Liposomes labeled with biotin and horseradish peroxidase: A probe for the enhanced amplification of antigen-antibody or oligonucleotide-DNA sensing processes by the precipitation of an insoluble product on electrodes

Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) a re used as a probe to amplify the sensing of antigen-antibody interactions or oligonucleotide-DNA binding. The HRP biocatalyzed oxidation of 4-chloro- 1-naphthol (1) in the presence of H2O2, and the precipitation of the insolu ble;product 2 ion electrode supports, are used as an amplification route fo r the sensing processes. The anti-dinitrophenyl antibody (DNP-Ab) is sensed by a dinitrophenyl-L-cysteine antigen monolayer associated with an Au elec trode. A biotinylated anti-IgG-antibody (Fc-specific) is linked to the anti gen-DNP-Ab complex, and the biotin-labeled HRP-liposomes associate with the assembly through an avidin bridge. The biocatalyzed precipitation of 2 on the electrode increases the electron-transfer resistances at the electrode- solution interface or the electrode resistance itself. The binding events o f the different proteins on the electrode and the biocatalyzed precipitatio n of 2 on the conductive support are followed by Faradaic impedance spectro scopy or constant-current chronopotentiometry, DNP-Ab concentrations as low as 1 x 10(-11) g.mL(-1) can be detected by this method. The labeled liposo mes were also used for the amplified detection of DNA 3, The oligonucleotid e 4, complementary to a part of the target DNA 3 that is a model nucleic ac id sequence for the Tay-Sachs genetic disorder, is assembled on an Au elect rode. Hybridization of the analyte 3 followed by the association of the bio tin-tagged oligonucleotide 5 yields a three-component double-stranded assem bly. Sensing of the analyte 3 is amplified by the,association of avidin, th e labeled liposomes, and the subsequent biocatalyzed precipitation of 2 on the electrodes. The DNA 3 is detected with a sensitivity that corresponds t o 6.5 x 10(-13) M. Faradaic impedance spectroscopy and chronopotentiometry were employed to follow the stepwise assembly of the systems and the electr onic transduction of the detection of the analyte DNA 3.