Hydrophobic DNA (H-DNA) nano-film was formed as the surface modifier on a thin glass plate working as a slab optical waveguide (SOWF). Cytochrom c (cytc) molecules were immobilized from aqueous solution with direct contacting to the H-DNA nano-film for 30 minutes. From SOWG absorption spectral changes during automated solution exchange (SE) processes, it was found that about 28.1% of cytc molecules was immobilized in the H-DNA nano-film with keeping their reduction functionality by reducing reagent.

Hydrophobic DNA (H-DNA) nano-film was formed on a thin glass plate of 50μm thick working as a slab optical waveguide. Bromothymol blue (BTB) molecules were immobilized from aqueous solution with direct contacting to the H-DNA nano-film for 20 minutes. From changes in absorption spectra observed with slab optical wave guide (SOWG) during automated solution exchange (SE) processes for 100 times, it was found that about 95% of bromothymol blue (BTB) molecules was immobilized in the H-DNA nano-film with keeping their functionality of color change responsible to pH change in the solution.

To immobilize cytochrome $c$ (cyt.,$c$) on ITO electrode with keeping its direct electron transfer (DET) activity, 10-carboxydecylphosphonic acid (10-CDPA) self-assembled monolayer (SAM) film was formed on ITO electrode. After 100 times washing process with exchanging phosphate buffer saline solution in the cell to fresh one, extit{in situ} slab optical waveguide (SOWG) absorption spectral measurement proved that about 80% of cyt.,$c$ immobilized on 10-CDPA modified ITO electrode was adsorbed on ITO electrode. Additionally SOWG spectral change of cyt.,$c$ between oxidized and reduced forms was observed with setting the ITO electrode potential at 0.3 and $-$0.3,V vs. Ag/AgCl, respectively showing DET reaction between cyt.,$c$ and ITO electrode occurred. About 30% of a monolayer coverage was estimated from the coulomb amount in the surface area of oxidation and reduction peaks on cyclic voltammetry (CV) data. CV peak current maintained 84% for ITO electrode modified with 10-CDPA SAM film after 60,min continuous scan with 0.1,V/sec from 0.3 and $-$0.3,V vs. Ag/AgCl.

In situ UV-vis. absorption spectra of cytochrome c adsorbed on ITO electrode was observed with slab optical waveguide spectroscopy combining pulse potential step (PPS) between 0.3 and -0.45 V vs. Ag/AgCl. The amount of cytochrome c adsorbed on ITO electrode was estimated from the amount of coulomb of the peaks in cyclic voltammogram to be about a monolayer coverage in this experimental condition. Spectral change between oxidized and reduced cytochrome c by PPS was finished in about 20 msec with phosphate buffer solution. The results strongly proved that SOWG spectroscopy should be effective for in situ observation of ET reaction kinetics of surface adsorbed molecules.

In situ observation of the adsorption process and the states of cytochrome c on glass/solution interfaces, and the functionality of the reduction reaction of adsorbed cytochorome c were performed by using slab optical waveguide (SOWG) spectroscopy. The peak position of the absorption band of cytochorome c adsorbed on a bare glass surface was almost the same as that of that in solution. The cytochorome c adsorbed on glass/solution interface was reduced by sodium dithionite solution. The adsorbed cytochorome c was still maintained its functionality after immobilization.

In situ observation of the adsorption process and reduction behavior of hemoglobin adsorbed on a bare glass surface was studied using slab optical waveguide (SOWG) spectroscopy. The peak position of the absorption band of hemoglobin adsorbed on the glass surface was almost the same as that of hemoglobin in solution. This result agrees with results previously reported by our group. The adsorbed hemoglobin molecules were also reduced by sodium dithionite solution. The adsorbed hemoglobin molecules still maintained their function in this experimental condition.

The adsorption behavior of cytochrome c was investigated using slab optical waveguide (SOWG) absorption spectroscopy at the near ultraviolet region utilizing thin quartz plates as planar waveguides. SOWG absorption spectra of cytochrome c measured at constant time intervals showed significant influence of surface hydrophilicity and solution chemistry on the adsorption of this important heme protein in quartz surface. Being polar and typically amphoteric, the protein preferred adsorption on hydrophilic surface than on hydrophobic surface as implied by the lower absorbance data obtained in the latter than in the former. At lower ionic strength and in the absence of buffer, the protein molecules tend to adsorb on the quartz surface. Plots of near steady-state absorbance versus protein concentration follow hyperbolic pattern in the absence of buffer or at low ionic strength and become more linear as the buffer concentration is increased. The results presented here are explained in terms of the general qualitative understanding of protein adsorption at solid-aqueous interfaces and further aids in elucidating the properties of protein monolayers and films.