Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences.
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 KD=13+/-4 nM at 295 K.
Kukol
Andreas
A
School of Life Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK. a.kukol@herts.ac.uk
Li
Peng
P
Estrela
Pedro
P
Ko-Ferrigno
Paul
P
Migliorato
Piero
P
D015374:Biosensing Techniques / Q000379:methods; D004247:DNA / Q000737:chemistry*; D017097:Electric Impedance; D005814:Genes, Viral; D006046:Gold; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics*; D008839:Microelectrodes; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D013057:Spectrum Analysis
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D015345:Oligonucleotide Probes; C109963:hemagglutinin, avian influenza A virus; D006046:Gold; D004247:DNA