A single-surface electrochemical biosensor for the detection of DNA triplet repeat expansion

Molecular diagnostics of inherited neurodegenerative disorders such as fragile X syndrome, myotonic dystrophy or Friedreich ataxia (FRDA) is based on analysis of the length of trinucleotide repetitive sequences in certain loci of genoimc DNA. The current methods employ PCR and electrophoretic determination of the amplified DNA fragment size. We have recently shown that length of a triplet repetitive DNA sequence can be determined using a double-surface electrochemical technique involving multiple hybridizaton of the expanded triplet repeat with short labeled reporter probe (spanning several trinucleotides). Here we propose a single-surface sensor employing an analogous principle. Target DNA (tDNA) is adsorbed onto surface of a carbon (pyrolytic graphite or screen-printed) electrode. Biotin-labeled reporter probe (RP) is hybridized with the immobilized tDNA followed by binding of streptavidin-alkaline phosphatase (ALP) Conjugate. The ALP catalyzes production of an electroactive indicator (1-naphtol) which is detected voltammetrically oil the same electrode. Signal resulting front this electrochemical enzyme-linked DNA hybridization assay is normalized to the amount of tDNA immobilized at the transducer surface either by measuring intrinsic tDNA voltammetric response, or using electrochemical labeling of the tDNA with omium tetroxide 2,2'-bipyridine complex. Detection of (GAA)(n) center dot (TTC)(n) triplet repeat expansion in nanogram quantities Of PCR-amplified tDNAs, including amplicons of patients' genomic DNA, is demonstrated. We show that our technique allow differentiation between normal and pathological alleles of X25 gene related to the FRDA.