Click-Chemistry-Enhanced Dynamic Light Scattering Immunoassay for Ultrasensitive GFAP Detection in Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) requires a rapid and objective assessment; however, current diagnostic methodologies are constrained by subjectivity, reliance on imaging techniques, and insufficient sensitivity for the early detection of biomarkers. In this study, we present a click-chemistry-enhanced dynamic light scattering (DLS) immunoassay for the ultrasensitive detection of glial fibrillary acidic protein (GFAP), a clinically significant blood biomarker for brain injury. Unlike conventional DLS immunoassays, which rely on weak antigen–antibody interactions to drive DLS probe aggregation, the proposed approach transforms immunorecognition into a rapid covalent amplification process through click chemistry. A systematic comparison of three click-chemistry-driven aggregation reactions demonstrated that faster association kinetics led to more efficient probe aggregation and stronger signal amplification. On this basis, the optimized inverse electron-demand Diels–Alder reaction (IEDDA)-enhanced DLS immunoassay exhibits the highest sensitivity for GFAP detection, achieving a limit of detection of 210 fg mL–1, which is 109-fold and 348-fold lower than that of conventional DLS immunoassays and ELISA methods, respectively. The detection time of the proposed method was only 26 min. Additionally, the IEDDA-enhanced DLS immunoassay exhibits favorable performance in the analysis of clinical serum samples. In conclusion, this study introduces an effective signal amplification strategy utilizing click chemistry to enhance the sensitivity of DLS immunoassays, thereby providing a promising analytical platform for the rapid assessment of TBI.