Daniel Mark

Versatile sample environments and automation for biological solution X-ray scattering experiments at the P12 beamline (PETRA III, DESY)

An integrated environment for biological small-angle X-ray scattering (BioSAXS) at the high-brilliance P12 synchrotron beamline of the EMBL (DESY, Hamburg) allows for a broad range of solution scattering experiments. Automated hardware and software systems have been designed to ensure that data collection and processing are efficient, streamlined and user friendly.

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 microm. 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 microm in the centrifugal field ranging from 93 to 452 g 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.

Dye-based coatings for hydrophobic valves and their application to polymer labs-on-a-chip

We provide a method for the selective surface patterning of microfluidic chips with hydrophobic fluoropolymers which is demonstrated by the fabrication of hydrophobic valves via dispensing. It enables efficient optical quality control for the surface patterning thus permitting the low-cost production of highly reproducible hydrophobic valves. Specifically, different dyes for fluoropolymers enabling visual quality control (QC) are investigated, and two fluoropolymer-solvent-dye solutions based on fluorescent quantum dots (QD) and carbon black (CB) are presented in detail. The latter creates superhydrophobic surfaces on arbitrary substrates, e.g. chips made from cyclic olefin copolymer (COC, water contact angle = 157.9°), provides good visibility for the visual QC in polymer labs-on-a-chip and increases the burst pressures of the hydrophobic valves. Finally, an application is presented which aims at the on-chip amplification of mRNA based on defined flow control by hydrophobic valves is presented. Here, the optimization based on QC in combination with the Teflon-CB coating improves the burst pressure reproducibility from 14.5% down to 6.1% compared to Teflon-coated valves.

Real-time PCR based detection of a panel of food-borne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification

We present an implementation of parallel, real-time PCR based detection of up to 6 different food-borne pathogens on our centrifugal microfluidic “LabDisk” platform. It has the following two novelties: (1) a microfluidic network for integration of positive controls (PCs), no-template controls (NTCs), and standards (STDs) into a centrifugal microfluidic PCR cartridge; (2) a microfluidic unit operation for sequential aliquoting of two liquids of highly different wetting characteristics into fourteen aliquots with 5.8 μL ± 0.3 μL (PCR mastermix) and 6.1 ± 0.8 μL (elution buffer), respectively. The presented “LabDisk” implementation can be used either in a qualitative or quantitative operation mode depending on the prestorage scheme of reagents. In qualitative mode, two DNA samples can be tested per cartridge for the presence of 6 food pathogens (Listeria monocytogenes, Salmonella typhimurium, EHEC, Staphylococcus aureus, Citrobacter freundii and Campylobacter jejuni), including PCs and NTCs. This was proofed for DNA concentrations of 10 pg, 1 pg, and 0.1 pg per pathogen. In quantitative mode, one DNA sample per cartridge can be analysed quantitatively for the presence of two pathogens by prestored and on-disk generated standard curves. 50 pg and 500 pg L. monocytogenes genomic DNA samples have been quantified to 83 ± 17 pg and 540 ± 116 pg DNA, respectively, while 50 pg and 500 pg S. typhimurium DNA samples have been quantified to 48 ± 4 pg and 643 ± 211 pg DNA. In both operation modes, the microfluidic routing of the liquids was done by spinning the cartridge on a low-cost centrifugal test rig. For real-time PCR amplification, the cartridge was then transferred into a commercially available thermocycler. The nucleic acid amplification and detection as presented here is fully compatible with upstream DNA extraction as presented previously (Strohmeier et al., Lab Chip, 2013, 13, 146-155). Concatenation of both fluidic structures will enable fully integrated sample-to-answer testing of food-borne pathogens in the future.

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.

IR thermocycler for centrifugal microfluidic platform with direct on-disk wireless temperature measurement system

We present an infrared (IR) thermocycler with closed loop temperature control for performing fast polymerase chain reactions (PCR) in centrifugal microfluidics [1]. It consists of an IR ring heater and an on-disk wireless temperature sensor module with a resolution of 0.1 K [2]. The closed loop system enables to precisely control the temperature of the reagents even at varying conditions e.g. manufacturing tolerances of the polymer film disks [3], different locations of the cavities, ambient temperature changes. Due to the direct heating of the reagents by IR absorption we achieve fast average heating gradients of up to 4 K/s. Average cooling gradients so far are limited to 1.3 K/s. Our system is superior in terms of energy efficiency, temperature accuracy and overall reproducibility and robustness.

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 %.

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.

The LabDisk – A Fully Automated Centrifugal Lab-on-a-Chip System for the Detection of Biological Threats

The world’s growing mobility, mass tourism and the threat of terrorism increase the risk of a fast spread of infectious microorganisms and toxins. Therefore, there is a growing demand for small, mobile, easy to use diagnostic systems for automated detection of those agents directly at the point of need. However, the state of the art for pathogen detection requires complex stationary devices and trained personal limiting the capability for a rapid and effective response. We present an alternative solution to this demand: the LabDisk platform, a portable fully automated Lab-on-a-Chip system which performs complex biochemical analyses at the point of need. We applied the LabDisk platform to an automated nucleic acid analysis for the detection of Bacillus anthracis and Francisella tularensis and to an immunoassay for the detection of ricin.

Microfluidic cartridges for DNA purification and genotyping processed in standard laboratory instruments

Two microfluidic cartridges intended for upgrading standard laboratory instruments with automated liquid handling capability by use of centrifugal forces are presented. The first microfluidic cartridge enables purification of DNA from human whole blood and is operated in a standard laboratory centrifuge. The second microfluidic catridge enables genotyping of pathogens by geometrically multiplexed real-time PCR. It is operated in a slightly modified off-the-shelf thermal cycler. Both solutions aim at smart and cost-efficient ways to automate work flows in laboratories. The DNA purification cartridge automates all liquid handling steps starting from a lysed blood sample to PCR ready DNA. The cartridge contains two manually crushable glass ampoules with liquid reagents. The DNA yield extracted from a 32 μl blood sample is 192 ± 30 ng which corresponds to 53 ± 8% of a reference extraction. The genotyping cartridge is applied to analyse isolates of the multi-resistant Staphyloccus aureus (MRSA) by real-time PCR. The wells contain pre-stored dry reagents such as primers and probes. Evaluation of the system with 44 genotyping assays showed a 100% specificity and agreement with the reference assays in standard tubes. The lower limit of detection was well below 10 copies of DNA per reaction.