Research review paperPCR microfluidic devices for DNA amplification
Introduction
MEMS technologies are being developed in the semiconductor industry and the characteristic dimensions of those small structures are on the order of 1–1000 μm. Microfluidics devices manufactured by MEMS will represent a central technology in many miniaturized systems used for biological, chemical, and medical applications, whose advances promise to revolutionize many processes of detection of pathogens or environmental pollutants (Chow, 2002, Felton, 2003, Stone and Kim, 2001). Among the microfluidics, the miniaturized PCR instrument (or called PCR microfluidics) has become a very important tool. The PCR process is widely used as a molecular biological tool to replicate DNA, and can create copies of specific fragments of DNA by cycling through three temperature steps. Each temperature cycle can double the DNA, and so 20–35 cycles can produce millions of DNA copies. However, conventional PCR instruments usually achieve a ramping rate of about 1–2 °C/s in the temperature range relevant for PCR, where a complete PCR analysis needs approximately 1–2 h. The resulting lower ramping rate is due to the high thermal capacity of the material of the PCR reaction system, which seemingly cannot meet the need of fast DNA amplification in spite of the fact that the ramping rates may be improved for the current PCR instruments in the market. Fortunately, since the introduction of the first PCR chip (Northrup et al., 1993, Northrup et al., 1995), all kinds of PCR microfluidic technologies have facilitated DNA amplification with much faster rates as the result of smaller thermal capacity and larger heat transfer rate between the PCR sample and temperature-controlled components, which have advantages of small sizes, fast ramping rates, low cost and high integration and so on. Here, we review the microfabrication, design, surface chemistry, temperature and fluid control, detection for PCR amplicons, integration with other functional components, and applications of PCR microfluidics. We begin with discussing the motivation for the development of PCR microfluidical systems. Next, we describe the aspects mentioned earlier in turn. It is noted here that we also emphasize the new substrate materials and the new ways to control the temperature cycling for PCR microfluidics, as well as the novel thermal convection-driven PCR thermocycling.
PCR microfluidics has a large potential for many applications, as will be discussed later. Miniaturization of PCR devices in these application fields leads to many improvements, mainly including decreased cost of fabrication and use; deceased time of DNA amplification; reduced consumption of biological sample necessary for PCR; reduced production of PCR dimer and other nonspecific products; increased portability and integration of the PCR device; acceptable disposal of the PCR reaction vessel; and avoidable cross-talk of the PCR reaction. In addition, large numbers of parallel amplification analyses on a single PCR microfluidic chip can lead to more accurate information and greater understanding necessary for some particular bioassays, which, however, are difficult, unpractical, or even impossible to perform on a macro-scale PCR device. Besides, single molecule PCR can be easily performed in PCR microfluidics, starting with a single-copy sequence in the PCR mixture (Belgrader et al., 2003, Burns et al., 1996, Lagally et al., 2000, Lagally et al., 2001b, Matsubara et al., 2005, Nagai et al., 2001a). Much smaller PCR reaction vessels can increase resolution while reducing the overall size of the PCR device, but effects related to the non-specific adsorption of biological samples to the surfaces of the vessel may become significant as a result of the increased Surface-to-Volume Ratio (SVR) upon miniaturization, which may inhibit PCR amplification. It is noted, here, that miniaturization of the PCR amplificaton is also needed to match microchip Capillary Electrophoresis (CE), which has to be employed to decrease the time required for the analysis of the amplicons to about several minutes or ever shorter times, along with very high sensitivity and the requirement of only 10− 12 or 10− 9 g sample levels. Though the time required for the DNA amplification is longer than that of the CE-chip separation in many cases, it has shown a strong trend to decrease so as to reduce the total time of a DNA analysis. In short, the concept of micro-Total Analysis Systems (“μ-TAS”) has shown the possibility of performing all the steps of a bioassay on a single chip leading to significant advantages in terms of speed, cost and automation, which will ultimately promote the further development of PCR microfluidics.
Let's trace the history of the PCR microfluidics development. About 30 years ago, the first miniaturized gas chromatograph was fabricated on a single silicon wafer at Stanford University (Terry, 1975). However, the response of the scientific community working on PCR (Mullis et al., 1986) research to this first microsilicon device has not been immediate, maybe because of the lack of technological experience with the use of silicon chips. Until 1993, several years after the introduction of PCR, and about a full 20 years after the first silicon gas chromatograph microinstrument, the first silicon-based stationary PCR chip was described by Northrup et al. (1993). Since then, the gate to the microfluidics-based PCR chip had been opened and many research groups began to develop chip-based PCR devices. Presumably on the basis of the fact that the significant concept of μ-TAS was presented in 1990 (Manz et al., 1990) and the CE-chip was developed in the early period (Manz et al., 1992, Harrison et al., 1993), the first PCR-CE integrated microfluidics device was presented in 1996 (Woolley et al., 1996). Although the PCR and CE steps were not performed in a single chip, it led the way to further integration of PCR with other pre- or post-PCR processing on a single silicon or glass chip. In addition, the integration of PCR with DNA microarray hybridization on a monolithic chip also was reported in 2000 (Anderson et al., 2000). Besides, it should be pointed out that flow-through PCR chip microfluidics has attracted great deal of interest and is exhibiting a rapid development, in parallel with stationary chamber PCR microfluidics, since it was introduced for the first time in 1998 (Kopp et al., 1998). In short, PCR microfluidics is expected to show a very strong life force in spite of its very short developing history of about 10 years.
