مطالعه تجربی و شبیه سازی اثر زبری پی پت در تشکیل مهر و موم گیگا در وصله بستن

The effect of pipette tip roughness on giga-seal formation of patch clamp recording has been studied through FEA simulation and patch clamp experiments. FEA simulation results show that the membrane cannot fill up all of the peaks and valleys of a rough pipette tip. As a result in three dimensions the seal between inside and outside is compromised by channels in the order of several nanometres. These channels increase the leakage current between two electrodes, increase the noise and decrease the seal resistance. In contrast focused-ion-beam polished pipettes have very flat tips. Single ion channel currents recorded by FIB polished pipettes show significantly smaller leakage current and noise than the currents recorded by conventional pipettes. Results of FEA simulation are consistent with the results of patch clamp experiments.

Introduced by Neher and Sakmman, patch clamp technique has been widely used for studying cellular ion channels [1]. Nowadays, it has been proven that many different diseases can be caused by the mis-function of ion channels [2]. In patch clamping a patch of membrane is isolated from external solution to record the currents flowing into the patch. To achieve this, small glass capillaries are heated and pulled to fabricate glass micropipettes with a tip diameter of 1–2 μm. The pipettes are then backfilled with a conductive solution and pressed against the surface of a cell. To improve the sealing condition a gentle suction is applied to the backend of the pipette. As it is shown in Fig. 1 there are two electrodes in patch clamp set-up: a recording electrode inside the pipette and a reference electrode in the bath solution. A high resistance seal between the glass and the patch of membrane reduces the leakage current between the two electrodes and completes the electrical isolation of the membrane patch. It also reduces the current noise of the recording, permitting good time resolution of single-channel currents which are in the order of 1 pA [3]. Since the electrical resistance of the seal is in the order of giga-ohms, it is called giga-seal. In this paper a study in the effect of the pipette tip roughness on giga-seal formation is presented. The study was conducted through repeated patch clamp experiments and finite element modelling and analysis.

In this research, the effect of pipette tip morphology on giga-seal formation in patch clamping technique has been studied through experiments and FEA simulation. High magnification SEM images revealed the surface nature of pipette tips to be in contact with cells. A DEM image of the tip is created using 3D reconstruction method. The real tip profiles are extracted from DEM image and used in FEA simulation. The results of the finite element modelling show that the cell cannot fill up all of the valleys of the tip and therefore in three-dimensions the inside of the pipette is connected to the outside by nanometre-high channels. Then the contact tips of pipettes were cut across, leaving a very smooth surface at the top of the pipettes. A large number of patch clamping experiments were conducted on HUVECs using the polished pipettes and 60% of the experiments achieved above 3 giga-ohm seals and the highest seal resistance reached 9 GΩ. The results show that nanomachined microglass pipettes have improved the giga-seal formation in patch clamping. Higher seal is obtained from better contact conditions of the smoother tip surface with membrane. This shows that the membrane cannot fill the valleys of the rough surface perfectly which could be possibly the reason for reports on lower seal resistance with rough surfaces in the literature.