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The engineering and manipulation of delocalized molecular exciton states is a key component for artificial biomimetic light harvesting complexes as well as alternative circuitry platforms based on exciton propagation. Here we examine the consequences of strong electronic coupling in cyanine homodimers on DNA duplex scaffolds. The most closely spaced dyes, attached to positions directly across the double-helix from one another, exhibit pronounced Davydov splitting due to strong electronic coupling. We demonstrate that the DNA scaffold is sufficiently robust to support observation of the transition from the lowest energy (J-like) one-exciton state to the nonlocal two-exciton state, where each cyanine dye is in the excited state. This transition proceeds via sequential photon absorption and persists for the lifetime of the exciton, establishing this as a controlled method for creating two-exciton states. Our observations suggest that DNA-organized dye networks have potential as platforms for molecular logic gates and entangled photon emission based on delocalized two-exciton states.

Structural DNA nanotechnology is poised to transform targeted therapeutic and theranostic delivery agents. Some of the most promising biomedical applications of DNA nanostructures include carriers for biosensing, imaging, and drug delivery. Additionally, the unique ability to precisely position inorganic and organic molecules on DNA-based substrates enables the spatially optimized high density interfacing of ligands with cell membrane receptors. To realize clinically viable biomedical products made from DNA nanostructures, it is necessary to fully understand the behavior of these systems inside and outside the cellular environment. To that end, cohesive and conclusive information on the physiological fate of DNA nanostructures at various time points - from the cell culture to the cell cytosol - is still lacking. In this highlight, we bring to attention efforts to understand DNA nanostructure behavior in vitro as well as some widespread disparities among studies on the subject. We also call for a discussion on the implementation of common standards and controls to address these disparities and consequently unify the scientific community's endeavours to build foundational knowledge on DNA nanostructure-cellular interaction.

Femtosecond (fs) laser pulsed excitation of plasmonic nanoparticle (NP)-biomolecule conjugates is a promising method to locally heat biological materials. Studies have demonstrated that fs pulses of light can modulate the activity of DNA or proteins when attached to plasmonic NPs; however, the precision over subsequent biological function remains largely undetermined. Specifically, the temperature the localized biomolecules "experience" remains unknown. We used 55 nm gold nanoparticles (AuNPs) displaying double-stranded (ds) DNA to examine how, for dsDNA with different melting temperatures, the laser pulse energy fluence and bulk solution temperature affect the rate of local DNA denaturation. A universal "template" single-stranded DNA was attached to the AuNP surface, and three dye-labeled probe strands, distinct in length and melting temperature, were hybridized to it creating three individual dsDNA-AuNP bioconjugates. The dye-labeled probe strands were used to quantify the rate and amount of DNA release after a given number of light pulses, which was then correlated to the dsDNA denaturation temperature, resulting in a quantitative nanothermometer. The localized DNA denaturation rate could be modulated by more than threefold over the biologically relevant range of 8-53 °C by varying pulse energy fluence, DNA melting temperature, and surrounding bath temperature. With a modified dissociation equation tailored for this system, a "sensed" temperature parameter was extracted and compared to simulated AuNP temperature profiles. Determining actual biological responses in such systems can allow researchers to design precision nanoscale photothermal heating sources.

The development of rapid, simple, and versatile biosensors for monitoring the activity of histone modifying enzymes (HMEs) is needed for the improvement of diagnostic assays, screening of HME inhibitors, and a better understanding of HME kinetics in different environments. Nanoparticles can play an important role in this regard by improving or complementing currently available enzyme detection technologies. Here, we present the development and application of a homogeneous methyltransferase (SET7/9) assay based on time-gated Förster resonance energy transfer (TG-FRET) between terbium complexes (Tb) and luminescent semiconductor quantum dots (QDs). Specific binding of a Tb-antibody conjugate to a SET7/9-methylated Lys4 on a histone H3(1-21) peptide substrate attached to the QD surface resulted in efficient FRET and provided the mechanism for monitoring the SET7/9 activity. Two common peptide-QD attachment strategies (biotin-streptavidin and polyhistidine-mediated self-assembly), two different QD colors (625 and 705 nm), and enzyme sensing with post- or pre-assembled QD-peptide conjugates demonstrated the broad applicability of this assay design. Limits of detection in the low picomolar concentration range, high selectivity tested against non-specific antibodies, enzymes, and co-factors, determination of the inhibition constants of the SET7/9 inhibitors SAH and (R)-PFI-2, and analysis of the co-factor (SAM) concentration-dependent enzyme kinetics of SET7/9 which followed the Michaelis-Menten model highlighted the excellent performance of this TG-FRET HME activity assay.

Although antibodies remain a primary recognition element in all forms of biosensing, functional limitations arising from their size, stability, and structure have motivated the development and production of many different artificial scaffold proteins for biological recognition. However, implementing such artificial binders into functional high-performance biosensors remains a challenging task. Here, we present the design and application of Förster resonance energy transfer (FRET) nanoprobes comprising small artificial proteins (αRep bidomains) labeled with a Tb complex (Tb) donor on the C-terminus and a semiconductor quantum dot (QD) acceptor on the N-terminus. Specific binding of one or two protein targets to the αReps induced a conformational change that could be detected by time-resolved Tb-to-QD FRET. These single-probe FRET switches were used in a separation-free solution-phase assay to quantify different protein targets at sub-nanomolar concentrations and to measure the conformational changes with sub-nanometer resolution. Probing ligand-receptor binding under physiological conditions at very low concentrations in solution is a special feature of FRET that can be efficiently combined with other structural characterization methods to develop, understand, and optimize artificial biosensors. Our results suggest that the αRep FRET nanoprobes have a strong potential for their application in advanced diagnostics and intracellular live-cell imaging of ligand-receptor interactions.

