Exploring the Trans-Cleavage Activity of CRISPR/Cas12a on Gold Nanoparticles for Stable and Sensitive Biosensing.
Anal Chem
; 93(11): 4967-4974, 2021 03 23.
Article
en En
| MEDLINE
| ID: mdl-33703873
ABSTRACT
Taking advantage of the excellent trans-cleavage activity, CRISPR-based diagnostics (CRISPR-Dx) has shown great promise in molecular diagnostics. However, the single-stranded DNA reporter of the current CRISPR-Dx suffers from poor stability and limited sensitivity, which make their application in complex biological environments difficult. Herein, we, for the first time, explore the trans-cleavage activity of CRISPR/Cas12a toward the substrate on gold nanoparticles and apply the new phenomenon to develop a spherical nucleic acid (SNA) reporter for stable and sensitive CRISPR-Dx biosensing. By anchoring the DNA substrate on gold nanoparticles, we discovered different trans-cleavage activities of different types of the Cas12a system (e.g., LbCas12a and AsCas12a) on a nanoparticle surface. The further study suggests that the trans-cleavage activity of LbCas12a on the nanoparticle surface is highly dependent on the density and length of DNA strands. Based on these interesting discoveries, we furthermore develop SNA reporter-based fluorescent CRISPR-Dx for stable and sensitive biosensing application. Compared to traditional ssDNA reporters, the SNA reporter exhibits improved stability, which enables the stable application in a complex serum environment. In addition, the SNA reporter system with tunable density exhibits high sensitivity with a detection limit of 10 fM, which is about 2 orders of magnitude lower than that of the ssDNA reporter system. Finally, the practical application of SNA reporter-based CRISPR-Dx in clinical serum was successfully achieved. These results indicate their significant potential in future research on biology science and medical diagnoses.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Nanopartículas del Metal
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
Tipo de estudio:
Diagnostic_studies
Idioma:
En
Revista:
Anal Chem
Año:
2021
Tipo del documento:
Article
País de afiliación:
China