(174ao) Towards Rapid Real-Time DNA Analysis Using Quantitative Fluorescence in Convective PCR

The demand for rapid, on-site diagnostics and DNA research has recently escalated. Microscale Rayleigh-Benard convective polymerase chain reaction (cPCR) holds promise to address these needs and provide real-time DNA analysis. In its current implementation, cPCR is performed in millimeter-scale cylindrical chambers of uniform circular cross-section subjected to fixed temperatures at the top (~58 °C) and bottom (~96 °C), ensuring an appropriate temperature gradient for the DNA molecules to go through the necessary sequence of denaturation, annealing, and extension for DNA amplification. However, post-processing steps like gel electrophoresis are still required to evaluate the success of the reaction. As an alternative, quantitative fluorescence measurements within cPCR chambers could enable real-time tracking of the progress of the reaction while offering detailed information about the spatial distribution of reaction products. For instance, such measurements can reveal profiles of double-stranded DNA (dsDNA) and its helicity state along the height of the chamber, providing access to sensitive DNA melting curve analysis using cPCR. Here we take initial steps towards this goal, developing methodologies to fabricate a cPCR chamber fully compatible with detailed quantitative fluorescence measurements. First, we note that precise and accurate chamber dimensions are needed to carry out cPCR, since small changes in the diameter of the chamber can cause important changes in the Rayleigh number, which governs the convective flow pattern of cPCR and its dynamics. To this end, computer numerical control (CNC) fabrication method, combined with surface polishing, was used to develop an optically clear acrylic reaction chamber of precise and reproducible dimensions that did not interfere with the collected fluorescence signal. Second, a non-destructive optical methodology was developed to measure the dimensions of the manufactured chamber diameter (~1.38 mm) with high accuracy. This methodology included ray tracing simulations, determination of a calibration factor using fluorescence beads to account for diffraction uncertainties, and semi-automated image processing and analysis implemented in MATLAB. Accurate diameter measurements allowed us to account for any offsets needed to produce chambers of the desired dimensions using CNC. Obtaining real-time fluorescence profiles during cPCR opens the path to cost-effective PCR diagnostics and, potentially, single-site mutation detection if sufficient sensitivity is achieved. Our research paves the way for a new era of accessible, sensitive, and efficient PCR technology using cPCR, transforming global healthcare and research.