Nanoscale CuO charge and morphology control Fusarium suppression and nutrient biofortification in field-grown tomato and watermelon.

Limited data exist on how surface charge and morphology impact the effectiveness of nanoscale copper oxide (CuO) as an agricultural amendment under field conditions. This study investigated the impact of these factors on tomatoes and watermelons following foliar treatment with CuO nanosheets (NS-) or nanospikes (NP+ and NP-) exhibiting positive or negative surface charge. Results showed plant species-dependent benefits. Notably, tomatoes infected with Fusarium oxysporum had significantly reduced disease progression when treated with NS-. Watermelons benefited similarly from NP+. Although disease suppression was significant and trends indicated increased yield, the yield effects weren't statistically significant. However, several nanoscale treatments significantly enhanced the fruit's nutritional value, and this nano-enabled biofortification was a function of particle charge and morphology. Negatively charged nanospikes significantly increased the Fe content of healthy watermelon and tomato (20-28 %) and Ca in healthy tomato (66 %), compared to their positively charged counterpart. Negatively charged nanospikes also outperformed negatively charged nanosheets, leading to significant increases in the content of S and Mg in infected watermelon (37-38 %), Fe in healthy watermelon (58 %), and Ca (42 %) in healthy tomato. These findings highlight the potential of tuning nanoscale CuO chemistry for disease suppression and enhanced food quality under field conditions.

Influence of CuO Nanoparticle Aspect Ratio and Surface Charge on Disease Suppression in Tomato (Solanum lycopersicum).

Nanoparticles (NPs) have been shown to deliver micronutrients to plants to improve health, increase biomass, and suppress disease. Nanoscale properties such as morphology, size, composition, and surface chemistry have all been shown to impact nanomaterial interactions with plant systems. An organic-ligand-free synthesis method was used to prepare positively charged copper oxide (CuO) nanospikes, negatively charged CuO nanospikes, and negatively charged CuO nanosheets with exposed (001) crystal faces. X-ray photoelectron spectroscopy measurements show that the negative charge correlates to increased surface concentration of O on the NP surface, whereas relatively higher Cu concentrations are observed on the positively charged surfaces. The NPs were then used to treat tomato (Solanum lycopersicum) grown in soil infested with Fusarium oxysporum f. sp. lycopersici under greenhouse conditions. The negatively charged CuO significantly reduced disease progression and increased biomass, while the positively charged NPs and a CuSO4 salt control had little impact on the plants. Self-assembled monolayers were used to mimic the leaf surface to understand the intermolecular interactions between the NPs and the plant leaf; the data demonstrate that NP electrostatics and hydrogen-bonding interactions play an important role in adsorption onto leaf surfaces. These findings have important implications for the tunable design of materials as a strategy for the use of nano-enabled agriculture to increase food production.

Use of Magnetic Modulation of Nitrogen-Vacancy Center Fluorescence in Nanodiamonds for Quantitative Analysis of Nanoparticles in Organisms.

The fluorescence intensity emitted by nitrogen-vacancy (NV) centers in diamond nanoparticles can be readily modulated by the application of a magnetic field using a small electromagnet. By acquiring interleaved images acquired in the presence and absence of the magnetic field and performing digital subtraction, the fluorescence intensity of the NV nanodiamond can be isolated from scattering and autofluorescence even when these backgrounds are changing monotonically during the experiments. This approach has the potential to enable the robust identification of nanodiamonds in organisms and other complex environments. Yet, the practical application of magnetic modulation imaging to realistic systems requires the use of quantitative analysis methods based on signal-to-noise considerations. Here, we describe the use of magnetic modulation to analyze the uptake of diamond nanoparticles from an aqueous environment into Caenorhabditis elegans, used here as a model system for identification and quantification of nanodiamonds in complex matrices. Based on the observed signal-to-noise ratio of sets of digitally subtracted images, we show that nanodiamonds can be identified on an individual pixel basis with a >99.95% confidence. To determine whether surface functionalization of the nanodiamond significantly impacted uptake, we used this approach to analyze the presence of nanodiamonds in C. elegans that had been exposed to these functionalized nanodiamonds in the water column, with uptake likely occurring by ingestion. In each case, the images show a significant nanoparticle uptake. However, differences in uptake between the three ligands were not outside of the experimental error, indicating that additional factors beyond the surface charge are important factors controlling uptake. Analysis of the number of pixels above the threshold in individual C. elegans organisms revealed distributions that deviate significantly from a Poisson distribution, suggesting that uptake of nanoparticles may not be a statistically independent event. The results presented here demonstrate that magnetic modulation combined with quantitative analysis of the resulting images can be used to robustly characterize nanoparticle uptake into organisms.