Lipases are an important class of lipid hydrolyzing enzymes that play significant roles in many aspects of cell biology and digestion; they also have large roles in commercial food and biofuel preparation and are being targeted for pharmaceutical development. Given these, and many other biotechnological roles, sensitive and specific biosensors capable of monitoring lipase activity in a quantitative manner are critical. Here, we describe a Förster resonance energy transfer (FRET)-based biosensor that originates from a custom-synthesized ester substrate displaying a peptide at one end and a dye acceptor at the other. These substrates were ratiometrically self-assembled to luminescent semiconductor quantum dot (QD) donors by metal affinity coordination using the appended peptide's terminal hexahistidine motif to give rise to the full biosensing construct. This resulted in a high rate of FRET between the QD donor and the proximal substrate's dye acceptor. The lipase hydrolyzed the intervening target ester bond in the peptide substrate which, in turn, displaced the dye acceptor containing component and altered the rate of FRET in a concentration-dependent manner. Specifics of the substrate's stepwise synthesis are described along with the sensors assembly, characterization, and application in a quantitative proof-of-concept demonstration assay that is based on an integrated Michaelis-Menten kinetic approach. The utility of this unique nanoparticle-based architecture within a sensor configuration is then discussed.

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.

DNA scaffolds enable base-pair-specific positioning of fluorescent molecules, allowing for nanometer-scale precision in controlling multidye interactions. Expanding on this concept, DNA-based molecular photonic wires (MPWs) allow for light harvesting and directional propagation of photonic energy on the nanometer scale. The most common MPW examples exploit Förster resonance energy transfer (FRET), and FRET between the same dye species (HomoFRET) was recently shown to increase the distance and efficiency at which MPWs can function. Although increased proximity between adjacent fluorophores can be used to increase the energy transfer efficiency, FRET assumptions break down as the distance between the dye molecules becomes comparable to their size (∼2 nm). Here we compare dye conjugation with single versus dimer Cy5 dye repeats as HomoFRET MPW components on a double-crossover DNA scaffold. At room temperature (RT) under low-light conditions, end-labeled uncoupled dye molecules provide optimal transfer, while the Cy5 dimers show ultrafast (<100 ps) nonradiative decay that severely limits their functionality. Of particular interest is the observation that through increased excitation fluence as well as cryogenic temperatures, the dimeric MPW shows suppression of the ultrafast decay, demonstrating fluorescence lifetimes similar to the single Cy5 MPWs. This work points to the complex dynamic capabilities of dye-based nanophotonic networks, where dye positioning and interactions can become critical, and could be used to extend the lengths and complexities of such dye-DNA devices, enabling multiparameter nanophotonic circuitry.

Researchers are continuously making progress towards diagnosis and treatment of numerous diseases. However, there are still major issues that are presenting many challenges for current medical diagnosis. On the other hand, DNA nanotechnology has evolved significantly over the last three decades and is highly interdisciplinary. With many potential technologies derived from the field, it is natural to begin exploring and incorporating its knowledge to develop DNA microsystems for biodiagnosis in order to help address current obstacles, such as disease detection and drug resistance. Here, current challenges in disease detection are presented along with standard methods for diagnosis. Then, a brief overview of DNA nanotechnology is introduced along with its main attractive features for constructing biodiagnostic microsystems. Lastly, suggested DNA-based microsystems are discussed through proof-of-concept demonstrations with improvement strategies for standard diagnostic approaches.

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.

The interfacing of nanoparticle (NP) materials with cells, tissues, and organisms for a range of applications including imaging, sensing, and drug delivery continues at a rampant pace. An emerging theme in this area is the use of NPs and nanostructured surfaces for the imaging and/or control of cellular membrane potential (MP). Given the important role that MP plays in cellular biology, both in normal physiology and in disease, new materials and methods are continually being developed to probe the activity of electrically excitable cells such as neurons and muscle cells. In this Review, we highlight the current state of the art for both the visualization and control of MP using traditional materials and techniques, discuss the advantageous features of NPs for performing these functions, and present recent examples from the literature of how NP materials have been implemented for the visualization and control of the activity of electrically excitable cells. We conclude with a forward-looking perspective of how we expect to see this field progress in the near term and further into the future.

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.

Developing reliable methods of constructing cell-free multienzyme biocatalytic systems is a milestone goal of synthetic biology. It would enable overcoming the limitations of current cell-based systems, which suffer from the presence of competing pathways, toxicity, and inefficient access to extracellular reactants and removal of products. DNA nanostructures have been suggested as ideal scaffolds for assembling sequential enzymatic cascades in close enough proximity to potentially allow for exploiting of channeling effects; however, initial demonstrations have provided somewhat contradictory results toward confirming this phenomenon. In this work, a three-enzyme sequential cascade was realized by site-specifically immobilizing DNA-conjugated amylase, maltase, and glucokinase on a self-assembled DNA origami triangle. The kinetics of seven different enzyme configurations were evaluated experimentally and compared to simulations of optimized activity. A 30-fold increase in the pathway's kinetic activity was observed for enzymes assembled to the DNA. Detailed kinetic analysis suggests that this catalytic enhancement originated from increased enzyme stability and a localized DNA surface affinity or hydration layer effect and not from a directed enzyme-to-enzyme channeling mechanism. Nevertheless, the approach used to construct this pathway still shows promise toward improving other more elaborate multienzymatic cascades and could potentially allow for the custom synthesis of complex (bio)molecules that cannot be realized with conventional organic chemistry approaches.

