We developed a nanobody-based homogeneous bioluminescent immunosensor to achieve a one-pot detection for point-of-care testing (POCT). This immunosensor was named BRET nano Q-body as its emission color changes via bioluminescence resonance energy transfer (BRET) upon antigen addition. NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminus of the nanobody, which was labeled with a fluorescent dye via thiol-maleimide Michael addition. The nanobody employed in this proof-of-principle experiment recognizes methotrexate (MTX), a chemotherapeutic agent. After optimizing the fluorescent dye and linker, the BRET nano Q-body dose-dependently exhibited a greater than 7-fold increase in emission ratio (TAMRA/NanoLuc). Moreover, we found its superior thermostability endurance in organic solvents, reducing agents, and detergents due to the robust structure of nanobody, as well as accommodation in biological fluids, such as milk, serum, and whole blood without dilution, with limits of detection of 0.50, 1.6, and 3.7 nM, respectively. Furthermore, the BRET nano Q-body was subjected to lyophilization and fabricated into a paper device, which markedly improved its portability and enabled more than one month of storage at 25 °C. The paper device also performed appropriate functions in the biological fluids without any dilution and can be used for on-site therapeutic drug monitoring of MTX. Altogether, we developed a powerful tool, the BRET nano Q-body, for POCT, and demonstrated its applicability in several biological fluids. In addition, we confirmed the feasibility of paper devices, which are expected to be transformative for in situ detection in therapeutic, diagnostic, and environmental applications.

中文翻译：

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BRET Nano Q-Body：一种基于纳米抗体的比率式生物发光免疫传感器，用于即时检测


我们开发了一种基于纳米抗体的均相生物发光免疫传感器，以实现即时检测 （POCT） 的一锅检测。该免疫传感器被命名为 BRET 纳米 Q 体，因为它在抗原添加后通过生物发光共振能量转移 （BRET） 发射颜色发生变化。将 NanoLuc 荧光素酶和含半胱氨酸的标签融合到纳米抗体的 N 端，通过硫醇-马来酰亚胺 Michael 加成用荧光染料标记。本原理验证实验中使用的纳米抗体识别化疗剂甲氨蝶呤 （MTX）。优化荧光染料和连接子后，BRET 纳米 Q 体剂量依赖性地表现出发射比 （TAMRA/NanoLuc） 增加 7 倍以上。此外，由于纳米抗体的稳健结构，我们发现它在有机溶剂、还原剂和去污剂中具有优异的热稳定性，并且在不稀释的情况下在生物体液（如牛奶、血清和全血）中的容纳性，检测限分别为 0.50、1.6 和 3.7 nM。此外，BRET 纳米 Q 体经过冻干并制成纸质装置，这显着提高了其便携性，并在 25 °C 下储存了一个多月。 该纸质装置在生物液中也发挥了适当的功能，无需任何稀释，可用于 MTX 的现场治疗药物监测。总之，我们开发了一种用于 POCT 的强大工具 BRET 纳米 Q 体，并证明了它在几种生物液体中的适用性。此外，我们证实了纸质设备的可行性，有望为治疗、诊断和环境应用中的原位检测带来变革性。