Hg²⁺-triggered cascade strand displacement assisted CRISPR-Cas12a for Hg²⁺ quantitative detection using a portable...

CRISPR-Cas12a is a powerful and programmable tool that has revolutionized the field of biosensing. However, the construction of a CRISPR-Cas12a-mediated portable system for on-site and quantitative detection of mercury ion (Hg²⁺) has yet to be explored. By integrating a target-triggered cascade toehold-mediated strand displacement reaction (TSDR) and CRISPR-Cas12a, we herein construct a portable on-site biosensor for the quantitative, sensitive, and selective detection of Hg²⁺ with a glucose meter. The Hg²⁺ initiates two cascade TSDRs through the T-Hg²⁺-T interaction to produce multiple double-stranded DNAs that can activate Cas12a's trans-cleavage activity. The Cas12a cleaves the sucrase-modified DNA on the electrode, resulting in the liberation of sucrase into the solution. The freed sucrase can catalyze sucrose to generate glucose, which can be quantitatively monitored by a glucometer. The developed portable biosensor provides a dynamic range of 5 orders of magnitude with a detection limit of 40 fM. This biosensor also displays excellent selectivity and stability for detecting Hg²⁺. Moreover, environmental water samples are utilized to further verify the robustness and effectiveness of the developed biosensor, highlighting its potential application in environmental monitoring and food safety analysis.