Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection

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 (PTE3), 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 PTE3 onto the AuNP surface via metal affinity coordination. This occurs between the PTE3-His6 termini and the AuNP-displayed Ni2+-nitrilotriacetic acid end groups and was confirmed with gel electrophoresis. The enzymatic efficiency of the resultant PTE3–AuNP bioconjugates was analyzed via enzyme progress curves acquired from two distinct assay formats that compared free unbound PTE3 with the following PTE3–AuNP bioconjugates: (1) fixed concentration of AuNPs while increasing the bioconjugate molar ratio of PTE3 displayed around the AuNP and (2) fixed concentration of PTE3 while increasing the bioconjugate molar ratio of PTE3–AuNP by decreasing the AuNP concentration. Both assay formats monitored the absorbance of p-nitrophenol that was produced as PTE3 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 (Vmax) increased by 17-fold over free enzyme for the lowest PTE3–AuNP ratio and the largest AuNP (i.e., ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]1, 20 nm dia. AuNP). This work provides a route to improve enzymatic OP detection strategies with enzyme–NP bioconjugates

Effects of shell thickness on the electric field dependence of exciton recombination in CdSe/CdS core/shell quantum dots

Here we examine the effects of shell thickness on the photophysical properties of CdSe/CdS core/shell quantum dots (QDs) in an electric field. Photoluminescence (PL) of QDs in an applied electric field is observed to decrease markedly with increasing shell thickness, with a thick-shelled (4.9 nm shell) sample exhibiting an order of magnitude greater PL suppression than a thin-shelled sample (1.25 nm shell) with the same core

Membrane Fluctuation and Polymer Adsorption

We investigate adsorption behaviors and desorption transitions of a flexible but self-avoiding polymer on a fluctuating (undulating) membrane (or interface). Incorporating hard-wall repulsion with polymer-membrane attraction of finite range, we find that the membrane fluctuation gives rise to an effective repulsion and enhances unbinding (desorption) transtition of polymer, lowering the transition temperature. Employing hard square-well model as an example, we calculate the desorption transition temperature and the critical behaviors of polymer and membrane fluctuations. Our results are qualitatively different from those of Garel, Kadar and Orland (Europhys. Lett. 29 (1995) 303) who employed a contact attractive potential which allows the polymer to permeate the other side of the surface

Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells

The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation

Meta-analysis of cellular toxicity for cadmium-containing quantum dots.

Understanding the relationships between the physicochemical properties of engineered nanomaterials and their toxicity is critical for environmental and health risk analysis. However, this task is confounded by material diversity, heterogeneity of published data and limited sampling within individual studies. Here, we present an approach for analysing and extracting pertinent knowledge from published studies focusing on the cellular toxicity of cadmium-containing semiconductor quantum dots. From 307 publications, we obtain 1,741 cell viability-related data samples, each with 24 qualitative and quantitative attributes describing the material properties and experimental conditions. Using random forest regression models to analyse the data, we show that toxicity is closely correlated with quantum dot surface properties (including shell, ligand and surface modifications), diameter, assay type and exposure time. Our approach of integrating quantitative and categorical data provides a roadmap for interrogating the wide-ranging toxicity data in the literature and suggests that meta-analysis can help develop methods for predicting the toxicity of engineered nanomaterials

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.status: publishe

Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly

Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing