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Target-induced duplex–triplex transition for ratiometric detection of adenosine triphosphate

Yao, Shujuan, Gao, Yanhua, Wang, Wen, Ni, Donghua, Zhang, Kaixian, Li, Ling, Du, Jianqiang, Cui, Xiujuan
Analytical methods 2017 v.9 no.21 pp. 3244-3248
adenosine triphosphate, analytical methods, antibodies, antigens, biosensors, blood serum, cattle, detection limit, fluorescence, oligonucleotides, signal-to-noise ratio, synthesis
Compared with conventional binding pairs of antigens and antibodies, facile chemical synthesis and generally impressive target selectivity make aptamers an ideal recognition tool for designing various biosensors. Herein, we developed a new ratiometric aptamer sensor based on an aptamer/target-binding-induced duplex–triplex transition. This sensor is composed of a capture strand (CS) modified with a BHQ₂ at the 3′ end, a triplex-forming middle strand (MS) modified with TAMRA at the 3′ end, and a target-specific aptamer strand (AS) modified with FAM at the 5′ end. In the absence of analyte, the CS probe could hybridize with both the AS probe at the 3′ end and the MS probe at the 5′ end. As BHQ₂ and FAM were in close proximity, the mixed solution showed low FAM fluorescence and high TAMRA fluorescence. After introducing the target molecule ATP, target/aptamer binding split from the duplex helix and liberated the end of the CS probe to fold back and form a triplex helix. Meanwhile, the FAM fluorescence could be recovered, and the TAMRA fluorescence could be quenched due to the close proximity of TAMRA and BHQ₂. The intensity ratio (FFAM/FTAMRA) was linear for ATP concentrations of 0.1–10 μM in buffer solution. The detection limit was determined as 65 nM, based on three times the signal-to-noise ratio. The sensor was also applied to ATP sensing in bovine serum, obtaining a good recovery and demonstrating the practicality of this ratiometric aptamer sensor.