Visible and red emissive molecular beacons for optical temperature measurements and quality control in diagnostic assays utilizing temperature-dependent amplification reactions

摘要

Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 A degrees C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their application for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA sequences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 A degrees C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 A degrees C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 A degrees C.