Smaller Gold Nanoparticles Release DNA More Efficiently During fs Laser Pulsed Optical Heating.

This work investigates the effect of plasmonic gold nanoparticle (AuNP) size on the rate of thermal release of single-stranded oligonucleotides under femtosecond (fs)-pulsed laser irradiation sources. Contrary to the theoretical predictions that larger AuNPs (50-60 nm diameter) would produce the most solution heating and fastest DNA release, it is found that smaller AuNP diameters (25 nm) lead to faster dsDNA denaturation rates. Controlling for the pulse energy fluence, AuNP concentration, DNA loading density, and the distance from the AuNP surface finds the same result. These results imply that the solution temperature increases around the AuNP during fs laser pulse optical heating may not be the only significant influence on dsDNA denaturation, suggesting that direct energy transfer from the AuNP to the DNA (phonon-phonon coupling), which is increased as AuNPs decrease in size, may play a significant role.

Investigating the dissipation of heat and quantum information from DNA-scaffolded chromophore networks.

Scaffolded molecular networks are important building blocks in biological pigment-protein complexes, and DNA nanotechnology allows analogous systems to be designed and synthesized. System-environment interactions in these systems are responsible for important processes, such as the dissipation of heat and quantum information. This study investigates the role of nanoscale molecular parameters in tuning these vibronic system-environment dynamics. Here, genetic algorithm methods are used to obtain nanoscale parameters for a DNA-scaffolded chromophore network based on comparisons between its calculated and measured optical spectra. These parameters include the positions, orientations, and energy level characteristics within the network. This information is then used to compute the dynamics, including the vibronic population dynamics and system-environment heat currents, using the hierarchical equations of motion. The dissipation of quantum information is identified by the system's transient change in entropy, which is proportional to the heat currents according to the second law of thermodynamics. These results indicate that the dissipation of quantum information is highly dependent on the particular nanoscale characteristics of the molecular network, which is a necessary first step before gleaning the systematic optimization rules. Subsequently, the I-concurrence dynamics are calculated to understand the evolution of the vibronic system's quantum entanglement, which are found to be long-lived compared to these system-bath dissipation processes.

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards.

DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.

Quantum Dot-Based FRET Nanosensors for Talin-Membrane Assembly and Mechanosensing.

Understanding the mechanisms of assembly and disassembly of macromolecular structures in cells relies on solving biomolecular interactions. However, those interactions often remain unclear because tools to track molecular dynamics are not sufficiently resolved in time or space. In this study, we present a straightforward method for resolving inter- and intra-molecular interactions in cell adhesive machinery, using quantum dot (QD) based Förster resonance energy transfer (FRET) nanosensors. Using a mechanosensitive protein, talin, one of the major components of focal adhesions, we are investigating the mechanosensing ability of proteins to sense and respond to mechanical stimuli. First, we quantified the distances separating talin and a giant unilamellar vesicle membrane for three talin variants. These variants differ in molecular length. Second, we investigated the mechanosensing capabilities of talin, i.e., its conformational changes due to mechanical stretching initiated by cytoskeleton contraction. Our results suggest that in early focal adhesion, talin undergoes stretching, corresponding to a decrease in the talin-membrane distance of 2.5 nm. We demonstrate that QD-FRET nanosensors can be applied for the sensitive quantification of mechanosensing with a sub-nanometer accuracy.

Determining interchromophore effects for energy transport in molecular networks using machine-learning algorithms.

Nature uses chromophore networks, with highly optimized structural and energetic characteristics, to perform important chemical functions. Due to its modularity, predictable aggregation characteristics, and established synthetic protocols, structural DNA nanotechnology is a promising medium for arranging chromophore networks with analogous structural and energetic controls. However, this high level of control creates a greater need to know how to optimize the systems precisely. This study uses the system's modularity to produce variations of a coupled 14-Site chromophore network. It uses machine-learning algorithms and spectroscopy measurements to reveal the energy-transport roles of these Sites, paying particular attention to the cooperative and inhibitive effects they impose on each other for transport across the network. The physical significance of these patterns is contextualized, using molecular dynamics simulations and energy-transport modeling. This analysis yields insights about how energy transfers across the Donor-Relay and Relay-Acceptor interfaces, as well as the energy-transport pathways through the homogeneous Relay segment. Overall, this report establishes an approach that uses machine-learning methods to understand, in fine detail, the role that each Site plays in an optoelectronic molecular network.

Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula.

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactions─especially cytosolic stability─remain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structures─crosshairs and a tetrahedron─were labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure's direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron's torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required.

FRET as a biomolecular research tool - understanding its potential while avoiding pitfalls.

The applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.

An Investigation into the Resistance of Spherical Nucleic Acids against DNA Enzymatic Degradation.

Nanoparticles coated with oligonucleotides, also termed spherical nucleic acids (SNAs), are at the forefront of scientific research and have been applied in vitro and in vivo for sensing, gene regulation, and drug delivery. They demonstrate unique properties stemming from the three-dimensional shell of oligonucleotides and present high cellular uptake. However, their resistance to enzymatic degradation is highly dependent on their physicochemical characteristics. In particular, the oligonucleotide loading of SNAs has been determined to be a critical parameter in SNA design. In order to ensure the successful function of SNAs, the degree of oligonucleotide loading has to be quantitatively determined to confirm that a dense oligonucleotide shell has been achieved. However, this can be time-consuming and may lead to multiple syntheses being required to achieve the necessary degree of surface functionalization. In this work we show how this limitation can be overcome by introducing an oligonucleotide modification. By replacing the phosphodiester bond on the oligonucleotide backbone with a phosphorothioate bond, SNAs even with a low DNA loading showed remarkable stability in the presence of nucleases. Furthermore, these chemically modified SNAs exhibited high selectivity and specificity toward the detection of mRNA in cellulo.

A Nanobody-on-Quantum Dot Displacement Assay for Rapid and Sensitive Quantification of the Epidermal Growth Factor Receptor (EGFR).

Biosensing approaches that combine small, engineered antibodies (nanobodies) with nanoparticles are often complicated. Here, we show that nanobodies with different C-terminal tags can be efficiently attached to a range of the most widely used biocompatible semiconductor quantum dots (QDs). Direct implementation into simplified assay formats was demonstrated by designing a rapid and wash-free mix-and-measure immunoassay for the epidermal growth factor receptor (EGFR). Terbium complex (Tb)-labeled hexahistidine-tagged nanobodies were specifically displaced from QD surfaces via EGFR-nanobody binding, leading to an EGFR concentration-dependent decrease of the Tb-to-QD Förster resonance energy transfer (FRET) signal. The detection limit of 80±20 pM (16±4 ng mL-1 ) was 3-fold lower than the clinical cut-off concentration for soluble EGFR and up to 10-fold lower compared to conventional sandwich FRET assays that required a pair of different nanobodies.

The Role of Ligands in the Chemical Synthesis and Applications of Inorganic Nanoparticles.

The design of nanoparticles is critical for their efficient use in many applications ranging from biomedicine to sensing and energy. While shape and size are responsible for the properties of the inorganic nanoparticle core, the choice of ligands is of utmost importance for the colloidal stability and function of the nanoparticles. Moreover, the selection of ligands employed in nanoparticle synthesis can determine their final size and shape. Ligands added after nanoparticle synthesis infer both new properties as well as provide enhanced colloidal stability. In this article, we provide a comprehensive review on the role of the ligands with respect to the nanoparticle morphology, stability, and function. We analyze the interaction of nanoparticle surface and ligands with different chemical groups, the types of bonding, the final dispersibility of ligand-coated nanoparticles in complex media, their reactivity, and their performance in biomedicine, photodetectors, photovoltaic devices, light-emitting devices, sensors, memory devices, thermoelectric applications, and catalysis.

Understanding Disorder, Vibronic Structure, and Delocalization in Electronically Coupled Dimers on DNA Duplexes.

