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

Abstract 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.