Abstract
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−1. 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.
Highlights
Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins
It is extremely attractive since its electron transfer properties can be recorded by means of a direct electrochemistry approach, and the loss/gain of specific conductive groups can be correlated with specific protein conformational modifications[20]
From the ferro-/ferri-cyanide analysis summarized in Fig. 2, it is highly apparent that the current enhancement due to nanostructures on the bare screen-printed electrodes (SPEs) WE (MWCNTs and NPTs deposited on C and Pt SPEs, respectively)
Summary
Protein electrochemistry represents a powerful technique for investigating the function and structure of proteins. The study of protein structures and chemical modifications could provide new insights into the complexity of their www.nature.com/scientificreports activities (e.g., redox regulation)[12] due to the strong correlation between protein conformational dynamics and their functions In this regard, the p53 protein represents an attractive metallo-redox sensitive protein, which has been widely investigated for its involvement in different pathophysiological processes. Several studies have demonstrated that conformationally altered p53 could occur in the absence of mutations[15], and recently, this denatured p53 isoform was found to be possibly implicated in the onset of neurodegenerative diseases[16,17,18,19] As a metalloprotein, it is extremely attractive since its electron transfer properties can be recorded by means of a direct electrochemistry approach, and the loss/gain of specific conductive groups can be correlated with specific protein conformational modifications[20]. The redox activity of different p53 redox products was investigated first on carbon-based electrodes, and the performances of different bulk and nanostructured materials (C and Pt) were compared, focusing on the quantification of the completely opened isoform of p53 (denatured p53)
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