Fig. 2: Preparation of ALBANC nanoprobe and its colorimetric detection assays.
a, Schematic illustration of the two-step synthesis of MMP-7 nanoprobe. First, AuNC bearing azide-functionalized peptides is synthesized using a one-pot protocol. Subsequently, AuNC is conjugated to DBCO-functionalized albumin via click chemistry reaction, resulting in the formation of the nanoprobe. Created in BioRender. Fruk, L. (2026) https://BioRender.com/r46tl5v. b,c, TEM image of AuNCs (b; mean diameter ± s.d., 1.6 ± 0.3 nm, n = 200 particles) and nanoprobe (c). Scale bars, 5, 10 or 50 nm. Experiment was repeated independently three times with similar results. d, Gel electrophoresis showing the assembly of nanoprobe with Coomassie stain (top) and luminol (bottom, stains for the AuNC). After conjugation, the AuNC is attached to albumin, as indicated by the presence of a single band around 70 kDa. NP, nanoprobe. Three independent experiments (three biological replicates) were performed, yielding similar results. e, Schematic illustration of two colorimetric assays for the detection of AuNC in synthetic urine: (1) peroxidase assay and (2) alloy formation assay, which involves buffer exchange of the AuNC into water via ultrafiltration. Created in BioRender. Fruk, L. (2026) https://BioRender.com/2id8x31. f, Representative UV-Vis spectra showing increase in absorbance at both 370 nm and 652 nm correlated to TMB oxidation in the presence of AuNCs. Inset shows photographs of substrate without AuNC (left) and substrate with AuNCs yielding blue color development (right). Three independent experiments were performed with similar results. g, The limit of detection (LoD) of AuNC in synthetic urine was measured by peroxidase assay, by plotting the initial velocity of TMB oxidation (A652 nm/s, n = 9 independent experiments, mean ± s.d., log-log fit). Peroxidase activity of AuNC is linear over 100 to 10,000 nM of AuNC concentration, with a log scale regression R2 = 0.9981. The dashed line indicates the LoD, calculated as 3 s.d. (3σ) above the mean background signal. h, TEM image of formed from AuNC-Ag alloy nanoparticles upon reaction with the substrate mix (AgNO3, CTAC and ascorbic acid). Three independent experiments were performed, yielding similar results. i, Elemental map photographs of Au-Ag alloy nanoparticles showing the presence of both Au and Ag. Three independent experiments were performed, yielding similar results. j, The formed Au-Ag alloy nanoparticles produced yellow color (λ peak = 414 nm). Three independent experiments were performed with similar results. k, The LoD of AuNC in synthetic urine measured by the alloy formation assay (n = 6 independent experimental replicates, involving independently synthesized AuNC batches, mean ± standard error of the mean (s.e.m.), log-log fit). Alloy formation assay is linear over 0.4 to 1,000 nM of AuNC concentration, with a log scale regression R2 = 0.9309. The dashed line indicates the LoD.
