Lysne Drew P, Stewart Michael H, Susumu Kimihiro, Leski Tomasz A, Stenger David A, Medintz Igor L, Díaz Sebastián A, Green Christopher M
National Research Council, 500 Fifth St NW, Washington, DC, 20001, USA.
U.S. Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Code 6900, Washington, DC, 20375, USA.
Sci Rep. 2025 Jul 31;15(1):27950. doi: 10.1038/s41598-025-09434-9.
CRISPR-Cas systems have revolutionized molecular diagnostics through their specificity and programmability, yet their broad adoption is hindered by the reliance on expensive and complex instrumentation. Here, we present an optimized quantum dot (QD) molecular beacon (QD-MB) platform that integrates Förster resonance energy transfer (FRET)-based detection with CRISPR-Cas functionality, achieving sub-picomolar sensitivity without the need for target amplification. By systematically tuning components, including His-tag modifications for improved QD conjugation, nucleic acid hairpin structures for enhanced enzyme interaction, and QD surface passivation strategies, we demonstrate a two-order-of-magnitude improvement in detection sensitivity. Using LwaCas13a and RNA targets, the limit of detection (LOD) decreased to under 1 pM with plate-reader-based fluorescence measurements and below 10 pM with a lamp-and-smartphone setup, establishing the feasibility of portable, field-ready applications. This work highlights the transformative potential of QD-MBs in biosensing and sets a foundation for further advances in CRISPR-based diagnostics and nanotechnology-enabled sensing platforms.
CRISPR-Cas系统凭借其特异性和可编程性，给分子诊断带来了变革，然而，对昂贵且复杂仪器的依赖阻碍了它们的广泛应用。在此，我们展示了一个经过优化的量子点（QD）分子信标（QD-MB）平台，该平台将基于荧光共振能量转移（FRET）的检测与CRISPR-Cas功能相结合，无需目标扩增即可实现亚皮摩尔级的灵敏度。通过系统地调整组件，包括用于改善量子点偶联的组氨酸标签修饰、用于增强酶相互作用的核酸发夹结构以及量子点表面钝化策略，我们证明检测灵敏度提高了两个数量级。使用LwaCas13a和RNA靶标，基于酶标仪的荧光测量的检测限（LOD）降至1 pM以下，使用灯和智能手机设置则降至10 pM以下，确立了便携式、现场可用应用的可行性。这项工作突出了QD-MB在生物传感中的变革潜力，并为基于CRISPR的诊断和纳米技术传感平台的进一步发展奠定了基础。
