A Biosensor for miRNA Detection By Electrorotation Rate of Glass Micro-Rods Modified with Peptide Nucleic Acid

[Introduction] MicroRNA (miRNA), which is RNA molecule with approximately 22 nucleotides in length, has attracted attention as novel biomarkers for the diagnosis of cancer, hepatitis, and other conditions. The miRNA has been commonly detected by RT-qPCR, used for synthesizing and quantifying cDNA. We can detect minute quantities of miRNA by this method. However, it requires laborious procedures, complex reagents, and fluorescent probes for labeling DNA. Thus, simple and rapid detection methods of miRNA have been required for point-of-care testing. When microparticles are exposed to a rotating electric field, the electrostatic interaction between the induced polar charges on the particles and the rotating electric field generates the torque on the particles. This phenomenon is called electrorotation (ROT). The rotation rate depends on the electrical properties of the particle surface. We developed the miRNA detection system based on the decrease of ROT rate of rod-shaped glass microparticles (micro-rods) by binding miRNA. The surface of micro-rods was modified by peptide nucleic acid (PNA) with a complementary sequence of target miRNA. The recognition of miRNA charged negatively to the modified PNA gives rise to the increase of surface conductivity of micro-rods and thereby the decrease of the rotation rate. In addition, the surface conductivity of micro-rods was almost constant by introducing PNA without the charge. Thus, the system could provide the simple detection of miRNA required no labeling with fluorescence molecules. [Experimental Methods] ROT measurements of micro-rods were conducted by a three-dimensional interdigitated array electrode device (3D-IDA) (Fig. A). The device consisted of two glass substrates with micropatterns of IDA (35 µm in electrode width and 70 µm in gaps between electrodes) made of indium-tin-oxide (ITO). A substrate was mounted orthogonally to another substrate via double adhesive tape (60 µm in thickness), resulting in the formation of microgrids (70 µm in length) surrounded by four microband electrodes. Applying AC voltages with a phase difference of 90 degrees each to the four microband electrodes generates a rotational electric field in microgrids. Micro-rods were treated with 100 mM 3-aminopropyltriethoxysilane for 1 hour, 10 mM 3-Sulfo-N-succinimidyl 4-(N-Maleimide-methyl) cyclohexane-1-carboxylate sodium salt for 1 hour, and 5 µM thiolated single-stranded PNA for 4 hours to modify the surface by PNA. The PNA-modified m