Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

Nanoparticle-based labels are emerging as simpler and more sensitive alternatives to traditional fluorescent small molecules and radioactive reporters in biomarker assays. The determination of biomarker levels is a recommended clinical practice for the assessment of many diseases, and detection of multiple analytes in a single assay, known as multiplexing, can increase predictive accuracy. While multiplexed detection can also simplify assay procedures and reduce systematic variability, combining multiple assays into a single procedure can lead to complications such as substrate cross-reactivity, signal overlap, and loss of sensitivity. By combining the specificity of biomolecular interactions with the tunability of quantum dot optical properties, we have developed a detection system capable of simultaneous evaluation of the activity of two critical enzyme classes, proteases and kinases. We avoid cross-reactivity and signal overlap by synthesizing enzyme-specific peptide sequences with orthogonal terminal functionalization for attachment to quantum dots with distinct emission spectra. Enzyme activity is reported via binding of either gold nanoparticle-peptide conjugates or FRET acceptor dye-labeled antibodies, which mediate changes in quantum dot emission spectra. To the best of our knowledge, this is the first demonstration of the multiplexed sensing of the activity of two different classes of enzymes via a nanoparticle-based activity assay. Using the quantum dot-based assay described herein, we were able to detect the protease activity of urokinase-type plasminogen activator at concentrations ≥ 50 ng/mL and the kinase activity of human epidermal growth factor receptor 2 at concentrations ≥ 7.5 nM, levels that are clinically relevant for determination of breast cancer prognosis. The modular nature of this assay design allows for the detection of different classes of enzymes simultaneously and represents a generic platform for high-throughput enzyme screening in rapid disease diagnosis and drug discovery.

Original publication




Journal article


ACS Nano

Publication Date





851 - 857


Enzyme Activation, Fluorescence Resonance Energy Transfer, Peptide Hydrolases, Peptides, Phosphotransferases, Quantum Dots