A Novel Electrochemical Biosensor Based on a Double-Signal Technique for d(CAG)_n Trinucleotide Repeats

Electrochemical sensors now play an important role in analysis and detection of nucleic acids. In this work, we present a novel double-signal technique for electrochemically measuring the sequence and length of the d(CAG)_n repeat. The double-signal technique used an electrochemical molecular beacon (a hairpin DNA labeled with ferrocene), which was directly modified on the surface of a gold electrode, while a reporter probe (a DNA sequence labeled with horseradish peroxidase) was hybridized to the target DNA. First a simple single-signal sensor was characterized in which d(CAG)_n repeats were detected using a short reporter DNA strand labeled with horseradish peroxidase. To obtain a reliable signal that was dependent on repeat number, a double-signal biosensor was created in which the single strand capture DNA in single-signal sensor was replaced by an electrochemical molecular beacon labeled with ferrocene. When the hairpin DNA hybridized to the target-reporter DNA complex, it opened, resulting in a decreased ferrocene current. Both electrochemical biosensors exhibited high selectivity and sensitivity with low detection limits of 0.21 and 0.15 pM, respectively, for the detection of d(CAG)_n repeats. The double-signal sensor was more accurate for the determination of repeat length, which was measured from the ratio of signals for HRP and ferrocene (H/F). A linear relationship was found between H/F and the number of repeats (n), H/F = 0.1398n + 9.89788, with a correlation coefficient of 0.974. Only 10 nM of target DNA was required for measurements based on the value of H/F in the double-signal technique. These results indicated that this new double-signal electrochemical sensor provided a reliable method for the analysis of CAG trinucleotide repeats.