A High-Performance Electrochemical Analysis Platform for Ultrasensitive Detection of HTLV-1 DNA: Integrating Cascade Signal Amplification with λ-Exonuclease-Assisted Target Recycling and Enzyme Catalysis
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 integrate λ-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 the λ-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 by λ-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.
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