Nanostructured micro-electrode arrays for electrophysiological measurements

Abstract

Elektrokemiske målinger og mling af membranpotentialer har afgrende betydning for forskning i den elektriske aktivitet af biologiske celler. Til dette forml bruger man traditionelt skarpe (i mikrometer-klassen) glaspipetter, der kan trnge ind i cellen og frer strm via en indvendig metaltrd eller strmfrende saltoplsning, hvilket gr det muligt at udfre intracellulre og ekstracellulre mlinger, samt mlinger af ionkanaler. Denne metode er dog upraktisk ved anvendelser, der krver flerkanal-mling. I stedet for, bruges matricer med mange flade metalelektroder, hvilket desvrre kun muliggr udfrelse af ekstracellulre mlinger. Med det forml at gre intracellulre flerkanal-mlinger mulige, arbejder flere forskergrupper mlrettet p at udvikle matricer med tredimensionale elektroder, der vil vre i stand til at penetrere cellemembranen. P trods af, at i den seneste tid er der blevet offentliggjort nogle prototyper af sdanne systemer, er det fortsat et ubesvaret sprgsml, hvorvidt de tredimensionale elektrodestrukturer i virkeligheden er i stand til at trnge ind gennem cellemembraner. I denne afhandling har jeg tilstrbt at gribe problemet an fra to sider. For det frste, har jeg arbejdet med udvikling af teknologi for fremstilling af tredimensionale elektroder, med fokus p parametre ssom ledningsevne, biokompatibilitet samt produktionslnsomhed. For det andet, har jeg arbejdet p en plidelig afbildningsmetode, der ville tillade en direkte visning af vekselvirkningen mellem cellemembranen og den tredimensionale nanostruktur. Som resultat af dette arbejde er der opfundet en nyskabende metode for maskels mnsterkontrol af kulstof-nanorr (CNT) skove, fremstillet og gennemtestet udstyr med multi-elektrode matricer med silicium nanotrde, samt udviklet en plidelig FIB-SEM afbildningsmetode, der tillader at skabe tredimensionale billeder af vekselvirkningen mellem nanotrde eller nanorr og biologiske celler.Potential and electrochemical measurements of biological cell electroactivity is crucial in cell biology research. The traditional technique with a micrometer-sharp glass pipette equipped with a metal wire within its core or electrically conductive saline solution allows extracellular, ion-channel and intracellular measurements. In applications that require multichannel measurements, this approach is, however, impractical and planar arrays of metal electrodes are usually employed. Yet, with planar geometry, they allow extracellular measurements only. Several approaches to developing functional three-dimensional electrode arrays with features able to penetrate cell membrane are currently investigated by various groups. While a number of experimental setups have been recently developed, the question remains whether the nanostructure is in fact penetrating the cellular membrane, and if the measurements are indeed intracellular. In my thesis, I approach the problem from two angles. Firstly, I worked on the development of stable and functional three-dimensional electrodes with focus on their electric connectivity, insulation, cell-penetration ability and investigated their electrochemical performance, biocompatibility, and cost-effectiveness of the fabrication. Secondly, I worked on a reliable imaging method that would be able to directly envision nanostructure-cell membrane interface. As a result, a novel maskless patterning method of CNT forests was invented, devices with multichannel arrays of electrodes with silicon nanowires were fabricated and tested, and a reliable FIBSEM method providing three-dimensional images of nanowire- and nanotube-cell interaction and membrane penetration was developed