Manufacture of chitosan microbeads using centrifugally driven flow of gel-forming solutions through a polymeric micronozzle

A centrifugally driven pulse-free flow has been used for generation of tripolyphosphate (TPP)-gelated chitosan beads with tunable diameters ranging from 148 to 257 lm. The production process requires a single motor as the sole actively actuated component. The 2% (w/w) chitosan solution was extruded through a polymeric nozzle with an inner diameter of 127 lm in the centrifugal field ranging from 93 to 452g and the drops were collected in an Eppendorf tube containing 10% (w/w) TPP solution at pH 4.0. The reproducibility of the bead diameters out of different nozzles was very good with overall CVs of the bead diameters down to 15% and the production rate was 45 beads per second per nozzle at 44 Hz rotor frequency. The production rate was proportional to the sixth power of the rotor frequency, which was explained by the non-Newtonian behaviour of the chitosan solution with a flow behaviour index of 0.466. An analytical model for the bead diameter and production rate has been presented and validated by the experimental data. The shrinkage of chitosan drops during gelation was estimated from the observations and the theoretical model

Porous Platinum Electrodes Fabricated by Cyclic Electrodeposition of PtCu Alloy: Application to Implantable Glucose Fuel Cells

We demonstrate the application of cyclic electrodeposition of PtCu alloy to fabricate porous platinum electrodes for implantable glucose fuel cells. Depending on the number of deposition cycles, electrodes with controllable specific surface area can be fabricated, their roughness factors ranging from 20 (slightly roughened) to 3100 (highly porous). Adjusting the specific surface area of the electrode from low to high determines whether it functions as cathode or anode, respectively. Compared to state of the art, this process is beneficial due to shorter fabrication times, lower temperatures, and the requirement of only one process for the fabrication of both electrodes. Correspondingly fabricated glucose fuel cells showed a power density of 5.1 μW cm^–2 under close to physiological conditions, which is an improvement by 16% compared to earlier designs. During continuous operation over 90 days the fuel cell showed a mean continuous decay of about 0.8% per day, which is related to catalyst poisoning at the anode. Future work will thus have to focus on the improvement of long-term stability instead of power density

Highly Flexible UV–Vis Radiation Sources and Novel Detection Schemes for Spectrophotometric HPLC Detection

The concept and performance of the first multiwavelength deep UV light-emitting-diode-based high-performance liquid chromatography (HPLC) absorbance detector are presented. In single-wavelength mode and with optical reference, the limit of detection (LOD) is comparable to conventional state-of-the-art HPLC absorbance detectors. In multiwavelength modeat present up to eight wavelengths without optical referencethe LOD is about 10 times higher than in single-wavelength mode. Multiplexing and demultiplexing methods are used to separate chromatographic signals in multiwavelength mode and keeps the detector configuration simple and yet flexible. Depending on the operation mode, stray light is either totally negligible or controlled electronically and digitally

Monochrome Multiplexing in Polymerase Chain Reaction by Photobleaching of Fluorogenic Hydrolysis Probes

Multiplexing in polymerase chain reaction (PCR) is a technique widely used to save cost and sample material and to increase sensitivity compared to distributing a sample to several singleplex reactions. One of the most common methods to detect the different amplification products is the use of fluorogenic probes that emit at different wavelengths (colors). To reduce the number of detection channels, several methods for monochrome multiplexing have been suggested. However, they pose restrictions to the amplifiable target length, the sequence, or the melting temperature. To circumvent these limitations, we suggest a novel approach that uses different fluorophores with the same emission maximum. Discrimination is achieved by their different fluorescence stability during photobleaching. Atto488 (emitting at the same wavelength as 6-carboxyfluorescein, FAM) and Atto467N (emitting at the same wavelength as cyanine 5, Cy5) were found to bleach significantly less than FAM and Cy5; i.e., the final fluorescence of Atto dyes was more than tripled compared to FAM and Cy5. We successfully applied this method by performing a 4-plex PCR targeting antibiotic resistance genes in <i>S. aureus</i> using only 2 color channels. Confidence of discrimination between the targets was >99.9% at high copy initial copy numbers of 100 000 copies. Cases where both targets were present could be discriminated with equal confidence for Cy5 channel and reduced levels of confidence (>68%) for FAM channel. Moreover, a 2-plex digital PCR reaction in 1 color channel was shown. In the future, the degree of multiplexing may be increased by adding fluorogenic probe pairs with other emission wavelengths. The method may also be applied to other probe and assay formats, such as Förster resonance energy transfer (FRET) probes and immunoassays

