One hundred spots parallel monitoring of DNA interactions by SPR imaging of polymer-functionalized surfaces applied to the detection of cystic fibrosis mutations

摘要

In the present paper, we report the detection of mutations implicated in human cystic fibrosis (CF). Nine different oligonucleotides are studied, including three possible mutations related to this specific genetic disease: a deletion of three bases, ΔF508, and two single-nucleotide polymorphisms 1540A/G and 1716G/A. We monitor, in real time and in parallel, hybridizations of a solution of unlabeled oligonucleotide targets to a matrix of 100 spots of oligonucleotide probes using surface plasmon resonance (SPR) imaging of a bio-functionalized gold slide. In order to functionalize our gold slide with the DNA probes, we have developed a self-assembled multilayer (SAM) based on electrostatic interactions and formed with 11-mercaptoundecanoic acid (MUA), poly(ethylenimine) (PEI) and ExtrAvidin layers. Probes are then linked to this SAM by the usual strong binding affinity of the avidin-biotin duplex. The 100 spots array deposited by a robot can be addressed either several times, sequentially, with the various oligonucleotide targets, or once, in parallel, with a mixture of some oligonucleotides. The specific response of our system is established along with the possibility of discriminating between a totally complementary sequence and its mutant form, even for a single base mismatch thus demonstrating the capacity of parallel diagnostic using patient like material.