Fig. 4: Biophysical analysis of MTX:VHH:nanoCLAMP complex interactions and its use to construct a methotrexate biosensor. | Nature Communications

Fig. 4: Biophysical analysis of MTX:VHH:nanoCLAMP complex interactions and its use to construct a methotrexate biosensor.

From: Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices

Fig. 4

a Microscale thermophoresis analysis of the VHH interaction with nanoCLAMP5. In the experiment, 0.5 μM solution of EGFP-VHH was titrated with the increasing concentrations of nanoCLAMP5. Fit of the data led to a Kd value of 3.7 μM. b As in (a) but using 10 nM solution of EGFP-VHH in the presence of 1 μM MTX. Fit of the data led to a Kd value of 8.2 nM. c A schematic representation of CaM-GDH-based two-component MTX biosensor. Conformational change in the VHH domain enables binding of nanoCLAMP8-CaM-BP fusion that induces an activating conformational change in CaM-GDH. d Titration of a 10 nM solution of VHH-CaM-GDH and 100 nM of nanoCLAMP-CaM-BP with increasing concentrations of methotrexate. e Fit of the data from (b) to a quadratic equation leading to a Kd value of 5.6 ± 1.5 nM. f Analyzing serum samples of patients receiving methotrexate therapy with the assay based on the developed MTX biosensor. The results of the assay were plotted against the values obtained using an Abbott Diagnostics immunochemistry station. The inset shows a separate plot for low concentration samples. Source Data are provided as a Source Data file.

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