School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa 226-8503, Japan.
Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology, Nagatsuta-cho, Yokohama, Kanagawa 226-8503, Japan.
ACS Sens. 2020 Nov 25;5(11):3457-3464. doi: 10.1021/acssensors.0c01404. Epub 2020 Nov 10.
"Quenchbody (Q-body)" is a quench-based fluorescent biosensor labeled with a fluorescent dye near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules quickly by simply mixing with a sample. However, the development of Q-bodies using V-nanobodies derived from camelid heavy-chain antibodies has not been reported despite their favorable characteristics. Here, we report a "mini Q-body" that can detect the chemotherapy agent methotrexate (MTX) by using anti-MTX nanobody. Three kinds of constructs each encoding an N-terminal Cys-tag and anti-MTX VHH gene with a different length of linker (GGGS) ( = 0, 2, and 4) between them were prepared followed by the expression in and labeling with several dye maleimides. When the fluorescence intensities in the presence of varied MTX concentrations were measured, TAMRA-labeled nanobodies showed a higher response than ATTO520- or R6G-labeled ones. Especially, TAMRA C6-labeled mini Q-body with no linker showed the highest response of ∼6-fold and a low detection limit of 0.56 nM. When each Trp residue in the mini Q-body was mutated to address the quenching mechanism, the major role of Trp34 at CDR1 in quenching was revealed. Furthermore, the mini Q-body could detect MTX in 50% human serum with a low detection limit of 1.72 nM, showing its applicability to therapeutic drug monitoring. This study is expected to become the basis of the construction of highly responsive mini Q-bodies for sensitive detection of many molecules from small haptens to larger proteins, which will lead to broader applications such as point-of-care tests.
“Quenchbody (Q-body)”是一种基于淬灭的荧光生物传感器，在抗体的抗原结合位点附近标记有荧光染料。Q-body 可以通过简单地与样品混合来快速检测一系列目标分子。然而，尽管 V 型纳米体具有良好的特性，但尚未报道使用源自骆驼重链抗体的 V 型纳米体来开发 Q-body。在这里，我们报告了一种“迷你 Q-body”，它可以通过使用抗 MTX 纳米体来检测化疗药物甲氨蝶呤 (MTX)。制备了三种构建体，每个构建体都在 N 端 Cys 标签和抗 MTX VHH 基因的编码序列之间带有不同长度的接头 (GGGS) ( = 0、2 和 4)，然后在大肠杆菌中表达并标记有几种染料马来酰亚胺。当测量存在不同 MTX 浓度时的荧光强度时，TAMRA 标记的纳米体比 ATTO520 或 R6G 标记的纳米体具有更高的响应。特别是，没有接头的 TAMRA C6 标记的迷你 Q-body 表现出最高的响应约 6 倍，检测限为 0.56 nM。当将迷你 Q-body 中的每个色氨酸残基突变为解决猝灭机制时，揭示了 CDR1 中的色氨酸 34 在猝灭中的主要作用。此外，迷你 Q-body 可以在 50%的人血清中检测到 MTX，检测限低至 1.72 nM，表明其适用于治疗药物监测。这项研究有望成为构建高响应迷你 Q-body 的基础，用于灵敏检测从小抗原到更大蛋白质的许多分子，从而将其应用扩展到即时检测等领域。
