%0 Journal Article
%T Unraveling the influence of CRISPR/Cas13a reaction components on enhancing trans-cleavage activity for ultrasensitive on-chip RNA detection.
%A He Q
%A Chen Q
%A Lian L
%A Qu J
%A Yuan X
%A Wang C
%A Xu L
%A Wei J
%A Zeng S
%A Yu D
%A Dong Y
%A Zhang Y
%A Deng L
%A Du K
%A Zhang C
%A Pandey V
%A Gul I
%A Qin P
%J Mikrochim Acta
%V 191
%N 8
%D 2024 07 17
%M 39017814
%F 6.408
%R 10.1007/s00604-024-06545-4
%X The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13's reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform.