A Translational Fluorescence Biosensing Strategy for Species-Specific Detection of Deinagkistrodon acutus Venom Using Complementary Time-Resolved and Rapid Diagnostic Platforms

Rapid and species-specific molecular detection remains a critical unmet need in the clinical management of snakebite poisoning, particularly when the offending species is uncertain or when residual circulating venom must be quantified after antivenom administration. Here, we developed a fluorescence-enabled dual-platform biosensing strategy for the ultrasensitive and selective detection of Deinagkistrodon acutus venom. High-titer rabbit anti-D. acutus IgG was generated using heat-detoxified whole venom, while a species-enriched single-specific equine antibody fragment (SSAb) was prepared by sequential immunoaffinity depletion of cross-reactive antibodies followed by D. acutus venom-specific enrichment. These antibody reagents were integrated into two complementary diagnostic formats: a two-step europium-based time-resolved fluorescence immunoassay (TRFIA) for laboratory quantification and a lateral flow assay (LFA) for rapid visual screening. The optimized TRFIA achieved a limit of detection (LOD) near 0.5 pg/mL and a limit of quantification (LOQ) of 1.0 pg/mL, a reportable range of 1.0–133.76 pg/mL, excellent linearity across independent experiments, and acceptable precision near the lower analytical boundary. The LFA enabled visual detection within approximately 10 min, with a visible test line appearing at 1.04 pg/mL and stronger signal development at higher venom concentrations. Neither platform showed detectable cross-reactivity with clinically relevant heterologous venoms, including Naja atra, Bungarus multicinctus, and Agkistrodon halys. In preliminary clinical serum testing, LFA positivity and TRFIA quantification were observed only in clinically diagnosed D. acutus envenomation cases, all of which were collected prior to antivenom administration; serial post-antivenom sampling will be explored in subsequent expanded clinical studies. This study establishes a clinically oriented fluorescence biosensing workflow that couples rapid point-of-care screening with ultrasensitive quantitative confirmation, providing a practical framework for precision diagnosis and treatment monitoring of D. acutus envenomation.