University thesis:
Dissertation, Universität Freiburg, 2020
Footnote:
Description:
Abstract: Centrifugal microfluidics is one among several microfluidic platforms to miniaturize, integrate, parallelize and automate biochemical assays for the point-of-need (PON). This work aims to reduce system complexity in molecular diagnostics on the centrifugal microfluidic platform, while maintaining integration density. Predominant applications cover the detection of pathogens causing infectious diseases, food safety or monitoring of cancer therapies. Moving complex workflows from a centralized lab to the PON is challenging, because it requires the integration of all process steps and reagents into a single cartridge (here: the LabDisk), including a sample interface, pre-storage and release of all required reagents, purification of nucleic acids, mixing with amplification reagents and performing the final analysis, allowing for an evident decision. In this framework, this thesis focusses on the reduction of complexity of two unit operations, to increase overall system robustness and performance: Automated nucleic acid extraction and dead-volume free mixing of eluted nucleic acids with dry reagents.<br>To maintain full control over liquid menisci in a rotating system, and thus, to increase the system’s robustness, continuous rotation is required for all processing steps. This applies especially for the highly wetting buffers in nucleic acid extraction. To provide continuous rotation during nucleic acid extraction, I introduce the concept of “magnetophoresis at continuous rotation”. A stationary magnetic field was provided by four permanent magnets located above the LabDisk, exerting a radially inwards pointing magnetic force on magnetic beads in the LabDisk. Magnetophoresis in radial inward direction was achieved in the present setup at a critical frequency of 5 Hz followed by azimuthal bead transfer to the adjacent chamber, which occurred by means of particle inertia, supported by deflection structures. By this, the beads were transported three times in azimuthal direction along four adjacent microfluidic chambers holding the extraction buffers. The process was verified by extracting nucleic acid from mosquito pools, whereas eluates from LabDisk and reference extractions (in tube) showed comparable results in terms of protein contamination (LabDisk: A260/A280 = 1.6±0.04; n = 4 vs. reference: A260/A280 = 1.8±0.17; n = 3) and threshold values (Ct) in real-time RT-PCR (LabDisk: Ct = 17.9±1.6, n = 4) falling in the 95 % confidence interval (CI) of the reference (Ct = 19.3±1.7, 95 % CI = [17.5 - 21.0], n = 3). Notably, ethanol concentration was quite high in the obtained eluate (cEthanol ~10 % (v/v)), here, corresponding to 18 μL ethanol carry-over.<br>Consequently, for the first time, I comprehensively investigated ethanol carry-over during nucleic acid extraction in the LabDisk, aiming to reduce it below an inhibitory level for downstream analysis (cEthanol, threshold ≤ 2.5 % (v/v)). While I could not observe significant carry-over of ethanol within the magnetic bead cluster, diffusion of ethanol vapor in the gas phase and condensation at the eluate was identified as the dominating effect. In the utilized LabDisk, ethanol carry-over by diffusion was measured to be as high as 2.1 nL s-1, 2.6 nL s-1 and 4.1 nL s-1 at 25 °C, 35 °C and 45 °C, respectively. To reduce the diffusion of ethanol vapor into the eluate passive countermeasures were taken. These included a reduction of the cross-section of the diffusion path (cEthanol = 4.2±0.5 % (v/v), equal to 7.6 μL carry-over, n = 3) and the introduction of a dynamic pressure pump inducing gas convection in the cartridge (cEthanol = 0.2±0.3 % (v/v), equal to 0.36 μL carry-over, n = 3).<br>In batch mode mixing, dead volumes during rehydration and mixing of lyophilized reagents are a big challenge, resulting in concentration gradients and thus inconsistent reaction conditions in downstream reactions. In this work, I introduce “thermo-pneumatic bubble mixing”, to compensate dead-volumes in downstream channels without the need for additional means. A high fluidic resistance vent channel acts as a low-pass filter with respect to pressure changes in the downstream air volume allowing for overpressure generation at high positive temperature change rates. Overpressure leads to bubble formation, pushing liquid from the downstream channel back into the mixing chamber. The concept was verified by rehydrating lyophilized reagents for loop-mediated isothermal amplification (LAMP). Mixing efficiency was characterized by the mean coefficient of variation (CV̅̅̅̅, n = 4 LabDisks) of the time-to-positivity (tp) of the LAMP reactions in 11 replicate reaction chambers on the LabDisk, resulting in a CV̅̅̅̅ reduction from 18.5 % to 3.3 %.<br>In summary, the presented unit operations are a major achievement towards less complex, and more robust integration. This strongly extends the toolbox for centrifugal microfluidic assay integration in molecular diagnostics. The work simplified system complexity by consequently exploiting the inherent advantages of the centrifugal microfluidic platform, while maintaining integration density