Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase.

TitleFluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase.
Publication TypeJournal Article
Year of Publication2013
AuthorsBartolini M, Naldi M, Nicolau DV, van Delft FCMJM, van Zijl J, Snijder J, van den Heuvel EFC, Naburgh EP, Calonghi N, Andrisano V
JournalAnalytical and bioanalytical chemistry
Volume405
Issue2-3
Pagination795-804
Date Published2013 Jan
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.

DOI10.3174/ajnr.A3505
Alternate JournalAnal Bioanal Chem