New techniques for the fabrication of biosensors based on nad (P) + dependent dehydrogenases

Identifier:  TDX:315

Handle: https://hdl.handle.net/20.500.11797/TDX315

Authors:  Paulov, Valeri

Abstract:
The objective of this work was the development of new configurations of reagentless biosensors based on NAD+ dependent dehydrogenases. These configurations are based on the immobilisation of enzyme, cofactor and the electrochemical catalyst used for its regeneration. In addition to being reagentless these configurations yielded biosensors with improved current density and operational stability compared to the state of the art. <br/> To achieve the objective two new NADH oxidising mediators were synthesised: a water insoluble polymer [Os(1,10-phenanthroline-5,6-dione)2(PVP)4Cl]Cl (Os-phendione-PVP) and an amphiphilic complex [Os(1,10-phenanthroline-5,6-dione)24,4'-(n-C18H37NHCO)2bpy)](PF6)2 (Os-phendione-surfactant). The electrochemical study of Os-phendione-PVP has revealed a rate constant for the heterogeneous electron transfer of the phendione redox couple ks = 25&#61617;2 s-1, and a second order rate constant for NADH oxidation k[NADH]=0=(1.1&#61617;0.1)x103 M-1 s-1. These constants are higher or of the same order of magnitude as those of previously described NADH oxidising mediators. The tensoactive mediators Os-phendione-surfactant and its analogue [Os(bpy)24,4'-(n-C18H37NHCO)2bpy)](PF6)2 (Os-bpy-surfactant) form very stable monolayers at the air-water interface collapsing at the surface pressure 60-65 mN m-1. <br/>The Os-phendione-surfactant was used for the construction of reagentless glutamate biosensors via the immobilisation of dehydrogenase and NAD+ between bilayers in lamellar phase formed by Os-phendione-surfactant and the lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine. The resulting glutamate biosensors demonstrated maximum current density of 3.5 &#61549;A cm-2 (RSD=25%), apparent Michaelis constant of 47 mM, and operational half life of 0.5 h. In addition graphite electrodes were modified by Os-phendione-PVP and utilised for (a) immobilisation of dehydrogenase and NAD+ in a hydrogel formed by crosslinking of poly(vinylpyridine) carrying amino groups with polyethylene glycol diglycidyl ether and (b) immobilisation of dehydrogenase and an NAD+-alginic acid derivative by adsorption. The configuration (a) yielded glutamate sensors with maximum current density of 8.7 &#61549;A cm-2 (RSD=5%), apparent Michaelis constant of 9.1 mM, operational half life of 12 h and glucose sensors with maximum current density of 37 &#61549;A cm-2 (RSD=14%), apparent Michaelis constant of 4.2 mM, the operational half life of 1 h. The glutamate sensors based on the configuration (b) showed maximum current density of 15.8 &#61549;A/cm2 (RSD=21%), apparent Michaelis constant of 17.6 mM and operational half life of 1.5 h. <br/>Glucose, glucose-6-phosphate, and glutamate biosensors were prepared and characterised. The employment of the thermophilic enzymes helps to dramatically increase the operational stability of biosensors at elevated temperatures higher than 60oC. The shelf life of glutamate electrodes built with the use of thermophilic dehydrogenase was eleven times longer than this of electrodes modified with the mesophilic enzyme. The addition of the copolymer of vinyl-pyrrolidone and dimethylamino ethyl methacrylate termed as Gafquat HS100 to the enzyme also significantly improved shelf life