Robotic Inside-out Recording Method Based on Vesicle Rupture Detection

The inside-out patch clamp technique is widely used in brain science and neuroscience research because of its ability to detect the extremely weak currents flowing through a single ion channel. In the inside-out patch clamp operations, a micrometer-scale dimensions pipette is used to aspirate part of the cell membrane, creating a giga omega-scale seal (usually termed as gigaseal) between the cell membrane and the inner wall of the micropipette to suppress the environmental electromagnetic noise. The aspirated cell membrane is then gently pulled away from the cell to form a vesicle, which is subsequently exposed to low-calcium liquid or air to induce its rupture, forming an inside-out seal for signal detection on a single ion channel. Current manual operations of inside-out patch clamp are complex and require a high level of operator expertise. Moreover, existing robotic inside-out patch clamp technologies are usually applicable to suspension cells, limiting the widespread application of this technique. This paper proposed a robotic inside-out recording patch clamp technique based on vesicle rupture detection to address these limitations. Firstly, impedance models were established to detect the state of the vesicle before and after the rupture. Further, the detection of vesicle rupture was performed based on transmembrane current responses under step inputs. Based on the above work, a robotic inside-out recording patch clamp method based on vesicle rupture detection was established in this paper. Experimental results demonstrated the effectiveness of the proposed method in achieving successful inside-out recording patch clamp mode on adherent HEK-293 cells with a success rate of 60 %, laying the foundation for subsequent studies on single ion channel functionality.