Abstract
The development of microscale analytical techniques has created an increasing demand for reliable and accurate heating at the microscale. Here, we present a novel method for calibrating the temperature of microdroplets using quenched, fluorescently labeled DNA oligomers. Upon melting, the 3' fluorophore of the reporter oligomer separates from the 5' quencher of its reverse complement, creating a fluorescent signal recorded as a melting curve. The melting temperature for a given oligomer is determined with a conventional quantitative polymerase chain reaction (qPCR) instrument and used to calibrate the temperature within a microdroplet, with identical buffer concentrations, heated with an infrared laser. Since significant premelt fluorescence prevents the use of a conventional (single-term) sigmoid or logistic function to describe the melting curve, we present a three-term sigmoid model that provides a very good match to the asymmetric fluorescence melting curve with premelting. Using mixtures of three oligomers of different lengths, we fit multiple three-term sigmoids to obtain precise comparison of the microscale and macroscale fluorescence melting curves using "extrapolated two-state" melting temperatures.
Highlights
The advent of microscale analytical technologies promises new advances in detection and quantitation in a variety of fields, from medicine to environmental monitoring to food safety [1,2,3]
To provide accurate thermometry for infrared laser-assisted heating in microdroplet PCR [11,12,13], we have developed a temperature calibration method that relies on the melting of contact-quenched fluorescently labeled DNA oligomers
Because premelting of DNA oligomers prevents modeling the fluorescence melting curve as a conventional sigmoid that applies to a two-state system, we have developed a model based on a three-term sigmoid that allows determination of precise melting temperatures from a fluorescence melting curve using curve fitting even when the premelting fluorescence is quite large
Summary
The advent of microscale analytical technologies promises new advances in detection and quantitation in a variety of fields, from medicine to environmental monitoring to food safety [1,2,3]. As greater reproducibility and sensitivity are required for commercialization, there is a greater demand for more accurate and efficient methods of temperature control Fulfilling this need is a non-trivial task, as many traditional techniques are incompatible with the confined spatial requirements of microfluidics [4, 5]. Contact heating methods typically rely on resistive Joule heating or other solid-state elements to provide heating combined with thermometry feedback control. These are typically limited to integrated “lab-on-a-chip” microfluidic technologies, where the critical elements are integrated into the chip architecture [6, 8,9,10]. Non-contact optical methods of temperature control are inherently more flexible and rely on radiation for heat delivery [7, 11,12,13,14,15,16]
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