Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay

• DOI: 10.1007/s00216-012-6476-7
• Publication Date: 2012-10-25T07:31:07Z
• Authors: Francesca Bettazzi; Ezat Hamid-Asl; Carla Lucia Esposito; Cristina Quintavalle; Nello Formisano; Serena Laschi; Silvia Catuogno; Margherita Iaboni; Giovanna Marrazza; Marco Mascini; Laura Cerchia; Vittorio De Franc...; Gerolama Condorelli; Ilaria Palchetti
• Keywords: Magnetic beads; MicroRNA; Electrochemical Techniques; 01 natural sciences; Electrochemical assay; 0104 chemical sciences; 3. Good health; miRNA-222; MicroRNAs; Cell Line, Tumor; Humans; Electrochemical assay; Magnetic beads; MicroRNA; miRNA-222; Biological Assay

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.