Revealing the impact of temperature on electrochemical, DNA-based sensors to correct signaling fluctuations

Developing convenient and easy-to-use analysis tools for real-world applications requires sensing approaches that enable rapid, reagentless, and in situ quantitation of diverse molecular targets. Electrochemical, DNA-based (E-DNA) sensors meet these needs by utilizing conformational changes in electrode-bound, redox reporter-modified DNA probes upon target binding. The recognition event alters the electron transfer rate from the redox reporter, which can be monitored using square wave voltammetry by synchronizing the excitation frequency with the charge transfer rate. However, the kinetic nature of the surface-bound sensing process makes signaling strongly temperature-dependent, an aspect that has been widely overlooked and that restricts the application of E-DNA sensors to temperature-controlled environments. Here, we explore the relationship between electrochemical signal, square wave frequency, and temperature across different DNA constructs. Doing so, we identified the architectures most susceptible to temperature-induced signal fluctuations and developed two straightforward correction strategies. These approaches are particularly effective for electrochemical, aptamer-based (E-AB) sensors, enabling accurate and stable measurements over a wide temperature range, from 22 ºC to 37 ºC. By eliminating the significant influence of temperature on signaling, we broaden the applicability of E-DNA sensors, enhancing their performance for point-of-care testing, continuous molecular monitoring, and other real-world scenarios where temperature fluctuations are unavoidable.