Binding of polar and hydrophobic molecules at the LiCoO2 (001)-water interface: force field development and molecular dynamics simulations.

A classical model in the framework of the INTERFACE force field has been developed for treating the LiCoO2 (LCO) (001)/water interface. In comparison to ab initio molecular dynamics (MD) simulations based on density functional theory, MD simulations using the classical model lead to generally reliable descriptions of interfacial properties, such as the density distribution of water molecules. Water molecules in close contact with the LCO surface form a strongly adsorbed layer, which leads to a free energy barrier for the adsorption of polar or charged molecules to the LCO surface. Moreover, due to the strong hydrogen bonding interactions with the LCO surface, the first water layer forms an interface that exhibits hydrophobic characters, leading to favorable adsorption of non-polar molecules to the interface. Therefore, despite its highly polar nature, the LCO (001) surface binds not only polar/charged but also non-polar solutes. As an application, the model is used to analyze the adsorption of reduced nicotinamide adenine dinucleotide (NADH) and its molecular components to the LCO (001) surface in water. The results suggest that recently observed redox activity of NADH at the LCO/water interface was due to the co-operativity between the ribose component, which drives binding to the LCO surface, and the nicotinamide moiety, which undergoes oxidation.

Cross-species transcriptomic signatures identify mechanisms related to species sensitivity and common responses to nanomaterials.

Physico-chemical characteristics of engineered nanomaterials are known to be important in determining the impact on organisms but effects are equally dependent upon the characteristics of the organism exposed. Species sensitivity may vary by orders of magnitude, which could be due to differences in the type or magnitude of the biochemical response, exposure or uptake of nanomaterials. Synthesizing conclusions across studies and species is difficult as multiple species are not often included in a study, and differences in batches of nanomaterials, the exposure duration and media across experiments confound comparisons. Here three model species, Danio rerio, Daphnia magna and Chironomus riparius, that differ in sensitivity to lithium cobalt oxide nanosheets are found to differ in immune-response, iron-sulfur protein and central nervous system pathways, among others. Nanomaterial uptake and dissolution does not fully explain cross-species differences. This comparison provides insight into how biomolecular responses across species relate to the varying sensitivity to nanomaterials.

Role of Foliar Biointerface Properties and Nanomaterial Chemistry in Controlling Cu Transfer into Wild-Type and Mutant Arabidopsis thaliana Leaf Tissue.

Seven Arabidopsis thaliana mutants with differences in cuticle thickness and stomatal density were foliar exposed to 50 mg L-1 Cu3(PO4)2 nanosheets (NS), CuO NS, CuO nanoparticles, and CuSO4. Three separate fractions of Cu (surface-attached, cuticle, interior leaf) were isolated from the leaf at 0.25, 2, 4, and 8 h. Cu transfer from the surface through the cuticle and into the leaf varied with mutant and particle type. The Cu content on the surface decreased significantly over 8 h but increased in the cuticle. Cu derived from the ionic form had the greatest cuticle concentration, suggesting greater difficulty in moving across this barrier and into the leaf. Leaf Cu in the increased-stomatal mutants was 8.5-44.9% greater than the decreased stomatal mutants, and abscisic acid to close the stomata decreased Cu in the leaf. This demonstrates the importance of nanomaterial entry through the stomata and enables the optimization of materials for nanoenabled agriculture.

