Novel ultrasensitive phosphate nanobiosensors based on co-integration of nanomaterials with pyruvate oxidase

The determination of phosphate is of environmental and clinical importance because it is one of the factors associated with eutrophication, which disrupts aquatic life cycles. However, phosphate is also an essential component of all living organisms. Various analytical methods, such as chromatography, colorimetry, optical fluorescent spectroscopy and electrochemical methods have been used for the determination of phosphate. However, these methods require skilled operators, expensive instrumentation, and, in some cases, involve usage of potentially carcinogenic chemicals. In recent years, the use of biosensors has attracted a lot of interest as a simple and rapid alternative to the traditional methods because of their enormous advantages, such as high selectivity, simplicity of use and the possibility for miniaturization. In this thesis, various configurations of phosphate nanobiosensors are reported. The thesis is structured into eight chapters, with Chapter 1 comprising of the general introduction. This Chapter focused on the environmental and biological significance of phosphates, the traditional methods used for the determination of phosphates, the use of nanomaterials for fabrication of phosphate nanobiosensors, and provides the aims of the study. In Chapter 2, a polypyrrole (PPy)-based phosphate potentiometric biosensor was fabricated by entrapment of pyruvate oxidase (PyOx) in PPy film during electropolymerization of pyrrole (Py). The optimised conditions for sensitive and reliable performance of the biosensor were 0.3 M Py, an applied current density of 0.05 mA/cm2, a polymerization time of 120 s, and a PyOx concentration of 2 U/mL. A minimum detectable concentration of 3 µM and a calculated detection limit of 0.63 µM phosphate were achieved with a sensitivity of 15 mV/decade. A wide linear range of 15-250 µM was achieved. The fabricated biosensor was used to detect phosphate in lake water and achieved 99-100% phosphate recovery. A huge reduction in enzyme usage (70 %) was achieved without compromising the performance of the biosensors compared to the enzyme requirement of other PyOx based phosphate biosensors. However, the sensitivity of the proposed sensor was low and further improvement was necessary. In Chapter 3, a more robust potentiometric phosphate biosensor was developed by the incorporation of gold nanoparticles (AuNPs) because of its bio-compatibility, large surface area for high enzyme loading, high catalytic activity. The nanocomposite biosensor was fabricated by co-entrapment of PyOx, its co-factors and AuNPs into the PPy film by galvanostatic polymerization. The entrapment of all components into a PPy film was accomplished with 0.3 M Py, applied current density of 0.1 mA/cm² and a polymerization time of 120 s. An enzyme concentration of 2 U/mL and 0.75 mg/L AuNPs were used for the fabrication of the composite nanobiosensor. Optimum potentiometric response for phosphate was obtained with cofactors concentration of 10 µM flavin adenine dinucleotide (FAD) and 150 µM thiamine pyrophosphate (TPP). A minimum detectable concentration of 1 µM and a detectable limit of 0.12 µM were achieved with a sensitivity of 39.8 mV/decade. A linear concentration range of 5-500 µM was obtained with the nanobiosensors for potentiometric of phosphate. Excellent recoveries for phosphate in lake water samples (97-106) were obtained with the nanobiosensor. The co-entrapment of PyOx, AuNPs, TPP and FAD into PPy film on a platinum electrode was also investigated for fabrication of a highly sensitive and robust amperometric phosphate nanobiosensors. The galvanostatic polymerization of Py was achieved using a polymerization time of 60 s, current density of 0.05 mA/cm2 and 0.3 M Py. Optimum amperometric response for phosphate was obtained with 2 U/mL PyOx, 0.75 mg/L AuNPs, 10 µM FAD and 150 µM TPP. A sensitivity of 213.4 µA mMˉ¹ cmˉ² was achieved with a minimum detectable concentration of 20 µM, a calculated detection limit of 1.9 µM and a linear concentration range of 20-350 µM (R² = 0.9905). The biosensor demonstrated good selectivity for phosphate in the presence of Clˉ, NO₃ˉ, SO₄²ˉ , Fˉ, acetate and urea. The fabrication of a superior amperometric phosphate nanobiosensor was accomplished in Chapter 5 by cross linking enzyme and co-factors on gold nanowires array (AuNWA) with glutaraldehyde (GLA) and bovine serum albumin (BSA). To fabricate the nanobiosensor, highly ordered AuNWA was synthesized by direct electrodeposition in combination with template approach. The nanobiosensor achieved a minimum detectable concentration of 1 µM, a detection limit of 0.1 µM, a wide linear range of 12.5 – 1000 µM and a sensitivity of 140.3 µA mMˉ¹ cmˉ². It also demonstrated very high selectivity against major interferants such as Cl-, NO₃ˉ, SO₄²ˉ and NO₂ˉ ions. The nanobiosensor was successfully applied to the determination of phosphate in water samples achieving a mean recovery of 96.5 %. The use of the AuNWA-GLA-BSA-FAD-TPP-PyOx nanobiosnsor for potentiometric detection of phosphate was also accomplished in Chapter 6 by co-immobilization of PyOx, FAD and TPP on the surface of AuNWAs by cross linking with GLA and BSA. Over 100 % improvement in the sensitivity was achieved with the constructed nanobiosensors when compared with PPy-PyOx biosensors. The resulting AuNWA-GLA-BSA-FAD-TPP-PyOx nanobiosensor enabled the achievement of a minimum detectable concentration of 1 µM, a detection limit of 0.11 µM, linear concentration range of 5-500 µM and a sensitivity of 31.1 mV/decade. The nanobiosensor was also successfully used to determine phosphate in lake water, achieving a percentage recovery of 96.6-102.6 %. The co-entrapment of of PyOx, FAD and TPP into PPy film during galvanostatic electropolymerization of Py on AuNWA was used in Chapter 7 to fabricate a novel potentiometric phosphate nanobiosensor. The optimum conditions for fabrication of the nanobiosensor were 0.15 M Py, 2 U/mL PyOx, an applied current density of 0.3 mA/cm², and a polymerization time of 120 s. The PPy-based potentiometric nanobiosensor achieved a linear concentration range from 5-825 µM, a sensitivity of 25.38 mV/decade, a minimum detectable concentration of 1 µM and a detection limit of 0.08 µM. Successfull determination of phosphate was achieved with the nanobiosensor in lake water, achieving a recovery of 96.9-103.4 %. Chapter 8 concluded by highlighting the important findings realized in the thesis for the fabrication of potentiometric and amperometric nanobiosensors. Some recommendations for future directions were made.