Subpicogram-Level Quantification of Trace Biomarkers Using an Automated High-Sensitivity Chemiluminescence Immunoassay Technology Based on a Novel Substrate Enhancement Strategy

Trace biomarkers showed great potential for improving the diagnosis and monitoring of various diseases. However, the sensitivity of the current high-throughput detection platforms is limited, posing a significant challenge for the reliable measurement and clinical application of trace biomarkers. We developed an automated high-sensitivity chemiluminescence immunoassay (CLIA) technology with subpicogram-level sensitivity based on the modification of the substrate molecular structure and the synergy of surfactant and fluorescein. Using this technology, we constructed assay kits for five trace biomarkers: leukemia inhibitory factor (LIF), high-sensitivity cardiac troponin I (hs-cTnI) and T (hs-cTnT), phosphorylated tau-217 (p-tau217), and estradiol (E2). The analytical and clinical performance was evaluated by examining the precision, linearity, limits of detection, and methodological comparison. The high-sensitivity chemiluminescent substrate demonstrated approximately 7-fold higher sensitivity and 2.5-fold greater luminescence efficiency compared to those of the traditional substrate, with a linear range exceeding 160 million relative light units (RLUs). The five trace biomarker assays exhibited excellent analytical sensitivity, with the LoD values of 0.070 pg/mL (LIF), 0.18 pg/mL (hs-cTnI), 1.1 pg/mL (hs-cTnT), 0.007 pg/mL (p-tau217), and 0.113 pg/mL (E2 II). Compared with other high-sensitivity detection methods, the Passing-Bablok linear correlation coefficients (r) ranged from 0.9767 to 0.9974. The proportions of cTnI, cTnT, and E2 II concentrations above the LoD in healthy populations exceeded 98%. We established an automated high-sensitivity CLIA technology based on a novel substrate enhancement strategy, improving the analytical sensitivity to the subpicogram level. An automated high-throughput detection method of various trace biomarkers was realized that may have broad application in the clinic.