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We have established a fast PCR-based micro flow-through process consisting of a helical constructed tube reactor. By this approach we can detect transcripts of measles and human papilloma virus (HPV) by continuous flow allowing for reverse transcription (RT) and amplification of cDNA. The micro reaction system consisted of two columnar reactors for thermostating the different reaction zones of the RT process and the amplification. The PCR reactor was built by asymmetric heating sections thus realizing different residence times and optimal conditions for denaturation, annealing and elongation. The system concept is based on low electrical power consumption (50-120 W) and is suited for portable diagnostic applications. The samples were applied in form of micro fluidic segments with single volumes between 65 and 130 nL injected into an inert carrier liquid inside a Teflon FEP tube with an inner diameter of 0.5 mm. Optimal amplification for template lengths of 292 bp (lambda-DNA), 127 bp (measles virus) and 95 bp (HPV) was achieved by maximal cycle times of 75 s.

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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.

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A microfluidic chip system for flow-through PCR reactions with an optimized thermal profile consisting of five temperature zones was presented. It allows the implementation of one PCR cycle in a half channel loop. In contrast, conventional systems with a three zones arrangement require a complete channel loop per cycle. Therefore, this arrangement increases the level of integration and allows the implementation of a 40 cycles flow-through thermocycler on the footprint of a microscope slide. To obtain a high throughput of samples in a small volume (10–100 nl), the fluidic chip device was designed to operate at segmented-flow conditions for PCR. That way, each droplet may contain a single sample that is independently processed while transported through the microchannel. To achieve stable fluidic conditions, the surface of the micro-channels was modified. In addition to the successful flow-through PCR reaction in the micro reactor, the detection of the tumor suppressor p53 for clinical diagnostics was demonstrated.