Monitoring Inhibition of Hemoglobin Peroxidase Activity After Exposure to Cigarette Smoke Using an Electrochemical Biosensor

This work presents a catalysis-based electrochemical biosensor to evaluate the peroxidase-like activity of methemoglobin (Hb-PLA) after exposure to cigarette smoke (CS) at different time intervals. The system consists of a microelectrode array coupled with a PDMS chamber containing a methemoglobin solution (biorecognition element). Hydrogen peroxide (H₂O₂) acts as the substrate, while 3,3',5,5'-tetramethylbenzidine (TMB) functions as the chromogenic substrate for the Hb-PLA through its oxidation reaction. A spectrophotometric technique is used as a reference method to assess the catalytic activity of methemoglobin. Positive control samples exhibited higher absorbance, indicating strong catalytic activity, whereas CS-exposed samples showed a marked reduction, which was confirmed by the negative control. Cyclic voltammetry revealed significant alterations in the oxidation and reduction peaks of the CS-exposed samples. Therefore, chronoamperometry was employed to quantify the charge transfer as the electrochemical response associated with Hb-PLA, yielding a sensitivity of 0.86 ± 0.06 (%Hb-PLA/mC) and a limit of detection (LOD) of 0.23 (mC). The results demonstrate that cigarette smoke impairs the Hb-PLA in a time-dependent manner, with longer exposure reducing the activity by up to 25%. The proposed biosensor provides a rapid, sensitive, and straightforward strategy for detecting functional alterations in solutions of methemoglobin induced by environmental pollutants such as cigarette smoke.