A DNA Walker-Based Biosensor for Ultrasensitive HIV DNA Detection

• A 3-D DNA walker biosensor was constructed for detection of HIV-DNA • The detection signal was amplified by DNAzyme-catalyzed interaction between particles • The 3-D walker biosensor could detect HIV-DNA as low as 12.8 pM • The good performance of biosensor was evaluated with serum samples Human Immunodeficiency Virus (HIV) compromises the human immune system and remains one of the deadliest infectious diseases globally. Nucleic acid testing is a powerful tool for the early detection of HIV infection, capable of real-time HIV DNA detection without the limitation of a window period. In this study, a DNA nanostructure-based biosensor that leveraged interactions between functionalized gold nanoparticles (AuNPs) and three-dimensional (3D) DNA walkers were developed for ultrasensitive fluorescence detection of HIV DNA. The biosensor comprised blocked-walking particles (BPs) with locked DNAzyme and substrate particles (SPs) with quenched carboxyfluorescein (FAM) fluorophores. Upon encountering HIV DNA, the blocking strands hybridized more strongly with the HIV DNA, exposing the DNAzyme, which subsequently cleaved FAM on the SPs from the AuNPs and restoring fluorescence. As all substrate strands on SPs were cleaved, the BPs interacted with new SPs, significantly amplifying the fluorescent signal. The fluorescence intensity showed a linear correlation with HIV DNA concentration ranging from 0.02 to 6.00 nM, with a detection limit of 12.8 pM. The performance of biosensor was evaluated using spiked serum samples, demonstrating good precision and recovery rates. The DNA walker biosensor leveraged particle-to-particle interactions for rapid signal amplification, offering great potential for sensitive biosensors.