Immune cells such as monocytes and neutrophils responds to external stimuli and during the invasion by foreign body undergoes phagocytosis during which reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−), hydrogen peroxide (H₂O₂) and hydroxyl radical (^•OH) are produced. The ROS generated in animal cells can then lead to oxidative degradation of biomolecules comprising of lipids, proteins and nucleic acid influencing the structural organization and metabolic functions within the cells. Reactive oxygen species are known to be involved in several diseases and ailments when either generated in excess or if the balance between the scavenging activities mediated by antioxidants and its production is disturbed. Therefore, specific and sensitive ROS detection methods is required for precise evaluation and monitoring of oxidative stress which can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed iron-porphyrin based modified carbon electrode as novel detection sensor with high specificity and sensitivity for detection of O₂^•−in animal cells.

Methods:

The detection of O₂^•− is based on amperometry comprising of counter electrode and working electrodes. The counter electrode is a platinum plate (2.5×2.5mm) while the working electrodes (φ1mm) was an iron-porphyrin modified carbon electrode. Ag/AgCl was used as a reference electrode.The oxidation current of O₂^•−  was measured at +0.5V veresus Ag/AgCl in phosphate buffer solution of containing 11.4mM glucose and THP-1(human monocytic cell line). The iron-porphyrin modified carbon electrode acts as O₂^•−detection sensor (scheme 1).

The iron-porphyrin modified carbon electrode is prepared by the electropolymerization of 1-methylimidazole-coordinated iron meso-tetra (3-thienyl) porphyrin ([Fe(im)₂(ttp)]Br). For the basics characterization of iron porphyrin modified carbon electrode, differential pulse voltammogram for the electropolymerized [Fe(im)2(ttp)]Br complex was recorded in an aqueous electrolyte solution containing phosphate buffer at pH 7.2 at a scan rate of 50 mV/sec using  a high performance potentiostat HZ-7000 (Hokuto denko Co., Ltd., Japan).

Results and Discussion

The O₂^•− generation was measured in THP-1 cells under the effect of differentiation inducer phorbol 12-myristate 13-acetate (PMA) in the absence and presence of exogenous addition 0.01 wt% Triton X at 25°C at a cell density of 3.0 × 10⁶ cells/well. Triton X is known to permeabilize the membranes of living cells. The results obtained showed no considerable changes in oxidation currents for O₂^•− under the effect of inducer (PMA). However, the addition of Triton-X showed a fast increase in oxidation current for O₂^•− by approximately 2 nA followed by a gradual drop which continued for a time span of more than 3 hours. To confirm the production of O₂^•−, the effect of superoxide dismutase (SOD) on oxidation current in PMA induced THP-1 cells was tested. The addition of 0.01 wt% Triton X100 in presence of SOD showed small change in  oxidation currents for O₂^•−.

Conclusion

The newly developed iron-porphyrin modified carbon electrode is claimed to be a useful tool for real-time monitoring and precise detection of O₂^•− in animal cells. It can claimed to bear potential for its wide application in biomedical research and clinical trials with high specificity and sensitivity.