14 research outputs found
Multiplexed Tracking of Protease Activity Using a Single Color of Quantum Dot Vector and a Time-Gated FoĢrster Resonance Energy Transfer Relay
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 FoĢrster 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 series of quantitative,
real-time kinetic assays of increasing biochemical complexity, including
multiplexed sensing, measuring inhibition in a multiplexed format,
and tracking the proteolytic activation of an inactive pro-protease
to its active form in a coupled, multienzyme system. These capabilities
were derived from a ratiometric analysis of the two FRET pathways
in the relay and permitted extraction of initial reaction rates, enzyme
specificity constants, and apparent inhibition constants. This work
adds to the growing body of research on multifunctional nanoparticles
and introduces multiplexed sensing as a novel capability for a single
nanoparticle vector. Furthermore, the ability to track both enzymes
within a coupled biological system using one vector represents a significant
advancement for nanoparticle-based biosensing. Prospective applications
in biochemical research, applied diagnostics, and drug discovery are
discussed
Understanding How Nanoparticle Attachment Enhances Phosphotriesterase Kinetic Efficiency
As a specific example of the enhancement of enzymatic activity that can be induced by nanoparticles, we investigate the hydrolysis of the organophosphate paraoxon by phosphotriesterase (PTE) when the latter is displayed on semiconductor quantum dots (QDs). PTE conjugation to QDs underwent extensive characterization including structural simulations, electrophoretic mobility shift assays, and dynamic light scattering to confirm orientational and ratiometric control over enzyme display which appears to be necessary for enhancement. PTE hydrolytic activity was then examined when attached to <i>ca.</i> 4 and 9 nm diameter QDs in comparison to controls of freely diffusing enzyme alone. The results confirm that the activity of the QD conjugates significantly exceeded that of freely diffusing PTE in both initial rate (ā¼4-fold) and enzymatic efficiency (ā¼2-fold). To probe kinetic acceleration, various modified assays including those with increased temperature, presence of a competitive inhibitor, and increased viscosity were undertaken to measure the activation energy and dissociation rates. Cumulatively, the data indicate that the higher activity is due to an acceleration in enzymeāproduct dissociation that is presumably driven by the markedly different microenvironment of the PTE-QD bioconjugateās hydration layer. This report highlights how a specific change in an enzymatic mechanism can be both identified and directly linked to its enhanced activity when displayed on a nanoparticle. Moreover, the generality of the mechanism suggests that it could well be responsible for other examples of nanoparticle-enhanced catalysis
Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology
Functionalizing Nanoparticles
with Biological Molecules:
Developing Chemistries that Facilitate Nanotechnolog
Achieving Effective Terminal Exciton Delivery in Quantum Dot Antenna-Sensitized Multistep DNA Photonic Wires
Assembling DNA-based photonic wires around semiconductor quantum dots (QDs) creates optically active hybrid architectures that exploit the unique properties of both components. DNA hybridization allows positioning of multiple, carefully arranged fluorophores that can engage in sequential energy transfer steps while the QDs provide a superior energy harvesting antenna capacity that drives a FoĢrster resonance energy transfer (FRET) cascade through the structures. Although the first generation of these composites demonstrated four-sequential energy transfer steps across a distance >150 Ć
, the exciton transfer efficiency reaching the final, terminal dye was estimated to be only ā¼0.7% with no concomitant sensitized emission observed. Had the terminal Cy7 dye utilized in that construct provided a sensitized emission, we estimate that this would have equated to an overall end-to-end ET efficiency of ā¤0.1%. In this report, we demonstrate that overall energy flow through a second generation hybrid architecture can be significantly improved by reengineering four key aspects of the composite structure: (1) making the initial DNA modification chemistry smaller and more facile to implement, (2) optimizing donorāacceptor dye pairings, (3) varying donorāacceptor dye spacing as a function of the FoĢrster distance <i>R</i><sub>0</sub>, and (4) increasing the number of DNA wires displayed around each central QD donor. These cumulative changes lead to a <i>2 orders of magnitude</i> improvement in the exciton transfer efficiency to the final terminal dye in comparison to the first-generation construct. The overall end-to-end efficiency through the optimized, five-fluorophore/four-step cascaded energy transfer system now approaches 10%. The results are analyzed using FoĢrster theory with various sources of randomness accounted for by averaging over ensembles of modeled constructs. Fits to the spectra suggest near-ideal behavior when the photonic wires have two sequential acceptor dyes (Cy3 and Cy3.5) and exciton transfer efficiencies approaching 100% are seen when the dye spacings are 0.5 Ć <i>R</i><sub>0</sub>. However, as additional dyes are included in each wire, strong nonidealities appear that are suspected to arise predominantly from the poor photophysical performance of the last two acceptor dyes (Cy5 and Cy5.5). The results are discussed in the context of improving exciton transfer efficiency along photonic wires and the contributions these architectures can make to understanding multistep FRET processes
Functionalization-Dependent Induction of Cellular Survival Pathways by CdSe Quantum Dots in Primary Normal Human Bronchial Epithelial Cells
