Lab-on-cell and cantilever-based sensors for gene analysis

Odorizzi, Lara (2010) Lab-on-cell and cantilever-based sensors for gene analysis. PhD thesis, University of Trento.

PDF - Doctoral Thesis


Nowadays, both gene mutations detection and function investigation are expected to assume a key role in diseases understanding and in many other biotechnological fields. In fact, gene mutations are often cause of genetic diseases and gene function analysis itself can help to have a broader vision on cells health status. Traditionally, gene mutations detection is carried out at pre-translational/sequence level (transcriptomic approach). On the other hand, the function of innumerable sequenced genes can be investigated by delivering them into cells through transfection methods and observing their expression result at post-translational level (proteomic approach). In this context, Micro-ElectroMechanical Systems (MEMSs) offer the intrinsic advantages of miniaturization: low sample and reagent consumption, reduction of costs, shorter analysis time and higher sensitivity. Their applications range from the whole cell assays to molecular biology investigations. On this subject, the thesis deals with two different tools for gene analysis: a Lab-on-Cell and cantilever-based sensors for in-vitro cell transfection and label-free Single Nucleotide Poly-morphisms (SNPs) detection, respectively. Regarding the first topic, an enhanced platform for single-site electroporation and controlled transfectants delivery has been presented. The device consists of a gold MicroElectrode Array (MEA) with multiple cell compartments, integrated microfluidics based on independent channels and nanostructured titanium dioxide (ns-TiO2) functionalized electrodes. Different activities have been reported, from the study of the microfabrication substrates bioaffinity and device development to the electroporation results. The functional characterization of the system has been carried out by electroporating HeLa cells with a small fluorescent dye and then, in order to validate the approach for gene delivery, with plasmid for the enhanced expression of the Green Fluorescent Protein (pEGFP-N1). The second research activity has been focused on a detection module aimed at the integration in a Lab-on-Chip (LOC) for the early screening of autoimmune diseases. The proposed approach consists of piezoresistive SOI-MEMS cantilever arrays operating in static mode. Their gold surface (aimed at the binding of specific thiolated DNA probes) has been deeply analyzed by means of Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) revealing an evident gold non-uniformity and low content together with oxygen and carbon contaminations. Different technological and cleaning solutions have been chosen in order to optimize the system. However, other improvements will be required. Moreover, the feasibility of the spotting technique has been demonstrated by verifying microcantilever mechanical resistance and good surface coverage without cross-contaminations. Finally, as future perspective, possible biological protocols and procedures have been also proposed and discussed starting from literature.

Item Type:Doctoral Thesis (PhD)
Doctoral School:Information and Communication Technology
Subjects:Area 09 - Ingegneria industriale e dell'informazione > ING-INF/07 MISURE ELETTRICHE E ELETTRONICHE
Area 05 - Scienze biologiche > BIO/11 BIOLOGIA MOLECOLARE
Area 05 - Scienze biologiche > BIO/13 BIOLOGIA APPLICATA
Uncontrolled Keywords:Lab-on-Cell; Cell Electroporation; Functionalization; Microcantilever Array; DNA Hybridization.
Repository Staff approval on:21 Jan 2011 12:30

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