Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with pre-amplification 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 μL-1. Moreover, Au-CFRET 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.
Keywords: CRISPR/dCas9; Fluorescence resonance energy transfer; Gold nanoparticles; Nucleic acid biosensing; Rapid detection; Recombinase polymerase amplification.
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