A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources ("feedstock flexibility"). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii , as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum . Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.

Synthetic DNA templated nanostructures offer an excellent platform for the precise spatial and orientational positioning of organic and inorganic nanomaterials. Previous reports have shown its applicability in the organization of plasmonic nanoparticles in a number of geometries for the purpose of realizing tunable nanoscale optical devices. However, translation of nanoparticle-DNA constructs to application requires additional efforts to increase scalability, reproducibility, and formation yields. Understanding all these factors is, in turn, predicated on in-depth analysis of each structure and comparing how formation changes with complexity. Towards the latter goal, we assemble seven unique plasmonic nanostructure symmetries of increasing complexity based on assembly of gold nanorods and nanoparticles on two different DNA origami templates, a DNA triangle and rhombus, and characterize them using gel electrophoresis, atomic force- and transmission electron microscopy, as well as optical spectroscopy. In particular, we focus on how much control can be elicited over yield, reproducibility, shape, size, inter-particle angles, gaps, and plasmon shifts as compared to expectations from computer simulations as structural complexity increases. We discuss how these results can contribute to establishing process principles for creating DNA templated plasmonic nanostructures.

Organophosphate compounds are ubiquitously employed as agricultural pesticides and maintained as chemical warfare agents by several nations. These compounds are highly toxic, show environmental persistence and accumulation, and contribute to numerous cases of poisoning and death each year. While their use as weapons of mass destruction is rare, these never fully disappear into obscurity as they continue to be tools of fear and control by governments and terrorist organizations. Beyond weaponization, their wide-scale dissemination as agricultural products has led to environmental accumulation and intoxication of soil and water across the globe. Therefore, there is a dire need for rapid and safe agents for environmental bioremediation, personal decontamination, and as therapeutic detoxicants. Organophosphate hydrolyzing enzymes are emerging as appealing targets to satisfy decontamination needs owing to their ability to hydrolyze both pesticides and nerve agents using biologically-derived materials safe for both the environment and the individual. As the release of genetically modified organisms is not widely accepted practice, researchers are exploring alternative strategies of organophosphate bioremediation that focus on cell-free enzyme systems. In this review, we first discuss several of the more prevalent organophosphorus hydrolyzing enzymes along with research and engineering efforts that have led to an enhancement in their activity, substrate tolerance, and stability. In the later half we focus on advances achieved through research focusing on enhancing the catalytic activity and stability of phosphotriesterase, a model organophosphate hydrolase, using various approaches such as nanoparticle display, DNA scaffolding, and outer membrane vesicle encapsulation., (Copyright © 2019 Thakur, Medintz and Walper.)

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.

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.

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 IC 50 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 IC 50 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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vaccines and immunotherapies have changed the face of health care. Biomaterials offer the ability to improve upon these medical technologies through increased control of the types and concentrations of immune signals delivered. Further, these carriers enable targeting, stability, and delivery of poorly soluble cargos. Inorganic nanomaterials possess unique optical, electric, and magnetic properties, as well as defined chemistry, high surface-to-volume- ratio, and high avidity display that make this class of materials particularly advantageous for vaccine design, cancer immunotherapy, and autoimmune treatments. In this review we focus on this understudied area by highlighting recent work with inorganic materials - including gold nanoparticles, carbon nanotubes, and quantum dots. We discuss the intrinsic features of these materials that impact the interactions with immune cells and tissue, as well as recent reports using inorganic materials across a range of emerging immunological applications.

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 Ni 2+ -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 k cat 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 k cat 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.

DNA self-assembly has proven to be a highly versatile tool for engineering complex and dynamic biocompatible nanostructures from the bottom up with a wide range of potential bioapplications currently being pursued. Primary among these is healthcare, with the goal of developing diagnostic, imaging, and drug delivery devices along with combinatorial theranostic devices. The path to understanding a role for DNA nanotechnology in biomedical sciences is being approached carefully and systematically, starting from analyzing the stability and immune-stimulatory properties of DNA nanostructures in physiological conditions, to estimating their accessibility and application inside cellular and model animal systems. Much remains to be uncovered but the field continues to show promising results toward developing useful biomedical devices. This review discusses some aspects of DNA nanotechnology that makes it a favorable ingredient for creating nanoscale research and biomedical devices and looks at experiments undertaken to determine its stability in vivo. This is presented in conjugation with examples of state-of-the-art developments in biomolecular sensing, imaging, and drug delivery. Finally, some of the major challenges that warrant the attention of the scientific community are highlighted, in order to advance the field into clinically relevant applications., (© 2019 U.S. Naval Research Laboratory. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

