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.

Extracellular vesicles (EVs) are promising biomarkers for diagnosing complex diseases such as cancer and neurodegenerative disorders. Yet, their clinical application is hindered by challenges in isolating cancer-derived EVs efficiently due to their broad size distribution in biological samples. This study introduces a microfluidic device fabricated using off-stoichiometry thiol-ene and cyclic olefin copolymer, addressing the absorption limitations of polydimethylsiloxane (PDMS). The device streamlines a standard laboratory assay into a semi-automated microfluidic chip, integrating sample mixing and magnetic particle separation. Using the microfluidic device, the binding kinetics between EVs and anti-CD9 nanobodies were measured for the first time. Based on the binding kinetics, already after 10 min the EV capture was saturated and comparable to standard laboratory assays, offering a faster alternative to antibody-based immunomagnetic protocols. Furthermore, this study reveals the binding kinetics of EVs to anti-CD9 nanobodies for the first time. Our findings demonstrate the potential of the microfluidic device to enhance clinical diagnostics by offering speed and reducing manual labor without compromising accuracy.