Digital Microfluidics-Powered Real-Time Monitoring of Isothermal DNA Amplification of Cancer Biomarker

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.

Combining sensors and actuators with electrowetting-on-dielectric (EWOD): advanced digital microfluidic systems for biomedical applications

“…Reproduced with permission from ref. 12. including cell culture, 87 polymerase chain reaction (PCR), 35,36,44,[88][89][90][91][92][93][94] isothermal amplification, 14,33,[95][96][97][98][99] and immunoassays. 100 Temperature control units are typically composed of microheaters/coolers, thermal sensors, and microcontrollers to form a close-looped control.…”

“…Hence, temperature control abilities are essential for DMF systems in integrated biomedical applications. So far, numerous temperature-sensitive purposes performed on EWOD platforms are present, including cell culture, 87 polymerase chain reaction (PCR), 35,36,44,88–94 isothermal amplification, 14,33,95–99 and immunoassays. 100…”

A Short Review on Miniaturized Biosensors for the Detection of Nucleic Acid Biomarkers

“…Coelho et al [ 115 ] reported a digital microfluidics (DMF) technology made explicitly for LAMP of nucleic acid; real-time quantification was accomplished to screen the cancer biomarker c-Myc, which is linked to 45% of all human malignancies. Herein, 90 pg of the target nucleic acid (0.5 ng/L) was successfully amplified in less than an hour after completely modifying the sample and chemicals on this proposed platform.…”

“… ( A ) Schematic representation of the components, including digitally barcoded nanoparticles, microfluidic cartridge, and an integrated instrument for the detection of molecular biomarkers [ 112 ] ( B ) Working principle of the nucleic acid-based nanotweezer sensor [ 113 ] ( C ) MTase activity uncovering by padlock probe with RCA technique [ 114 ] ( D ) Multi-view of the DMF device and accumulation [ 115 ]. …”

Microfluidic SERS devices: brightening the future of bioanalysis

“…Since their invention, microfluidics systems have attracted attention in a variety of biodiagnostic methods including PCR [ 194 ], LAMP [ 195 ], chemiluminescence-based assays [ 196 ], and immunosensors [ 24 , 197 – 200 ] platforms. Microfluidic immunosensors can be much more efficient and rapid than conventional immunoassay methods, due to the increase of surface-to-volume ratio that maximises mass transport in immunoreactions which, in turn, leads to a more rapid analysis [ 24 , 201 ].…”