DNA-nanoparticle conjugates are important tools in nanobiotechnology. Knowing the orientation, function, and length of DNA on nanoparticle surfaces at low nanomolar concentrations under physiological conditions is therefore of great interest. Here, we investigate the conformation of a 31 nucleotides (nt) long DNA attached to a semiconductor quantum dot (QD) via Förster resonance energy transfer (FRET) from Tb-DNA probes hybridized to different positions on the QD-DNA. Precise Tb-to-QD distance determination from 7 to 14 nm along 26 nt of the peptide-appended QD-DNA was realized by time-resolved FRET spectroscopy. The FRET nanoruler measured linear single-stranded (ssDNA) and double-stranded (dsDNA) extensions of ∼0.15 and ∼0.31 nm per base, reflecting the different conformations. Comparison with biomolecular modeling confirmed the denser conformation of ssDNA and a possibly more flexible orientation on the QD surface, whereas the dsDNA was fully extended with radial orientation. The temporally distinct photoluminescence decays of the different DNA-FRET configurations were used for prototypical DNA hybridization assays that demonstrated the large potential for extended temporal multiplexing. The extensive experimental and theoretical analysis of 11 different distances/configurations of the same QD-DNA conjugate provided important information on DNA conformation on nanoparticle surfaces and will be an important benchmark for the development and optimization of DNA-nanobiosensors.

Although a fundamental understanding of the pathogenicity of most biothreat agents has been elucidated and available treatments have increased substantially over the past decades, they still represent a significant public health threat in this age of (bio)terrorism, indiscriminate warfare, pollution, climate change, unchecked population growth, and globalization. The key step to almost all prevention, protection, prophylaxis, post-exposure treatment, and mitigation of any bioagent is early detection. Here, we review available methods for detecting bioagents including pathogenic bacteria and viruses along with their toxins. An introduction placing this subject in the historical context of previous naturally occurring outbreaks and efforts to weaponize selected agents is first provided along with definitions and relevant considerations. An overview of the detection technologies that find use in this endeavor along with how they provide data or transduce signal within a sensing configuration follows. Current "gold" standards for biothreat detection/diagnostics along with a listing of relevant FDA approved in vitro diagnostic devices is then discussed to provide an overview of the current state of the art. Given the 2014 outbreak of Ebola virus in Western Africa and the recent 2016 spread of Zika virus in the Americas, discussion of what constitutes a public health emergency and how new in vitro diagnostic devices are authorized for emergency use in the U.S. are also included. The majority of the Review is then subdivided around the sensing of bacterial, viral, and toxin biothreats with each including an overview of the major agents in that class, a detailed cross-section of different sensing methods in development based on assay format or analytical technique, and some discussion of related microfluidic lab-on-a-chip/point-of-care devices. Finally, an outlook is given on how this field will develop from the perspective of the biosensing technology itself and the new emerging threats they may face.

Multistep enzymatic cascades are becoming more prevalent in industrial settings as engineers strive to synthesize complex products and pharmaceuticals in economical, environmentally friendly ways. Previous work has shown that immobilizing enzymes on nanoparticles can enhance their activity significantly due to localized interfacial effects, and this enhancement remains in place even when that enzyme's activity is coupled to another enzyme that is still freely diffusing. Here, we investigate the effects of displaying two enzymes with coupled catalytic activity directly on the same nanoparticle surface. For this, the well-characterized enzymes pyruvate kinase (PykA) and lactate dehydrogenase (LDH) were utilized as a model system; they jointly convert phosphoenolpyruvate to lactate in two sequential steps as part of downstream glycolysis. The enzymes were expressed with terminal polyhistidine tags to facilitate their conjugation to semiconductor quantum dots (QDs) which were used here as prototypical nanoparticles. Characterization of enzyme coassembly to two different sized QDs showed a propensity to cross-link into nanoclusters consisting of primarily dimers and some trimers. Individual and joint enzyme activity in this format was extensively investigated in direct comparison to control samples lacking the QD scaffolds. We found that QD association enhances LDH activity by >50-fold and its total turnover by at least 41-fold, and that this high activation appears to be largely due to stabilization of its quarternary structure. When both enzymes are simultaneously bound to the QD surfaces, their colocalization leads to >100-fold improvements in the overall rates of coupled activity. Experimental results in conjunction with detailed kinetic simulations provide evidence that this significant improvement in coupled activity is partially attributable to a combination of enhanced enzymatic activity and stabilization of LDH. More importantly, experiments aimed at disrupting channeled processes and further kinetic modeling suggest that the bulk of the performance enhancement arises from intermediary "channeling" between the QD-colocalized enzymes. A full understanding of the underlying processes that give rise to such enhancements from coupled enzymatic activity on nanoparticle scaffolds can provide design criteria for improved biocatalytic applications.

