Beschreibung:
Zusammenfassung: Medical and environmental diagnostics often rely on absolute quantification of nucleic acids using digital nucleic acid amplification techniques. In recent years, several major companies have entered the digital amplification market and the number of applications has greatly increased. However, the existing digital amplification systems suffer from a low degree of integration and complicated handling steps. This thesis introduces centrifugal step emulsification as a new droplet generation method enabling droplet generation on a centrifugal microfluidic platform. In the generated droplets three digital nucleic acid amplification techniques are performed (polymerase chain reaction (PCR), recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)). The ability to produce large numbers of monodisperse compartments in the picoliter to nanoliter range is a key requirement for digital nucleic acid amplification. Centrifugal step emulsification offered robust and monodisperse droplet generation (coefficient of variation 1.4–2.2%) on a centrifugal microfluidic setup with only one flowing phase. The droplet diameter could be tuned in the range of 68–220 µm resulting in a droplet volume of 164 pl to 5.6 nl. The droplet volume was independent of rotational frequency in the region of 5–50 Hz. Moreover, centrifugal step emulsification allowed to produce water-in-oil-emulsions with an internal volume fraction of 97.2%. That is to the best of my knowledge the highest ratio reported so far for a microfluidic system. Parallelization allowed for droplet generation rates of up to 3.7 kHz, emulsifying 20 µl in less than 15 s.