Label-Free Resonance Rayleigh Scattering Amplification for Lipopolysaccharide Detection and Logical Circuit by CRISPR/Cas12a-Driven Guanine Nanowire Assisted Non-Cross-Linking Hybridization Chain Reaction

Abstract

Although the CRISPR/Cas system has pioneered a new generation of analytical techniques, there remain many challenges in developing a label-free, accurate, and reliable CRISPR/Cas-based assay for reporting the levels of low abundance biomolecules in complex biological samples. Here, we reported a novel CRISPR-derived resonance Rayleigh scattering (RRS) amplification strategy and logical circuit based on a guanine nanowire (G-wire) assisted non-cross-linking hybridization chain reaction (GWancHCR) for label-free detection of lipopolysaccharide (LPS). In the presence of a target, the protospacer-adjacent motif-inserted aptamer is rationally designed to specifically combine with LPS rather than Cas12a, suppressing the trans-cleavage activity of CRISPR/Cas12a and retaining the reporter probes to trigger non-cross-linking aggregation. Owing to the automatic hybridization chain reaction (HCR), in the presence of Mg2+, the released G-quadruplex sequence aggregated to assemble the G-wire superstructure through non-cross-linking. As a result, a dramatically amplified RRS intensity is observed, allowing for reporting LPS levels in a low detection limit of 0.17 pg/mL and a wide linear range among 1.0–100.0 ng/mL. Moreover, this reaction event is capable of programming to perform classical Boolean logic tree analysis, including basic logic computing and complex integrated logic circuits. This study comprehensively analyzed with respect to information flow, matter (molecular events), and energy (RRS), revealing the potential promise in designing of molecular-level “Internet of Things”, intelligent computing, and sensing systems.

Aptamer-based CRISPR-Cas powered diagnostics of diverse biomarkers and small molecule targets

“…Gao et al introduced a G-wire assisted non-cross-linking HCR reaction to create a label-free resonance Rayleigh scattering (RRS) CRISPR-effector powered aptameric sensor system that could reveal the molecular size, shape, conformation, and interfacial features [ 81 ]. When the target was present, the aptamer containing the PAM segment specifically recognized the target rather than crRNA/Cas12a system, suppressing trans-cleavage activity and triggering the HCR reaction by automatically aggregating reporter probes.…”

Recent advances in cascade isothermal amplification techniques for ultra-sensitive nucleic acid detection

“…Copyright © 2020 Elsevier B.V. (E) Schematic illustration of CRISPR-HCR for miRNA detection [ 67 ]. Copyright © 2022 Elsevier B.V. (F) Schematic illustration of GWancHCR amplified CRISPR/Cas12a system for LPS detection by RRS [ 68 ]. Copyright © 2022 American Chemical Society.…”

“…Gao et al [ 68 ] reported a novel CRISPR-derived RRS amplification strategy and logical circuit based on G-wire assisted non-cross-linking HCR (GWancHCR) for label-free detection of lipopolysaccharide (LPS, Fig. 5 F).…”

Dually Amplified Electrochemical Aptasensor for Endotoxin Detection via Target-Assisted Electrochemically Mediated ATRP

“…Specifically, the changes in pH, temperature, and incubation time and the presence of thrombin, glucans, polynucleotides, thromboplastin, and some other components can cause a positive response . With this in mind, several approaches based on the specific recognition by neutralizing proteins, antimicrobial peptides, and aptamers , have been established. By in vitro selection via a protocol called SELEX (systematic evolution of ligands by exponential enrichment), aptamers are single-stranded oligonucleotides that are capable of binding to various targets with high affinity and specificity comparable to antibodies .…”