Engineered scaffold affinity proteins are used in many biological applications with the aim of replacing natural antibodies. Although their very small sizes are beneficial for multivalent nanoparticle conjugation and efficient Förster resonance energy transfer (FRET), the application of engineered affinity proteins in such nanobiosensing formats has been largely neglected. Here, it is shown that very small (≈6.5 kDa) histidine-tagged albumin-binding domain-derived affinity proteins (ADAPTs) can efficiently self-assemble to zwitterionic ligand-coated quantum dots (QDs). These ADAPT-QD conjugates are significantly smaller than QD-conjugates based on IgG, Fab', or single-domain antibodies. Immediate applicability by the quantification of the human epidermal growth factor receptor 2 (HER2) in serum-containing samples using time-gated Tb-to-QD FRET detection on the clinical benchtop immunoassay analyzer KRYPTOR is demonstrated here. Limits of detection down to 40 × 10 -12 m (≈8 ng mL -1 ) are in a relevant clinical concentration range and outperform previously tested assays with antibodies, antibody fragments, and nanobodies., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ability to control the intracellular release of drug cargos from nanobioconjugate delivery scaffolds is critical for the successful implementation of nanoparticle (NP)-mediated drug delivery. This is particularly true for hard NP carriers such as semiconductor quantum dots (QDs) and gold NPs. Here, we report the development of a QD-based multicomponent drug release system that, when delivered to the cytosol of mammalian cells, is triggered to release its drug cargo by the simple addition of a competitive ligand to the extracellular medium. The ensemble construct consists of the central QD scaffold that is decorated with a fixed number of maltose binding proteins (MBPs). The MBP binding site is loaded with dye or drug conjugates of the maltose analogue beta-cyclodextrin (βCD) to yield a QD-MBP-βCD ensemble conjugate. The fidelity of conjugate assembly is monitored by Förster resonance energy transfer (FRET) from the QD donor to the dye/drug acceptor. Microplate-based FRET assays demonstrated that the βCD conjugate was released from the MBP binding pocket by maltose addition with an affinity that matched native MBP-maltose binding interactions. In COS-1 cells, the microinjected assembled conjugates remained stably intact in the cytosol until the addition of maltose to the extracellular medium, which underwent facilitated uptake into the cell. Live cell FRET-based confocal microscopy imaging captured the kinetics of realtime release of the βCD ligand as a function of extracellular maltose concentration. Our results demonstrate the utility of the self-assembled QD-MBP-βCD system to facilitate intracellular drug release that is triggered extracellularly through the simple addition of a well-tolerated nutrient and is not dependent on the use of light, magnetic field, ultrasound, or other traditional methods of stimulated drug release. We expect this extracellularly triggered drug release modality to be useful for the in vitro characterization of new drug candidates intended for systemic delivery/actuation and potentially for on-demand drug release in vivo.

Molecular photonic wires (MPWs) are tunable nanophotonic structures capable of capturing and directing light with high transfer efficiencies. DNA-based assembly techniques provide a simple and economical preparation method for MPWs that allows precise positioning of the molecular transfer components. Unfortunately, the longest DNA-based MPWs (∼30 nm) report only modest transfer efficiencies of ∼2% and have not been demonstrated on solid-state platforms. Here, we demonstrate that DNA-based MPWs can be spin-coated in a polymer matrix onto silicon wafers and exhibit a 5-fold increase in photonic transfer efficiency over solution-phase MPWs. Cooling these MPWs to 5 K led to further efficiency increases ranging from ∼40 to 240% depending on the length of the MPW. The improvement of MPW energy transport efficiencies advances prospects for their incorporation in a variety of optoelectronics technologies and makes them an ideal test bed for further exploration of nanoscale energy transfer.

We examine the effect of electronic coupling on the optical properties of Cy3 dimers attached to DNA duplexes as a function of base pair (bp) separation using steady-state and time-resolved spectroscopy. For close Cy3-Cy3 separations, 0 and 1 bp between dyes, intermediate to strong electronic coupling is revealed by modulation of the absorption and fluorescence properties including spectral band shape, peak wavelength, and excited-state lifetime. Using a vibronic exciton model, we estimate coupling strengths of 150 and 266 cm -1 for the 1 and 0 bp separations, respectively, which are comparable to those found in natural light-harvesting complexes. For the strongest electronic coupling (0 bp separation), we observe that the absorption band shape is strongly affected by the base pairs that surround the dyes, where more strongly hydrogen-bonded G-C pairs produce a red-shifted absorption spectrum consistent with a J-type dimer. This effect is studied theoretically using molecular dynamics simulation, which predicts an in-line dye configuration that is consistent with the experimental J-type spectrum. When the Cy3 dimers are in a standard aqueous buffer, the presence of relatively strong electronic coupling is accompanied by decreased fluorescence lifetime, suggesting that it promotes nonradiative relaxation in cyanine dyes. However, we show that the use of a viscous solvent can suppress this nonradiative recombination and thereby restore the dimer fluorescent emission. Ultrafast transient absorption measurements of Cy3 dimers in both standard aqueous buffer and viscous glycerol buffer suggest that sufficiently strong electronic coupling increases the probability of excited-state relaxation through a dark state that is related to Cy3 torsional motion.

While technologies for the remediation of chemical contaminants continue to emerge, growing interest in green technologies has led researchers to explore natural catalytic mechanisms derived from microbial species. One such method, enzymatic degradation, offers an alternative to harsh chemical catalysts and resins. Recombinant enzymes, however, are often too labile or show limited activity when challenged with nonideal environmental conditions that may vary in salinity, pH, or other physical properties. Here, we demonstrate how phosphotriesterase encapsulated in a bacterial outer membrane vesicle can be used to degrade the organophosphate chemical warfare agent (CWA) simulant paraoxon in environmental water samples. We also carried out remediation assays on solid surfaces, including glass, painted metal, and fabric, that were selected as representative materials, which could potentially be contaminated with a CWA.

