基于裂开型核酸适体非标记荧光法检测ATP

A New Label-Free Fluorometric Assay for ATP Based on Split Aptamer

基于裂开型核酸适体序列短、能有效降低因探针形成二级结构产生假阳性信号等优点,选择裂开型核酸适体作为特异性识别探针,核酸染料噻唑橙(TO)为信号探针,用单壁碳纳米管(SWCNTs)降低背景信号,利用“适配体-目标分子-适配体”的“三明治”夹心方式,建立了一种检测ATP的新方法。在pH8.0的Tris-HCl缓冲溶液中,裂开成两段的ATP适体特异性识别ATP分子,生成稳定的“适配体-ATP-适配体”复合结构。单壁碳纳米管对该复合结构的吸附力较弱,因此该复合物游离在溶液中,TO与其结合而产生强荧光。当不存在ATP时,核酸适体探针以单链状态存在,可通过π—π共轭作用结合到SWCNTs表面,进而不能与TO结合,TO游离在溶液中荧光非常微弱。反应体系中ATP浓度越高,形成的“适配体-ATP-适配体”夹心识别结构复合物越多,检测到的荧光强度越大,据此实现对ATP的检测。在优化实验条件下,在最大荧光发射波长550nm处,ATP的浓度在9.0×10^-9~1.0×10^-7mol·L^-1范围内与ΔF/F0值成线性关系,r=0.9964。该方法加标回收率为95.2%~104%,相对标准偏差(RSD)为1.02%~4.54%,检出限达到2.67×10^-9mol·L^-1。该方法基于功能核酸对目标物亲合力强、选择识别性高的特点,对ATP的检测表现出很好的选择性,实验结果表明,当相对误差控制在±5%以内时,200倍的UTP,GTP和CTP均不干扰ATP的测定。另外,该方法操作简单、快速、无需标记、灵敏准确,可用于血清样品中ATP的测定,在快速检测小分子物质领域中有较好的应用前景。

A novel Label-Free Fluorometric Assay based on the recombination of split aptamer chip was developed for thedetection of adenosine triphosphate (ATP). In this strategy, the split aptamer was selected as a specific capture probe for the split two fragments aptamers could specifically form a ternary assembly in the presence of ligand and the two separate oligonucleotides lack secondary structures, thus not yielding false-positive or nonspecific signals, while the Thiazoleorange(TO), an almost non-fluorescence dye in buffer solution, was used as signal probe, and the single-walled carbon nanotubes (SWCNTs) was applied to reduce the background signals. In the pH 8.0 Tris-HCl buffer solution, those two split aptamer fragments will be combined with each other to form a stable “aptamer-ATP-aptamer” composite structure upon interacting with its target ATP. The “sandwich” structure can’t wrap the sidewalls of the SWCNTs and is freed in solution, and TO shows agreat fluorescence enhancement when binding to the“aptamer-ATP-aptamer” composite structure. In the absence of ATP, the split aptamers, existing in a single-stranded state, bind to the surface of the SWCNT svia a π-π-conjugate interaction, and TO shows weak fluorescence because “sandwich”structure is not formed. In the system, the higher the ATP concentration is, the more the “aptamer-ATP-aptamer” sandwich recognition structure complex obtained, sois the fluorescence. Under the optimized experimental conditions, the ATP concentration in the range from 9.0×10^-9 mol·L^-1 to 1.0×10^-7 mol·L^-1 was linear with the Δ F/F 0 value at the maximum fluorescence emission wavelength of 550 nm, r =0.996 4, with a low detection limit of 2.67×10^-9 mol·L^-1 . The recoveries of the method were 95.2%~104%, and the relative standard deviation (RSD) was 1.02%~4.54%, respectively. Based on the specific molecular recognition and high affinity of twosplit aptamers, the reaction product was shown that a “turn-on” fluorescence response to ATP with good selectivity, only a slight fluorescence change could be observed by GTP, CTP, and UTP (at a 200-fold higher concentration than that of ATP), indicating that UTP, CTP, and GTP could not interact with P1 and P1 to initiate the reaction. The method is simple, rapid, free-label, sensitive and accurate, and can be used for the determination of ATP in serum samples. Therefore, the present strategy has a great potential application prospect in the field of rapid detection of small molecular substances.