Microfluidic Tools for DNA Analysis - DNA Nanotechnology: From Structure to Function - page 126

7.2.4 Toward Simplicity

One result of the recent development in the field of TAS is that the chip and the

main functional structures for analysis can be made very small, but external supplies
are still bulky and complicated. In particular, the growing needs on point-of-care
testing (POCT) or the on-site detection for resource-poor settings call for the low-
cost and easy-to-use microfluidic chips without the loss of the “sample-to-answer”
capability.
The complexity of an analytical microfluidic chip is primarily determined by the
fundamental mechanisms of a reaction. For PCR, all reactants including template,
primers, dNTPs (deoxyribonucleotide triphosphates), polymerase, and salts can be
premixed into a stand-alone tube. It should be noted that this commonplace in
PCR may not be so common for isothermal amplifications. Many amplification
technologies termed with “isothermal” are not truly isothermal during the whole
process, such that the structure of chips integrated with isothermal amplifications
is not simpler than that of chips integrated with PCR. Many kinds of isothermal
amplifications, such as strand displacement amplification (SDA), nucleic acid
sequence-based amplification (NASBA), rolling cycle amplification (RCA), single
primer isothermal amplification (SPIA), or nicking endonuclease-mediated ampli-
fication (NEMA), require initial denaturation at high temperature (e.g., 95 ı C) or
require different temperatures at different reaction steps (e.g., signal amplification
RCA [ 55 ]). The so-called “different reaction steps” reflect that some kinds of
isothermal technologies do not allow for the all-in-one premixing operation, i.e.,
one or more components must be added into the reaction system in order. Surveying
existing reports, only loop-mediated isothermal amplification (LAMP) [ 56 ], smart
amplification process version 2 (SMAP 2) modified from LAMP [ 57 ], helicase-
dependent amplification (HDA) [ 58 ], and recombinase polymerase amplification
(RPA) [ 59 ] enabled genuine isothermal process under single temperature, and all
components necessary for reactions can be premixed entirely. LAMP or SMAP
2 only need one enzyme, which intrinsically avoids the disruption of coordinated
catalytic activity of multiple enzymes [ 60 ]. We think that the current preferential
choice for an as simple on-chip DNA amplification as possible is LAMP (SMAP
2 is highly proprietary and still under fundamental investigations regarding its
performance [ 61 ]), based on our comparison herein and existing extensive studies
by researchers.
LAMP is not only isothermal, fast, and specific but also highly sensitive and
robust so that it does not require the extracted DNA to be highly purified or
denaturized [ 62 , 63 ]. But the real cost for performing LAMP per each reaction is
still high, compared to PCR, which results from the reagents, consumables, and
the specialized instrument. We miniaturized the traditional LAMP into parallel
microfluidic channels and embedded cost-effective optical fibers with the chip [ 64 ].
We validated that the optical fibers can monitor the turbidity in real time instead of
using the commercially available but quite expensive turbidimeter.