Multiplexed electrochemical immunoassay of phosphorylated proteins based on enzyme-functionalized gold nanorod labels and electric field-driven acceleration.

A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53(392)), Ser15 (phospho-p53(15)), Ser46 (phospho-p53(46)), and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes, and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by a multienzyme amplification strategy using gold nanorods (AuNRs) as nanocarrier for coimmobilization of horseradish peroxidase (HRP) and detection antibody (Ab(2)) at a high ratio of HRP/Ab(2), which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min; thus, the whole sandwich immunoreactions could be completed in less than 5 min. Under optimal conditions, this method could simultaneously detect phospho-p53(392), phospho-p53(15), phospho-p53(46), and total p53 ranging from 0.01 to 20 nM, 0.05 to 20 nM, 0.1 to 50 nM, and 0.05 to 20 nM with detection limits of 5 pM, 20 pM, 30 pM, and 10 pM, respectively. Accurate determinations of these proteins in human plasma samples were demonstrated by comparison to the standard ELISA method. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

[1]  Dan Du,et al.  Functionalized graphene oxide as a nanocarrier in a multienzyme labeling amplification strategy for ultrasensitive electrochemical immunoassay of phosphorylated p53 (S392). , 2011, Analytical chemistry.

[2]  J. Vaqué,et al.  Ultrasensitive electrochemical immunosensor for oral cancer biomarker IL-6 using carbon nanotube forest electrodes and multilabel amplification. , 2010, Analytical chemistry.

[3]  Jun Liu,et al.  Sensitive immunosensor for cancer biomarker based on dual signal amplification strategy of graphene sheets and multienzyme functionalized carbon nanospheres. , 2010, Analytical chemistry.

[4]  Feng Yan,et al.  Dual signal amplification of glucose oxidase-functionalized nanocomposites as a trace label for ultrasensitive simultaneous multiplexed electrochemical detection of tumor markers. , 2009, Analytical chemistry.

[5]  J. Bar,et al.  Expression of p53 Protein Phosphorylated at Serine 20 and Serine 392 in Malignant and Benign Ovarian Neoplasms: Correlation With Clinicopathological Parameters of Tumors , 2009, International Journal of Gynecologic Cancer.

[6]  James F Rusling,et al.  Ultrasensitive immunosensor for cancer biomarker proteins using gold nanoparticle film electrodes and multienzyme-particle amplification. , 2009, ACS nano.

[7]  Yafeng Wu,et al.  Enzyme-functionalized silica nanoparticles as sensitive labels in biosensing. , 2009, Analytical chemistry.

[8]  Jun-Jie Zhu,et al.  Gold Nanoparticle–Colloidal Carbon Nanosphere Hybrid Material: Preparation, Characterization, and Application for an Amplified Electrochemical Immunoassay , 2008 .

[9]  Feng Yan,et al.  Electric field-driven strategy for multiplexed detection of protein biomarkers using a disposable reagentless electrochemical immunosensor array. , 2008, Analytical chemistry.

[10]  Guodong Liu,et al.  Quantum-dot-based electrochemical immunoassay for high-throughput screening of the prostate-specific antigen. , 2008, Small.

[11]  Dakrong Pissuwan,et al.  Prospects for Gold Nanorod Particles in Diagnostic and Therapeutic Applications , 2008, Biotechnology & genetic engineering reviews.

[12]  Feng Yan,et al.  A disposable multianalyte electrochemical immunosensor array for automated simultaneous determination of tumor markers. , 2007, Clinical chemistry.

[13]  Ruo Yuan,et al.  Magnetic control of an electrochemical microfluidic device with an arrayed immunosensor for simultaneous multiple immunoassays. , 2007, Clinical chemistry.

[14]  Karen H. Vousden,et al.  p53 in health and disease , 2007, Nature Reviews Molecular Cell Biology.

[15]  C. D. Geddes,et al.  Microwave triggered metal enhanced chemiluminescence: Quantitative protein determination. , 2006, Analytical chemistry.

[16]  G. Wahl,et al.  Regulating the p53 pathway: in vitro hypotheses, in vivo veritas , 2006, Nature Reviews Cancer.

[17]  Yuehe Lin,et al.  Electroactive silica nanoparticles for biological labeling. , 2006, Small.

[18]  Guodong Liu,et al.  Sensitive immunoassay of a biomarker tumor necrosis factor-alpha based on poly(guanine)-functionalized silica nanoparticle label. , 2006, Analytical chemistry.

[19]  Guodong Liu,et al.  Apoferritin-templated synthesis of metal phosphate nanoparticle labels for electrochemical immunoassay. , 2006, Small.

[20]  Joseph D. Gong,et al.  Carbon nanotube amplification strategies for highly sensitive immunodetection of cancer biomarkers. , 2006, Journal of the American Chemical Society.

[21]  H. Ju,et al.  Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers. , 2006, Analytical chemistry.

[22]  Michael S. Wilson,et al.  Multiplex measurement of seven tumor markers using an electrochemical protein chip. , 2006, Analytical chemistry.

[23]  T. Morozova,et al.  Active bead-linked immunoassay on protein microarrays. , 2006, Analytica chimica acta.

[24]  Michael S Wilson,et al.  Electrochemical multianalyte immunoassays using an array-based sensor. , 2006, Analytical chemistry.

[25]  H. Imagawa,et al.  Rapid and reagent-saving immunoassay using innovative stirring actions of magnetic beads in microreactors in the sequential injection mode. , 2005, Talanta.

[26]  Michael S Wilson,et al.  Electrochemical immunosensors for the simultaneous detection of two tumor markers. , 2005, Analytical chemistry.

[27]  Guodong Liu,et al.  Electrochemical coding for multiplexed immunoassays of proteins. , 2004, Analytical chemistry.

[28]  Zigang Dong,et al.  Post-translational modification of p53 in tumorigenesis , 2004, Nature Reviews Cancer.

[29]  Xin Lu,et al.  Ser392 Phosphorylation Regulates the Oncogenic Function of Mutant p53 , 2004, Cancer Research.

[30]  T. Morozova,et al.  Electrophoresis-assisted active immunoassay. , 2003, Analytical chemistry.

[31]  C. Harris,et al.  Distinct pattern of p53 phosphorylation in human tumors , 2001, Oncogene.

[32]  E. Appella,et al.  Post-translational modifications and activation of p53 by genotoxic stresses. , 2001, European journal of biochemistry.

[33]  A. Levine,et al.  Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Kapoor,et al.  Functional activation of p53 via phosphorylation following DNA damage by UV but not γ radiation , 1998 .

[35]  W. Heineman,et al.  Simultaneous immunoassay using electrochemical detection of metal ion labels. , 1994, Analytical chemistry.

[36]  A. Tichý,et al.  Gamma-radiation-induced phosphorylation of p53 on serine 15 is dose-dependent in MOLT-4 leukaemia cells. , 2009, Folia biologica.