Thermal Optimization for Enabling PCR Compatibility in Acoustic MICROPLATE SYSTEMS

The Polymerase Chain Reaction (PCR) is a principal technique in molecular biology that relies on precise thermocycling to amplify specific DNA sequences. Echo-qualified acoustic microplates, designed with flat-bottom wells to allow acoustic dispensing, offer limited thermal contact area compared to traditional PCR microplates, which have deeper well profiles and larger surface contact with heating blocks. This geometric constraint contributes to poor heat transfer, leading to deviations in enzyme activity, reduced specificity, and diminished amplification efficiency— posing a significant constraint when utilizing those microplates for PCR thermocycling. To overcome this obstacle and make the Echo microplate compatible for PCR thermocyclers, five different thermal configurations were systematically developed and evaluated. These configurations consisted of the following:: (1) the baseline air gap with no interface, (2) a graphite-only thermal interface to improve conduction, (3) a modified heating block to enhance heat transfer and transient response, (4) a graphite setup augmented with an overshoot and undershoot thermal control algorithm, and (5) a nano-graphite thermal adaptor coated with nanodiamond and hexagonal boron nitride (h-BN) for advanced thermal management.

Each design was assessed based on ramp rate, heat uniformity, and structural stability. The preliminary results showed that the air gap configuration yielded the weakest performance, with a ramp rate of ~0.31°C/s, temperature deviation of ±1.2°C, and structural deformation up to ~0.50 mm.

The graphite interface moderately improved these metrics, achieving a ramp rate of approximately 0.42°C/s and a deviation of no more than 0.5°C. The modified heating block improved heat transfer, achieving a ramp rate of ~0.47 °C/s, ±0.6 °C deviation, and ~0.18 mm deformation due to better thermal contact. However, it is costly (~$549 USD) and may not be compatible with all PCR platforms. Installation and calibration require hardware-level modification, making it less adaptable in flexible or high-throughput workflows.

Incorporating the overshoot and undershoot algorithm with the graphite configuration allowed better tracking and prediction of thermal behavior across cycles, reaching a similar ramp rate ~0.42 °C/s. By assessing temperature transients using the control strategy, fast thermal transitions were achieved. However, a rapid increase in temperature, especially in the denaturation stage, can negatively impact on the preservation of enzyme activity. For instance, Taq DNA polymerase, frequently used in PCR, retains its optimal functional activity at a range of 95 °C for about 40 minutes only, whereas the time decreases to 5–6 minutes at 97.5 °C, dramatically reducing enzyme activity and loss of amplification efficiency. Also, the activity of psychrophilic DNA polymerases and mesophilic recombinant DNA polymerase is prone to error at higher temperatures where their performance paradoxically declines. Furthermore, overshoot during the annealing phase will create mismatches or off-target amplification which compromise qPCR reliability. These findings highlight that while overshoot/undershoot algorithms are valuable predictive tools, they must be precisely calibrated based on the thermal sensitivity of the enzymes involved.

Eventually, the nano-graphite thermal adaptor outperformed all previous configurations, achieving the highest ramp rate (~1.37 C/s), the lowest temperature deviation (±0.3°C), and minimal structural deformation (~0.05 mm). This configuration provided the most reliable and reproducible conditions for PCR.

This research demonstrated a successful adaptation of Echo microplates to thermally demanding PCR protocols through engineered interface solutions. It outlines both the predictive potential of control-based algorithms and the practical advantages of nano-enhanced thermal adapters. Taking together, these findings illustrate the way in which molecular diagnostics can be improved through advanced material design and simulation-driven thermal optimization.