Electrochemical DNA Hybridization Assay: Enzyme‐Labeled Detection of Mutation in p53 Gene

Abstract

AbstractThis paper discusses a new electrochemical DNA hybridization sensing approach based on the detection of a linked enzyme label. In this method we employ enzyme that is attached to a tethered ssDNA oligomer on the surface and the target analyte is a complementary ssDNA oligomer that does not require any pre‐treatment. The advantage of using of enzyme label is in its amplification of the registration of the hybridization event due to the catalytic reaction facilitated in the process. One particular novelty is associated with the use of enzymes that directly communicate with the electrode surface thus allowing for minimizing the need of additional reagents in the assay. The electrochemical assay was demonstrated when using mixed self‐assembled monolayers from thiolated oligonucleotide and 6‐mercapto 1‐hexanol on gold surfaces. Horseradish peroxidase (HRP) is attached to the surface tethered oligonucleotide using streptavidin‐biotin chemistry, and the enzyme successfully established direct electron transfer (DET) with the electrode or mediated electron transfer (MET) using a mediator. Hybridization results in increasing the angle of contact between electrode and DNA and also the stiffness of the ds DNA, which results in displacing the enzyme away from the electrode surface, and thereby reducing the occurrence of direct electron transfer between the enzyme and the electrode. The cyclic voltammetry showed a clear distinction in response between the complete complementary sequence and the two‐base mismatch sequence. Ellipsometric measurements show that the thickness of the thiol modified oligonucleotide on gold surfaces changes before and after hybridization for the complementary sequence, where as a minimal change in thickness was observed for the noncomplementary sequence. The model target analyte in this study was TP53 gene where a specific mutation is a marker for a list of cancers. Mutations of the TP53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and many other organs. Analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.