Fabrication and application of nanostructured materials for sulfite biosensing

A biosensor as an integrated miniaturized device, exploits the modern microelectronics with specific sensing probe through signal transduction. The challenge for new generation biosensors is to achieve specific analyte detection at very low concentrations, which is possible by tailoring the materials used for fabrication of these devices based on nanoscience and nanotechnology. The new approach is explored in this thesis for fabrication of novel nanobiosensors for ultrasensitive detection of sulfite. The literature review in chapter 1 describes the long history of sulfite usage as preservative, its benefits and health concerns. This chapter also outlines the existing approaches for fabrication of sulfite nanobiosensors, and explores the new and emerging fabrication strategies based on the use of nanomaterials. The resulting new constructive matrices for electroelectrochemical detection provides amplified redox signals, enhanced direct electron transfer and an enlarged catalytic surface area for increased enzyme immobilization. The integration of all these properties certainly improved the performance of the resulting sulfite nanobiosensor with ultrahigh sensitivity and selectivity, as compared with the traditional methods used for sulfite detection. In chapter 2, the use of composite PPy and PtNPs for entrapment of sulfite oxidase (SOx) was considered for improvement of sulfite detection. The properties of the resulting nanobiosensor were dramatically improved due to increased surface area compared to those achieved in absence of PtNPs. Furthermore, the deposition of PtNPs on the electrode surface improved the chemical kinetics of redox reactions, as this nanomaterial acted in providing catalytic centres. The morphology of the exposed surface of the fabricated PtNPs-PPy-SOx demonstrated a clear difference compared to in the absence of PtNPs decoration, the electrochemical behaviour revealed by CV and potentiometric measurements further indicates fast and efficient redox kinetics. Optimization of the PtNPs-PPy-SOx biosensor enabled achievement of a detection limit of 12.35 nM, a linear range of 0.75 - 65.50 µM with a sensitivity of 57.5 mV/decade, and was successfully applied to real samples. The fabrication and use of PPy nanowires array was considered in chapter 3 to achieve better mass and electron transfer with improved surface area for SOx immobilization. Simple and low cost Anodic Aluminium Oxide (AAO) templates were prepared by a two-step anodization method. Pyrrole was deposited into the nanochannels of AAO templates by electropolymerization to obtain highly ordered PPyNWA with desired dimensions. The improved catalytic performance was achieved by decorating the nanowire surface with PtNPs. The resulting geometrical structure provided enlarged activated surface area for increased enzyme loading with free space available for the mobility of analyte. SEM images revealed the morphology of PPyNWA and their highly ordered features. Furthermore, EIS and CV were employed to gain a better understanding of the electrochemical performance in terms of sensitivity and stability for sulfite detection. The amperometric response was used for the optimization and evaluation of electrode modification. The modified electrode with PPyNWA improved detection limit (7.4 nM), and the linear concentration range (0.12 - 1200 μM) with a sensitivity of 57.33 μA mMˉ¹cmˉ². The resulting nanobiosensor was applied to real samples and also indicated improved stability of 120 days. In chapter 4, the use of AAO templates was considered for fabrication gold nanowires array. The one dimensional features of the AuNWA were modified with PtNPs to activate the surface morphology three dimensionally. The nanostructural assembly with high surface to volume ratio for SOx immobilization boosted the direct electron transfer (DET) of redox reactions and increased the sensitivity of the resulting sulfite nanobiosensor. Furthermore, cross-linking of SOx with a mixture of glutaraldehyde (GLA) and bovine serum albumin (BSA) on the AuNWA modified with PtNPs provided an opportunity to enhance the sensitivity of the nanobiosensor. Fast electrochemical kinetics achieved with the nanobiosensor enabled an improved detection limit of 3.5 nM and linear range of 4.9 µM – 1.5 mM for low concentrations and 2 mM – 5.5 mM for higher with high sensitivities of 61.5 µA mMˉ¹cmˉ² and 90.00 µA mMˉ¹cmˉ², respectively. The achieved sensitivities in low and high sulfite concentration ranges were 61.5 µA mMˉ¹cmˉ² and 90.00 µA mMˉ¹cmˉ². Long term storage stability was 140 days. A novel sulfite nanobiosensor based on the use of nanostructure composite material, involving encapsulation of SOx into Nafion membrane and PtNPs decorated hybrid matrix of MWCNTs and PPy is described in chapter 5. The large surface provided by inclusion of MWCNTs enabled immobilization of more enzyme molecules, resulting in faster direct electron transfer and enhanced response towards H₂O₂. The achieved sensitivity was strongly influenced by enzyme loading and catalytic activation of the matrix with PtNPs. The homogeneity of the resulting Naf-SOx-PtNPs-MWCNTs/PPy nanocomposite matrix was characterized by SEM, EIS and CV. The optimized sulfite nanobiosensor gave a linear range of 20 nM – 6 mM with a high sensitivity of 71 mA mMˉ¹cmˉ², detection limit of 5.4 nM, and response time of <3 s. The sulfite nanobiosensor was also successfully applied to real samples and demonstrated a long term stability of 112 days. Chapter 6 describes the importance of nanobiosensor for the detection of sulfite, and a brief review of all the strategies used for fabrication of sulfite nanobiosensor. It also provides a comparison of all the approaches in terms of fabrication process, and performance of the resulting nanobiosensors. Overall, the use of nanomaterials gave the best sensitivity of 89.97 mA mMˉ¹cmˉ², detection limit of 3.5 nM, and a linear range of 20 nM to 6 mM. Furthermore, it describes future research directions for fabrication of sulfite nanobiosensors based on careful nanoengineering of various matrices, and their integration on screen printed electrodes.