13 research outputs found
Colloidal Stability of Gold Nanoparticles Coated with Multithiol-Poly(ethylene glycol) Ligands: Importance of Structural Constraints of the Sulfur Anchoring Groups
Gold nanoparticles (AuNPs) coated
with a series of polyÂ(ethylene
glycol) (PEG) ligands appended with four different sulfur-based terminal
anchoring groups (monothiol, flexible dithiol, constrained dithiol,
disulfide) were prepared to explore how the structures of the sulfur-based
anchoring groups affect the colloidal stability in aqueous media.
The PEG-coated AuNPs were prepared by ligand exchange of citrate-stabilized
AuNPs with each ligand. The colloidal stability of the AuNPs in different
harsh environmental conditions was monitored visually and spectroscopically.
The AuNPs coated with dithiol- or disulfide-PEG exhibited improved
stability under high salt concentration and against ligand replacement
competition with dithiothreitol compared with those coated with their
monothiol counterpart. Importantly, the ligands with structurally
constrained dithiol or disulfide showed better colloidal stability
and higher sulfur coverage on the Au surface compared to the ligands
with more flexible dithiol and monothiol. X-ray photoelectron spectroscopy
also revealed that the disulfide-PEG ligand had the highest S coverage
on Au surface on the Au surface among the ligands studied. This result
was supported by energy minimization modeling studies: the structurally
more constrained disulfide ligand has the shortest S–S distance
and could pack more densely on the Au surface. The experimental results
indicate that the colloidal stability of the AuNPs is systematically
enhanced in the following order: monothiol < flexible dithiol <
constrained dithiol < disulfide. The present study indicates that
the colloidal stability of thiolated ligand-functionalized AuNPs can
be enhanced by (i) a multidentate chelating effect and (ii) use of
the constrained and compact structure of the multidentate anchoring
groups
Synthesis and Characterization of PEGylated Luminescent Gold Nanoclusters Doped with Silver and Other Metals
Doping
of fluorescent noble metal nanoclusters is being pursued
to manipulate the structure of such materials along with improving
physicochemical characteristics such as long-term stability and photoluminescence
quantum yield. Here, we synthesize metal-doped and alloyed ultrasmall
gold nanoclusters (AuNCs) directly in water using a facile one-step
coreduction reaction with bidentate dithiolane PEGylated ligands that
terminate in different functional groups including a methoxy, carboxy,
amine, and azide. Two primary types of cluster materials were the
focus of synthesis and characterization: first, a series of doped/alloyed
Ag-doped AuNCs, where the ratio of Au:Ag was varied across a wide
range including 99:1, 98:2, 90:10, 80:20, 50:50, 20:80, 10:90, and
2:98 along with pure AuNC and AgNC controls; second, doped Au:D NCs,
where D included Pt, Cu, Zn, and Cd. Physical characterization of
the modified AuNCs included TEM analysis of size, XPS/EDX analysis
of dopant content, and a detailed analysis of photophysical properties
including absorption and photoluminescence profiles, quantum yields
over time, photoluminescence lifetimes, and examination of energy
levels for selected materials. The addition of just a few Ag dopant
atoms per AuNC yielded significant enhancement in quantum yield along
with improving long-term photostability especially in comparison to
materials with a very high Ag content. Preliminary cell imaging applications
of the Ag-doped AuNCs were also investigated. Facilitated cellular
uptake by mammalian cells via endocytosis following modification with
cell penetrating peptides was confirmed by colabeling with specific
cellular markers. Long-term intracellular photostability and lack
of aggregation were confirmed with microinjection studies, and cytoviability
assays showed the doped clusters to be minimally toxic
Purple‑, Blue‑, and Green-Emitting Multishell Alloyed Quantum Dots: Synthesis, Characterization, and Application for Ratiometric Extracellular pH Sensing
We
report the synthesis of a series of Cd<sub><i>x</i></sub>Zn<sub>1–<i>x</i></sub>Se/Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S/ZnS and ZnSe/Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S/ZnS
multishell alloyed luminescent semiconductor quantum dots (QDs) with
fluorescence maxima ranging from 410 to 530 nm which cover the purple,
blue, and green portion of the spectrum. Their subsequent surface
modification to prepare water-soluble blue-emitting QDs, characterization,
and application for ratiometric pH sensing in aqueous buffers and
in an extracellular environment are further described. QDs were synthesized
starting from ZnSe cores, and the fluorescence peak positions were
tuned by (i) cation exchange with cadmium ions and/or (ii) overcoating
with Cd<sub><i>y</i></sub>Zn<sub>1–<i>y</i></sub>S layers. The as-prepared QDs had reasonably high fluorescence
quantum yields (∼30–55%), narrow fluorescence bands
(fwhm ∼25–35 nm), and monodispersed semispherical shapes.
