Pretreatment Strategies for Optimizing Multilayer Coatings on Additively Manufactured Ti6Al4V for Sensing Platforms

The layer-by-layer (LbL) assembly technique is a versatile method for fabricating multilayer coatings with precise control over composition and structure. Its ability to create functionalized surfaces makes it highly suitable for biomedical diagnostics and biosensors. Multilayer films composed of chitosan (CHI), a polycation, and hyaluronic acid (HA), a polyanionic glycosaminoglycan, present biocompatibility and the specific interaction of HA with CD44 receptors, commonly overexpressed on circulating tumor cells (CTCs). This interaction represents a potential strategy for early cancer detection. This study applies the LbL technique to enhance CTC adhesion on Ti6Al4V alloy electrodes, produced via additive manufacturing for biosensor applications. A key focus is optimizing the deposition process to improve film performance by investigating plasma pretreatment and a polyethyleneimine (PEI) precursor layer. These methods aim to refine electrode surface properties, enhancing the selectivity and sensitivity of CHI/HA coatings for oncological diagnostics. The Ti6Al4V electrodes were polished with sandpaper and cleaned with acetone and isopropyl alcohol. The electrodes were subjected to cold air plasma (Plasma Cleaner PDC-32G, Harrick Plasma) for 15 min and followed by PEI monolayer deposition. To evaluate the treatments, electrodes that were not treated with plasma and PEI or received only one treatment were also tested. CHI/HA multilayer films were then deposited via LbL assembly. Surface hydrophilicity and topography were evaluated, and PC3 prostate cancer cells were cultured to study cell adhesion. The CHI/HA films increased electrode hydrophilicity, primarily due to HA's water-affinity properties. Atomic force microscopy (AFM) revealed reduced surface roughness after coating, attributed to the filling of grooves by the polyelectrolytes. Electrodes with plasma pretreatment and PEI pre-layer showed intermediate roughness values, suggesting enhanced adhesion properties. These surface modifications improved PC3 cell adhesion, particularly on pretreated electrodes, aligning with the HA-CD44 receptor interaction. Plasma and PEI pretreatments further improved film deposition and surface characteristics, creating conditions for cell attachment. In conclusion, integrating plasma pretreatment and PEI pre-layer deposition into the LbL process significantly improved the adhesion and functionality of CHI/HA films on Ti6Al4V electrodes. The pretreatments applied to the metallic substrates significantly improved the effectiveness of LbL functionalization, enhancing this technique as a more efficient and accessible approach for electrode modification in sensor development.