A MXene-Au Nanosheets-based Fluorescent-SERS Dual-Mode Biosensor Integrated with CRISPR/Cas12a System for Endotoxin Detection

Abstract

Endotoxins or lipopolysaccharides (LPS) are potent inflammatory agents that pose serious health risks even at low concentrations, thus requiring sensitive and reliable detection methods. Current methods for endotoxin detection, primarily the Limulus Amebocyte Lysate (LAL) assay and Enzyme-linked Immunosorbent Assays (ELISA) are limited by complex procedures, prolonged assay time, and susceptibility to interference. To address these challenges, we developed a single-reporter probe-based dual-mode biosensor integrating MXene-Au nanosheets with the CRISPR/Cas12a system for endotoxin detection. The sensing platform was constructed by conjugating ssDNA-Cy5 reporters onto MXene-Au nanosheets. MXene-Au nanosheets served dual functions, including efficient quenching of fluorescence and strong enhancement of SERS signals. In the presence of target endotoxins, CRISPR/Cas12a remains inactive, leaving the ssDNA-Cy5 reporters intact on the MXene-Au surface. The close proximity of Cy5 to the MXene-Au surface results in quenched fluorescence (“FL off”) but enhanced SERS signals (“SERS on”). Conversely, in the absence of endotoxins, activated CRISPR/Cas12a cleaves the ssDNA-Cy5, releasing Cy5 from the surface, thus leading to fluorescence recovery (“FL on”) but diminished SERS signals (“SERS off”). By measuring the changes in SERS and FL signals and analysing the ratiometric I_SERS/I_FL signals, the biosensor achieved a lower limit of detection (LOD) of 15.9 pg/mL within 30 minutes compared to single detection modes. Validation studies in complex matrices, including spiked water and milk samples, showed performance comparable to commercial endotoxin detection kits. Overall, our CRISPR-Cas12a-mediated fluorescent-SERS dual-mode biosensor not only improves detection sensitivity but also provides built-in quality control, improving the reliability of results through cross-validation.