Feature Article Electron Transfer Reaction of Cytochrome c at the Electrode Surface Analyzed with Noncontact Optical Waveguide Spectroscopy Kyoko Fukuda and Hiroyuki Ohno* Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan e-mail: ohnoh@cc.tuat.ac.jp Received: June 11, 2001 Final version: September 21, 2001 Abstract We proposed a unique application of optical waveguide (OWG) spectroscopy under noncontact condition to analyze the electrochemical redox reaction of molecules on the electrode surface. The spectral change of cytochrome c (cyt.c), induced by the potential switching, was clearly detected. In addition, the absorbance change at the Soret band and Q- band was recorded dynamically with this apparatus. The absorbance change was revealed to simply reflect the electron transfer reaction of cyt.c at the electrode surface. The magnitude of the intensity change was gradually diminished suggesting some cyt.cs were adsorbed on the electrode surface and suppressed further redox reaction of the dissolved cyt.cs. The noncontact measurement enables to take the absorption spectra of only the adsorbed cyt.c on the electrode. The adsorbed cyt.c was revealed to be denatured containing ferric ion in the heme and the redox activity was completely lost. Keywords: Noncontact, Optical waveguide, Cytochrome c, Redox reaction 1. Introduction In solution, proteins dissolved frequently adsorbed on several substrates nonspecifically. The adsorption often followed the structural change and the loss of biological activityoftheproteins[1].Therefore,itisimportanttostudy the adsorption phenomena especially to design a surface which prevent the protein adsorption or the denaturation of proteinsafteradsorption.Theinformationonthecharacter- istics of the adsorbed proteins should also be useful in medical, biomaterial, biosensor research, and related fields. Severalmethodshavebeenreportedtoanalyzetheproteins on the electrode surface [2, 3]. However, there was no convenientmethodtoanalyzethevisiblespectraofproteins onthesubstratesurface.UV-visiblespectroscopy,whichisa general and potential method to analyze the transition energy of electrons for molecules reflecting the chemical environment. However, it is difficult to observe the absorp- tion spectra of molecules that were monomolecularly dispersed on the substrate even optically transparent ones such as ITO electrode. Some techniques to analyze the molecules on the substrate by using the interaction of an evanescent field were developed [4 ± 7]. Optical waveguide (OWG) spectroscopy has recently been recognized as a useful method to analyze the adsorbed molecules on the waveguidesurface[1,7±14].OWGspectroscopyisakindof internal refraction spectroscopy, and detects absorption of the evanescent wave by samples on the waveguide surface. OWG spectroscopy is expected to realize a nondestructive, sensitive and in situ analysis. There are some articles on the UV-visible [9, 10] or IR spectra [11] and the near-field florescence [12] of the samples adsorbed on the waveguide surface.Allsuchpreviousreportsilluminatedtheanalysisof the molecules directly adsorbed on the waveguide. Further- more, the spectral change followed by the electrochemical response of the molecules was reported to be detectable withOWGspectroscopy[13].Inthiscase,theITOlayerwas formed onto the quartz plate to use this as both the waveguide and the working electrode. There is a strict limit of measurement for this, i.e., only optically transparent electrode enables this kind of OWG analysis. Since, most of solid materials for the analysis of protein adsorption were optically opaque, the dynamic information on the proteins at the interface cannot be developed. The purpose of our study is to provide convenient OWG measurement for the analysis of molecules on the electrode regardless of optical transparency. It was revealed that the OWG spectrum of the molecules adsorbed on the substrate, which is not in contact with the waveguide surface, could be detected when the gap is properly controlled. In a previous article, we determined the relation between the absorbance of the spectrum and the gap between the target substrate and the waveguide [14]. Latex beads with appropriate diameter were found to be effective as the gap controller. According to these basic data, we constructed an electro- chemical cell system on the waveguide in order to analyze the electron transfer reaction. In the present work, after the confirmation of this cell system, cytochrome c (cyt.c) was used as a target molecule and analyzed the behavior at the electrode. This is the first report to analyze the redox 605 Electroanalysis 2002, 14, No. 9 ¹WILEY-VCH Verlag GmbH, 69469 Weinheim, Germany, 2002 1040-0397/02/0905-0605 $ 17.50+.50/0