Electrochemical Impedance Spectroscopy for studying fluorescence loss in immobilized Green Fluorescent Protein

Electron transport and transition within a molecule are involved in some of the major cellular and biochemical processes in an organism. In proteins, electron transfer is generally characterized by two mechanisms, electron tunnelling and electron hopping. Green Fluorescent Protein (GFP) is the most widely used fluorescent protein in various biological studies. We know that folding occurs in GFP followed by an autocatalytic cyclization and finally oxidation to form a chromophore. The fluorescence phenomenon would cease in proteins which have lost their native structure. In this paper, we have immobilized Strep-TMR (Streptavidin-TMR), BSA (Bovine Serum Albumin) and GFP on a solid surface and checked its functionality under a fluorescence microscope. We also measured the impedance of these bound proteins and found out that & omega;max of the Nyquist plot in GFP was less than that of Strep-TMR and BSA. However, when GFP was photobleached by light exposure at various time intervals, an increase in & omega;max was observed. This photobleaching and impedimetric correlation observed in case of GFP was compared with the other two proteins as well. Studies have suggested that a number of mechanisms are involved in photobleaching, like structural changes due to photodegradation, ceasing of electron transport or the completion of absorption emission cycles of a fluorophore. In our study, it seems that the electrons responsible for fluorescence in GFP might be responsible for the changes in the & omega;max of the Nyquist plot of EIS system. This study is one of the first few approaches that has tried to establish a relationship between fluorescence and electrochemical changes within a fluorescent protein upon photoexposure.

Automated summary

Electron transport and transition within a molecule play important roles in cellular and biochemical processes. In this study, we immobilized Strep-TMR, BSA, and GFP on a solid surface and examined their functionality under a fluorescence microscope. We found that electron transfer in GFP affects the impedance of the system, and this relationship was further explored through photobleaching experiments.