Showing papers by "Michael H. Stewart published in 2012"
TL;DR: The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event.
Abstract: The unique photophysical properties of semiconductor quantum dot (QD) bioconjugates offer many advantages for active sensing, imaging, and optical diagnostics. In particular, QDs have been widely adopted as either donors or acceptors in Forster resonance energy transfer (FRET)-based assays and biosensors. Here, we expand their utility by demonstrating that QDs can function in a simultaneous role as acceptors and donors within time-gated FRET relays. To achieve this configuration, the QD was used as a central nanoplatform and coassembled with peptides or oligonucleotides that were labeled with either a long lifetime luminescent terbium(III) complex (Tb) or a fluorescent dye, Alexa Fluor 647 (A647). Within the FRET relay, the QD served as a critical intermediary where (1) an excited-state Tb donor transferred energy to the ground-state QD following a suitable microsecond delay and (2) the QD subsequently transferred that energy to an A647 acceptor. A detailed photophysical analysis was undertaken for each step of the FRET relay. The assembly of increasing ratios of Tb/QD was found to linearly increase the magnitude of the FRET-sensitized time-gated QD photoluminescence intensity. Importantly, the Tb was found to sensitize the subsequent QD-A647 donor-acceptor FRET pair without significantly affecting the intrinsic energy transfer efficiency within the second step in the relay. The utility of incorporating QDs into this type of time-gated energy transfer configuration was demonstrated in prototypical bioassays for monitoring protease activity and nucleic acid hybridization; the latter included a dual target format where each orthogonal FRET step transduced a separate binding event. Potential benefits of this time-gated FRET approach include: eliminating background fluorescence, accessing two approximately independent FRET mechanisms in a single QD-bioconjugate, and multiplexed biosensing based on spectrotemporal resolution of QD-FRET without requiring multiple colors of QD.
237 citations
TL;DR: A "hopping" mode of proteolysis at the nanoparticle was identified, confirming enhanced activity, and mechanistic details, including deviations from classic MM-behavior were revealed.
Abstract: Recent studies show that polyvalent, ligand-modified nanoparticles provide significantly enhanced binding characteristics compared to isolated ligands. Here, we assess the ability of substrate-modified nanoparticles to provide enhanced enzymatic activity. Energy transfer assays allowed quantitative, real-time measurement of proteolytic digestion at polyvalent quantum dot-peptide conjugates. Enzymatic progress curves were analyzed using an integrated Michaelis–Menten (MM) formalism, revealing mechanistic details, including deviations from classic MM-behavior. A “hopping” mode of proteolysis at the nanoparticle was identified, confirming enhanced activity.
114 citations
TL;DR: The ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing and introduces multiplexed sensing as a novel capability for a single nanoparticle vector.
Abstract: Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Forster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a...
93 citations
TL;DR: Using a polyampholyte coating, in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand), results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures, suggesting a novel and robust way of delivering neurotherapeutics to neurons.
Abstract: We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP(9)GGH(6) (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.
63 citations
TL;DR: Energy transfer between a range of CdSe-ZnS core-shell QDs and a redox-active osmium(II) polypyridyl complex is examined, finding that the QD donors engage in relatively efficient Förster resonance energy transfer with the Os complex acceptor despite relatively low overall spectral overlap.
Abstract: The ability of luminescent semiconductor quantum dots (QDs) to engage in diverse energy transfer processes with organic dyes, light-harvesting proteins, metal complexes, and redox-active labels continues to stimulate interest in developing them for biosensing and light-harvesting applications. Within biosensing configurations, changes in the rate of energy transfer between the QD and the proximal donor, or acceptor, based upon some external (biological) event form the principle basis for signal transduction. However, designing QD sensors to function optimally is predicated on a full understanding of all relevant energy transfer mechanisms. In this report, we examine energy transfer between a range of CdSe–ZnS core–shell QDs and a redox-active osmium(II) polypyridyl complex. To facilitate this, the Os complex was synthesized as a reactive isothiocyanate and used to label a hexahistidine-terminated peptide. The Os-labeled peptide was ratiometrically self-assembled to the QDs via metal affinity coordination,...
53 citations
Patent•
16 Nov 2012
TL;DR: A peptide attached to a nanoparticles (such as quantum dots) selectively directs the nanoparticles to neurons in a tissue or organism as mentioned in this paper, and the peptide can selectively direct neurons to neurons.
Abstract: A peptide attached to a nanoparticles (such as quantum dots) selectively directs the nanoparticles to neurons in a tissue or organism.
