Ab102500

Steady-state kinetics were obtained using a commercial hydrogen peroxide assay kit (Abcam, ab102500), ThcOx [100 nM (Figure S8)], and varying concentrations of the heterocycle containing full-length and leaderless PatE3′ or full-length ThcE1. The substrates were added to a Corning 96-well half-area black flat bottom polystyrene microplate. In the presence of horseradish peroxidase (HRP), the OxiRed probe produces a product with red fluorescence (excitation and emission at 535 and 587 nm, respectively) by reaction with H2O2, which is the other product of azole formation (Scheme S2). Fluorescence was measured with a SpectraMax 2e microplate reader (Molecular Devices). All experiments were performed at 25 °C. Data were corrected for the autofluorescence of ThcOx, and the substrate was used for background correction. Initial rates were plotted against substrate concentration and fitted to the Michaelis–Menten equation (below) in GraphPad Prism. $v_{\mathrm{int}}=\frac{V\max [S]}{K_m+[S]}$

To fabricate specific nanoprobes for different targets, we conjugated citrate-capped PtNPs with periodate-oxidized specific monoclonal antibodies, using the heterobifunctional cross-linking reagent 3-[2-pyridyldithio]-propionyl hydrazide (Thermo Fisher Scientific, cat. no. 22301).^{25 (link),26 (link)} Conjugation of the monoclonal antibodies to the PtNPs and functionality of the nanoprobes were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), UV—visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR; Cary 630 ATR-FTIR), H₂O₂ decomposition assay (hydrogen peroxide assay kit, ab102500, Abcam, USA), dynamic light scattering and zeta-potential (Malvern Zetasizer Nano, Malvern Instruments, UK), transmission electron microscopy (JEM-2100Plus, JEOL, Japan), and field-emission scanning electron microscopy (Ultra 55, Zeiss, Germany).