Luminescent Quantum Dots Fluorescence Resonance Energy Transfer-Based Probes for Enzymatic Activity and Enzyme Inhibitors

Abstract

The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 microg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency.

Aqueous Near‐Infrared Fluorescent Composites Based on Apoferritin‐Encapsulated PbS Quantum Dots

“…[363,371] Scheme 5.2: Two-step FRET mechanism (adapted from reference [363] Moreover, some groups reported the application of FRET between II-VI QDs and fluorescent dyes linked together by different biomolecules such as antibody, [369,373,378] BSA, [374,377] peptide, [367,375,376,382,383] and DNA [379][380][381].…”

“…Bioluminescence resonance energy transfer (BRET) with PbS quantum dots 90 the peptide linker. [382,383] …”

Quantum Clusters of Gold Exhibiting FRET

“…Since semiconductor nanoparticles or quantum dots are strongly fluorescent, they are widely used in FRET-based techniques. [5][6][7][8][9][10][11][12][13][14] CdSe/ZnS quantum dot-aptamer conjugates are used in targeted cancer imaging, sensing, and therapy based on FRET. 5 Cds/ZnS quantum dots can be used as protease sensors by employing FRET.…”

Monitoring of Enzymatic Proteolysis Using Self‐Assembled Quantum Dot‐Protein Substrate Sensors

“…Other dye-labeled biological molecules such as peptides, DNA, RNA, or aptamers that are responsive to enzymes, drugs, or other chemicals can be easily adapted to this format and indeed a few preliminary qualitative enzymatic studies using QD-substrates have been reported, including a QD-sensor for monitoring lactamase activity associated with antibiotic resistance [55][56][57][58]. With the large number of putative proteases in the human genome (∼560) as well as those utilized by pathogens, many will require intact protein substrates for recognition and cleavage/digestion [1][2][3][4].…”