Technical Concept of a Flow‐through Microreactor for In‐situ RT‐PCR

Abstract

This paper presents a novel flow-through reverse transcription-polymerase chain reaction (RT-PCR) microreactor with optimized thermal and fluidic characteristics. It integrates the steps of reverse transcription of the initially applied RNA sample and the amplification of specific DNA fragments of the cDNA formed. The microreactor comprises a heating plate with different temperature zones and an interchangeable fluidic chip with serpentine microchannels. The heating plate provides temperature zones for reverse transcription, hot start activation, denaturation, annealing and extension. The fluidic chip contains a serpentine microchannel with integrated features for segmented sample stream generation and dosing operations, which is guided over the temperature zones according to the thermal protocol of RT-PCR. Reverse transcription is performed in an integrated microchannel section immediately before the amplification of the cDNA's, generated from the primarily applied RNA sample. One feature of this chip system is the generation of a segmented flow for high-throughput analysis of RT-PCR samples. It is shown that RT-PCR reactions can be performed successfully in the microreactor with continuous and segmented flow regimes. The aim of the experiments was the detection of the HPV 16 DNA genome and of viral oncogene transcripts (E6/E7), respectively. Both markers are of importance in medical diagnostics and will be applied in further studies for the identification of oncogene positive cells in cell populations by means of flow-through in-situ RT-PCR.

Use of a Corona Discharge to Selectively Pattern a Hydrophilic/Hydrophobic Interface for Integrating Segmented Flow with Microchip Electrophoresis and Electrochemical Detection

“…Droplets can be generated in a microchip device through perpendicular channels 3–5 or off-chip with a micro-mixing tee. 6 On-chip generation of segmented flow has been used to promote reagent mixing for applications such as nanoscale PCR, 7 studying reaction kinetics, 5 and trapping single cells. 2,8 Recently, there has also been an interest in using segmented flow to help improve temporal resolution when samples from off-chip processes are transferred to the microchip for subsequent analysis.…”

Enzyme synthesis and activity assay in microfluidic droplets on a chip

“…Ultimately, it needs to work with only one DNA template molecule provided per droplet (so that there is a simple physical connection between the performance of an enzyme variant and the copy of the gene that it encoded it). There are certainly ways of amplifying DNA from a single copy of a plasmid using PCR technology within the droplet 20, 21, 38. But it remains true that efficient transcription and translation and sensitive enzyme assay will be key to successful use of the technology.…”

“…The system allows monodispersed pico‐ to nano‐liter volume droplets to be produced at high rates 6, 7 and manipulated in a highly controlled manner 8–10. These droplets can be used as separate bioreactors for a wide range of new biological applications 11–14, such as cell‐based assays 15, 16, the synthesis of DNA molecules 17–19 and polymerase chain reactions (PCRs) 20, 21. Critically for in vitro evolution, the system also allows for in vitro protein expression, via in vitro transcription and translation (IVTT) from coding DNA 22, 23.…”

Micro segmented flow-functional elements and biotechnical applications

“…For DNA amplification, a thermocycler made of silicon and glass chips has been developed for miniaturized rapid PCR by use of flow-through two phases system, in which system the amplifications of various DNA templates of different sources and properties were achieved in less than half an hour (108). A novel microfluidic segmented flow system for reverse transcription-polymerase chain reaction (RT-PCR) has been demonstrated by the amplification of cell-isolated, HPV 16 target ontogeny expressing RNA (109). Transcripts of measles and human papilloma virus (HPV) by reverse transcription (RT) and amplification of cDNA have been successfully complemented and detected in droplet based microfluidic system which holds a great promise for portable diagnostic biomedical applications (110).…”