Electrochemical differentiation of native-denatured hemoglobin using a same-metal PCB-based three-electrode system

Denaturation statistically decreases a protein's structural stability and alters its reactivity, particularly in its redox reactions. One such protein is the redox-active hemoglobin (Hb) molecule, which is among the most extensively studied due to its importance in respiration. To investigate its redox activity, the standard electrochemical sensing method using a three-electrode platform has been widely employed. In this work, however, an unconventional approach was used whereby a same-metal (gold) three-electrode sensor was utilized. Interestingly, despite the absence of dissimilar electrode materials, the current PCB-based same-metal three-electrode (PCB-3T) sensor successfully produced characteristic Hb electrochemical profiles. In phosphate-buffered saline, closely spaced anodic and cathodic peak potentials were observed, indicating enhanced redox reversibility; an outcome that is difficult to achieve in in vitro studies of conventional systems without extensive surface modification. Additionally, a cathodic potential peak was detected when Hb was dissolved in ultrapure H₂O, consistent with previous in vivo research. The miniaturized architecture of PCB-3T enables microliter-scale analysis with minimal activation cycles, making it suitable for characterizing unstable proteins, such as denatured Hb. Cyclic voltammetry (CV) results indicated that denatured Hb exhibited irregular peak potential shifts and higher peak currents than native Hb; consequently, electrochemical impedance spectroscopy revealed differences between native and denatured Hb in Nyquist and Bode plots. p-values of 0.003 and 0.02 (α = 0.05, n = 5) were obtained for CV anodic potential differentiation, indicating improved resolution, and the reproducibility offers potential for further protein electrochemistry studies.