Development Of Automated Micro-Biomanipulation System For Electrophysiology Study

The robotic and automation techniques increase the productivity for the development of novel therapeutics and give novel method for the study for biological studies. A significant demand for both accuracy and productivity in cell drug discovery highlights the need for the automated drug delivery and recording for single cells with integrated robotics and micro/nano manipulation technologies. Most of the existing methods for studying ion channel and drugs focus on studying extracellular drugs and suspended cells. It has been challenging to achieve a high-yield micro-biomanipulation because the contact between the micro-scale end-effector and the cell has to be precisely controlled. The process of the micro-biomanipulation of single cell should be analyzed, and novel automated techniques which can be used to study the intracellular drugs and the electrophysiology at the single-cell level for adherent cells are needed. In this study, the approaches and details for automated bio-micromanipulation systems are developed, which can be used for the automated ion channel drug delivery and drug screening at the individual cell level for adherent cells. This thesis study includes the following three aspects.First, an automated patch clamp recording strategy is developed by utilizing the resistance feedback, which can be used to perform automated patch clamp recording on adherent cells. The basic idea of the proposed control research is that the whole process of the patch clamp recording can be monitored by the resistance feedback. By analyzing the electrical resistance changes of electrode, cell condition can be algorithmically detected. By combination of a visual identification approach, the cells and patch clamp pipette can be identified and the whole process of the patch clamp recording can be monitored by the visual feedback and resistance feedback. By this approach the suspension procedures of adherent cells can be avoided, and the seal condition can be improved compared to the recent commercial patch clamp system. Experiments performed on neuroblastoma cells and dorsal root ganglion cells demonstrate the effectiveness of the proposed approach.Second, a novel strategy for extracellular and intracellular drug delivery system is investigated, which enables the precise volume drug delivery to small adherent cells (diameter<30μm). A Polydimethylsiloxane based chamber is developed for extracellular drug delivery. A novel electric feedback for observe the penetration during the intracellular delivery is proposed, and the electrical model and parameters is analyzed. The measurement of the voltage is mainly governed by the change of resistances during the whole process of intracellular drug injection. After the discussion of these resistances, theoretically, the voltage measurement is an efficient indicator to monitor the process of intracellular injection. A control algorithm for single cell injection is developed with micro precision, which enables the drug delivery system to perform drug delivery rapidly and precisely. Experimental results are consistent with the electrical model analysis, and the system works well for performing extracellular and intracellular drug delivery on small adherent cells.Third, the automated micro-biomanipulation system is used to study the activation of ion channels and the related electrophysiology properties. Cell line (neuroblastoma cells) and primary cells (dorsal root ganglion neurons) are studied, and ion channel related chemicals and drugs (N-Methyl-D-aspartate, Amyloid-P, Paclitaxel) are recorded by the system. The studies of these chemicals and drugs demonstrate that the system and approaches have a perfect performance for electrophysiology studies. The experiment result shows that the system performing a consistent result compared to the well-known published results, and the system has the ability to perform cell-attached recording which requires a high seal resistance. Besides, it is shown that the adherent primary cells can be easily recorded by this system which is a difficult task for the commercial systems.In summary, the automated micro-biomanipulation system and the novel feedback control strategies provide novel solutions for electrophysiology study at single-cell level for adherent cells. The study of several ion channel related chemicals and the activation of ion channels demonstrate that the system have high yield and rapid performance.