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Exposing a carbon electrode to hemoglobin (Hb) and alcoholic solvents, such as methanol, ethanol or 1-propanol, drastically changes Hb electroactivity, but until this work, the important underlying mechanisms were unclear. For the first time, we show that these alcohols impact Hb electroactivity via three mechanisms: modification of the carbon surface oxides on the glassy carbon (GC) electrode, Hb film formation, and structural changes to Hb. C X-ray photoelectron spectroscopy provided evidence for significant alcohol-induced modification of the carbon surface oxides, and differential pulse voltammetry showed links between these modifications and Hb electroactivity. Spectroscopic ellipsometry showed that Hb films formed during exposure to Hb- and alcohol-containing electrolytes increased in thickness with increasing alcohol content, although film thickness played only a minor role in Hb electroactivity. Alcohol-induced structural changes in Hb are confirmed with UV-visible absorption and fluorescence data, showing that Hb denaturation also was a significant factor in increasing Hb electroactivity. Carbon-surface-oxide modification and Hb denaturation worked in tandem to maximally increase the Hb electroactivity in 60% methanol. While in ethanol and 1-propanol, the significant increases in Hb electroactivity caused by Hb denaturation were offset by an increase in Hb-inhibiting carbon surface oxides. Knowledge of these mechanisms shows the impact of alcohols on both Hb and carbon electrodes, allows for thoughtful design of the Hb-sensing system, is vital for proper analysis of Hb electroactivity in the presence of these alcohols (e.g., when used as binder solvents for immobilizing Hb into films), and provides fundamental understanding of the Hb-carbon interactions.

The electrochemical biosensing of hemoglobin (Hb) may provide a relatively fast method of detection with the possibility of developing point-of-care diagnostics or at-home monitoring; however, obtaining a Hb electrochemical signal is often slow and difficult because the four redox active heme groups are buried in the interior hydrophobic regions of the protein. Ideally, the biosensor electrode would be inexpensive and formed from environmentally sustainable materials, such as carbon. However, the electroactivity of Hb is inconsistent on different carbon electrode materials. Additionally, Hb electroactivity is strongly dependent on whether the Hb is immobilized on the electrode surface or is in a solution-based sample. Clearly, for a point-of-care diagnostic, it would be useful to be able to measure the Hb concentrations within a liquid phase, and therefore a carbon material which evidences Hb-electroactivity is required. Until this work, there was little understanding of the role that carbon-oxygen surface functional groups (from the carbon electrode) played in Hb electroactivity. We examine Hb electroactivity of several carbons are examined through cyclic voltammetry and differential pulse voltammetry in a neutral phosphate buffered electrolyte. Carbon-oxygen surface functionalities were characterized using X-ray photoelectron spectroscopy (XPS), thermal programmed desorption (TPD) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). Our findings show that Hb electroactivity is inhibited by ether and carbonyl surface groups present on the carbon electrode. Using this information, a Hb-inactive carbon (Vulcan XC-72) was made active for Hb detection by removal of these surface groups (see figure). Ultrasonication removed the ether functionalities, resulting in a significant increase in the Hb electroreduction. The amount of reduction was increased further if the ultrasonication was followed by a relatively simple 15 minute electroreduction at -1.95 V vs. Hg/Hg2SO4 (saturated K2SO4) in stirred 10 mM phosphate electrolyte (pH 5.03) purged with N2. The electroreduction was shown to selectively remove the carbonyl and quinone functionalities, resulting in the increase Hb electroactivity. The knowledge of carbon-oxygen surface functionalities is essential to better understand hemoglobin’s electroactivity on carbon and influences the choice of carbon electrode materials for further development of hemoglobin electrochemical biosensors. Figure: Representative CVs (a) and DPVs (b) of glassy carbon (dotted green curve), ultrasonicated Vulcan XC-72 on glassy carbon (dashed purple curve) and ultrasonication+electrochemically reduced Vulcan XC-72 on glassy carbon (solid purple curve) in 0.1 M PB containing 0.2 g L-1 BHb from at least three replicate trials. Figure 1

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