Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD 4811, Australia.
Biosensors (Basel). 2023 Mar 3;13(3):338. doi: 10.3390/bios13030338.
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 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.
准确控制生物和化学反应样品的温度以及仪器校准对于诊断、制药和化学工业至关重要。对于实时 PCR 应用和差示扫描荧光法中常用的微升规模反应，这尤其具有挑战性。在这里，我们描述了一种简单、廉价的比率双荧光蛋白温度生物传感器 (DFPTB) 的开发。循环三代绿色荧光蛋白和单体红色荧光蛋白的组合能够定量相对温度变化，并识别宽温度范围（4-70°C）内的温度差异。在标准磷酸盐缓冲盐条件下，在生物学相关的温度范围 25-42°C 内，在 pH 值为 7.2 时，达到了最大灵敏度 6.7%°C 和精度 0.1°C。在从 pH 值 4.8 到 9.1 的各种生物缓冲液中均能实现良好的温度灵敏度。DFPTB 可用于纯化或混合细菌封装的格式，为体外和体内拓扑精确温度测量的应用铺平了道路。
