VOLTAMMETRIC SENSORS FOR THE DETERMINATION OF BIOMOLECULES/DOPING AGENTS

Abstract

The need of highly sensitive and selective sensing probes having enormous practical applications and significant benefits over conventional technologies attracted researchers towards nanomaterials based technologies. Last few years have seen a worldwide outburst of research interest of electrochemists at the interface of nanotechnology and electrochemistry. The implementation of nanoscience and nanotechnology achievements in bioelectrochemistry ameliorates the current electroanalytical techniques and methodologies by improving sensitivity and selectivity along with detection limit. Application of carbon nanomaterials in sensor research offers excellent prospects for designing novel sensing systems and enhancing the performance of bioanalytical assay. The fabrication of electrode surface with nanomaterials imparts functionality distinct from the base electrode and has been found to show consistently good results with high sensitivity and selectivity. In view of the large number of biomolecules in human body and the ability of nanomaterial modified electrodes to measure extremely small amounts of specific biomarkers at molecular levels, an endeavour, has been made in the present investigation to develop simple and sensitive electroanalytical methods using nanomaterial based sensors for the qualitative and quantitative analysis of biologically important compounds, drugs and doping agents. The first chapter of the thesis is "General Introduction" giving a recapitulate review of the relevant work and, highlights the significance of electrochemical studies in biological systems along with its relevance in assorted areas. The brief account of various electroanalytical techniques employed for present dissertation has also been described. The• chapter also discusses the concise outline of advantages rendered by nanomaterial modified electrodes over conventional electrodes. The second chapter of the thesis describes the simultaneous determination of relative concentrations of guanine and 8-hydroxyguanine in oxidatively damaged calf thymus DNA sample. Guanine is highly susceptible to oxidative stress in the genomic DNA due to having the lowest oxidation potential hence; guanine plays a key role in the oxidation of DNA by various types of oxidants and free radicals. 8-Hydroxyguanine is one of the prominent lesions generated during DNA damage by oxidative processes and most extensively investigated due to its miscoding properties and potential role in mutagenesis, iii carcinogenesis and aging. A quantitatively important alteration occurring during oxidative damage to DNA consists of oxidation of guanine residues into 8-hydroxyguanine. The bare and single walled carbon nanotubes modified edge plane pyrolytic graphite electrodes are utilized to examine such type of vital alteration. It has been found that remarkable enhancement in the oxidation peak current of both compounds was observed along with the negative shift of peak potentials using modified electrode as compared to bare electrode. Oxidative damage to DNA bases by isotope dilution mass spectrometry required isotopically labelled oxidized bases as internal standards and GC-MS requires a derivatization procedure which can cause `artifactual' oxidation of some undamaged bases leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore, using proposed method which requires no internal standards and derivatization steps and, hence, eliminates possible `artifactual' oxidation of DNA bases; the level of guanine and 8-hydroxyguanine can be measured in calf thymus DNA with high rank of accuracy. .0 H N N H2N "'N N H 0 H2N N Guanine 8-Hydroxyguanine The third chapter of the thesis presents the novel approach using square wave voltammetry with the combination of multi-walled carbon nanotubes modified edge plane pyrolytic graphite electrode (MWNT/EPPGE) as a sensitive and selective sensor for the detection of catecholamines — epinephrine (EP) and norepinephrine (NE) in human body fluids. The chapter is divided into two sections -- first section deals with the determination of epinephrine while the second section describes the simultaneous determination of epinephrine and norepinephrine. Catecholamines are produced by sympathetic nervous system activation and act as hormones and neurotransmitter to monitor heart rate, brain muscles activity, glycogenolysis, fatty acid mobilization and body temperature. These important actions also make them potent doping agents and hence, epinephrine is banned in competitive games by World Anti. Doping Agency. Studies show that changes of their iv concentration in nervous tissues and body fluids are diagnostic symptoms of several diseases. The amount of catecholamines present in blood, plasma or serum is considered as a diagnostic aid to monitor therapeutic administration or to identify the causative agent in potential poisoning victims. Hence, the quantitative determination of catecholamines is quite helpful for developing nerve physiology, clinical diagnosis of some diseases and controlling medicine in pharmacological research. The release of catecholamines in human system also depends on smoking and exercise because these stimulants activates the sympathetic nervous system acting via splanchnic nerves to the adrenal medulla and thus stimulates the release of catecholamines into the blood stream. Therefore, blood plasma and urine samples of smokers ' and athletes were analyzed to determine catecholamines — epinephrine and norepinephrine concentrations. A broad bump at — 250 mV is appeared for the oxidation of epinephrine and norepinephrine at bare EPPGE whereas at MWNT/EPPGE two well-separated peaks at —150 and —215 mV are appeared for the oxidation of EP and NE, respectively. The oxidation peak current of both the neurotransmitters increased significantly along with the negative shift of peak potentials using MWNT/EPPGE. The oxidation of both the compounds occurred in a pH dependent, 2e and 2H+ process and the electrode reaction followed diffusion controlled pathway. Linear calibration curves were obtained for epinephrine and norepinephrine in the range 0.5 — 100 nM with limit of