In the life sciences current search for speed and automation, sensing surfaces which operate with immobilised biomolecules have found a broad range of applications. In particular, miniaturization and automation of in vitro screening systems may be advantageous in the drug discovery process, in which a large number of new chemical entities needs to be screened for affinity towards specific target proteins. Specifically, in drug discovery programs, acetylcholinesterase (AChE) inhibitors acting at the catalytic binding site (CAS) of the enzyme are of great interest for treatment of cholinergic deficiencies in the central nervous system (e.g. Alzheimer's disease). Other then CAS binders, more recently, AChE’s peripheral binding site (PAS) binders have attracted the attention of several research groups.(1) The rational basis of this interest is the experimental evidence that the interaction of soluble amyloid-beta (Aβ) peptide with AChE’s PAS may promote the deposition of the neurotoxic Aβ oligomers/fibrils and accelerate the onset and progression of the AD pathology.(2) The available in vitro screening assay for the selection of PAS binders implies the use of a large amount of the target enzyme (a micro-molar concentration is needed).(3) Therefore, in the attempt of keeping the screening costs within an academic laboratory budget, the assay is usually performed with electric eel AChE instead of the human isoform, making the selection of a proper drug candidate more difficult. On the light of these premises, and in view of the advantages in terms of miniaturization and increased stability and efficiency of the immobilized enzyme, in this talk the initial development of a new AChE-based fluorescence sensing surface for the identification of PAS binders through propidium displacement studies will be presented. To achieve this goal, different micro-patterned silicon wafers (diameter 4 in) with a reflective layer (either platinum or silicon) and SiO2 pillars, lines, and holes were fabricated and tested. Indeed, reflective surfaces may have the advantage of higher output signals when fluorescence detection is used. Therefore, an initial selection was performed to obtain a suitable multi-layered material of optimal pattern thickness, required to maximize fluorescence signal and maintain chemical stability. Then, the selective immobilization of recombinant human AChE on the SiO2 architectures with optimal geometry and chemistry was achieved. Measurements with CLSM, AFM and scanning Auger microscopy–scanning electron microscopy (SAMSEM) supported the conclusion that AChE was mostly confined to the top of SiO2 structures. This confinement might be because of chemical contrast which resulted from either the structuring of the wafer or the cleaning procedures used before derivatisation.
Development of a new micro-patterned bio-sensing surface to screen for acetylcholinesterase PAS binders
BARTOLINI, MANUELA;NALDI, MARINA;CALONGHI, NATALIA;ANDRISANO, VINCENZA
2013
Abstract
In the life sciences current search for speed and automation, sensing surfaces which operate with immobilised biomolecules have found a broad range of applications. In particular, miniaturization and automation of in vitro screening systems may be advantageous in the drug discovery process, in which a large number of new chemical entities needs to be screened for affinity towards specific target proteins. Specifically, in drug discovery programs, acetylcholinesterase (AChE) inhibitors acting at the catalytic binding site (CAS) of the enzyme are of great interest for treatment of cholinergic deficiencies in the central nervous system (e.g. Alzheimer's disease). Other then CAS binders, more recently, AChE’s peripheral binding site (PAS) binders have attracted the attention of several research groups.(1) The rational basis of this interest is the experimental evidence that the interaction of soluble amyloid-beta (Aβ) peptide with AChE’s PAS may promote the deposition of the neurotoxic Aβ oligomers/fibrils and accelerate the onset and progression of the AD pathology.(2) The available in vitro screening assay for the selection of PAS binders implies the use of a large amount of the target enzyme (a micro-molar concentration is needed).(3) Therefore, in the attempt of keeping the screening costs within an academic laboratory budget, the assay is usually performed with electric eel AChE instead of the human isoform, making the selection of a proper drug candidate more difficult. On the light of these premises, and in view of the advantages in terms of miniaturization and increased stability and efficiency of the immobilized enzyme, in this talk the initial development of a new AChE-based fluorescence sensing surface for the identification of PAS binders through propidium displacement studies will be presented. To achieve this goal, different micro-patterned silicon wafers (diameter 4 in) with a reflective layer (either platinum or silicon) and SiO2 pillars, lines, and holes were fabricated and tested. Indeed, reflective surfaces may have the advantage of higher output signals when fluorescence detection is used. Therefore, an initial selection was performed to obtain a suitable multi-layered material of optimal pattern thickness, required to maximize fluorescence signal and maintain chemical stability. Then, the selective immobilization of recombinant human AChE on the SiO2 architectures with optimal geometry and chemistry was achieved. Measurements with CLSM, AFM and scanning Auger microscopy–scanning electron microscopy (SAMSEM) supported the conclusion that AChE was mostly confined to the top of SiO2 structures. This confinement might be because of chemical contrast which resulted from either the structuring of the wafer or the cleaning procedures used before derivatisation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
