Hybridisation mix synthesis in a spiral lab-on-chip device for fast-track microarray genotyping of human pathogens

摘要

DNA microarrays can provide bacterial identification, which is crucial for targeted therapy. However they lack rapidness, because of multiple analysis steps. Therefore a fast one-step method for synthesising a hybridisation-ready reagent out of initial bacterial DNA is required. This work presents the combination and acceleration of PCR and fluorescent labelling within a disposable microfluidic chip, fabricated by injection moulding. 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 and a high-yield polymerase leading to a one-step process accelerated by cyclic-flow PCR. Three different bacterial samples (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa) were processed and the bacterial DNA was successfully amplified and labelled with detection limits down to 102 cells per reaction. The specificity of species identification was comparable to the approach of separate PCR and labelling. Furthermore the overall processing time was decreased from 6 hours to 1.5 hours. We showed that a disposable polycarbonate chip, fabricated by injection moulding is suitable for the significant acceleration of DNA microarray assays. The reaction output lead to high-sensitivity bacterial identification in a short time, which is crucial for an early and targeted therapy against infectious diseases.