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A gold nanoparticle-enhanced dCas9-mediated fluorescence resonance energy transfer for nucleic acid detection

文献类型： 外文期刊

作者： Yang, Yao 1 ; Zhai, Shanshan 1 ; Zhang, Li 2 ; Wu, Yuhua 1 ; Li, Jun 1 ; Li, Yunjing 1 ; Li, Xiaofei 1 ; Zhu, Longjiao 3 ; Xu, Wentao 3 ; Wu, Gang 1 ; Gao, Hongfei 1 ;

作者机构： 1.Chinese Acad Agr Sci, Oil Crops Res Inst, Key Lab Agr Genet Modified Organisms Traceabil, Minist Agr & Rural Affairs, Wuhan 430062, Peoples R China

2.South Cent Minzu Univ, Coll Life Sci, Hubei Prov Key Lab Protect & Applicat Special Plan, Wuhan 430074, Peoples R China

3.China Agr Univ, Key Lab Precis Nutr & Food Qual, Dept Nutr & Hlth, Beijing 100083, Peoples R China

关键词： CRISPR/dCas9; Fluorescence resonance energy transfer; Gold nanoparticles; Recombinase polymerase amplification; Nucleic acid biosensing; Rapid detection

期刊名称：TALANTA （ 影响因子：6.1； 五年影响因子：5.5 ）

ISSN： 0039-9140

年卷期： 2025 年 282 卷

页码：

收录情况： SCI

摘要： Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with preamplification have shown great potential in molecular diagnoses. However, the current CRISPR-based methods require additional reporters and time-consuming process. Herein, a gold nanoparticle (AuNP)enhanced CRISPR/dCas9-mediated fluorescence resonance energy transfer (FRET) termed Au-CFRET platform was proposed for rapid, sensitive, and specific detection of nucleic acid for the first time. In the Au-CFRET sensing platform, AuNP was functionalized with dCas9 and used as nanoprobe. Target DNA was amplified with FAM-labeled primers and then precisely bound with AuNP-dCas9. The formed complex rendered the distance between AuNP acceptor and FAM donor to be short enough for the occurrence of FRET, thus resulting in fluorescence quenching. Moreover, AuNPs were demonstrated to enhance binding efficiency of dCas9 to target DNA in Au-CFRET system. The key factors regarding the FRET efficiency were analyzed and characterized in detail, including the length of donor/acceptor and the size of AuNPs. Under the optimal conditions, Au-CFRET could determinate CaMV35S promoter of genetically modified rice as low as 21 copies mu L-1. Moreover, AuCFRET sensing system coupled with one-step extraction and recombinase polymerase amplification can identify the genuine plant seeds within 30 min from sampling to results at room/body temperature without expensive equipment or technical expertise, and requires no additional exogenous reporters. Therefore, the proposed sensing platform significantly simplified the system and shortened the assay time for nucleic acid diagnoses.

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