Structural DNA nanotechnology is a promising approach to create chromophore networks with modular structures and Hamiltonians to control the material's functions. The functional behaviors of these systems depend on the interactions of the chromophores' vibronic states, as well as interactions with their environment. To optimize their functions, it is necessary to characterize the chromophore network's structural and energetic properties, including the electronic delocalization in some cases. In this study, parameters of interest are deduced in DNA-scaffolded Cyanine 3 and Cyanine 5 dimers. The methods include steady-state optical measurements, physical modeling, and a genetic algorithm approach. The parameters include the chromophore network's vibronic Hamiltonian, molecular positions, transition dipole orientations, and environmentally induced energy broadening. Additionally, the study uses temperature-dependent optical measurements to characterize the spectral broadening further. These combined results reveal the quantum mechanical delocalization, which is important for functions like coherent energy transport and quantum information applications.

A Multiparametric Evaluation of Quantum Dot Size and Surface-Grafted Peptide Density on Cellular Uptake and Cytotoxicity.

Despite the progress in nanotechnology for biomedical applications, great efforts are still being employed in optimizing nanoparticle (NP) design parameters to improve functionality and minimize bionanotoxicity. In this study, we developed CdSe/CdS/ZnS core/shell/shell quantum dots (QDs) that are compact ligand-coated and surface-functionalized with an HIV-1-derived TAT cell-penetrating peptide (CPP) analog to improve both biocompatibility and cellular uptake. Multiparametric studies were performed in different mammalian and murine cell lines to compare the effects of varying QD size and number of surface CPPs on cellular uptake, viability, generation of reactive oxygen species, mitochondrial health, cell area, and autophagy. Our results showed that the number of cell-associated NPs and their respective toxicity are higher for the larger QDs. Meanwhile, increasing the number of surface CPPs also enhanced cellular uptake and induced cytotoxicity through the generation of mitoROS and autophagy. Thus, here we report the optimal size and surface CPP combinations for improved QD cellular uptake.

Gold-Nanoparticle-Mediated Depolarization of Membrane Potential Is Dependent on Concentration and Tethering Distance from the Plasma Membrane.

The photoactivation of plasma-membrane-tethered gold nanoparticles (AuNPs) for the photothermally driven depolarization of membrane potential has recently emerged as a new platform for the controlled actuation of electrically active cells. In this report, we characterize the relationship between AuNP concentration and AuNP-membrane separation distance with the efficiency of photoactivated plasma membrane depolarization. We show in differentiated rat pheochromocytoma (PC-12) cells that AuNPs capped with poly(ethylene glycol) (PEG)-cholesterol ligands localize to the plasma membrane and remain resident for up to 1 h. The efficiency of AuNP-mediated depolarization is directly dependent on the concentration of the NPs on the cell surface. We further show that the efficiency of AuNP-mediated photothermal depolarization of membrane potential is directly dependent on the tethering distance between the AuNP and the plasma membrane, which we control by iteratively tuning the length of the PEG linker. Importantly, the AuNP conjugates do not adversely affect cell viability under the photoactivation conditions required for membrane depolarization. Our results demonstrate the fine control that can be elicited over AuNP bioconjugates and establishes principles for the rational design of functional nanomaterials for the control of electrically excitable cells.

Bioluminescence-Based Energy Transfer Using Semiconductor Quantum Dots as Acceptors.

Bioluminescence resonance energy transfer (BRET) is the non-radiative transfer of energy from a bioluminescent protein donor to a fluorophore acceptor. It shares all the formalism of Förster resonance energy transfer (FRET) but differs in one key aspect: that the excited donor here is produced by biochemical means and not by an external illumination. Often the choice of BRET source is the bioluminescent protein Renilla luciferase, which catalyzes the oxidation of a substrate, typically coelenterazine, producing an oxidized product in its electronic excited state that, in turn, couples with a proximal fluorophore resulting in a fluorescence emission from the acceptor. The acceptors pertinent to this discussion are semiconductor quantum dots (QDs), which offer some unrivalled photophysical properties. Amongst other advantages, the QD's large Stokes shift is particularly advantageous as it allows easy and accurate deconstruction of acceptor signal, which is difficult to attain using organic dyes or fluorescent proteins. QD-BRET systems are gaining popularity in non-invasive bioimaging and as probes for biosensing as they don't require external optical illumination, which dramatically improves the signal-to-noise ratio by avoiding background auto-fluorescence. Despite the additional advantages such systems offer, there are challenges lying ahead that need to be addressed before they are utilized for translational types of research.

