MicroRNA detection using magnetic separation and zinc-based nanolabels as signal transducers

In this work, a new microplate based assay for the detection of microRNA (miRNA) is described. The assay uses suspensions of paramagnetic beads immobilized with locked nucleic acid (LNA) oligonucleotide probes to capture miRNA. Poly(A) tails are generated on the captured miRNA using the poly(A) polymerase reaction. Poly(T)(30) oligonucleotides hybridize with poly(A) tails and are subsequently labeled with ZnS nanoparticles through biotin-NeutrAvidin chemistry. For signal transduction, zinc ions from the ZnS nanoparticles are released to bind with and activate zinc-ion depleted carbonic anhydrase. The activated kinetics of carbonic anhydrase converts fluorescein diacetate to fluorescein for fluorescence measurement. This work presents a method combining polyadenylation, ion release of nanolabels, and enzyme activation for miRNA detection in a convenient, magnetic bead-based microplate assay format.