A rapid and sensitive “add-mix-measure” assay for multiple proteinases based on one gold nanoparticle–peptide–fluorophore conjugate

We report here a homogeneous fluorometric method for the assays of multiple proteinases based on one AuNPs–peptide–fluorophore conjugate as the substrate. The peptide portion of conjugates has cleavage sites for multiple proteinases. Once nanoprobes are fabricated by end-immobilizing fluorophore labeled peptide onto the surface of gold nanoparticles, proteinase is introduced and enzymatic cleavage of residues in the peptide portion of the conjugates occurs as a consequence of the specific substrate recognition by the proteinase, manifesting in the form of strong fluorescence signal recovery. The method offers a sensitive and rapid evaluation of proteinases activity operating either in an endpoint or real-time format. The “add-mix-measure” format of this method eliminates tedious nanoprobes processing, significantly reduces the assay time and makes it have more generality. Moreover, this assay can be used to detect proteinase activity in biological media. The developed assay is ideal for industrial routine tests, clinical assay and high-throughput screening proteinase inhibitors. As far as we know, this is the first report that a single substrate can be used to assess multiple proteinases which recognize different cleavage motifs.