𝑎𝑐𝑡 = 𝑆 𝑝𝑒𝑎𝑘 × 6,24 • 10 18 × 𝑆 1 𝜈 × 𝑛 (2.3)
wh. S peak -area of the MG peak on the voltammogram, AV;

ν -potential scan rate, V/s; n -number of electrons transferred in the electrochemical reaction (n = 2 for MG);

6,2410 18 -number of electrons for the charge of 1 C; S 1 -area occupied by one adsorbed molecule of dye (for MG 0,8 nm 2 , or 810 -13 mm 2 [249]), mm 2 .

Conclusions to the chapter

The chapter lists and argues the choice of reagents and methods for carrying out the necessary experiments, describes the objects of study.

The choice of interfering substances investigated in developing amperometric methods for detection of sorbitol and choline was argued. The techniques of sol-gel proceeding and generation of bio-composite SiO 2 -films on the electrode surface by electrodeposition method were described. The method of the formation of CNTcoatings by electrophoretic deposition on the surface of glassy carbon electrode was defined, including macroporous CNT-layers. The techniques of voltammetric measurements, spectroscopic studies and formulas for calculations were listed.

CHAPTER 3. ENCAPSULATION OF OXYDASES BY ELECTROCHEMICALLY-ASSISTED DEPOSITION OF SOL-GEL

BIOCOMPOSITE

One of the needs of electroanalytical chemistry is the development of new, simple and effective methods of enzyme immobilization on the surface of electrodes.

The EAD method allows to encapsulate biomolecules in SiO 2 -film on the electrode surface by simple one-step process, which has currently been applied only for certain enzymes. Meanwhile, the immobilization of less active enzymes, such as ChOx, requires ways to increase the sensitivity of such modified electrodes.

As it was mentioned in the literature survey (paragraph 1. One possible approach for achievement of this goal is use of platinum-based nanomaterials, including nanoparticles and nanofibers [START_REF]Platinum nanowire nanoelectrode array for the fabrication of biosensors[END_REF]253]. Such materials can exhibit electrocatalytic properties towards hydrogen peroxide oxidation due to their small size and large surface area.

This chapter covers the results of the EAD method application for the immobilization of oxidases in bio-composite SiO 2 -film on electrode surface. It was shown the prospects of the combination of this method with nanomaterials to improve the sensitivity of modified electrodes. The influence of platinum nanostructured materials on the analytical signal of modified electrodes was studied.

Immobilization of glucose oxidase into the SiO2-film on the surface of platinum nanofibers

Platinum nanofibers (Pt-Nfb), forming a conductive network with a large number of intersections can be quite easily obtained by the method electrospinning, which allows to control the thickness and density of the network [242,254,255].

Despite the attractive characteristics such as small diameter and high density of fibers there is no information in the literature about the use of electrospun Pt-Nfb for the development of biosensors. The first part of this section presents the results of modification of Pt-Nfb with bio-composite SiO 2 -film, containing an enzyme from the class of oxidases.

As a model, we have chosen the enzyme glucose oxidase (GOx), which is often used in amperometric biosensors [122]. This is due to the acute need of quick monitoring of the glucose concentration in the blood (and food) for people with diabetes. Another reason for the popularity of GOx is its high stability, which allows application of different ways of immobilization without loss of enzyme activity.

Given this, we have chosen this particular enzyme as a model, to verify the applicability of Pt-Nfb network for the oxidases immobilization in the SiO 2 -film by electrochemically-assisted deposition method. In this case, the advantage of this method is the ability to selectively modify only Pt-Nfb, without affecting the glass substrate.

Morphological and electrochemical characteristics of platinum nanofibers network

Given that electrospun Pt-Nfb was not previously used as an electrode, we have investigated their morphological and electrochemical properties, as well as response of hydrogen peroxide on the electrodes of this type. (b) AFM pictures of fibers with scratch and cross-section profile.

Electrochemical properties of platinum nanofibers network.

The mechanical stability of the assembly is good enough in solution to perform electrochemical measurements. As shown in Fig. 3.2, the fiber density affects the electroactive surface area. Samples displaying various densities of Pt nanofibers have been characterized by cyclic voltammetry in sulfuric acid solution [256]. The electroactive surface area measurement was performed by the area calculation of anodic oxidation peak of adsorbed hydrogen (Fig. 3.2a, shaded), which corresponds to the charge, using the formula:

The estimation made from the integration of the hydrogen desorption peak

shows that one fiber layer exhibits an electroactive surface area (0.29 cm 2 ) similar to the geometric surface area defined by the O-ring of the electrochemical cell (0.28

Immobilization of glucose oxidase using electrochemically-assisted deposition method

In an attempt to cover only the surface of Pt-Nfb sol-gel electrochemicallyassisted deposition was preferred over the classical evaporation method, which is basically restricted to film deposition onto flat surfaces. We carried out an immobilization of model enzyme GOx on the surface of Pt-Nfb by EAD method, and the influence of EAD parameters on the thickness of the formed SiO 2 -film and the electrochemical response of immobilized enzyme was studied.

Voltammetric characteristics of platinum nanofibers modified with SiO 2 -glucose oxidase film

The cyclic voltammogram of Pt-Nfb in phosphate buffer has a complex shape, inherent in platinum electrodes (Fig. 3.4, curve 1). The current increase is noted in the anodic region at potential 0.7 V, which can be attributed to the formation of oxides of platinum. The significant peak at the potential of 0.0 V in the cathodic region can be noticed caused by several reactions, including the reduction of platinum As mentioned in the literature survey (paragraph 1.2.3), the properties of the formed film, and therefore the response of immobilized enzyme are affected by electrochemically-assisted deposition parameters, such as potential and duration.

Therefore, further optimization of EAD duration was conducted to achieve the best response of immobilized enzyme.

Influence of the duration of electrochemically-assisted deposition on the amperometric response of immobilized glucose oxidase

To assess the optimal conditions for obtaining electrogenerated film SiO 2 -GOx, we investigated the relationship of modified electrode amperometric response to glucose with the duration of EAD at a constant potential of -1.2 V. At 1-2 s the response is negligible, further signal sharply increases for the duration of electrodeposition 3 s, and then the sensitivity gradually decreases, approaching zero at 10 s (Fig. 3.5). To conclude this section, it can be stated that silica deposition occurs essentially on the Pt nanofibers for very short deposition times (<3 s). More prolonged electrolysis resulted in larger amounts of OH -catalyst diffusing onto the overall surface, thereby inducing film formation on both the platinum nanofibers and the glass substrate, which can then lead to the full encapsulation of the fibers into the silica material. Such morphological variations affect the electroactivity of the immobilized GOx. the biocomposite film totally covers the nanofiber assembly (i.e., 10 s deposition, Fig. 3.6f), it totally blocks the electrochemical detection of glucose.

Comparison of platinum nanofibers and platinum macroelectrode for the immobilization of glucose oxidase

A comparison was performed with Pt-Nfbs and bare Pt electrode with using 3 s electrochemically-assisted deposition in the same conditions. Similar results were obtained which suggests that here the electrochemical response of the assembly is mainly limited by diffusion of glucose in the sol-gel layer (Fig. 3.8). At these conditions Pt-Nfbs behave as partially-blocked electrode [257] and diffusion layers of individual nanofibers overlap to form a common layer, similar to the diffusion layer of macroelectrode [258,260,261]. However, such a control experiment has to be taken into account very carefully as the electrolysis of the sol occurs at more negative potential on the Pt bare electrode than on Pt-Nfbs, potentially affecting the thickness of the deposit. In any case, these results highlight the interest of the low-dimensional Pt-Nfbs and well-controlled solgel electrochemically assisted deposition to improve the response of biomoleculedoped thin silica films produced by the electrochemically-assisted deposition method.

Immobilization of choline oxidase into the SiO2-film

Choline oxidase (ChOx), as well as glucose oxidase, belongs to the class of oxidases, which makes possible the use of common approach of sensitive hydrogen peroxide detection to develop a biosensor based on it. However, unlike GOx, ChOx is characterized by much lower stability, which makes necessary finding ways to increase its stability in the immobilized state. Thanks to biocompatibility of SiO 2based materials, they are promising for use as a matrix for encapsulation of the enzyme on the surface of the electrode. The use of rapid one-step EAD method allows immobilization in mild conditions and shortening of the time of unfavorable conditions exposure on the enzyme molecule.

Electrochemical generation of the platinum nanoparticles on the surface of glassy carbon electrode for the choline oxidase immobilization

Pt-Nfb did not demonstrate significant advantages of sensitivity and selectivity compared with planar platinum electrode and method for their preparation requires sophisticated equipment. Therefore, in order to obtain choline-based biosensor, we decided to investigated another type of electrode -glassy carbon electrode modified with platinum nanoparticles (Pt-Nps). Nanoparticles were chosen due to the fact that they can be easily obtained on the electrode surface by electrochemical generation. At the same time, the size of such particles can be much smaller than the diameter of Pt-Nfb, which may contribute to an increase of electrocatalytic effect, compared with nanofibers.

Optimization of the conditions of the glassy carbon electrode modification with platinum nanoparticles

We used a method described in work [START_REF] Stefanie | Local electrocatalytic induction of sol-gel deposition at Pt nanoparticles[END_REF] for electrochemical generation of nanoparticles on the surface of GCE, which involves alternation of reducing and dumping pulses of short duration. While imposing negative potential the reduction of platinum precursor occurs on the electrode surface, which leads to the formation of platinum nanoparticles, at dumping pulse no electrochemical reaction occurs. SEMimage of modified electrode (Fig. 3.9a) reveals a presence of nanoparticles that are uniformly distributed over the electrode surface.

nm a

It is noticeable that unmodified GCE is almost inactive to hydrogen peroxide in the range of potentials -0.5 -1.0 V. There is no difference in voltammograms of this electrode in the presence and in the absence of H 2 O 2 (Fig. 3.9b, curves 1,2). A slight increase of current is seen from the potential 0.9 V you, which apparently precedes the peak of H 2 O 2 , oxidation observed at potentials higher than 1 V. In contrast, the voltammogram of modified GCE-Pt-NPs in the presence of hydrogen peroxide is significantly different from voltammogramms in the buffer solution (Fig. 3.9c). Two half-wave, cathodic and anodic, are visible at potential about 0.35 V corresponding to the reduction and oxidation of hydrogen peroxide, respectively (Fig. 3.9). They appear due to the presence of platinum nanoparticles on the surface of GCE, catalyzing the electrochemical reaction of H 2 O 2 . Thus, the electrodeposition of Pt-NPs on the surface of GCE significantly shifts the potential of H 2 O 2 oxidation in the negative direction and improves the electrochemical response. Such behavior makes the electrode a promising sensor for hydrogen peroxide.

The assumption can be made that the pulse duration affects the size of nanoparticles and the number of pulses affects their quantity. The change of these parameters may affect their catalytic properties. Therefore, to increase the sensitivity of GCE-Pt-NPs to hydrogen peroxide the number and duration of pulses have been optimized. Sensitivity was calculated as described in paragraph 2.3.3). The results are shown in Table 3.1. Table 3.1 This can be explained by the fact that smaller particles are likely to exhibit stronger catalytic properties. Sensitivity also increases with the number of pulses from one to three, and further increase leads to a decrease in the signal, possibly due to the formation of a large number of particles forming agglomerates. Thus, the optimal parameters of electrochemical generation of Pt-NPs were three pulses of 0.3 sec each.

