Screening and SPR-guided maturation of anti-ricin cyclononapeptide for high-sensitivity fluorescent LFA integrated with ultracompact handheld reader.

Ricin is a highly lethal plant-derived biotoxin posing severe threats to public security and necessitating rapid, sensitive, and on-site detection methods. To overcome the inherent batch-to-batch variability of traditional antibodies, synthetic peptides have emerged as promising alternative recognition elements. Herein, we report a de novo screening and surface plasmon resonance (SPR)-guided affinity maturation of novel anti-ricin cyclononapeptides for the development of a highly sensitive fluorescent lateral flow assay (LFA). Through a virtual-experimental iterative strategy integrating phage display, in silico molecular docking, alanine/histidine scanning, and double-site saturation mutagenesis, we evolved an optimum variant cyclopeptide (F7) with a 100-fold enhanced binding affinity (KD = 165 nM). Based on F7, we constructed two novel sandwich LFAs involving cyclopeptide-monoclonal antibody (mAb) or cyclopeptide-glycoprotein using time-resolved fluorescent microspheres as sensitive signal reporters. Facilitated by a self-developed ultracompact handheld reader, this optimized LFA achieved a high sensitivity of 0.1 ng/mL without requiring complicated signal amplification. Crucially, the developed LFA method demonstrated excellent matrix tolerance, enabling reliable semi-quantification of trace ricin in complex authentic samples, including human plasma, beverages, castor plant tissues, and suspected white powder mixtures. Combined with the self-developed ultracompact handheld LFA reader, the LFA platform exhibited satisfactory capture efficiency in simulated bioterrorism surface-swabbing scenarios. It is expected that this field-deployable, high-performance LFA platform could prove instrumental in the rapid risk assessment, on-site screening of complex environmental matrices, and emergency monitoring of severe biological hazards.