Extracellular vesicles (EV) have many attributes important for biomedicine; however, current EV isolation methods require long multi-step protocols that generally involve bulky equipment that cannot be easily translated to clinics. Our aim was to design a new cyclic olefin copolymer–off-stoichiometry thiol-ene (COC–OSTE) asymmetric flow field fractionation microfluidic device that could isolate EV from high-volume samples in a simple and efficient manner. We tested the device with large volumes of urine and conditioned cell media samples, and compared it with the two most commonly used EV isolation methods. Our device was able to separate particles by size and buoyancy, and the attained size distribution was significantly smaller than other methods. This would allow for targeting EV size fractions of interest in the future. However, the results were sample dependent, with some samples showing significant improvement over the current EV separation methods. We present a novel design for a COC–OSTE microfluidic device, based on bifurcating asymmetric flow field-flow fractionation (A4F) technology, which is able to isolate EV from large volume samples in a simple, continuous-flow manner. Its potential to be mass-manufactured increases the chances of implementing EV isolation in a clinical or industry-friendly setting, which requires high repeatability and throughput.

Extracellular vesicles are small membrane-bound structures that are released by cells and play important roles in intercellular communication garnering significant attention in scientific society recently due to their potential as diagnostic and therapeutic tools. However, separating EVs from large-volume samples remains a challenge due to their small size and low concentration. In this manuscript, we presented a novel method for separating polystyrene beads as control and extracellular vesicles from large sample volumes using bifurcated asymmetric field flow fractionation in PDMS-free microfluidic devices. Separation characteristics were evaluated using the control system of polystyrene bead mix, which offers up to 3.7X enrichment of EV-sized beads. Furthermore, in the EV-sample from bioreactor culture media, we observed a notable population distribution shift of extracellular vesicles. Herein presented novel PDMS-free microfluidic device fabrication protocol resulted in devices with reduced EV-loss compared to size-exclusion columns. This method represented an improvement over the current state of the art in terms of EV separation from large sample volumes through the use of novel field flow fractionation design.

Current in vitro models have significant limitations for new respiratory disease research and rapid drug repurposing. Lung on a chip (LOAC) technology offers a potential solution to these problems. However, these devices typically are fabricated from polydimethylsiloxane (PDMS), which has small hydrophobic molecule absorption, which hinders the application of this technology in drug repurposing for respiratory diseases. Off-stoichiometry thiol–ene (OSTE) is a promising alternative material class to PDMS. Therefore, this study aimed to test OSTE as an alternative material for LOAC prototype development and compare it to PDMS. We tested OSTE material for light transmission, small molecule absorption, inhibition of enzymatic reactions, membrane particle, and fluorescent dye absorption. Next, we microfabricated LOAC devices from PDMS and OSTE, functionalized with human umbilical vein endothelial cell (HUVEC) and A549 cell lines, and analyzed them with immunofluorescence. We demonstrated that compared to PDMS, OSTE has similar absorption of membrane particles and effect on enzymatic reactions, significantly lower small molecule absorption, and lower light transmission. Consequently, the immunofluorescence of OSTE LOAC was significantly impaired by OSTE optical properties. In conclusion, OSTE is a promising material for LOAC, but optical issues should be addressed in future LOAC prototypes to benefit from the material properties.

Indium tin oxide (ITO) is a transparent conducting material with exceptional electrical and optical properties, widely used in biosensing and bioelectronics. Functionalization of ITO with linker molecules enables covalent attachment of biomolecules, allowing for dual-mode optical and electrochemical detection. Carbonic anhydrase IX (CA IX), a transmembrane enzyme overexpressed in hypoxic tumors, is a promising biomarker for cancer diagnostics due to its restricted expression in normal tissues. However, conventional detection methods are time-intensive and unsuitable for point-of-care applications. In this study, ITO surfaces were functionalized using silane-based chemistry to immobilize CA IX-specific antibodies, creating a novel biosensing platform. The biosensor utilized a secondary horseradish peroxidase (HRP)-conjugated antibody to catalyze the oxidation of luminol in the presence of hydrogen peroxide, producing a chemiluminescent and electrochemical signal. Characterization of the biosensor via a dual-mode optical and electrochemical approach revealed efficient antibody immobilization. Due to the high variation observed in the optical approach, limit of detection (LOD) experiments were conducted exclusively with electrochemistry, yielding an LOD of 266.4 ng/mL. These findings demonstrate the potential of ITO-based electrochemical biosensors for sensitive and selective CA IX detection, highlighting their applicability in cancer diagnostics and other biomedical fields.

Surface modification is essential in microfluidic applications due to the inherent hydrophobicity of polymers, which can lead to biofouling and reagent denaturation. Despite the development, challenges such as hydrophobic molecule absorption and limitations in scaling are still present. Off-stoichiometry thiol-ene (OSTE) materials have emerged as a promising alternative, offering advantages like rapid prototyping, minimal hydrophobic absorption, and customizable surface chemistries. While the thiol-ene polymerization mechanism is well understood, the fundamental understanding of thiol group binding on OSTE surfaces remains limited. Existing techniques to analyze surface groups lack the capability to confirm the stable presence of thiol groups on the surface. In our study, using Raman and X-ray photoelectron spectroscopy techniques, we investigated a potential method for enhancing the surface properties of OSTE polymer—the attachment of novel linkers to the surface. We have demonstrated our synthesized compound efficiency by binding gold nanoparticles to the OSTE surface. Our findings indicate that chemical reactions involving double bonds with the material surface hold the most potential for effective surface modification for gold binding.