DEVELOPMENT OF VOLTAMMETRIC SENSORS FOR THE DETERMINATION OF BIOMOLECULES AND DOPING AGENTS

Abstract

Over the last decade numerous developments in nanoscience and nanotechnology have contributed significantly to the electrochemistry. Nanotechnology based electrochemical platform offers a promising tool for attainment of multiple aims in biomolecular analysis. Nanomaterials prepared from metal, semiconductors and carbon or polymeric species have allured great attention due to their widespread applications in different areas of science. There has been a substantial progress in construction of highly efficient nanomaterials based electrochemical sensors for monitoring of biologically important molecules and pharmaceutical drugs. It is observed that the sensitive and selective detection of specific biomolecules and drugs is mandatory for elucidating the physiological processes as well as for early diagnosis and therapy of diseases. The recent upcoming of new forms of carbon based nanomaterials such as fullerene, graphene and carbon nanotubes (CNT) have revolutionalized the electrochemical research and brought many potential applications in nanoscience. These carbon based nanomaterials have enticed many researchers due to their attractive electronic, optical, thermal and electro-catalytic properties over other conductive materials. These properties together with their nanometric size and high aspect ratio make them suitable for electrochemical sensing of verities of organic compounds. Considering the significance of nanomaterials in the area of electrochemistry, in this thesis, an attempt has been made to systematically utilize the different modification approaches employing carbon based nanomaterials with a focus on the development of highly sensitive electrochemical sensors for the investigations of biomolecules and doping agents. The thesis is divided in six chapters. In chapter 1 an overview on conventional electrodes, types of nanomaterials and modification of electrodes using nanomaterials with particular emphasis on carbon nanotubes modified electrode is presented. This chapter also deals with the illustration of methodology employed in the present investigation comprising some theoretical aspects of voltammetric techniques. Chapter 2 describes the application of multi-walled carbon nanotubes (MWCNT) modified edge plane pyrolytic graphite electrode (EPPGE) for studying the electrochemical reduction of Norfloxacin (NFX). The modified electrode was subjected to the determination of NFX in biological fluids and pharmaceutical tablets. The modified electrode exhibited excellent electro-catalytic properties towards the reduction of NFX by decreasing the reduction peak potential and ii increasing the cathodic peak current. Moreover, nanotubes modified electrode was also used to investigate the effect of NFX on catabolism of caffeine through its determination in urine sample. It is found that the long term administration of NFX reduces the catabolism of caffeine by preventing its demethylation, which may lead to accumulation of caffeine in human system. It is suggested that patient on medication with NFX should avoid the excess consumption of caffeine. This is the first time detection of NFX using nanotubes modified electrode based on its electrochemical reduction. The advantage of present method for detection of NFX is the large negative potential window at which the common metabolites of urine do not interfere with the analyte due to their non-reducible nature. The modified electrode determined the NFX concentration ranged between 1.2 and 1000 μM with detection limit (3σ/slope) and sensitivity of 40 ± 3.3 nM and 0.072 μA μM-1 Chapter 3 deals with the use of concept of swift heavy ion irradiation of carbon nanotubes and its effect on structural properties of nanotubes. MWCNT was irradiated by Ag ions of high energy (~ 120 MeV) at different fluences of 1e12, 3e12 and 1e13 ions cm, respectively. The method is simple, sensitive and can be readily applied to monitor the NFX in urine samples and also its effect on catabolism of caffeine. -2 with 15 UD Pelletron Accelerator. After optimizing the experimental parameters, the irradiated sensor was employed for the simultaneous determination of two important neurotransmitters; Epinephrine and 5-Hydroxytryptamine (serotonin). Simultaneous determination of both neurotransmitters was carried out in phosphate buffer of pH 7.2 (as supporting electrolyte) using square wave voltammtery (SWV) and cyclic voltammetry (CV). [Nanotubes before and after heavy ion irradiation] iii [Epinephrine] [Serotonin] Irradiation by Ag ions caused an enhancement in electrocatalytic activities of nanotubes due to the increased conductivity with insertion of Ag ions and an increment in effective surface area after irradiation. The irradiated sensor was applied for the determination of epinephrine and serotonin in real samples such as blood and urine and exhibited good accuracy and precision of the method. The coexisting metabolites present in urine such as uric acid, ascorbic acid and dopamine did not show any interference during the simultaneous determination of epinephrine and serotonin. In