VOLTAMMETRIC SENSORS FOR HIGHLY SENSITIVE DETERMINATION OF BIOMOLECULES AND DRUGS

Abstract

The analysis of biomolecules and drugs has great importance in medical science and in the pharmaceutical industries. In both the cases accuracy is the main factor, which can affect the health of living mankind. From the previous literature, it has been concluded that small concentration of biomolecules and drugs is responsible to regulate the various biological activities. Any imbalance in their concentration can disturb the human physiological system, which leads to various serious diseases. Therefore, rapid, sensitive and highly selective methods are needed for the selective determination of biomolecules and drugs in clinical diagnosis and quantification of drugs in pharmaceutical analysis. Voltammetric sensors have many advantages in term of excellent sensitivity and selectivity for the determination of electro-active biomolecules, drugs, organic compounds and inorganic compounds. As a result of this, several unmodified and modified voltammetric sensors have been developed for the analysis of the trace amount of biologically significant biomolecules and drugs. In the present thesis, various voltammetric sensors have been fabricated, characterized and used for the determination of biomolecules and drugs. The fabrication, characterization, behavior, results and applications of these sensors are summarized into five chapters. The first chapter of the thesis is ―General Introduction‖. It describes important aspects of electrochemical, voltammetric techniques, conventional electrodes, types of surface modifiers and analytes of interest. This chapter also presents the methodology applied in the present studies. The second chapter describes the fabrication of unmodified and nanopalladium grained polymer nanocomposite based sensors for the sensitive determination of a biomolecule, Melatonin (MEL) in human biological samples and pharmaceutical samples. This chapter has been divided into two sections. In the first section, the unmodified glassy carbon sensor is employed for the determination of MEL using cyclic voltammetry (CV) and square wave voltammetry (SWV). Irreversible oxidation of MEL was found to proceed through an adsorption controlled pathway at the surface of glassy carbon. The quantitative estimation of MEL was performed using SWV in a linear concentration range of 5–200 μM with a sensitivity of 0.0491 μM/μA and a limit of detection of 0.3432 μM was observed. The proposed protocol was also applied for determining MEL content in pharmaceutical samples and recovery studies in human urine samples were also ii performed. The observed results demonstrated a good agreement with the stated values. In the second section, the electrocatalytic properties of the palladium nanoparticles based polymer nanocomposite towards sensing of MEL are presented. The modification protocol involved a single step fabrication and was achieved using CV. The sensing surface was characterized using voltammetry, Field Emission Scanning Electron Microscopy (FE-SEM), Energy dispersive X-ray analysis (EDX) and Electron Impedance Spectroscopy (EIS). Quantitative estimation of MEL has also been carried out using SWV in the linear concentration range of 5–100 μM and sensitivity and limit of detection of 0.1235 μA/μM and 0.09 μM respectively have been achieved. The surface modified sensor exhibited almost 3 fold improvement in the sensing properties in comparison to the unmodified surface. The proposed method was also successfully extended for the determination of MEL in commercially available pharmaceutical formulations and human urine samples. The third chapter of the thesis presents the development of melamine based molecularly imprinted, palladium nanoparticles decorated multi-walled carbon nanotubes and silver nanoparticles decorated graphene nanorribon modified sensor for the determination of biomolecules. For the sake of clarity, this chapter has been divided into three sections. The first section, describes a sensitive and facile molecularly imprinted sensor for the determination of 8- hydroxydeoxyguanosine (8-OHdG), an important oxidative DNA damage product. The molecularly imprinted polymer film was fabricated by electropolymerization of melamine in the presence of 8-OHdG, on glutaraldehyde/poly-1,5-diaminonaphthalene modified edge plane pyrolytic graphite (EPPG) surface. The imprinted sensor surface was characterized by using FESEM, EIS, CV, SWV and UV-visible spectroscopy. The calibration response was linear over a concentration range of 0.020–3 μM for 8-OHdG with sensitivity and limit of detection (3σ/b) as 10.59 μM/μA and 3 nM respectively. The common metabolites in urine, like uric acid, ascorbic acid, xanthine and hypoxanthine did not interfere up to 100-fold concentration. The imprinted sensor is also successfully employed for the determination of 8-OHdG in human urine sample of a renal failure patient. The second section, deals with the nano palladium decorated multi walled carbon nanotubes (PdNP:MWCNT) modified sensor for the determination of 5- hydroxytryptophan, an important serotonin (5-HT) precursor. PdNP:MWCNT was synthesized in a simple single step, followed by the characterization using FE-SEM, EDX, Film-X-ray diffraction (XRD), EIS and voltammetry. The PdNP:MWCNT was then used for the surface modification of iii glassy carbon electrode (GCE) and applied for the electrochemical analysis