title: Optimization of label-free DNA detection with electrochemical impedance spectroscopy using PNA probes.
abstract: DNA biosensors, especially those based upon detection of the intrinsic negative charge of target DNA, can be greatly improved by the use of uncharged peptide nucleic acid (PNA) probes. Hybridization causes an increased electrostatic barrier for the negatively charged ferri/ferrocyanide redox couple, resulting in an increase in charge transfer resistance R(ct) that is measured using electrochemical impedance spectroscopy. We report on the optimization of PNA probe surface density by the simultaneous co-immobilization of thiol-modified probes and mercaptohexanol, with the PNA surface density controlled by the thiol mole ratio in solution. Maximum R(ct) change upon hybridization is obtained with 10% PNA mole fraction. The effect of the measurement buffer ionic strength is investigated. The electrostatic barrier for charge transfer to the ferri/ferrocyanide redox couple is approximately independent of ionic strength with PNA probes, but greatly increases with decreasing ionic strength, after hybridization with target DNA. This significantly enhances the R(ct) change upon hybridization. The optimization of PNA surface density and measurement buffer ionic strength leads to a 385-fold increase in R(ct) upon hybridization, a factor of 100 larger than previously reported results using either PNA or DNA probes.
authors.affiliation: Electrical Engineering Division, Department of Engineering, University of Cambridge, 9 JJ Thomson Avenue, Cambridge CB3 0FA, UK.
authors.forename: Simon D
authors.initials: SD
authors.lastname: Keighley
authors.forename: Pedro
authors.initials: P
authors.lastname: Estrela
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Piero
authors.initials: P
authors.lastname: Migliorato
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D004247:DNA / Q000032:analysis / Q000737:chemistry* / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry*; D017097:Electric Impedance; D004563:Electrochemistry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D015342:DNA Probes; D020135:Peptide Nucleic Acids; D004247:DNA