Chemical Engineering Journal 101 (2004) 151–156 Microfabricated flow-through device for DNA amplification—towards in situ gene analysis Tatsuhiro Fukuba a , Takatoki Yamamoto a , Takeshi Naganuma b , Teruo Fujii a,∗ a Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba Meguro-ku, Tokyo 153-8505, Japan b School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 739-8528, Japan Abstract This study presents a microfabricated device for polymerase chain reaction (PCR) with a flow-through manner for use in environmental microbiology. The device was developed utilizing photolithography and softlithography techniques and evaluated as one component of a totally integrated in situ gene analysis system. The developed device was composed of a glass-based “temperature control chip” and polydimethylsiloxane (PDMS)-based “microchannel chip”. For the temperature control chip, six heaters made from indium tin oxide (ITO) are placed on a glass substrate to define three uniform temperature zones for flow-through PCR. On the each heater, a platinum (Pt) line was placed as a temperature sensor. The PDMS microchannel structure was fabricated by using a molding method with a negatively patterned mold master. The width and depth of folded microchannel was 100 m and total length for 30 cycles of flow-through PCR was approximately 3.0 m. With the flow-through PCR device, 580 and 1450 bp (base pairs) of DNA fragments were successfully amplified from Escherichia coli genomic DNA and directly from untreated cells. © 2003 Elsevier B.V. All rights reserved. Keywords: Flow-through PCR; Microfabrication; PDMS 1. Introduction In recent years, gene-based analysis with molecular bio- logical techniques has become essential for most research field relating to any living organisms. In particular, poly- merase chain reaction (PCR) and its various derivatives are the most important techniques to amplify targeted DNA fragments from temperate DNA, or complementary DNA (cDNA) samples. To perform PCR, appropriate tempera- ture cycling is necessary for denaturation (e.g. 96 ◦ C) of double-stranded DNA, annealing of oligonucleotide primer pairs (e.g. 50 ◦ C) and enzymatic elongation of new DNA strands catalyzed by DNA polymerase (e.g. 72 ◦ C). With properly designed primer pairs, the desired region of DNA fragments can be amplified with high selectivity and sensi- tivity by performing 20–30 times temperature cycling. How- ever, conventional techniques of performing PCR require much time and manpower. Thus, automation of routine work such as sample preparation (e.g. DNA extraction and purifi- cation), PCR and analysis (e.g. electrophoresis) is strongly demanded. Especially for the field of environmental micro- biological study, totally automated and miniaturized devices ∗ Corresponding author. Tel.: +81-3-5452-6211; fax: +81-3-5452-6212. E-mail address: tfujii@iis.u-tokyo.ac.jp (T. Fujii). for in situ gene analysis are essential to analyze unique ecosystems in extreme environments such as deep-sea [1], deep subsurface [2] and under-glacial lakes [3] as challeng- ing targets. In most of the cases, sampling-based methods have been employed to this day, but some serious prob- lems are caused by contamination or time lag between sam- pling and analysis with these methods. Well-automated and miniaturized in situ gene analysis systems can solve such problems and completely novel knowledge will be obtained about the frontiers of life. These years, many examples of miniaturized PCR devices are reported in the field of research called “micro-total anal- ysis systems (Micro-TAS)” or “Lab-on-a-chip” [4–6]. One of the unique alternative methods for PCR is flow-through (continuous-flow) PCR [7] and some miniaturized devices for flow-through PCR are also reported [8–10]. A system for flow-through PCR is composed of several constant and uni- form temperature zones and folded or looped long capillary to carry PCR reagents on each temperature zones instead of heat blocks and reaction tubes (Fig. 1). This method was primarily proposed for high-speed PCR because its small thermal mass makes it capable of rapid temperature cycles compared with a conventional method [7]. Furthermore, con- tinuous amplification for variable samples is also possible by supplying samples sequentially [11] and this feature is 1385-8947/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.cej.2003.11.016