DNA immobilisation procedures for surface plasmon resonance imaging (SPRI) based microarray systems.
Two different surface chemistries have been studied for the development of surface plasmon resonance imaging (SPRI) based DNA microarray affinity sensors: (1) 11-mercaptoundecanoic acid-poly(ethylenimine) (MUA-PEI) and (2) dextran procedures. The MUA-PEI method consists of assembling a multilayer on the basis of electrostatic interactions formed with: 11-mercaptoundecanoic acid (MUA), poly(ethylenimine) (PEI) and extravidin layers. The dextran procedure involves assembling a multilayer formed with 11-mercaptoundecanol, dextran and streptavidin layers, which are linked by covalent bonds. The oligonucleotide probes are immobilised onto the sensor surface as spots forming a matrix 14x14, which is spotted by a robot, while the target sequences are free in solution. The system allows the interaction (hybridisation) monitoring, in real-time and in parallel, of unlabeled oligonucleotide solution targets to oligonucleotide probes immobilised on a 196 spots matrix. Using oligonucleotides as probes and targets, both functionalised surfaces have been evaluated in view of their application to the diagnosis of gene mutations involved in human diseases. In particular, we demonstrate the ability to detect, in parallel, several mutations causing human cystic fibrosis (CF), which lie within exon 10 of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene. The immobilised probes were complementary to sequences corresponding the mutant or wild type alleles. Two deletions of three bases (DeltaF508 and DeltaI507) and four single nucleotide polymorphisms (M470V, Q493X, V520F and 1716 G>A) were investigated. In both functionalised surfaces, the system showed the capacity to discriminate normal and mutant sequences differing by a single base.