Solution phase printing of graphene-based electrodes has recently become an attractive low-cost, scalable manufacturing technique to create in-field electrochemical biosensors. Here, we report a graphene-based electrode developed via inkjet maskless lithography (IML) for the direct and rapid monitoring of triple-O linked phosphonate organophosphates (OPs); these constitute the active compounds found in chemical warfare agents and pesticides that exhibit acute toxicity as well as long-term pollution to soils and waterways. The IML-printed graphene electrode is nano/microstructured with a 1000 mW benchtop laser engraver and electrochemically deposited platinum nanoparticles (dia. ∼25 nm) to improve its electrical conductivity (sheet resistance decreased from ∼10 000 to 100 Ω/sq), surface area, and electroactive nature for subsequent enzyme functionalization and biosensing. The enzyme phosphotriesterase (PTE) was conjugated to the electrode surface via glutaraldehyde cross-linking. The resulting biosensor was able to rapidly measure (5 s response time) the insecticide paraoxon (a model OP) with a low detection limit (3 nM), and high sensitivity (370 nA/μM) with negligible interference from similar nerve agents. Moreover, the biosensor exhibited high reusability (average of 0.3% decrease in sensitivity per sensing event), stability (90% anodic current signal retention over 1000 s), longevity (70% retained sensitivity after 8 weeks), and the ability to selectively sense OP in actual soil and water samples. Hence, this work presents a scalable printed graphene manufacturing technique that can be used to create OP biosensors that are suitable for in-field applications as well as, more generally, for low-cost biosensor test strips that could be incorporated into wearable or disposable sensing paradigms.

Success in the projects aimed at providing an advanced understanding of the brain is directly predicated on making critical advances in nanotechnology. This Perspective addresses the unique interface of neuroscience and nanomaterials by considering the foundational problem of sensing neuron membrane voltage and offers a potential solution that may be facilitated by a prototypical nanomaterial. Despite substantial improvements, the visualization of instantaneous voltage changes within individual neurons, whether in cell culture or in vivo, at both the single-cell and network level at high speed remains complex and problematic. The unique properties of semiconductor quantum dots (QDs) have made them powerful fluorophores for bioimaging. What is not widely appreciated, however, is that QD photoluminescence is exquisitely sensitive to proximal electric fields. This property should be suitable for sensing voltage changes that occur in the active neuronal membrane. Here, we examine the potential role of QDs in addressing the important challenge of real-time optical voltage imaging.

The growing emphasis on green chemistry, renewable resources, synthetic biology, regio-/stereospecific chemical transformations, and nanotechnology for providing new biological products and therapeutics is reinvigorating research into enzymatic catalysis. Although the promise is profound, many complex issues remain to be addressed before this effort will have a significant impact. Prime among these is to combat the degradation of enzymes frequently seen in ex vivo formats following immobilization to stabilize the enzymes for long-term application and to find ways of enhancing their activity. One promising avenue for progress on these issues is via nanoparticle (NP) display, which has been found in a number of cases to enhance enzyme activity while also improving long-term stability. In this feature article, we discuss the phenomenon of enhanced enzymatic activity at NP interfaces with an emphasis on our own work in this area. Important factors such as NP surface chemistry, bioconjugation approaches, and assay formats are first discussed because they can critically affect the observed enhancement. Examples are given of improved performance for enzymes such as phosphotriesterase, alkaline phosphatase, trypsin, horseradish peroxidase, and β-galactosidase and in configurations with either the enzyme or the substrate attached to the NP. The putative mechanisms that give rise to the performance boost are discussed along with how detailed kinetic modeling can contribute to their understanding. Given the importance of biosensing, we also highlight how this configuration is already making a significant contribution to NP-based enzymatic sensors. Finally, a perspective is provided on how this field may develop and how NP-based enzymatic enhancement can be extended to coupled systems and multienzyme cascades.

Water soluble metallic nanoparticles are being developed for a variety of roles ranging from catalysis to drug delivery and as potential contrast agents. We demonstrate direct synthesis of high-quality water-soluble Au, Ag, Pt, Pd, Cu and alloyed AuPt nanoparticles as well as ligand-exchange of FePt, cubic Pt and Au/Pt core/shell nanoparticles using bidentate dithiolane PEG as a universal ligand. Simple chemistry can greatly expand the applications of metal nanoparticles.

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.

DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution., Competing Interests: The authors declare no competing financial interest.

Ultrasmall noble metal and especially gold nanoclusters (AuNCs, ≤2 nm diameter in size) display a range of unique quantum confined and photophysical properties which are far different from their larger-sized nanoparticle counterparts or that of the bulk parent material. Amongst these properties, the photoluminescence of gold AuNCs has stimulated much interest for biological applications due to a combination of their small size, high photostability and range of emissions depending on size and surface stabilizing ligands, including near-IR emission. The dearth of robust Förster resonance energy transfer (FRET) donors and acceptors available to populate the latter spectral range is also driving strong interest in applying AuNCs for similar utility and especially for biosensing. However, the exact mechanism of how AuNCs engage in this type of energy transfer (ET) is still not defined and accumulating evidence indicates that it is not by a classical Förster process although, interestingly, many of the same characteristics and photophysical requirements seem to be present and even many-times required. Here, we summarize the state of the art in AuNC ET studies with a special emphasis on relevance to biological utility ranging from diagnostics to distance measurements along with describing the different ET mechanisms that have been ascribed with their use. Due to its corresponding importance in this discussion, we provide a brief overview of how these materials are synthesized, the current understanding of how their photoluminescence originates, and some related information on silver nanocluster (AgNC) ET along with related processes such as chemically-induced ET. A perspective and outlook on how this area will develop in the future is also provided.

