A hybrid planar patch-clamp system for the characterization of ion channels in biological cells.

摘要

Ion-channels are proteins embedded in cell membranes that regulate the flow of ions across the cellular boundary, thereby maintaining the cell's delicate chemical balance. More than 40% of the known human diseases are directly or indirectly related to dysfunctions in ion channels. At present, a method called patch-clamping is the gold standard for studying ion-channel activity; however, it suffers from a lack of high-throughput-screening (HTS) and high cost per data point. As the Human Genome Project unravels a wealth of genetic information and combinatorial chemistry produces a vast database of compounds, we need to address novel HTS schemes for ion channel research.; In this dissertation, we have taken a preliminary step in the direction of automation and HTS with research on a miniaturized hybrid planar patch-clamp system constructed in silicon-based technology. This system is designed and fabricated using MEMS, microfluidics and CMOS silicon-based technologies. The advantage lies in the eventual ease of investigating ion-channels as drug targets for medical research. We describe three major aspects of research on this hybrid system: MEMS and microfluidics, integrated electronics and ion-channel modeling.; A MEMS micropore membrane structure is described, with attached microfluidics and integrated dielectrophoretic and mechanical forces to isolate and position a single cell automatically over the micropore. Suction and electric fields are applied to create a high seal resistance for whole cell and single ion channel investigations. The hybrid system contains a novel, low-noise, integrated CMOS instrumentation amplifier to process ion-channel signal currents. The amplifier employs Correlated Double Sampling for noise suppression and uses a unique integration-discrete differentiation technique to process both whole cell (1-10nA) and single ion-channel currents (5-10pA) at 10-100 kHz rates. In addition, the dissertation describes the simulation of ion-transport through a voltage-gated KcsA ion channel with the use of a technology computer-aided design (TCAD) simulation program. The model incorporates ion-transport factors, including the ion-ion interaction, protein surface charges, and the transmembrane potential to simulate ion-channel I-V characteristics. Furthermore, analytical models are derived to describe the ion-transport through a cylindrical ion channel with the probability density function and power spectral density of ion number fluctuations.