Acetylcholinesterase oriented immobilization on nanopatterned bilayered surfaces for biosensing devices

Fabrication and implementation of nanoposts on which biomolecules, (DNA and proteins) are immobilized to allow a better representation to complementary biomolecules and thus more sensitive devices, are the focus of intense research towards solutions for modulating bioactivity. The expected benefit is protein and DNA diagnostics devices with improved response time, sensitivity and efficiency. Numerous interdisciplinary fields, such as biosensing and bioelectronics, require methods to immobilize biomolecules on miniaturized solid supports with complementary geometry and chemistries. In particular, the fabrication of advanced integrated "lab-on-chips", useful for the achievement of rapid and accurate analyses, is strictly connected with progress in the micro- and nano-technology field. Indeed, the possibility of binding biomolecules, such as proteins, antibodies and enzymes, on a chip allows the fabrication of innovative biodevices, such as potentially automated micro and nanosensors. Accidental intoxication by either organophosphate pesticides or lethal poisoning by organophosphate nerve agents (such as sarin) or chemical weapons is related to massive inhibition of acetylcholinesterase (AChE), the enzyme involved in the degradation of the cholinergic neurotransmitter acetylcholine [1]. A rapid detection of low levels of exposure to poisoning agents is essential for the administration of a proper antidote. Therefore the aim of the present work is the spatially addressed immobilization of human AChE on purposely fabricated structures with spatially controlled geometries in view of the development of a microdevice biosensing tools with increased sensitivity and efficiency for environmental monitoring in risk areas. The interest in this field is underlined by the large amount of related projects financed by the US army [2]. Another potential application of AChE-based devices is the fast screening of new inhibitors of this enzyme as a potential drug candidate for Alzheimer’s disease treatment. Reusable micro-biodevices containing a low amount of an expensive enzyme, such as recombinant human AChE, also offer the advantage of cost reduction. A square centimeter sample, diced from a silicon wafer with 100 nm high SiO2 microstructures (posts, holes and lines), has been used for the initial optimization of the AChE immobilization procedure trough the activation of the SiO2 surface and aminosilanization by aminopropyltriethoxysilane (APTES), in mild conditions [3]. Since the protein-flat surface interaction was previously reported to alter the AChE biological behavior, a chain spacer was introduced by letting the primary amino groups react with a 12.5% glutaraldehyde solution. In this way, stable AChE immobilization was achieved by a covalent linkage between the enzyme and the aldehydic residues on the surface of the wafer. Covalent linkage to a chemically defined pattern is a pre-requisite to obtain integrated "lab-on-chips". The enzyme distribution was evaluated by confocal microscopy analyses, using propidium as fluorescent marker. Confocal fluorescence micrographs (Fig. 1) showed an enzyme distribution which perfectly matches the initial SiO2 pattern. The yield of the immobilized enzyme has been determined, as well as the residual activity. It was measured that 10 μg of AChE was retained on the surface, corresponding to 46% of the initial amount. The immobilized AChE showed to be still active and its activity was evaluated by classical Ellman’s assay [4]. The kinetic constant could also be calculated by building a Michaelis-Menten plot (Fig. 2). A highly dense homogeneous enzyme distribution on SiO2 features and preservation of the enzymatic activity were the main outcomes of this study, in view of a purposely developed micro-device.