Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase
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- Bartolini, M., Naldi, M., Nicolau, D.V. et al. Anal Bioanal Chem (2013) 405: 795. doi:10.1007/s00216-012-6237-7
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Human acetylcholinesterase (AChE) is a widely studied target enzyme in drug discovery for Alzheimer’s disease (AD). In this paper we report evaluation of the optimum structure and chemistry of the supporting material for a new AChE-based fluorescence sensing surface. To achieve this objective, multilayered silicon wafers with spatially controlled geometry and chemical diversity were fabricated. Specifically, silicon wafers with silicon oxide patterns (SiO2/Si wafers), platinum-coated silicon wafers with SiO2 patterns (SiO2/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO2 and SiO2 (SiO2/TiO2/Pt/Ti/Si wafers) were labelled with the fluorescent conjugation agent HiLyte Fluor 555. Selection of a suitable material and the optimum pattern thickness required to maximize the fluorescence signal and maintain chemical stability was performed by confocal laser-scanning microscopy (CLSM). Results showed that the highest signal-to-background ratio was always obtained on wafers with 100 nm thick SiO2 features. Hence, these wafers were selected for covalent binding of human AChE. Batch-wise kinetic studies revealed that enzyme activity was retained after immobilization. Combined use of atomic-force microscopy and CLSM revealed that AChE was homogeneously and selectively distributed on the SiO2 microstructures at a suitable distance from the reflective surface. In the optimum design, efficient fluorescence emission was obtained from the AChE-based biosensing surface after labelling with propidium, a selective fluorescent probe of the peripheral binding site of AChE.