Beijing Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceuticals, School of Life Science, Beijing Institute of Technology, Beijing 100081, China.
Anal Methods. 2023 Jun 1;15(21):2641-2649. doi: 10.1039/d3ay00526g.
Rapid detection of nucleic acids is integral for clinical diagnostics, especially if a major public-health emergency occurs. However, such detection cannot be carried out efficiently in remote areas limited by medical resources. Herein, a dual-labeled fluorescence resonance energy transfer (FRET) lateral flow assay (LFA) based on one-pot enzyme-free cascade amplification was developed for rapid, convenient, and sensitive detection of open reading frame (ORF)1ab of severe acute respiratory syndrome-coronavirus-2. The catalyzed hairpin assembly (CHA) reaction of two well-designed hairpin probes was initiated by a target sequence and generated a hybridization chain reaction (HCR) initiator. Then, HCR probes modified with biotin were initiated to produce long DNA nanowires. After two-level amplification, the cascade-amplified product was detected by dual-labeled lateral flow strips. Gold nanoparticles (AuNPs)-streptavidin combined with the product and then ran along a nitrocellulose membrane under the action of capillary force. After binding with fluorescent microsphere-labeled-specific probes on the T line, a positive signal (red color) could be observed. Meanwhile, AuNPs could quench the fluorescence of the T line, and an inverse relationship between fluorescence intensity and the concentration of the CHA-HCR-amplified product was formed. The proposed strategy achieved a satisfactory limit of detection of 2.46 pM for colorimetric detection and 174 fM for fluorescent detection, respectively. Benefitting from the features of being one-pot, enzyme-free, low background, high sensitivity, and selectivity, this strategy shows great potential in bioanalysis and clinical diagnostics upon further development.
核酸的快速检测对于临床诊断至关重要，尤其是在发生重大公共卫生紧急情况时。然而，在医疗资源有限的偏远地区，这种检测无法有效地进行。在此，开发了一种基于一锅酶免级联扩增的双标记荧光共振能量转移（FRET）侧流分析（LFA），用于快速、方便、灵敏地检测严重急性呼吸综合征冠状病毒 2 的开放阅读框（ORF）1ab。两条精心设计的发夹探针的催化发夹组装（CHA）反应由目标序列引发，并生成杂交链式反应（HCR）引发子。然后，用生物素修饰的 HCR 探针被引发以产生长的 DNA 纳米线。经过两级扩增，级联扩增产物通过双标记侧流条进行检测。金纳米粒子（AuNPs）-链霉亲和素与产物结合，然后在毛细作用力下沿硝酸纤维素膜运行。与 T 线上荧光微球标记的特异性探针结合后，可观察到阳性信号（红色）。同时，AuNPs 可以猝灭 T 线的荧光，并且荧光强度与 CHA-HCR 扩增产物的浓度之间形成反比关系。该策略分别实现了比色检测 2.46 pM 和荧光检测 174 fM 的令人满意的检测限。受益于一锅法、无酶、背景低、高灵敏度和选择性的特点，该策略在进一步发展后在生物分析和临床诊断中具有很大的潜力。
