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.