SYNTHESIS & CHARACTERIZATION OF IMMOBILIZING MATERIALS FOR BIOSENSING APPLICATION

Abstract

Urea and uric acid are the most important end product of protein degradation and purine metabolism, thus their proper balance in blood is essential for overall well being and for renal health specifically. The optimal concentration of urea in blood is an indication of proper renal functioning, its high level in blood causes urinary tract obstruction, dehyradation, shock, burns and gastrointestinal bleeding, whereas a substantial low level of urea concentration causes hepatic failure, nephrotic syndrome, cachexia. Similarly abnormal uric acid levels lead to gout, chronic renal disease, some organic acidemias, leukemia, pneumonia and Lesch–Nyhan syndrome. Hence, the detection of these analytes in body fluids is clinically important indicator. Although direct spectroscopic methods can be used for their determination, but these methods are dependent on the pre-treatment of sample and cannot be used for onsite monitoring. The primary focus of this work is to develop urea and uric acid biosensors with improved properties such as sensitivity and response time by using different substrates on indium tin-oxide (ITO) glass plates and different methods of immobilization. The first part of the dissertation focuses on the development of urea biosensor with improved properties based on conducting polymer polypyrrole (PPy). PPy is synthesized electrochemically on ITO glass plates using ptoluene sulphonic acid as dopant. However, the absence of active binding sites in PPy, which can bind the biomolecule, limits its application to some extent. Also simultaneous electrochemical deposition of enzyme, during polymerization showed decrease in background current. This degradation of the polymer was attributed to the denaturing of the enzyme. This problem can be overcome by incorporating an actively binding group containing substances into the polymer film that provides free NH2 group to enhance the immobilization as well as electrochemical properties. In this investigation, electrodes are fabricated by embedding bovine serum albumin (BSA) in PPy during electrochemical synthesis on an ITO glass electrode. The free NH2 groups of embedded BSA at the surface of the PPy film were exploited for the covalent binding of enzyme via carbodiimide coupling reaction. The advantage of using BSA embedded surface modified PPy films for efficient enzyme loading is described. PPy film was characterized with scanning electron microscopy, infrared spectroscopy and UV visible spectroscopy before and after each step of surface modification and enzyme immobilization. Potentiometric and spectrophotometric response of the enzyme electrode (Urs/BSA-PPy/ITO) were measured as a function of urea concentration. In the following section, the use of carbon nanotubes in a silica matrix is studied to develop urea biosensor. Tetraethyl orthosilicate (TEOS) is used to prepare SiO2 solution which provides a network for incorporation of carbon nanotubes. Functionalized multiwalled carbon nanotubes are used to supply free COOH groups, on which the enzyme can covalently immobilize. The synergistic effect of silica matrix, F-MWCNTs and biocompatibility of Urs/MWCNTs/SiO2 made the biosensor to have the excellent electro catalytic activity and high stability. Electrodes were characterized at each step of formation and potentiometric response was measured as a function of urea concentration. The next part of the dissertation focuses on the development of uric acid biosensor by immobilizing enzyme uricase through a self assembled monolayer (SAM) of 3-aminopropyltriethoxysilane (APTES) using a crosslinker, Bis[sulfosuccinimidyl]suberate (BS3) on an indium-tin-oxide (ITO) coated glass plate. Electrodes fabricated were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical techniques. Chronoamperometric response was measured as a function of uric acid concentration in aqueous solution (pH 7.4). Last part of the theisi focuses on the modification of above SAM layer on ITO with gold nanoparticles. Chronoamperometric response was measured as a function of uric acid concentration in aqueous solution. The results indicates better reproducibility and response time of the electrode. This research work provides a comprehensive study of fabricating different biosensors by using different matrixes and different techniques of transducing. The development of biosensors has come from the rapid advances in health care technology as a frequent measurement of biochemical parameters such as blood cations, gases and metabolites required for effective patient care. The need for cheap and reliable sensors for monitoring such parameters has lead to exponential increase in the research and development of biosensors. Electrochemical biosensors have emerged as the most commonly used biosensors as they have been found to overcome most of the disadvantages, which inhibit the use of other types of biosensors. They are rapid, easy to handle, simple and low cost. The basic fact behind this bio-interaction process is that the electrochemical species such as electrons are consumed or generated producing an electrochemical signal, which can be measured by the detector. These biosensors are usually based on potentiometry and amperometry. This study identifies the already established materials for developing different biosensor which may be used for the detection of urea and uric acid in aqueous solution. The main objective of this dissertation is to use modified electrode such as BSA/PPy, MWCNT/SiO2, APTES/BS3 and GNPs/APTES as matrix for the development of urea and uric acid biosensors.