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Abstract

Here, we prove how capillary assembly in combination with soft lithography can be used to perform DNA (yeast genomic and phage lambda) molecular combing to generate highly ordered arrays of isolated and perfectly aligned DNA strands for genetic analysis and diagnosis.

Abstract

A simple surface chemical bi-functionalization procedure, involving micro contact printing of silane molecules is presented. The printing process has been optimized for obtaining high quality layers of good homogeneity. The produced chemical templates are used in order to spatially localize the adsorption of different entities diluted in liquid solution. By combining optical and Atomic Force microscopy we confirm the selective adsorption of gold nanoparticles, dendrimers and bacteria.

Mots-Clés / Keywords

Biochips; Surface chemistry; Dendrimers; Chemical patterning; Soft lithography;

Abstract

The main purpose of this research work is the demonstration that soft-lithography, very often called Micro-Contact Printing (µCP) is an efficient patterning technique for arranging biomolecules on a surface in the perspective of biochip applications. For DNA Micro-arrays applications, we demonstrate that µCP is a competitive method compared to the conventional spotting technology, commonly used today. The cost of the technology is much lower, the surface density of the chip is drastically increased and the quality and definition of the biopatterns are greatly improved. A systematic study of the inking mechanisms of the elastomeric stamps is provided together with the study and comprehension of transfer mechanisms of molecules from the surface of the stamp to the substrate. The crucial role played by the free fragments of polymers not cross-linked during the polymerisation of the stamp is highlighted. In a second section we investigate the possibility of using µCP for generating single biomolecule biochips. We show how this printing technique can be optimized for reaching sub-micrometric scale down to 50 nanometers features. Two technological processes involving soft-lithography are proposed: the first one for combing long DNA molecules on spatially organized and registered positions for genetic applications, the second one for assembling individual DNA molecules in ordered arrays compatible with the dynamic tracking of individual DNA molecules over large populations.

Résumé

L'objectif des travaux est de démontrer que la lithographie douce, quelquefois baptisée " Micro-Contcat Printing (µCP)", constitue une méthode de dépôt de biomolécules présentant de nombreux avantages pour des applications de type Biopuces. Pour la fabrication de puces à ADN, nous démontrons que le µCP est une technique compétitive par rapport au dépôt robotisé de gouttes traditionnellement utilisé. Le coût est inférieur, la densité des puces est augmentée et la qualité et la définition des motifs biomoléculaires sont supérieures. Une étude complète des mécanismes d'encrage des timbres élastomères d'impression ainsi que des mécanismes de transfert par contact des molécules vers le substrat est présentée. Le rôle prépondérant des fragments de polymère non réticulés présents à la surface des timbres est mis en évidence. Dans un second volet nous étudions la possibilité de générer par la même méthode des puces à biomolécules uniques. Nous montrons comment le µCP peut être poussé jusqu'à une résolution sub-micrométrique proche de 50 nm. Deux voies technologiques originales impliquant la lithographie douce sont proposées : l'une pour peigner individuellement en des sites organisés précisément sur la surface des longs brins d'ADN pour des études de génétique, l'autre pour fixer des molécules individuelles d'ADN par une extrémité rendant possible l'étude dynamique de molécules uniques (ADN) sur de larges populations.

Mots-Clés / Keywords

Lithographie douce; Microcontact printing; Microtransfer molding; ADN; Biopuces; Microarrays; DNA; Biochips;

Abstract

It is well-established that, during microcontact printing (CP) using poly(dimethylsiloxane) (PDMS)-based stamps, some unexpected siloxane fragments can be transferred from the stamp to the surface of the sample. This so-called contamination effect coexists with the delivery of the molecules constituting the ink and by this way influences the printing process. The real impact of this contamination for the CP technique is still partially unknown. In this work, we investigate the kinetics of this contamination process through the surface characterization of both the sample and the stamp after imprinting. The way both the curing conditions of the PDMS material and the contact time influence the degree of contamination of the surface is investigated on silicon and glass substrates. We propose a cleaning process of the stamp during several hours which eliminates any trace of contamination during printing. We show that hydrophobicity recovery of PDMS surfaces after hydrophilic treatment using oxygen plasma is considerably slowed down when the PDMS material is cleaned using our procedure. Finally, by comparing cleaned and uncleaned PDMS stamps, we show the influence of contamination on the quality of CP using fluorescent DNA molecules as an ink. Surprisingly, we observe that the amount of DNA molecules transferred during CP is higher for the uncleaned stamp, highlighting the positive impact of the presence of low molecular weight siloxane fragments on the CP process. This result is attributed to the better adsorption of oligonucleotides on the stamp surface in presence of these contaminating molecules.