Peptide-based digital ELISA with single-molecule arrays for ultrasensitive and specific endotoxin detection in clinical plasma and pharmaceutical products.

Endotoxin detection is critical for the diagnosis and management of sepsis and for the safety evaluation of pharmaceutical products. Here, we report a peptide-based digital enzyme-linked immunosorbent assay (dELISA) that integrates two high-affinity, phage-displayed peptides into a single-molecule array platform, enabling highly sensitive and specific endotoxin detection without the use of antibodies. The carboxylated activation of magnetic beads was optimized using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) at 10 °C, and L-lysine (Lys) was identified as an effective blocking agent that maintained bead monodispersity through electrostatic repulsion. Under optimized conditions, the assay achieved a detection limit of 0.2 pg mL-1 with a linear range spanning 0-200 pg mL-1. The method exhibited strong accuracy (coefficient of variation (CV) < 15%) and interference resistance (recovery rates of 76.9%-110.1%) when applied to pharmaceutical formulations such as saline, 5% dextrose, and commercial ophthalmic solutions. In clinical validation, the platform successfully quantified endotoxin in plasma samples from septic and non-septic patients and demonstrated excellent recovery (78.0%) in whole blood spiked with exogenous endotoxin. By eliminating antibody-related limitations, this peptide-based dELISA offers a robust and translational tool for direct endotoxin detection in complex biological matrices.