Supraparticle Assemblies of Magnetic Nanoparticles and Quantum Dots for Selective Cell Isolation and Counting on a Smartphone-Based Imaging Platform.

There are numerous diagnostic and therapeutic applications for the detection and enumeration of specific cell types. Flow cytometry is the gold standard technique for this purpose but is poorly suited to point-of-need assays. The ideal platform for these assays would combine the immunocytochemical capabilities of flow cytometry with low-cost, portable instrumentation, and a simple and rapid assay workflow. Here, we present a smartphone-based imaging platform (SIP) in tandem with magnetic-fluorescent suprananoparticle assemblies as a step toward these ideal criteria. The assemblies (MNP@QD) are magnetic iron oxide nanoparticles surrounded by a dense corona of many brightly luminescent semiconductor quantum dots (QDs), where both the assemblies and their immunoconjugates are prepared by self-assembly. As proof of concept, we show that the MNP@QD and SIP pairing is able to selectively isolate, fluorescently immunolabel, and count breast cancer cells that are positive for human epidermal growth factor receptor 2 (HER2). These results are an important foundation for future point-of-need diagnostics capable of multiplexed isolation, counting, and immunoprofiling of cells on a smartphone, enabled by the highly advantageous optical properties of QDs.

Bayesian Network Resource for Meta-Analysis: Cellular Toxicity of Quantum Dots.

A web-based resource for meta-analysis of nanomaterials toxicity is developed whereby the utility of Bayesian networks (BNs) is illustrated for exploring the cellular toxicity of Cd-containing quantum dots (QDs). BN models are developed based on a dataset compiled from 517 publications comprising 3028 cell viability data samples and 837 IC50 values. BN QD toxicity (BN-QDTox) models are developed using both continuous (i.e., numerical) and categorical attributes. Using these models, the most relevant attributes identified for correlating IC50 are: QD diameter, exposure time, surface ligand, shell, assay type, surface modification, and surface charge, with the addition of QD concentration for the cell viability analysis. Data exploration via BN models further enables identification of possible association rules for QDs cellular toxicity. The BN models as web-based applications can be used for rapid intelligent query of the available body of evidence for a given nanomaterial and can be readily updated as the body of knowledge expands.

Transducing Protease Activity into DNA Output for Developing Smart Bionanosensors.

DNA can process information through sequence-based reorganization but cannot typically receive input information from most biological processes and translate that into DNA compatible language. Coupling DNA to a substrate responsive to biological events can address this limitation. A two-component sensor incorporating a chimeric peptide-DNA substrate is evaluated here as a protease-to-DNA signal convertor which transduces protease activity through DNA gates that discriminate between different input proteases. Acceptor dye-labeled peptide-DNAs are assembled onto semiconductor quantum dot (QD) donors as the input gate. Addition of trypsin or chymotrypsin cleaves their cognate peptide sequence altering the efficiency of Förster resonance energy transfer (FRET) with the QD and frees a DNA output which interacts with a tetrahedral output gate. Downstream output gate rearrangement results in FRET sensitization of a new acceptor dye. Following characterization of component assembly and optimization of individual steps, sensor ability to discriminate between the two proteases is confirmed along with effects from joint interactions where potential for cross-talk is highest. Processing multiple bits of information for a sensing outcome provides more confidence than relying on a single change especially for the discrimination between different targets. Coupling other substrates to DNA that respond similarly could help target other types of enzymes.

Nanoparticle Size Influences Localized Enzymatic Enhancement-A Case Study with Phosphotriesterase.

