Investigations on the Q and CT Bands of Cytochrome c Submonolayer Adsorbed on an Alumina Surface Using Broadband Spectroscopy with Single-Mode Integrated Optical Waveguides

Abstract

In this work, we report experimental results on the molar absorptivity of cytochrome c adsorbed at different submonolayer levels onto an aluminum oxide waveguide surface; our data show a clear dependence of the protein optical properties on its surface density. The measurements were performed using the broadband, single-mode, integrated optical waveguide spectroscopic technique, which is an extremely sensitive tool able to reach submonolayer levels of detection required for this type of studies. This investigation focuses on the molar absorptivity at the Q-band (centered at 525 nm) and, for the first time to our knowledge, the weak charge transfer (CT) band (centered at 695 nm) of surface-adsorbed cyt c. Polarized light in the spectral region from 450 to 775 nm was all-coupled into an alumina thin film, which functioned as a single-mode planar optical waveguide. The alumina thin-film waveguide used for this work had a thickness of 180 nm and was deposited on a glass substrate by the atomic layer deposition process. The protein submonolayer was formed on the alumina waveguide surface through electrostatic adsorption from an aqueous buffer solution at neutral pH. The optical properties of the surface-adsorbed cyt c were investigated for bulk protein concentrations ranging from 5 nM to 8200 nM in the aqueous buffer solution. For a protein surface density of 2.3 pmol/cm(2), the molar absorptivity measured at the charge transfer band was 335 M(-1) cm(-1), and for a surface density of 15 pmol/cm(2) was 720 M(-1) cm(-1), which is much closer to the value of cyt c dissolved in an aqueous neutral buffer (830 M(-1) cm(-1)). The modification of the protein molar absorptivity and its dependence on the surface density can most likely be attributed to conformational changes of the surface-adsorbed species.

Figure 1

Schematic drawing of the waveguide from the lateral view with the flowcell mounted on the top surface. The black dots represent the protein adsorbed to the surface. The light with TE polarization was coupled to the waveguide by the diffraction gratings patterned in the substrate.

Figure 2

Sensitivity values as function of light wavelength for an alumina waveguide with 180 nm of thickness and a propagation length of 3.4 cm.

Figure 3

Light intensity against wavelength measured with the single-mode waveguide spectrometer at different steps of an experiment to determine the optical absorbance spectrum of a protein film.

Figure 4

(a) Absorbance for cytochrome c adsorbed to the alumina waveguide surface at different concentrations. (b) Magnified view of the charge transfer band. The legends show the protein concentration in the bulk solution measured after being adsorbed to the waveguide surface.

Figure 5

(a) Example of straight line subtraction from the charge transfer absorption band. (b) Absorbance of the CT band after straight-line subtraction for variable solution concentrations of cytochrome c.

Figure 6

Surface coverage for cytochrome c adsorbed on an alumina waveguide surface for different bulk concentrations. An adsorption equilibrium constant, K_ad, of (10 ± 2) × 10⁶ M⁻¹ was determined from our experimental results.

Figure 7

(a) Molar absorptivity for cytochrome c adsorbed to an aluminum oxide surface. The curves represent different surface densities calculated for the different concentrations in bulk solution. (b) Magnified view of the 670–720 nm region to better visualize the CT band.

Figure 8

Molar absorptivity for cyt c in solution of 10 μM cytochrome c in buffer solution (7 mM phosphate, 10 mM NaCl, pH 7.2). The black curve was obtained by placing the 1 cm cuvette in the optical path of our waveguide-based spectrometer, and the red curve was acquired using a commercial UV–vis spectrophotometer.

Figure 9

Ratio of the second derivative of the molar absorptivity with respect to the wavelength divided by the molar absorptivity. The quantity R in the y-axis is independent of the sensitivity factor and surface density calculated for the waveguide. Therefore, it can directly address if a particular transition band is changing its spectral behavior.