chapter 4, the application of single-walled carbon nanotubes (SWCNT) for the modification of EPPGE is documented. The modified sensor was used for electrochemical reduction of a topical corticosteroid; Halobetasol propionate (HBP). At SWCNT-coated EPPGE, the electrochemical response of HBP increases significantly as reduction peak current increases and peak potential shifts to less negative direction as compared to bare EPPGE, showing the electrocatalytic ability of SWCNT towards the reduction of HBP. The controlled potential electrolysis was performed to obtain the product of HBP formed during its electrochemical reduction. For this purpose, the solution of HBP was electrolyzed by applying constant potential ~ 70 mV more negative than reduction peak potential of HBP using potentiostat. The product of reduction was characterized by FT-IR and 1H-NMR spectroscopic measurements and the possible site of reduction was deduced as >C=O. The proposed methodology was also used for the determination of HBP in various pharmaceutical preparations. The results obtained from the determination of HBP in tablet samples did not show any interference from the excipients, viz KCl, NaCl and petroleum jelly because all these compounds are not reducible. The modified electrode showed good stability and reproducibility with relative standard deviation of 2.21 % and 3.32 %, respectively, confirming that this approach can be successfully used for the determination of HBP in various pharmacological samples. iv Chapter 5 illustrates the utilization of SWCNT modified EPPGE for the investigation of an important β2-agonist; Salbutamol, which is used as bronchodilator in the treatment of asthmatic disorders and chronic obstructive pulmonary diseases. Salbutamol is found to be able to increase muscle protein, reduce total body fat and promote muscle growth, hence, it is abused by athletes in competitive games. Its use in sports by athletes has been banned by World Anti Doping Agency (WADA) and it has been listed as a doping agent. The threshold concentration of this drug is 1000 ng mL-1, and gives an indication of oral administration according to WADA rules. In the proposed work, modified electrode showed improved voltammetric response towards the oxidation of salbutamol with well-defined peak at ~ 600 mV with enhanced peak current in comparison to bare electrode. The CNT increased the electrochemical performance of electrode due to their excellent conductivity and high surface area. The proposed sensor showed a good linear range, low detection limit, high sensitivity with good stability and reproducibility. The sensing of salbutamol was carried out in pharmaceutical tablets and human body fluids which make the proposed method of significant interest for doping control purposes at the site of competitive games. [Salbutamol] The last chapter of thesis chapter 6 describes the application of EPPGE for the determination of two analgesic drugs. In the first section of this chapter, the electrochemical study of non-steroidal anti-inflammatory drug; Diclofenac has been presented. Diclofenac has analgesic, antipyretic and anti-inflammatory properties and is widely prescribed in clinical medicine for the treatment of several diseases. The investigation of this drug was carried out at EPPGE using SWV and CV in phosphate buffer of pH 7.2. The oxidation peak current increased linearly with the concentration of diclofenac in the range 10 – 1000 nM and detection limit and sensitivity of proposed method were 6.2 nM and 69 nA nM-1, respectively. The controlled potential electrolysis was performed and the product formed from the oxidation of diclofenac was characterized using v 1H-NMR. The developed method was applied for the determination of diclofenac in pharmaceutical formulation and urine samples obtained from the patients undergoing treatment with diclofenac. [Diclofenac] [Caffeine] [Aspirin] The second section of this chapter presents the results on the determination of two analgesic drugs; Caffeine and Aspirin. Caffeine is a stimulant drug which arouses the central nervous system and cardiovascular system. It is prescribed as an analgesic adjuvant in pharmaceutical preparations for the treatment of headache and pain. Caffeine is used by professional athletes to give them alertness and extra energy for their work so that they could improve their physical performance. Therefore, caffeine has been reported as a doping agent at a level of 12 μg mL-1 by WADA. Aspirin is an important analgesic that reduces the pain without interfering the functions of other sense organs. Bare EPPGE was used for the determination of caffeine and aspirin at pH 7.2. The electrode showed two well-defined peaks having peak potential of ~ 1225 mV and ~ 1335 mV for aspirin and caffeine, respectively. The peak current of oxidation peaks was found to increase with increase in the concentration of caffeine and aspirin in the range 0.02 – 100 μM and detection limits of 0.01 μM and 0.08 μM, respectively are observed. The proposed sensor was successfully applied for the determination of caffeine and aspirin in urine samples, pharmaceutical preparations and coffee beverages.