Enhancements in enzymatic catalytic activity are frequently observed when an enzyme is displayed on a nanoparticle (NP) surface. The exact mechanisms of how this unique interfacial environment gives rise to this phenomenon are still not understood, although evidence suggests that it can help alleviate some of the enzyme's rate-limiting mechanistic steps. The physicochemical limitations that govern when this process arises are also not known including, in particular, the range of NP size and curvature that are associated with it. To investigate the latter, we undertook a case study using the enzyme phosphotriesterase (PTE) and a series of differentially sized gold NPs (AuNPs). PTE, expressed with a terminal hexahistidine sequence, was ratiometrically coordinated to a series of increasing size AuNPs (diameter ≃ 1.5, 5, 10, 20, 55, 100 nm) surface-functionalized with Ni2+-nitrilotriacetic acid ligands and its activity assayed in a comparative format versus that of equivalent amounts of free enzyme controls. PTE-AuNP samples were prepared where the total PTE concentration and NP surface density were kept fixed by varying AuNP concentration along with the converse format. Assembly to the AuNPs increased PTE kcat ca. 3-10-fold depending upon NP size, with the smaller-sized particles showing the highest increase, while enzyme efficiency only increased 2-fold. Further kinetic testing suggested that the PTE enhancement again arose from alleviating its rate limiting step of enzyme-product release and not from a change in the activation energy. Comparison of kcat and enzyme specificity with AuNP diameter revealed that enhancement was directly correlated to AuNP size and curvature with the smaller NPs showing the largest kinetic enhancements. Kinetic simulations showed that almost all of the PTE enhancement variation across AuNP sizes could be reproduced by adjusting only the rate of enzyme-product dissociation. Understanding how NP size directly affects the enhancement of an attached enzyme can provide a rational basis for designing hybrid enzyme-NP materials that specifically exploit this emergent property.

Nanoparticle-Peptide-Drug Bioconjugates for Unassisted Defeat of Multidrug Resistance in a Model Cancer Cell Line.

Multidrug resistance (MDR) is a significant challenge in the treatment of many types of cancers as membrane-associated transporters actively pump drugs out of the cell, limiting therapeutic efficacy. While nanoparticle (NP)-based therapeutics have emerged as a mechanism for overcoming MDR, they often rely on the delivery of multiple anticancer drugs, nucleic acid hybrids, or MDR pump inhibitors. The effectiveness of these strategies, however, can be limited by their off-target toxicity or the need for genetic transfection. In this paper, we describe a NP-peptide-drug bioconjugate that achieves significant cell killing in MDR-positive cancer cells without the need for additional drugs. We use a quantum dot (QD) as a central scaffold to append two species of peptide, a cell-uptake peptide to facilitate endocytic internalization and a peptide-drug conjugate that is susceptible to cleavage by esterases found within the endocytic pathway. This approach relies on spatiotemporal control over drug release, where endosomes traffic drug away from membrane-resident pumps and release it closer to the nucleus. Cellular internalization studies showed high uptake of the NP-drug complex and nuclear localization of the drug after 48 h in MDR-positive cells. Additionally, cellular proliferation assays demonstrated a 40% decrease in cell viability for the NP-drug bioconjugate compared to free drug, confirming the utility of this system in overcoming MDR in cancer cells.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications.

Luminescent semiconductor quantum dots (QDs) are one of the more popular nanomaterials currently utilized within biological applications. However, what is not widely appreciated is their growing role as versatile energy transfer (ET) donors and acceptors within a similar biological context. The progress made on integrating QDs and ET in biological configurations and applications is reviewed in detail here. The goal is to provide the reader with (1) an appreciation for what QDs are capable of in this context, (2) how this field has grown over a relatively short time span, and, in particular, (3) how QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates. An initial discussion of QD materials along with key concepts surrounding their preparation and bioconjugation is provided given the defining role these aspects play in the QDs ability to succeed in subsequent ET applications. The discussion is then divided around the specific roles that QDs provide as either Förster resonance energy transfer (FRET) or charge/electron transfer donor and/or acceptor. For each QD-ET mechanism, a working explanation of the appropriate background theory and formalism is articulated before examining their biosensing and related ET utility. Other configurations such as incorporation of QDs into multistep ET processes or use of initial chemical and bioluminescent excitation are treated similarly. ET processes that are still not fully understood such as QD interactions with gold and other metal nanoparticles along with carbon allotropes are also covered. Given their maturity, some specific applications ranging from in vitro sensing assays to cellular imaging are separated and discussed in more detail. Finally a perspective on how this field will continue to evolve is provided.