BRET Nano Q-body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing

Yang, Yinghui; Inoue, Akihito; Yasuda, Takanobu; Ueda, Hiroshi; Zhu, Bo; Kitaguchi, Tetsuya

doi:10.51094/jxiv.763

##article.authors##

Yang, Yinghui Graduate School of Life Science and Technology, Tokyo Institute of Technology
Inoue, Akihito Graduate School of Life Science and Technology, Tokyo Institute of Technology
Yasuda, Takanobu Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Ueda, Hiroshi Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Zhu, Bo Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology
Kitaguchi, Tetsuya Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology

DOI:

https://doi.org/10.51094/jxiv.763

キーワード:

immunosensor、 BRET、 Quenchbody、 nanobody、 point-of-care testing

抄録

We developed a nanobody-based homogeneous bioluminescent immunosensor whose emission color changes by the bioluminescence resonance energy transfer (BRET) upon antigen addition to realize the one-pot analysis for point-of-care testing (POCT) and named it BRET nano Q-body. The NanoLuc luciferase and a cysteine-containing tag were fused to the N-terminal of the nanobody, which was subsequently labeled with fluorescent dye through a thiol-maleimide reaction. The nanobody employed in this proof-of-principle experiment recognizes methotrexate (MTX), a chemotherapy agent for cancer treatment. The BRET nano Q-body after fluorescent dye and linker optimization exhibited a more than 8-fold increase in emission ratio (TAMRA/Nluc) in a dose-dependent manner. We also found that 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 0.5, 1.6, and 3.7 nM, respectively. Furthermore, we performed lyophilization on BRET nano Q-body and made it into the paper device, greatly improving portability and allowing more than one month of storage in 25 ℃. The paper device also functioned properly in the aforementioned biological fluids without dilution and can be applied to therapeutic drug monitoring of MTX on site. We provided a powerful tool, BRET nano Q-body for POCT, and demonstrated its applicability in several biological fluids and the feasibility of paper devices, which is greatly expected as the pioneer for in situ detection in therapeutic, diagnostic, and environmental applications.

利益相反に関する開示

T.Y., H.U., B.Z. and T.K. received honoraria from HikariQ Health, Inc. for another unrelated project.

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