ARTICLE pubs.acs.org/ac
Integrated Glass Microdevice for Nucleic Acid Purification, Loop-Mediated Isothermal Amplification, and Online Detection Qingqing Wu, Wei Jin, Chao Zhou, Sihai Han, Wenxiu Yang, Qiangyuan Zhu, Qinhan Jin, and Ying Mu* Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310058, People's Republic of China ABSTRACT: A microdevice made of glass for genetic analysis has been fabricated, for the first time, for integration of extraction of nucleic acids and loop-mediated isothermal amplification (LAMP), followed by online fluorescence detection of amplification products on a single chip. The nucleic acid (NA) extraction region consists of a microfabricated serpentine channel in which micropillars were etched to increase the channel surface area and the capture efficiency of NAs. Nucleic acid molecules were bound to these pillars and channel surface in the presence of the chaotropic salt guanidine hydrochloride and eluted into a downstream amplification chamber with low ionic strength buffer where loop-mediated isothermal amplification was efficiently performed. Amplification can be detected online by the increase of fluorescence intensity at 540 nm when a low concentration of SYBR Green I, a fluorescent dsDNA intercalating dye, is employed. Flow control was accomplished by using laminar flow and differential channel flow resistances. Through passivation of the LAMP chamber and the channel between the extraction region and amplification domain, effective nucleic acid extraction and amplification were performed by just using a double-channel syringe pump and a heating block. By using this integrated microdevice, the purification of nucleic acids from complex biological matrixes and their subsequent amplification and detection online could be finished within 2 h.
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he purification of nucleic acids (NAs) is a precondition for most genetic analyses or diagnostics and their forensic applications. However, the conventional methods for NA extraction, such as phenolchloroform extraction, have the disadvantage of being time-consuming and difficult to scale down to small sample volumes. Compared to conventional methods, the micro total analysis system (μ-TAS) offers several remarkable advantages, such as low reagent and sample consumption, increased speed, enhanced sensitivity, etc.14 Microfluidic approaches to nucleic acid isolation have therefore received great attention in recent years. There are many investigations concerning the extraction of nucleic acids in microfluidic systems; these works concern mostly packing microchannels with silica beads510 or with solgel and hybrid silica beads/solgel11,12 or fabricating micropillars and microposts in silicon microfluidic channels.13,14 NAs adsorb to silica in the presence of a high concentration of the chaotropic agent,15 and the extracted nucleic acids are eluted with an aqueous low-salt buffer and concentrated into a very small volume. One of the motivations for the development of on-chip NA extraction is to integrate other genetic analysis units such as nucleic acid amplification and detection into a single microchip. Legendre et al.16 demonstrated integrated online DNA purification and polymerase chain reaction (PCR) in a valveless glass microdevice. The chromatography required for solid-phase extraction (SPE) in the microfluidic sample preparation device was carried out in a silica bead/solgel SPE bed, where the purified r 2011 American Chemical Society
DNA was eluted directly into a downstream chamber where conventional thermocycling allowing for PCR amplification of specific DNA target sequences took place. Easley et al.17 developed a fully integrated microfluidic system that can accept whole blood as a crude biological sample. Upon loading the sample, the glass microfluidic genetic analysis system device carries out onchip DNA purification and PCR-based amplification, followed by separation and detection in a manner that allows for microliter samples to be screened for infectious pathogens with the sampleinanswer-out mode in
