Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase.
Anal Bioanal Chem
; 405(2-3): 795-804, 2013 Jan.
Article
em En
| MEDLINE
| ID: mdl-22814970
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Acetilcolinesterase
/
Técnicas Biossensoriais
/
Enzimas Imobilizadas
Limite:
Humans
Idioma:
En
Revista:
Anal Bioanal Chem
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Itália