Gold nanoparticles (GNPs) conjugated to biomolecules are crucial for various applications in biotechnology, medicine, and diagnostics . The distinctive optical properties of gold nanoparticles, including their strong surface plasmon resonance and intense scattering and absorption properties, make them useful as contrast agents for various imaging techniques, including electron microscopy, optical microscopy, and lateral flow assays . The conjugation with antibodies, antigens, proteins, or nucleic acids enables to bind the GNPs to desired biomolecular targets minimizing interference from other components present in the sample. When conjugated with specific biological molecules, gold nanoparticles (GNPs) provide high sensitivity, and multiplexing capabilities in biosensing facilitating the detection of various targets through colorimetric, fluorescent, or surface-enhanced Raman scattering (SERS) detection methods . GNP-based sensor platforms with microfluidic devices result in the development of portable, point-of-care diagnostic tools applicable across healthcare, food safety, and environmental monitoring sectors . The necessary stage in incorporating GNPs into any sensing platform requires their modification with biomolecular ligands. However, the process of conjugation presents numerous obstacles. Preserving the functionality of biomolecules during conjugation is paramount to maintain assay performance and accuracy. The functionalization of GNPs with large biomolecules or multiple ligands may introduce steric hindrance effects, which could impact the accessibility of binding sites or the efficiency of target recognition. Therefore, optimizing the spacing and orientation of biomolecules on GNPs is crucial for minimizing steric hindrance effects and maximizing assay sensitivity .