Interface-Stabilized Logic Nanoprobe Enables High-Fidelity Orthogonal Sensing of MMP-1 and HNE in Thiol-Rich...

Reliable multiplexed sensing in thiol-rich biological environments remains challenging because nonspecific ligand displacement, background leakage, and partial channel corruption can compromise the analytical meaning of multichannel fluorescence outputs. Here, we report an interface-stabilized logic-gated fluorescent nanoprobe for high-fidelity orthogonal sensing of matrix metalloproteinase-1 (MMP-1) and human neutrophil elastase (HNE). Two fluorogenic peptide substrates were co-immobilized on gold nanoparticles through an Au-C≡C nano-biointerface to generate independent FITC and 5-TAMRA fluorescence outputs upon target-specific cleavage. This interface design preserved a low-background OFF state under glutathione-rich conditions and supported stable dual-channel transduction in biologically relevant environments. Under optimized conditions, the nanoprobe exhibited linear fluorescence responses toward MMP-1 from 0 to 800 ng/mL and HNE from 0 to 1000 ng/mL, with regression equations of F = 0.32[MMP-1] + 19.12 (R2 = 0.993) and F = 0.18[HNE] + 43.54 (R2 = 0.993), respectively. The corresponding detection limits were 8.03 ng/mL for MMP-1 and 9.44 ng/mL for HNE. The nanoprobe also showed strong selectivity against interfering proteins, ions, oxidants, and reductants, together with good tolerance to physiologically relevant pH variation. Compared with an Au-S-linked control, the Au-C≡C nanoprobe exhibited substantially lower temperature- and GSH-induced fluorescence leakage. Evaluation under four protease-input states further demonstrated channel-specific activation and enabled an AND-gated dual-positive output. In live-cell studies, dual-channel fluorescence activation clearly distinguished A549 cells from BEAS-2B cells, and integration of the two channels into a dual-enzyme activity index improved analytical discrimination between malignant and nonmalignant states. Local tumor imaging in a subcutaneous LLC model provided proof-of-concept validation that the orthogonal sensing concept remains operable in a biologically complex environment. This work establishes an interface-enabled strategy for preserving signal fidelity in multiplexed protease sensing and provides a logic-gated platform for orthogonal biochemical analysis in thiol-rich biological systems.