Influence of the duration (1) and number of pulses (2) of Pt-Nps electrogeneration on the sensitivity of GCE-Pt-Nps to H2O2

Investigation of the stability of amperometric response of glassy carbon electrode modified with platinum nanoparticles

In addition to high sensitivity to hydrogen peroxide electrochemical transducer must demonstrate the stability and reproducibility of the response. Therefore, we have tested the stability of amperometric and voltammetric responses of GCE-Pt-Nps to hydrogen peroxide (Fig. 3.10). In the efforts to improve stability and to prevent leaching of nanoparticles from the electrode surface we have coated the GCE-Pt-Nps electrode with biocomposite film SiO 2 -ChOx with the same parameters of electrodeposition as explained in section 3.1. This modified electrode was active to choline (Appendix C), but its stability was still quite low.

Thus, the glassy carbon electrode modified with platinum nanoparticles is characterized by poor stability of the response. This situation can be explained by the gradual leaching of platinum nanoparticles from the electrode surface or their deactivation. Therefore, given the high demands on the stability of amperometric sensors, GCE-Pt-NPs was not desirable for using as a transducer in amperometric sensor for choline.

The choice of electrode for the choline oxidase immobilization

Given the poor stability of nanoparticles a comparative study of the other types of electrodes was conducted in order to find a new type of transducer for the immobilization of ChOx (Fig. 3.11). As objects of comparison we selected screen-printed electrodes as they have low cost and easy fabrication, as well as small size including both working, auxiliary and reference electrodes. At the same time, specially designed for use in the sensors, they usually have high electrochemical activity. The type working electrode material (gold and platinum) was chosen because of their high sensitivity to H 2 O 2 .

Cyclic voltammograms of platinum (Fig. 3.11c) and gold (Fig. 3.11d) screenprinted electrodes (AuSPE) shows active oxidation of hydrogen peroxide on themthe current increase in both cases is noticeable starting from potential 0.2 V (Fig.

3.11c, d curve 2

). The value of current is approximately the same for these two electrodes and commensurate with the current on GCE modified with platinum nanoparticles (Fig. 3.11b). Given this, any of the two screen-printed electrodes can be used as a transducer for the immobilization of ChOx. However, taking into account the slightly higher response to H 2 O 2 , lower cost and advantages of gold electrode

[251], we have chosen the AuSPE for the immobilization of ChOx.

Immobilization of choline oxidase into SiO 2 -film on the surface of gold screen-printed electrode

For the one-step immobilization of ChOx in the SiO 2 -film on the surface of AuSPE the EAD method was proposed with parameters similar to those used for the immobilization of GOx on the surface of Pt-Nfb. However, to achieve a significant response the duration of deposition has been increased to 20 seconds.

The AuSPE modified with film SiO 2 -ChOx demonstrates significant increase of anodic current on the voltammogram in the presence of choline (Fig. 3.12a). The method of hydrodynamic voltammetry (Fig. 3.12b) confirms this finding.

Indeed, the voltammogram of AuSPE-SiO 2 -ChOx in the presence of choline (Fig.

3.12b, curve 2) depicts two half-waves, whose presence can be explained by a complex mechanism of hydrogen peroxide oxidation on gold electrodes [262]. From this voltammogram the optimum working potential (0.7 V) was selected.

Choice of the deposition potential of the SiO 2 -choline oxidase film

Negative potential imposed during EAD, affects the rate of formation of electrogenerated catalyst affecting the parameters of formed SiO 2 -films, such as thickness and porosity. In turn, these characteristics influence the response of The results shown in Fig. 3.13 indicate that the change of deposition potential leads to changes in the sensitivity of the modified electrode. The greatest response observed for the EAD at potential -1.1 -1.2 V, which correlates with the optimal parameters of deposition of GOx-containing film on the surface of Pt-Nfb (section 3.1). At the same time, at higher potential (Fig. 3.13b, curve 1) the response is almost absent due to slow speed of electrogeneration resulting in the absence of SiO 2 -film on the electrode. However, more negative potentials (-1.3 B) (Fig. 3.13b, curve 4) lead to a too violent reaction of electrolysis of water and active hydrogen bubbles formation which prevent the formation of the film, causing its destruction.

Investigation of the response stability of gold screen-printed electrode modified with SiO 2 -choline film

Stability is an important parameter of biosensors, so we performed a study of long-term stability of AuSPE-SiO 2 -ChOx. The study was conducted by recording the calibration curves every 4 days for 2 weeks and comparing the sensitivity of the modified electrode to choline relative to the initial value immediately after modification.

As known, the main causes of sensitivity loss of biosensors is inactivation of enzymes in the film, as well as their leaching, including as a result of the film destruction. The presence of surfactants in sol during EAD significantly affects the last factor, enhancing the interaction of the enzyme with silanol groups of film as well as acting as structuring agent and improving its morphological properties (paragraph 1.2, 2.1.2). Therefore, we have investigated the effect of concentration of CTAB in the sol on the long-term stability of AuSPE-SiO 2 -ChOx.

To assess the stability of the biosensor the relative sensitivity of the electrode modified with film SiO 2 -CTAB-ChOx was calculated, using the formula:

𝑆 = 𝑆 𝑥 𝑆 1 × 100% (3.5)
wh. S -relative sensitivity, %; S 1 -sensitivity of the electrode to substrate at first day after modification, μA/mM; S х -sensitivity of the electrode to substrate at x-th day after modification, μA/mM.

The modified electrode was stored in a refrigerator at +4 C in the interim between experiments. It should be noted that the free enzyme in buffer solution under these conditions completely lost the activity in week, which contrasts with preservation of activity of immobilized enzyme. The data presented in Table 3.2. The data obtained shows that the biggest stability of modified electrode is observed at concentrations of CTAB in sol 12.2 mM, which is much greater than its CMC in aqueous solutions (≈ 1 mM [START_REF]Surfactant-Induced Modification of Dopants Reactivity in Sol-Gel Matrixes / C. Rottman[END_REF]). This correlates with data on a range of CTAB concentrations in sol with the highest analytical response of immobilized biomolecules [START_REF] Nadzhafova | Heme proteins sequestered in silica sol-gels using surfactants feature direct electron transfer and peroxidase activity[END_REF][START_REF]silica-nanocomposite films electrogenerated on pyrolitic graphite electrode[END_REF]. High concentrations of CTAB leads to a high foaming and flotation effect, and may lead to the restructuring of micelle and film structure reducing amperometric response of immobilized enzyme.

The operational stability of AuSPE-SiO 2 -ChOx which is necessary during continuous operating of biosensor was investigated in voltammetric and amperometric modes at optimum concentration of CTAB in sol (Fig. The constant response is also observed in amperometric mode. Catalytic oxidation current of hydrogen peroxide, which is formed by the enzymatic oxidation of choline after 50 minutes of continuous measurements while mixing decreases no more than at 6% (Fig. 3.14b).

Thus, these data indicate high operational stability of AuSPE-SiO 2 -ChOx, which is achieved through the use of sol-gel encapsulation of enzymes and addition of surfactant CTAB. The resulting stability is quite sufficient for the application of developed modified electrode as a sensitive element of biosensor and its use in the analysis different objects.

Further, the optimum parameters were the EAD at potential -1.1 V for 20 s, and the presence of 12.2 mM CTAB in sol.

Dependence of the amperometric response of the gold screen-printed electrode modified with SiO 2 -choline oxidase film from the choline concentration

Dependence of the amperometric response of AuSPE-SiO 2 -ChOx on the concentration of choline resembles the saturation curve (Fig. (mM) (R 2 = 0/998). The detection limit, calculated by 3S-criteria was 5 μM choline (0.7 mg/L). Thus, the sensitivity of the modified electrode is sufficient for the determination of choline in foods and biological fluids.

Conclusions to chapter 3

The oxidation current of hydrogen peroxide on the surface of platinum

Characterization of the electrophoretically deposited carbon nanotubes layers

The electrophoretic deposition method allows the accumulation of charged particles from their suspension onto one of the two electrodes used in the device. For this reason, carbon nanotubes bearing a high content of negatively-charged carboxylic groups on their surface have been chosen to facilitate their dis-persion (as non-modified CNT of this sort are almost insoluble in the pure water) and to make them likely to move in the electric field in order to get fast deposition of high quality CNT layers. The rate of the precipitation of charged particles depends on the applied potential difference, surface area of electrodes, distance between them and the process duration [266]. Therefore, the quantity of deposited nanotubes can be finely controlled by varying the time of potential application while keeping other parameters constant. The optimal applied potential difference of 60 V was used throughout because it constitutes a good compromise between a high speed of deposition and limited decomposition of ultra-pure water (which would generate oxygen bubbles that may affect CNT assembly).

Morphological characteristics

A necessary condition for the efficiency of electrode modification with CNT is the formation of a homogeneous and reproducible coating. The application of EPD method allows obtaining of such coatings. All images can clearly show the presence of distinct horizontally-oriented nanotubes, indicating the absence of aggregation, occuring, for example, when CNT dispersion is simply dropped on the electrode.

The influence of the duration of deposition on the CNT-layer thickness

The formation of CNT assemblies was also monitored by atomic force microscopy. In spite of the difficulties to recognize single nanotubes (for more detailed image see Appendix D), the overall thickness and uniformity of film can be The equation of this dependence is following:

That gives the possibility to control the quantity of deposited carbon nanotubes and, consequently, the film thickness by tuning the electrophoretic deposition time.

Such variation could influence the electrochemical characteristics of modified electrode.

Measurement of the electroactive surface area of CNT-layer

Electroactive surface area is one of the most important parameters of the electrodes, whose increase usually leads to an increase in current density, and hence the sensitivity of such electrodes. To estimate the surface area of GCE-CNT electrodes, we have used the adsorption of the dye methylene green (MG). It is known that phenothiazine dyes can easily adsorb onto the surface of carbon nanotubes owing to the favorable electrostatic and π-π stacking interactions [233].

We have thus exploited the adsorption of methylene green, which is Thus, the present electrophoretic method allows obtaining in a controlled way a porous conductive matrix with extended electroactive surface area, which might be of interest for the immobilization of a large quantity of enzyme.

According to the formula (2.3), by integrating the peak area of adsorbed MG one may calculate the approximate area of electroactive surface of electrode modified with CNT. For the electrode modified with CNT by EPD duration of 120 s (Fig. 4.5a, curve 5) electroactive surface area was 1033 mm 2 , while the geometric area of the electrode of the same diameter (6 mm) is 28 mm 2 (according to the formula S = πr 2 ).

Thus, modification of electrode with CNT by EPD method increases electroactive surface area of the electrode for almost 40 times, leading to the improvement of its sensitivity to electroactive substances.

Electrocatalytic properties of CNT assemblies towards NADH oxidation

To reveal the outlook for the application of hereby deposited carbon nanotubes film for the bioelectrode development, we examined its electrocatalytic effect toward the oxidation of NADH, an enzymatic cofactor mostly required for enzymes from dehydrogenases group. showing no significant anodic current below 0.6 V (actually a peak (not shown on the figure) can be observed at ca. 0.8 V), in agreement with previous literature [START_REF] Musameh | Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes[END_REF]). On the opposite, the cyclic voltammogram of NADH recorded using the GC-CNT electrode (Fig. Comparing parts a and b in Fig. 4.6 and curves 1 and 2 in Fig. 4.7 reveals significant decrease in overpotentials for NADH oxidation on carbon electrodes mainly due to the expected electrocatalytic action of CNT. These results are similar to those obtained in work [START_REF] Musameh | Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes[END_REF]. The potential of NADH oxidation is not so low as ca. 0.0 V observed by some researchers on the nanotubes particularly treated to induce the formation of quinone-like functional groups [219,220]. The absence of visible peaks in the low-potential range argues that here the high-voltage excitation during the EPD does not produce quinone species and the catalytic effect of carbon nanotubes relies mostly on their edge plane sites exposed to the solution [217].

Comparison of the voltammetric characteristics of NADH oxidation on the modified with CNT and non-modified glassy carbon electrods

A comparison of the electrochemical behavior of CNT simply drop-coated on the electrode surface was also performed. One has to comment that it is not obvious to obtain a homogeneous film by drop-coating on the surface of the electrode because carbon nanotubes demonstrated a tendency to form aggregates during the dryingprocess. Here, we deposited the CNTs from aqueous solu tion, by dropping 10 μL of the same water suspension as for EPD (deposition has also been performed with DMF for the dispersion of the CNT with a similar result). The electrochemical behavior of the drop-coated film CNTs was then compared to the film deposited by EPD. On the voltammogram in PBS of GC-electrode with drop-coated CNTs (Fig.

4.6c

) an anodic peak at potential about -0,05 V can be noticed that is absent in case of electrophoretically-deposited CNTs (Fig. 4.6b). This peak can be attributed to the presence of catalytic impurities or to some easily-oxidized groups on the surface of as-produced CNTs (e.g. quinone-like). One should notice also that the quantity of CNTs (indirectly compared from the magnitude of the background current) deposited in case of drop-coating was here several times higher than those deposited electrophoretically. But the current of NADH oxidation at 0.42 V was higher in case of electrophoretically-deposited CNTs, possibly because of the better distribution of the CNT in the porous structure obtained with EPD allowing a good access to the internal surface of the electrode.

Stability of the NADH voltammetric response

The stability of the signal is an important biosensors parameter. Oxidation of NADH at non-modified electrodes often leads to contamination of surface with reaction products and the decrease of response over time [START_REF] Musameh | Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes[END_REF]. The stability of NADH oxidation peak at GCE and GCE-CNT have been investigated (Fig. 4.8). Thus, the NADH oxidation current on the unmodified GCE is significantly reduced after the second scan, and after the 3rd cycle the signal is only 50% of the original one (Fig. 4.8a). At the same time, current on the GCE-CNT decreases much more slowly, after the 3-d potential scan the current decrease is not more than 15% of the original response ((Fig. 4.8b). It should be noted that for the subsequent scans the current approaches to a constant value and remains constant over time.

Summing up the above, we can conclude that the modification of GCE with CNT by EPD method significantly improves its electrochemical characteristics toward NADH oxidation. In particular, the potential peak is shifted by 0.25 -0.30 V toward negative values, that facilitates the process of coenzyme oxidation on electrode. In addition, the stability of the electrode response to NADH increases about 3 times due to the absence of surface electrode contamination with reaction products. It should be emphasized that the EPD method enables the obtaining of more homogeneous and porous coatings than conventional drop-coating method, which leads to increased in about two and a half times sensitivity of the electrode to NADH.

Electrochemically-assisted deposition of SiO2-sorbitol dehydrogenase film on the CNT-modified electrode

To highlight the potential interest of such assemblies in designing bioelectrochemical devices, the electrophoretically-deposited CNT layer was used as a support for deposition of a sol-gel biocomposite comprising dehydrogenase enzymes (d-sorbitol dehydrogenase, DSDH). Due to the heterogeneous topography of the porous CNT layer, the evaporation-based sol-gel deposition approach is limited, at least as far as uniform film formation around CNT is expected, so that the EAD method (which already led to successful sol-gel film formation on non flat supports) was applied here.

One of the advantages of the method of sol-gel EAD is the possibility of partial modification of the required part of the electrode only. For instance, we showed earlier the feasibility of controlled modification by sol-gel biocomposite of platinum nanofibers lying on a glass substrate (see paragraph 3.1). The situation of the GC-CNT electrode is more complex as the glassy carbon used as a substrate is conductive and the formation of silica film could be triggered not only by the carbon nanotubes but also by the underlying support. Therefore, the linear sweep voltammograms were recorded at the different types of electrodes dipped in the silica sol in order to examine the activity of the reactions responsible for the generation of the catalyst for the silica condensation (i.e., water and/or oxygen reduction (1.1)).

Comparing the curves obtained for bare GC and GC-CNT electrodes (see curve 1 and 2 on Fig. 4.9), clearly indicates much higher reduction rates on the carbon nanotubes than on the bare electrode (e.g., more than one order of magnitude at -1.3 V). The reason for this has to be found in the much larger electroactive surface area for GC-CNT as well as catalytic effect of carbon nanotubes leading to faster reaction rate.

Thus, by applying the negative potential of -1.3 V to the GC-CNT electrode, one would be able to induce the formation of hydroxyl-ions mainly in the vicinity of the nanotubes walls and not so much onto the glassy carbon support, which should lead to preferable CNT covering with DSDH-doped silica as it was demonstrated before for the localized sol-gel deposition on Pt nanoparticles lying on a glassy carbon surface [START_REF] Stefanie | Local electrocatalytic induction of sol-gel deposition at Pt nanoparticles[END_REF].

- using either bare GC or the GC-CNT electrode covered with a silica film containing DSDH generated by the method of EAD. Note that to make the data comparable, it was necessary to apply a more cathodic potential to the bare GC electrode (i.e., -1.8

V, with respect to -1.3 V for GC-CNT) to ensure the generation of the same electrolysis current and thus the same amount of so-produced hydroxyl ions catalysts

[268] (which would lead to similar quantities of deposited biocomposite on both electrodes). Thus NADH oxidation was facilitated when performed using the GC-CNT electrode (by ca. 0.3 V). Also, significantly larger bioelectrocatalytic currents were obtained in the presence of CNT. Assuming a same amount of deposited materials in both cases (as discussed above) and thus a same quantity of enzyme in the film, the smaller current observed with bare GC can be explained by some diffusional restrictions for the analyte to reach the active enzymatic centers through the silica layer, whereas the porous structure of the carbon nanotube assemblies resembles more to a large surface area 3D-electrode ensuring fast diffusion of reactants to the proximal electrode surface. Thereby, the biocomposite modified electrode constructed on the basis of electrophoretically deposited carbon nanotubes offers the advantages of lower potential of detection and larger current due to its porous structure associated to the intrinsic electrocatalytic properties of nanotubes.

Optimization of the parameters of the biocomposite film

In order to receive the best response of the immobilized enzyme and to study the influence of the main parameters affecting the characteristics of either the silicabased biocomposite layer or the underlying carbon nanotube assemblies, the two-step optimization of the deposition parameters was performed. First, we examined the effect of the electrodeposition time (in the range 5-25 s), which directly affects the quantity of deposited biocomposite, while keeping the same parameters of carbon nanotubes layer (time of electrophoretic deposition 60 s). The naked-eyes appearance of the electrode was not changed while the potential was applied for 5 s, nevertheless a clearly visible silica layer could be remarked for 25 s deposition. Analyzing the amperometric response of such electrodes to the d-sorbitol analyte (Fig. 4.11a) revealed a very strong impact of the deposition time on the bioelectrode sensitivity.

The highest responses only appear in a narrow time window, i.e., between 12 and 20 s, with a maximum reached at 16 s (Fig. 4.11b). Such great influence of the EAD process duration relies on the catalytic nature of this sol-gel deposition process. As previously assessed using AFM and EQCM techniques [269], the sol-gel deposition can be basically divided in two successive regimes, the first one being characterized by a slow rate of deposition and the second one by the fast deposition of much larger quantities of sol-gel material. As a consequence, the particular shape of the variation depicted in Fig. 4.11b can be rationalized as follows: short biocomposite deposition time resulted in low amounts of incorporated enzyme and thus to low bioelectrochemical response, whereas long deposition times led to thick deposits preventing fast mass transport into the biocomposite films resulting thereby in sensitivity loss as it was described before for the sol-gel deposition on the Pt-Nfbs (see paragraph 3.1) and macroporous gold electrode [START_REF]the functionalization of macroporous electrodes / F. Qu[END_REF]. The optimal situation arises thus from the best compromise of sufficiently high amount of incorporated enzyme while maintaining a porous structure open enough to ensure fast diffusion of reactants. A second important point to be investigated is the impact of the carbon nanotubes film thickness on the electrochemically assisted bioencapsulation. Fig. 4.12 shows the dependence of the bioelectrode sensitivity on the time of electrophoretic deposition of the CNT assemblies while keeping constant the parameters of sol-gel deposition (-1.3 V, for 16 s). The relation has also a sharpened shape with better sensitivities in the range from 20 to 60 s electrophoretic deposition.

According to the equation (4.1) this corresponds to a layer thickness of CNT from 100 to 150 nm. In fact, not only the time of sol-gel electrodeposition but also the quantity of carbon nanotubes on the glassy carbon electrode affect the quantity of sol-gel materials deposited because sol electrolysis is facilitated on carbon nanotubes (see Fig. 4.9). A higher quantity of carbon nanotubes on the electrode surface leads to faster sol-gel deposition. A great care has thus to be taken when optimizing the solgel electrodeposition on porous and catalytically active materials. At the same time, the results are reproducible, while maintaining constant parameters.

Analytical performance of the modified electrode

The d-sorbitol dehydrogenase immobilized into silica film on the surface of carbon nanotubes showed typical Michaelis-Menten kinetics and the gradual saturation of response after concentration of d-sorbitol 5 mM (Fig. 4.13a). The apparent Michaelis-Menten constant (K m ) was calculated from the Lineweaver-Burk plot and its value was found to be 4.1 mM which is very similar to one measured in the solution [270] indicating a good activity of DSDH entrapped in the electrogenerated silica film and rapid mass transfer of reagents. Finally, the analytical characteristics of the bioelectrode have been determined by recording its amperometric response at the applied potential of +0.5 V, as a function of the d-sorbitol concentration. Using the optimal thickness of carbon nanotubes layer and the best parameters of sol-gel EAD, the calibration plot for the oxidation of d-sorbitol was obtained and found to be linear in the range from 0.5 mM to 3.5 mM. The linear regression equation was I(ΔA) = (-0.09 ± 0.15) + (2.77 ± 0.07) × C (mM) with the correlation coefficient 0.995. The detection limit by 3S criteria was found to be 0.16 mM. The modification method can be reproduced on the different electrodes and on different days with a precision of 9% (n = 3). The bioelectrode demonstrated sufficient stability for the measurements during one month as it was shown before for the film with same composition [241], the signal drop after 20 consecutive measurements did not exceed 10%.

Electrophoretic deposition of macroporous CNT-assemblies for electrochemical applications

The application of macroporous electrodes is a promising approach to increase the sensitivity of electrochemical analysis [271]. They can be used in particular for the immobilization of biomolecules [START_REF]the functionalization of macroporous electrodes / F. Qu[END_REF][272][273][274]. Due to the porous structure, the electrode has higher electroactive surface area and the pore size is large enough to allow the quick and unobscured diffusion of reagents from solution to the electrode.

In particular, macroporous gold electrode was used to immobilize the enzyme with coenzyme that was possible thanks to the large pore volume [275].