Completely Superhydrophobic PDMS Surfaces for Microfluidics

This study presents a straightforward two-step fabrication process of durable, completely superhydrophobic microchannels in PDMS. First, a composite material of PDMS/PTFE particles is prepared and used to replicate a master microstructure. Superhydrophobic surfaces are formed by subsequent plasma treatment, in which the PDMS is isotropically etched and PTFE particles are excavated. We compare the advancing and receding contact angles of intrinsic PDMS samples and composite PTFE/PDMS samples (1 wt %, 8 wt %, and 15 wt % PTFE particle concentration) and demonstrate that both the horizontal and vertical surfaces are indeed superhydrophobic. The best superhydrophobicity is observed for samples with a PTFE particle concentration of 15 wt %, which have advancing and receding contact angles of 159° ± 4° and 158° ± 3°, respectively

Illustration of microfluidic process flow of a <i>GeneSlice</i> for nested PCR and melt curve analysis on a Rotor-Gene Q.

<p>The microfluidic structures of interest at different points in time (A-E) are shown on top, indicating liquid movement from the dark blue to the light blue position. Corresponding temperatures (red) and rotational speed (black) at each depicted point in time can be read from the given diagram at the bottom. The liquid stays in the position depicted in B during pre-amplification, which consists of 10 thermal PCR cycles at constant 400 RPM. During main-amplification and melt curve analysis, which follow the last (E) depicted fluidic operation, no further fluidic operations take place and liquids stay in the main-amplification cavities (E, light blue).</p

Melt curve analysis of sheep and goat amplimers.

<p>1 ng of sheep and goat DNA were amplified on two <i>GeneSlices</i>. The melt curve analysis shows the successful amplification of the <i>12S rRNA</i> and <i>cytb</i> genes for both species. The different melting behavior is caused by inter-species sequence differences within the Caprinae.</p

Schematic description of a microfluidic disk segment (“<i>GeneSlice”</i>) with pre-stored reagents (A), and a compatible rotor holder (“<i>GeneSlice 100 Rotor”</i>), which can hold up to four <i>GeneSlices</i> in one Rotor-Gene Q run (B).

<p>The <i>GeneSlice</i> comprises pre-amplification chambers for sample and no-template control (NTC) liquids, capillary siphon valves for transfer of the pre-amplification product and a centrifugo-thermopneumatic two-stage aliquoting structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131845#pone.0131845.ref031" target="_blank">31</a>] for 14 (sample) and 1 (NTC) main-amplification(s), respectively. The aliquoting structures guide excess liquid into waste chambers. All required reagents are pre-stored in lyophilized or air-dried format. The air vent for pressure equalization is covered with a membrane. Siphon valves are rendered hydrophilic by coatings.</p

Melt curve analysis of a mixture of human and pig DNA in different ratios.

<p>Artificial mixtures of human and pig DNA were created from 0% to 100%, each, and amplified. Analysis of the respective animal-group-specific main-amplification and universal <i>12S rRNA</i> cavities shows resolution of the DNA components down to 1% (<i>12S rRNA</i>) for pig and human; 5% both for pig, and 10% both for human as minor components.</p

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3–4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, <i>n</i> = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; <i>n</i> = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels