Kinetically Tunable, Subzero-Active, Visual Time-Temperature Indicators Based on the Permanganate-Oxalate Reaction

Biological products and specimens often require consistent ultracold storage to preserve their integrity. Existing time-temperature indicators (TTIs) are inadequate for monitoring ultracold conditions at the individual aliquot level. We adapted the autocatalytic permanganate-oxalate reaction to create visual TTIs functional below 0 °C. Using eutectic compositions of LiClO₄, NaClO₄, and Mg(ClO₄)₂, we depressed the melting points of the reaction mixtures to -18 °C, -37 °C, and -67 °C, respectively. The incorporation of perchlorate salts as antifreeze systems did not derail the kinetic behavior of the permanganate-oxalate reaction and allowed the reactions to pause below their melting points. Here, we developed and characterized eight customized TTIs, running from five minutes at 25 °C to 7 days at -20 °C. Temperature sensitivity was consistent with Arrhenius behavior (i.e., exponential increases in run time with linear decreases in temperature). The TTIs exhibited good accuracy and reproducibility, with within-batch and between-batch run-time precision at the targeted temperatures of ≤ 4.8% CV and ≤ 7.5% CV, respectively. The average absorbance vs. time trajectories, expressed as RMSD %CVs, were 4.5% for intra-batch and 10.4% for inter-batch runs. Indicators withstood multiple freeze/thaw cycles or extended pre-freezing periods with minimal impact on reaction kinetics. Once activated and stored below their melting points, TTIs maintained color intensity for at least 12 months. This work establishes the permanganate-oxalate system in eutectic perchlorate-based antifreeze solutions as a simple, inexpensive approach for ultracold-active TTIs, offering customizable kinetics and robust performance. The described TTIs can serve to improve quality monitoring of biologicals and biospecimens during ultracold storage and handling.