Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7-mu m-diameter microelectrode

Citation
Dj. Caruana et A. Heller, Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7-mu m-diameter microelectrode, J AM CHEM S, 121(4), 1999, pp. 769-774
Citations number
24
Categorie Soggetti
Chemistry & Analysis",Chemistry
Journal title
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
ISSN journal
00027863 → ACNP
Volume
121
Issue
4
Year of publication
1999
Pages
769 - 774
Database
ISI
SICI code
0002-7863(19990203)121:4<769:EADOHA>2.0.ZU;2-3
Abstract
A single base pair mismatch in an 18-base oligonucleotide was detected ampe rometrically with a 7-mu m-diameter carbon microelectrode. The hybridizatio n was followed directly and in real time by steady-state amperometry. The m icroelectrode was coated with a hybridization-sensing layer in a two-step e lectrophoretic process, which yielded microelectrodes with reproducible dim ensions. In the first step, a thin film of an electron-conducting redox pol ymer was deposited electrophoretically at constant potential in a low ionic strength solution. In the second step, a carbodiimide-activated single-str anded probe was reactively electrophoretically deposited and covalently att ached to the redox polymer film. The labeling enzyme, thermostable soybean peroxidase (SBP), was covalently bound to the 5'-end of the target single-s tranded oligonucleotide. When the redox polymer and the enzyme were brought to close proximity by hybridization of the target and probe oligonucleotid es, the film on the electrode switched from being a noncatalyst to a cataly st for H2O2 electroreduction at -0.06 V vs Ag/AgCl. The current observed co rresponded to that generated by similar to 40 000 surface-bound and electri cally connected SEP molecules.