Methylene Blue as a Novel Electrochemical Hybridization Indicator for DNA Biosensor
Described here are the investigation of the interaction of methylene blue (MB) with calf thymus double-stranded DNA (dsDNA) and calf thymus single-stranded DNA (ssDNA) by means of differential pulse voltammetry (DPV) and cyclic voltammetry (CV) and a novel assay for the voltammetric detection of oligonucleotides (1,2) at carbon paste electrode (CPE) using MB as the hybridization indicator. The monitoring of 20 µM MB by CV was also accomplished due to the enhanced voltammetric signal obtained with ssDNA modified CPE. The 21-mer probes were then hybridized with complementary ("target") sequences at the CPE. MB was investigated at the hybrid-modified CPE, the bare CPE, and the probe-modified CPE and the increased peak currents were observed, in respect to the order of electrodes. Numerous factors affecting the probe immobilization, target hybridization and indicator binding reactions were optimized to maximize the sensitivity and speed the assay time.
The response of the hybridization of the probe with the single-base mismatch oligonucleotide at CPE is detected by MB. In the presence of an oligonucleotide containing a single-base mismatch , the hybrid contains unbound two guanine bases. These accessible guanines increased the voltammetric signal obtained with the hybrid-modified CPE.
Control experiments were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. Following the exposure of the probe-modified CPE to the noncomplementary oligonucleotide, we observed a peak which was smaller than the signal of the probe, but higher than the signal of the hybrid formed with the target sequence.
The detection of hybridization between poly[G] and poly[C] was accomplished by using MB in connection with CPE. The difference between the electrochemical responses, observed due to the MB association with the poly[G] modified surface thus served as the hybridization signals. These results show that MB holds great promise for use in DNA biosensors.
 A. Erdem, B. Meric, K. Kerman, T. Dalbasti, M. Ozsoz, Electroanalysis, 1999, 11, 1372.
Arzum Erdem, Kagan Kerman, Burcu Meric, Dilsat Ozkan and Mehmet Ozsoz
Faculty of Pharmacy, Department of Analytical Chemistry,