Development of a nanofluidic particle size sorter and its biomedical applications

Immunoassays are methods that allow for highly specific and sensitive assessment of a large variety of analytes in different bio-fluids, such as cell lysate, serum, or urine. Unfortunately, conventional immunoassay-associated techniques are characterized by lengthy operational protocols, high-reagent consumption, and require the use of centralized laboratories. To this extent, micro- and nanofluidic devices have gained significant interest in biomedical research due to their rapid sample processing with precise fluid control and low volume requirements. Their reduced dimensions enable the miniaturization of the whole device for lab-on-a-chip applications. In typical two-dimensional microfluidic devices, the height of the channel (out-of-plane dimension) remains constant. However, a new degree of freedom is obtained when the height is varied, enabling more complex functionalities. Furthermore, traditional microfluidic devices, to operate, often require peripheral infrastructures, such as a syringe or pressure pump. This significantly hinders their portability and applicability in resource-limited environments.
The research presented within this doctoral dissertation investigated the use of micro- and nanofluidic systems with channel variations in all three dimensions to overcome the drawbacks of conventional laboratory-based immunoassays and those of active fluidic systems. More specifically, a thermoplastic device with a vertically tapered channel was developed for size-dependent separation and immobilization of bio-functionalized particles. The immobilized particles were, in turn, used for on-chip fluorescent immunosorbent assays in the framework of various biomedical applications. The device is passively operated to reduce operational requirements and uses surface-tension effects in conjunction with specialized capillary constructs to provide a steady, homogeneous, and reproducible liquid flow.
A key device component is its nano functionality in the vertical rather than lateral axis. This unique feature allows for low aspect ratio patterning, reduces resolution requirements, and greatly simplifies downstream fabrication processes. However, the integration of these uniquely defined fluidic geometries required the use and adaptation of state-of-the-art nanofabrication protocols. An in-depth experimental characterization of grayscale e-beam lithography (g-EBL) enabled highly controllable 3D structuring of the resist material with nanoscale topography variations but on the millimeter length scale. The accuracy of the adapted g-EBL protocol was used in conjunction with thermoplastic patterning methods, such as nanoimprint lithography and injection molding, to fabricate nanofluidic devices with extremely shallow channel dimensions in an upscalable and cost-effective manner. The developed process was optimized to go from device ideation to fluidic application within a single day, enabling rapid prototyping of various geometrical parameters.
The 3D fluidic devices were used to address various shortcomings in different biomedical applications. Firstly, as the doctoral research was performed during the COVID-19 pandemic, a critical technological development was the device’s application toward rapid and quantifiable antibody detection. It was shown that the size-dependent particle trapping properties could be leveraged to perform multiplexed antibody detection of SARS-CoV-2 and Influenza A associated antibodies on distinctly functionalized particles with a high degree of sensitivity and specificity. The multiplexing capabilities of the device were extended by using secondary antibodies labeled with different fluorophores that targeted either short-term (IgM) or long-term (IgG) antibodies. This fluidic platform provided a viable alternative for the widely implemented rapid antibody and antigen tests, characterized by a limited sensitivity and only provided a binary (yes/no) result.