Graphene oxide (GO) has immense potential for widespread use in diverse in vitro and in vivo biomedical applications owing to its thermal and chemical resistance, excellent electrical properties and solubility, and high surface-to-volume ratio. However, development of GO-based biological nanocomposites and biosensors has been hampered by its poor intrinsic biocompatibility and difficult covalent biofunctionalization across its lattice. Many studies exploit the strategy of chemically modifying GO by noncovalent and reversible attachment of (bio)molecules or sole covalent biofunctionalization of residual moieties at the lattice edges, resulting in a low coating coverage and a largely bioincompatible composite. Here, we address these problems and present a facile yet powerful method for the covalent biofunctionalization of GO using colamine (CA) and the poly(ethylene glycol) cross-linker that results in a vast improvement in the biomolecular coating density and heterogeneity across the entire GO lattice. We further demonstrate that our biofunctionalized GO with CA as the cross-linker provides superior nonspecific biomolecule adhesion suppression with increased biomarker detection sensitivity in a DNA-biosensing assay compared to the (3-aminopropyl)triethoxysilane cross-linker. Our optimized biofunctionalization method will aid the development of GO-based in situ applications including biosensors, tissue nanocomposites, and drug carriers.

The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning is pivotal. Here, we demonstrate highly selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO₂ substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.

Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.