Facile integration of NiO nanoflowers and Ketjen Black as a robust electrochemical aptasensing platform for tramadol detection in human biofluids.

Given the high potential for abuse and significant toxicological risks of tramadol (TRM), accurate monitoring of this opioid in human biofluids is of critical medico-legal importance. Despite substantial progress in analytical technologies, few biosensing platforms successfully combine high selectivity, operational simplicity, and clinical applicability. Herein, we report a novel electrochemical aptasensor based on differential pulse voltammetry (DPV), constructed using a hybrid nanocomposite of Ketjen Black (KB) and structurally disrupted nickel oxide nanoflowers (NiO NFs). The synergistic integration of conductive KB and defect-rich NiO nanostructures provides a robust matrix for stable aptamer immobilization and efficient signal amplification. The proposed aptasensor exhibited an exceptionally broad linear range (10 pM-750 nM) with an ultra-low limit of detection of 37 pM. It maintained excellent performance in complex biological matrices, including human serum, urine and saliva, offering high recovery rates and negligible cross-reactivity with structurally related opioids. Importantly, the fabrication strategy aligns with the principles of green analytical chemistry, utilizing microliter-scale reagent volumes, avoiding hazardous chemicals, and enabling a one-pot, scalable process. Collectively, these combined features underscore the platform's practical utility and ecological soundness, making it highly promising for real-time TRM detection in both clinical diagnostics and forensic investigations.