Creation of a Nanobody-Based Fluorescent Immunosensor Mini Q-body for Rapid Signal-On Detection of Small Hapten Methotrexate.

"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 VHH-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)n (n = 0, 2, and 4) between them were prepared followed by the expression in Escherichia coli 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.