CRISPR-Cas12a-Based Aptasensor for On-Site and Highly Sensitive Detection of Microcystin-LR in Freshwater

Abstract

On-site monitoring of trace organic pollutants with facile methods is critical to environmental pollutant prevention and control. Herein, we proposed a CRISPR-Cas12a-based aptasensor platform (named as MC-LR-Casor) for on-site and sensitive detection of microcystin-LR (MC-LR). After hybridization with blocker DNA, the MC-LR aptamers were conjugated to magnetic beads (MBs) to get the MB aptasensor. In the presence of MC-LR, their interactions with aptamers were triggered and the specific binding caused the release of blocker DNA. Using the programmability of the CRISPR-Cas system, the released blocker DNA was designed to activate a Cas12a-crRNA complex. Single strand DNA reporters were rapidly cleaved by the complex. Signal readout could be achieved by fluorometer or lateral flow strips, which were positively correlated to MC-LR concentration. Benefiting from the CRISPR-Cas12a amplification system, the proposed sensing platform exhibited high sensitivity and reached the limit of detection of ∼3 × 10–6 μg/L (fluorescence method) or 1 × 10–3 μg/L (lateral flow assay). In addition, the MC-LR-Casor showed excellent selectivity and good recovery rates, demonstrating their good applicability for real water sample analysis. During the whole assay, only two steps of incubation at a constant temperature were required and the results could be visualized when employing flow strips. Therefore, the proposed assay offered a simple and convenient alternative for in situ MC-LR monitoring, which may hold great promise for future environmental surveillance.

CRISPR/Cas12a-Assisted Chemiluminescence Sensor for Aflatoxin B1 Detection in Cereal Based on Functional Nucleic Acid and In-Pipet Rolling Circle Amplification

“…The binding between the aptamer and AFB 1 is driven by molecular forces, such as the van der Waals forces, hydrogen bonding, electrostatic interactions, and hydrophobic forces, all of which are influenced by the incubation conditions. 37 Several key parameters were optimized to obtain the optimal analytical performance, including the competitive reaction mode, the ratio of the aptamer to the activator, and the reaction temperature and time between AFB 1 and the aptamer− activator duplex. For the competitive reaction mode, indirect and direct ways were investigated (Figure S7 of the Supporting Information).…”

Insight into the Sensing Behavior of DNA Probes Based on MOF–Nucleic Acid Interaction for Bioanalysis

“…By this way, a rapid colorimetric bioassay was fabricated. It can be seen from Figure B that the absorbance at 416 nm was indeed dependent on the concentration of AFB1 and based on which the detection limit (LOD) of the assay was calculated to be 0.97 nM using the 3σ/ k rule (where σ denotes the standard deviation obtained by conducting blank samples and k is the slope of the calibration curve) (Figure C). Meanwhile, the blocker DNA was employed to trigger the HCR events between H1 and H2, thus producing fluorescent responses (Figure A,b).…”

CRISPR/Cas System: The Accelerator for the Development of Non-nucleic Acid Target Detection in Food Safety

“…Kang et al proposed a CRISPR/Cas12a-powered aptasensor platform for on-site and sensitive detection of microcystin-LR (MC-LR) . The MC-LR aptamers were first conjugated to magnetic beads (MBs) after hybridization with blocker DNA.…”