[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
Giant plasma membrane vesicles (GPMVs) incorporating connexin proteins, referred to as connectosomes, serve as promising tools for studying cell membrane properties and intercellular communication. This study aimed to evaluate the...
Introduction Channel proteins in the plasma membrane play important roles in regulating the transport of specific molecules and ions in response to concentration differences between inside and outside cells. Therefore, the simple and rapid analysis of channel function and screening of agents for regulating the channel functions are desired in the research fields for pharmacology and drug discovery. Patch-clamp technique provides a unique and precise analysis for channel function. However, it has low measurement throughput to requires precise manipulation of a glass electrode to contact with a single cell. In this study, we applied the manipulation technique by dielectrophoresis (DEP) for easy and rapid evaluation of the opening and closing of channels embedded in Giant Plasma Membrane Vesicles (GPMVs). The time dependence of the fluorescence intensity of the internal dye of GPMVs, which were ordered well on the microwell array electrode by DEP was used to investigate the open/closed state of connexin, that is a channel protein expressed on the membrane of GPMVs. Furthermore, we revealed that the DEP behavior of GPMVs depends on the applied frequency and the open/closed state of connexins on GPMVs. Methods HeLa cells were transfected to express connexin 43 fused with FusionRed at the C-terminal (Cx-HeLa). The Cx-HeLa cells stained with Calcein-AM were incubated for 6 hours in an active buffer (10 mM HEPES, 2 mM CaCl 2 , 150 mM NaCl, 25 mM paraformaldehyde, 2 mM dithiothreitol, 125 mM glycine, pH 7.4). The active buffer containing GPMVs was exchanged to DEP medium (270 mM sucrose, 2 mM CaCl 2 , 50 mS m -1 in conductivity) by dialysis. The DEP device consisted of a bottom indium-tin-oxide (ITO) substrate with 10,000 microwells (16 µm in diameter and 10 µm in height) and a top ITO substrate mounted on the bottom substrate via a double adhesion tape (15 µm in thickness) (Fig. A). After GPMVs suspension was introduced in the DEP device, sine waves were applied to the top and bottom ITO substrates to induce DEP force to GPMVs. Results and Conclusion GPMVs were isolated from Cx-HeLa cells, and the mean diameter of GPMVs was 5.6 µm (S.D. 2.1 µm). It is known that connexin channels maintain the close state in the solution containing Ca 2+ [1]. When an AC voltage of 800 kHz was applied, the GPMVs with the connexin closed were quickly trapped in the microwells. This indicates the attractive force to higher electric field regions, called positive dielectrophoresis (p-DEP), acted on the GPMVs. The outer solution of GPMVs arrayed by p-DEP was exchanged to the buffer without Ca 2+ for opening connexins. The fluorescence intensity of GPMVs in the open connexin state decreased rapidly compared to that in the closed connexin state (Fig. B). This result indicates that the fluorescent dye diffused outside through the opened connexin. Next, the dielectrophoretic behavior of the GPMVs introduced into the device was investigated. When an AC voltage with 500 kHz was applied, the GPMVs with closed connexins moved to the center of four microwells (Fig. C). This result indicates that negative-DEP (n-DEP), the repulsive force from the higher electric field region was induced to GPMVs. The critical factor for identifying the electric properties of GPMVs is the cross-over frequency at which the n-DEP and p-DEP switch. The cross-over frequency was found to be between 500 kHz and 700 kHz for GPMVs with closed channels, while it was shifted to the region between 700 kHz and 800 kHz with open channels. The shift of cross-over frequency could be attributed to the decrease in membrane capacitance caused by the opening of the connexin channels. In conclusion, DEP manipulation of GPMVs could be useful for tracking single vesicles and easily discriminating the open/closed state of connexins embedded in the membrane. References [1] J. Thimm, A. Mechler, H. Lin, S. Rhee, R. Lal, J. Biol. Chem. 2005 , 280 (11), 10646–10654. Figure 1
To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique.