detections 0.15 x 10 9 and 0.90 x 10-10 M, respectively. The fourth chapter of the thesis deals with the comparative study of the electrocatalytic activity of two types of CNTs — single walled carbon nanotubes (SWNTs) and multi walled carbon nanotubes (MWNTs) towards electrochemical determinations. A detailed comparison has been made between the response of S WNT and MWNT modified edge plane pyrolytic graphite electrode in terms of several important analytical parameters viz, sensitivity, selectivity and detection limit in order to understand that which one have enhanced electrocatalytic activity. The chapter also discussed the effect of surfactant — cetyltrimethyl ammonium bromide as electrode surface modifier along with single walled carbon nanotubes. The chapter is divided into three sections — first section deals with the voltammetric determination of a new potent calcium antagonist, amlodipine besylate (ADB), in human body fluids mainly focused on the comparison of electrocatalytic activity of MWNTs and SWNTs towards the oxidation of ADB. The second section describes the v simultaneous determination of two important synthetic corticosteroids — prednisolone and prednisone in human body fluids and pharmaceutical preparations at single walled carbon nanotubes modified edge plane pyrolytic graphite electrode (SWNT/EPPGE). The urine and plasma samples of patient undergoing treatment with prednisolone were analyzed so that the method can be used to determine prednisolone and prednisone in doping cases and other clinical purposes. The third section discusses the use of single .walled carbon nanotubes-cetyltrimethylammonium bromide nanocomposite film modified edge plane pyrolytic graphite electrode (S WNTs-CTAB/EPPGE) for the determination of betamethasone, a potent pharmaceutical ingredient and doping agent, in urine samples of pregnant women who are undergoing treatment with betamethasone. IIaH I CH3 OH H3Ci ■ Amlodipine besylate Prednisolone The aim of fifth chapter of the thesis is to resolve the conflicting views and find out the actual reason of electrocatalytic property of fullerene. The effect of embedded metallic impurities (Fe, Cu, Co, Ni) of fullerene, which are accessible to fluids on which fullerene is casted is studied. The fullerene modified glassy carbon electrode was also employed to determine concentration of adenine and 2'-deoxyadenosine (2'-dAdo) in urine samples of carcinoma patient. The chapter is divided into two sections— in the first section the effect of embedded metallic impurities of fullerene; substrate and the application of fullerene-C60_ modified electrodes for the determination of nandrolone have been discussed. Second section deals with the simultaneous determination of adenine and 2'-deoxyadenosine in human urine and plasma samples. Nandrolone, an anabolic androgenic steroid banned in sports by the International Olympic Committee and World Anti-Doping Agency, as it is extensively misused by vi bodybuilders and athletes for the purpose of enhancing athletic performance, has been discussed in the first part of this chapter. Clinically, it is prescribed in the treatment of anaemia, neoplasia including breast cancer, rebuilding of muscles after debilitating disease and treatment of osteoporosis in postmenopausal women. A comparison of edge plane pyrolytic graphite substrate is made with other substrates like indium tin oxide, glassy carbon, gold and basal plane pyrolytic graphite as a substrate for fullerene modification for the determination of nandrolone by square wave voltammetry in phosphate buffer media. Comparative study of voltammetric response of nandrolone at untreated, purified and super-purified fullerene modified edge plane pyrolytic graphite electrode is also carried out to determine the role of embedded metallic impurities of fullerene on determination of nandrolone. It is observed that edge plane pyrolytic graphite electrode serves as best substrate among the studied for casting fullerene. The removal of embedded metals from fullerene shifts the peak potential of nandrolone to more positive potentials and peak current decreases. A linear calibration curve is obtained in the concentration range of 0.01 — 50 nM with a detection limit and sensitivity of 1.5 x 10-11 M and 1.838 μA nM-1, respectively. The developed method was satisfactorily applied to the determination of nandrolone in several commercially available medicinal samples at untreated fullerene — C60 — modified edge plane pyrolytic graphite electrode. Adenine is one of the two purine nucleobases and 2'-deoxyadenosine (2'-dAdo) is one of the purine 2'-deoxyribonucleosides present in deoxyribonucleic acid (DNA) and therefore, both are essential molecules of life and evolution. Adenine is of tremendous biological significance as it is one of the nitrogenous bases found in deoxyribonucleic acid and ribonucleic acid to make up genetic information. It is a component of adenosine triphosphate which is a major energy releasing molecule in cells. Adenine is.also a part of various coenzymes and being a part of nucleic acids it plays an important role in protein synthesis. 2'-dAdo is a carbohydrate derivative of adenine and the conversion of 2'-dAdo to adenine represent a protective device to control the plasma level of 2'-dAdo when the activity of adenosine deaminase (ADA) is inhibited. In the case of hepatocellular carcinoma the level of adenine has been found to increase considerably. Thus, the simultaneous determination of 2'-dAdo and adenine is the subject of considerable interest especially in human body fluids particularly urine in case of carcinoma. Thus, keeping in consideration the importance of adenine concentration in body fluids studies have been performed to vii determine its concentration in urine sample of carcinoma patient using fullerene — C60 — modified glassy carbon electrode. Two well-defined anodic peaks at potential of 1248 mV and 994 mV were observed for 2'-dAdo and adenine, respectively. Linear calibration curves were obtained within the concentration range 10 nM — 100 μM for both compounds in 0.1 M phosphate buffer. solution having the limit of detection 1.5 x 10-8 M and 4.5x 10-8 M for 2'-dAdo and adenine, respectively.