Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection

We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.