A In-Pipette Pneumatic Micro-pressure Control System For Giga-Ohm Seal Of Patch Clamp

As a powerful tool in electrophysiological research, the patch clamp technology has seen numerous applications in the recording of channels ion currents embedded in the cell membrane, cellular secretion, pharmacology, pathological physiology, neuroscience, brain research, cellular and molecular biology as well as the physiological study of plant cells, etc.In patch clamp experiment, the formation of giga-ohm seal is a critical step, which is usually fulfilled through controlling the air pressure within pipette and the holder. Up to now, that process is commonly performed with experimenter's blowing and inhaling, resulting in some unavoidable problems. At first, the value of artificially obtained air pressure is just based on the operators' individual experiences, making the accurate calculation of that value impossible. Secondly, the pressures supported by experimenter's blowing and inhaling is unstable, wherein the adsorption to cell often need to be repeated several times due to the insufficiency of the in-pipette negative pressure, affecting the quality and efficiency. Thirdly, the method of experimenter's blowing and inhaling for sealing makes impossible the automatic giga-ohm seal in patch clamp experiment, the autocontrol for subsequent experiments and the high throughput measurement.In this paper, we developed a novel in-pipette pneumatic micro-pressure control system for giga-ohm seal of patch clamp. We describe this system in three main sections. In the first section, we analyzed the calculation of the pneumatic pressures and fluxes, which are the fundamental parameters for a micro-pressure control system, and choose the suitable pneumatic elements according to the calculated parameters. The second section was set out from view of the circuit design of the control system, illustrating the control and parameters of each parts of the circuit and the PI (Proportion Integral) control of the whole system in detail, respectively. Further, the open-loop test and close-loop test were carried out in detail. In the last section, some giga-ohm seals in patch clamp experiments were successfully performed on rat pancreatic using this pneumatic micro-pressure control system. As for rat pancreatic cells, the positive pressure of 0.5 kPa was introduced into the pipette as it immerged in the buffer, the average pipette resistance was 5.04 M?? and after attached the cell membrane it increased to 5.44 M?. Then the in-pipette pressure was changed from a positive pressure to a negative pressure one of 3.5 kPa, Changing the pipette –40 mV to –90 mV often helps to obtain or speed the seal formation. Then, the average seal-resistance was 2.98 G.?. As a further check, some artificial giga-ohm seal results were set as the control and compared with the results of our pneumatic micro-pressure control system. As a result, both results were very close, indicating our system could provide good-quality seal giga-ohm for patch clamp experiments despite the merits of the accuracy, high efficiency, and easy operation. This system will be helpful for the realization of the automatization patch clamp experiment as well as the high throughput measure.