Biossensor nanoestruturado construído através da imobilização multicamada de enzimas

Abstract

Biosensors design study has increased in the last years, especially for enzymatic biosensors, witch among the biosensors, are the most mature and the ones that have reached bigger industrial scale. The use of nanotechnology has opened new perspectives in the biosensor construction. The molecular capacity control brings the possibility of design of the structure, sometimes foreseeing the best possible configuration. The objective of this research was to build enzymatic multilayer biosensors with nanometric structures, using three enzymes: glucose oxidase from Aspergillus Niger (GOx), horseradish peroxidase (HRP) and invertase from bakers yeast (INV), previously submitted to biotinylation, using a layer-by-layer deposition approach and through covalent immobilization of the 1st layer and affinity immobilization in subsequent layers. Multiple layers biosensor constructions were successful obtained. Comparison between them was made and the tested assemblies were significantly altered by the designed sequence of enzymes within the structures and the pH of the system: - Bi-enzymatic structures built with GOx in the first layer and HRP in the second layer have presented enzymatic activity results of approximately 1,7 times greater than structures built with HRP in the first layer and GOx in the second layer, when evaluated at PBS buffer (pH = 7.4). This behavior was inverted when the structures were evaluated with citrate buffer (pH = 5.3); - Tri-enzymatic structures built with HRP in the first layer, GOx in the second layer and INV in the third layer have presented enzymatic activity results of approximately 1,5 times greater than structures built with GOx in the first layer, HRP in the second layer and INV in the third layer, when evaluated at citrate buffer. The differences among these assemblies were credited to the microenvironments in the enzymes vicinities due to electrostatic interactions between enzymes aminoacids, substrate and solid support. Bi-enzymatic structures were submitted to regular (sugar-sweetened) and sugar-free soft drinks real samples, as a proof of concept, and they have presented catalytic response in the detection of glucose in regular samples. The designed three dimensional structure used to build the bi-enzymatic and tri-enzymatic structures has a potential for nano-sensing in food and beverage industries, detection of allergenic species, toxic components and contamination and also this strategy can potentially be used to mimic cell metabolic channels.