Article

Functionalized Graphene Oxide as a Nanocarrier in a Multienzyme Labeling Amplification Strategy for Ultrasensitive Electrochemical Immunoassay of Phosphorylated p53 (S392)

Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, People's Republic of China.
Analytical Chemistry (Impact Factor: 5.64). 02/2011; 83(3):746-52. DOI: 10.1021/ac101715s
Source: PubMed

ABSTRACT

P53 phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We report a new electrochemical immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53(392)) based on graphene oxide (GO) as a nanocarrier in a multienzyme amplification strategy. Greatly enhanced sensitivity was achieved by using the bioconjugates featuring horseradish peroxidase (HRP) and p53(392) signal antibody (p53(392)Ab(2)) linked to functionalized GO (HRP-p53(392)Ab(2)-GO) at a high ratio of HRP/p53(392)Ab(2). After a sandwich immunoreaction, the HRP-p53(392)Ab(2)-GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide. The increase of response current was proportional to the phospho-p53(392) concentration in the range of 0.02-2 nM with the detection limit of 0.01 nM, which was 10-fold lower than that of the traditional sandwich electrochemical measurement for p53(392). The amplified immunoassay developed in this work shows acceptable stability and reproducibility, and the assay results for phospho-p53(392) spiked in human plasma also show good recovery (92-103.8%). This simple and low-cost immunosensor shows great promise for detection of other phosphorylated proteins and clinical applications.

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    • "The fist graphene-based immunosensor reported was dedicated to the specific detection of phosphorylated p53 on serine 392. For immunosensor fabrication it was used screen-printed carbon electrode (SPCE) modified with AuNPs to self-assemble a layer of N-hydroxysuccinimideactivated hexa (ethylene glycol) undecane thiol (NHS) for primary attachment of phospho-p53392 capture antibody (Ab1/NHS/AuNPs-SPCE)[110]. The HRP-p53392Ab2-GO conjugate was synthetized by the functionalization of carboxylated GO with HRP and the immobilization of capture antibody p53392 (Ab2) by amidization reaction. "
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