Polyethylene glycol passivated nanoplasmonic biosensor for sensitive detection in complex biological samples

Abstract

Nanoplasmonic biosensors hold immense potential for diagnostics, yet their application in complex biological fluids is significantly hampered by non-specific binding (NSB). This study reports the development and systematic optimization of a robust gold nanoparticle (AuNP)-based localized surface plasmon resonance (LSPR) platform on a 96-well plate format, engineered to minimize NSB for reliable sensing. For this purpose, ~30 nm citrate-stabilized AuNPs were first synthesized and immobilized onto polyethylenimine (PEI) coated well plate bottoms. To mitigate surface fouling, PEI-AuNP platform was passivated using various concentrations of thiol-polyethylene glycol 2000 (PEG2K)-N-Hydroxysuccinimide (NHS). The anti-fouling efficacy was rigorously evaluated against both bovine serum albumin (BSA) and the more challenging fetal bovine serum (FBS). The platform's functionality for specific detection was then validated using a model IgG/anti-IgG immunoassay. The optimized PEI-AuNP platform exhibited a characteristic LSPR peak at ~527 nm and demonstrated a refractive index sensitivity of 55.6 nm/RIU. Critically, surface passivation with heterobifunctional PEG at concentrations of 50 µM and higher effectively resisted biofouling, resulting in statistically insignificant LSPR shifts upon exposure to both BSA and FBS (p gt; 0.1). The platform's quantitative performance was validated with a model immunoassay in undiluted FBS, yielding a limit of detection (LOD) of 0.73 nM. This study presents a well-characterized and robust nanoplasmonic biosensor platform on a standard 96-well plate, in which a single-molecule, heterobifunctional PEG-based surface chemistry is systematically optimized to achieve resistance to non-specific binding in complex biological media, representing an important step toward the development of high-throughput, label-free, and sensitive diagnostic assays suitable for point-of-care applications.