Lab-on-a-chip technology for protein detection in bio-reactors.

Lab-on-a-chips and micro total analysis systems have been around for more than three decades. They find applications in medical, environmental and food diagnostics and appear in many different configurations and complexities. They all have in common that fluids are transported in small volumes through micrometer sized structures. A promising and growing subcategory of these microfluidic systems are the multiphase, droplet or segmented flow microfluidics. Discrete volumes of reagents are transportedinside microfluidic channels, surrounded with an immiscible fluid or a gas, which prevents cross-contamination between the plugs or droplets. These droplets with a typical volume between 1 microliter and 1 picolitercan be used as reaction vessels for (bio)chemical reactions or for bioassays such as ELISA and PCR. They can be generated at high frequencies, up to 10000 per second, which is especially promising for high-throughput applications.
Many commonly applied bioassays (e.g., ELISA) use a solid support to anchor the antibodies or capture probes, allowing the separation of targets from the complex biological matrices. Magnetic nano-or microparticles are perfectly suited for this task due to their high surface-to-volume ratio and the potential to magnetically separate them from the sample matrix. In that context droplet-based segmented flow microfluidics (DBSF) and on‑chip manipulation of magnetic microparticles appears a logic and attractive combination. However, integrating both technologies requires a good understanding of micro-engineering, microfluidics and bioassay development to come to a successful diagnostic device. The objective of this PhD research was to study the integration of magnetic microparticle manipulations in a DBSF microfluidic chip.
Thefirst part of this work consisted mainly of capacity building. First, existing fabrication protocols for polydimethylsiloxane (PDMS) microfluidic chips were adjusted to match the available equipment and the design requirements. Next, DBSF was implemented in the microchannels. In order to minimize the absorption and migration of biomolecules, specific fluorocarbon was introduced, replacing traditional hydrocarbon-based oil. Thisin turn required corresponding surfactants and surface coating and madeit necessary to repeat much previously reported work. The basic unit operations such as droplet generation, mixing and splitting were studied before novel concepts were attempted.
In the second part, a novel microfluidic concept was developed to actively control the splitting ratio of droplets. A computational fluid dynamics model was composed, validatedand implemented to simulate the effect of different designs and flow parameters. With the final design, droplet splitting ratios between 50/50 and 95/5 were achieved solely by controlling the operational parameters.This considerably reduces the time necessary to match the splitting ratio with potential (bio)assays in future work. The dynamic control especially accelerated the experimental phase of microparticle separation in the follow-up research.
In the third part, the magnetic configuration to separate the microparticles from the sample matrix was studied using mathematical simulations and experimental tests. A detailed three-dimensional model of the magnetic field in proximity of a cubic permanent magnet was applied to accurately calculate the magnetic force on superparamagnetic particles. Using these results, the conditions for particle aggregation, attraction and immobilization were studied and good separation conditions were determined. In the improved setup, up to 90% of the droplet volume was removed from the microparticles while the non-separated particle fraction remained below 5%. Only when more than 95% of the original sample volume was removed in a single purification step, 10% of the microparticles were not correctly separated.Finally, in a last part, a selective DNA extraction assay with microparticles was studied in the DBSFmicrofluidic system to evaluate the overall performance of the novel design. It was demonstrated that the hybridization and capture efficiency of the biofunctionalized particles was identical for off‑chip and on‑chip methods. Also, the effect of the particle separation efficiency on the extraction efficiency was tested for different splitting regimes. Finally, the impact of separation at a higher splitting ratio forthe repeated washing of the particles was discussed. The successful implementation of selective DNA extraction is new to the field of DBSF microfluidics and is very promising for future applications.</>

Keywords:Microfluidics, Aptamers, Bioreactors

Disciplines:Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering, Analytical chemistry, Pharmaceutical analysis and quality assurance

Project type:PhD project