Nanofluidic platforms for sensing applications in biomedical and environmental fields

Nowadays, nanofluidic platforms are powerful tools for carrying out fundamental studies on molecular-scale phenomena. Typically, the use of these systems results in being crucial both in biomedical and environmental fields. In fact, they are widely exploited for many applications such as detection, concentration, sorting, counting and sizing of several nano-objects such as nanoplastics, viruses, antibodies and DNA. This is the context in which my Ph.D. research project is inserted. I have worked on the development of elastomeric nanofluidic platforms, equipped with different nanostructures, that are the functional areas of the entire fluidic system, useful for different applications among which single nanoparticles detection, high-sensitivity immunoassay analysis and DNA sensing. Typically, nanofluidic platforms are composed of two U-shaped microchannels connected by nanostructures with suitable geometries. All devices were fabricated starting from a pre-patterned silicon mold on which nanostructures were etched using the Focus Ion Beam (FIB) milling technique. Then, the molds were replicated through a, Poly(DiMethylSiloxane)(PDMS) based, double REplica Molding (REM) technique. Although FIB is a high-resolution but expensive technology with REM technique, that is a low-cost and simple approach, I was able to fabricate many polymeric replicas with high precision re-using the mold for several times. This combination allowed obtaining high-resolution nanofluidic platforms reducing fabrication costs, a method that is potentially applicable to processes with high production rate. However, when the dimension shrinks from micro to nanoscale, PDMS presents significant limits. In particular, polymeric nanostructures suffer from the “roof collapse” phenomenon that occurs when the replica is sealed with a glass substrate, a necessary procedure to obtain watertight devices. It is possible to overcome this problem both by exploiting the Junction Gap Breakdown (JGB) technique and by using hard-PDMS (h-PDMS) during the fabrication process. During my Ph.D. research activity, I have initially worked on an asymmetric structure that was a funnel-shaped nanochannel in which the tip, after experiencing “roof-collapse”, was re-opened, thanks to the Junction Gap Breakdown procedure. From an electrical investigation of the devices fabricated with this strategy, we observed an ion current rectification characteristic and analyzing the electro-kinetic transport properties we observed that, in few minutes, intra-funnel accumulation occurs, and this phenomenon results in being stronger for low ionic strength solutions. Combining intra-funnel accumulation of biomolecules, governed by electro-hydrokinetic phenomena, that occurs applying high voltage across the device, and an appropriate functionalization of nanochannel polymeric surface with antibodies, it was possible to decrease sensing limit for the detection of one or several targeted antigens for clinical diagnostics. It was possible to identify through fluorescence optical microscopy and electrical measurements, the uptake of a specific antigen, diluted in solution (down to 1 pg/ml), to the nanochannel surface when functionalized with antibodies. So, in this condition, we successfully detected antigen-antibody binding on the nanostructure surface, a promising step for realizing a high-sensitivity nanofluidic immuno-assay sensor. Successively, I have developed other nanofluidic devices equipped with symmetric nanostructures for single-particle sensing. These devices were made using h-PDMS (hard- PDMS) in order to confer higher rigidity to the nanostructures, i.e. the functional part of the device, avoiding collapse problems. H-PDMS was used in exploiting a “focused drop-casting” approach in order to make only the nanostructure region stiffer, while leaving the other regions of the device flexible enough to avoid the formation of cracks along the device. Combining the nanoscale dimension of the sensing gate with the Resistive Pulse Sensing (RPS) technique, it was possible to analyze single nanoparticles (NPs) and the motion of single λ-DNA molecules through the nanochannel as transient variations in ionic current during the translocation events, allowing a real-time, label-free and high-sensitivity detection. In particular, it was possible to demonstrate the possibility of counting nano-objects depending on selected characteristics (i.e. charge and size ranging from 40 nm to 100 nm) that is a crucial step, useful in many fields such as medicine (drug delivery, imaging, cell-secreted carriers), environment (groundwater remediation, nanoplastics detection) and food production (nano-agrochemicals, nano-encapsulated additives, anti-microbials).