Ligand exchange with hydrophilic compact ligands was successfully
carried out to prepare a series of water-soluble blue-emitting QDs.
QDs coated with the hydrophilic compact ligands preserved the intrinsic
photophysical properties well and showed excellent colloidal stability
in aqueous buffers for over a year. The blue-emitting QDs were further
conjugated with the pH-sensitive dye, fluorescein isothiocyanate (FITC),
to construct a fluorescence resonance energy transfer-based ratiometric
pH sensing platform, and pH monitoring with the QD-FITC conjugates
was successfully demonstrated at pHs ranging between 3 and 7.5. Further
assembly of the QD-FITC conjugates with membrane localization peptides
allowed monitoring of the pH in extracellular environments. High quality,
water-soluble blue-emitting QDs coated with compact ligands can help
expand the practical fluorescence range of QDs for a variety of biological
applications
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 Förster 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 Å, the exciton transfer efficiency reaching the final, terminal dye was estimated to be only ∼0.7% 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 ≤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 donor–acceptor dye pairings, (3) varying donor–acceptor dye spacing as a function of the Förster 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 Förster 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 × <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
Quantum Dots as Simultaneous Acceptors and Donors in Time-Gated Förster 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 Förster 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 terbiumÂ(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
Intracellularly Actuated Quantum Dot–Peptide–Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway
Nanoparticle
(NP)-mediated drug delivery (NMDD) has emerged as
a novel method to overcome the limitations of traditional systemic
delivery of therapeutics, including the controlled release of the
NP-associated drug cargo. Currently, our most advanced understanding
of how to control NP-associated cargos is in the context of soft nanoparticles
(e.g., liposomes), but less is known about controlling the release
of cargos from the surface of hard NPs (e.g., gold NPs). Here we employ
a semiconductor quantum dot (QD) as a prototypical hard NP platform
and use intracellularly triggered actuation to achieve spatiotemporal
control of drug release and modulation of drug efficacy. Conjugated
to the QD are two peptides: (1) a cell-penetrating peptide (CPP) that
facilitates uptake of the conjugate into the endocytic pathway and
(2) a display peptide conjugated to doxorubicin (DOX) via three different
linkages (ester, disulfide, and hydrazone) that are responsive to
enzymatic cleavage, reducing conditions, and low pH, respectively.
Formation of the QD–[peptide–DOX]–CPP complex
is driven by self-assembly that allows control over both the ratio
of each peptide species conjugated to the QD and the eventual drug
dose delivered to cells. Förster resonance energy transfer
assays confirmed successful assembly of the QD–peptide complexes
and functionality of the linkages. Confocal microscopy was employed
to visualize residence of the QD–[peptide–DOX]–CPP
complexes in the endocytic pathway, and distinct differences in DOX
localization were noted for the ester linkage, which showed clear
signs of nuclear delivery versus the hydrazone, disulfide, and amide
control. Finally, delivery of the QD–[peptide–DOX]–CPP
conjugate resulted in cytotoxicity for the ester linkage that was
comparable to free DOX. Attachment of DOX via the hydrazone linkage
facilitated intermediary toxicity, while the disulfide and amide control
linkages showed minimal toxicity. Our data demonstrate the utility
of hard NP–peptide bioconjugates to function as multifunctional
scaffolds for simultaneous control over cellular drug uptake and toxicity
and the vital role played by the nature of the chemical linkage that
appends the drug to the NP carrier
Complex Förster Energy Transfer Interactions between Semiconductor Quantum Dots and a Redox-Active Osmium Assembly
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 <i>via</i> metal affinity coordination, bringing the Os complex into close proximity of the nanocrystal surface. QDs displaying different emission maxima were assembled with increasing ratios of Os–peptide complex and subjected to detailed steady-state, ultrafast transient absorption, and luminescence lifetime decay analyses. Although the possibility exists for charge transfer quenching interactions, we find that the QD donors engage in relatively efficient Förster resonance energy transfer with the Os complex acceptor despite relatively low overall spectral overlap. These results are in contrast to other similar QD donor–redox-active acceptor systems with similar separation distances, but displaying far higher spectral overlap, where charge transfer processes were reported to be the dominant QD quenching mechanism
Optimizing Protein Coordination to Quantum Dots with Designer Peptidyl Linkers
Semiconductor quantum dots (QDs) demonstrate select optical
properties
that make them of particular use in biological imaging and biosensing.
