Label-free fluorescence dual-amplified detection of adenosine based on exonuclease III-assisted DNA cycling and hybridization chain reaction.

In this work, we constructed a label-free and dual-amplified fluorescence aptasensor for sensitive analysis of adenosine based on exonuclease III (Exo III)-assisted DNA cycling and hybridization chain reaction (HCR). Firstly, we fabricated a trifunctional probe that consisting of the catalytic strand, the aptamer sequence and a streptavidin-magnetic nanobead (streptavidin-MNB). The streptavidin-MNB played a role of enrichment and separation to achieve a low background. The aptamer sequence was employed as a recognition element to bind the target adenosine, leading to the releasing of the catalytic stand. Then, the catalytic strand induced the Exo III-assisted DNA cycling reaction and produced a large amount of DNA fragments, which got a primary amplification. Subsequently, the DNA fragments acted as trigger strands to initiate HCR, forming nicked double helices with multiple G-quadruplex structures, which achieved a secondary amplification. Finally, the G-quadruplex structures bonded with the N-nethyl mesopor-phyrin IX (NMM) and yielded an enhanced fluorescence signal, realizing the label-free detection. In the proposed strategy, a small amount of adenosine can be converted to a large amount of DNA triggers, leading to a significant amplification for the target. This method exhibited a high sensitivity toward adenosine with a detection limit of 4.2×10(-7) mol L(-1), which was about 10 times lower than that of the reported label-free strategies. Moreover, this assay can significantly distinguish the content of adenosine in urine samples of cancer patients and normal human, indicating that our method will offer a new strategy for reliable quantification of adenosine in medical research and early clinical diagnosis.