(248d) Selecting Optimal Flow Regimes for Rapid Microscale PCR Using Natural Convection

The lack of rapid, affordable, and easy to use medical diagnostic technologies is one of the most critical issues confronting global public health, particularly in resource-limited settings where dedicated laboratory facilities do not exist. Many assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But a major challenge to the development of instrumentation to meet these needs is the highly inefficient design of conventional PCR thermocycling hardware that is slow, expensive, and consumes considerable electrical power to repeatedly heat and cool the reagent mixture. Natural convection has emerged as a promising alternative thermocycling approach that has the potential to address these needs using an inherently simple design (similar in principle to a lava lamp) that consumes minimal power and is well-suited for use in portable applications.

One of the challenges facing design of convective thermocyclers, particularly at the micro-scale, is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time. Here we describe results of a new effort to probe the full 3-D velocity and temperature distributions in miniaturized convective thermocyclers. This analysis is challenging because the thermal and geometric conditions of convective PCR can produce flow profiles exhibiting characteristics that can range from laminar, to transitional, to turbulent. Here, we highlight two reactor geometries that exhibit very different flow characteristics ranging from stable closed circulatory loops extending between the two temperature extremes to more complex paths that continually evolve over time and provide increased exposure to intermediate temperatures.

A surprising and unexpected result of this analysis is the discovery of a subset of complex flow trajectories that appear to be highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone--the rate limiting step of PCR. This hypothesis is confirmed by experiments that show PCR amplification is achievable in under 10 minutes in reactors designed to generate these flow conditions. Our results suggest that by selecting the optimal range of flow trajectories, it is possible to achieve orders of magnitude enhancements in cycling time (~ 20 s per cycle), a capability that is an inherent feature of the underlying physics of convective flow.