Block-Copolymer Derived Nanoporous Thin Films for the Development of a L-BMAA Aptamer-Based Impedimetric Biosensor

摘要

Produced by diverse cyanobacteria, b-N-methylamino-l-alanine (L-BMAA) is a non-protein neurotoxic cyanotoxin that has been linked to an elevated incidence of neurodegenerative diseases such Parkinson's and Alzheimer's disease and Multiple Lateral Sclerosis. The continuing rising of water temperatures and eutrophication of the water bodies propitiate the increment and size of harmful algal blooms, subsequently increasing the production of L-BMAA and other cyanotoxins. This toxin is known to bioaccumulate in plants, animals and humans. Currently, the detection of L-BMAA in water is limited by its hydrophilicity, absence of ultraviolet and fluorescent properties, and the isomers that cause false positives. Given the threat that this cyanotoxin could represent to the long-term human health, it is imperative to develop new analytical techniques for its detection in water. Therefore, this project proposes the development of an impedimetric aptamer-based biosensor, using block-copolymer (BCP's) derived nanoporous thin films as the electrode, for the detection of L-BMAA. Our hypothesis is that the development of such aptasensor will lead to the advancement of an innovative device for a sensitive, portable, economic and flexible way to achieve the detection of the cyanotoxin in water. To achieve this, we used BCP polystyrene-poly(methylmethacrylate) (PS-b-PMMA), known to form cylinder-like structures, to create recessed nanodisk-array electrodes (RNEs). These cylindrical nanostructures provide primary mass transport pathways for ionic and redox active species which changes upon analyte binding, making them ideal to use in biosensing applications. The nanoporous electrodes were prepared by spin-coating a PS-b-PMMA solution in toluene over gold-coated silicon wafers, followed by thermal annealing and UV etching. Different annealing times, temperatures and UV exposure has been used in order to produce the alignment of the cylindrical polymer microdomains in a vertical fashion over the surface of the electrode. The prepared films were characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), to confirm the formation of RNE's. Under specific sets of conditions, CV data shows sigmoidal curves at high scan rates characteristic of RNE's, suggesting the formation of this type of nanopores with a sufficiently large distance among them to attain radial diffusion. Atomic force microscopy (AFM) images of the samples evidence the formation of vertical nanopores but mixed with horizontal alignments. Furthermore, grazing incident small angle x-ray scattering (GISAXS) suggests that the polymer was well-dispersed among the surface although the expected scattering profile for RNEs is missing. Future work includes the optimization of the electrode preparation methods and the selection of a L-BMAA specific aptamer through graphene oxide-assisted selection evolution of ligands by exponential enrichment (GO-SELEX).