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

Area of Science:

• Biotechnology
• Molecular Biology
• Biophysics

Background:

• Accurate temperature control is critical for biological and chemical reactions in diagnostic, pharmaceutical, and chemical industries.
• Microlitre-scale reactions, common in real-time PCR and differential scanning fluorometry, present challenges for precise temperature monitoring.
• Existing methods may lack the sensitivity or applicability for diverse biological and chemical environments.

Purpose of the Study:

• To develop a simple, inexpensive, and accurate temperature biosensor for microlitre-scale reactions.
• To quantify relative temperature changes and identify discrepancies across a broad temperature range.
• To enable precise temperature measurements in various biological buffers and pH conditions, including in vitro and in vivo applications.

Main Methods:

• Development of a ratiometric dual fluorescent protein temperature biosensor (DFPTB).
• Utilized a combination of cycle three green fluorescent protein and monomeric red fluorescent protein for temperature sensing.
• Tested the biosensor across a temperature range of 4-70 °C in various buffer conditions and pH levels.

Main Results:

• The DFPTB accurately quantified relative temperature changes from 4-70 °C.
• Maximal sensitivity of 6.7% °C^-1 and precision of 0.1 °C were achieved between 25-42 °C in phosphate-buffered saline (pH 7.2).
• The biosensor demonstrated good sensitivity across a pH range of 4.8 to 9.1 and in various biological buffers.

Conclusions:

• The developed DFPTB is a simple, inexpensive, and effective tool for precise temperature monitoring in microlitre-scale reactions.
• Its versatility in different buffer conditions and pH levels makes it suitable for diverse applications.
• The biosensor's potential for both in vitro and in vivo use opens new avenues for topologically precise temperature measurements.