Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends

Zhang, Chunsun; Xing, Da

The continuous-flow PCR concept of using a capillary passing through different temperature baths was introduced by Nakano et al. in 1994 (85) . In 1998, Kopp et al. reported a continuous-flow PCR chip using a serpentine channel passing through three thermostable copper blocks ( Figure 2A ) (86) . Since then, especially during the last two years, such PCR chips have undergone substantial improvements (4, 30, 44, 50, 55, 58, 60, 66, 67, 87, 88) . However, continuous-flow PCR poses several problems during thermal cycling: (i) Gas bubbles are easily generated in the microchannels, which adversely affect PCR amplification. (ii) Pressure-driven flow easily produces a hyperbolic flow profile that may lead to progressive sample dispersion (56) , and often requires an external bulky syringe pump (23, 30, 41, 42, 44, 50, 58, 66, 67, 71, 72, (74) (75) (76) 88) , which adversely affect the development of compact, portable and integrated continuous-flow PCR chips. (iii) The rate at which the PCR solution travels between different temperature zones is difficult to regulate, and thus most of the continuous-flow PCR systems lack the flexibility to regionally control fluid flow velocity so as to meet different PCR requirements. (iv) Highly integrated continuous-flow PCR chips are rarely reported due to high fabrication cost and difficulties in controlling the continuous liquid flow. In order to overcome these problems, some alternative approaches have been utilized. For example, by introducing a highly viscous fluorinated oil cap immediately before the introduction of the PCR sample, Nakayama et al. have overcome the generation of air bubbles in the continuous-flow microchannel (50) . Gui and Ren have proposed the possibility of performing a novel EOF-driven continuous-flow PCR on a serpentine channel chip on the basis of numeric simulation (87) . Similar work was also reported by Chen et al. (56) . Li et al. have reported a continuous-flow PCR chip with a serpentine channel of varying width for 'regional velocity control' (30) . Most recently, a hybrid chip incorporating the continuous-flow PCR, LDR and hybridization assays has been proposed by Hashimoto et al. to perform analysis of single base mutations in genomic DNA (60) . This is a significant work because it opens the door to the integration of the continuous-flow PCR with pre-PCR or post-PCR analytical step.