Analysis of Intracellular Molecules Using Nanoneedles Functionalized with FRET-based Molecular Probes

Abstract

We have been developing a low invasive cell manipulation technology using a nanoneedle of diameter 200 nm and length 10 μm. The nanoneedle insertion is performed by using an atomic force microscope (AFM) to sense a force response when inserting the needle into a living cell body. In this study, nanoneedles functionalized with FRET-based molecular probe have been developed to detect the intracellular mRNA or protein of a living cell. A molecular beacon which targets on human GAPDH mRNA or a FRET-sensor protein which contains substrate motif of caspase-3 was immobilized on a nanoneedle and each functionalized nanoneedle was inserted into a healthy HeLa cell or an apoptotic HeLa cell, respectively. FRET disruptions on the nanoneedles caused by hybridizing with mRNA or by digesting of the sensor protein with caspase-3 could be detected with a confocal scanning fluorescent microscopy.