Fig. 5: Electrochemical analysis of MTX biosensor and thereon based electrodes.

a A schematic representation of solution electrochemistry measurements where electrons generated by the MTX biosensor are transported to the electrode surface via an electron mediator (shown as golden balls). The functional elements are drawn as in Fig. 4c. b Electrochemical response of 0.5 μM VHH-GDH-CaM and 0.75 μM nanoCLAMP-CaM-BP solutions in the absence and presence of 1 μM of MTX. Sensor enzymatic activity is reported as maximum μA increase per minute using disposable DropSense gold electrode polarized at +0.1 V vs. Ag reference strip and mPMS as an electron transfer mediator. The data represents reading of individual electrodes tested sequentially. c A schematic of an electrode covalently modified with the VHH-GDH-CaM fusion component of MTX biosensor. The second component nanoCLAMP-CaM-BP is present in solution and associates with the electrode in the presence of the ligand leading to bioelectrode activation. d Typical cyclic voltammograms for MTX biosensor-based bioelectrode: black—buffer A solution (25 mM Tris–H₂SO₄ buffer, pH 7.2, 100 mM Na₂SO₄ and 1 mM Ca(CH₃COO)₂); blue—in presence of 20 mM glucose; green—in presence of 20 mM glucose and 200 nM nanoCLAMP-CaM-BP; red—in presence of 20 mM glucose, 200 nM nanoCLAMP-CaM-BP and 200 nM MTX. The electrode was scanned at the rate of 2 mV/s vs. Ag/AgCl/3 M KCl reference electrode at room temperature. e Increase in the electric current on the MTX bioelectrode shown in (c) following its exposure to the increased concentration of MTX. The upper inset shows reversibility of the activation after rinsing of the electrode. The lower inset compares the response of the electrode to the specific ligand MTX or unspecific ligand rapamycin. The data points represent average of three independent measurements performed on the same electrode. Data are presented as mean values ± standard error of mean. f as in (e) but using 50% human serum diluted in buffer A, 20 mM glucose and the indicated concentrations of MTX. g Rapamycin dose response of the bio-electrode constructed by cross-linking FKBP-CaM-GDH to the electrode and providing FRB-CaM-BP in solution. The inset provides a fit of the linear part of the titration curve. The data points represent average of three independent measurements performed on the same electrode. Data are presented as mean values ± standard error of mean. h Analysis of the MTX and rapamycin bio-electrodes for their ability to selectively recognize their cognate analytes in the presence of the component of the orthogonal GDH-biosensor. The bars represent values of average of three independent measurements performed on the same electrode. The error bars denote positive and negative boundaries of the standard error of mean. Source Data are provided as a Source Data file.