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
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
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.