Thin-film heater technology for probing DNA hybridization kinetics

Abstract

Monitoring of structural changes in biomolecules is crucial for unraveling the mechanisms underlying essential biological functions. Controlled temperature modulation enables precise characterization of the kinetics of processes such as DNA hybridization and melting by allowing dynamic perturbation and observation of molecular transitions. However, existing methods for thermal control in fluorescence microscopy and microfluidic settings are often limited by slow thermal response, high costs, or fabrication complexity. To address this, we developed a tape-based microfluidic device incorporating a thin-film printed circuit board (PCB) heater, providing direct and rapid temperature control within a microfluidic environment compatible with real-time fluorescence microscopy. DNA duplexes and molecular beacons were loaded into dual microchannels, and sinusoidal thermal perturbations ranging from 0.05 to 8 Hz were applied. Continuous fluorescence measurements enabled extraction of phase shifts as a function of modulation frequency, allowing quantitative analysis of DNA strand dissociation and reassociation kinetics. Relaxation times for perfectly matched and single-base mismatched duplexes were determined by identifying the cutoff frequency at a 45◦ phase lag, consistent with firstorder kinetic models. Both frequency-sweep and chirped-modulation experiments produced robust, reproducible results across multiple devices. The accessible design, based on inexpensive materials and straightforward fabrication, supports high-throughput or parallel analysis of biomolecular kinetics. Beyond DNA hybridization studies, this system can be adapted to investigate other thermally sensitive biological reactions, such as protein folding and enzyme catalysis. Overall, this approach provides a practical and versatile tool for probing biomolecular kinetics with high temporal resolution in a microfluidic format.