F. von Stetten

A highly efficient buckypaper-based electrode material for mediatorless laccase-catalyzed dioxygen reduction

The redox enzyme laccase from Trametes versicolor efficiently catalyzes the oxygen reduction reaction (ORR) in mediatorless biofuel cell cathodes when adsorbed onto multi-walled carbon nanotubes (MWCNTs). In this work we demonstrate that the fabrication of MWCNTs in form of buckypaper (BP) results in an excellent electrode material for laccase-catalyzed cathodes. BPs are mechanically stable, self-entangling mats with high dispersion of MWCNTs resulting in easy to handle homogeneous layers with highly mesoporous structures and excellent electrical conductivities. All biocathodes have been electrochemically investigated in oxygen-saturated buffer at pH 5 by galvanostatic polarization and potentiodynamic linear sweep voltammetry. Both methods confirm an efficient direct interaction of laccase with BP with a high open circuit potential of 0.882 V vs. normal hydrogen electrode (NHE). The high oxygen reduction performance leads to high current densities of 422±71 μA cm(-2) at a typical cathode potential of 0.744 V vs. NHE. When the current density is normalized to the mass of the electrode material (mass activity), the BP-based film electrodes exhibit a 68-fold higher current density at 0.744 V vs. NHE than electrodes fabricated from the same MWCNTs in a non-dispersed agglomerated form as packed electrodes. This clearly shows that MWCNTs can act more efficiently as cathode when prepared in form of BP. This can be attributed to reduced diffusional mass transfer limitations and enhanced electrical conductivity. BP is thus a very promising material for the construction of mediatorless laccase cathodes for ORR in biofuel cells. In addition we demonstrated that these electrodes exhibit a high tolerance towards glucose, the most common bioanode fuel.

Healthy Aims: Developing New Medical Implants and Diagnostic Equipment

Healthy Aims is a 23- million, four-year project, funded under the EUs information society technology sixth framework program to develop intelligent medical implants and diagnostic systems (www.healthyaims.org). The project has 25 partners from 10 countries, including commercial, clinical, and research groups. This consortium represents a combination of disciplines to design and fabricate new medical devices and components as well as to test them in laboratories and subsequent clinical trials. The project focuses on medical implants for nerve stimulation and diagnostic equipment based on strain-gauge technology.

Automated nucleic acid extraction from whole blood, B. subtilis, E. coli, and Rift Valley fever virus on a centrifugal microfluidic LabDisk

We present total nucleic acid extraction from whole blood, Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, and Rift Valley fever RNA virus on a low-cost, centrifugal microfluidic LabDisk cartridge processed in a light-weight (<2 kg) and portable processing device. Compared to earlier work on disk based centrifugal microfluidics, this includes the following advances: combined lysis and nucleic acid purification on one cartridge and handling of sample volumes as large as 200 μL. The presented system has been validated for logarithmic dilutions of aforementioned bacteria and viruses from various sample matrices including blood plasma and culture media and extraction of human DNA from whole blood. Recovered DNA and RNA concentrations in the eluate were characterized by quantitative PCR to: 58.2–98.5%, 45.3–102.1% and 29.5–34.2% versus a manual reference for Bacillus subtilis, Escherichia coli and Rift Valley fever virus, respectively. For extraction of human DNA from whole blood, similar results for on-disk ((10.1 ± 7.6) × 104 DNA copies) and manual reference extraction ((10.2 ± 6.3) × 104 DNA copies) could be achieved. Eluates from on-disk extraction show slightly increased ethanol concentrations of 4.1 ± 0.3% to 5.5 ± 0.2% compared to a manual reference (2.0 ± 0.5% to 3.6 ± 0.6%). The complete process chain for sample preparation is automatically performed within ∼30 minutes, including ∼15 minutes lysis time. It is amenable to concatenation with downstream modules for multiplex nucleic acid amplification as recently demonstrated for panel testing of various pathogens at the point of care.

A versatile miniature bioreactor and its application to bioelectrochemistry studies

Often, reproducible investigations on bio-microsystems essentially require a flexible but well-defined experimental setup, which in its features corresponds to a bioreactor. We therefore developed a miniature bioreactor with a volume in the range of a few millilitre that is assembled by alternate stacking of individual polycarbonate elements and silicone gaskets. All the necessary supply pipes are incorporated as bore holes or cavities within the individual elements. Their combination allows for a bioreactor assembly that is easily adaptable in size and functionality to experimental demands. It allows for controlling oxygen transfer as well as the monitoring of dissolved oxygen concentration and pH-value. The system provides access for media exchange or sterile sampling. A mass transfer coefficient for oxygen (k(L)a) of 4.3x10(-3) s(-1) at a flow rate of only 15 ml min(-1) and a mixing time of 1.5s at a flow rate of 11 ml min(-1) were observed for the modular bioreactor. Single reactor chambers can be interconnected via ion-conductive membranes to form a two-chamber test setup for investigations on electrochemical systems such as fuel cells or sensors. The versatile applicability of this modular and flexible bioreactor was demonstrated by recording a growth curve of Escherichia coli (including monitoring of pH and oxygen) saturation, and also as by two bioelectrochemical experiments. In the first electrochemical experiment the use of the bioreactor enabled a direct comparison of electrode materials for a laccase-catalyzed oxygen reduction electrode. In a second experiment, the bioreactor was utilized to characterize the influence of outer membrane cytochromes on the performance of Shewanella oneidensis in a microbial fuel cell.

