(B-78) Visual Time-Temperature Indicators of Biospecimen Exposure to Thawed Conditions

Introduction: Many biological analytes of interest to biomedical research can show limited stability when exposed to thawed or partially frozen conditions. Although specific guidelines are available for proper sample handling and storage of biospecimens, improprieties and inconsistencies can occur unwittingly. This can directly lead to costly false leads in biomedical research. Implementing a system to track the exposure of biospecimens to thawed conditions or improper storage is necessary to avoid using compromised samples. Unfortunately, there are very few tools and not yet an accepted approach for evidenced-based biospecimen quality control.

Toward this end, we are developing an inexpensive visual time-temperature indicator (TTI) that can be used with biospecimens at the individual aliquot level to track exposure to thawed or non-ideal frozen conditions. The proposed TTIs are based on the kinetic control of the autocatalytic permanganate/oxalate redox reaction, which goes from an intense pink solution to a colorless solution. The kinetic control of the reactions allows for the design of TTIs to run for specific time-temperature intervals, depending on specific handling or storage requirements.


Materials and Methods: A MATLAB script to simulate the reaction kinetics was developed. The concentrations of each chemical involved in the reaction are used as input in the MATLAB script, which numerically solves the multi-component differential rate equations and then displays the concentrations of the components over time. The reaction is run experimentally by mixing the solutions at specific temperatures and monitoring the absorbance at 525 nm over time. For experiments at room temperature (25 °C), a plate reader is used to monitor the absorbance of the solution. For temperatures below 25 °C, a spectrophotometer combined with a chilled circulating bath system is used to monitor the absorbance of the solution. Eutectic compositions of LiClO₄, NaClO₄, and Mg(ClO₄)₂ solutions are used as antifreeze solutions in the development of the TTIs.


Results, Discussion and Conclusions: In this work, we manipulate the components of the autocatalytic permanganate/oxalate redox reaction to design TTIs for specific time-temperature intervals, depending on specific handling or storage requirements. With the help of a MATLAB script, the reaction kinetics can be simulated facilitating the design of the TTIs for specific times at room temperature. The aqueous-based reaction can be adjusted to run between a few seconds and 2 h at room temperature, matching with simulated data. Additionally, we have experimentally demonstrated that the reaction time exhibits a non-linear dependency on temperature.

Furthermore, eutectic perchlorate solutions are used for depressing the system’s freezing and melting point (mp), allowing TTIs to remain active at subzero temperatures warmer than the proper biospecimen storage and handling guidelines indicate, e.g., when storage at -20 °C or -40 °C is unacceptable. We have characterized LiClO₄ (mp: -18 °C), NaClO₄ (mp: -37 °C), and Mg(ClO₄)₂ (mp: -67 °C) as antifreeze solutions, showing the potential of keeping the reaction active at subzero temperatures, while maintaining kinetic control. Antifreeze-based TTIs can be designed to run for specific time at temperatures above their respective freezing point. In this way, TTIs can be kept at typical storage freezer temperatures (-20 °C, -40 °C, and -80 °C) without any color change until exposed to warmer temperatures. This is helpful, for example, to track potential thawed state exposure over the entire life of any archived biospecimen.

In conclusion, we have demonstrated the capability to control the kinetics of a permanganate/oxalate redox reaction not only at room temperature but also at subzero temperatures, facilitating the design of TTIs for specific applications. We expect that the implementation of the TTIs currently being designed can contribute to avoiding the use of unstable biospecimens, helping to minimize false discoveries in biomedical and clinical research.