Journal
ANALYTICAL CHEMISTRY
Volume 83, Issue 3, Pages 746-752Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac101715s
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Funding
- National Institutes of Health CounterACT program through the National Institute of Neurological Disorders and Stroke [NS058161-01]
- PNNL Laboratory Directed Research and Development program
- U.S. Department of Energy (DOE) by Battelle [DE-AC05-76RL01830]
- DOE's office of Biological and Environmental Research located at PNNL
- National Natural Science Foundation of China [21075047]
- Program for Chenguang Young Scientist for Wuhan [200950431184]
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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 p53392. The amplified immunoassay developed in this work shows acceptable stability and reproducibility, and the assay results for phospho-p53392 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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