Quantum dots (QDs) are semiconductor nanocrystals exhibiting unique optical properties that can be exploited for many practical applications ranging from photovoltaics to biomedical imaging and drug delivery. A significant number of studies have alluded to the cytotoxic potential of these materials, implicating Cd-leaching as the causal factor. Here, we investigated the role of heavy metals in biological responses and the potential of CdSe-induced genotoxicity. Our results indicate that, while negatively charged QDs are relatively noncytotoxic compared to positively charged QDs, the same does not hold true for their genotoxic potential. Keeping QD core composition and size constant, 3 nm CdSe QD cores were functionalized with mercaptopropionic acid (MPA) or cysteamine (CYST), resulting in negatively or positively charged surfaces, respectively. CYST-QDs were found to induce significant cytotoxicity accompanied by DNA strand breakage. However, MPA-QDs, even in the absence of cytotoxicity and reactive oxygen species formation, also induced a high number of DNA strand breaks. QD-induced DNA damage was confirmed by identifying the presence of p53 binding protein 1 (53BP1) in the nuclei of exposed cells and subsequent diminishment of p53 from cytoplasmic cellular extracts. Further, high-throughput real-time PCR analyses revealed upregulation of DNA damage and response genes and several proinflammatory cytokine genes. Most importantly, transcriptome sequencing revealed upregulation of the metallothionein family of genes in cells exposed to MPA-QDs but not CYST-QDs. These data indicate that cytotoxic assays must be supplemented with genotoxic analyses to better understand cellular responses and the full impact of nanoparticle exposure when making recommendations with regard to risk assessment
Synthesis and Characterization of PbS/ZnS Core/Shell Nanocrystals
We
demonstrate a synthetic method to add a ZnS shell, with controlled
thickness, to PbS nanocrystals using Zn oleate and thioacetamide as
Zn and S precursors. The ZnS shell reaction is self-limiting and deposits
approximately a monolayer of ZnS per shell reaction without causing
the PbS nanocrystals to Ostwald ripen. The reaction is self-limiting
because the sulfur precursor, thioacetamide, is less reactive toward
the PbS/ZnS core/shell nanocrystal surface as compared to the Zn oleate
precursor present in the reaction solution. To increase the ZnS shell
thickness beyond a monolayer, subsequent ZnS shell reactions are modified
by adding the thioacetamide 10 minutes before the Zn oleate. This
gives the thioacetamide time to react at the PbS/ZnS core/shell nanocrystal
surface before the Zn oleate is added. High angle annular dark field
scanning transmission electron microscopy (HAADF-STEM) shows most
ZnS shells lack crystalline order. However, select core/shell nanocrystals
have epitaxial crystalline (zinc-blende) ZnS shells or crystalline
(zinc-blende) shells with no obvious epitaxial relationship to the
PbS core. The PbS core 1S<sub>h</sub>ā1S<sub>e</sub> absorbance
and photoluminescence peak energies redshift upon shell addition due
to relief of a ligand-induced tensile strain and wave function leakage
into the shell. The photoluminescence quantum yield decreases after
ZnS shell addition likely due to nonradiative defect states at the
core/shell interface
Multimodal Characterization of a Linear DNA-Based Nanostructure
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 by a variety of chemical handles, dyes, semiconductor quantum dots, gold nanoparticles, and electroactive labels. To this we apply a combination of physical/optical characterization methods including electrophoresis, atomic force microscopy, transmission electron microscopy, dynamic light scattering, FoĢrster resonance energy transfer, voltammetry, and structural modeling. In general, the results indicate that the differences among the techniques 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
Proteolytic Activity at Quantum Dot-Conjugates: Kinetic Analysis Reveals Enhanced Enzyme Activity and Localized Interfacial āHoppingā
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 MichaelisāMenten
(MM) formalism, revealing mechanistic details, including deviations
from classic MM-behavior. A āhoppingā mode of proteolysis
at the nanoparticle was identified, confirming enhanced activity
Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated FoĢrster Resonance Energy Transfer Relays: Characterization and Biosensing
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 FoĢrster 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 terbiumĀ(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 QDāA647 donorāacceptor 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
Probing the Quenching of Quantum Dot Photoluminescence by Peptide-Labeled Ruthenium(II) Complexes
Charge transfer processes with semiconductor
quantum dots (QDs)
have generated much interest for potential utility in energy conversion.
Such configurations are generally nonbiological; however, recent studies
have shown that a redox-active rutheniumĀ(II)āphenanthroline
complex (Ru<sup>2+</sup>-phen) is particularly efficient at quenching
the photoluminescence (PL) of QDs, and this mechanism demonstrates
good potential for application as a generalized biosensing detection
modality since it is aqueous compatible. Multiple possibilities for
charge transfer and/or energy transfer mechanisms exist within this
type of assembly, and there is currently a limited understanding of
the underlying photophysical processes in such biocomposite systems
where nanomaterials are directly interfaced with biomolecules such
as proteins. Here, we utilize
redox reactions, steady-state absorption, PL spectroscopy, time-resolved
PL spectroscopy, and femtosecond transient absorption spectroscopy
(FSTA) to investigate PL quenching in biological assemblies of CdSe/ZnS
QDs formed with peptide-linked Ru<sup>2+</sup>-phen. The results reveal
that QD quenching requires the Ru<sup>2+</sup> oxidation state and
is not consistent with FoĢrster resonance energy transfer, strongly
supporting a charge transfer mechanism. Further, two colors of CdSe/ZnS
core/shell QDs with similar macroscopic optical properties were found
to have very different rates of charge transfer quenching, by Ru<sup>2+</sup>-phen with the key difference between them appearing to be
the thickness of their ZnS outer shell. The effect of shell thickness
was found to be larger than the effect of increasing distance between
the QD and Ru<sup>2+</sup>-phen when using peptides of increasing
persistence length. FSTA and time-resolved upconversion PL results
further show that exciton quenching is a rather slow process consistent
with other QD conjugate materials that undergo hole transfer. An improved
understanding of the QDāRu<sup>2+</sup>-phen system can allow
for the design of more sophisticated charge-transfer-based biosensors
using QD platforms