Electrochemical detection of DNA hybridization using DNA intercalators has been received much attention due to its much more simplicity compared with that using redox label and its higher sensitivity compared with that based on direct oxidation of guanine base. The key step for fabrication of DNA electrochemical biosensors is effective combination of ssDNA layers with electrochemical transducers. To improve the performance of biosensors, effective immobilization of probe ssDNA onto the transducer surface through suitable method is of great significant. Increasing the immobilization amount and controlling over the molecular orientation of probe oligonucleotides upgrade the detection limit of DNA biosensor. During the past decade, many efforts have been devoted to explore new supporting materials and immobilization methods for the fabrication of DNA biosensors. Metal oxide nanostructures are suitable matrixes and novel candidates for immobilization of enzymes and proteins due to their high electrical conductivity, wide electrochemical working window, high biocompatibility, large surface area, no toxicity, chemical and photochemical stability, electrochemical activity, ease of preparation and excellent substrate adhesion. In the present study ssDNA probe sequences related to the taxon: 32630Tumor necrosis factor (TNF) was immobilized on the surface of GC electrode modified with nickel oxide nanoparticles and Ru[(NH3)5Cl]PF6 complex was used as an electroactive indicator. The sensor showed excellent and selective response toward complementary target at nanomolar or lower concentration range. DNA immobilization and hybridization were characterized by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry using K3Fe(CN)6/K4Fe(CN)6 and [Ru(NH3)5Cl]PF6 as probe and indicator. The Ru-complex current response indicates only the complementary sequence gave an obvious current signal in comparison to non-complementary and three-point mismatched sequences