Expression Patterns of Energy-Related Genes in Single Cells Uncover Key Isoforms and Enzymes That Gain Priority Under Nanoparticle-Induced Stress.

Cellular responses to nanoparticles (NPs) have been largely studied in cell populations, providing averaged values that often misrepresent the true molecular processes that occur in the individual cell. To understand how a cell redistributes limited molecular resources to achieve optimal response and survival requires single-cell analysis. Here we applied multiplex single molecule-based fluorescence in situ hybridization (fliFISH) to quantify the expression of 10 genes simultaneously in individual intact cells, including glycolysis and glucose transporter genes, which are critical for restoring and maintaining energy balance. We focused on individual gill epithelial cell responses to lithium cobalt oxide (LCO) NPs, which are actively pursued as cathode materials in lithium-ion batteries, raising concerns about their impact on the environment and human health. We found large variabilities in the expression levels of all genes between neighboring cells under the same exposure conditions, from only a few transcripts to over 100 copies in individual cells. Gene expression ratios among the 10 genes in each cell uncovered shifts in favor of genes that play key roles in restoring and maintaining energy balance. Among these genes are isoforms that can secure and increase glycolysis rates more efficiently, as well as genes with multiple cellular functions, in addition to glycolysis, including DNA repair, regulation of gene expression, cell cycle progression, and proliferation. Our study uncovered prioritization of gene expression in individual cells for restoring energy balance under LCO NP exposures. Broadly, our study gained insight into single-cell strategies for redistributing limited resources to achieve optimal response and survival under stress.

Detection of Aqueous Solvated Electrons Produced by Photoemission from Solids Using Transient Absorption Measurements.

Solvated electrons in water have long been of interest to chemists. While readily produced using intense multiphoton excitation of water and/or irradiation with high-energy particles, the possible role of solvated electrons in electrochemical and photoelectrochemical reactions at electrodes has been controversial. Recent studies showed that excitation of electrons to the conduction band of diamond leads to barrier-free emission of electrons into water. While these electrons can be inferred from the reactions they induce, direct detection by transient absorption measurements provides more direct evidence. Here, we present studies demonstrating direct detection of solvated electrons produced at diamond electrode surfaces and the influence of electrochemical potential and solution-phase electron scavengers. We further present a more detailed analysis of experimental conditions needed to detect solvated electrons emitted from diamond and other solid materials through transient optical absorption measurements.

Ag-Diamond Core-Shell Nanostructures Incorporated with Silicon-Vacancy Centers.

Silicon-vacancy (SiV) centers in diamond have attracted attention as highly stable fluorophores for sensing and as possible candidates for quantum information science. While prior studies have shown that the formation of hybrid diamond-metal structures can increase the rates of optical absorption and emission, many practical applications require diamond plasmonic structures that are stable in harsh chemical and thermal environments. Here, we demonstrate that Ag nanospheres, produced both in quasi-random arrays by thermal dewetting and in ordered arrays using electron-beam lithography, can be completely encapsulated with a thin diamond coating containing SiV centers, leading to hybrid core-shell nanostructures exhibiting extraordinary chemical and thermal stability as well as enhanced optical properties. Diamond shells with a thickness on the order of 20-100 nm are sufficient to encapsulate and protect the Ag nanostructures with different sizes ranging from 20 nm to hundreds of nanometers, allowing them to withstand heating to temperatures of 1000 °C and immersion in harsh boiling acid for 24 h. Ultrafast photoluminescence lifetime and super-resolution optical imaging experiments were used to study the SiV properties on and off the core-shell structures, which show that the SiV on core-shell structures have higher brightness and faster decay rate. The stability and optical properties of the hybrid Ag-diamond core-shell structures make them attractive candidates for high-efficiency imaging and quantum-based sensing applications.

Energy Starvation in Daphnia magna from Exposure to a Lithium Cobalt Oxide Nanomaterial.

