Gap Junctions Study Using a Microfluidic Sensor

Cells in tissues and organs coordinate their activities by communicating with each other, and this communication is mediated by specialized channels, called gap junctions. There are two types of gap junction communications both governed by potential gradients: molecular diffusion and electrolyte transport. Most cells are known to express multiple connexins that form different types of gap junctions. To date, there is no known gap junction blocker which specifically blocks one kind of gap junction channel. Therefore, we have developed a microfluidic sensor for studying intercellular communication in a high throughput manner. The concept of the microfluidic sensor is based on the ability for a multi-stream channel to partition a cell monolayer with different chemically loaded solutions. Numerical simulations were conducted to characterize the performance of the chip in terms of multiple flow control, interface stability and fluid exchange time. The flexibility of the sensor allowed the characterization of molecular diffusion and electrical coupling. The study of intercellular diffusion of fluorescent probes in Normal Rat Kidney cells confirmed the size dependent permeability through the Cx43. With the aid of a numerical algorithm, we were able to extract the diffusion rate of molecular probes from fluorescent microscopy recordings. The testing of different molecular size fluorescent dyes showed increasing diffusion rates with decreasing molecular weight. A similar experimental configuration gave us the ability to screen different gap junction blockers. The testing of showed homogeneous reduction of the diffusion rate for all tested dyes, while MFA displayed a charge dependent potency. The ability for the sensor to test different dyes or different blockers was also demonstrated in detail. We also measured the electrical intercellular communication via the same microfluidic chip using a sucrose gap configuration. The use of a non-conductive solution in the middle stream of the tri-stream channel allowed the constriction of electrolyte transport through gap junction channels. Both 1-Heptanol and 2-APB reversibly reduced gap junction communication. The gap junction blockers 1-Heptanol and 2-APB provided distinct kinetics of electrolyte transport alteration, suggesting the presence of two different blocking mechanisms. We also demonstrated the ability to screen candidate blockers such as the ion channel inhibitor, GsMTx-4. The application GsMTx-4 did not show an effect on the gap junction channels. Finally, we studied the effect of the cell substrate stiffness on the gap junctions' activity. The substrate was coated with a layer of Polydimethylsiloxane (PDMS), presenting different stiffness, prior to experimentation. The study of the molecular diffusion through cells cultured on substrates of different stiffness showed an increase of the molecular permeability with decreasing the substrate stiffness. To understand the link between intercellular diffusion of molecular probes and substrate stiffness, we immunostained proteins playing a role in substrate sensing, such as actin filaments and in the cell-cell junction such as ZO-1. The results showed an increased number of ZO-1 complexes for softer substrates and an increase of the Cx43 complexes at the cell-cell junction, which was responsible for facilitating gap junction mediated molecular diffusion

DEVICE AND METHOD FOR SINGLE CELL SCREENING BASED ON INTER-CELLULAR COMMUNICATION

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An electrochemical-sensor system for real-time flow measurements in porous materials

Flow monitoring in porous materials is critical for the engineering of paper-based microfluidic bioassays. Here, we present an electrochemical-sensor system that monitors the liquid flow in porous materials without affecting the real flow in paper-strip samples. The developed microfluidic sensor records an amperometric signal created by the solution movement mediated by paper wicking. This approach allows the in situ monitoring of the different hydrodynamic conditions of a specific paper geometry or composition. In addition, the method proposed in this work was employed to characterise the fluid flow of different nitrocellulose paper strips after oxygen-plasma treatment or dextran coating. The dextran fluid-flow modifiers were further used on the paper strip-based assays as means of signal enhancement. The proposed electrochemical-sensing method offers a valuable alternative to existing optical-based monitoring techniques for flow measurement in paper-based microfluidic systems.close1