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.

中文翻译：

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基于 MXene-Au 纳米片的荧光-SERS 双模式生物传感器，与 CRISPR/Cas12a 系统集成，用于内毒素检测


内毒素或脂多糖 （LPS） 是强效炎症因子，即使在低浓度下也会对健康造成严重风险，因此需要灵敏可靠的检测方法。目前的内毒素检测方法，主要是鲎试剂细胞裂解物 （LAL） 测定和酶联免疫吸附测定 （ELISA），受到程序复杂、检测时间长和易受干扰的限制。为了应对这些挑战，我们开发了一种基于单报告探针的双模式生物传感器，将 MXene-Au 纳米片与 CRISPR/Cas12a 系统集成在一起，用于内毒素检测。通过将 ssDNA-Cy5 报告基因偶联到 MXene-Au 纳米片上来构建传感平台。MXene-Au 纳米片具有双重功能，包括高效猝灭荧光和强烈增强 SERS 信号。在存在靶内毒素的情况下，CRISPR/Cas12a 保持无活性，使 ssDNA-Cy5 报告基因在 MXene-Au 表面保持完整。Cy5 与 MXene-Au 表面非常接近，导致荧光猝灭（“FL 关闭”）但增强 SERS 信号（“SERS 打开”）。相反，在没有内毒素的情况下，激活的 CRISPR/Cas12a 会裂解 ssDNA-Cy5，从表面释放 Cy5，从而导致荧光恢复（“FL 打开”）但 SERS 信号减弱（“SERS 关闭”）。通过测量 SERS 和 FL 信号的变化并分析比率式 I_SERS/I_FL 信号，与单一检测模式相比，生物传感器在 30 分钟内实现了 15.9 pg/mL 的检测下限 （LOD）。对复杂基质（包括加标水和牛奶样品）的验证研究表明，其性能与市售内毒素检测试剂盒相当。 总体而言，我们的 CRISPR-Cas12a 介导的荧光 SERS 双模式生物传感器不仅提高了检测灵敏度，而且还提供了内置的质量控制，通过交叉验证提高了结果的可靠性。