Hu Tingting, Yan Ying, Tang Zhenwei, Liu Xinfa, Ma Changbei
School of Life Sciences, Central South University, Changsha 410013, China.
Polymers (Basel). 2021 Feb 27;13(5):725. doi: 10.3390/polym13050725.
In this paper, a fast and simple strategy for sensitive detection of streptavidin (SA) was proposed based on terminal protection of small molecule-linked DNA and cationic conjugated polymer-mediated fluorescence resonance energy transfer (FRET). In principle, we designed a biotin-labelled DNA probe (P1) as the recognitive probe of SA, along with a complementary DNA probe (P2) to form double-stranded DNA (dsDNA) with P1. SYBR Green I (SG I) as a fluorescent dye was further used to specifically bind to dsDNA to emit stronger fluorescence. The cationic poly[(9,9-bis(6'-N,N,N-triethy-lammonium)hexyl) fluorenylene phenylene dibromide] (PFP) acted as the donor to participate in the FRET and transfer energy to the recipient SG I. In the absence of SA, P1 could not hybridize with P2 to form dsDNA and was digested by exonuclease I (Exo I); thus, only a weak FRET signal would be observed. In the presence of SA, biotin could specifically bind to SA, which protected P1 from Exo I cleavage. Then, P1 and P2 were hybridized into dsDNA. Therefore, the addition of SG I and PFP led to obvious FRET signal due to strong electrostatic interactions. Then, SA can be quantitatively detected by monitoring FRET changes. As the whole reagent reaction was carried out in 1.5 mL EP and detected in the colorimetric dish, the operation process of the detection system was relatively simple. The response time for each step was also relatively short. In this detection system, the linear equation was obtained for SA from 0.1 to 20 nM with a low detection limit of 0.068 nM (S/N = 3). In addition, this strategy has also achieved satisfactory results in the application of biological samples, which reveals the application prospect of this method in the future.
本文基于小分子连接DNA的末端保护和阳离子共轭聚合物介导的荧光共振能量转移（FRET），提出了一种快速简便的链霉亲和素（SA）灵敏检测策略。原理上，我们设计了一种生物素标记的DNA探针（P1）作为SA的识别探针，以及一种互补DNA探针（P2）与P1形成双链DNA（dsDNA）。进一步使用荧光染料SYBR Green I（SG I）特异性结合dsDNA以发出更强的荧光。阳离子聚[（9,9-双（6'-N，N，N-三乙基铵）己基）芴撑亚苯基二溴化物]（PFP）作为供体参与FRET并将能量转移给受体SG I。在没有SA的情况下，P1不能与P2杂交形成dsDNA并被核酸外切酶I（Exo I）消化；因此，只能观察到微弱的FRET信号。在有SA的情况下，生物素可以特异性结合SA，保护P1不被Exo I切割。然后，P1和P2杂交形成dsDNA。因此，由于强烈的静电相互作用，加入SG I和PFP会导致明显的FRET信号。然后，可以通过监测FRET变化来定量检测SA。由于整个试剂反应在1.5 mL离心管中进行并在比色皿中检测，检测系统的操作过程相对简单。每个步骤的响应时间也相对较短。在该检测系统中，得到了SA在0.1至20 nM范围内的线性方程，检测限低至0.068 nM（S/N = 3）。此外，该策略在生物样品应用中也取得了满意的结果，揭示了该方法未来的应用前景。
