6 research outputs found
Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries that Facilitate Nanotechnology
Functionalizing Nanoparticles
with Biological Molecules:
Developing Chemistries that Facilitate Nanotechnolog
Cytotoxicity of Quantum Dots Used for <i>In Vitro</i> Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores
Interest
in taking advantage of the unique spectral properties
of semiconductor quantum dots
(QDs) has driven their widespread use in biological applications such
as <i>in vitro</i> cellular labeling/imaging and sensing.
Despite their demonstrated utility, concerns over the potential toxic
effects of QD core materials on cellular proliferation and homeostasis
have persisted, leaving in question the suitability of QDs as alternatives
for more traditional fluorescent materials (e.g., organic dyes, fluorescent
proteins) for <i>in vitro</i> cellular applications. Surprisingly,
direct comparative studies examining the cytotoxic potential of QDs
versus these more traditional cellular labeling fluorophores remain
limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay
material, we present a comprehensive study in which we characterize
the influence of QD dose (concentration and incubation time), QD surface
capping ligand, and delivery modality (peptide or cationic amphiphile
transfection reagent) on cellular viability in three human cell lines
representing various morphological lineages (epithelial, endothelial,
monocytic). We further compare the effects of QD cellular labeling
on cellular proliferation relative to those associated with a panel
of traditionally employed organic cell labeling fluorophores that
span a broad spectral range. Our results demonstrate the important
role played by QD dose, capping ligand structure, and delivery agent
in modulating cellular toxicity. Further, the results show that at
the concentrations and time regimes required for robust QD-based cellular
labeling, the impact of our in-house synthesized QD materials on cellular
proliferation is comparable to that of six commercial cell labeling
fluorophores. Cumulatively, our results demonstrate that the proper
tuning of QD dose, surface ligand, and delivery modality can provide
robust <i>in vitro</i> cell labeling reagents that exhibit
minimal impact on cellular viability
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 poly(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 poly(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
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
A New Family of Pyridine-Appended Multidentate Polymers As Hydrophilic Surface Ligands for Preparing Stable Biocompatible Quantum Dots
The growing utility of semiconductor
quantum dots (QDs) in biochemical
and cellular research necessitates, in turn, continuous development
of surface functionalizing ligands to optimize their performance for
ever more challenging and diverse biological applications. Here, we
describe a new class of multifunctional polymeric ligands as a stable,
compact and high affinity alternative to multidentate thiolated molecules.
The polymeric ligands are designed with a poly(acrylic acid) backbone
where pyridines are used as anchoring groups that are not sensitive
to degradation by air and light, along with short poly(ethylene glycol)
(PEG) pendant groups which are coincorporated for aqueous solubility,
biocompatibility and colloidal stability. The percentages of each
of the latter functional groups are controlled during initial synthesis
along with incorporation of carboxyl groups which serve as chemical
handles for subsequent covalent modification of the QD surface. A
detailed physicochemical characterization indicates that the multiple
pyridine groups are efficiently bound on the QD surface since they
provide for relatively small overall hydrodynamic sizes along with
good colloidal stability and strong fluorescence over a wide pH range,
under high salt concentration and in extremely dilute conditions at
room temperature under room light over extended timeframes. Covalent
conjugation of dyes and metal-affinity coordination with functional
enzymes to the QD surfaces were also demonstrated. Biocompatibility
and long-term stability of the pyridine polymer coated QDs were then
confirmed in a battery of relevant assays including cellular delivery
by both microinjection and peptide facilitated uptake along with intracellular
single QD tracking studies and cytotoxicity testing. Cumulatively,
these results suggest this QD functionalization strategy is a viable
alternative that provides some desirable properties of both compact,
discrete ligands and large amphiphilic polymers
Elucidating Surface Ligand-Dependent Kinetic Enhancement of Proteolytic Activity at Surface-Modified Quantum Dots
Combining
biomolecules such as enzymes with nanoparticles has much to offer
for creating next generation synergistically functional bionanomaterials.
However, almost nothing is known about how these two disparate components
interact at this critical biomolecular-materials interface to give
rise to improved activity and emergent properties. Here we examine
how the nanoparticle surface can influence and increase localized
enzyme activity using a designer experimental system consisting of
trypsin proteolysis acting on peptide-substrates displayed around
semiconductor quantum dots (QDs). To minimize the complexity of analyzing
this system, only the chemical nature of the QD surface functionalizing
ligands were modified. This was accomplished by synthesizing a series
of QD ligands that were either positively or negatively charged, zwitterionic,
neutral, and with differing lengths. The QDs were then assembled with
different ratios of dye-labeled peptide substrates and exposed to
trypsin giving rise to progress curves that were monitored by Förster
resonance energy transfer (FRET). The resulting trypsin activity profiles
were analyzed in the context of detailed molecular dynamics simulations
of key interactions occurring at this interface. Overall, we find
that a combination of factors can give rise to a localized activity
that was 35-fold higher than comparable freely diffusing enzyme–substrate
interactions. Contributing factors include the peptide substrate being
prominently displayed extending from the QD surface and not sterically
hindered by the longer surface ligands in conjunction with the presence
of electrostatic and other productive attractive forces between the
enzyme and the QD surface. An intimate understanding of such critical
interactions at this interface can produce a set of guidelines that
will allow the rational design of next generation high-activity bionanocomposites
and theranostics