An automated microfluidic platform for the screening and characterization of novel hepatitis B virus capsid assembly modulators.

To date, hepatitis B virus (HBV) capsid assembly modulators (CAMs), which target the viral core protein and induce the formation of non-functional viral capsids, have been identified and characterized in microtiter plate-based biochemical or cell-based in vitro assays. In this work, we developed an automated microfluidic screening assay, which uses convection-dominated Taylor-Aris dispersion to generate high-resolution dose-response curves, enabling the measurements of compound EC50 values at very short incubation times. The measurement of early kinetics down to 7.7 seconds in the microfluidic format was utilized to discriminate between the two different classes of CAMs known so far. The CAM (-N), leading to the formation of morphologically normal capsids and the CAM (-A), leading to aberrant HBV capsid structures. CAM-A compounds like BAY 41-4109 and GLS4 showed rapid kinetics, with assembly rates above 80% of the core protein after only a 7 second exposure to the compound, whereas CAM-N compounds like ABI-H0731 and JNJ-56136379 showed significantly slower kinetics. Using our microfluidic system, we characterized two of our in-house screening compounds. Interestingly, one compound showed a CAM-N/A intermediate behavior, which was verified with two standard methods for CAM classification, size exclusion chromatography, and anti-HBc immunofluorescence microscopy. With this proof-of-concept study, we believe that this microfluidic system is a robust primary screening tool for HBV CAM drug discovery, especially for the hit finding and hit-to-lead optimization phases. In addition to EC50 values, this system gives valuable first information about the mode of action of novel CAM screening compounds.

Microfluidic Chamber Design for Controlled Droplet Expansion and Coalescence.

The defined formation and expansion of droplets are essential operations for droplet-based screening assays. The volumetric expansion of droplets causes a dilution of the ingredients. Dilution is required for the generation of concentration graduation which is mandatory for many different assay protocols. Here, we describe the design of a microfluidic operation unit based on a bypassed chamber and its operation modes. The different operation modes enable the defined formation of sub-µL droplets on the one hand and the expansion of low nL to sub-µL droplets by controlled coalescence on the other. In this way the chamber acts as fluidic interface between two fluidic network parts dimensioned for different droplet volumes. Hence, channel confined droplets of about 30-40 nL from the first network part were expanded to cannel confined droplets of about 500 to about 2500 nL in the second network part. Four different operation modes were realized: (a) flow rate independent droplet formation in a self-controlled way caused by the bypassed chamber design, (b) single droplet expansion mode, (c) multiple droplet expansion mode, and (d) multiple droplet coalescence mode. The last mode was used for the automated coalescence of 12 droplets of about 40 nL volume to produce a highly ordered output sequence with individual droplet volumes of about 500 nL volume. The experimental investigation confirmed a high tolerance of the developed chamber against the variation of key parameters of the dispersed-phase like salt content, pH value and fluid viscosity. The presented fluidic chamber provides a solution for the problem of bridging different droplet volumes in a fluidic network.

Development of a lab-on-a-chip device for diagnosis of plant pathogens.

A lab-on-a-chip system for rapid nucleic acid-based analysis was developed that can be applied for diagnosis of selected Phytophthora species as a first example for use in plant pathology. All necessary polymerase chain reaction process (PCR) and hybridization steps can be performed consecutively within a single chip consisting of two components, an inflexible and a flexible one, with integrated microchannels and microchambers. Data from the microarray is collected from a simple electrical measurement that is based on elementary silver deposition by enzymatical catalyzation. Temperatures in the PCR and in the hybridization zone are managed by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply for use on site. The specificity of the lab-on-a-chip system could be demonstrated for the tested five Phytophthora species. The two Pythium species gave signals below the threshold. The results of the electrical detection of the microarray correspond to the values obtained with the control method (optical grey scale analysis).

Planar chip device for PCR and hybridization with surface acoustic wave pump.

We have developed a microfluidic device operating at a planar surface instead of a closed channel network. The fluid is transported in single droplets using surface acoustic waves (SAW) on a piezoelectric LiNbO(3) substrate. The surface of the piezo is chemically structured to induce high contact angles of the droplets or enclose areas where the liquid can wet the substrate. Combining the SAW technique with thin film resistance heaters, a biological analysis chip with integrated DNA amplification by PCR and hybridization was designed. To prevent evaporation of the PCR reagents at high temperatures the sample is enclosed in droplets of mineral oil. On this chip the SAW resolves dried primers, shifts the oil capped liquid between the two heaters and mixes during hybridization. The chip is able to perform a highly sensitive, fast and specific PCR with a volume as low as 200 nl. During the temperature cycles an online monitoring of the DNA concentration is feasible with an optical unit, providing a sensitivity of 0.1 ng. After PCR the product is moved to the second heater for the hybridization on a spotted DNA array. With our chip we were able to detect a single nucleotide polymorphism (SNP) responsible for the Leiden Factor V syndrome from human blood.