Using time-shared scanning optical tweezers assisted two-photon fluorescence imaging to establish a versatile CRISPR/Cas12a-mediated biosensor.

Based on the admirable precision to identify target nucleic acids and the particular trans-cleavage feature, CRISPR/Cas12a system is a useful means to further improve the sensing accuracy and the design flexibility of fluorescence biosensors. However, the current construction concepts still suffer from insufficient sensitivity, unsuitable for complicated real samples and limited detection species. In this work, much efforts are achieved to address these obstacles. At first, we adopt a microsphere sustained signal enrichment, under which a home-made time-shared scanning optical tweezers assisted fluorescence imaging is employed to guarantee a stable excitation and also realize multiflux measurement. Furthermore, by taking advantage of the low background merit of the near-infrared light excited two-photon fluorescence, a commendable anti-interference capability is endowed to operate in complex media. After utilizing a functional DNA (e.g. aptamer and DNAzyme) regulated mediation pathway to respond non-nucleic acid analytes (alpha fetal protein and Pb), the newly-established CRISPR/Cas12a-mediated fluorescence biosensor is found to display favorable assay performance. More importantly, our analytical methodology can act as a versatile and reliable toolbox in various applications such as disease diagnosis and environmental analysis, propelling the development of CRISPR system in biosensing field.