The application of carbon nanotubes as nanoscale building blocks for the construction of analogous macroporous electrodes appears as a promising way to combine the advantages belonging to the macroporous structure [276] and the electrocatalytic properties of individual nanotube [START_REF] Lourdes | Role of carbon nanotubes in electroanalytical chemistry: a review[END_REF]. This notwithstanding, there are only a few examples dealing with three-dimensional 3D carbon nanotube ensembles with engineered and controlled macroporosity. The method of direct synthesis allows creation of 3D CNT-ensembles with long tube-to-tube distance [277,278] but it gives rather insufficient possibility of porosity control. Interesting results can be obtained by the method of ice-segregation induced self-assembly (ISISA) that was applied for the fabrication of self-supported porous CNT monoliths [279].

Another widespread method is a template approach that consists in mixing singlewalled carbon nanotubes with sacrificial particles, formation of a composite and further removal of the particles [280]. A two-dimensional network can also be obtained by using patterning methods from the solution [281]. To date, the above mentioned ensembles have not been applied in the area of electrochemical biosensors.

The method of vacuum filtration allows obtaining rather thick porous CNT films [282], but it can lead to the segregation of particle and nanotube layers during the filtration process [283] and does not permit to get the films on the solid support.

A possible way to overcome these limitations could be the resort to an electrophoretic deposition method for the fabrication of macroporous CNT-electrode. One drawback of such CNT-assemblies can be the uncontrolled packing of the material that hinders the access to the CNT at the bottom of the film, especially for biosensors applications. In this chapter the formation of 3D-macroporous CNT-assemblies in the form of thin films on electrode surfaces by means of electrophoretic deposition is described. With a view to evaluate the prospects of such kind of films for possible application in biosensing, their electrochemical response to hydrogen peroxide and nicotinamide adenine dinucleotide have been investigated and the first example of enzyme immobilization using electrophoretically deposited 3D macroporous CNTelectrode have been shown.

Fabrication of macroporous CNT-layers on the electrode surface

The method of the electrophoretic deposition basically allows the formation of deposits from suspensions of charged particles. The carbon nanotubes are negatively charged in a broad pH range due to the presence of carboxylic groups on their surface. On the other hand, the polystyrene beads are also negatively charged owing to the presence of sulfonate groups on their surface [284] (potassium persulfate was used as initiator of the polymerization in their synthesis) and can be electrophoretically deposited as well. However, we did not succeed in fabricating a uniform 3D-film consisting of PS beads only as they tended to form a monolayer with big gaps between the deposited beads (see Appendix A). This likely occurs because of the weak adhesion between the polystyrene and glassy carbon together with unfavorable interaction originated from their spherical shape and the electrostatic repulsion between particles. Considering the above observations, the deposition of a mixture of carbon nanotubes and PS beads could contribute to overcome the lacks of each individual component by bringing a volume from polymeric particles while nanotubes contribute to the reinforcement of the composite. In contrast, the appearance of the film formed in the presence of larger amounts of PS beads is different, demonstrating the absence of well-defined 500 nmmacropores but showing rather a disordered sparse packing of nanotubes (Fig. 4.15C and D), suggesting the collapsing of such thick macroporous film.

Optimization of PS-beads concentration in the suspension

To investigate the optimal concentration of PS beads leading to thick and highly porous CNT films, we analyzed different samples obtained with the same 135 s deposition time and PS beads contents varying from 0.05 and 0.5 mg/mL. The dependence of the deposit thickness on the PS beads concentration represents a linear correlation with quite big slope since higher amounts of PS beads increase significantly the volume of deposit (Fig. 4.16a). At the same time, when the PS template is removed, this dependence has a dramatically different shape (compare curves 1 and 2 in Fig. 4.16a) and can be basically divided into three parts. At the initial part of the plot the thickness of the template-free macroporous CNT deposit is close to the CNT-PS one (yet lower), demonstrating that burning of the template does not have significant influence on the macrostructure of deposit due to a small quantity of beads. For the same reason, such films suffer from rather small thicknesses and low pore volumes. At the second part, the rate of the thickness increasing slows down and reaches the maximum at a PS beads concentration of 0.2 mg/mL. At higher concentrations the thickness suddenly drops lower than 1 μm and does not change significantly in the third part of the plot. This demonstrates that too large concentrations of polystyrene have to be avoided as they lead to pore collapse during the calcination. In conclusion, the maximal thickness of the macroporous film can be obtained at a concentration of PS beads of about 0.2 mg/mL, defining a maximal PS beads / CNT ratio above which collapsing of macropores starts to occur. As a rule, the quantity of the electrophoretically deposited material depends strongly on the deposition time [266]. We verified thereby this approach and its impact on the feasibility of the macroporous film formation. Fig. 4.16b shows a dependence of the film thickness on the time of electrophoretic deposition. This dependence has linear type when only carbon nanotubes (no PS beads) are present in the dispersion (see curve 3 in Fig. 4.16b). In the same manner, but with much bigger slope, grows the deposit thickness when the PS beads have been introduced in the mixture (Fig. 4.16, curve 1). But this increase occurs until 100 s of deposition, and then tends to level off and could even decrease at much longer deposition times. We suppose that the rate of the PS beads deposition significantly decreases above 100 s because of a significant depletion of the particles concentrations consumed during the deposition. The loss in thickness at longer time is more difficult to explain but it might be due to the film destruction by oxygen bubbles formed on a larger time window during the deposition (they were clearly observed at the naked eye).

In agreement with the observation made in Fig. 4.16a for a PS-beads content of 0.2 mg/mL, the thickness of the deposit decreases by ca. a factor 2 after template removal (Fig. 4.16b, curve 2) but still remains much bigger than that of the deposit fabricated without PS beads (Fig. 4.16b, curve 3). At low deposition time (i.e., less than 75 s), the thickness of the macroporous CNT film is below 500 nm, which is likely to correspond to a sparse thin film similar to the one that can be obtained by the patterning from solution. After 75 s of deposition, the thickness of the deposit considerably increases, up to 2 lm, indicating a formation of true macroporous film with cellular structure.

Methylene green adsorption and electrochemical characterization

The strong adsorption on the CNT-surface and the electroactive nature of MG was exploited to compare the surface area of the different films prepared here. Fig.

Co-immobilization of sorbitol dehydrogenase and coenzyme on the macroporous CNT-assembly

Though the macroporous structure does not provide true direct advantage for the direct detection of NADH in the solution, it still could be useful for the development of biosensors based on the NAD-dependent enzymes. In that case, the enhancement can be achieved by immobilization of both the enzyme and the cofactor inside the porous film. The larger inner surface area together with the bigger pore volume would lead to increasing of the number of active sites where the enzymatic reaction is allowed to take place.

We used here a recently described protocol for the immobilization of NAD+ Thus, the porous structure of the macroporous CNT film provides higher sensitivity for the detection of D-sorbitol when the NAD + and DSDH are immobilized together into the silica film and this could be further exploited for the construction of advanced dehydrogenase-based biosensors.

Conclusions to chapter 4

Modification of glassy carbon electrode with carbon nanotubes by electrophoretic deposition method significantly improves the efficiency of the latter when detecting coenzyme NADH. The presence of carbon nanotubes shifts NADH oxidation potential at 0.25-0.30 V in negative potentials direction, and significantly increases the stability of the response.

Electrochemically-assisted deposition method allows immobilization of dehydrogenases in SiO 2 -film with preservation of enzymatic activity, which was illustrated by sorbitol dehydrogenase example. The electrode modified with this enzyme, shows stable and reproducible response to changes in the concentration of sorbitol in solution.

The electrode modified with carbon nanotubes and sorbitol dehydrogenase demonstrates significant advantages in comparison with the electrode without carbon nanotubes. In particular, the sensitivity and selectivity of sorbitol detection are increased thanks to the use of low working potential 0.5 V. The response of the modified electrode depends on the thickness of carbon nanotubes layer and SiO 2 -film deposition parameters. Best analytical characteristics were obtained when using carbon nanotube layer thickness of 100 -150 nm and electrochemically-assisted deposition parameters -1.3 V, 16 s.

Macroporous layer of carbon nanotubes obtained by the method of electrophoretic deposition is a promising matrix for developing biosensors based on oxidases and dehydrogenases. In particular, it allows to improve the sensitivity and selectivity of the electrode modified with film SiO 2 -dehydrogenase when combined with adsorbed methylene green dye and immobilized coenzyme.

CHAPTER 5. APPLICATION OF MODIFIED ELECTRODES AS SENSITIVE ELEMENTS OF BIOSENSORS FOR THE DETERMINATION OF SORBITOL AND CHOLINE

Selective determination of biologically-active organic substances is an important task of modern analytical chemistry. Such substances are often used in food production, resulting in their significant intake with food. However, excess or deficiency of such substances in the human body can cause serious physiological disorders.

Currently, analytical determination of these substances is a complex task due to the similarity of the chemical properties of individual substances in homologous series (e.g., polyhydric alcohols, monosaccharides, polysaccharides have OH - groups). In fact, the only method that allows sensitive and selective analysis of organic substances is chromatography. However, it had disadvantages, such as considerable length analysis, complex sample preparation, high cost and complexity of the equipment, the need of pure reagents. All this makes impossible screening of organic substances. However, the sensitivity at the level of 10 -5 -10 -4 M is usually quite sufficient for analysis of substances, particularly in foods [285]. Examples of substances that require determination in the food are sorbitol and choline, important members of metabolism in the human body.

Electrochemical biosensors are promising alternative for the determination of organic compounds in complex objects with minimal sample preparation. They are characterized by sufficient sensitivity, speed of analysis, and that is the most important, high selectivity of determination thanks to the use of enzymes as recognition elements. Price of one analysis is rather low due to the possibility of multiple use, a small amount of enzyme and cheapness of electrodes (e.g. screenprinted electrodes).

This section describes the results of the application of developed modified electrodes for the determination of choline and sorbitol. The determination of sorbitol was carried out using the glassy carbon electrode modified with non-porous CNTlayer and biocomposite film SiO 2 -DSDH with optimal parameters (as described in chapter 4, paragraph 4.3). The choline analysis was conducted using gold screenprinted electrode modified with biocomposite film SiO 2 -ChOx with optimal parameters (as described in chapter 3, paragraph 3.2). The deposition of silicabiocomposite in both cases was performed by electrochemically-assisted deposition method.

Determination of sorbitol using glassy carbon electrode modified with CNT and SiO2-sorbitol dehydrogenase film

Sorbitol is an alcohol contained in fruits that has a sweet flavor. It is widely used in the food industry (food additive E 420) as a sweetener and [286] as well as in the cosmetic and pharmaceutical industries. Very often it is used along with fructose as a sweetener in diabetic foods for people with diabetes.

In human body, sorbitol is formed from glucose and is converted to fructose with DSDH. Its abundance in the tissues leads to the swelling that occurs in diabetes [287]. It also fermented by the bacteria of large intestine to acetate and H 2 , which leads to diarrhea, severe disorders of the gastrointestinal tract and weight loss when its excessive consumption [288]. These symptoms have been reported for the abuse of sorbitol in amounts of more than 10 g/day.

Thus, monitoring of sorbitol in the food is an important analytical task. In For the verification, the method of "added -found" was used. Results of the determination of sorbitol additions in buffer solution are presented in Table 5.1.

