Department of Information Engineering, University of Brescia, Brescia, Italy.
Integrated Systems Laboratory (LSI), EPFL, Lausanne, Switzerland.
Sci Rep. 2019 Nov 22;9(1):17347. doi: 10.1038/s41598-019-53994-6.
Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins. Currently available biochemical assays provide limited information related to the conformational state of proteins and high costs. This work provides novel insights into the electrochemical investigation of the metalloprotein p53 and its redox products using label-free direct electrochemistry and label-based antibody-specific approaches. First, the redox activities of different p53 redox products were qualitatively investigated on carbon-based electrodes. Then, focusing on the open p53 isoform (denatured p53), a quantitative analysis was performed, comparing the performances of different bulk and nanostructured materials (carbon and platinum). Overall, four different p53 products could be successfully discriminated, from wild type to denatured. Label-free analysis suggested a single electron exchange with electron transfer rate constants on the order of 1 s. Label-based analysis showed decreasing affinity of pAb240 towards denatured, oxidized and nitrated p53. Furthermore, platinum nanostructured electrodes showed the highest enhancement of the limit of detection in the quantitative analysis (100 ng/ml). Overall, the obtained results represent a first step towards the implementation of highly requested complex integrated devices for clinical practices, with the aim to go beyond simple protein quantification.
蛋白质电化学代表了一种强大的技术，可用于研究蛋白质的功能和结构。目前可用的生化分析方法仅提供与蛋白质构象状态相关的有限信息，且成本高昂。本工作通过无标记直接电化学和基于标记的抗体特异性方法，为研究金属蛋白 p53 及其氧化还原产物的电化学提供了新的见解。首先，在碳基电极上定性研究了不同 p53 氧化还原产物的氧化还原活性。然后，聚焦于开放型 p53 同工型（变性 p53），进行了定量分析，比较了不同体相和纳米结构材料（碳和铂）的性能。总体而言，成功区分了四种不同的 p53 产物，从野生型到变性型。无标记分析表明存在单电子交换，电子转移速率常数在 1 s 数量级。基于标记的分析表明，pAb240 对变性、氧化和硝化 p53 的亲和力降低。此外，铂纳米结构电极在定量分析中表现出最高的检测限增强（100 ng/ml）。总体而言，所得结果代表了朝着实施高度要求的复杂集成设备迈出的第一步，旨在超越简单的蛋白质定量。