of 5- hydroxytryptophan (5-HTP), a serotonin precursor. A significant increment in the peak current response was observed and peak potential also shifted towards less positive potentials indicating the facilitated oxidation process at PdNP:MWCNT/GCE. The quantitative determination of 5-HTP was carried out by using SWV and the anodic peak current was found to increase with increasing 5-HTP concentration in the linear range of 2–400 μM with a sensitivity and limit of detection of 0.2122 μA/μM and 77 nM respectively. The fabricated sensor further displayed excellent selectivity for 5-HTP in the presence of major interfering biomolecules in the urine with excellent recovery. The third section, of this chapter demonstrates the simple, rapid and sensitive voltammetric sensor for the determination of histamine (HTM); a neurotransmitter. The graphene nanoribbons were prepared by chemical method and silver nanoparticles (AgNPs) were prepared by the reduction of silver nitrate. The composite of graphene nanoribbons (GNRs) and AgNPs was drop casted on the surface of EPPG and the GNRs-AgNP exhibited superior catalytic activity towards the oxidation of HTM. To characterize the modified surface film-XRD, EDX, Raman Spectroscopy, Transmission Electron Microscopy (TEM), High Resolution Transmission Electron Microscopy (HRTEM), FE-SEM, EIS and CV have been used. The SWV has been used for the quantitative analysis of HTM and the GNRs-AgNP modified sensor demonstrated a linear calibration plot in the concentration range of 1–500 μM with sensitivity 0.158 μA/μM and the limit of the detection was found to be 0.049 μM. The GNRs-AgNPs sensor was highly selective in the presence of common interfering compounds present in biological fluids. The good recoveries (> 99%) were found in blood plasma samples. The determination of HTM was carried out in red wine by using standard addition method and the results were validated by using HPLC. The proposed sensor can be effectively applied for the determination of HTM in real samples. The fourth chapter deals with the fabrication of a gold-palladium nanoparticles decorated electrochemically reduced graphene oxide (AuNP-PdNP-ErGO) modified glassy carbon sensor for the individual and simultaneous determination of lomefloxacin (LMF) and amoxicillin (AMX). A new sensing platform exploiting the beneficial interaction of gold, palladium and ErGO has been prepared involving a one-step electrochemical process, and is characterized using FE-SEM, Elemental Mapping, TEM, Raman Spectroscopy, film-XRD and EIS. The calibration curves for LMF and AMX have been constructed using square wave voltammetry and exhibited a linear response in the concentration range of 4–500 μM and 30–350 μM respectively. The sensitivity and iv limit of detection were 0.0773 μA/μM and 81 nM; 0.0376 μA/μM and 9 μM for LMF and AMX respectively. The proposed protocol was successfully applied for detecting the presence of LMF and AMX in the complex matrix like urine and in the solutions containing excess of potentially interfering substances like ascorbic acid, uric acid, hypoxanthine etc. The fifth chapter is divided into two sections, first section describes a simple, facile and sensitive method for the fabrication of molecularly imprinted sensor for the determination of hydrochlorothiazide (HCTZ). The sensor is fabricated by the deposition of iron oxide nanoparticles followed by the electropolymerization of melamine monomer in the presence of HCTZ at the surface of EPPG. The surface of the imprinted sensor was characterized by using FE-SEM, EDX, EIS, film-XRD, CV and SWV. The oxidation of HCTZ occurred in a single, well-defined peak and the peak current was dependent on the concentration of HCTZ in the range 0.025–10 μM. The sensitivity and limit of detection (3σ/b) were found to be 2.342 μM/μA and 4 nM respectively. The proposed method was applied for the determination of HCTZ in the presence of common metabolites present in the human system. The obtained results indicated that uric acid, ascorbic acid, hypoxanthine and xanthine did not interfere up to 100 fold concentration. The imprinted sensor was successfully applied for the determination of HCTZ in real samples. The second section of this chapter deals with a simple, facile, selective and cost effective electrochemical method for the determination of telmisartan (TMS); a drug used for the treatment of hypertension. A sodium dodecyl sulfate (SDS) modified EPPG is prepared by the simple immersion of EPPG in SDS solution at concentration greater than critical micelle concentration (CMC). The modified sensor exhibited superior sensing properties towards the oxidation of TMS. The modified surface was characterized by using the EDX, FE-SEM, EIS and CV. The quantitative investigations of the TMS were performed by applying the SWV. The micelles of SDS form a pseudo complex with cation radical of TMS and catalyse the oxidation. The proposed sensor showed the linear calibration plot in the concentration range of 5–100 μM with sensitivity 0.2983 μA/μM and limit of the detection 0.082 μM. The specificity of the developed sensor was also evaluated in the presence of commonly present interfering substances in biological samples. The amount of TMS excreted in urine of the patients undergoing treatment has also been determined. The proposed method can be effectively applied for the investigation of TMS in pharmaceutical formulations and biological samples.