Reverse transcription-polymerase chain reaction (RT-PCR) in flow-through micro-reactors: Thermal and fluidic concepts

Felbel, Jana; Reichert, Anett; Kielpinski, Mark; Urban, Matthias; Henkel, Thomas; Häfner, Norman; Weber, J. L.

doi:10.1016/j.cej.2007.07.019

Cited by 22 publications

(19 citation statements)

References 11 publications

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“…developed a microfabricated glass chip for the functional integration of RT and PCR in a serpentine-channel continuous-flow mode. Similar studies were also performed by Tsai and Sue (2006) and Felbel et al (2008) using the serpentine-channel continuous-flow RT-PCR system. However, the detection method was off-line agarose gel electrophoresis, which was time-consuming and laborintensive.…”

Section: Segmented-flow Rt-pcr For Simultaneous Detection Of Pathogenmentioning

confidence: 86%

“…The developed microfluidic system not only overcomes the thermal cross-talk effects of radiation and/or conduction but also utilizes fluorescence microscopy to realize the on-line product detection. Up to now, the amplification products of most microfluidic RT-PCR (Felbel et al 2008;Hartung et al 2009;Lee et al 2008;Liao et al 2005;Lien et al 2007;Lien et al 2009;Tsai and Sue 2006) have been analyzed by agarose gel electrophoresis and ethidium bromide staining, which is time-consuming and labor-intensive. Therefore, our current efforts are focused on the on-line fluorescence detection, which offers several obvious advantages.…”

Section: Discussionmentioning

confidence: 99%

“…This design advantage is achieved by maintaining the zones at preset constant temperatures . Currently, the structural styles of the continuousflow RT-PCR microfluidics can be divided into two main categories: the serpentine-channel continuous-flow RT-PCR (Felbel et al 2008;Tsai and Sue 2006) and the spiralchannel continuous-flow RT-PCR (Hartung et al 2009). In the latter configuration, PCR device has an underlying advantage of a circular arrangement of temperature zones in the sequence of denaturation, annealing, and extension, which could increase the PCR efficiency compared with the former.…”

Section: Introductionmentioning

confidence: 99%

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Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

Zhang

Xing

2010

Microfluid Nanofluid

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Being beneficial from dramatic process in biochemical and micro-electro-mechanical technologies, this study presents an integrated microfluidic system capable of performing a continuous-flow reverse transcription-polymerase chain reaction (RT-PCR) combined with fluorescence microscopy for rapid diagnosis of RNA-based viruses. The device consists of two heated cylinders for heating the different reaction zones of the reverse transcription and the amplification. In the amplification cylinder, bath refrigerating is used for thermal protection of the annealing region, which is heated possibly due to the thermal effects of radiation, conduction, and/or convection from denaturation and extension regions, thus resulting in a space-saving design of the whole device. Detection of the amplified products is performed on-line by a fluorescence microscopy with SybrGreen I, a widely used intercalating dye. In this article, the proposed miniature RT-PCR system is used to amplify and detect two RNA-based viruses (Noroviruses (NVs) and Rotaviruses (RVs)), which are now recognized as the most common etiological agents of acute viral gastroenteritis causing numerous outbreaks worldwide. The experimental data have demonstrated the ability of the presented system to perform a two-step or one-step RT-PCR process. On this device, the NVs and RVs RNA samples were successfully reverse transcribed and amplified within 1 h, and the limit of detection of the RNA concentration was 6.4 9 10 4 copies ll -1 using onestep RT-PCR process. Consequently, the developed microfluidic system can provide a promising platform for fast diagnosis of RNA-based viruses.

