Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

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• 作者：Andreas Kukol ; Peng Li ; Pedro Estrela ; Paul Ko-Ferrigno ; Piero Migliorato
• 关键词：DNA ; Biosensor ; Avian influenza ; Label-free detection ; Electrochemical impedance spectroscopy ; Sulfhydryl chemistry ; Spacer molecule ; Oligonucleotide
• 刊名：Analytical Biochemistry
• 出版年：2008
• 出版时间：1 March 2008
• 年：2008
• 卷：374
• 期：1
• 页码：143-153
• 全文大小：381 K

文摘

Microarrays based on DNA–DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of K_D = 13 ± 4 nM at 295 K.