High-Resolution Melting Curve Analysis with Linear Temperature Gradient in Digital Microfluidics Platform

Precise temperature control and measurement are crucial for in vitro nucleic acid denaturation, especially in melting curve analysis (MCA). However, the inherent cumbersome multi-step calibration process required to obtain an accurate spatial temperature gradient can be a major limitation for the study of MCA on existing microfluidic chips. This work proposes to obtain a highly linear temperature gradient with a functional substrate for achieving high-resolution MCA on a digital microfluidics (DMF) platform. Taking advantage of the high thermal conductivity of aluminum, we employ a thin trapezoidal aluminum plate as the DMF substrate to compensate for the uneven heat transfer caused by temperature differences at different substrate positions and the ambient environment. We further pattern a banded superhydrophilic thread on the superhydrophobic surface to passively achieve a liquid thread by driving the mother droplet through the electrowetting on dielectric (EWOD) effect. With a heater and a cooler positioned at the opposite ends of the substrate, we can create a highly linear (simulated R 2 = 1 and experimental R 2 = 0.9998) temperature gradient from 40°C to 90°C across the liquid thread, requiring only temperature calibration at the two end points. The elimination of laborious temperature calibration steps can effectively enable rapid, continuous and reliable nucleic acid amplification as well as MCA on DMF. The high temperature accuracy also ensures simultaneous multiple hybrid DNA detection for rapid one-time discrimination of single nucleotide mutations in KRAS gene targets. This innovation underscores the DMF platform's potential in the realms of biomedical research and clinical diagnostics.