Enzyme-free and sensitive electrochemical determination of the FLT3 gene based on a dual signal amplified strategy: Controlled nanomaterial multilayers and a target-catalyzed hairpin assembly

An isothermal, enzyme-free and sensitive electrochemical DNA sensor was developed for the detection of the FLT3 gene in acute myeloid leukemia (AML). First, aminated multi-walled carbon nanotubes (AMWNTs) and gold nanoparticles (AuNPs) were alternately self-assembled on a gold electrode using a layer-by-layer strategy. Then, the hairpin DNA probe 1 (H1), with a thiol group at the 3′ end and a ferrocenyl moiety (Fc) at the 5′ end, was immobilized on the AMWNTs/AuNPs multilayer films through Au-S bonding. When the target DNA (TD) appeared, it hybridized with and opened the hairpin structure of H1, and Fc was forced away from the electrode surface, leading to a significant decrease in the current peak of square wave voltammetry. Subsequently, the hairpin DNA probe 2 (H2) bound to H1, freeing the TD to trigger another reaction cycle. The combination of this target-catalyzed hairpin assembly and the LBL assembly of nanomaterials achieved a detection limit of 0.1 pM with a wide linear range of 0.1-1000 pM. The sensor discriminated between mismatched DNA and the target DNA with high selectivity. This dual signal amplification strategy is relatively simple and inexpensive because it does not need any enzymes or sophisticated equipment and successfully assayed the FLT3 gene from real samples.