A Novel Ficoll400-Based Signal Amplification Platform for High-Sensitive Cardiac Troponin I Immunoassay

This study reports the development of a novel signal amplification carrier based on the soluble macromolecular polysaccharide Ficoll400 to enhance the sensitivity of chemiluminescent immunoassays (CLIA), with a particular focus on detecting low-abundance biomarkers, such as high-sensitivity cardiac troponin I (hs-cTnI). The carrier was constructed via the reductive amination of Ficoll400, followed by conjugation with streptavidin (SA), thereby generating dense SA binding sites capable of attaching numerous biotinylated horseradish peroxidase (Bio-HRP) molecules to amplify the chemiluminescent signal. Comparative analyses with other polysaccharides (Dextran T500 and hyaluronic acid) demonstrated that Ficoll400 conjugates, especially when aminated with l-lysine, provided superior signal enhancement with minimal background noise, improving the signal-to-noise ratio by up to 140-fold. Characterization by SDS-PAGE, dynamic light scattering, and transmission electron microscopy confirmed the formation of large macromolecular structures conducive to signal amplification. Optimization of experimental parameters, such as conjugation ratios, pH, buffer composition, and surfactant presence, further enhanced the assay performance. The developed hs-cTnI CLIA method exhibited excellent analytical sensitivity, with a limit of detection of 0.5 pg·mL^-1 and a limit of quantitation of 0.9 pg·mL^-1, alongside robust stability under accelerated and onboard conditions and high precision, with coefficients of variation below 10%. Clinical sample testing demonstrated strong correlation and consistency with a commercial hs-cTnI assay, confirming the method's reliability and potential for clinical application. Overall, the Ficoll400-SA amplification carrier provides a stable, biocompatible, and easily prepared platform that significantly enhances immunoassay sensitivity, with promising applications for the detection of various low-abundance biomarkers in vitro.