Interference of some substances on the determination of sorbitol

As objects of study we selected products of the food and cosmetic industries that may contain sorbitol. These products are dietary sweets, chewing gum, and

Carbohydrates and polyhydric alcohols do not interfere with determination of 1 mM sorbitol, including mannitol, which is a stereoisomer of sorbitol, indicating the high selectivity of the modified electrode.

Ascorbic acid does quite significant interfering influence due to the nonenzymatic oxidation on the electrode at operating potential 0.5 V. But lower working potential (0.5 V) enables an approach for its masking -the introduction of Fe (III). It was investigated that even fivefold excess of Fe(III) does not interfere with determination of sorbitol (Table 5.2). Given the fact that the objects of study contain sorbitol in significantly higher amounts than ascorbic acid, 3 mM of Fe(NO 3 ) 3 will be sufficient for the masking and will not interfere with the determination of sorbitol.

According to the literature data [292,293], the masking effect Fe(III) can be explained by two factors: the formation of the transition complex of Fe(III) with ascorbic acid and subsequent oxidation of ascorbic acid by ferric iron in the complex forming Fe (II) and dehydroascorbic acid .

The presence of anionic surfactant leads to a gradual decrease in signal with time during the time of contact of the electrode with the solution more than 10 minutes, probably due to leaching of the enzyme from the film [START_REF]silica-nanocomposite films electrogenerated on pyrolitic graphite electrode[END_REF]. Consequently, when analyzing cosmetic products containing surfactants one should avoid prolonged (over 10 minutes) contact of modified electrode with test solutions.

Thus, given the absence of interfering effects of most of the investigated compounds and the ability to mask ascorbic acid the developed modified electrode can be used for determination of sorbitol in the food and cosmetic samples.

Determination of sorbitol in the food and cosmetic samples

As objects of study we used toothpaste for children «Oral B Stages berry bubble» (manufacturer «Procter & Gamble Co.», Germany), chewing gum «Orbit» (manufacturer LLC «Wrigley», Russia), biscuits "Diabetic" (producer PJSC "Kharkiv biscuit Factory", Ukraine), shower gel "White honey" (produced by "Yaka", Ukraine). The composition of the objects is given in Appendix G. Results of sorbitol determination in the objects of food and cosmetic industries are shown in Table 5.3. The obtained data are characterized by satisfactory accuracy and reproducibility. The found sorbitol content in the object "Shower gel" is perhaps somewhat underestated due to the presence of anionic surfactants, which lead to a decrease in the signal. For determination of sorbitol the electrochemical, titrimetric, polarimetric, chromatographic and enzymatic methods are used.

The titrimetric method of sorbitol analysis, which is used in the pharmacopoeia is based on the formation of its complex with copper, the concentration of which is determined by iodometric titration [295]. There is also a method of reverse iodometric titration of periodate excess that remains after sorbitol oxidation in sulfuric acid medium [296]. However, these methods are not sensitive and selectivein fact they determine the total content of polyols and glucose.

One of the methods is direct electrochemical detection of sorbitol on platinum [297,298] Although chromatographic methods of analysis provide selective and sensitive determination of sorbitol in many objects, their common drawback is an expensive equipment, complex sample preparation and significant duration of analysis, as well as the need for special training and high requirements for reagents purity.

In enzymatic methods of analysis DSDH is most often used with diaphorase which catalyzes reduction of iodonitrotetrazolium chloride to iodonitrotetrazolium formazan (Fig. 5.2), while measuring the value of the absorption of the latter at λ = 492 nm photometrically [303]. There are also ready test kits for sorbitol and xylitol determination by this method in food [304,305].

Sorbitol + NAD + 𝑠𝑜𝑟𝑏𝑖𝑡𝑜𝑙 𝑑𝑒ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛𝑎𝑠𝑒 → Fructose + NADH Fig. 5.2

. Schemes of reactions of enzymatic determination of sorbitol

This method is very selective and sensitive, but the significant consumption of enzymes and cofactors, including their high cost (especially diaphorase), makes it not viable from an economic point of view. In addition, this method is not express, and the error of determination increases because of the use of complex two-enzyme system.

The advantages of the developed method for the sorbitol determination using modified GCE-CNT-SiO 2 -DSDH electrode compared with titrimetric, polarimetric and electrochemical methods are the high selectivity of determination in the presence of other polyhydric alcohols and carbohydrates, as well as the lower limit of detection. Compared with chromatographic methods, the technique requires minimal sample preparation, characterized by expressivity and the possibility of using by unqualified personnel. The sensitivity of this method is sufficient to determine the sorbitol in the majority of objects. The method can be applied for screening sorbitol content in the objects of food and cosmetics industries, as well as for the analysis of biological fluids if sensitivity increased.

To date there are only a few examples of the development of amperometric biosensors based on DSDH to determine sorbitol (Table 5.4). However, in most cases, these sensors were used only for the analysis of model solutions, not real objects. Compared with them the developed GCE-CNT-SiO 2 -DSDH electrode has high stability and reproducibility of results, limit of detection is sufficient to determine sorbitol in most objects. The advantage is also a simple modification and the absence of mediator.

Determination of choline using gold screen-printed electrode modified with film SiO2-choline oxidase

Choline -a quaternary ammonium base, which belongs to the vitamin B (B 4 ) group, although the human body is able to synthesize it. However, it should be necessarily present in the diet of humans; its recommended dose is 425 and 550 mg per day for women and men respectively [306]. In the body choline participates in the synthesis of important neurotransmitter acetylcholine, it also participates in the regulation of insulin levels, in the transport of fat in the liver (as the part of some phospholipids) and in the formation of cell membranes. The deficiency of choline in the body leads to serious violations: lipopexia and liver damage, kidney damage, bleeding. However, excessive consumptionof choline leads to the so-called "fish odor syndrome", sweating, excessive salivation, reduced pressure [129].

Foods rich in choline are veal liver, egg yolks, milk, spinach and cauliflower.

In addition, choline is artificially added to some specialized food such as baby food, vitamin formula, sports drinks. Control of choline content in these foods is an important analytical task. For example, infant vitamin mixture is almost the only source of choline for infants, and its lack in food can lead to severe developmental Results of choline additions determination in phosphate buffer solution are presented in Table 5.5. They are characterized by satisfactory accuracy and reproducibility.

Table 5.5

Results of choline additions determination in phosphate buffer

Interference of some substances on the determination of choline

The influence of the substances contained in the food and able to interfere with the determination of choline by the modified electrode was studied. The effect of substances at their average concentrations in the corresponding objects was investigated (Appendix G). Since urea and uric acid are lacking in food, their

As seen from the results, the main macrocomponents do not interfere with the determination of choline. One should note the absence of interfering effects of ethanol in relatively high concentrations. The presence of equimolar amounts of Cu(II) leads to the signal decrease, it is known from the literature that copper cations inhibit ChOx [309], but the presence of copper in such high concentrations in the studied objects is ruled out. Ascorbic acid significantly interferes with the determination of choline due to its nonenzymatic oxidation on the electrode at 0.7 V.

Elimination of interfering influence of ascorbic acid

Given the fact that ascorbic acid is a common interfering component in the development of biosensors, the literature offers several ways to eliminate its impact.

In particular, the most common way is the use of semipermeable membranes that limit the access of interfering substances to the electrode due to the size or charge of their molecules [310,311]. However, this approach often leads to a decrease in the sensitivity of the biosensor to analyte [176]. Therefore, another promising approach is the eliminatation of the reductants effect by their prior oxidation on the membraneoxidants [175]. The composition of such membranes can vary, but one of the best oxidants that can be used for this purpose is the oxide of manganese (IV) [173,174].

The disadvantage of such membranes is the low reproducibility of the signal and slow response time. Therefore, to simplify the procedure of the modified electrode fabrication, we decided to add MnO 2 powder directly into the solution of the analyte so that he could oxidize ascorbic acid before determination.

The dry powder of MnO 2 was added (10 mg per 10 ml of solution) to the solution that was stirred with a magnetic stirrer for 30 min. Then the sample was filtered through a paper filter and used for the determination of choline as described in paragraph 5.2.1. Applied potential +0.7 V.

To test the impact of MnO 2 on choline signal its determination was conducted using AuSPE-SiO2-ChOx in a series of model solutions (Table 5.7). From the obtained data it can be concluded that MnO 2 does not interact with choline because found choline amount remains constant before and after treatment with MnO 2 (solutions 1, 2, Table 5.7). At the same time, the presence of ascorbic acid leads to an overestimation of the expected results of choline determination (solution 3, Table 5.7). However, after the treatment of this solution with MnO 2 (solution 4, Table 5.7), the found amount of choline correlates with added. Thus, treatment of 0.1 mM ascorbic acid solution with MnO 2 leads to the elimination of its interfering impact in choline determination using AuSPE-SiO 2 -ChOx. According to the stoichiometry of the reaction [312], the amount of MnO 2 1 mg/ml is sufficient to eliminate the interfering effects of ascorbic acid up to its concentration in a solution 10 mM. The ascorbic acid content should not exceed 5 mM after sample preparation, so such quantity of MnO 2 is enough to mask its influence.

Determination of choline in food products

The infant formula «Bebi» was chosen as an object of study, its composition is listed in Appendix G. However, attempts to determine choline in food mixture using a standard photometric method with Reinecke salt [314] were unsuccessful due to the low sensitivity and reproducibility of the method, and chromatographic technique requires the use of special columns.

Comparison of the standard methods of choline determination with the developed technique

For determination of choline in foods the spectrophotometric, chromatographic and enzymatic methods of analysis are used. Its analysis is complicated because it can be either in free form or in the ester derivatives of phosphoric acid, so its preliminary hydrolysis is necessary before determining total choline [129].

Historically, the first photometric method for choline determination is based on formation of insoluble colored compound with Reinecke salt, whose absorption is detected in methanolic solution at 520 nm [314]. The gravimetric variant of this method is also possible [315]. Despite its simplicity, this method is time-consuming, low-sensitive (the loss of choline during washing is possible), requires the use of toxic reagents. In addition, this method is not selective, determination of choline is prevented by most other amines.

Chromatographic determination of choline is possible using liquid [316][317][318],

gas [318,319] NMR spectroscopy can be also used for determination of choline [322]. The method allows to determine choline content with great accuracy in various forms, but is unsuitable for most laboratories because of the high cost of equipment and low linearity and sensitivity.

Enzymatic methods for choline determination are based on its oxidation with ChOx (after preliminary hydrolysis of esters by phospholipase) with the release of H 2 O 2 . The hydrogen peroxide reacts with phenol and 4-aminoantypyrine contained in the mixture in the presence of peroxidase, giving a colored reaction product, which is detected photometrically at a wavelength of 505 nm [313,323]. There are also modifications of this method with other dyes [324] [324]. This method of choline determination is quite expensive, given the high cost of the enzyme, with a significant error of determination by photometric method of signal detection. In addition, the determination of choline by this method is influenced by many reductants that can react with hydrogen peroxide. Their influence can be removed by using activated carbon [325].

The developed method of choline determination with AuSPE-SiO 2 -ChOx has significant advantages over existing methods. In particular, the use of planar technology, relatively cheap reagents and small quantities of enzyme allow producing large number of electrodes with little cost. Sensitivity and selectivity of the modified electrode is sufficient to determine choline in foods and biological fluids with minimal sample preparation. Small time of analysis and its simplicity allows the use of the developed technique by unskilled personal.