Growing evidence across organisms points to altered energy metabolism as an adverse outcome of metal oxide nanomaterial toxicity, with a mechanism of toxicity potentially related to the redox chemistry of processes involved in energy production. Despite this evidence, the significance of this mechanism has gone unrecognized in nanotoxicology due to the field's focus on oxidative stress as a universal─but nonspecific─nanotoxicity mechanism. To further explore metabolic impacts, we determined lithium cobalt oxide's (LCO's) effects on these pathways in the model organism Daphnia magna through global gene-expression analysis using RNA-Seq and untargeted metabolomics by direct-injection mass spectrometry. Our results show that a sublethal 1 mg/L 48 h exposure of D. magna to LCO nanosheets causes significant impacts on metabolic pathways versus untreated controls, while exposure to ions released over 48 h does not. Specifically, transcriptomic analysis using DAVID indicated significant enrichment (Benjamini-adjusted p ≤0.0.5) in LCO-exposed animals for changes in pathways involved in the cellular response to starvation (25 genes), mitochondrial function (70 genes), ATP-binding (70 genes), oxidative phosphorylation (53 genes), NADH dehydrogenase activity (12 genes), and protein biosynthesis (40 genes). Metabolomic analysis using MetaboAnalyst indicated significant enrichment (γ-adjusted p <0.1) for changes in amino acid metabolism (19 metabolites) and starch, sucrose, and galactose metabolism (7 metabolites). Overlap of significantly impacted pathways by RNA-Seq and metabolomics suggests amino acid breakdown and increased sugar import for energy production. Results indicate that LCO-exposed Daphnia respond to energy starvation by altering metabolic pathways, both at the gene expression and metabolite levels. These results support altered energy production as a sensitive nanotoxicity adverse outcome for LCO exposure and suggest negative impacts on energy metabolism as an important avenue for future studies of nanotoxicity, including for other biological systems and for metal oxide nanomaterials more broadly.

High-Density Covalent Grafting of Spin-Active Molecular Moieties to Diamond Surfaces.

Functionalization of diamond surfaces with TEMPO and other surface paramagnetic species represents one approach to the implementation of novel chemical detection schemes that make use of shallow quantum color defects such as silicon-vacancy (SiV) and nitrogen-vacancy (NV) centers. Yet, prior approaches to quantum-based chemical sensing have been hampered by the absence of high-quality surface functionalization schemes for linking radicals to diamond surfaces. Here, we demonstrate a highly controlled approach to the functionalization of diamond surfaces with carboxylic acid groups via all-carbon tethers of different lengths, followed by covalent chemistry to yield high-quality, TEMPO-modified surfaces. Our studies yield estimated surface densities of 4-amino-TEMPO of approximately 1.4 molecules nm-2 on nanodiamond (varying with molecular linker length) and 3.3 molecules nm-2 on planar diamond. These values are higher than those reported previously using other functionalization methods. The ζ-potential of nanodiamonds was used to track reaction progress and elucidate the regioselectivity of the reaction between ethenyl and carboxylate groups and surface radicals.

Influence of Surface Ligand Molecular Structure on Phospholipid Membrane Disruption by Cationic Nanoparticles.

Cationic nanoparticles are known to interact with biological membranes and often cause serious membrane damage. Therefore, it is important to understand the molecular mechanism for such interactions and the factors that impact the degree of membrane damage. Previously, we have demonstrated that spatial distribution of molecular charge at cationic nanoparticle surfaces plays an important role in determining the cellular uptake and membrane damage of these nanoparticles. In this work, using diamond nanoparticles (DNPs) functionalized with five different amine-based surface ligands and small phospholipid unilamellar vesicles (SUVs), we further investigate how chemical features and conformational flexibility of surface ligands impact nanoparticle/membrane interactions. 31P-NMR T2 relaxation measurements quantify the mobility changes in lipid dynamics upon exposing the SUVs to functional DNPs, and coarse-grained molecular dynamics simulations further elucidate molecular details for the different modes of DNP-SUV interactions depending on the surface ligands. Collectively, our results show that the length of the hydrophobic segment and conformational flexibility of surface ligands are two key factors that dictate the degree of membrane damage by the DNP, while the amount of surface charge alone is not predictive of the strength of interaction.

