Human T-cell lymphophilic virus type 1(HTLV-1),the known retrovirus causing cancer in humans,is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis.Due to its ability to evade the host's defense mechanisms,early tracking of HTLV-1 becomes crucial.In this study,we integrateλ-Exonuclease(λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase(TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA.During theλ-Exo-assisted target recycling,HTLV-1 DNA is recognized by hairpin DNA(Hp-DNA),forming double-stranded DNA(dsDNA)through DNA hybridization.The dsDNA,featuring blunt 5'terminal phosphorylation,is cleaved byλ-Exo,generating abundant short output sequence(sDNA).HTLV-1 DNA is released,initiating a cyclic hybridization-cleavage process.Subsequently,thiol-labelled capture DNA(CP-DNA)assembled on gold electrode surface captures a substantial amount of the generated sDNA,forming CP-DNA-sDNA nanostructures.When TdT and dNTPs are present on the electrode surface,the 3'-OH terminal of sDNA extends to generate long single-stranded DNA(ssDNA)structure.Methylene blue(MB)is selected as the electrochemical signal molecule.MB not only binds with ssDNA but also interacts specifically with dsDNA,resulting in a significantly enhanced electrochemical signal on modified electrode surface.The detection limit of HTLV-1 DNA is as low as 19 amol/L(S/N=3)when the two signal amplification strategies are combined.The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.