Compared with other known biosensors based on ChOx, the developed modified electrode is characterized by fast response and low detection limit, which can be explained by the porous structure of SiO 2 -film that facilitates the diffusion of reactants inside (Table 5.9). Modification procedure is simple and, unlike most other biosensors, does not use mediator, which could worsen the analytical characteristics and reproducibility of biosensors. The sensitivity developed electrode slightly lower than in some works [168,307,326], but it is quite sufficient to determine choline in foods that has been shown in the analysis of real objects. It should be noted that immobilized on the electrode surface ChOx is characterized by low apparent Michaelis constant (Table 5.9), indicating the preservation of its native structure due to biocompatibility of SiO 2 -materials. In addition, immobilization into SiO 2 -film increases the stability of the ChOx structure, for example, large amounts of ethanol do not lead to its deactivation. The developed electrode is stable and can be used repeatedly (as was shown in section 3.2.5), which is particularly important for biosensors.

Conclusions to chapter 5.

The prospects of the developed modified electrodes application as amperometric biosensors for the analysis of real objects were shown.

The technique of amperometric determination of sorbitol using glassy carbon electrode modified with biocomposite film SiO 2 -sorbitol dehydrogenase was developped. The calibration graph was linear in the range of concentrations of 510 -4 -3,510 -5 M, the detection limit was 1,610 -4 M. The equimolar amounts of sucrose, glucose, urea, mannitol, and glycerol do not interfere with the determination of

74 ABBREVIATIONS

 74 ABBREVIATIONS .......................................................................................................

Fig. 1 . 1 .

 11 Fig. 1.1. Schemes of amperometric biosensors of 1st (a), 2nd (b) and 3d (c) generations.

Fig. 1 . 2 .

 12 Fig. 1.2. Scheme of reactions occurring during the sol-gel synthesis (on the example of TEOS): hydrolysis (a), condensation (b), polycondensation (c).

Fig. 1 . 3 .

 13 Fig. 1.3. Scheme of different biosensor configurations based on the sol-gelmaterials [33].

Fig. 1 . 4 .

 14 Fig. 1.4. Scheme of electrochemically-assisted deposition.

Fig. 1 . 5 .

 15 Fig. 1.5. Structure of single-walled (a) and multi-walled (b) carbon nanotubes

  Fig. 1.6. Scheme of electrophoretic deposition of CNT.

  reactions of dehydrogenases, turning to the reduced form NADH. Due to the electrochemical activity of pair NAD + /NADH the amperometric detection of concentration changes of any component of this pair could be linked to the initial concentration of the substrate-analyte. However, the NADH interacts with the electrode, causing the dependency of the position and height of the peak on the material and the structure of the electrode as well as on the pH and the buffer solution[213]. Although the formal redox potential of pair NAD + /NADH is quite low (approximately -0.515 V vs. Ag/AgCl[213][214][215]), the process is characterized by significant overvoltage on conventional electrodes (0.7 V -on platinum, 1.0 V -on gold) because of irreversible oxidation of NADH[215]. Electrochemical detection at such high potentials would have led to significant interfering effect from other electroactive substances and even water electrolysis, preventing any analytical application of such biosensors.The electrochemical oxidation of NADH at carbon electrodes has been reported to lead to the electrode poisoning due to contamination of surface by reaction products[216], including adsorbed NAD + dimers[217]. This leads to a decrease of the anodic current of NADH oxidation and consequently to the loss of electrode sensitivity with time, greatly complicating the development of stable biosensors.Therefore, research efforts are aimed at searching of new materials and ways of NADH oxidation potential reduction. The carbon nanotubes established a reputation as a promising material for such enzyme electrodes[START_REF] Joseph | Carbon-Nanotube Based Electrochemical Biosensors[END_REF] 100]. Thus, the use of an electrode modified with carbon nanotubes reduces the potential of NADH oxidation by almost 0.5 V compared to the unmodified carbon electrode[START_REF] Musameh | Low-potential stable NADH detection at carbon-nanotube-modified glassy carbon electrodes[END_REF]. Even better results can be obtained by performing pre-activation of carbon nanotubes by anodic oxidation[218] or microwave processing[219]. This treatment leads to partial destruction of the nanotubes structure and the emergence of highly-active centers[217, 218] as well as to the appearance of quinone-like groups on the surface of the nanotubes, which act as mediators[219, 220].Another way to solve the problem of high oxidation potential of NADH may be the use of electrochemical mediators, including various quinones, aromatic diamines, phthalocyanine, ruthenium complexes and other[215, 221]. Scheme of such mediators application may include simple addition to the solution or immobilization on the electrode by adsorption, covalent binding or (electro)polymerization[221, 222]. Phenothiazine and phenoxazine redox dyes[223] attract attention as mediators due to the high rate of heterogeneous electron transfer reactions of NADH and are considered as one of the most promising electrochemical mediators for coenzyme oxidation[222, 224]. The biosensors based on such dyes have already been developed: Meldola Blue [225, 226], Nile Blue [227], Methylene Blue [228-230] and Methylene Green [231, 232].

  3.4, «Sigma») from Aspergillus niger (M w ≈ 160000, activity 15 -50 units/mg). Isoelectric point 4,2; -Choline oxidase (ChOx, EC 1.1.3.17, «Sigma») from Alcaligenes sp. (M w ≈ 95000, activity ≥ 10 units/mg). Isoelectric point 4,1; -D-Sorbitol dehydrogenase (DSDH, EC 1.1.1.15), synthesized at the Department of Microbiology of the University of Saarland (Germany).

(

  dimethyldiallylammonium chloride) (PDDA, low molecular weight, 20 wt% in water, «Aldrich»), poly-(ethyleneimine) (PEI, high molecular weight, water-free, «Aldrich»), and cationic surfactant cetyltrimethylammonium bromide (CTAB, «Sigma»).

  electrochemical experiments were carried out on potentiostat/galvanostat PalmSens («Palm Instruments BV», Netherlands) potentiostat EmStat2 («Palm Instruments BV», The Netherlands) and bipotentsiostat/galvanostat μStat 400 («DropSens», Spain). The three-electrode cell was used containing (except gold screen-printed electrode) corresponding working electrode, an Ag/AgCl reference electrode (Ag / AgCl, 3 M KCl, «Metrohm», Germany) and a platinum auxiliary electrode. Data from the potentiostat was transferred to a personal computer and processed using software «PSTrace», «DropView» and «Origin». Following electrodes were used as working: -Platinum nanofibers (Pt-Nfb) were synthesized and deposited on the surface of the glass substrate in Physico-Chemical Institute of the University of Giessen (Germany) by the method of electrospinning [242]. There were one-, two-, and four-layers Pt-Nfb with resistance 10, 2 and 0.5 kΩ accordingly; -Glassy carbon electrode (GCE) in the form of plates (Sigradur ®, «HTW Hochtemperatur-Werkstoffe», Germany), modified with CNT; -Gold screen-printed electrode (AuSPE) («DropSens», Spain). This type of electrode contained both gold working electrode, silver reference electrode and gold auxiliary electrode.

(

  Ag/AgCl), all potentials are mentioned versus it. All voltammogramms were obtained in buffer solutions of appropriate pH (unless otherwise specified) in static mode (without stirring). Potential scan rate varied from 20 to 100 mV/s, in some cases the extended range from 5 to 200 mV/s was used. Coenzyme NAD + (1 mM) was added into solution during measurement in the study of the voltammetric characteristics of immobilized DSDH. Amperograms were obtained at constant stirring, applied potential was kept constant throughout the measurement. At the beginning of the measurement dumping time was at least 200 s. Hydrodynamic voltammetry was carried out in the dynamic mode (with constant stirring). Potential changed stepwise with interval 50 mV every 30 s. The equilibrium current at the end of the time interval was measured. If necessary voltammetric measurements were performed in an anaerobic mode. Oxygen removal for the anaerobic experiments was achieved by bubbling argon for 15 min prior to experiment and this atmosphere was kept in the cell during the whole measurement.

  4.2), the general feature of all oxidases is release of hydrogen peroxide in the enzymatic reaction. The initial concentration of the substrate can be determined by registering the change of the H 2 O 2 concentration. One of the best materials for electrochemical detection of H 2 O 2 is platinum [250]. The oxidation of hydrogen peroxide on platinum surface occurs with high signal reproducibility and at low potential due to the catalytic action of platinum oxides. This fact can be exploited to avoid interfering effects of reductants, increasing thereby the selectivity of platinum electrodes in comparison with other types of electrodes [251]. However, even with relatively low reaction potential [252], one would desire further reduction of the H 2 O 2 oxidation potential.

3. 1 . 1 . 1 .

 111 Morphological characterization of platinum nanofibers assemblies.

Fig. 3 .

 3 Fig.3.1 depicts typical microscopic characterization data of the Pt-Nfb assemblies. SEM (Fig.3.1a) and AFM (Fig.3.1b) imaging reveals that the diameter of individual fibers is in the range 30-60 nm. Under the conditions used here, the overall thickness of the two-layers assembly remains limited to ca. 100 nm, as shown in the AFM profile (Fig.3.1b). Platinum nanofibers deposited by electrospinning are highly interconnected, forming a 2Dnetwork of Pt nanoelectrodes on the glass substrate, suggesting good conductivity. This is indeed the case, as the conductivity of the assembly varied typically from 0.5 to 10 kΩ/cm (two points measurement), depending on the film density (which can be tuned by adjusting either the deposition time and/or the number of deposited layers). As also shown, individual Pt nanofibers can be easily evidenced by AFM, and their diameter can be evaluated quite accurately when they are deposited directly onto the glass support (see the two first features around X =1 μm in the line scan in Fig.3.1b).

Fig. 3 . 1 .

 31 Fig. 3.1. Characterization of platinum nanofibers. (a) SEM picture;

cm 2 )Fig. 3 . 2 .Fig. 3 . 3 .

 23233 Fig. 3.2. Cyclic voltammogram of Pt-Nfbs in 0.5 M H 2 SO 4 solution. Scan rate 100 mV/s; (b) Dependence of estimated electroactive surface area of Pt-Nfbs as a function of the number of nanofibers layers.

Fig. 3 .

 3 Fig. 3.4. (a) Cyclic voltammograms of Pt-Nfb (1), Pt-Nfb-SiO 2(2) and Pt-Nfb-SiO 2 -GOx(3) in PBS (рН 6,0) containing 10 mM glucose(2, 3). Scan rate 20 mV/s;(б) Amperometric response of Pt-Nfb (1), Pt-Nfb-SiO 2(2) and Pt-Nfb-SiO 2 -GOx(3) to the additions of glucose (on the graph). Supporting electrolyte: PBS рН 6,0.Applied potential +0,6 V.

Fig. 3 .

 3 Fig. 3.5. (a) Amperometric response of Pt-Nfb-SiO 2 -GOx, modified with time of EAD 1 s (1), 2 s (2), 3 s (3), 4 s (4), 5 s (5) and 10 s (6) to the additions of glucose (on the graph). Supporting electrolyte: PBS рH 6,0. Applied potential +0,6 V; (b) Dependence of electrode Pt-Nfb-SiO 2 -GOx sensitivity on glucose at potential +0,6 V as a function of time of sol-gel electrodeposition.

Fig. 3 . 6 .

 36 Fig. 3.6. AFM images and cross-section profiles (along white lines shown in the images) of silica-modified Pt-Nfbs prepared with using increasing electrolysis times: 1 s (a), 2 s (b), 3 s (c), 4 s (d), 5 s (e) and 10 s (f).