Microthermoforming of microfluidic substrates by soft lithography (µTSL): optimization using design of experiments

We present a detailed analysis of microthermoforming by soft lithography (μTSL) for replication of foil-based microfluidic substrates. The process was systematically optimized by design of experiments (DOE) enabling fabrication of defect-free lab-on-a-chip devices. After the assessment of typical error patterns we optimized the process toward the minimum deviation between mold and thermoformed foil substrates. The following process parameters have most significant impact on the dimensional responses (p 40% relative impact. The DOE results in an empirical process model with a maximum deviation between the prediction and experimental proof of 2% for the optimum parameter set. Finally, process optimization is validated by the fabrication and testing of a microfluidic structure for blood plasma separation from human whole blood. The optimized process enabled metering of a nominal volume of 4.0 μl of blood plasma with an accuracy deviation of 3% and a metering precision of ±7.0%. The μTSL process takes about 30 min and easily enables the replication of 300 μm wide microchannels having vertical sidewalls without any draft angles in a well-controllable way. It proves to be suitable for multiple applications in the field of microfluidic devices. S Online supplementary data available from stacks.iop.org/JMM/21/115002/mmedia (Some figures in this article are in colour only in the electronic version)

Magnetic chemiluminescent immunoassay for human C-reactive protein on the centrifugal microfluidics platform

Human C-reactive protein (CRP) has been reported as an inflammatory biomarker with the highest reference for use in clinical practice. However, the existing analytical techniques are lacking automation and simplicity, as desired for a prospective immunoassay format for point-of-care (PoC) analysis. We have developed an automated magnetic chemiluminescent immunoassay (MCIA) on a mobile analyser for rapid PoC determination of CRP. The MCIA is fully automated after the initial loading of sample and immunoreagents at the inlet ports. The automated protocol involves the transportation of magnetic capture microparticles between adjacent reaction compartments using a set of stationary magnets, a microfluidic polymer disposable and a specific centrifugal protocol. The developed MCIA has a sample-to-answer time of 25 min and hands-on time of approximately 5 min. It detects the entire pathophysiological range of CRP, as desired for clinically-relevant high sensitivity CRP immunoassay format, i.e. 3–81 ng mL−1 in diluted human serum with a limit of detection (LOD) and limit of quantification (LOQ) of 1.5 ng mL−1 and 1.8 ng mL−1, respectively.

Real-time PCR probe optimization using design of experiments approach.

Graphical abstract

Aliquoting structure for centrifugal microfluidics based on a new pneumatic valve

We present a new microvalve that can be monolithically integrated in centrifugally driven lab-on-a-chip systems. In contrast to existing operation principles that use hydrophobic patches, geometrically defined capillary stops or siphons, here we present a pneumatic principle. It needs neither additional local coatings nor expensive micro sized geometries. The valve is controlled by the spinning frequency and can be switched to be open when the centrifugal pressure overcomes the pneumatic pressure inside an unvented reaction cavity. We designed and characterized valves ranging in centrifugal burst pressure from 6700 Pa to 2100 Pa. Based on this valving principle we present a new structure for aliquoting of liquids. We experimentally demonstrated this by splitting 105 muL volumes into 16 aliquots with a volume CV of 3 %.

Centrifugo-thermopneumatic fluid control for valving and aliquoting applied to multiplex real-time PCR on off-the-shelf centrifugal thermocycler

We introduce microfluidic automation of geometrically multiplexed real-time PCR to off-the-shelf Rotor-Gene Q thermocyclers (RGQ, QIAGEN GmbH, Hilden, Germany). For centrifugal fluid control the RGQ provides low and constant rotation of 400 rpm, only. Compatibility to this very limited flexibility of centrifugal actuation is achieved by using thermal gas compression and expansion for valving and aliquoting. In contrast to existing thermo-pneumatic actuation, centrifugo-thermopneumatic (CTP) fluid control employs the induced change of partial vapor pressure by global temperature control as actuation parameter for two new unit operations: CTP siphon valving and CTP two-stage aliquoting. CTP siphon valving was demonstrated to reliably transfer sample liquid in all cases (n = 35) and CTP two-step aliquoting transfers metered aliquots of 18.2 ± 1.2 μl (CV 6.7%, n = 8) into reaction cavities within 5 s (n = 24). Thermal characteristics of CTP two-stage aliquoting were found to be in good agreement with an introduced analytical model (R2 = 0.9876, n = 3). A microfluidic disk segment comprising both new unit operations was used for automation of real-time PCR amplification of Escherichia coli DNA. Required primers and probes were pre-stored in the reaction cavities and a comparison with reference reactions in conventional PCR tubes yielded the same PCR efficiency, repeatability, and reproducibility.

Liquid reagent storage and release for centrifugally operated Lab-on-a-Chip systems based on a burstable seal

We present a new approach for the pre-storage and release of liquid reagents in centrifugally operated Lab-on-a-Chip (LoaC) cartridges. Liquids are stored in sealed cavities which are separated from the fluidic system by a weak-bonded interface. During centrifugal rotation, the liquid exerts an inertial force onto the predetermined area. This delaminates the sealing foil locally, resulting in a fluidic connection to the downstream channel. Time-to-release could be adjusted to a range between 31 s and 143 s by geometrical variations of the structure, enabling time controlled release. In sum, the burstable seal is a universal and robust valve: it is vapor tight, independent on wetting properties of liquids, and time controllable. Hence it excels siphon based valving concepts often used in centrifugal microfluidics and is most valuable for the design of LoaC cartridges for point of care applications.