Comparison of electron transfer of choline oxidase enzyme by three types of carbon nanotubes

Dissertation to receive a master's degree in the field of medical nanotechnology

Abstract

Choline is one of the components of phospholipids and plays an important role in fat metabolism. It is also necessary for the formation of acetylcholine, which is involved in the transmission of nerve impulses. Lack or increase of choline in the body causes complications and diseases in humans; Therefore, it seems necessary to detect its amount in the body. To detect choline, electrochemical biosensors based on choline oxidase enzyme are often used. Since the redox group of the enzyme is buried inside it, to make an electrochemical sensor using it, electrodes modified with factors such as carbon nanotubes/ionic liquid composites should be used. In this research, three types of bamboo-like, multi-walled and single-walled carbon nanotubes were used for this purpose and the results were compared. The ionic liquid used was 1-allyl 3-methylimidazolium bromide.

Electrochemical parameters, including the apparent electron transfer constant ks, were obtained by cyclic voltammetry. The value of ks for bamboo-like, multi-walled and single-walled nanotubes was 23.11 s-1, 1.75 s-1 and 1.49 s-1, respectively. In the next step, the analytical parameters of the biosensor for choline detection were obtained. The detection limit for biosensors made with bamboo-like, multi-walled and single-walled carbon nanotubes was 0.28 Mµ, 3.08 Mµ and 1.97 Mµ, respectively. The linear range of these three designed biosensors is respectively 1-10×0.7-3-10×2.1 mM for Bombo nanotubes, 1-10×8.2-2-10×2.1 mM and 1-10×3-1-10×3.1 mM for multi-walled nanotubes and 1-10×9.5-1-10×9.1 mM and 2-10×4.6-2-10×2.1 was obtained for single-walled nanotubes. The sensitivity of these three biosensors was calculated as 294 ?A/mM.cm2 for Bombo, 51.67 ?A/mM.cm2 and 136.276/mM.cm2 for multi-walled nanotubes and 45.226.?A/mM.cm2 and 56.213.?A/mM.cm for single-walled nanotubes. The results showed that the biosensor designed using Bombo-like carbon nanotube has better electrocatalytic activity and direct electron transfer.

Key words

Carbon nanotube, choline oxidase enzyme, ionic liquid and direct electron transfer

1-1. Choline oxidase enzyme

1-1-1. Enzyme introduction

Choline oxidase enzyme is one of the types of oxidoreductase enzymes. Oxidoreductases, which constitute about 25% of known enzymes, play an important role in the metabolism of living cells. This group of enzymes includes all enzymes that catalyze oxidation and reduction reactions. From the enzymes of this group, we can mention oxidases, peroxidases, dehydrogenases, catalase and many other enzymes [3]. The importance of studying oxidoreductases lies in the nature of the reactions they catalyze. In fact, because these enzymes catalyze difficult reactions, such as selective oxidation and reduction of organic molecules, they are of particular importance in the environmental field. In addition, since oxidoreductases are widely used in the synthesis of organic compounds, they have applications in pharmaceuticals [4-5]. Among oxidoreductases, oxidases are particularly important because they use oxygen as an oxidizing agent and do not need cofactors. The choline oxidase enzyme that is examined in this treatise is one of the types of oxidases.

1-1-2. History

Choline oxidase enzyme was first discovered in 1933 by Bernheim [1] in animal tissue [6]. Among animal tissues, choline oxidase is found mostly in mouse liver. This form of the enzyme was also found in the kidney, but not in the blood, muscle or heart [7]. In 1952, Kelly [2] proved the existence of the prosthetic group of flavin adenine dinucleotide (FAD) in this enzyme.. Despite the fact that the first samples of choline oxidase were extracted from animal tissues with great difficulty and impurities, later this enzyme was also obtained from other species of micro-organisms, including the extraction of the enzyme in 1977 from Arthrobacter [3] [8].

1-1-3. Enzyme reaction

choline oxidase enzyme catalyzes the oxidation reaction of choline to glycine betaine. For the first time in 1952, the presence of the flavin adenine dinucleotide (FAD) prosthetic group in this enzyme was proved. In fact, choline oxidase enzyme oxidizes the substrate with the help of its FAD prosthetic group. Choline oxidation is done in two stages; In the first step, by transferring the hydride ion (H- from choline to molecular oxygen, the substrate becomes an intermediate product (betaine aldehyde). In the next step, betaine aldehyde produces the final product betaine by transferring two more electrons. The overall scheme of this reaction is shown in the figure below [9]. Figure 1-1. Two-step oxidation reaction scheme of choline to glycine Betaine catalyzed by choline oxidase enzyme

The active site of enzyme

choline oxidase enzyme is in dimer form and there are two domains important for substrate and FAD binding. Meanwhile, the 23Ser sequence creates many interactions with the flavin. The active site of the enzyme is located between the flavin binding domain and a conserved loop structure.

Figure 1-2. Schematic of the active site of choline oxidase enzyme

1-1-5. The importance of the study

The enzyme choline oxidase has received much attention due to its biotechnological and medical applications. The importance of studying this enzyme can be mentioned as follows:

1) From a chemical point of view, the oxidation reaction catalyzed by choline oxidase enzyme is of particular importance. Because breaking the covalent carbon-hydrogen bond of the substrate without the presence of the enzyme is not energetically favorable [10]. Glycine betaine is a compatible dissolved substance that accumulates in the cytoplasm of pathogenic bacteria and plants cells and causes them to resist osmotic pressure and heat stress. This reaction is very important in biotechnology and medicine, because it prevents dehydration and cell death of plants and pathogenic bacteria [11]. The study of choline oxidase enzyme is of great interest in the field of genetic engineering, especially during osmotic stress; Because in this way, the effective factors that increase the synthesis of this enzyme in cultivated plants such as rice and potatoes can be identified and used, which is very important from an economic point of view.

3) Chaperones are proteins that maintain the real structure of proteins, and glycine betaine is one of these proteins [12]. Glycine betaine protects the translation and transcription machinery by reducing the melting temperature of two DNA strands [13].

4) Another application of this enzyme is the measurement of organic phosphorus substances. These substances are increasingly used as insecticides, pesticides and chemical warfare agents. Organic phosphorus inhibits the choline oxidase enzyme. Based on this, biosensors for measuring organic phosphorus substances can be made based on the inhibition of choline oxidase enzyme [14].

5) Another clinical application of choline oxidase is its use in making enzyme biosensors for choline measurement [15]. Choline is found abundantly in nature in free form, acetylcholine or in more complex compounds such as phospholipids and their metabolic intermediates. This substance is a necessary and complementary part of lestin, which exists in all plant and animal cells. Choline in unsupplemented diets is also mainly in the form of lecithin, less than 10% of which is released in free form or in the combination of sphingomyelin [15].