Poregenic - Patch on Chip System for Adherent Cellular Networks

Abstract

through a synthetic bilayer lipid membrane (BLM) are measured to obtain a rapid and sensitive signature of BLM disruption. In this study, we describe application of electrophysiology methods to investigate molecular interactions between a variety of ENM and BLM. To explore the effect of ENM surface properties on ENM-BLM interactions, BLM were challenged with customfabricated polypropargyl glycolide nanoparticles having a variety of surface functional groups. Polyethylene glycol functionalized ENM triggered ionic current spikes having a duration of milliseconds, while carboxylfunctionalized ENM generated both rapid current spikes and extended integral conductance. Ion selectivity of the ENM-generated conductance was investigated by measuring reversal potential of transmembrane currents at various applied potentials in the presence of a 10:1 KCl gradient. Reversal potentials for different types of ENM varied from 0 mV, indicating no ion preference, to 37 mV, indicating preference for K þ . To explore the effect of BLM composition on ENM-BLM interactions, two BLM compositions were tested: pure DOPC, and a 3:1 ratio of POPC:POPE. For a variety of ENM, the DOPC BLM consistently exhibited ion currents at lower concentrations and lower transmembrane voltages than did the POPC:POPE BLM. These results demonstrate the utility and versatility of electrophysiology methods to characterize molecular interactions between ENM and BLM. Automated patch-clamp setups are applied to investigate dose response relationships and target kinetics in the development of new pharmaceutical agents. Currently, automated systems are limited to investigations of suspended single cells. We pursue the development of assays for detecting the membrane properties in adherent networks because the majority of cells in humans grow adherently. In a first step, we developed PoreGenicÒ, a novel patch-clamp system for cells growing on a sensor chip with micro-structured needle electrodes arranged in an 8Â8 multi-electrode array with a pitch of 100 mm. PoreGenicÒ allows for the electrical cell manipulation as well as for extra-and intracellular potential measurements. Four types of needle electrodes of different shapes and materials were tested with heights of less than 10 mm. For intracellular detection, electroporation pulses were applied to form membrane pores for the introduction of the electrodes into the cytoplasm. Fluorescence and scanningelectron microscopy in combination with focused ion beam preparation were used to characterize the success of electroporation. In a number of experiments, we could access the cytoplasm and detect intracellular potentials. Our current system features 16 hollow needle structures with fluidic connections for patch-clamp experiments. Gold nanoparticles are of great interest to the scientific community owing to their potential applications in biology and medicine, earned by their extraordinary optical and chemical properties; counting their excellent biocompatibility, colloidal stability and suitable surface properties fo...