A Biosensor for miRNA Detection By Electrorotation Rate of Glass Micro-Rods Modified with Peptide Nucleic Acid
Authors: Matsumoto Misato , Isozaki Yushi , Suzuki Masato , Yasukawa Tomoyuki 
Published: December 2024

[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 micro-rods were then treated with miRNA for 1 hour. The temperature gradually decreased from 45 ºC to 20 ºC at 0.83 ºC min-1 and then kept at room temperature for 30 min. The treated micro-rods were injected into the 3D-IDA and subjected to the ROT measurement.
[Results and Discussion]
The application of voltage (10 Vpp, 100 kHz) to the 3D-IDA caused the micro-rods to rotate at the center of each grid (Fig. B). The rotation rate decreased with increasing the length of micro-rods. The micro-rods with the length of 40 µm that is the maximum frequency value of the length were selectively observed in this work. The PNA-modified micro-rods introduced in the device adsorbed on the IDA substrate. The adsorption could be due to the electrostatic interaction between the positive charge on micro-rods derived from unreacted amino group introduced for the PNA modification and the negative charge on the IDA substrate. Most of micro-rods treated with miRNA were rotated at the center of grids. For the micro-rods treated with 5 nM miRNA, the rotation rate slightly increased with increasing the applied frequency up to 50 kHz, followed by a decrease of the rate (Fig. C). Rotation rate decreased with increasing the miRNA concentration. Furthermore, the frequency with maximum rate shifted to higher-frequency region with increasing the concentration. These results were attributed to the increase of the surface conductivity by binding miRNA with the negative charge on the micro-rods. However, the micro-rods treated with non-complementary miRNA absorbed on the IDA substrate to observe no electrorotation. In addition, the rotation was inhibited by the shift of the rotation center of microrods to the edge of electrodes by applying AC voltage with over 200 kHz. The shift of the center is due to the force of the negative dielectrophoresis. These results indicate that the rotation rate of micro-rods in lower frequency region allows to the determination of miRNA with target sequence without fluorescence label.

Figure 1