33 citations
TL;DR: Results indicate that the differences among the techniques for analyzing DNA-organized nanostructures are not so large as to prevent their effective use in combination, that the data tend to be corroborative, and that differences observed among them can actually be quite informative.
Abstract: Designer DNA structures have garnered much interest as a way of assembling novel nanoscale architectures with exquisite control over the positioning of discrete molecules or nanoparticles. Exploiting this potential for a variety of applications such as light-harvesting, molecular electronics, or biosensing is contingent on the degree to which various nanoarchitectures with desired molecular functionalizations can be realized, and this depends critically on characterization. Many techniques exist for analyzing DNA-organized nanostructures; however, these are almost never used in concert because of overlapping concerns about their differing character, measurement environments, and the disparity in DNA modification chemistries and probe structure or size. To assess these concerns and to see what might be gleaned from a multimodal characterization, we intensively study a single DNA nanostructure using a multiplicity of methods. Our test bed is a linear 100 base-pair double-stranded DNA that has been modified ...
31 citations
TL;DR: QD-mCherry hybrid materials can be utilized for in vitro biosensing of caspase-3 enzymatic activity and it is shown that QDs and fluorescent proteins can be conjugated together intracellularly with strong potential for live-cell imaging and biosensing applications.
Abstract: There has been considerable recent interest in the creation of nanoparticle-biomolecule hybrid materials for uses such as in vitro and in vivo biosensing, biological imaging, and drug delivery. Nanoparticles have a high surface to volume ratio, making them capable of being decorated with various biomolecules on their surface which retain their biological activity. Techniques to bind these biomolecules to nanoparticle surfaces are also advancing rapidly. Here we demonstrate hybrid materials assembled around CdSe/ZnS core/shell semiconductor quantum dots (QDs). These intrinsically fluorescent materials are conjugated to the fluorescent proteins YFP, mCherry and the light harvesting complex b-phycoerythrin (b-PE). QDs have fluorescent properties that make them ideal as donor fluorophores for Forster resonance energy transfer (FRET) while the fluorescent proteins are able to act as FRET acceptors displaying many advantages over organic dyes. We examine FRET interactions between QDs and all three fluorescent proteins. Furthermore, we show QD-mCherry hybrid materials can be utilized for in vitro biosensing of caspase-3 enzymatic activity. We further show that QDs and fluorescent proteins can be conjugated together intracellularly with strong potential for live-cell imaging and biosensing applications.
28 citations
TL;DR: Palm-1, a palmitylated peptide that is capable of both cellular uptake and rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for cytosolic delivery of QDs.
Abstract: Currently there is considerable interest in using bioconjugated nanoparticles for in vivo imaging, biosensing and
theranostics. Luminescent CdSe/ZnS core shell semiconductor quantum dots (QDs) have unique optical properties
and bioconjugation capabilities that make them ideal prototypes for these purposes. We have previously described
the metal-affinity association between the imidazole groups of terminal hexahistidine residues of peptides and
proteins and the ZnS shell of quantum dots as a useful bioconjugation technique. We have also demonstrated that
QDs labeled with an oligohistidine-tagged cell penetrating peptide (CPP) derived from the HIV TAT-protein could
undergo specific endocytosis-mediated cellular uptake in both HEK293T/17 and COS-1 cells. However, the QDs
were predominantly sequestered in the endosomes. This remains a significant hindrance to future potential cellular
imaging applications which require the QDs to access other subcellular organelles. Here we describe the testing of
several cytosolic QD delivery modalities including microinjection, the commercial cytosolic delivery agent PULSin,
and the cytosolic delivery peptide Palm-1. Palm-1, a palmitylated peptide that is capable of both cellular uptake and
rapid endosomal escape in multiple cell lines without concomitant toxicity, is shown to be the superior method for
cytosolic delivery of QDs. Potential intracellular applications for this peptide are discussed.
Patent•
04 Apr 2012TL;DR: In this paper, quantum dots are modified with varying amounts of a redox-active moiety effective to quench charge transfer quenching and a fluorescent dye effective to perform fluorescence resonance energy transfer, so that the modified quantum dots have a plurality of photophysical properties.
Abstract: Quantum dots are modified with varying amounts of (a) a redox-active moiety effective to perform charge transfer quenching, and (b) a fluorescent dye effective to perform fluorescence resonance energy transfer (FRET), so that the modified quantum dots have a plurality of photophysical properties The FRET and charge transfer pathways operate independently, providing for two channels of control for varying luminescence of quantum dots having the same innate properties