Development of next-generation microfluidic systems for enhanced, faster, and cost-effective immunoassays for tissue diagnostics - Ac electrothermal flow & Acoustofluidics

The application of microfluidics in the field of surface-based assays and more specifically, the spatial molecular profiling of tumor tissues has gained a lot of interest, especially with the increased interest in personalized medicine and targeted therapy. During a static incubation of the immunoreaction reagents with the tissue section, the reagents begin to adsorb and bind to the target epitopes on the tissue sample. However, the laminar fluid flow associated with the small dimensions of microfluidic systems and the domination of the diffusional mass transport of reagents can limit their transport to the tissue. Furthermore, during the reaction time, a depletion region is formed around the target area with a much lower abundance of the reagent. At this point, the reaction rate becomes limited due to the scarcity of the detection reagents and their diffusion-limited transport, which can lead to very lengthy experiments. Moreover, uniform staining of the tissue plays an important role in the analysis, since it ensures that all relevant antigen epitopes on the tissue are equally exposed to the detection reagents, thereby enhancing the signal contrast and facilitating quantifiable detection.In the current work, we have investigated and integrated two microfluidic mixing techniques, including AC electrothermal flow (ACET), and acoustofluidic mixing (AF). First, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume and close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. As a proof of concept, the new design has been used in ACET-enhanced immunoassays to improve the immunostaining signal of the HER2 cancer biomarker (Human epidermal growth factor receptor 2) on breast cancer cells. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time. Moreover, we also provide detailed insight into the working mechanism of the new ACET design, allowing us to propose several new designs for the ACET in-plane microfluidic motion. Finally, a preliminary in-plane design is investigated for the application of our design in microfluidic pumping.In the second topic of our research, AF mixing could be easily integrated into an industrial prototype system for fast and automatic tissue diagnostics. Our new proposed AF technology has the advantage of the ease of integration, and operation at low frequencies (< 10 KHz range) thus omitting the need for advanced and high-frequency electronic devices, low cost of the core piezo-electric actuation elements, and retaining the possibility of microscopic imaging over the complete microfluidic chamber, and, finally, the absence of thermal interference with the conducted staining experiments. AF mixing was successfully used to achieve uniform staining on large cancer cell pellet sections, even when using small fluid flows and short incubation times. AF mixing could reduce by 80% the incubation time of the Her2 and CK (Cytokeratins) biomarkers on breast cancer cell pellets with respect to the existing protocol of spatial immunostaining, and reduce their concentration by 66% while achieving a higher signal-to-background ratio than obtained in comparable spatially resolved immunoassays with static incubation.