Comparison of pathogen DNA isolation methods from large volumes of whole blood to improve molecular diagnosis of bloodstream infections.

For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml), the novel non-enzymatic procedure (Polaris, 1-5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67%) and EasyMAG (58-79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.

Preparation and characterization of structured hydrogel microparticles based on cross-linked hyperbranched polyglycerol.

The aim of this work was to obtain well-defined HyPG-MA (methacrylated hyperbranched polyglycerol) microparticles with uniform sizes. Therefore, three different preparation methods were evaluated. First, we assessed a micromolding technique using rigid SU-8 (a photoresist based on epoxies) grids. Independent of the surface treatment of the SU-8 grid or the type of polymer used, approximately 50% of the microgels remained attached to the SU-8 grid or broke into smaller particles during the release process in which drying of the gels was followed by a sonication process. Although 90% methacrylate conversion could be obtained, this method has some additional drawbacks as the obtained dried microgels did not rehydrate completely after the drying step. Second, a soft micromolding technique was evaluated using elastomeric PDMS (poly(dimethyl siloxane)) grids. The use of these flexible grids resulted in a high yield (80-90% yield; >90% methacrylate conversion) of microgels with a well-defined size and shape (squares 100 microm x 100 microm x 50 microm or hexagons with Ø 30 microm and a thickness of 20 microm) without the occurrence of water evaporation. However, a number of particles showed a less-defined shape as not all grids could be filled well. The microgels showed restricted swelling, implying that these gels are dimensionally stable. Third, an alternative method referred to as photolithography was evaluated. This method was suitable to tailor accurately the size and shape of HyPG-MA microgels and additionally gained 100% yield. Well-defined HyPG-MA microgels in the size range of 200-1400 microm (thickness of 6, 20, or 50 microm), with a methacrylate conversion of >90%, could easily be prepared by adding an inhibitor (e.g., 1% (w/v) of vitamin C) to the polymer solution to inhibit dark polymerization. Microgels in the size range of 30-100 microm (>90% conversion) could only be obtained when applying the photomask in direct contact with the polymer solution and using a higher (i.e., 2% (w/v)) concentration of vitamin C. Additionally, the microgels showed limited swelling, indicating that rather dimensionally stable particles were obtained. In conclusion, this paper shows that photolithography and soft micromolding, as compared to rigid micromolding, are the most appropriate techniques to fabricate structured HyPG-MA microgels with a tailorable and well-defined size and shape. These microgels have great potential in tissue engineering and drug delivery applications.

Rapid prototyping of microfluidic devices with a wax printer.

We demonstrate a rapid and inexpensive approach for the fabrication of high resolution poly(dimethylsiloxane) (PDMS)-based microfluidic devices. The complete process of fabrication could be performed in several hours (or less) without any specialized equipment other than a consumer-grade wax printer. The channels produced by this method are of high enough quality that we are able to demonstrate the sizing and separation of DNA fragments using capillary electrophoresis (CE) with no apparent loss of resolution over that found with glass chips fabricated by conventional photolithographic methods. We believe that this method will greatly improve the accessibility of rapid prototyping methods.

Single-substrate liquid-crystal displays by photo-enforced stratification.

Data visualization plays a crucial role in our society, as illustrated by the many displays that surround us. In the future, displays may become even more pervasive, ranging from individually addressable image-rendering wall hangings to data displays integrated in clothes. Liquid-crystal displays (LCDs) provide most of the flat-panel displays currently used. To keep pace with the ever-increasing possibilities afforded by developments in information technology, we need to develop manufacturing processes that will make LCDs cheaper and larger, with more freedom in design. Existing batch processes for making and filling LCD cells are relatively expensive, with size and shape limitations. Here we report a cost-effective, single-substrate technique in which a coated film is transformed into a polymer-covered liquid-crystal layer. This approach is based on photo-enforced stratification: a two-step photopolymerization-induced phase separation of a liquid-crystal blend and a polymer precursor. The process leads to the formation of micrometre-sized containers filled with a switchable liquid-crystal phase. In this way, displays can be produced on a variety of substrates using current coating technology. The developed process may be an important step towards new technologies such as 'display-on-anything' and 'paintable displays'.