Protease functional assay on membrane.

• A facile and sensitive protease assay on a paper membrane is reported. • The paper based assay yields a LOD that is four times better than the conventional solution-based assays. • Naked eye observable responses is obtained for the active form of proteases. • MMP-7 assay in synthetic urine is demonstrated with responses comparable to that of responses in buffer. • The proposed approach is generic for detection of a wide range of proteases. Targeting enzymes, proteases in particular, using various assay formats has attracted considerable interest for early disease diagnosis. While affinity-based assays that report the presence of proteases in samples have been widely explored, it is of utmost importance to develop functional assays that provide information on the activity of proteases for a more precise indication of the disease state and progression. Herein, we report a paper-based functional assay that yields naked eye observable and concentration dependent responses for the active form of proteases. The paper-based assay involves a facile single step fabrication process of depositing protease specific peptide functionalized gold nanoparticles on paper membranes. Matrilysin is used as a model protease to validate the proposed methodology. The proteolysis-driven aggregation of nanoparticles on the membrane yields a colorimetric response that has limit of detection that is ∼4 times lower (3.1 μg/mL) than for the same assay performed in homogeneous solution (12.5 μg/mL). To the best of our knowledge, this is the first report on heterogeneous protease assay on paper relying on aggregation of peptide-functionalized nanoparticles. The approach easily can be extended to assay other enzymes by functionalizing the gold nanoparticles using specific peptides. [ABSTRACT FROM AUTHOR]