Influence of Sensor Coating and Topography on Protein and Nanoparticle Interaction with Supported Lipid Bilayers.

Supported lipid bilayers (SLBs) have proven to be valuable model systems for studying the interactions of proteins, peptides, and nanoparticles with biological membranes. The physicochemical properties (e.g., topography, coating) of the solid substrate may affect the formation and properties of supported phospholipid bilayers, and thus, subsequent interactions with biomolecules or nanoparticles. Here, we examine the influence of support coating (SiO2 vs Si3N4) and topography [sensors with embedded vs protruding gold nanodisks for nanoplasmonic sensing (NPS)] on the formation and subsequent interactions of supported phospholipid bilayers with the model protein cytochrome c and with cationic polymer-wrapped quantum dots using quartz crystal microbalance with dissipation monitoring and NPS techniques. The specific protein and nanoparticle were chosen because they differ in the degree to which they penetrate the bilayer. We find that bilayer formation and subsequent non-penetrative association with cytochrome c were not significantly influenced by substrate composition or topography. In contrast, the interactions of nanoparticles with SLBs depended on the substrate composition. The substrate-dependence of nanoparticle adsorption is attributed to the more negative zeta-potential of the bilayers supported by the silica vs the silicon nitride substrate and to the penetration of the cationic polymer wrapping the nanoparticles into the bilayer. Our results indicate that the degree to which nanoscale analytes interact with SLBs may be influenced by the underlying substrate material.

Protein Fe-S Centers as a Molecular Target of Toxicity of a Complex Transition Metal Oxide Nanomaterial with Downstream Impacts on Metabolism and Growth.

Oxidative stress is frequently identified as a mechanism of toxicity of nanomaterials. However, rarely have the specific underlying molecular targets responsible for these impacts been identified. We previously demonstrated significant negative impacts of transition metal oxide (TMO) lithium-ion battery cathode nanomaterial, lithium cobalt oxide (LCO), on the growth, development, hemoglobin, and heme synthesis gene expression in the larvae of a model sediment invertebrate Chironomus riparius. Here, we propose that alteration of the Fe-S protein function by LCO is a molecular initiating event leading to these changes. A 10 mg/L LCO exposure causes significant oxidation of the aconitase 4Fe-4S center after 7 d as determined from the electron paramagnetic resonance spectroscopy measurements of intact larvae and a significant reduction in the aconitase activity of larval protein after 48 h (p < 0.05). Next-generation RNA sequencing identified significant changes in the expression of genes involved in 4Fe-4S center binding, Fe-S center synthesis, iron ion binding, and metabolism for 10 mg/L LCO at 48 h (FDR-adjusted, p < 0.1). We propose an adverse outcome pathway, where the oxidation of metabolic and regulatory Fe-S centers of proteins by LCO disrupts metabolic homeostasis, which negatively impacts the growth and development, a mechanism that may apply for these conserved proteins across species and for other TMO nanomaterials.

Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function.

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.

Copper Nanomaterial Morphology and Composition Control Foliar Transfer through the Cuticle and Mediate Resistance to Root Fungal Disease in Tomato (Solanum lycopersicum).

Two copper nanomaterials (CuO nanoparticles [NPs] and Cu3(PO4)2·3H2O nanosheets) and CuSO4 were applied to tomato (Solanum lycopersicum) leaves, and elemental Cu movement from the leaf surface through the cuticle and into the interior leaf tissue was monitored over 8 h. Two forms of nanoscale Cu were used to foliar treat tomato on a weekly basis in greenhouse and field experiments in the presence of the pathogen Fusarium oxysporum f. sp. lycopersici. For CuSO4, Cu accumulation and retention in the cuticle was over 7-fold greater than the nanomaterials, demonstrating that nanoscale morphology and composition mediate Cu accumulation in leaf tissue. In the greenhouse, weekly foliar applications of the nanosheets and NPs increased seedling biomass by 90.9 and 93.3%, respectively, compared to diseased and ionic Cu controls. In the field, Cu3(PO4)2·3H2O nanosheets reduced disease progress by 26.0% and significantly increased fruit yield by over 45.5% per plant relative to the other treatments in diseased soil. These findings suggest that nanoscale nutrient chemical properties can be tuned to maximize and control movement through the cuticle and that interactions at the seedling leaf biointerface can lead to season-long benefit for tomato growing in the presence of Fusarium spp.

