Gold nanoparticles-based protease assay

Abstract

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...

Applications of Nanoparticles in Biology

“…In a representative study, Stevens et al reported a two-stage approach by using Fmoc-protected peptides (substrate of thermolysin) with a cysteine amino acid attached to gold nanoparticles. [70] In presence of thermolysin the peptides are fragmented and the assembly changes color from blue to red with a sensitivity of 90 zg mL À1 (i.e., less than 380 molecules of protease). [71] This approach has been extended to kinases [72] and phosphatases [73,74] at low concentration.…”

Polypeptide functionalized gold nanoparticles for bioanalytical applications

“…5 This strategy enabled detection of the proteases thrombin and lethal factor Xa in the nanomolar range. Another approach was described by Li et al where alkaline phosphatase activity could be detected based on that the substrate adenosine 5'-triphosphate (ATP) and its hydrolyzed derivates could function as a stabilizing capping agent and prevent salt induced aggregation to different extents.…”

“…Nanomaterials, and in particular functionalized nanoparticles, have consequently been extensively used for development of novel and highly sensitive detection strategies for important biomolecular analytes providing valuable assays for diagnostics and drug development. The nanoparticles can be designed to be responsive to a wide range of biomolecules including proteases, which can be disease markers for the common cold to cancers and ADIS, [4][5] or to significantly smaller entities such as toxic metal ions. 6 The assay response is most often but not always directly linked to the molecular recognition event.…”

Functionalisation of Colloidal Transition Metal Sulphides Nanocrystals: A Fascinating and Challenging Playground for the Chemist

“…Among the most employed ligands for metal nanoparticles, thiols [39,[322][323][324][325][326][327][328] have to be mentioned, whereas metal oxides NP can be conveniently derivatised by carboxylates or, even stronger, by multifunctional ligands such as dopamine-derivatives and cathecols [280,283,292,[329][330][331]. A further distinction can be carried out accordingly to the chemical nature of the surface to be derivatised, which can be noble metal NPs [9,141,163,269,318, or metal oxides, the most commonly functionalised colloids, but also of other composition (halogenides, chalcogenides, phosphates, carbonates etc.…”