Laboratoire d'Electrochimie Moléculaire, UMR CNRS 7591, Université Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Baïf, F-75205 Paris Cedex 13, France.
J Am Chem Soc. 2012 Apr 18;134(15):6834-45. doi: 10.1021/ja301193s. Epub 2012 Apr 9.
Nanoporous films of indium tin oxide (ITO), with thicknesses ranging from 250 nm to 2 μm, were prepared by Glancing Angle Deposition (GLAD) and used as highly sensitive transparent 3D-electrodes for quantitatively interrogating, by time-resolved spectroelectrochemistry, the reactivity of microperoxidase-11 (MP-11) adsorbed within such films. The capacitive current densities of these 3D-electrodes as well as the amount of adsorbed MP-11 were shown to be linearly correlated to the GLAD ITO film thickness, indicating a homogeneous distribution of MP-11 across the film as well as homogeneous film porosity. Under saturating adsorption conditions, MP-11 film concentration as high as 60 mM was reached. This is equivalent to a stack of 110 monolayers of MP-11 per micrometer film thickness. This high MP-11 film loading combined with the excellent ITO film conductivity has allowed the simultaneous characterization of the heterogeneous one-electron transfer dynamics of the MP-11 Fe(III)/Fe(II) redox couple by cyclic voltammetry and cyclic voltabsorptometry, up to a scan rate of few volts per second with a satisfactory single-scan signal-to-noise ratio. The potency of the method to unravel complex redox coupled chemical reactions was also demonstrated with the catalytic reduction of oxygen by MP-11. In the presence of O(2), cross-correlation of electrochemical and spectroscopic data has allowed us to determine the key kinetics and thermodynamics parameters of the redox catalysis that otherwise could not be easily extracted using conventional protein film voltammetry. On the basis of numerical simulations of cyclic voltammograms and voltabsorptograms and within the framework of different plausible catalytic reaction schemes including appropriate approximations, it was shown possible to discriminate between different possible catalytic pathways and to identify the relevant catalytic cycle. In addition, from the best fits of simulations to the experimental voltammograms and voltabsorptograms, the partition coefficient of O(2) for the ITO film as well as the values of two kinetic rate constants could be extracted. It was finally concluded that the catalytic reduction of O(2) by MP-11 adsorbed within nanoporous ITO films occurs via a 2-electron mechanism with the formation of an intermediate Fe(III)-OOH adduct characterized by a decay rate of 11 s(-1). The spectroelectroanalytical strategy presented here opens new opportunities for characterizing complex redox-coupled chemical reactions not only with redox proteins, but also with redox biomimetic systems and catalysts. It might also be of great interest for the development and optimization of new spectroelectrochemical sensors and biosensors, or eventually new photoelectrocatalytic systems or biofuel cells.
通过掠入射沉积（GLAD）制备了厚度为 250nm 至 2μm 的氧化铟锡（ITO）纳米多孔薄膜，并将其用作高度灵敏的透明 3D 电极，通过时间分辨光谱电化学定量研究吸附在这些薄膜中的微过氧化物酶-11（MP-11）的反应性。这些 3D 电极的电容电流密度以及吸附的 MP-11 的量与 GLAD ITO 薄膜厚度呈线性相关，表明 MP-11 在薄膜中均匀分布以及薄膜具有均匀的多孔性。在饱和吸附条件下，达到了高达 60mM 的 MP-11 薄膜浓度。这相当于每微米薄膜厚度有 110 层 MP-11 单层。这种高浓度的 MP-11 薄膜负载量与优异的 ITO 薄膜导电性相结合，允许通过循环伏安法和循环伏安吸光法同时对 MP-11 Fe(III)/Fe(II) 氧化还原偶的非均相单电子转移动力学进行特征描述，扫描速率高达每秒几伏，具有令人满意的单扫描信号噪声比。该方法还通过 MP-11 对氧气的催化还原来证明其能够揭示复杂的氧化还原偶联化学反应。在 O2 存在下，电化学和光谱数据的互相关允许我们确定氧化还原催化的关键动力学和热力学参数，否则使用常规的蛋白质薄膜伏安法很难提取这些参数。基于循环伏安图和伏安吸光图的数值模拟，并在包括适当近似的不同可能的催化反应方案的框架内，有可能区分不同的可能催化途径并识别相关的催化循环。此外，从模拟对实验循环伏安图和伏安吸光图的最佳拟合中，可以提取 O2 的 ITO 薄膜的分配系数以及两个动力学速率常数的值。最后得出结论，吸附在纳米多孔 ITO 薄膜中的 MP-11 催化还原 O2 是通过 2 电子机制进行的，形成了特征衰减速率为 11s-1 的 Fe(III)-OOH 中间产物。这里提出的光谱电化学分析策略不仅为氧化还原蛋白，而且为氧化还原仿生系统和催化剂的复杂氧化还原偶联化学反应的表征开辟了新的机会。对于新的光谱电化学传感器和生物传感器的开发和优化，或者最终对于新的光电催化系统或生物燃料电池，它也可能具有很大的兴趣。
