Porous reduced graphene oxide modified electrodes for the analysis of protein aggregation. Part 1: Lysozyme aggregation at pH 2 and 7.4
Porous reduced graphene oxide modified electrodes for the analysis of protein aggregation. Part 1: Lysozyme aggregation at pH 2 and 7.4
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DOI：
10.1016/j.electacta.2017.09.083
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发表时间：
2017-11
影响因子：
6.6
通讯作者：
A. Vasilescu;Samia Boulahneche;F. Chekin;S. Gáspár;M. Medjram;A. Diagne;Santosh K. Singh;Sreekumar Kurungot;R. Boukherroub;S. Szunerits
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文献类型：
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作者：
A. Vasilescu;Samia Boulahneche;F. Chekin;S. Gáspár;M. Medjram;A. Diagne;Santosh K. Singh;Sreekumar Kurungot;R. Boukherroub;S. Szunerits
由于错误折叠和聚集导致的蛋白质不稳定性是基于蛋白质的疗法的一个重要问题，因为它会影响此类药物的生物利用度和免疫原性。因此，开发简单且经济有效的方法来分析药物制剂，表明蛋白质聚集体的存在或不存在，具有非常重要的意义。这项工作提出了一种基于多孔还原氧化石墨烯涂层玻碳电极（GC/prGO）的新型电化学界面，可以通过跟踪蛋白质氧化电流的变化来早期、灵敏地识别蛋白质聚集。这项工作的新颖之处在于探索 GC/prGO 接口捕获不同聚合行为的能力。溶菌酶被用作模型，通过电化学跟踪其在两个 pH 值（pH 2 和 pH 7.4）下的聚集，分别导致淀粉样蛋白和无定形聚集体的形成。通过微分脉冲伏安法 (DPV) 比较不同电极结构的溶菌酶氧化峰，可以验证 GC/prGO 界面与裸玻璃电极或涂有无孔还原氧化石墨烯的电极相比具有更高的灵敏度。通过硫黄素 T 荧光、尺寸排阻色谱和原子力显微镜 (AFM) 成像进行平行实验。这些测试进一步强调了 GC/prGO 电极以快速可靠的方式可视化蛋白质结构的变化以及 pH 2 和 pH 7.4 下发生的过程之间的差异的有用性。特别是，能够强调与聚集第一步相关的变化，这可能表明聚集过程，建议将 GC/prGO 电极与 DPV 结合作为生物制药聚集研究的新分析工具。这项工作的第 2 部分稍后将展示该方法在分析速效注射人胰岛素制剂 Humulin R（用于糖尿病治疗以及降钙素）方面的实用性。
Protein instability due to misfolding and aggregation is of big concern for protein based therapeutics because it impacts the bioavailability and immunogenicity of such drugs. The development of simple and cost-effective methods for the analysis of pharmaceutical formulations, indicating the presence or absence of protein aggregates, is consequently of high importance. This work proposes a novel electrochemical interface based on porous reduced graphene oxide coated glassy carbon electrode (GC/prGO) allowing for the early and sensitive identification of protein aggregation by following the change in the oxidative current of the proteins. The novelty of this work lies in the exploration of the ability of GC/prGO interfaces to capture different aggregation behaviors. Lysozyme is used as a model to follow by electrochemistry its aggregation at two pH values, pH 2 and pH 7.4, leading to the formation of amyloid and amorphous aggregates, respectively. Comparing the oxidation peak of lysozyme by differential pulse voltammetry (DPV) for different electrode architectures allowed validating the higher sensitivity of the GC/prGO interface versus bare glassy electrodes or electrodes coated with non-porous reduced graphene oxide. Parallel experiments were performed by fluorescence with thioflavin T, size exclusion chromatography and Atomic Force Microscopy (AFM) imaging. These tests further highlighted the usefulness of GC/prGO electrode to visualize in a fast and reliable manner the changes in the protein structure and the differences between the processes occurring at pH 2 and pH 7.4. In particular, the ability to emphasize changes related to the first steps in aggregation that could be indicative of the aggregation course, recommend the GC/prGO electrode in combination with DPV as a new analytical tool for aggregation studies of biopharmaceuticals. Part 2 of this work will demonstrate later the utility of this approach for the analysis of a fast acting injectable human insulin formulation, Humulin R, used for diabetes treatment as well as for calcitonin.