title: Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay.
abstract: MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin-alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L(-1) and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.
authors.affiliation: Dipartimento di Chimica Ugo Schiff, Università degli Studi di Firenze, Sesto Fiorentino (Fi), Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Bettazzi
authors.forename: Ezat
authors.initials: E
authors.lastname: Hamid-Asl
authors.forename: Carla Lucia
authors.initials: CL
authors.lastname: Esposito
authors.forename: Cristina
authors.initials: C
authors.lastname: Quintavalle
authors.forename: Nello
authors.initials: N
authors.lastname: Formisano
authors.forename: Serena
authors.initials: S
authors.lastname: Laschi
authors.forename: Silvia
authors.initials: S
authors.lastname: Catuogno
authors.forename: Margherita
authors.initials: M
authors.lastname: Iaboni
authors.forename: Giovanna
authors.initials: G
authors.lastname: Marrazza
authors.forename: Marco
authors.initials: M
authors.lastname: Mascini
authors.forename: Laura
authors.initials: L
authors.lastname: Cerchia
authors.forename: Vittorio
authors.initials: V
authors.lastname: De Franciscis
authors.forename: Gerolama
authors.initials: G
authors.lastname: Condorelli
authors.forename: Ilaria
authors.initials: I
authors.lastname: Palchetti
mesh_terms: D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D045744:Cell Line, Tumor; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics
chemical_list: D035683:MicroRNAs