Key Laboratory for Organic Electronics & Information Displays (KLOEID) and Institute of Advanced Materials (IAM), Nanjing University of Posts & Telecommunications , Nanjing 210023, People's Republic of China.
ACS Appl Mater Interfaces. 2014 Mar 12;6(5):3406-12. doi: 10.1021/am405550j. Epub 2014 Feb 27.
Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein's large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules.
蛋白质的快速灵敏检测对生物医学研究和临床诊断至关重要。然而，到目前为止，大多数检测方法都依赖于抗体检测，通常繁琐且耗时，并且灵敏度较差。在此，我们开发了一种简单灵敏的荧光蛋白检测策略，该策略基于分裂适体片段和水溶性聚阳离子聚合物（聚{[9,9-双（6'-(N,N,N-二乙基甲基铵)己基）-2,7-芴撑乙炔基]-交替-共-[2,5-双（3'-(N,N,N-二乙基甲基铵)-1'-氧代丙基）-1,4-亚苯基]四碘化物}（PFEP））。凝血酶结合 DNA 适体被分裂成两个片段用于靶标识别。具有高荧光发射的 PFEP 用作染料标记 DNA 探针信号放大的能量供体。在不存在靶标的情况下，通过强静电相互作用形成三个 DNA/PFEP 复合物，导致两个荧光团之间发生有效的Förster 共振能量转移（FRET）。而当存在靶标时，两个分裂适体片段的连接形成 G-四链体，并随后与 PFEP 组装形成 G-四链体/凝血酶/PFEP 复合物。由于蛋白质的大尺寸，PFEP 与染料之间的距离增加，导致 FRET 信号显著降低。与完整适体相比，使用较短的分裂适体片段增加了形成 G-四链体的可能性。因此，加入靶标前后 FRET 信号的变化率显著提高，从而获得了更高的灵敏度（检测限（LOD）=2 nM）。该策略快速、成本低且具有均相检测，不需要加热以避免 DNA 探针的不利二级结构，具有显著优势。通过进一步的努力，这种方法可以扩展到用于简单灵敏地检测各种生物分子的通用方法。
