Characterization of Agarose Microdroplet Properties for Acoustic Trapping of Encapsulated Bacteria and On-chip DNA Extraction

Sammanfattning: The aim of this project was to investigate if the translation of a DNA extraction protocol to on-chip, for the purpose of reducing the total assay time, would be possible. This was to be enabled by encapsulating bacteria in hydrogel microdroplets that would then be acoustically trapped in a microfluidic system. Further, the suitability of using agarose as the hydrogel for bacteria encapsulation and acoustic trapping was to be investigated. Agarose microdroplets had previously proven unsuitable for acoustic trapping due to the exhibition of a negative contrast factor that was believed to be caused by the presence of microbubbles in the gel. The removal of these microbubbles was achieved by introducing a degas step into the preparation procedure of the gels and the effect of the degassing was confirmed by density measurements of the gels. Agarose microdroplets, at five different agarose concentrations, with an average droplet diameter of 78.05 ± 15.12μm were produces using a microfluidic chip utilizing a cross-flow design made from PDMS and glass. The microfluidic chip containing the acoustic trap was made from Si and glass where the acoustic trap was 400μm3. The designs of both microfluidic chips were based on a design by B. Stenestam. The three-dimensional, acoustic trapping of the agarose droplets, at all different concentrations used, was successful. The agarose droplets exhibited a positive contrast factor and the droplets could be retained against flows allowing for the full exchange of liquids in the trap tens of times per minute. In the end, the DNA extraction protocol was never run on-chip. It was however run on bacteria encapsulated in agarose microdroplets in an Eppendorf tube. Bacteria were encapsulated into agarose microdroplets at a 1% agarose concentration and DNA was successfully extracted from the bacteria at both reduced incubation temperatures and times. The DNA extracted was later analyzed and the results showed that there was no difference in the quality of the DNA extracted following the traditional DNA extraction protocol or the modified protocol that was run on the microdroplets. The total assay time for the DNA extraction protocol run on the agarose microdroplets was three hours and 30 minutes, which is a 30% reduction in total assay time compared to the traditional DNA extraction protocol that has a total assay time of five hours. The final conclusion, based on the results of this project, was that it will be possible to translate the preexisting DNA extraction protocol to on-chip using the methods presented in this project, introducing a few alterations, and that it is likely that this will result in a reduction of the total assay time.