Fig. 3 . 7 .

 37 Fig. 3.7. SEM images of silica-modified Pt-Nfbs prepared for various electrodeposition times: 1 s (a), 2 s (b), 3 s (c) and 10 s (d).

Fig. 3 . 8 .

 38 Fig. 3.8. Amperometric response of Pt-bare electrode (1), 2-layers Pt-Nfb (2) and 4-layers Pt-Nfb (3) modified with SiO 2 -GOx film by EAD method (-1,2 V, 3 s), to the additions of glucose (on the graph). Supporting electrolyte: PBS pH 6,0. Applied potential +0,6 V.

Fig. 3 .

 3 Fig. 3.9. (a) SEM-image of GCE, modified with with Pt-Nps by means of electrochemical generation; (b, c) Cyclic voltammograms of GCE (b) and GCE-Pt-Nps (c) in 0.025 М PBS (рН 7.0) in the absence (1) and presence of 3 mM H 2 O 2 (2).Scan rate 50 mV/s.

1 Fig. 3 .

 13 Fig. 3.10. (a) Amperometric response of GCE-Pt-Nps to the additions of H 2 O 2 (on the graph). Supporting electrolyte: 0.025 PBS (рН 7.0), applied potential +0.7 V; (b) Cyclic voltammograms (20 cycles) of GCE-Pt-Nps in the presence of H 2 O 2 (1 mM). Supporting electrolyte: 0.025 PBS (рН 7.0), scan rate 50 mV/s.

Fig. 3 . 11 .

 311 Fig. 3.11. Comparative cyclic voltammograms of GCE (a), GCE-Pt-Nps (b), platinum (c) and gold (d) screen-printed electrodes in the absence (1) and presence (2) of 1 mM H 2 O 2 . Supporting electrolyte: 0.025 PBS (рН 7.0), scan rate 50 mV/s. The dotted line shows proportionality of scales of (a, b) and (c, d).

Fig. 3 .

 3 Fig. 3.12. Cyclic voltammograms (a) and hydrodynamic voltammograms (b) of AuSPE modified with SiO 2 -ChOx film in the absence (1) and presence (2) of 2 mM choline. Supporting electrolyte: 0.025 М PBS (рН 7.5), scan rate 50 mV/s.

Fig. 3 . 13 .

 313 Fig. 3.13. Amperometric curves (a) and corresponding dependences of current from choline concentration in the initial range (b) for AuSPE-SiO 2 -ChOx, obtained at potential -1.0 (1), -1.1 (2), -1.2 (3), -1.3 (4) V and time of deposition 20 s. Supporting electrolyte: 0.025 М PBS (рН 7.5), applied potential +0.7 V.

Fig. 3 .

 3 Fig. 3.14. (a) Cyclic voltammograms of AuSPE-SiO 2 -ChOx (at optimum CTAB concentration) in the absence (1) and presence of 2 mM choline: 1st(2), 10th (3), 20th (4) and 30th (5) cycles of potential scan (50 mV/s); (b) Amperometric curve of AuSPE-SiO 2 -ChOx in the presence of choline (0.5 mM). Applied potential +0.7 V. Supporting electrolyte: 0.025 М PBS (рН 7.5)

Fig. 3 .

 3 Fig. 3.15. (a) Dependence of the amperometric response of AuSPE-SiO 2 -ChOx at potential +0.7 V on the choline concentration. Supporting electrolyte: 0.025 М PBS (рН 7.5); (b) Lineweaver-Burk plot for the determination of apparent Michaelis constant.

Fig. 3 .

 3 Fig. 3.16. Dependence of the amperometric response value of AuSPE-SiO 2 -ChOx at potential +0.7 V on the choline concentration in the solution. Supporting electrolyte: 0.025 М PBS (рН 7.5).

  nanofibers is increases in about 10 times compared with bare platinum electrode, making the use of such nanofibers promising for the development of oxidase-based biosensors. At the same time platinum fibers coated with SiO 2 -film do not have advantages over bare platinum electrode. The response of glucose oxidase immobilized in SiO 2 -film on the surface of platinum nanofibers significantly depends on the thickness of biocomposite film.Electrogeneration of platinum particles on the surface of the glassy carbon electrode increases the sensitivity of the latter to hydrogen peroxide. However, response of such modified electrode is unstable due to the gradual leaching of nanoparticles from the electrode surface, showing little promise of its application in the development of biosensors.The use of gold screen-printed electrode is promising for the immobilization of choline oxidase in SiO 2 -film. The application of simple one-step method of electrochemically-assisted deposition allows the immobilization of the enzyme on the surface of the gold electrode with preservation of its activity. The increase of the stability and sensitivity of the modified electrode to choline can be achieved by altering the deposition parameters and by introduction of the surfactants in the sol.The best performance of modified electrode was obtained using deposition at -1.1 V for 20 s and CTAB concentration in sol 12.2 mM. The developed sensitive element of the choline biosensor has a wide linear range and low detection limit.CHAPTER 4. IMMOBILIZATION OF SORBITOL DEHYDROGENASE ON THE ELECTROPHORETICALLY-DEPOSITED CARBON NANOTUBESThe use of dehydrogenases in biosensors are usually less common than corresponding oxidases due to their low stability and difficulty of coenzyme NAD + /NADH detection. For example, there are only a few works about the development of biosensors based on sorbitol dehydrogenase (DSDH) -enzyme that can be used to identify the substrate sorbitol. However, these works are limited to the definition of sorbitol in model solutions, data on the analysis of real objects are missing. Encapsulation in thin silica film can increase the stability of enzymes in the immobilized state, making appropriate the use of this method for the development of dehydrogenases-based biosensors. Given the lack of literature data on the application of EAD method to immobilize dehydrogenases, the point of interest is the development of methodological approaches of the EAD method application for the immobilization of DSDH into SiO 2 -film, as well as the use of developed biosensor for sorbitol determination in the real objects.The development of sensitive dehydrogenase-based biosensors requires the selective, sensitive and reliable detection of coenzyme NADH formed in the enzymatic reaction (see paragraph1.5.2). As noted in the literature review, the metal electrodes (including platinum and gold) are unsuitable for such purposes because of the high potential of NADH oxidation and poisoning of the electrode surface by reaction products, leading to a sensitivity decrease. The better choice is electrodes based on allotropic modification of carbon. For example, carbon nanotubes (CNT) are promising for use as a matrix for such biosensors due to the low potential oxidation of NADH on them. However, their handling is difficult because of small size, negligible solubility in inorganic solvents and the tendency to aggregation. As previously stated in the literature review (paragraph 1.3.2), electrophoretic deposition method is able to solve these problems allowing controlled electrode modification with CNT from low-concentrated dispersions. CNT-coatings obtained therefore, have not been used as a matrix for enzyme immobilization, but they have great potential and are promising for electrode modification including the sol-gel method. This chapter presents the results of study of CNT-layer, obtained by EPD on the surface of GCE, including the activity towards NADH oxidation. The prospects of its combination with EAD method for the immobilization of dehydrogenases are shown.

  Fig. 4.1. SEM image of nanotubes deposit on the surface of GCE with (a) short time of potential applying (t = 5 s) and with (b) long time of potential applying (t = 60 s).

Fig. 4 .

 4 Fig. 4.2. (a) AFM image and (b) cross-section profile (along white line) of the CNT-layer with 30 s of deposition. Image size 20 х 20 μm.

Fig. 4 . 3 .

 43 Fig. 4.3. Dependence of the thickness of carbon nanotubes layer measured by AFM from the time of electrophoretic deposition.

Fig. 4 .Fig. 4

 44 Fig. 4.4. (a) Cyclic voltammograms in the anaerobic conditions of GCE-CNT with adsorbed methylene green at different scan rates: (1) 5 mV/s, (2) 10 mV/s, (3) 20 mV/s, (4) 50 mV/s, (5) 100 mV/s, (6) 200 mV/s. (b) Oxidation (1) and reduction (2) peaks currents of methylene green versus scan rate.

Fig. 4 . 6 .

 46 Fig. 4.6. Cyclic voltammograms of the (a) GCE, (b) GCE-CNT (prepared by EPD) and (c) GCE-CNT (drop-coated) in the 0.067 M PBS (pH 7.5) without (solid lines) and with (dashed lines) 5 mM NADH. Scan rate 20 mV/s.

Fig. 4 . 7 .

 47 Fig. 4.7. Hydrodynamic voltammograms for 5 mM NADH in 67 mM PBS (pH 7.5) at (1) GCE and (2) GCE-CNT electrode.

Fig. 4 . 8 .

 48 Fig. 4.8. Cyclic voltammograms of GCE (a) and GCE-CNT (b) in the presence of 5 mM NADH: 1-st (1), 2-nd (2) and 3-d (3) cycle. Supporting electrolyte: 0,067 М PBS (рН 7,5), scan rate 20 мV/s.

1 Fig. 4 . 9 .Fig. 4 .

 1494 Fig. 4.9. Linear sweep voltammograms of GC electrode (1) and GC-CNT electrode (2) in the sol-gel solution. Scan rate 100 mV/s.

1 Fig. 4 .

 14 Fig. 4.10. Cyclic voltammograms of GCE (1) and GCE-CNT electrode (2) modified with thin film SiO 2 -DSDH by sol-gel EAD in the 0.1 M Tris-HCl buffer (pH 9.0) containing 5 mM D-sorbitol and 1 mM NAD + . Scan rate 20 mV/s. The conditions of sol-gel EAD were: -1.8 V, 16 s for GCE and -1.3 V, 16 s for GCE-CNT electrode.

Fig. 4 .

 4 Fig. 4.11. (a) Amperometric response of the GCE-CNT electrode modified by SiO 2 -DSDH film with different time of EAD to the additions of D-sorbitol: (a) 0 s, (b) 10 s, (c) 14 s, (d) 16 s, (e) 17 s, (f) 25 s. E = +0.5 V. (b) Sensitivity of the GCE-CNT electrode modified with SiO 2 -DSDH film to the D-sorbitol at +0.5 V as a function of the time used for the sol-gel EAD. Supporting electrolyte 0.1 M Tris-HCl buffer (pH 9.0) contains 1 mM NAD + . The time of electrophoretic deposition of carbon nanotubes was 60 s for all samples.

Fig. 4 . 12 .

 412 Fig. 4.12. Electrochemical response of the GCE-CNT electrode modified by SiO 2 -DSDH with optimal parameters (-1.3 V, 16 s) to the D-sorbitol at +0.5 V as a function of the electrophoretic deposition time and corresponding layer thickness (from equation (4.1)) of carbon nanotubes. Supporting electrolyte 0.1 M Tris-HCl buffer (pH 9.0) contains 1 mM NAD + .

Fig. 4 .

 4 Fig. 4.13. (a) Dependence of the amperometric response of GCE-CNT-SiO 2 -DSDH from the D-sorbitol concentration at potential +0,5 V. Supporting electrolyte: 0,1 М Tris-HCl (рН 9,0) and 1 mM NAD + .

Fig. 4 .

