Analytical and Bioanalytical Chemistry

, Volume 405, Issue 2–3, pp 795–804 | Cite as

Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase

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


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 (SiO2/Si wafers), platinum-coated silicon wafers with SiO2 patterns (SiO2/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO2 and SiO2 (SiO2/TiO2/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 SiO2 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 SiO2 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.


Micropatterned 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


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



This work was supported by a European Union-funded FP7 grant (Bio-Inspired Self-assembled Nano-Enabled Surfaces—BISNES, grant number 214538). Financial support from the University of Bologna (RFO) and MIUR (PRIN 2009) is also gratefully acknowledged. Davide Cavalletti is gratefully acknowledged for technical assistance with CLSM.

Supplementary material

216_2012_6237_MOESM1_ESM.pdf (750 kb)
ESM 1 (PDF 749 kb)


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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Manuela Bartolini
    • 1
  • Marina Naldi
    • 1
  • Dan V. Nicolau
    • 2
  • Falco C. M. J. M. van Delft
    • 3
  • Jeroen van Zijl
    • 3
  • Jaap Snijder
    • 3
  • Eric F. C. van den Heuvel
    • 3
  • Emile P. Naburgh
    • 4
  • Natalia Calonghi
    • 5
  • Vincenza Andrisano
    • 1
  1. 1.Department of Pharmaceutical SciencesUniversity of BolognaBolognaItaly
  2. 2.Department of Electrical Engineering & ElectronicsUniversity of LiverpoolLiverpoolUK
  3. 3.Technology Labs., Philips Innovation ServicesEindhovenThe Netherlands
  4. 4.Q&R Surface Analysis, Philips Innovation ServicesEindhovenThe Netherlands
  5. 5.Department of BiochemistryUniversity of BolognaBolognaItaly

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