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Most aptasensors are designed based on aptamers' conformational change or structural transformation upon interaction with their targets. A limitation of these strategies is the unpredictability of such structural alterations that may yield false-positive or nonspecific signals. In bioanalysis, a dominant strategy for protein determination is the sandwich assay because of its high specificity and sensitivity resulting from the dual recognition mechanism. However, the approach is poorly suited to detection of low molecular weight targets, which unlikely bind to two aptamers simultaneously due to steric hindrance. To circumvent this drawback, a split aptamer strategy was recently developed, in which nucleic acid aptamers were split into two fragments that could specifically form a ternary assembly in the presence of ligand. This elegant approach has been extensively adopted for detection of various targets, especially small molecular weight targets, with different transduction methods including colorimetric, fluorescence, and electrochemical techniques. This review first introduced split aptamers and how to obtain split aptamers, and then summarized recent advances in the development of sandwich-format biosensing assays based on split aptamer fragments, aiming at providing a general guide for the design of split-aptamer-based sandwich assays. Furthermore, we discussed possible challenges in the development of split-aptamer-based sandwich assay regarding stability, sensitivity and multiplicity, so as to offer future opportunities of this assay. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) systems, regarded as "God's scissors", have recently received considerable attention in performing genome editing, transcriptional regulation and biosensor construction, benefiting from their adjustable mechanical properties, easy to operate and design, good biocompatibility, and the collateral cleavage activity. Since 2019, aptamers have been becoming an attractive star integrated into CRISPR/Cas systems as newly emerging molecular recognition elements and flexible signal transduction modules, with their significant advantages such as high sensitivity, remarkable specificity, in vitro synthesis, base-pairing, labeling and modification, and programmability capability. In this review, the recent progress of CRISPR/Cas system-based aptasensors (Cas-aptasensors) is comprehensively summarized including their design principles and superior applications. Firstly, we briefly introduce the essential features of aptamers and the CRISPR/Cas system and then outline the composition of Cas-aptasensors involving the Cas proteins, crRNA, reporter probes, as well as the target analytes and their specific aptamers. In detail, we emphasize their fabrication methods, bio-recognition mechanism, as well as the detection evaluation of four major Cas-aptasensors types of fluorescent, electrochemical, colorimetric, and upconversion luminescent resonance energy transfer, and other four novel Cas-aptasensors including inductively light-up RNA aptamer-based sensors coupled plasma mass spectrometry, resonance Rayleigh scattering, and surface-enhanced Raman scattering, along with. Finally, the challenges and future directions are concluded and depicted for aptamers, CRISPR/Cas systems, and their accelerating applications in Cas-aptasensors. This review is expected to inspire more researchers to have insight into Cas-aptasensors and to help to improve the bioanalytical efficiency and probability of success. [Display omitted] • The review summarizes the recent progress of CRISPR/Cas system-based aptasensors including their design principles and superior applications. • The review emphasizes eight types of Cas-aptasensor on their respects of fabrication methods, bio-recognition mechanism, as well as detection evaluation. • The challenges and future directions are concluded and depicted for more applications of Cas-aptasensors. [ABSTRACT FROM AUTHOR]