Interfacial water and ion distribution determine ζ potential and binding affinity of nanoparticles to biomolecules.

The molecular features that dictate interactions between functionalized nanoparticles and biomolecules are not well understood. This is in part because for highly charged nanoparticles in solution, establishing a clear connection between the molecular features of surface ligands and common experimental observables such as ζ potential requires going beyond the classical models based on continuum and mean field models. Motivated by these considerations, molecular dynamics simulations are used to probe the electrostatic properties of functionalized gold nanoparticles and their interaction with a charged peptide in salt solutions. Counterions are observed to screen the bare ligand charge to a significant degree even at the moderate salt concentration of 50 mM. As a result, the apparent charge density and ζ potential are largely insensitive to the bare ligand charge densities, which fall in the range of ligand densities typically measured experimentally for gold nanoparticles. While this screening effect was predicted by classical models such as the Manning condensation theory, the magnitudes of the apparent surface charge from microscopic simulations and mean-field models are significantly different. Moreover, our simulations found that the chemical features of the surface ligand (e.g., primary vs. quaternary amines, heterogeneous ligand lengths) modulate the interfacial ion and water distributions and therefore the interfacial potential. The importance of interfacial water is further highlighted by the observation that introducing a fraction of hydrophobic ligands enhances the strength of electrostatic binding of the charged peptide. Finally, the simulations highlight that the electric double layer is perturbed upon binding interactions. As a result, it is the bare charge density rather than the apparent charge density or ζ potential that better correlates with binding affinity of the nanoparticle to a charged peptide. Overall, our study highlights the importance of molecular features of the nanoparticle/water interface and underscores a set of design rules for the modulation of electrostatic driven interactions at nano/bio interfaces.

Influence of the Spatial Distribution of Cationic Functional Groups at Nanoparticle Surfaces on Bacterial Viability and Membrane Interactions.

While positively charged nanomaterials induce cytotoxicity in many organisms, much less is known about how the spatial distribution and presentation of molecular surface charge impact nanoparticle-biological interactions. We systematically functionalized diamond nanoparticle surfaces with five different cationic surface molecules having different molecular structures and conformations, including four small ligands and one polymer, and we then probed the molecular-level interaction between these nanoparticles and bacterial cells. Shewanella oneidensis MR-1 was used as a model bacterial cell system to investigate how the molecular length and conformation of cationic surface charges influence their interactions with the Gram-negative bacterial membranes. Nuclear magnetic resonance (NMR) and X-ray photoelectron spectroscopy (XPS) demonstrate the covalent modification of the nanoparticle surface with the desired cationic organic monolayers. Surprisingly, bacterial growth-based viability (GBV) and membrane damage assays both show only minimal biological impact by the NPs functionalized with short cationic ligands within the concentration range tested, yet NPs covalently linked to a cationic polymer induce strong cytotoxicity, including reduced cellular viability and significant membrane damage at the same concentration of cationic groups. Transmission electron microscopy (TEM) images of these NP-exposed bacterial cells show that NPs functionalized with cationic polymers induce significant membrane distortion and the production of outer membrane vesicle-like features, while NPs bearing short cationic ligands only exhibit weak membrane association. Our results demonstrate that the spatial distribution of molecular charge plays a key role in controlling the interaction of cationic nanoparticles with bacterial cell membranes and the subsequent biological impact. Nanoparticles functionalized with ligands having different lengths and conformations can have large differences in interactions even while having nearly identical zeta potentials. While the zeta potential is a convenient and commonly used measure of nanoparticle charge, it does not capture essential differences in molecular-level nanoparticle properties that control their biological impact.