Handheld energy-efficient magneto-optical real-time quantitative PCR device for target DNA enrichment and quantification

Abstract

A PCR provides not only valuable genetic information that enables precise differential disease diagnosis but also quantitative data to assess various clinical states. We report the successful integration of novel dual-mode magnetic Fe3O4 nanoclusters that deliver photothermal conversion. The clusters can be excited with pulsed laser light for precision thermal cycle modulation to develop an ultrafast quantitative PCR system. Traditional PCR heats and cools the sample from outside the PCR tube; the heat needs to pass through the heat block, tube wall and transfer to the DNA through water molecules. In contrast, our system uses nanoparticles inside the liquid phase as numerous ‘nanoheaters’; thus the thermal transfer between particles and adjacent water or DNA molecules becomes extremely efficient because of proximity at the molecular level. Moreover, the defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome, a mitochondrial disease, was efficaciously detected. The system has a simple design, is extremely energy efficient and is faster than traditional qPCR. Our finding provides new insight into rapid and accurate quantitative diagnostics for future point-of-care applications. Iron nanoparticles play double duty as ‘nanoheaters’ for DNA amplification and as molecular manipulators in a new point-of-care prototype. Polymerase chain reaction (PCR) devices create multiple copies of small DNA strands for genetic testing, but usually require bulky heating and cooling mechanisms. Dar-Bin Shieh and colleagues from National Cheng Kung University in Taiwan have found that tiny clusters of iron oxide nanocubes can improve heat transfer in PCR devices by entering the liquid phase next to the target DNA. When irradiated by laser light, the iron nanoclusters release heat to precisely modulate DNA temperatures. Furthermore, functionalizing the nanoparticles with nucleotide probes enables them to attach to and extract specific disease-indicating mitochondria using magnetic fields. The portable, battery-powered prototype can wirelessly interface with tablet computers and is twice as efficient at isolating mitochondria as commercial kits. A novel dual-mode magnetic Fe3O4 nanoclusters was developed that exhibit efficient photothermal conversion and superparamagnetism. The particles served as numerous ‘nanoheaters’ in a PCR reaction mixture while enabling effective magnetic-based macromolecular manipulation. The thermal transfer between particles and adjacent water and DNA molecules thus become extremely efficient because of molecular-level proximity and allowed rapid amplification cycle progression. The defective mitochondrial DNA from cybrid cell lines of a patient with chronic progressive external ophthalmoplegia syndrome was efficaciously quantified by fluorescence intensity detection. Our finding provides new insight into rapid and accurate quantitative DNA diagnostics for future point-of-care applications.