DESIGN AND DEVELOPMENT OF MICROARRAYS FOR FUNCTIONAL GENOMICS

LUCHI, MASSIMILIANO

DESIGN AND DEVELOPMENT OF MICROARRAYS FOR FUNCTIONAL GENOMICS

LUCHI, MASSIMILIANO

2007-03-09

http://thesis2.sba.units.it/store/handle/item/12262

http://hdl.handle.net/10077/11555

Doctoral Thesis

Contributor(s)

TOMMASINI, FERNANDO

Abstract

Several studies explore the possibilities offered by the Surface Plasmon Resonance (SPR) as a possible technique to develop a biosensor devoted to DNA analysis. The aim of this study is to explore the possibility to improve the performances of the SPR using nanofabrication of plasmonic crystals, in order to develop a new tool for functional genomics studies with new biological architecture. This could be a possible base for a new type of array DNA platforms according to a direct nucleic acids measurement protocol that will avoid the time consuming and error prone reversed-transcriptase Polymerase Chain Reaction, where genes can only be analyzed individually and the measurements are affected by the polymerase activity and selectivity. The SPR technique was not yet established in our group, therefore the aim of this study is to introduce from scratch this method in our group in order to establish a new platform that could be used also for other applications. Consequently, an in dept understanding of the theory behind the technique is required and the study starts with a thorough investigation of DNA hybridization based on functional selfassembled monolayer systems. In particular, the Surface Plasmon Fluorescence Spectroscopy (SPFS) will be explored as the main detection method. The SPFS technique was firstly introduced by the Knoll' s group at the Max Planck Institute for Polymer research in 2000 Extensive DNA hybridization studies have been carried out since then The need of the fluorescence to detect the DNA hybridization is due to the fact that currently the SPR itself cannot detect such small molecules. This is not true for example for proteins since, possessing a larger size than oligonucleotides, the protein binding can be visible in both the SPR. This dual-channel sensing ability of SPFS can be fully used for uncovering more interfacial information which could be of great interest if possible also for the DNA. Therefore this study tries to improve this aspect of the technique taking into account the possibilities offered by the nanofabrication with the goal in mind to realize, in the future, a completely label-free system, in order to be able to perform DNA studies directly on the raw samples, reducing thus also the time needed to prepare the samples. Based on the understanding that fluorescence suffers severe quenching if the fluorophores are too dose to the metal, it has been designed an architecture to extend the interaction platform out of the 'quenching' region that is simpler than those previously adopted. The present study is made by two separated parts converging upon the same aim that is to exploit new analytical methodologies for molecular biomedicine applications. The power of these new technologies comes from the potentiality that nanofabrication gives to molecular biology once the two fields are joint and that may allot to overcome the present issues related to the traditional molecular biology' s techniques. Therefore, a nanofabrication approach has been applied to a nucleic acids sequence analysis. There are different ways to analyze nucleic acids but for several reasons, SPR has been the choice we decided to choose. It is an in-situ optical method that allow to work both in air and in water. The possibility to work in aqueous environment and being a non destructive method, make SPR a particularly well suited method for biological applications. In addition, the possibility in the future to develop label-free technologies may allow, once the proper substrates are realized, to perform direct analysis on biological samples without the need to process them in order to label them or purify them, step which reduce the amount of the analyte since the yield it is never 100%. Furthermore, there is the possibility to improve the SPR methods using appropriately pattemed substrates that can be realized with x-ray lithographic techniques. An optical sensor is a sensing device which, by optical means, converts the quantity measured (measurand) to another quantity ( output) which is typically encoded into one of the characteristics of a light wave. In SPR sensors, a surface plasmon is excited at the interface between a metal film and a dielectric medium (superstrate), changes in the refractive index of which are to be measured. A change in the refractive index of the superstrate produces a change n the propagation constant of the surface plasmon. This change alters the coupling condition between a light wave and the surface Plasmon, which can be observed as a change in one of the characteristics of the optical wave interacting with the surface plasmon. Based on which characteristic of the light wave interacting with the surface plasmon is measured, SPR sensors can be classified as SPR sensor with angular, wavelength, intensity, phase, or polarization modulation. In this study, Surface Plasmon Resonance' s potential for nucleic acid analysis has been explored in arder to develop the knowledge for new microarray DNA platforms. In particular, Surface Plasmon Fluorescence Spectroscopy (SPFS) has been investigated with a new indirect labeling system that allows avoiding the labeling of the targets but still making use of the high sensitivity allowed by SPFS. Moreover, we have strengthen the believe that optical methods, and especially those surface plasmon based, are well suited for DNA analysis due to the speed of the measurement, the possibility to have label-free and indirect-label methods, the possibility to work in liquid, change the temperature and, last but not least, doing a non-destructive measurement allowing the sample to be eventually further analyzed. In this study the interfacial hybridization of a plasmid has been investigated using SPR and SPFS. Two different architectures have been used for the molecular recognition layer, direct binding of the probe on the gold surface via sulfur-gold bond and functionalization of the surface using a biotinilated oligonucleotide bonded on polystyrene. The second architecture has been proven to be able to avoid the fluorescence quenching observed with the former one. Moreover, this architecture can be easily produced faster than other architectures found in literature. The detection of the plasmid, being indirect, has a very good feature: there is no need to directly label the target. Therefore no labeling efficiency issue is encountered, since the secondary probe can be purified after labeling. The work done shows that there is still a huge potential to be uncovered, in arder to improve the current state of the art. These improvements are base on the nanofabrication of plasmonic crystals. Substrates to improve the SPR technique have been indeed produced using the x-ray lithography. The bidimensional patterned substrates have shown the characteristic to induce the coupling of the light with the metal layer and they will be useful in further studies to improve the technique. Indeed, preliminary experiments performed using the new VASE® Research Spectroscopic Ellipsometer (J. A. Woollam Co., Inc.) show that using plasmonic crystals coupled with this setup may lead to an increase of sensitivity of one order of magnitude of the label free SPR. In addition, this study had the fundamental importance to introduce Surface Plasmon Resonance (SPR) and Surface Plasmon Fluorescence Spectroscopy (SPFS) techniques in our laboratory, opening countless opportunities for a great number of new projects and research lines.