Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates

Medintz, Igor L.; Clapp, Aaron R.; Brunel, Florence M.; Tiefenbrunn, Theresa; Uyeda, H. Tetsuo; Chang, Eddie L.; Deschamps, Jeffrey R.; Dawson, Philip E.; Mattoussi, Hedi

doi:10.1038/nmat1676

Cited by 524 publications

(613 citation statements)

References 46 publications

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“…18 As such, we chose to utilize the sample with four peptides/QD as substrate in the assay. Triplicate samples at concentrations ranging from 7.5 to 50 pmols of QD decorated with four peptides each were exposed to 65 units of caspase 3 per reaction, while samples containing no enzyme were used as negative controls.…”

Section: Resultsmentioning

confidence: 99%

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Prasuhn¹,

et al. 2010

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The nanoscale size and unique optical properties of semiconductor quantum dots (QDs) have made them attractive as central photoluminescent scaffolds for a variety of biosensing platforms. In this report we functionalize QDs with dye-labeled peptides using two different linkage chemistries to yield Förster resonance energy transfer (FRET)-based sensors capable of monitoring either enzymatic activity or ionic presence. The first sensor targets the proteolytic activity of caspase 3, a key downstream effector of apoptosis. This QD conjugate utilized carbodiimide chemistry to covalently link dye-labeled peptide substrates to the terminal carboxyl groups on the QD’s surface hydrophilic ligands in a quantitative manner. Caspase 3 cleaved the peptide substrate and disrupted QD donor-dye acceptor FRET providing signal transduction of enzymatic activity and allowing derivation of relevant Michaelis−Menten kinetic descriptors. The second sensor was designed to monitor Ca2+ ions that are ubiquitous in many biological processes. For this sensor, Cu+-catalyzed [3 + 2] azide−alkyne cycloaddition was exploited to attach a recently developed azide-functionalized CalciumRuby-Cl indicator dye to a cognate alkyne group present on the terminus of a modified peptide. The labeled peptide also expressed a polyhistidine sequence, which facilitated its subsequent metal-affinity coordination to the QD surface establishing the final FRET sensing construct. Adding exogenous Ca2+ to the sensor solution increased the dyes fluorescence, altering the donor−acceptor emission ratio and manifested a dissociation constant similar to that of the native dye. These results highlight the potential for combining peptides with QDs using different chemistries to create sensors for monitoring chemical compounds and biological processes.

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Section: Resultsmentioning

confidence: 99%

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Prasuhn¹,

et al. 2010

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“…Although a noncovalent conjugation strategy is used here, the ratio of His 6 peptide and thus acceptor DNA acceptor per QD is also easily controlled through the molar ratio added for self-assembly with the QD as repeatedly demonstrated in Refs. [1,2,6,7,11,13,14,21,23] and discussed in detail in references [13,21,23].…”

Section: Discussionmentioning

confidence: 99%

“…The FRET analysis also accounted for the contribution and any deviation due to Poisson distribution effects on low valence during the selfassembly process as described in Ref. [13,21,23]. …”

Section: Fluorescence Resonance Energy Transfer Analysismentioning

confidence: 99%

Improved peptidyl linkers for self-assembly of semiconductor quantum dot bioconjugates

Berti

D’Agostino²,

Boeneman

et al. 2009

Nano Res.

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We demonstrate improved peptide linkers which allow both conjugation to biomolecules such as DNA and self-assembly with luminescent semiconductor quantum dots. A hexahistidine peptidyl sequence was generated by standard solid phase peptide synthesis and modified with the succinimidyl ester of iodoacetamide to yield a thiol-reactive iodoacetyl polyhistidine linker. The reactive peptide was conjugated to dye-labeled thiolated DNA which was utilized as a model target biomolecule. Agarose gel electrophoresis and fl uorescence resonance energy transfer analysis confi rmed that the linker allowed the DNA to self-assemble with quantum dots via metal-affi nity driven coordination. In contrast to previous peptidyl linkers that were based on disulfi de exchange and were thus labile to reduction, the reactive haloacetyl chemistry demonstrated here results in a more stable thioether bond linking the DNA to the peptide which can withstand strongly reducing environments such as the intracellular cytoplasm. As thiol groups occur naturally in proteins, can be engineered into cloned proteins, inserted into nascent peptides or added to DNA during synthesis, the chemistry demonstrated here can provide a simple method for self-assembling a variety of stable quantum dot bioconjugates.

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“…1-5 These properties make QDs powerful tools for labeling and optical imaging in biological, 3,6-11 biomedical, 12-18 and chem-/bio-sensing applications. [19][20][21][22] Imaging cellular events at the single-molecule level is a particular area where the superior brightness and photostability of QDs excel as compared to more typical dye and fluorescent protein imaging reagents. 11 Notwithstanding, the optimal design of QDs for single-molecule imaging in live cells presents a unique set of challenges.…”

Section: Introductionmentioning

confidence: 99%

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

et al. 2008

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We present a family of water-soluble quantum dots (QDs) that exhibit low nonspecific binding to cells, small hydrodynamic diameter, tunable surface charge, high quantum yield, and good solution stability across a wide pH range. These QDs are amenable to covalent modification via simple carbodiimide coupling chemistry, which is achieved by functionalizing the surface of QDs with a new class of heterobifunctional ligands incorporating dihydrolipoic acid, a short poly(ethylene glycol) (PEG) spacer, and an amine or carboxylate terminus. The covalent attachment of molecules is demonstrated by appending a rhodamine dye to form a QD-dye conjugate exhibiting fluorescence resonance energy transfer (FRET). High-affinity labeling is demonstrated by covalent attachment of streptavidin, thus enabling the tracking of biotinylated epidermal growth factor (EGF) bound to EGF receptor on live cells. In addition, QDs solubilized with the heterobifunctional ligands retain their metal-affinity driven conjugation chemistry with polyhistidine-tagged proteins. This dual functionality is demonstrated by simultaneous covalent attachment of a rhodamine FRET acceptor and binding of polyhistidine-tagged streptavidin on the same nanocrystal to create a targeted QD, which exhibits dual-wavelength emission. Such emission properties could serve as the basis for ratiometric sensing of the cellular receptor's local chemical environment.

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Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot–peptide conjugates

Cited by 524 publications

References 46 publications

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Quantum Dot Peptide Biosensors for Monitoring Caspase 3 Proteolysis and Calcium Ions

Improved peptidyl linkers for self-assembly of semiconductor quantum dot bioconjugates

Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging

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