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Section: Segmented-flow Rt-pcr For Simultaneous Detection Of Pathogenmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

Zhang

Xing

2010

Microfluid Nanofluid

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“…The reduction of PCR volume and the use of highly heat conducting materials as silicon allowed to reduce the times for thermocycling considerably [45]. In addition, miniaturization opened the possibility of continuous flow PCR [46][47][48][49] and of realizing highly parallelized thermocycling processes. Meanwhile, a lot of different strategies reaching from micro-and nanotiterplates over chip arrays to emulsions and gel droplets is used for microscaled PCR in highly parallelized systems [50][51][52].…”

Section: Pcr In Micro Fluid Segmentsmentioning

confidence: 99%

Droplet-Based Microfluidics as a Biomimetic Principle: From PCR-Based Virus Diagnostics to a General Concept for Handling of Biomolecular Information

Köhler

2012

Microdroplet Technology

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The ability of lithographic fabrication of microchannels for fluid transport on the one hand and the continuously decreasing critical dimensions during the development of microlithography over the last decades generated high expectations on the progress of automated processing of small amounts of substances. In particular, it was expected that large numbers of well distinguished chemical entities-atoms, molecules, nano particles-could become manipulated highly parallel and with high speed. This hope was fed by the imagination of an analogy between the electron transport in integrated circuits and the transport of chemical objects inside microfluidic networks. The experiences of the last both decades had shown that such ideas of a complete analogy are not realistic. The progress of handling of substance-coupled information is much slower as the progress of hard ware development in the electronic devices. But, beside this general disappointment, microfluidics is still promising the arise and growth-up of very important new strategies for organizing chemical systems at the microscale and for supplying functional interfaces between the complex information inside the world of molecules and particles on the one hand and electronic systems on the other hand.Whereas clouds of electrons are manipulated in digital electronic devices, clouds of molecules are transported through microchannels by a convective flow. But, the special conditions in microfluidics cause low Reynolds numbers which reflect the small ratio of channel diameter and volume flow rate to the viscous forces. The microfluidic conditions cause always a laminary structure of flow with steep flow velocity gradients and results in to very high fluidic dispersion of concentration signals if homogeneous fluids are applied. So, the principle high potential of J.M. K€ ohler

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“…Monodispersed microbubbles and microdroplets can be controllably prepared in microfluidic devices (Choi et al 2010;Marmottant and Raven 2009) and it has been proved that the microdispersed emulsion systems have good transport and reacting properties (Panic et al 2004;Xu et al 2008a). Using these properties, microfluidic devices can be applied to enhance chemical reactions (Razzaq et al 2009), prepare microsphere materials (Park et al 2009), synthesize nanoparticles (Sevonkaev and Matijevic 2009), provide biological analysis (Felbel et al 2008), develop fuel cells (Kjeang et al 2009), and so on. For the purpose of giving accurate control of the emulsification process, the basic studies on droplet/bubble generation rules in microfluidic devices are very important.…”

Section: Introductionmentioning

confidence: 99%

Droplet generation in micro-sieve dispersion device

Wang

Lü

et al. 2010

Microfluid Nanofluid

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Microfluidic devices with micro-sieve plate as the dispersion medium have been widely used for the mass production of emulsions. While unfortunately, few studies have so far been made for the droplet generation rules in those devices. In this work, the droplet generation processes in micro-sieve dispersion devices are investigated with specially designed micro-sieve pore arrays. The effects of channel structure, pore arrangement, and feeding method of dispersed phase on the average size and distribution of droplets are studied carefully. It is found the dimensionless average droplet diameters (d av /d e ) in microsieve dispersion devices can be represented by a linear relation with Ca -1/4 of continuous phase, the same as the scaling law in T-junction microchannels. The flow distribution among pores and the steric hindrance between droplets affect the diameter distribution of generated droplet very much. Monodispersed droplets with polydispersity index less than 5% can be made at Ca number larger than 0.01 and phase ratio (Q D /Q C ) less than 1/6 in the present investigation.

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Reverse transcription-polymerase chain reaction (RT-PCR) in flow-through micro-reactors: Thermal and fluidic concepts

Cited by 22 publications

References 11 publications

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

Fast identification of foodborne pathogenic viruses using continuous-flow reverse transcription-PCR with fluorescence detection

Droplet-Based Microfluidics as a Biomimetic Principle: From PCR-Based Virus Diagnostics to a General Concept for Handling of Biomolecular Information

Droplet generation in micro-sieve dispersion device

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