Real-Time Temperature Sensing Using a Ratiometric Dual Fluorescent Protein Biosensor

Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development of a simple, inexpensive ratiometric dual fluorescent protein temperature biosensor (DFPTB). A combination of cycle three green fluorescent protein and a monomeric red fluorescent protein enabled the quantification of relative temperature changes and the identification of temperature discrepancies across a wide temperature range of 4–70 °C. The maximal sensitivity of 6.7% °C(−1) and precision of 0.1 °C were achieved in a biologically relevant temperature range of 25–42 °C in standard phosphate-buffered saline conditions at a pH of 7.2. Good temperature sensitivity was achieved in a variety of biological buffers and pH ranging from 4.8 to 9.1. The DFPTB can be used in either purified or mixed bacteria-encapsulated formats, paving the way for in vitro and in vivo applications for topologically precise temperature measurements.