Virtual reaction chambers as a tool for polymerase chain reaction and protein thermal shift assays

摘要

IIn the frame of this thesis Virtual reaction chambers (VRCs) were analysed as a tool for polymerase chain reaction and protein analysis. VRCs are digital microfluidic reactors, consisting of a aqueous sample volume (100nL - 300 nL) encapsulated by an oil droplet ( 1microL). Firstly a portable system for the temperature manipulation and fluorescence measurement of VRCs was constructed for this thesis. Subsequently the device was used to perform polymerase chain reactions (PCR) from DNA as well as reverse transcription PCR from RNA. Furthermore two methods of multiplexed PCR were developed, one of which specifcally exploits the advantages of VRCs. Thus it can be concluded that virtual reaction chambers are a suitable tool for polymerase chain reaction of DNA and RNA especially for point-of-care applications. Furthermore a mathematical model based on chain growth was constructed to describe the PCR, with particular focus on the extension step. Additionally the heating device and fluorescence measurement were used to measure the thermal stability of proteins. Here advantage was taken of the fact that VRCs are easily superheatable. Thus proteins still stable at temperatures above 100°C can be measured using this system. This and the low sample consumption makes VRCs an ideal tool for screening purposes in protein analysis.