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

• Published in: Analytical chemistry (Washington) Vol. 73; no. 1; pp. 91 - 102
• Main Authors: Alfonta, Lital, Singh, Anup K, Willner, Itamar
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01-01-2001
• Subjects: 4-chloro-1-naphthol; Antigen-Antibody Reactions; Biological and medical sciences; Biosensors; Biotechnology; Biotin - chemistry; DNA - analysis; Electrochemistry; Electrodes; Fundamental and applied biological sciences. Psychology; Horseradish Peroxidase - chemistry; Liposomes - chemistry; Methods. Procedures. Technologies; Oligonucleotides - analysis; Various methods and equipments; Molecular hybridization; Enzyme; Antibody; Liposome; Molecular interaction; Biotin; Chemical labelling; Immunosensor; Amplification; Antigen; Peroxidases; Biosensor; Oligonucleotide; Peroxidase; Oxidoreductases; Enzymatic labelling
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac000819v

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 acid sequence for the Tay-Sachs genetic disorder, is assembled on an Au electrode. Hybridization of the analyte 3 followed by the association of the biotin-tagged 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.