Controlled attachment of biomolecules such as proteins to the QD surface
is thus critically necessary for development of these functional nanobiomaterials.
QD surface coatings such as polyÂ(ethylene glycol) impart colloidal
stability to the QDs, making them usable in physiological environments,
but can impede attachment of proteins due to steric interactions.
While this problem is being partially addressed through the development
of more compact QD ligands, here we present an alternative and complementary
approach to this issue by engineering rigid peptidyl linkers that
can be appended onto almost all expressed proteins. The linkers are
specifically designed to extend a terminal polyhistidine sequence
out from the globular protein structure and penetrate the QD ligand
coating to enhance binding by metal-affinity driven coordination.
α-Helical linkers of two lengths terminating in either a single
or triple hexahistidine motif were fused onto a single-domain antibody;
these were then self-assembled onto QDs to create a model immunosensor
system targeted against the biothreat agent ricin. We utilized this
system to systematically evaluate the peptidyl linker design in functional
assays using QDs stabilized with four different types of coating ligands
including polyÂ(ethylene glycol). We show that increased linker length,
but surprisingly not added histidines, can improve protein to QD attachment
and sensor performance despite the surface ligand size with both custom
and commercial QD preparations. Implications for these findings on
the development of QD-based biosensors are discussed
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 rutheniumÂ(II)–phenanthroline
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 Förster 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 QD–Ru<sup>2+</sup>-phen system can allow
for the design of more sophisticated charge-transfer-based biosensors
using QD platforms
Competition between Förster Resonance Energy Transfer and Electron Transfer in Stoichiometrically Assembled Semiconductor Quantum Dot–Fullerene Conjugates
Understanding how semiconductor quantum dots (QDs) engage in photoinduced energy transfer with carbon allotropes is necessary for enhanced performance in solar cells and other optoelectronic devices along with the potential to create new types of (bio)sensors. Here, we systematically investigate energy transfer interactions between C<sub>60</sub> fullerenes and four different QDs, composed of CdSe/ZnS (type I) and CdSe/CdS/ZnS (quasi type II), with emission maxima ranging from 530 to 630 nm. C<sub>60</sub>-pyrrolidine tris-acid was first coupled to the N-terminus of a hexahistidine-terminated peptide <i>via</i> carbodiimide chemistry to yield a C<sub>60</sub>-labeled peptide (pepC<sub>60</sub>). This peptide provided the critical means to achieve ratiometric self-assembly of the QD-(pepC<sub>60</sub>) nanoheterostructures by exploiting metal affinity coordination to the QD surface. Controlled QD-(pepC<sub>60</sub>)<sub><i>N</i></sub> bioconjugates were prepared by discretely increasing the ratio (<i>N</i>) of pepC<sub>60</sub> assembled per QD in mixtures of dimethyl sulfoxide and buffer; this mixed organic/aqueous approach helped alleviate issues of C<sub>60</sub> solubility. An extensive set of control experiments were initially performed to verify the specific and ratiometric nature of QD-(pepC<sub>60</sub>)<sub><i>N</i></sub> assembly. Photoinitiated energy transfer in these hybrid organic–inorganic systems was then interrogated using steady-state and time-resolved fluorescence along with ultrafast transient absorption spectroscopy. Coordination of pepC<sub>60</sub> to the QD results in QD PL quenching that directly tracks with the number of peptides displayed around the QD. A detailed photophysical analysis suggests a competition between electron transfer and Förster resonance energy transfer from the QD to the C<sub>60</sub> that is dependent upon a complex interplay of pepC<sub>60</sub> ratio per QD, the presence of underlying spectral overlap, and contributions from QD size. These results highlight several important factors that must be considered when designing QD-donor/C<sub>60</sub>-acceptor systems for potential optoelectronic and biosensing applications