Cystic fibrosis: a label-free detection approach based on thermally modulated electrochemical impedance spectroscopy.
Cystic fibrosis is one of the most common genetically inherited diseases in northern Europe, with the DF508 mutation being the most common, and among the Caucasian population being responsible for almost 70% of cases. In this work, we report on the use of thermally modulated electrochemical impedance spectroscopy for the discrimination of the DF508 mutation from the wild-type sequence. DNA probes (15 and 21 bases long) were immobilised on the surface of gold electrodes and the variation of the charge-transfer resistance was monitored as a function of hybridisation. Two sets of targets were used in this work: synthetic 15-mer sequences and two single-stranded synthetic analogues of PCR products 82 (mutant) and 85 (wild type) bases long. Hybridisation with short targets resulted in very sequence specific charge-transfer-resistance variation with a discrimination factor at room temperature between fully complementary and mismatched sequences of approximately fivefold. However, in the case of the single-stranded synthetic PCR product analogues, a lower discrimination factor was recorded (1.5-fold). The effect of temperature was investigated to improve discrimination and the use of a posthybridisation wash at elevated temperature resulted in a fivefold improvement in the discrimination factor. Using an electrode array with probes immobilised against each of the mutant and wild-type sequences, we achieved an unequivocal detection of the DF508 mutation.
Nanobiotechnology & Bioanalysis Group, Departament d'Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain.
Hany
H
Nasef
Valerio
V
Beni
Veli C
VC
Ozalp
Ciara K
CK
O'Sullivan
D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D017097:Electric Impedance; D004563:Electrochemistry / Q000379:methods*; D006361:Heating / Q000379:methods; D006801:Humans; D008969:Molecular Sequence Data; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013057:Spectrum Analysis / Q000379:methods*; D013194:Staining and Labeling
D004247:DNA