Phenothiazine-Modified Electrodes: A Useful Platform for Protein Adsorption Study

Abstract

Using glucose oxidase (GOx) as a target protein, we studied the adsorption of protein on the phenothiazine-modified electrodes and assessed the potential of using the electrodes in biochemical applications. Experiment results showed that thionine chloride (TC) and its structural analogues, such as toluidine blue and methylene blue, fluoresced under photochemical excitation after being immobilized on indium-doped tin oxide (ITO) electrodes fabricated using either diazotization-reduction or oxidative polymerization. The surface-bound phenothiazines exhibited substantial binding affinities to the protein. At a pH > 5, the adsorbate showed no sign of desorption even the electrodes were electrically biased with voltages between ±0.3 V vs SCE. Thus, emission decay occurred while GOx was injected over the electrodes, which was consistent with the observations made using conductive-mode atomic force microscopy (CM-AFM). Under a quiescent condition, the protein interacted with the immobilized TC via a pseudo-first-order kinetic mechanism. The reaction reached a maximum rate at a pH > 5, at which the rate constant was approximately 7 × 10(-8) L/(U s). Under this condition, the adsorption rate increased as the level of the protein increased, regardless of pH, revealing application potential for GOx quantitation. The adsorption rate, however, decreased with a decrease in pH if the pH < 5. We concluded that static interactions played a crucial role. By monitoring Fe(CN)6(3-/4-) taking place at the TC-modified electrodes in pH 7 solutions, we observed that the adsorption of GOx imposed impedance on Fe(CN)6(3-/4-). The resulting charge-transfer resistance (RCT) increased as the amount of the protein increased, leading to a conclusion that the protein could reach the maximum surface coverage when its concentrations were greater than 100 U/mL. The protein molecules were likely repel each other as approaching the TC sites. Despite this, they maintained the native bioactivity after being adsorbed, enabling the TC-modified electrodes to function as glucose sensors. Glucose concentrations between 1 and 60 mM could be detected. Long-term analysis, in addition, showed that the electrode responses to the analyte were consistent and reproducible. Phenothiazine-modified electrodes are evident as a useful tool for understanding the adsorption of protein on solid surfaces and quantifying proteins.