Minimizing Ethanol Carry-Over in Centrifugal Microfluidic Nucleic Acid Extraction by Advanced Bead Handling and Management of Diffusive Mass Transfer

We present three concepts for centrifugal microfluidics reducing ethanol carry-over in magnetic bead-based nucleic acid (NA) extraction. Ethanol carry-over is critical regarding inhibition of downstream NA amplification. We identified two possible carry-over pathways: Liquid co-transport within bead-clusters and vapor diffusion. For the first time, we integrated magnetic bead handling in centrifugal microfluidics at continuous rotation aiming to avoid liquid co-transport within bead-clusters. Consequently, no significant contribution to ethanol carry-over could be assigned anymore to liquid co-transport. Major carry-over was attributed to diffusive transport of ethanol vapor. Countermeasures reduced this from 9.7 % (v/v) to 0.4 % (v/v), below the critical level for inhibition of downstream amplification reactions.