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. FigureMicropatterned silicon wafers containing covalently bound human acetylcholinesterase. The binding and displacement of the fluorescent probe propidium (red areas) from the enzyme's peripheral binding site is visualized by scanning laser microscopy.

Medienart:

E-Artikel

Erscheinungsjahr:

2012

Erschienen:

2012

Enthalten in:

Zur Gesamtaufnahme - volume:405

Enthalten in:

Analytical and bioanalytical chemistry - 405(2012), 2-3 vom: 20. Juli, Seite 795-804

Sprache:

Englisch

Beteiligte Personen:

Bartolini, Manuela [VerfasserIn]
Naldi, Marina [VerfasserIn]
Nicolau, Dan V. [VerfasserIn]
van Delft, Falco C. M. J. M. [VerfasserIn]
van Zijl, Jeroen [VerfasserIn]
Snijder, Jaap [VerfasserIn]
van den Heuvel, Eric F. C. [VerfasserIn]
Naburgh, Emile P. [VerfasserIn]
Calonghi, Natalia [VerfasserIn]
Andrisano, Vincenza [VerfasserIn]

Links:

Volltext [lizenzpflichtig]

BKL:

35.23$jAnalytische Chemie: Allgemeines

35.71$jBiochemische Methoden

42.03$jMethoden und Techniken der Biologie

Themen:

Acetylcholinesterase
Atomic-force microscopy
Biosensing surface
Confocal scanning microscopy
Fluorescence labelling
Micropatterned silicon wafer

RVK:

RVK Klassifikation

Anmerkungen:

© Springer-Verlag 2012

doi:

10.1007/s00216-012-6237-7

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

OLC2090249773

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