A Single-Step Digital Nucleic Acid Amplification Platform by Digital Plasma Separation on a Chip

Abstract

Quantitative nucleic acid detection is the future for precision molecular medicine. However, multiple sample preparation steps and thermal heat cycles of PCR are necessary for nucleic acid (NA) analysis. We developed a single-step isothermal digital NA amplification microfluidic platform by a technique termed digital plasma separation. We invented an innovative biophysical microcliff design that allows the removal of blood cells based on inertia; it can skim plasma into 224 wells (100nl/well) in one-step (>99% separation efficiency for 6μm particles). By separating the blood cells that obstruct optical detection, the microcliff design enabled quantitative DNA detection via an isothermal DNA amplification technique called Recombinase Polymerase Amplification (RPA) in 30 minutes directly from human blood samples. We were able to perform endpoint quantitative digital RPA with a dynamic range of 10∼1000 copies/μl via automatic compartmentalization of the samples. The precise control of fluid flow on chip is accomplished by passive degas-driven flow actuation, which does not require external tubing or pumps, thus making the chip portable. A large amount of sample (∼100μl of blood samples) can be processed. We envision this single-step digital blood plasma separation platform will benefit low cost quantitative molecular diagnostics in both developed and developing countries.