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  • Title: Electrochemical DNA sensors based on enzyme dendritic architectures: an approach for enhanced sensitivity.
    Author: Domínguez E, Rincón O, Narváez A.
    Journal: Anal Chem; 2004 Jun 01; 76(11):3132-8. PubMed ID: 15167793.
    Abstract:
    The modification of enzymes with multiple single-stranded oligonucleotides opens up a new concept for the development of DNA sensors with enhanced sensitivity. This work describes the generation of reporter sequences labeled with an enzyme for the demonstration of their ability to specifically hybridize and to permit signal amplification by successive hybridization steps. The synthetic pathway for the labeling of GOx with oligonucleotide sequences is based on the oxidation of the glycosidic residues of the enzyme and their covalent binding with 5'-end amine-modified oligonucleotides. Spectrophotometric characterization of these functionalized sequences results in an average number of three linked oligonucleotides per enzyme molecule. Their specificity is demonstrated in both a direct and a sandwich-type hybridization assay. The transduction of the enzyme-linked DNA sensors is based on self-assembled multilayers, including a chemically modified anionic horseradish peroxidase electrochemically connected to a water-soluble cationic poly[(vinylpyridine)Os(bpy)(2)Cl] redox polymer in an electrostatic ordered assembly. The sensing layer is constructed by the covalent binding of the DNA probe over the redox polymer through the 3'-phosphate group, enabling the capture of the target sequence. Upon addition of glucose, hybridization results in the production of H(2)O(2), which readily diffuses to the electrocatalytic assembly, giving rise to a cathodic current at 100 mV vs Ag/AgCl. Hybridization is always performed at room temperature, and after 30 min of incubation, an amperometric response is obtained that is proportional to DNA concentration. The simultaneous sandwich assay enables the quantification of a free-label 44-mer oligonucleotide at 1 nM concentration. Signal amplification is realized by a new hybridization step over the free sequences, giving rise to a dendritic architecture that accumulates enzyme molecules per hybridization event.
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