Author:

Abstract:

In this work, we present a miniaturized device that performs uniform droplet generation and fast temperature cycling, for digital droplet Polymerase Chain Reaction (ddPCR). The device was fabricated using deep silicon etch for sculpting the microfluidic structures that were then sealed by anodic bonding with a Pyrex wafer. Droplets of an aqueous solution containing PCR reagents and templates were generated in oil using a simple T-junction. Droplet volume and generation rate were respectively 60 pL and 6400 droplets per second. Volume reproducibility in different experiments was about 5%. A standard two-step Taqman protocol was used for DNA amplification. After each PCR cycle a fluorescent image of the reactor was recorded using a conventional inverted microscope equipped with a CCD camera. The analysis of these images allowed to count dark and fluorescent droplets and to reconstruct the real time fluorescence intensity curve for each droplet. To demonstrate the functionality of the device, human genomic DNA with seven nominal starting concentrations ranging from 20 to 13000 copies per μl was amplified. The determined template concentration was in agreement with the nominal one in the full dynamic range of the system (almost three orders of magnitude). In the current design the dynamic range is limited by the reactor volume ( 3mm x 3mm x 30 m) that can be extended by a factor of hundred by increasing both depth and footprint.