Abstract
Proteases are enzymes that catalyse the breaking of specific peptide bonds in proteins and polypeptides. They are heavily involved in many normal biological processes as well as in diseases, including cancer, stroke and infection. In fact, proteolytic activity is sometimes used as a marker for some cancer types. Here we present luminescent quantum dot (QD) bioconjugates designed to detect proteolytic activity by fluorescence resonance energy transfer. To achieve this, we developed a modular peptide structure which allowed us to attach dye-labelled substrates for the proteases caspase-1, thrombin, collagenase and chymotrypsin to the QD surface. The fluorescence resonance energy transfer efficiency within these nanoassemblies is easily controlled, and proteolytic assays were carried out under both excess enzyme and excess substrate conditions. These assays provide quantitative data including enzymatic velocity, Michaelis–Menten kinetic parameters, and mechanisms of enzymatic inhibition. We also screened a number of inhibitory compounds against the QD–thrombin conjugate. This technology is not limited to sensing proteases, but may be amenable to monitoring other enzymatic modifications.
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Acknowledgements
The authors acknowledge NRL and L. Chrisey at the Office of Naval Research (ONR grant No N001404WX20270) and A. Krishnan at DARPA for support. A.R.C. is supported by a National Research Council Fellowship through NRL. P.E.D. acknowledges the Skaggs Institute for Chemical Biology. The authors thank E. Alnemri of the Kimmel Cancer Institute, Thomas Jefferson University for advice about caspase recognition sequences. The authors also thank J.M. Mauro (Invitrogen) for useful discussions.
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Medintz, I., Clapp, A., Brunel, F. et al. Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates. Nature Mater 5, 581–589 (2006). https://doi.org/10.1038/nmat1676
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DOI: https://doi.org/10.1038/nmat1676
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