Key Laboratory of Natural Products Chemical Biology, Ministry of Education, School of Pharmacy, Shandong University, Jinan 250012, PR China.
School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China.
Biosens Bioelectron. 2016 Dec 15;86:966-970. doi: 10.1016/j.bios.2016.07.108. Epub 2016 Jul 30.
Herein, a sensitive and enzyme-free assay for adenosine detection has been developed on the basis of binding induced colocalization activated hybridization chain reaction (HCR) strategy on the surface of magnetic nanobead. First, the recognition probe was fabricated and divided into two parts: the Apt-1 that composed a part of adenosine aptamer and toehold domain, and the Apt-2 that consisted of another part of adenosine aptamer and branch migration domain. The Apt-1 was immobilized on a streptavidin-magnetic nanobead (streptavidin-MNBs) that played the roles of enrichment and separation. Then the recognition event of adenosine could bring the two parts of aptamer together and induce the colocalization of toehold domain and branch migration domain, which could serve as an integrated initiator to trigger the HCR, producing a long nicked double-stranded polymer. Finally, the intercalating dye SYBR Green I was inserted into the polymer, generating an enhanced fluorescence signal. In this strategy, the initiator was divided into two parts and could be suppressed effectively in the absence of adenosine. Utilizing the separated function, the spontaneous hybridization of H1 and H2 could be avoided, and a low background could be acquired. Moreover, through the double amplification of HCR and multimolecules binding of SYBR Green I, highly sensitive and enzyme-free detection were achieved. The detection limit for adenosine detection was 2.0×10(-7)mol/L, which was comparable or superior to the previous aptasensors. Importantly, adenosine analysis in human urines has been performed, and this strategy could significantly distinguish the adenosine content in normal human urines and cancer patient urines, suggesting that this proposed assay will become a reliable and sensitive adenosine detection method in early clinical diagnosis and medical research.
在此，我们基于结合诱导的杂交链式反应（HCR）策略在磁性纳米珠表面上开发了一种用于腺苷检测的灵敏且无需酶的测定法。首先，制备了识别探针，并将其分为两部分：Apt-1 由腺苷适体的一部分和结合域组成，Apt-2 由腺苷适体的另一部分和分支迁移域组成。Apt-1 固定在链霉亲和素-磁性纳米珠（streptavidin-MNBs）上，该珠起到了富集和分离的作用。然后，腺苷的识别事件可使适体的两部分结合在一起，并诱导结合域和分支迁移域的共定位，这可作为触发 HCR 的集成引发子，产生长的缺口双链聚合物。最后，嵌入染料 SYBR Green I 插入聚合物中，产生增强的荧光信号。在该策略中，将引发子分为两部分，在没有腺苷的情况下可以有效地抑制。利用分离功能，可以避免 H1 和 H2 的自发杂交，并获得低背景。此外，通过 HCR 的双重扩增和 SYBR Green I 的多分子结合，实现了高灵敏度和无需酶的检测。腺苷检测的检测限为 2.0×10(-7)mol/L，可与以前的适体传感器相媲美或更优。重要的是，已经对人尿中的腺苷进行了分析，该策略可以明显区分正常人尿和癌症患者尿中的腺苷含量，表明该方法将成为早期临床诊断和医学研究中可靠且灵敏的腺苷检测方法。
