title: Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase.
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.
authors: Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy.
authors: Manuela
authors: M
authors: Bartolini
authors: Marina
authors: M
authors: Naldi
authors: Dan V
authors: DV
authors: Nicolau
authors: Falco C M J M
authors: FC
authors: van Delft
authors: Jeroen
authors: J
authors: van Zijl
authors: Jaap
authors: J
authors: Snijder
authors: Eric F C
authors: EF
authors: van den Heuvel
authors: Emile P
authors: EP
authors: Naburgh
authors: Natalia
authors: N
authors: Calonghi
authors: Vincenza
authors: V
authors: Andrisano
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D012822:Silicon Dioxide / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D005456:Fluorescent Dyes; D012822:Silicon Dioxide; D000110:Acetylcholinesterase