Accumulating studies by many groups have found consistent enhancement in a wide variety of enzyme activities when they are displayed around nanoparticles. However, the underlying mechanism(s) that give rise to this phenomenon are still largely unknown. Herein, we develop a detailed reaction scheme that considers many of the various possible interactions between a substrate and a given enzyme-nanoparticle bioconjugate. The properties and some functional predictions that emanate from the reaction scheme were then tested using a model system where the homotetrameric beta-galactosidase enzyme complex was assembled with luminescent semiconductor nanocrystalline quantum dots displayed around its periphery. This type of assembly occurs as the ∼465 kDa enzyme complex is significantly larger than the 4.2 nm diameter green emitting quantum dots utilized. This unique architecture, in conjunction with the fact that this enzyme functions at or near the diffusion limit, provided a unique opportunity to selectively probe certain aspects of enzyme enhancement when attached to a nanoparticle with minimal potential perturbations to the native enzyme structure. Experimental assays were conducted where both free enzymes and quantum dot-decorated enzymes were compared directly in side-by-side samples and included formats where the kinetic processes were challenged with increasing viscosity and competitive inhibitors. The results strongly suggest that it is possible for there to be significant enhancements in an enzyme's catalytic rate or k cat after attachment to a nanoparticle even when it is apparently diffusion limited without requiring any gross changes to the enzyme's structure. A discussion of how this reaction scheme and model can be applied to other systems is provided.

Molecular logic devices (MLDs) constructed from DNA are promising for applications in bioanalysis, computing, and other applications requiring Boolean logic. These MLDs accept oligonucleotide inputs and generate fluorescence output through changes in structure. Although fluorescent dyes are most common in MLD designs, nontraditional luminescent materials with unique optical properties can potentially enhance MLD capabilities. In this context, luminescent lanthanide complexes (LLCs) have been largely overlooked. Here, we demonstrate a set of high-contrast DNA photonic logic gates based on toehold-mediated strand displacement and time-gated FRET. The gates include NAND, NOR, OR, and AND designs that accept two unlabeled target oligonucleotide sequences as inputs. Bright "true" output states utilize time-gated, FRET-sensitized emission from an Alexa Fluor 546 (A546) dye acceptor paired with a luminescent terbium cryptate (Tb) donor. Dark "false" output states are generated through either displacement of the A546, or through competitive and sequential quenching of the Tb or A546 by a dark quencher. Time-gated FRET and the long luminescence lifetime and spectrally narrow emission lines of the Tb donor enable 4-10-fold contrast between Boolean outputs, ≤10% signal variation for a common output, multicolor implementation of two logic gates in parallel, and effective performance in buffer and serum. These metrics exceed those reported for many other logic gate designs with only fluorescent dyes and with other non-LLC materials. Preliminary three-input AND and NAND gates are also demonstrated. The powerful combination of an LLC FRET donor with DNA-based logic gates is anticipated to have many future applications in bioanalysis.

The rapid detection of organophosphates (OPs), a class of strong neurotoxins, is critically important for monitoring acute insecticide exposure and potential chemical warfare agent use. Herein, we improve the enzymatic activity of a phosphotriesterase trimer (PTE 3 ), an enzyme that selectively recognizes OPs directly, by conjugation with distinctly sized (i.e., 5, 10, and 20 nm diameter) gold nanoparticles (AuNPs). The number of enzymes immobilized on the AuNP was controlled by conjugating increasing molar ratios of PTE 3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE 3 -His 6 termini and the AuNP-displayed Ni 2+ -nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE 3 -AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE 3 with the following PTE 3 -AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE 3 displayed around the AuNP and (2) fixed concentration of PTE 3 while increasing the bioconjugate molar ratio of PTE 3 -AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE 3 hydrolyzed the substrate paraoxon, a commercial insecticide and OP nerve agent simulant. Results demonstrate a general equivalent trend between the two formats. For all experiments, a maximum enzymatic velocity (V max ) increased by 17-fold over free enzyme for the lowest PTE 3 -AuNP ratio and the largest AuNP (i.e., ratio of 1 : 1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme-NP bioconjugates.

Sequence-selective bis-intercalating dyes exhibit large increases in fluorescence in the presence of specific DNA sequences. This property makes this class of fluorophore of particular importance to biosensing and super-resolution imaging. Here we report ultrafast transient anisotropy measurements of resonance energy transfer (RET) between thiazole orange (TO) molecules in a complex formed between the homodimer TOTO and double-stranded (ds) DNA. Biexponential homo-RET dynamics suggest two subpopulations within the ensemble: 80% intercalated and 20% non-intercalated. Based on the application of the transition density cube method to describe the electronic coupling and Monte Carlo simulations of the TOTO/dsDNA geometry, the dihedral angle between intercalated TO molecules is estimated to be 81° ± 5°, corresponding to a coupling strength of 45 ± 22 cm -1 . Dye intercalation with this geometry is found to occur independently of the underlying DNA sequence, despite the known preference of TOTO for the nucleobase sequence CTAG. The non-intercalated subpopulation is inferred to have a mean inter-dye separation distance of 19 Å, corresponding to coupling strengths between 0 and 25 cm -1 . This information is important to enable the rational design of energy transfer systems that utilize TOTO as a relay dye. The approach used here is generally applicable to determining the electronic coupling strength and intercalation configuration of other dimeric bis-intercalators.

