Impact of some Layer-by-layer assembly conditions on the enzymatic activity of glucose oxidase immobilized in nanopores

Enzyme immobilization is a prosperous approach to enzymatic application because it enhances its in vitro performance by increasing stabilization, providing higher operational time, better control of the process, and enables its reuse. In specific, the elaboration of enzymatic nanotubes is especially interesting due to the creating of a nano-confinement environment (slightly mimicking the in vivo environment), the creating of a protective shell that will isolate the enzyme from direct contact with the medium, and the possibility of tailoring the nanotube’s components and parameters. These nanotubes may be prepared by a template-assisted synthesis combined with a bottom-up Layer-by-layer (LBL) assembly technique to create nanotubes inside the pores of a nanoporous membrane. The LBL technique consists of depositing alternating layers of materials that are oppositely charged, in this project this is performed by immersing the deposition support (polycarbonate nanoporous membrane) in the solutions of the materials (weak polyelectrolytes (PE)). The enzyme chosen to be immobilized in the assembled nanotubes was the Glucose Oxidase (GOx - negatively charged polyelectrolyte), this choice was mainly moved because it is a well-studied enzyme, with a high number of literature references and its 3D structure is well determined by science, and it was partially moved because of its importance in the industry and its vast fields of application. The second deposition material chosen was Branched-Polyethyleneimine (bPEI - positively charged polyelectrolyte), a choice motivated due to literature resources that prove that this polyelectrolyte combined with GOx, and LBL, will prevent the enzyme desorption. The aim of this thesis is to analyze how the assembly parameters influence the enzymatic activity of the (bPEI/GOx) nanotubes and possibly finding a most optimal assembly setup to maximize the catalyst performance. Three parameters were chosen as the variables: PE assembly concentration, pore diameter of the membranes, and number of bilayers deposition. To measure the GOx activity of the samples (i.e., membrane containing nanotubes) a cascade reaction was performed between GOx and Peroxidase. Cascade reaction means that the product of the first reaction is reagent of the second reaction, in this case, the product of the second enzyme is a colored product, which color intensity (i.e., spectrometry absorbance) is measured because it is directly correlated with GOx’s activity. The samples’ activity measurement was performed in static (no flows) and dynamic (income and outcome flows) regimes, to obtain combined & complementary information on the GOx activity behavior. Results have shown that, for static applications, the decrease of the pore diameter decreases the specific activity (i.e., activity per GOx mass) results, possibly due to diffusional restriction for the substrate to enter the pore. This result is no longer observed in the dynamic, possibly due to the flow. Regarding the PE influence, samples assembled with higher & even PE concentration (both GOx and PEI with same solution concentration) present higher specific activity results (static and dynamic) than samples assembled with lower & even PE concentrations. Regarding the bilayers parameter, for the static and dynamic applications the increase of bilayers has provoked a decrease of the specific activity for samples assembled with high & even PE concentrations and has provoked the contrary effect on samples assembled with low & even PE concentrations. The PE and bilayer influence responses are due to the same reason, samples assembled with high & even PE concentrations present layers with a high amount of GOx, this dense layer block the substrate access to the deeper GOx layers and will decrease the activity. For low & even PE concentrations the layers are less crowded, allowing the substrate to diffuse and increasing the total activity; the increase of the specific activity is possibly a response to the nanoconfinement effect and due to an enhanced GOx immobilization configuration (e.g., facilitating the contact between enzyme-substrate). Samples were also assembled with high & uneven PE concentration, however, the interpretation of its results was limited to hypothetical explanations. This project has concluded that the assembly parameters indeed affect the specific activity of the immobilized enzymes and that the optimal setup to maximize enzyme activity may be achieved by the combination of parameter factors (just one factor is not sufficient). However, further studies and analysis are needed for the achievement of this maximum enzymatic activity due to assembly scenario.