Capture of p53 by electrodes modified with consensus DNA duplexes and amplified voltammetric detection using ferrocene-capped gold nanoparticle/streptavidin conjugates.
p53, a tumor suppressor protein and a transcription factor, is capable of inhibiting the growth of tumor cells by eliciting either cell-cycle arrest or apoptosis through a cascade of events. p53 binds sites within the promoters of several genes that conform to a sequence commonly defined as the consensus site. In more than 50% of cancer cases, the p53 gene has been found to be mutated and the p53 protein loses its ability to bind the consensus DNA. In this work, double-stranded (ds-) oligonucleotides (ODNs) containing the consensus site are immobilized onto gold electrodes to capture wild-type p53. The cysteine residues on the exterior of the p53 molecule were derivatized for the attachment of gold nanoparticle/streptavidin conjugates capped with multiple ferrocene (Fc) groups. Well-defined voltammetric peaks of high signal intensity were obtained, and p53 concentration as low as 2.2 pM was measured. The peak heights were found to be dependent on the surface density of the consensus ds-ODN, the sequence of the immobilized ODNs, and the p53 concentration. With base pair(s) in the full consensus binding sequence altered, the level of p53 binding was found to decrease sharply, and no p53 binding occurred at electrodes covered with nonconsensus ds-ODNs. The amenability of this method to the analyses of p53 from normal and cancer cell lysates was also demonstrated. Owing to the p53 mutation in the cancer cells, the concentration of the wild-type p53 was found to decrease significantly (by about 50-182 times). The sensitivity and amenability for real sample analysis of the method compared well with enzyme-linked immunosorbant assay (ELISA), and complements ELISA in that wild-type p53, instead of total p53 (wild-type and mutant p53) concentration, is measured. The method described herein is simple and selective and does not require the use of p53 antibodies.
Wang
Jianxiu
J
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People's Republic of China 410083.
Zhu
Xu
X
Tu
Qiuyun
Q
Guo
Qin
Q
Zarui
Chikneyan S
CS
Momand
Jamil
J
Sun
Xiu Zhu
XZ
Zhou
Feimeng
F
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods; D004566:Electrodes; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005296:Ferrous Compounds / Q000737:chemistry*; D016158:Genes, p53; D006801:Humans; D000075163:Metallocenes; D009154:Mutation; D053758:Nanoparticles / Q000737:chemistry*; D009841:Oligonucleotides / Q000032:analysis / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin / Q000737:chemistry*
D005296:Ferrous Compounds; D000075163:Metallocenes; D009841:Oligonucleotides; D004247:DNA; D019809:Streptavidin; D003545:Cysteine; C004998:ferrocene