Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization |
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Authors: | Wang Yiran Stanzel Manfred Gumbrecht Walter Humenik Martin Sprinzl Mathias |
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Institution: | Laboratorium für Biochemie, Universit?t Bayreuth, Universit?tsstr. 30, 95440 Bayreuth, Germany. |
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Abstract: | A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol-gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 x 10(-18)mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 microL. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter. |
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