The present paper describes an alternative approach to the traditionally used covalent immobilization methods that require cost-intensive and complicated chemistry modification of a single-stranded DNA (ssDNA) capture probe. The low-cost pencil graphite electrode (PGE) modified with carbon black (CB) and gold nanoparticles (AuNPs) was used as an electrochemical platform and the non-modified ssDNA was immobilized on a self-assembled cysteamine modified AuNPs/CB-PGE through a phosphoramidate bond between the 5'-terminal phosphate group of ssDNA and the primary amine group of cysteamine. The microRNA-21 was used as a target model in the fabrication of this electrochemical DNA biosensor and the hybridization process with the complementary probe was monitored by differential pulse voltammetry using methylene blue (MB) as an electrochemical hybridization indicator. The decreased reduction peak current of MB shows a good linear correlation with the increased concentration of microRNA-21 target sequences because the MB signal is determined by the amount of exposed guanine bases. The linear range of the fabricated DNA biosensor was from 1.0 × 10-8 to 5.0 × 10-7 M with a detection limit of 1.0 × 10-9 M. These results show that the covalent immobilization of a non-modified ssDNA capture probe through a phosphoramidate-bonding strategy could serve as a cost-effective and versatile approach for the fabrication of DNA biosensors related to a wide range of applications that cover the fields of medical diagnostic and environmental monitoring. The fabricated electrochemical DNA biosensor was used to analyze microRNA-21 in a (spiked) human serum sample and it showed satisfactory and encouraging results as an electrochemical DNA biosensor platform.
Keywords: DNA immobilization; differential pulse voltammetry; electrochemical DNA biosensors; methylene blue; pencil graphite electrode; phosphoramidate-bonding.