Label-free DNA detection of hepatitis C virus based on modified conducting polypyrrole films at microelectrodes and atomic force microscopy tip-integrated electrodes.
We present a new strategy for the label-free electrochemical detection of DNA hybridization for detecting hepatitis C virus based on electrostatic modulation of the ion-exchange kinetics of a polypyrrole film deposited at microelectrodes. Synthetic single-stranded 18-mer HCV genotype-1-specific probe DNA has been immobilized at a 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole film established by electropolymerization at the previously formed polypyrrole layer. HCV DNA sequences (244-mer) resulting from the reverse transcriptase-linked polymerase chain reaction amplification of the original viral RNA were monitored by affecting the ion-exchange properties of the polypyrrole film. The performance of this miniaturized DNA sensor system was studied in respect to selectivity, sensitivity, and reproducibility. The limit of detection was determined at 1.82x10(-21) mol L(-1). Control experiments were performed with cDNA from HCV genotypes 2a/c, 2b, and 3 and did not show any unspecific binding. Additionally, the influence of the spacer length of 2,5-bis(2-thienyl)-N-(3-phosphoryl-n-alkyl)pyrrole on the behavior of the DNA sensor was investigated. This biosensing scheme was finally extended to the electrochemical detection of DNA at submicrometer-sized DNA biosensors integrated into bifunctional atomic force scanning electrochemical microscopy probes. The 18-mer DNA target was again monitored by following the ion-exchange properties of the polypyrrole film. Control experiments were performed with 12-base pair mismatched sequences.
dos Santos Riccardi
Carla
C
School of Chemistry and Biochemistry, Georgia Institute of Technology, 901 Atlantic Dr, Atlanta, Georgia 30332-0400, USA.
Kranz
Christine
C
Kowalik
Janusz
J
Yamanaka
Hideko
H
Mizaikoff
Boris
B
Josowicz
Mira
M
D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D015342:DNA Probes / Q000032:analysis* / Q000737:chemistry*; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007474:Ion Exchange; D008567:Membranes, Artificial; D008839:Microelectrodes; D018625:Microscopy, Atomic Force; D009693:Nucleic Acid Hybridization; D011076:Polycarboxylate Cement / Q000737:chemistry; D011108:Polymers / Q000737:chemistry*; D016062:Porosity; D011758:Pyrroles / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015342:DNA Probes; D008567:Membranes, Artificial; D011076:Polycarboxylate Cement; D011108:Polymers; D011758:Pyrroles; C005506:polycarbonate; C067635:polypyrrole