A Single-Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

Abstract

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 genomic 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 doublesurface electrochemical technique involving multiple hybridization 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 streptavidinalkaline phosphatase (ALP) conjugate. The ALP catalyzes production of an electroactive indicator (1-naphthol) which is detected voltammetrically on the same electrode. Signal resulting from 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 osmium tetroxide 2,2'-bipyridine complex. Detection of (GAA) n · (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.

DNA hybridization biosensors using polylysine modified SPCEs

“…la Escosura Muñiz et al, 2007;Fotja et al, 2006;Del Pozo et al, 2005;Erdem et al, 2004;Pividori et al, 2003;De los Santoś Alvarez et al, 2002), covalent attachment (Zhu et al, 2005;Teh et al, 2005;Kara et al, 2005), film entrapment (Zhang et al, 2004(Zhang et al, , 2003 or affinity binding (Lee et al, 2005;Mir and Katakis, 2005;Hernández Santos et al, 2005). An ideal method of immobilization should guarantee the orientation of the probes in the immobilized layers to facilitate the recognition event.…”

Sequence-specific electrochemical detection of nucleic acids in real samples

“…This method also worked for DNA samples extracted from human whole blood. This group presented a similar approach in 2006 [70] when they used a single-surface electrochemical biosensor to detect FRDA. The tDNA was adsorbed to the electrode surface (PGE or screen-printed carbon electrode) and hybridized with RP-biot to enable the enzyme-linked assay.…”

“…They used the same approach as demonstrated in [70] and were able to detect 15 μg/ml of tDNA, which corresponds with 0.75 pmol of specific sequence. Furthermore, they demonstrated the combination of this electrochemical enzyme-linked DNA hybridization with reverse transcription of RNA followed by PCR amplification of the resulting cDNA with specific primers (RT-PCR) to examine the expression of gene rbcL encoding a subunit of the enzyme Rubisco.…”

“…Combining the double-surface technique [70] with RT-PCR and PEX [76], they were able to detect single-nucleotide polymorphism in samples received from cell cultures, differentiating mutant and wild-type genotypes where mutant genotypes indicate the inability of p53 to act as a tumor suppressor.…”

Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme‐Linked Electrochemical Assay

“…The enzymelinked electrochemical techniques employing biotin DNA labeling and streptavidin-enzyme conjugates subsequently attached to the biotin tags have taken the advantage of biocatalytic signal amplification and have been applied by many authors involved in electrochemical DNA sensing (reviewed in [1,2,9]). Application of biotinylated (and subsequently enzyme-labeled) reporter probes was combined with both classical (single -surface) electrochemical biosensor concept [10] and the double -surface [1,2,9] techniques. Similarly, using biotinylated dNTPs, PEx-based electrochemical assays have been developed and applied for the detection of various DNA targets [6,11], including PCR-amplified genomic DNA sequences.…”