Pustaka Ilmiah Universitas Padjadjaran

Abstrak

Sensitive Detection of Mitochondrial DNA A3243G tRNALeu Mutation via an Electrochemical Biosensor Using Meldola’s Blue as a Hybridization Indicator

An indicator based electrochemical DNA biosensor for detection of the mitochondrial DNA A3243G tRNALeu point mutation related to maternally inherited diabetes (MID), maternally inherited diabetes and deafness (MIDD) and type 2 diabetes mellitus (DM) is analyzed both with synthetic and real samples for the first time in this study. Meanwhile, MID, MIDD, DM and control samples were verified with agarose gel electrophoresis which is the gold standard methodology for real samples. For this purpose, capture probes were adsorbed onto the disposable pencil graphite electrode (PGE) and the interaction with complementary or non-complementary sequences was carried out by passive adsorption on the surface of the graphite electrodes. The discrimination of probe, probe/complementary target (hybrid), and probe/single base mis match target was achieved by measuring changes of the peak currents of Meldola’s Blue (MDB) as a hybridization indicator by using 20 base-pair synthetic oligonucleotides and Polymerase Chain Reaction (PCR) amplicons. MDB reduction signal was measured by Differential Pulse Voltammetry (DPV). The sensor was able to clearly distinguish total match from mismatch DNA in a 30 min. detection time. The results obtained DPV experiments for hybridization detection were compared with agarose gel electrophoresis in terms of being alternative as a non-toxic, inexpensive and rapid method. The limit of detection (S/N = 3) was calculated as 2.35% in heteroplasmy which has both wild and mutant type sequences.