A microfluidic cartridge for fast and accurate diagnosis of Mycobacterium tuberculosis infections on standard laboratory equipment.

We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml-1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.

Rapid Tuberculosis Diagnostics Including Molecular First- and Second-Line Resistance Testing Based on a Novel Microfluidic DNA Extraction Cartridge.

Xpert MTB/RIF testing has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing worldwide. However, it has weaknesses, such as its restriction to Rif resistance testing and the inability to use extracted DNA for further testing. Herein, a holistic diagnostic workflow, including TB detection and resistance testing toward Rif, isoniazid, and important second-line drugs (SLDs), based on a novel microfluidic DNA extraction cartridge (TB-Disk), is presented. DNA from 73 precharacterized sputum samples was extracted with TB-Disk, including 45 clinical and bacteriologically confirmed TB samples, nine TB-negative samples, and 19 sputum samples spiked with twofold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus), and GenoType MTBDRsl. A total of 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as Mycobacterium tuberculosis complex positive. A total of 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as multidrug-resistant TB by GT-plus and further tested for resistance toward SLDs using GenoType MTBDRsl, yielding 75% (6/8) valid results. FT-MTB with cartridge-based DNA extraction (Disk-DNA) and DNA extracted with FluoroLyse yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB enables sensitive TB detection. The Disk-DNA can be further used for screening resistance toward first-line drugs and SLDs.

Liquids on-chip: direct storage and release employing micro-perforated vapor barrier films.

Liquids on-chip describes a reagent storage concept for disposable pressure driven Lab-on-Chip (LoC) devices, which enables liquid storage in reservoirs without additional packaging. On-chip storage of liquids can be considered as one of the major challenges for the commercial break through of polymer-based LoC devices. Especially the ability for long-term storage and reagent release on demand are the most important aspects for a fully developed technology. On-chip storage not only replaces manual pipetting, it creates numerous advantages: fully automated processing, ease of use, reduction of contamination and transportation risks. Previous concepts for on-chip storage are based on liquid packaging solutions (e.g. stick packs, blisters, glass ampoules), which implicate manufacturing complexity and additional pick and place processes. That is why we prefer on-chip storage of liquids directly in reservoirs. The liquids are collected in reservoirs, which are made of high barrier polymers or coated by selected barrier layers. Therefore, commonly used polymers for LoC applications as cyclic olefin polymer (COP) and polycarbonate (PC) were investigated in the context of novel polymer composites. To ensure long-term stability the reservoirs are sealed with a commercially available barrier film by hot embossing. The barrier film is structured by pulsed laser ablation, which installs rated break points without affecting the barrier properties. A flexible membrane is actuated through pneumatic pressure for reagent release on demand. The membrane deflection breaks the barrier film and leads to efficient cleaning of the reservoirs in order to provide the liquids for further processing.