title: Electrochemiluminescent hybridization chip with electric field aided mismatch discrimination.
abstract: This paper describes a heterogeneous DNA-hybridization assay based on electrochemiluminescence (ECL) detection on gold electrodes. Short, 15-mer oligonucleotides were conjugated with a synthesized electrochemiluminescent label, bis(2,2'-bipyridine)-5-isothiocyanato-1,10-phenanthroline ruthenium(II) at the amino-modified 5'-end. Gold electrodes were derivatized with 15-mer oligonucleotide probes via 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking reaction and hybridized with Ru-labeled strands. Two types of self-assembled-monolayers have been utilized for the immobilization reaction, 3-mercaptopropanoic acid (3-MHA) and 16-mercaptohexadecanoic acid (16-MHA). Longer thiols were more stable at high electrode potentials needed for the ECL generation. The system was sensitive down to 1 fmol of labeled complementary strand, detected in 30 microL of buffer. Mismatch discrimination was achieved both passively by washing and actively by application of negative electrode potential on electrodes prior to detection, but active denaturing lead to better results. Two base-pair mismatches were discriminated at room temperature.
authors.affiliation: Laboratory of Sensors, Actuators and Microsystems, Institute of Microtechnology, University of Neuchâtel, Rue Jaquet-Droz 1, CH-2007 Neuchâtel, Switzerland. anna-maria.spehar@hut.fi
authors.forename: Anna-Maria
authors.initials: AM
authors.lastname: Spehar-Deleze
authors.forename: Laurette
authors.initials: L
authors.lastname: Schmidt
authors.forename: Reinhard
authors.initials: R
authors.lastname: Neier
authors.forename: Sakari
authors.initials: S
authors.lastname: Kulmala
authors.forename: Nico
authors.initials: N
authors.lastname: de Rooij
authors.forename: Milena
authors.initials: M
authors.lastname: Koudelka-Hep
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics*; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017403:In Situ Hybridization / Q000295:instrumentation* / Q000379:methods; D008163:Luminescent Measurements / Q000295:instrumentation*
chemical_list: D004247:DNA