Switchable nanozyme-based colorimetric assay for protamine and heparin using silver citrate nanoparticles

Monitoring protamine and heparin is essential to maintaining the delicate balance between anticoagulation and its reversal during surgical and cardiovascular procedures. Disruptions in this balance can result in severe complications such as excessive bleeding or thrombosis, highlighting the critical need for precise, real-time monitoring tools. In this study, we present a novel, switchable colorimetric sensor based on silver citrate nanozymes with laccase-like catalytic activity. The sensing mechanism relies on electrostatic modulation: protamine, a positively charged biomolecule, inhibits nanozyme activity, while heparin, a negatively charged polyanion, reverses this effect—producing a distinct and reversible color change. This interaction enables the ultra-sensitive detection of protamine (LOD: 9.3 ng/mL) and heparin (LOD: 36.0 ng/mL). The sensor operates across clinically relevant ranges for both analytes (protamine: 0.08–350 μg/mL; heparin: 0.2–380 μg/mL), matching therapeutic plasma levels observed in perioperative and anticoagulation contexts. The platform was successfully validated in complex biological matrices, including human serum and urine, achieving high recovery rates (96.0–105.8 %) and excellent reproducibility (RSD < 3.27 %). Owing to its simplicity, cost-effectiveness, and robustness, this assay offers considerable promise for integration into clinical diagnostic workflows and perioperative anticoagulant management.