High dynamic range digital assay enabled by dual volume centrifugal step emulsification

Inhalt:
We implement dual volume centrifugal step emulsification on a single chip to extend the dynamic range of digital assays. Compared to published single volume approaches, the range between the lower detection limit and the upper limit of quantification increases by two orders of magnitude. In comparison to existing multi volume approaches, the dual volume centrifugal step emulsification requires neither complex manufacturing nor specialized equipment. Sample metering into two sub-volumes, droplet generation and alignment of the droplets in two separate monolayers are performed automatically by microfluidic design. Digital quantification is demonstrated by an exemplary droplet digital loop mediated isothermal amplification (ddLAMP). Within 5 minutes, the reaction mix is split into sub volumes of 10.5 mul and 2.5 mul, and 2,5k and 176k droplets are generated with diameters of 31.6 ± 1.4 µm and 213.9 ± 7.5 µm, respectively. After 30 minutes incubation, quantification over 5 log steps is demonstrated with a linearity of R2 ≥ 0.992.