(134g) Toward Concurrent DNA Amplification and Analysis through Quantitative Fluorescence in Isothermal Convective PCR (cPCR)

Most polymerase chain reaction (PCR) platforms, including conventional and microfluidic systems, require separate post-processing steps like melting curve analysis (MCA) or gel electrophoresis to confirm amplification and identify target sequences, adding cost, complexity and limiting portability. While isothermal convective PCR (cPCR) enables rapid, real-time, low-power DNA amplification via thermally driven (Top: 58 °C, Bottom: 96 °C) 3-dimensional flow in a millimeter-scale cylindrical chamber (8 mm height, 1.38 mm diameter), it still relies on external methods for sequence verification. The lack of compact, fully integrated systems capable of real-time, sequence-specific detection remains a key barrier. To address this, we present a unified approach that enables concurrent DNA amplification and MCA within a single platform, advancing beyond the constraints of both conventional and cPCR technologies. The approach seamlessly combines intrinsic characteristics of MCA (DNA fluorescence as a function of temperature) and cPCR (spatial temperature distribution) to enable quantitative fluorescence measurements along the cPCR chamber’s height that offer access to sensitive MCA data directly within cPCR systems.

Working toward the unified approach, we demonstrated the fabrication of a cPCR chamber specifically designed for detailed quantitative fluorescence measurements, featuring an octagonal geometry with precise and accurate dimensions (95% confidence interval: 1.3833-1.3933 mm) to enable different viewing angles and repeatable performance in cPCR. This achievement addressed a fabrication gap at scales too large for microfabrication yet too small for conventional machining by using precise fluorescent ring deposition in confined cylindrical geometries. Thereafter, experimental steps towards incorporating MCA into cPCR were outlined. First, a specialized, large field-of-view fluorescence imaging setup was developed (9.18 μm/pixels resolution) to capture fluorescence signals generated by asymmetric flow patterns within the chamber from top to bottom in a single image. The imaging system avoided volumetric illumination to minimize out-of-focus noise and collected fluorescence emissions from multiple viewing angles by rotating the chamber, addressing the complexity introduced by 3D flow asymmetries. The optical setup was initially used for fluorescent thermometry, enabling comparison between experimental and CFD-predicted gradients to better understand the temperature profile driving flow patterns and spatial distribution of reaction components in cPCR. Then, experimental fluorescence measurements during cPCR were performed and investigated, aided by key insights from sufficiently detailed CFD simulations of amplicon-specific reaction and fluorescence models, to allow for DNA analysis in ongoing cPCR reaction.

This work presents a biosensing platform for simultaneous DNA amplification and MCA with 0.04 °C average resolution, exceeding conventional systems (0.1 °C), with preliminary analysis indicating a two-thirds reduction in detection time and the potential for mutation-sensitive, portable diagnostics using smartphone cameras and LED excitation.