Immobilization-free sequence-specific electrochemical detection of DNA using ferrocene-labeled peptide nucleic acid.
An electrochemical method for sequence-specific detection of DNA without solid-phase probe immobilization is reported. This detection scheme starts with a solution-phase hybridization of ferrocene-labeled peptide nucleic acid (Fc-PNA) and its complementary DNA (cDNA) sequence, followed by the electrochemical transduction of Fc-PNA-DNA hybrid on indium tin oxide (ITO)-based substrates. On the bare ITO electrode, the negatively charged Fc-PNA-DNA hybrid exhibits a much reduced electrochemical signal than that of the neutral-charge Fc-PNA. This is attributed to the electrostatic repulsion between the negatively charged ITO surface and the negatively charged DNA, hindering the access of Fc-PNA-DNA to the electrode. On the contrary, when the transduction measurement is done on the ITO electrode coated with a positively charged poly(allylamine hydrochloride) (PAH) layer, the electrostatic attraction between the (+) PAH surface and the (-) Fc-PNA-DNA hybrid leads to a much higher electrochemical signal than that of the Fc-PNA. The measured electrochemical signal is proportional to the amount of cDNA present. In terms of detection sensitivity, the PAH-modified ITO platform was found to be more sensitive (with a detection limit of 40 fmol) than the bare ITO counterpart (with a detection limit of 500 fmol). At elevated temperatures, this method was able to distinguish fully matched target DNA from DNA with partial mismatches. Unpurified PCR amplicons were detected using a similar format with a detection limit down to 4.17 amol. This detection method holds great promise for single-base mismatch detection as well as electrochemistry-based detection of post-PCR products.
Luo
Xiaoteng
X
Bioengineering Graduate Program, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Lee
Thomas Ming-Hung
TM
Hsing
I-Ming
IM
D020137:Base Pair Mismatch; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D005296:Ferrous Compounds / Q000737:chemistry*; D000075163:Metallocenes; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D011073:Polyamines / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity; D017971:Tin Compounds / Q000737:chemistry
D018076:DNA, Complementary; D005296:Ferrous Compounds; D000075163:Metallocenes; D020135:Peptide Nucleic Acids; D011073:Polyamines; D017971:Tin Compounds; C063994:polyallylamine; C109984:indium tin oxide; C004998:ferrocene