Microfluidic Tools for DNA Analysis - DNA Nanotechnology: From Structure to Function - page 115

hydrophobic octadecyltrichlorosilane inside the well [ 16 ]. They also showed the
applicability of real-time reverse transcription PCR reactions with hundreds of
nanoliters. To prevent the evaporation of aqueous solution and potential cross-
contamination among individual wells, both works covered the entire area with
mineral oil before thermal cycling.
The nature of micro-well is a separate space in which a certain reaction takes
place. This separation can also be realized by the droplet technique that can confine
the reaction inside an emulsion of droplets in an immiscible phase of liquid (often
hydrophobic). Microfluidic generation and manipulation of emulsions in spatially
defined arrays enabled high-throughput and multiplexed nucleic acid amplifications.
Droplet-based microfluidics creates discrete volumes with the use of immiscible
phases. Surfactants can play a crucial role for stabilizing droplets during the
formation and reaction process [ 17 ]. Single template molecule in 10 nL volume
produces the same concentration of product produced by 10 3

starting template in

a10 L volume, which was used for PCR of single template DNA in the reduced

volume [ 18 ]. Droplets can be made into small volumes, so researchers have been
exploring convenient and reliable droplet formation techniques.
Emulsion PCR in droplets could reduce cross-contamination between droplets
and enable a fast amplification with rapid thermal cycling. One/two/three dimen-
sional, ordered or unordered droplets have been constructed on many different
types of miniaturized devices. Faris and coworkers printed water-in-oil droplets
on the hydrophobic surface of an unmodified normal polystyrene Petri dish in an
array format and covered the PCR droplets array with mineral oil [ 7 ]. Nakano
and coworkers showed a strategy by which the numerous water-in-oil droplets
were formed in bulk oil phase in a tube [ 19 ]. In this study, PCR mixture as the
aqueous phase was simply added into a silicone oil while stirring with a magnetic
bar. An integrated microfluidic platform performing droplet PCR requires elaborate
chip design such that droplets can pass through different temperature zones within
the device. Viovy and coworkers presented a fully automated system performing
continuous sampling, reagent mixing, and PCR in droplets transported in immiscible
oil inside a Teflon capillary that was wrapped 35 times around a heating cylinder
that is divided in three temperature zones [ 20 ]. The ingenious design based on
three-way pinch valves enabled a consecutive injection and downstream analysis
out of the capillary (Fig. 7.3 a). They paid particular attention to surface treatment
for the inner wall of tubing and wells, which ensures a cross-contamination free
amplification among droplets. The droplets are formed in the capillary by a series
of cyclic operations including vertical and lateral movement of the aspirating tip
(Fig. 7.3 b). This movement makes the tip subject to an aqueous solution (containing
PCR reactants) and carrier oil, sequentially, resulting in a one-dimensional droplet
array separated by immiscible oil. They demonstrated the absence of drop-to-drop
contamination.
Toward droplet generation with higher efficiency, microfluidic channels with
T-junction configuration (one channel is introduced with one fluid, and the other
channel is introduced with another immiscible fluid) or flow-focusing configuration
(one middle channel is introduced with a fluid, and the other two outside channels