Wiederkehr Rodrigo S, Hoops Geoffrey C, Aslan Mustafa M, Byard Courtney L, Mendes Sergio B
Department of Physics and Astronomy, University of Louisville, Louisville, Kentucky 40292, Department of Chemistry, Butler University, 4600 Sunset Avenue, Indianapolis, Indiana 46208.
J Phys Chem C Nanomater Interfaces. 2009 May 14;113(19):8306-8312. doi: 10.1021/jp810845e.
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.
在这项工作中，我们报告了细胞色素c在不同亚单层水平吸附到氧化铝波导表面上的摩尔吸光率的实验结果；我们的数据表明蛋白质光学性质对其表面密度有明显的依赖性。测量是使用宽带、单模集成光波导光谱技术进行的，这是一种极其灵敏的工具，能够达到这类研究所需的亚单层检测水平。本研究重点关注Q波段（中心波长为525 nm）的摩尔吸光率，据我们所知，这也是首次研究表面吸附的细胞色素c的弱电荷转移（CT）波段（中心波长为695 nm）。450至775 nm光谱区域的偏振光全部耦合到氧化铝薄膜中，该薄膜用作单模平面光波导。用于这项工作的氧化铝薄膜波导厚度为180 nm，通过原子层沉积工艺沉积在玻璃基板上。蛋白质亚单层通过在中性pH值的水性缓冲溶液中静电吸附在氧化铝波导表面形成。研究了水性缓冲溶液中本体蛋白质浓度范围为5 nM至8200 nM时表面吸附的细胞色素c的光学性质。对于蛋白质表面密度为2.3 pmol/cm²，在电荷转移波段测得的摩尔吸光率为335 M⁻¹ cm⁻¹，对于表面密度为15 pmol/cm²，摩尔吸光率为720 M⁻¹ cm⁻¹，这与溶解在中性水性缓冲溶液中的细胞色素c的值（830 M⁻¹ cm⁻¹）更为接近。蛋白质摩尔吸光率的变化及其对表面密度的依赖性很可能归因于表面吸附物种的构象变化。
