A dual-toehold-initiated strand displacement reaction-based fluorescent sensor for HIV DNA detection

Sensitive and rapid detection of human immunodeficiency virus (HIV) DNA is crucial for the effective prevention and treatment of acquired immunodeficiency syndrome (AIDS). The entropy-driven catalytic strand displacement reaction (SDR), an enzyme-free and hairpin-free DNA self-assembly amplification strategy, offers several advantages such as minimal nonspecific amplification, carrier-free operation, and simplified procedures, rendering it a promising approach for developing highly sensitive, rapid HIV DNA detection assays. In nucleic acid testing, longer target sequences typically yield more accurate detection results but require stronger driving forces, which can prolong reaction times. Introducing a second toehold in the SDR offers an additional driving force, presenting a potential solution to this limitation. Based on this concept, this study developed an analytical method that integrates the SDR with fluorescence resonance energy transfer (FRET) to amplify fluorescence signals, enabling the detection of a 33-base pair (bp) HIV DNA target. This method achieved a low detection limit of 0.0200 nM and a wide linear detection range of 0.05-10 nM. The successful detection of the 33 bp target demonstrates the potential of this dual-toehold SDR strategy for highly sensitive and specific HIV diagnostics.