 4 Fig. 4.14A and B show edge-views of a typical CNT-PS film obtained by electrophoretic deposition on the surface of a glassy carbon electrode. The PS beads and carbon nanotubes are homogeneously mixed together forming a uniform deposit with thickness of about 4.5 μm. The apparent small amount of carbon nanotubes in the interspace of beads is a visual artifact that can be explained by low contrast of the image. The real quantity of nanotubes is much bigger as proved on the images of the film after template removal (see part C and D in Fig. 4.14). After calcination of the PS beads, the film structure is maintained, forming a cellular 3D network with pore sizes corresponding to those of the removed beads (≈ 500 nm). These pores are separated by a 40-60 nm thick CNT layer. The thickness of the template-free macroporous-CNT film (Fig. 4.14C) was only slightly smaller than the one of CNT-PS film (Fig. 4.14A).

Fig. 4 .

 4 Fig. 4.14. SEM-images on the edge (left column) and under the angle of 35 (right column) of a CNT-PS composite film deposited in the presence of PS-beads at 0.2 mg/mL, before (A, B) and after (C, D) template removal. Time of electrophoretic deposition was 135 s.

Fig. 4 .

 4 Fig. 4.16. (a) Variation of the CNT-PS layer thickness (135 s deposition) as a function of the concentration of PS-beads in the initial dispersion before (1) and after (2) template removal. (b) Variation of the CNT-PS (0.2 mg/mL) deposit thickness as a function of the electrophoretic deposition time, as measured with AFM or profilometry, before (1) and after template removal (2), as well as for the samples deposited without PS beads (3).

4 .Fig. 4 .Fig. 4 . 19 .Fig. 4 .Fig. 4 .

 4441944 Fig. 4.17. (a) Cyclic voltammograms of MG adsorbed on the macroporous CNT layers ([PS-beads] = 0.1 mg/mL) with different duration of electrophoretic deposition: (1) 15 s, (2) 45 s, (3) 75 s, (4) 105 s, (5) 135 s. Supporting electrolyte: 0,067 М PBS (рН 7,5), scan rate 20 mV/s. (b) Peak current of MG as a function of electrophoretic deposition time of non-macroporous CNT layers (1,1') and macroporous CNT layers ([PS beads] = 0.1 mg/mL) (2,2').

Fig. 4 .

 4 Fig. 4.22. Anodic peak current values corresponding to the adsorbed MG alone or in the presence of 1mM NADH, as a function of the electrophoretic deposition time applied to prepare the macroporous CNT electrodes.

Fig. 4 . 23 .

 423 Fig. 4.23. Cyclic voltammograms of (a, b) non-macroporous and (c, d) macroporous CNT film ([PS beads] = 0.2 mg/mL) prepared by electrophoretic deposition for (a, c) 15 s and (b, d) 135 s and modified with adsorbed MG and SiO 2 -

Fig. 5 . 1 .

 51 Fig. 5.1. Typical amperogram obtained during D-sorbitol determination with GCE-CNT-SiO 2 -DSDH using method of standard addition. Applied potential +0,5 V.

  Sample preparation was performed in accordance with State Standard[294].The samples of objects was weighed on an analytical balance so that in the final volume the concentration of sorbitol was between 20-50 mM. Sample was manually crushed in a porcelain mortar, quantitatively transferred into a 100 mL beaker and 40 mL of heated to 60 -70 C bidistilled water was added and stirred for one hour on a heated magnetic mixer. The additions of standard 50 mM solution of sorbitol were injected in some samples. The resulting suspension was filtered through a paper filter in a 50 mL flask and adjusted to the mark on the flask. Further determination of sorbitol in the resulting solution was performed by the method of standard additions, as described in paragraph 5.1.1.

*

  The stability of the electrode is expressed in the format X% / Y, where Xthe percentage of response (from the original) that persists after use of the electrode during the time interval Y.

7 VFig. 5 . 3 .

 753 Fig. 5.3. Typical amperogram obtained during choline determination with AuSPE-SiO 2 -ChOx using method of standard addition. Applied potential +0,7 V.

Fig. 5 .Fig. 5 . 4 .

 554 Fig. 5.4 shows that the current increase on the AuSPE after addition of ascorbic acid solution after contact with MnO 2 is very small (Fig.5.4a). For comparison, the

  Sample preparation was carried out according to the[313]. The sample was weighed on analytical balance, transferred to a beaker and 30 ml of 1 M HCl was added. The beaker was covered with a glass and heated at 60 -70 C in a water bath for 4 hours, stirring occasionally. Heating with acid promotes separation of milk proteins and hydrolysis of bound in the form of esters of choline to the free form.After cooling, the mixture was filtered through filter paper in 50 ml flask. The pH of the solution was adjusted to 3.0 -4.0 with 10 M NaOH. Treatment with 50 mg of MnO 2 was performed as indicated in section 5.2.2.1, the volume of the solution was adjusted to the mark with water. The resulting solution was stored in a refrigerator at 4 C up to one week. Determination of choline in the resulting solution was performed by the method of standard additions as described in paragraph 5.2.1. Results of determination are given in Table5.8. The data obtained are characterized by satisfactory accuracy and reproducibility and correlated with the content of choline, declared by the manufacturer (Appendix G).

  , ion chromatography [320] and capillary electrophoresis [321]. The mass detector [316, 317], ion exchange membrane detector [320] and indirect UV detection [321] can be applied. The general drawbacks of chromatographic techniques are long and complex sample preparation, high cost and the inability of analysis in the field.

*

  The stability of the electrode is expressed in the format X% / Y, where Xthe percentage of response (from the original) that persists after use of the electrode during the time interval Y.

CONCLUSIONS

  sorbitol. Interfering effect of ascorbic acid can be eliminated by the introduction of trivalent iron. Results of sorbitol determination by the developed technique in the food and cosmetic products are characterized by satisfactory accuracy and reproducibility.The technique of amperometric determination of choline using gold screenprinted electrode modified with film SiO 2 -choline oxidase was proposed. The calibration graph was linear in the concentration range of 110 -5 -610 -4 M, the detection limit was 510 -6 M. The 10-fold excess of sucrose, lactose, urea and heavy metals except Cu (II) do not interfere with the choline determination. Interfering effect of ascorbic acid may be eliminated by prior mixing with MnO 2 . Results of choline determination in the food using developed technique are characterized by satisfactory accuracy and reproducibility. The advantages of the developed techniques in comparison with known methods of sorbitol and choline determination are simple sample preparation, higher selectivity and expressivity of analysis, and low cost of a single determination. The simple one-step procedure for immobilization of oxidases and dehydrogenases in SiO 2 -film on the surface of different types of electrodes by electrodeposition method was unified. The examples of choline oxidase and sorbitol dehydrogenase showed that immobilized enzymes more actively bind to the substrate and retain their activity longer than in solution.  Electrodes modified with platinum nanomaterials are more sensitive to hydrogen peroxide than platinum macroelectrodes. At the same time, these electrodes coated with biocomposite SiO 2 -enzyme film exhibit analytical characteristics similar to platinum macroelectrodes.  Modification of glassy carbon electrode with carbon nanotubes (CNT) by electrophoretic deposition method increases the sensitivity and stability of the amperometric response of coenzyme NADH, which is promising for the development of dehydrogenase-based biosensors. The use of CNT shifts NADH oxidation potential at 0.25-0.3 V towards negative values, increasing thereby the selectivity of its detection.  The use of gold screen-printed electrode is promising for the immobilization of choline oxidase in SiO 2 -film. The increase of modified electrode sensitivity to choline is achieved by optimization of the parameters of electrochemically-assisted deposition, the maximum response was obtained during the deposition for 20 s at potential of -1.1 V.  Electrode modified with CNT and sorbitol dehydrogenase, shows significant advantages in terms of sensitivity and selectivity of sorbitol detection compared with electrode containing no CNT. Analytical signal of modified electrode depends on the parameters of electrodeposition and thickness of CNT-layer. Best characteristics were obtained using -1.3 V, 16 s of electrochemically-assisted deposition and CNT-layer thickness from 100 to 150 nm.

Fig

  Fig. D.1. AFM-image of the surface of GCE, modified with CNT by EPD.

TABLE OF CONTENTS

 OF

TABLE

Table 1 .1 Main methods of sol-gel films obtaining

 1

	Method	Advantages	Drawbacks

Table 3 .2 Influence of CTAB concentration in the sol on the long-term response stability of AuSPE-SiO2-ChOx

 3

	С (CTAB), mM	Relative sensitivity in two weeks, %
	0.0	< 1
	6.1	9.8
	12.2	52.7
	21.3	18.9
	42.7	8.5

Table 5 .1 Results of the determination of sorbitol additions in the buffer solution using GCE-CNT-SiO2-DSDH. n = 3, P = 0,95

 5

	Sorbitol concentration, mM
	Added	Found	S r
	0.40	0.41 ± 0.04	0.04
	0.60	0.60 ± 0.05	0.03
	0.90	0.90 ± 0.04	0.02

Table 5 .3 Results of sorbitol determination in the food and cosmetics samples. n = 3, P = 0,95

 5

			Sorbitol content, мг/г
	Sample	Added	Found	S r
	Toothpaste for children «Oral B	0	117 ± 20	0,06
	Stages berry bubble»	296	411 ± 33	0,04
		0	549 ± 46	0,03
	Chewing gum «Orbit»
		694	1251 ± 122	0,04

Table 5 .4 Comparative characteristics of known amperometric biosensors based on DSDH

 5

	Electrode	Modifier	Response time, s	Linear range, mM	Limit of detection, μM	Potential of detection, mV	Stability*	RSD, %	Real object analysis	Link

Table 5 .7 Influence of MnO2 on the results of 0.8 mM choline determination using AuSPE-SiO2-ChOx

 5

	№	Solution content	Choline found, mM
	1	Choline	0.81 ± 0.06
	2	Choline + 10 mg MnO 2	0.81 ± 0.06
	3	Choline + 0.1 mM ascorbic acid	0.91 ± 0.09
	4	Choline + 0.1 mM ascorbic acid + 10 mg MnO 2	0.81 ± 0.07

Table 5 .8 Results of choline determination in model solution and food products.

 5

		n = 3, P = 0,95
		Choline content, mM
	sample	Added	Found, х ± Δх	S r
		0.30	0.34 ± 0.04	0.05
	Model solution*
		1.00	1.09 ± 0.12	0.05
		Choline content, mg/g
		0	0.95 ± 0.14	0.06
	Infant formula «Bebi»
		1.12	1.99 ± 0.19	0.04

Table 5 .9 Comparative characteristics of known amperometric biosensors based on ChOx

 5

	Electrode	Modifier	Response time, s	К м , mM	Linear range, mM	Limit of detection, μM	Potential of detection, mV	Stability*	RSD, %	Real object analysis	Link
	Pt	Diaminobenze	30 1.2	0.05 ÷	50	0	85% /	8	-[327]
		n-Prussian blue			2			2 m.
	Pt	Polyvinylferroc	70 2.32 0.004 ÷	4	750 -	4,6 -[328]
		ene chlorate			1.2
	Carb	Prussian blue	30 2	0.02 ÷	20	50	1 m.	14	-[329]
	on				2
	paste
	Pt	Au-Nps-PVA-	20 0.78 0.02 ÷	10	400 80% /	7,4 -[264]
		Glutaraldehyde			0.4			14 d.
	GCE PDDA-FePO 4 -	2	0.47 0.002 ÷	0.4 0	95% /	3,2 -[307]
		Prussian blue			3.2			14 d.
	Pt	SiO 2 -CNT	15 -	0.005 ÷	0.1 160 75% /	4,8 + [168]
					0.1			1 m.

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