This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip. The utilised geometry consists of a spiral meander with 40 turns, representing a cyclic-flow PCR system. The used reaction chemistry includes Cy3-conjugated primers leading to a one-step process accelerated by cyclic-flow PCR. DNA of three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) was processed and successfully amplified and labelled with detection limits down to 10 2 cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. The overall processing time was decreased from 6 to 1.5 h. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output led to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.

In this article we present a long target droplet polymerase chain reaction (PCR) microsystem for the amplification of the 16S ribosomal RNA gene. It is used for detecting Gram-positive and Gram-negative pathogens at high-throughput and is optimised for downstream species identification. The miniaturised device consists of three heating plates for denaturation, annealing and extension arranged to form a triangular prism. Around this prism a fluoropolymeric tubing is coiled, which represents the reactor. The source DNA was thermally isolated from bacterial cells without any purification, which proved the robustness of the system. Long target sequences up to 1.3 kbp from Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa have successfully been amplified, which is crucial for the successive species classification with DNA microarrays at high accuracy. In addition to the kilobase amplicon, detection limits down to DNA concentrations equivalent to 10(2) bacterial cells per reaction were achieved, which qualifies the microfluidic device for clinical applications. PCR efficiency could be increased up to 2-fold and the total processing time was accelerated 3-fold in comparison to a conventional thermocycler. Besides this speed-up, the device operates in continuous mode with consecutive droplets, offering a maximal throughput of 80 samples per hour in a single reactor. Therefore we have overcome the trade-off between target length, sensitivity and throughput, existing in present literature. This qualifies the device for the application in species identification by PCR and microarray technology with high sample numbers. Moreover early diagnosis of infectious diseases can be implemented, allowing immediate species specific antibiotic treatment. Finally this can improve patient convalescence significantly.

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