ABSTRACT
We introduce a digital microfluidics (DMF) platform specifically designed to perform a loop-mediated isothermal amplification (LAMP) of DNA and applied it to a real-time amplification to monitor a cancer biomarker, c-Myc (associated to 40% of all human tumors), using fluorescence microscopy. We demonstrate the full manipulation of the sample and reagents on the DMF platform, resulting in the successful amplification of 90 pg of the target DNA (0.5 ng/µL) in less than one hour. Furthermore, we test the efficiency of an innovative mixing strategy in DMF by employing two mixing methodologies onto the DMF droplets-low frequency AC (alternating current) actuation as well as back-and-forth droplet motion-which allows for improved fluorescence readouts. Fluorophore bleaching effects are minimized through on-chip sample partitioning by DMF processes and sequential droplet irradiation. Finally, LAMP reactions require only 2 µL volume droplets, which represents a 10-fold volume reduction in comparison to benchtop LAMP.
Subject(s)
Microfluidics , Neoplasms , Biomarkers, Tumor , DNA , Fluorescent Dyes , Humans , Microfluidics/methods , Neoplasms/diagnosis , Nucleic Acid Amplification Techniques/methods , Oligonucleotide Array Sequence AnalysisABSTRACT
Digital microfluidics (DMF) arises as the next step in the fast-evolving field of operation platforms for molecular diagnostics. Moreover, isothermal schemes, such as loop-mediated isothermal amplification (LAMP), allow for further simplification of amplification protocols. Integrating DMF with LAMP will be at the core of a new generation of detection devices for effective molecular diagnostics at point-of-care (POC), providing simple, fast, and automated nucleic acid amplification with exceptional integration capabilities. Here, we demonstrate for the first time the role of coupling DMF and LAMP, in a dedicated device that allows straightforward mixing of LAMP reagents and target DNA, as well as optimum temperature control (reaction droplets undergo a temperature variation of just 0.3 °C, for 65 °C at the bottom plate). This device is produced using low-temperature and low-cost production processes, adaptable to disposable and flexible substrates. DMF-LAMP is performed with enhanced sensitivity without compromising reaction efficacy or losing reliability and efficiency, by LAMP-amplifying 0.5 ng/µL of target DNA in just 45 min. Moreover, on-chip LAMP was performed in 1.5 µL, a considerably lower volume than standard bench-top reactions.
