The Permanganate-Oxalate Reaction as a Visual Time-Temperature Indicator: Reaction Systems for Use Above 0 °C

Biological molecules are often unstable at non-ideal temperatures, causing product inefficacy or false leads in research. Cost-effective, easily implemented tools are needed to ensure the quality of biological products and specimens requiring cold storage. Here, we developed a series of inexpensive visual time-temperature indicators (TTIs) based on the autocatalytic permanganate/oxalate reaction. This reaction transitions from intense pink to colorless in a non-linear manner, preserving color for an extended period followed by rapid decolorization, making it ideal for TTIs. Through extensive modeling and experimentation, TTIs were formulated that run for predefined periods at temperatures from 0 °C to 25 °C. They exhibited high run-time accuracy and reproducibility, with intra-batch and inter-batch run time precisions of ≤ 4.0% and ≤ 6.8% CV, respectively, at targeted temperatures. Average intra-batch and inter-batch absorbance vs. time trajectories, expressed as RMSD %CVs were 5.1% and 9.6%, respectively. The TTIs displayed temperature sensitivity consistent with Arrhenius behavior, making them suitable for monitoring biomolecular stability during temperature fluctuations. Pre-activation stability tests demonstrated the stability of solution components (excluding activating KMnO₄) for at least two months. Long-term stability studies demonstrated that activated TTIs maintained color intensity when frozen at -80 °C for at least 24 months-though they were not designed to withstand freeze-thaw cycles as their antifreeze-containing counterparts that will be described elsewhere. The permanganate/oxalate reaction provides a platform for creating inexpensive visual TTIs with customizable run times, reproducible kinetics, and appropriate temperature sensitivity. These TTIs have potential applications in monitoring the integrity of biological products and biospecimens during storage and handling.