Fig. 2. Dual-probe 3 was cleaved by target (i) thrombin and (ii) MMPs, specificity was confirmed by MALDI TOF MS. (a) Dual-probe 3 was incubated with target and off-target enzymes, with or without inhibitor. Activation was determined by measuring the fluorescence increase (compared to enzyme-free control) at 10 min at two different wavelengths (ex/em 485/528 nm (FAM) and 640/670 nm (Cy5)). (b) The fold-change in fluorescence intensity of fragments b, f and dual-probe 3 were compared following activation with MMP-9 or thrombin, with probe 3 showing specific FAM activation with thrombin and Cy5 increasing with MMP-9 (c) structure of dual-probe 3 showing the theoretical molecular weights of the fragments obtained following cleavage; (d) MALDI-TOF MS spectra of 3 before and after treatment with thrombin or MMP-9. The initial peak for 3 at m/z 6182.001 Da (calc. for C₃₀₇H₄₂₇N₆₄O₆₇S₃⁺ [M⁺]: 6182.353 Da) disappears following treatment showing two new peaks with the expected m/z. (e) MALDI-TOF MS (m/z 1500–3000 Da) correlates well with the expected fragments even when the two enzymes act together. Data is the mean of three independent replicates performed in duplicate. Error bars represent s.e.m. Statistical analysis was performed with one-way ANOVA test. (a) * P = 0.0225; ** P = 0.0026; **** P < 0.0001. (b) P** = 0.0018; *** P = 0.0004; **** P < 0.0001.