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

L. Alfonta, A. K. Singh, I. Willner

Research output: Contribution to journalArticlepeer-review

213 Scopus citations

Abstract

Liposomes labeled with biotin and the enzyme horseradish peroxidase (HRP) are 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 insoluble product 2 on electrode supports, are used as an amplification route for the sensing processes. The anti-dinitrophenyl antibody (DNP-Ab) is sensed by a dinitrophenyl-L-cysteine antigen monolayer associated with an Au electrode. A biotinylated anti-IgG-antibody (Fc-specific) is linked to the antigen-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 of the different proteins on the electrode and the biocatalyzed precipitation of 2 on the conductive support are followed by Faradaic impedance spectroscopy or constant-current chronopotentiometry. DNP-Ab concentrations as low as 1 × 10-11 g·mL-1 can be detected by this method. The labeled liposomes were also used for the amplified detection of DNA 3. The oligonucleotide 4, complementary to a part of the target DNA 3 that is a model nucleic add sequence for the Tay-Sachs genetic disorder, is assembled on an Au electrode. Hybridization of the analyte 3 followed by the association of the biotintagged oligonucleotide 5 yields a three-component double-stranded assembly. Sensing of the analyte 3 is amplified by the association of avidin, the labeled liposomes, and the subsequent biocatalyzed precipitation of 2 on the electrodes. The DNA 3 is detected with a sensitivity that corresponds to 6.5 × 10-13 M. Faradaic impedance spectroscopy and chronopotentiometry were employed to follow the stepwise assembly of the systems and the electronic transduction of the detection of the analyte DNA 3.

Original languageEnglish
Pages (from-to)91-102
Number of pages12
JournalAnalytical Chemistry
Volume73
Issue number1
DOIs
StatePublished - 1 Jan 2001
Externally publishedYes

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