Quantitative evaluation of biomolecule-gold nanoparticle conjugation with varying morphologies and application of monodispersed conjugates for SPR biosensing

The conjugation of biomolecules to gold nanoparticles (AuNPs) is fundamental to applications in nanobiotechnology, including plasmonic biosensors for diagnostics, therapeutics, and nanoassemblies. Despite the importance of anisotropic and larger AuNPs for achieving stronger optical response and novel functionalities, conjugation remains challenging due to reduced colloidal stability. Conventional characterization methods are hindered by demanding sample preparation, lack of in situ capability, and size estimations biased by particle heterogeneity and spherical assumptions, leading to inaccuracies for anisotropic nanoparticles. Here, we present a rapid and sensitive method for quantitative evaluation of DNA-AuNP conjugation using UV–vis spectroscopy aided with Assembly Degree Metric (ADM), a parameter derived from localized surface plasmon resonance spectral characteristics. ADM integrates the analysis of spectral shifts and peak broadening to assess conjugation efficiency, nanoparticle stability, and nanoassembly formation. We validated our approach against Dynamic Light Scattering and Transmission Electron Microscopy and showed its adaptability across different AuNP morphologies by testing spherical and star-shaped AuNPs of varying sizes (50–80 nm). Our method enabled the production of monodisperse large anisotropic bioconjugates, which we utilized these conjugates as signal amplification elements in a surface plasmon resonance biosensor. The ADM method can be further applied to nanobiotechnology areas, including high-performance nanoplasmonic biosensors, photothermal heating and functional DNA nanostructures.