(604a) Real Time Detection and Imaging of mRNA/Proteins in Individual Cells by Nanoelectroporation (NEP)

New technologies that can detect, localize, quantify and monitor the specific genes in individual cells are required for advancement in disease therapy, drug discovery and medical diagnostics. We recently developed a novel nanoelectroporation (NEP) technique that can deliver detecting/imaging agents, including molecular beacons (MBs), quantum-dots (QDs), aptamers and their combinations (i.e. QD based MB FRET and QD based aptamer FRET) into isolated individual cells with controlled dose and location. The delivery is achieved by the focused electric field through a nanochannel connecting two microscale channels with a single cell located at the outlet of the nanochannel in one microchannel and detecting/imaging agents located in the other microchannel. By adjusting the magnitude of electric field, pulse length and pulse number, dose and location control of the delivery can be precisely controlled.

Molecular beacons are dual-labeled oligonucleotides probes which have a fluorophore at one end and a quencher at the other end of a stem-hairpin structure. After successful hybridization with complementary targets, GAPDH mRNA in this work, the fluorescence will be restored by separating the fluorophore and the quencher, which makes MB one of the best candidates to visualize in real-time the expression and localization of specific mRNA inside the cells. Compared with organic fluorescent dyes, QD based MB was used here for enhanced signal to noise ratio.

Aptamers have attracted a lot of research interest in the development of sensors for proteins because of their high specificity and affinity. In our work, QDs based aptamer beacons (QD-AS1411) are used to detect proteins in single cells. Aptamer was first conjugated to QDs and an antisense oligonulecotide was used to quench the QDs. In the presence of target protein, nucleolin in this work, the binding of aptamer to nucleolin results in the dissociation of the antisense oligonuclotide quencher and the fluorescence of QDs is then restored and detected using confocal microscopy. NEP is a powerful tool that can provide sensitive detection and mapping of m-RNA/protein inside cells with real-time monitoring.