Section snippets
New materials for PCR microfluidics
The development of PCR microfluidics has occurred at an exponential rate. If we have a glance through the literature on PCR microfluidics in the 1990s and currently, we will find that almost all PCR microchambers (or microchannels) were constructed from silicon (Belgrader et al., 1998a, Belgrader et al., 1998b, Belgrader et al., 1999a, Belgrader et al., 1999b, Belgrader et al., 2001, Benett et al., 2000, Bu et al., 2003, Cheng et al., 1996, Cheng et al., 1998, Chaudhari et al., 1998, Cui et
Fabrication of chip PCR microfluidics
It has to be stressed that the fabrication methods are chosen if the substrate material of the PCR microfluidics is given. As a whole, the diversity of fabricating methods available for the PCR microfluidics can be classified into silicon/glass-based and polymer-based microfabricating methods. The former have greatly contributed to the advance in the early work on the PCR microfluidics and until now have grown into the more mature technologies, whereas the latter is continuously developing and
Designs for PCR microfluidics
Since the introduction of the thermostable thermos aquatics (Taq) DNA polymerase as a substitute for the Klenow fragment of Escherichia coli DNA polymerase I, considerable efforts have been made to promote the automation of PCR amplification. Furthermore, the miniaturization of PCR devices can offer an opportunity to improve them further in terms of shorter amplification times, higher sample throughput, and minimum human/world-to-PCR intervention and contamination. Up to now, PCR microfluidics
Temperature kinetics, heating and temperature sensing methods, and simulation for PCR microfluidics
PCR is a typical temperature-controlled and enzyme-catalyzed biochemical reaction system that consists of the periodical repetition of three different temperatures (melting, annealing and extension temperature). Alternatively, only two temperatures can be applied, combining annealing and extension temperature thus further reducing the complexity of the thermal cycling profile and increasing the speed and efficiency of the PCR reaction (Belgrader et al., 1998a). As a rule, the PCR system
Flow control methods of microfluidics for PCR microfluidics
In PCR microfluidics, especially flow-through PCR microfluidics, the flow of the DNA sample within microchannels must be precisely controlled for successfully performing the PCR. Convection-driven PCR microfluidics does not need an external force to drive the fluid through the different temperature zones. Nevertheless, the sample needs to be carefully introduced into the microchannels which must be accurately sealed to prevent bubble formation that is a significant concern as the presence of
Necessity of surface passivation for PCR microfluidics
For PCR, biocompatibility may be the most sensitive and delicate issue because a PCR solution is in permanent contact with a certain amount of the materials. Currently, the PCR microfluidics have been mostly micromachined from silicon, glass or polymer substrate materials. Early work concerning silicon/glass PCR microfluidics have revealed the problems of deleterious surface interactions (Wilding et al., 1994, Wilding et al., 1995, Shoffner et al., 1996), which are mainly caused by the
Integration of PCR with other analytic steps on a single microfluidics
Since the onset of the “μ-TAS” concept in the early 1990s, great interest has been taken in the development of the μ-TAS technique by many research groups. Microfluidic analytical components, such as micropump, microvalve, microheater, microsensor, microflowmeter, microdetector, etc., have been constructed onto silicon/glass/polymer substrate materials using MEMS technology in order to integrate the steps constituting the analytical process (for example sample preparation, chemical reaction and
Detection of PCR amplification products for PCR microfluidics
As is seen from the development history of PCR microfluidics, another “bottleneck” blocking the realization of a truly integrated DNA analyzer may be a portable detection module for on-line PCR product detection. The most common detection scheme is off-line or on-line CE separation of the PCR product (see above), usually followed by laser-induced fluorescence detection or in some cases by EC detection (Zhao et al., 2002). However, optical detection systems are difficult to miniaturize onto a
Applications of PCR microfluidics
Seen from the reported literature concerning PCR microfluidics, a significantly wide range of target DNA samples has been amplified within the PCR microfluidics with a variety of architectures, which indicates that the PCR microfluidics have a beautiful future of wide applications although few chip-based PCR microfluidics have been commercialized. Presently, as far as the amplified target DNAs are concerned, PCR microfluidics have had a great potential for biomedical and bioanalytical
Outlook and conclusions
The PCR microfluidics, in particular microchip-based PCR microfluidics, have been developed and nowadays have become an important domain of application of miniaturization technology. The organic combination of both science and technology in terms of MEMS has contributed to advances in many aspects of PCR microfluidics, including new substrate materials and correspondingly adopted microfabrication technologies, various architectures for PCR microfluidics, heating and temperature sensing,
Acknowledgements
The authors would like to thank the National Natural Science Foundation of China for funding this study (Contract No. 10272102).
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