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 (SiO(2)/Si wafers), platinum-coated silicon wafers with SiO(2) patterns (SiO(2)/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO(2) and SiO(2) (SiO(2)/TiO(2)/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 SiO(2) 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 SiO(2) 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.

Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase

BARTOLINI, MANUELA;NALDI, MARINA;CALONGHI, NATALIA;ANDRISANO, VINCENZA
2013

Abstract

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 (SiO(2)/Si wafers), platinum-coated silicon wafers with SiO(2) patterns (SiO(2)/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO(2) and SiO(2) (SiO(2)/TiO(2)/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 SiO(2) 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 SiO(2) 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.
M. Bartolini; M. Naldi; D. V. Nicolau; F. C. M. J. M. Delft; J. Zijl; J. Snijder; E. F. C. Heuvel; E. P. Naburgh; N. Calonghi; V. Andrisano
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/149004
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