Analysis of Protease Activity Using Quantum Dots and Resonance Energy Transfer

Abstract

This review demonstrates the detection of protease activity based on the energy transfer of quantum dots (QDs). By incorporation of varying protease substrates into designed QD probes both in fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) system, proteolytic activity led to changes in the energy transfer efficiency. Especially due to the superior properties of QDs, it can be served as an excellent probe for a multiplexed and high-throughput protease assay with high sensitivity. It is anticipated that the QD-based FRET/BRET probes will have a great potential for dissecting the fundamental roles of proteases and designing potential protease inhibitors as therapeutic drugs in biology and nanomedicine.

Materials for FRET Analysis: Beyond Traditional Dye–Dye Combinations

“…Although less common, BL enzymes have also been coupled with traditional organic dyes and increasingly NMs, especially QDs as acceptors [283,286,[319][320][321]. Currently QD-BRET has been used for sensing protease activity, protein-protein interactions and in vivo imaging, as reviewed in Ref.…”

“…Enhanced BRET between the firefly Photinus pyralis luciferase variant PpyGRTS quantum rods (QRs) as the energy acceptor has also been described recently, with BRET ratios dependent upon enzyme loading, rod aspect ratio, and donor-acceptor distances (Figure 6.18b) [320]. Protease activity sensing in a QD-BRET system relies on a peptide substrate linkage between the luciferase and the QD that is cleaved in the presence of the protease of interest, reducing the BRET-based QD emissions [321]. QD-BRET has also been used to study protein-protein and receptor-ligand interactions.…”

“…This was first shown in a mouse model where superficial and deep tissues were imaged using QDs with a 655 nm emission, which were coupled to Luc8 [319]. As highlighted later in the QD section (Section 6.5.5), the relatively narrow emission profiles open up the possibility of multiplexed sensing of proteinprotein interactions using a number of QD-BRET pairs [286,321].…”

“…The broad absorption spectra and large Stokes shift found in QDs is also of benefit for FRET studies, as it allows excitation of mixed QD donor populations at one wavelength far removed from their emissions and also facilitates selection of an excitation wavelength that corresponds to the acceptors absorption minima, thus reducing direct excitation background signals. The ability to excite multiple QD donors using a single excitation wavelength, combined with their narrow and symmetric emission (which makes deconvolution of multiple fluorescent signals simpler), makes them attractive labels for multiplex applications [32,321,513,[524][525][526][527][528][529].…”

Gold nanoparticle-based fluorescence quenching via metal coordination for assaying protease activity

“…The ability of AuNPs to induce fluorescence quenching of proximal dyes is reported to be directed by a surface energy transfer process [7][8][9]; the rate of energy transfer from a dye to AuNP depends on the inverse of fourth power of the donor-acceptor separation, which triggers a much longer working distance (up to 22 nm) than that observed in a traditional fluorescence resonance energy transfer system (up to 10 nm, due to the inverse sixth power distance dependency). To this end, AuNP-organic dye couples have been implemented for the highly sensitive detection of oligonucleotides [10][11][12], proteins [13][14][15][16][17], and other small molecules [18][19][20].…”