Significant efforts are being undertaken to optimize the cargo carrying capacity and especially the cellular delivery efficiency of functionalized nanoparticles for applications in biological research and pharmacological delivery. One approach to increasing nanoparticle surface cargo display capacity is to decrease the number of moieties required for mediating cellular delivery by improving their efficiency. We describe a series of multivalent cell penetrating peptide (CPP) dendrimers that facilitate rapid cellular delivery of prototypical nanoparticle-semiconductor quantum dots (QDs). The modular CPP dendrimers were assembled through an innovative convergent oxime ligation strategy between (Arg 9 ) n motifs and a dendritic QD-coordination scaffold. Dendrimeric peptides sequentially incorporate a terminal (His) 6 motif for metal-affinity QD coordination, a Pro 9 spacer, a branching poly-lysine scaffold, and wedged display of (Arg 9 ) n binding motifs with n = 1×, 2×, 4×, 8×, 16× multivalency. QD dendrimer display capacity was estimated using structural simulations and QD-(Arg 9 ) 1-16 conjugates characterized by dynamic light scattering along with surface plasmon resonance-based binding assays to heparan sulfate proteoglycan surfaces. Cellular uptake via endocytosis was confirmed and peptide delivery kinetics investigated as a function of QD-(Arg 9 ) 1-16 conjugate exposure time and QD assembly ratio where cellular viability assays reflected no overt cytotoxicity. The ability of single dendrimer conjugates to facilitate cellular uptake was confirmed for QD-(Arg 9 ) 2-16 repeats along with the ability to deliver >850 kDa of protein cargo per QD. Minimizing the number of CPPs required for cellular uptake is critical for expanding nanoparticle cargo carrying capacity and can allow for inclusion of additional sensors, therapeutics and contrast agents on their surface.

Biotechnology has revolutionized therapeutics for the treatment of a wide range of diseases. Recent advances in protein engineering and material science have made the targeted delivery of enzyme therapeutics using nanocarriers (NCs) a new model of treatment. Several NCs have been approved for clinical use in drug delivery. Despite their advantages, few NCs have been approved to deliver enzyme cargo in a targeted manner. This review details the current arsenal of platforms developed to deliver enzyme therapeutics as well as the advantages and challenges of using enzymes as drugs, with examples from the literature, and discusses the benefits and liabilities of a given approach. We conclude by providing a perspective on how this field may evolve over the near and long-term.

Multiplexed photoluminescence (PL) detection plays an important role in chemical and biological sensing. Here, it is shown that time-gated (TG) detection of a single terbium-donor-based Förster resonance energy transfer (FRET) pair can be used to selectively quantify low nanomolar concentrations of multiple DNAs or microRNAs in a single sample. This study demonstrates the applicability of single-TG-FRET-pair multiplexing for molecular (Tb-to-dye) and nanoparticle (Tb-to-quantum-dot) biosensing. Both systems use acceptor-sensitization and donor-quenching for quantifying biomolecular recognition and modification of the donor-acceptor distance for tuning the PL decays. TG intensity detection provides extremely low background noise and a quick and simple one-step assay format. Single-TG-FRET-pair multiplexing can be combined with spectral and spatial resolution, paving the way for biosensing with unprecedented high-order multiplexing capabilities., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report the development of a quantum dot (QD)-peptide-fullerene (C 60 ) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C 60 electron acceptor. The photoexcited QD donor engages in ET with the C 60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C 60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C 60 bioconjugate labels the plasma membrane by insertion of the peptide-C 60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F 0 of 2-20% depending on the QD-C 60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C 60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C 60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C 60 construct displays 20- to 40-fold greater ΔF/F 0 than VSDs. The tractable nature of the QD-peptide-C 60 system offers the advantages of ease of assembly, large ΔF/F 0 , enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

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.

Treatments for autoimmunity - diseases where the immune system mistakenly attacks self-molecules - are not curative and leave patients immunocompromised. New studies aimed at more specific treatments reveal development of inflammation or tolerance is influenced by the form self-antigens are presented. Using a mouse model of multiple sclerosis (MS), we show for the first time that quantum dots (QDs) can be used to generate immunological tolerance by controlling the density of self-antigen on QDs. These assemblies display dense arrangements of myelin self-peptide associated with disease in MS, are uniform in size (<20 nm), and allow direct visualization in immune tissues. Peptide-QDs rapidly concentrate in draining lymph nodes, co-localizing with macrophages expressing scavenger receptors involved in tolerance. Treatment with peptide-QDs reduces disease incidence 10-fold. Strikingly, the degree of tolerance - and the underlying expansion of regulatory T cells - correlates with the density of myelin molecules presented on QDs. A key discovery is that higher numbers of tolerogenic particles displaying lower levels of self-peptide are more effective for inducing tolerance than fewer particles each displaying higher densities of peptide. QDs conjugated with self-antigens could serve as a new platform to induce tolerance, while visualizing QD therapeutics in tolerogenic tissue domains.