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We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C-and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range.lethal factor ͉ plasmon surface band ͉ thrombin E nzymes analytical detection is a key tool in enzymology, extremely important for the screening of noxious toxins and pathologies associated with their presence, and for the development of effective and selective therapeutics. Among enzymes, proteases (1, 2) are particularly relevant because proteolytic processing is the final step in the expression of the activity of a great variety of proteins (3). Standard assays for proteases include those based on radioisotopes or on fluorogenic substrates. A protease assay system that uses functionalized, supermagnetic nanoparticles as magnetic relaxation switches and bi-biotinylated peptide substrates for particles clustering has been reported (4). All of these techniques require specific instrumentation and hence an equipped laboratory. We report here an assay based on nanometer-size gold colloids. Citrate stabilized gold colloids of Ն4 nm diameter present an absorption band at Ϸ520 nm due to plasmon resonance (5, 6) with a very high molar absorptivity. This band is shifted to longer wavelengths upon clustering of the colloids, thus leading to color changes of the solution, from pink-red to violet-blue (7). Clustering may be induced by physical methods (like the increase of the ionic strength of the solution) (8) or chemically by addition of molecules able to connect one nanoparticle to another (9). By taking advantage of this phenomenon, very sensitive detection procedures have been introduced for analytes ranging from DNA to proteins and metal ions (10). Because thiols interact strongly with gold nanoparticles, a molecule featuring a head and tail thiol causes such a process (11). Indeed, when we treat a gold colloid solution with a peptide of the general formula Cys-(AA) n -Cys (where AA is any amino acid but cysteine), the color of the solution turns from pink-red to violet-blue. No change of color, however, is observed with a peptide lacking one of the terminal cysteines. Accordingly, we reasoned that the cleavage of a Cys-(AA) n -Cys peptide in two fragments, each containing a single cysteine, would result in a system unable to induce aggregation of the gold nanoparticles and, hence, failing to induce the color change of the s...

We report here the first example of peptide-functionalized gold nanoparticles hydrolytically active against carboxylate esters. The active units are constituted by His-Phe-OH terminating thiols. The confinement of the catalytic units in the monolayer covering the nanoparticles triggers a cooperative hydrolytic mechanism operative at pH < 7 in which a carboxylate and an imidazolium ion act as general base and general acid, respectively. Such a mechanism is absent with an analogous monomeric dipeptide, and this results in a more than 300-fold rate acceleration of the hydrolytic process at low pH in the presence of the functional nanoparticles.