Achievement of 15-Minute Adaptive PCR Benchmark with 1370 nm Laser Heating.

In low-resource and point-of-care settings, traditional PCR often faces challenges of poor sample preparation, adverse environmental conditions, and long assay times. We have previously described a laboratory-based instrument to achieve "adaptive" PCR, a PCR thermocycling control system that replaces preset cycling times and temperatures with the optical monitoring of added L-DNA stereoisomers matching the sequences of the reaction primers and target. These L-DNA biosensors directly monitor DNA hybridization, compensating for ambient environmental conditions and poor sample preparation. This report describes instrument simplifications and a comparative evaluation of both direct photothermal and plasmonic laser heating to reduce the assay time to 15 min. Instrument performance was assessed using a split sample design to compare reaction performances of 1370 and 808 nm adaptive PCR heating modalities to a standard PCR instrument. Both the novel 1370 nm direct heating and the 808 nm plasmonic method achieved target amplification similar to the traditional PCR system within 15 min. However, a major disadvantage of 808 nm heating was nanorod optical interference that reduced the fluorescence signal from PCR probes and optical cycling components. Further characterization of the 1370 nm direct heating method found comparable limits of detection of 10 copies/µL and reaction efficiencies of approximately 2 for both the 1370 nm system and the traditional PCR instrument. These results suggest that a field-deployable PCR instrument design incorporating both adaptive optical control and 1370 nm laser heating can achieve 15 min sample assay times without sacrificing analytical sensitivity.