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.

[1]  I. Willner,et al.  Probing Biomolecular Interactions at Conductive and Semiconductive Surfaces by Impedance Spectroscopy: Routes to Impedimetric Immunosensors, DNA‐Sensors, and Enzyme Biosensors , 2003 .

[2]  I. Donatelli,et al.  A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control , 2006, BMC infectious diseases.

[3]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[4]  Terry C. Chilcott,et al.  Impedance spectroscopy of interfaces, membranes and ultrastructures , 1996 .

[5]  Y. Kawaoka,et al.  Emerging influenza viruses: past and present. , 2005, Current molecular medicine.

[6]  Niall Johnson,et al.  Updating the Accounts: Global Mortality of the 1918-1920 "Spanish" Influenza Pandemic , 2002, Bulletin of the history of medicine.

[7]  M. Vey,et al.  Influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin‐like endoprotease. , 1992, The EMBO journal.

[8]  M. Beer,et al.  Rapid and Highly Sensitive Pathotyping of Avian Influenza A H5N1 Virus by Using Real-Time Reverse Transcription-PCR , 2006, Journal of Clinical Microbiology.

[9]  Yoshio Umezawa,et al.  Electrochemical Detection of a One‐Base Mismatch in an Oligonucleotide Using Ion‐Channel Sensors with Self‐Assembled PNA Monolayers , 2000 .

[10]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[11]  N. Cox,et al.  Global epidemiology of influenza: past and present. , 2000, Annual review of medicine.

[12]  Giulio Milazzo,et al.  Topics in Bioelectrochemistry and Bioenergetics , 1978 .

[13]  A. Amonsin,et al.  Single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) for influenza A virus subtype H5N1 detection. , 2004, Viral immunology.

[14]  C. Naeve,et al.  Large-Scale Sequence Analysis of Avian Influenza Isolates , 2006, Science.

[15]  B. Érshler.  Investigation of electrode reactions by the method of charging-curves and with the aid of alternating currents , 1947 .

[16]  M. D. Rooij,et al.  Electrochemical Methods: Fundamentals and Applications , 2003 .

[17]  D. Tang,et al.  Protective avian influenza in ovo vaccination with non-replicating human adenovirus vector. , 2007, Vaccine.

[18]  Shu Chen,et al.  Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR , 2001, Journal of Clinical Microbiology.

[19]  James A. Smagala,et al.  MChip: a tool for influenza surveillance. , 2006, Analytical chemistry.

[20]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[21]  G. Whitesides,et al.  Self-Assembled Monolayers That Resist the Adsorption of Proteins and the Adhesion of Bacterial and Mammalian Cells , 2001 .

[22]  N. Cox,et al.  Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. , 1998, Science.

[23]  T. Hien,et al.  Avian influenza A (H5N1) , 2005, Journal of Clinical Virology.

[24]  Y. Umezawa,et al.  Voltammetric anion responsive sensors based on modulation of ion permeability through Langmuir-Blodgett films containing synthetic anion receptors , 1990 .

[25]  A. Steel,et al.  Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly. , 2000, Biophysical journal.

[26]  K. Rhodes.  Essentials of Diagnostic Virology , 2000 .

[27]  T. A. Hall,et al.  BIOEDIT: A USER-FRIENDLY BIOLOGICAL SEQUENCE ALIGNMENT EDITOR AND ANALYSIS PROGRAM FOR WINDOWS 95/98/ NT , 1999 .

[28]  R. Hintsche,et al.  Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes. , 2005, Biosensors & bioelectronics.

[29]  James A. Smagala,et al.  Experimental Evaluation of the FluChip Diagnostic Microarray for Influenza Virus Surveillance , 2006, Journal of Clinical Microbiology.

[30]  K. Lohman,et al.  Development of a Real-Time Reverse Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7 Hemagglutinin Subtypes , 2002, Journal of Clinical Microbiology.

[31]  R. Hintsche,et al.  DNA hybridization detection on electrical microarrays using coulostatic pulse technique. , 2006, Biosensors & bioelectronics.

[32]  C. Hsu,et al.  Technical Note: Concerning the Conversion of the Constant Phase Element Parameter Y0 into a Capacitance , 2001 .

[33]  R. Webster,et al.  Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus , 1998, The Lancet.

[34]  Shaojun Dong,et al.  Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy , 2007 .

[35]  Eric C J Claas,et al.  Human influenza A H 5 N 1 virus related to a highly pathogenic avian influenza virus , 2005 .

[36]  Kia Peyvan,et al.  CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection. , 2007, Biosensors & bioelectronics.

[37]  Yoshihiro Kawaoka,et al.  Influenza: lessons from past pandemics, warnings from current incidents , 2005, Nature Reviews Microbiology.

[38]  R. Webby,et al.  Use of Semiconductor-Based Oligonucleotide Microarrays for Influenza A Virus Subtype Identification and Sequencing , 2006, Journal of Clinical Microbiology.

[39]  Yong Poovorawan,et al.  Single step multiplex real-time RT-PCR for H5N1 influenza A virus detection. , 2006, Journal of virological methods.

[40]  M. Borca,et al.  Rapid Detection of Classical Swine Fever Virus by a Portable Real-Time Reverse Transcriptase PCR Assay , 2003, Journal of Clinical Microbiology.

[41]  Robin H. Liu,et al.  Validation of a fully integrated microfluidic array device for influenza A subtype identification and sequencing. , 2006, Analytical chemistry.

[42]  J. Randles.  Kinetics of rapid electrode reactions , 1947 .