期刊
ANALYTICAL BIOCHEMISTRY
卷 374, 期 1, 页码 143-153出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2007.10.035
关键词
DNA; biosensor; avian influenza; label-free detection; electrochemical impedance spectroscopy; sulfhydryl chemistry; Spacer molecule; oligonucleotide
资金
- BBSRC [BB/D523094/1] Funding Source: UKRI
- MRC [MC_U105359876] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D523094/1] Funding Source: researchfish
- Medical Research Council [MC_U105359876] Funding Source: researchfish
- Biotechnology and Biological Sciences Research Council [BB/D523094/1, bb/d523094/1] Funding Source: Medline
- Medical Research Council [MC_U105359876] Funding Source: Medline
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 I 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. (c) 2007 Elsevier Inc. All rights reserved.
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