Synthesis, characterization and fabrication of polypyrrole and novel 3-substituted polypyrrole biosensors

This thesis presents the results of the investigations of the use of conducting polymers of unsubstituted and substituted pyrroles for the fabrication of sulfite and catechol biosensors. Part-I of this thesis consists of two chapters. Chapter 1 presents a brief introduction to biosensors, conducting polypyrrole, polypyrrole based biosensors, sulfite and phenolic biosensors, as well as the aims and objectives of this project. The extensive use of polypyrrole (PPy) in the development of electronic noses (ENs) and electronic tongues (ETs) as multi analytical devices was highlighted. A review of the use of PPy based ENs for environmental and industrial analysis is presented in the Chapter 2. The diverse use of PPy in these devices as a transduction material for a wide range of toxic and non-toxic substances, such as ammonia, nitrogen oxides, carbon monoxide, sulphur dioxide, hydrogen sulphide, methane, oxygen, hydrogen, alcohols, phenol, benzene and water vapours, is presented. The development, fabrication and characterization of unsubstituted pyrrole biosensors for sulfite and catechol is presented in Part-II of this thesis. Chapter 3 describes the use of immobilisation of sulfite oxidase (SOx) into a 54 nm thick PPy film for the fabrication of a polypyrrole-based amperometric nanobiosensor for reliable sulfite determination. Optimum immobilisation was accomplished by galvanostatic polymerization in a monomer solution which contained 0.1 M pyrrole and 5 units of SOx with a polymerization period of 120 seconds and an applied current density of 0.2 rnA cm-2• The presence of SOx in the PPy film was confirmed by scanning electron microscopy and amperometric measurement of sulfite. The effect of pH and interferants on sulfite determination with the nanobiosensor was also investigated. A linear concentration range of 0.9 to 400 JlM sulfite was achieved with the nanobiosensor and the minimum detectable sulfite concentration was 0.9 JlM (0.1 ppm). The successful application of the nanobiosensor to the determination of sulfite in wine and beers samples is reported. Chapter 4 further explores the use ofunsubstituted pyrrole for the development of a polypyrrole-tyrosinase (PPy-Tyr) potentiometric biosensor. The entrapment of tyrosinase into the polypyrrole film was carried out by galvanostatic polymerization on a platinum electrode. The optimum conditions for the formation of the PPy-Tyr film include a current density of 0.5 rnA cm·2 , a polymerization period of 150 seconds, 0.1 M pyrrole and 50 U mL-1 tyrosinase. Scanning electron microscopy (SEM), cyclic voltammetry (CV) and potentiometric measurements were used to confirm the presence of tyrosinase in the polymer film. The PPy-Tyr biosensor gave a sensitivity of 10 m VI J..LM for catechol, with a response time of 80 seconds and a linear concentration range of 1-50 J..LM catechol and the minimum detectable concentration was 1.0 J..LM. The PPy-Tyr biosensor was stable for at least 1 month when stored in a buffer at about 4 • C. The use of 3-substituted pyrroles for the fabrication of sulfite and phenol biosensor was explored in Part-III of this thesis by synthesizing 3-ethylpyrrole (EtPy). The final and intermediate products for the synthesis of EtPy were confirmed by fourier transform infrared (FTIR), nuclear magnetic resonance eH NMR and 13C NMR) spectroscopy and mass spectrometry (MS). The synthesized EtPy was employed in Chapter 5 for the fabrication of an amperometric sulfite biosensor. Sulfite oxidase was immobilized by galvanostatic polymerization of an adsorbed EtPy-SOx layer. The optimum conditions for the formation ofpoly(3-ethylpyrrole)-sulifte oxidase (PEtPy-SOx) biosensor include physical adsorption of 0.2 mg EtPy, 0.5 U of SOx, a current density of 0.75 rnA cm·2 and a polymerization period of 10 min. The PEtPy-SOx biosensor can detect as little as 0.1 J..LM sulfite and gave a linear concentration range of0.1-400 J..LM sulfte with a response time of 40 seconds. The electropolymerization of an adsorbed 3-ethylpyrrole-tyrosinase layer on a platinum electrode was investigated in Chapter 6 for the fabrication of a novel poly(3- ethylpyrrole)-tyrosinase (PEtPy-Tyr) based potentiometric catechol biosensor. The optimum conditions for the formation ofthe PEtPy-Tyr biosensor include 0.038 mg of3- ethylpyrrole (EtPy), 15 U of tyrosinase, a current density of 0.7 rnA cm·2 and a polymerization period of 15 min. The PEtPy-Tyr biosensor produced a reliable and sensitive response to catechol with a linear concentration range of 1-350 J!M, a minimum detectable catechol concentration of 0.1 J!M and a response time of 50 seconds. Chapter 7 concludes that both unsubstituted and substituted pyrroles (in this case 3-ethylpyrrole) are useful for fabrication ofbiosensors for sulfite and catechol. However, it was, noted that the use of the substituted pyrrole resulted in a substantial improvement of the sensitivity and minimum detectable concentration achieved for both sulfite and catechol biosensor. Also it resulted in a considerable reduction in the required enzyme concentrations.