Single-Molecule Methane Sensing Using Palladium-Functionalized nIR Fluorescent Single-Walled Carbon Nanotubes.

There has been considerable interest in detecting atmospheric and process-associated methane (CH4) at low concentrations due to its potency as a greenhouse gas. Nanosensor technology, particularly fluorescent single-walled carbon nanotube (SWCNT) arrays, is promising for such applications because of their chemical sensitivities at single-molecule detection limits. However, the methodologies for connecting the stochastic molecular fluctuations from gas impingement on such sensors require further development. In this work, we synthesize Pd-conjugated ss(GT)15-DNA-wrapped SWCNTas near-infrared (nIR) fluorescent, single-molecule sensors of CH4. The complexes are characterized using X-ray photoelectron spectroscopy (XPS) and spectrophotometry, demonstrating spectral changes between the Pd2+ and Pd0 oxidation states. The nIR fluctuations generated upon exposure from 8 to 26 ppb of CH4 were separated into high- and low-frequency components. Aggregating the low-frequency components for an array of sensors showed the most consistent levels of detection with a limit of 0.7 ppb. These results advance the hardware and computational methods necessary to apply this approach to the challenge of environmental methane sensing.

Single-Walled Carbon Nanotube-Guided Topical Skin Delivery of Tyrosinase to Prevent Photoinduced Damage.

When the skin is exposed to ultraviolet radiation (UV), it leads to the degradation of the extracellular matrix (ECM) and results in inflammation. Subsequently, melanocytes are triggered to induce tyrosinase-mediated melanin synthesis, protecting the skin. Here, we introduce a proactive approach to protect the skin from photodamage via the topical delivery of Streptomyces avermitilis-derived tyrosinase (SaTy) using single-walled carbon nanotube (SWNT). Utilizing a reverse electrodialysis (RED) battery, we facilitated the delivery of SaTy-SWNT complexes up to depths of approximately 300 µm, as analyzed by using confocal Raman microscopy. When applied to ex vivo porcine skin and in vivo albino mouse skin, SaTy-SWNT synthesized melanin, resulting in 4-fold greater UV/vis absorption at 475 nm than in mice without SaTy-SWNT. The synthesized melanin efficiently absorbed UV light and alleviated skin inflammation. In addition, the densification of dermal collagen, achieved through SaTy-mediated cross-linking, reduced photoinduced wrinkles by 66.3% in the affected area. Our findings suggest that SWNT-mediated topical protein delivery holds promise in tissue engineering applications.

Bioengineered liver crosslinked with nano-graphene oxide enables efficient liver regeneration via MMP suppression and immunomodulation.

Decellularized extracellular matrix scaffold, widely utilized for organ engineering, often undergoes matrix decomposition after transplantation and produces byproducts that cause inflammation, leading to clinical failure. Here we propose a strategy using nano-graphene oxide to modify the biophysical properties of decellularized liver scaffolds. Notably, we demonstrate that scaffolds crosslinked with nano-graphene oxide show high resistance to enzymatic degradation via direct inhibition of matrix metalloproteinase activity and increased mechanical rigidity. We find that M2-like macrophage polarization is promoted within the crosslinked scaffolds, which reduces graft-elicited inflammation. Moreover, we show that low activities of matrix metalloproteinases, attributed to both nano-graphene oxide and tissue inhibitors of metalloproteinases expressed by M2c, can protect the crosslinked scaffolds against in vivo degradation. Lastly, we demonstrate that bioengineered livers fabricated with the crosslinked scaffolds remain functional, thereby effectively regenerating damaged livers after transplantation into liver failure mouse models. Overall, nano-graphene oxide crosslinking prolongs allograft survival and ultimately improves therapeutic effects of bioengineered livers, which offer an alternative for donor organs.

In vivo surface-enhanced Raman scattering nanosensor for the real-time monitoring of multiple stress signalling molecules in plants.

When under stress, plants release molecules to activate their defense system. Detecting these stress-related molecules offers the possibility to address stress conditions and prevent the development of diseases. However, detecting endogenous signalling molecules in living plants remains challenging due to low concentrations of these analytes and interference with other compounds; additionally, many methods currently used are invasive and labour-intensive. Here we show a non-destructive surface-enhanced Raman scattering (SERS)-based nanoprobe for the real-time detection of multiple stress-related endogenous molecules in living plants. The nanoprobe, which is placed in the intercellular space, is optically active in the near-infrared region (785 nm) to avoid interferences from plant autofluorescence. It consists of a Si nanosphere surrounded by a corrugated Ag shell modified by a water-soluble cationic polymer poly(diallyldimethylammonium chloride), which can interact with multiple plant signalling molecules. We measure a SERS enhancement factor of 2.9 × 107 and a signal-to-noise ratio of up to 64 with an acquisition time of ~100 ms. To show quantitative multiplex detection, we adopted a binding model to interpret the SERS intensities of two different analytes bound to the SERS hot spot of the nanoprobe. Under either abiotic or biotic stress, our optical nanosensors can successfully monitor salicylic acid, extracellular adenosine triphosphate, cruciferous phytoalexin and glutathione in Nasturtium officinale, Triticum aestivum L. and Hordeum vulgare L.-all stress-related molecules indicating the possible onset of a plant disease. We believe that plasmonic nanosensor platforms can enable the early diagnosis of stress, contributing to a timely disease management of plants.

Structural and Electronic Modulations of Imidazolium Covalent Organic Framework-Derived Electrocatalysts for Oxygen Redox Reactions in Rechargeable Zn-Air Batteries.

Covalent organic frameworks (COFs) are promising candidates for the controllable design of electrocatalysts. However, bifunctional electrocatalytic activities for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) remain challenging in COFs. In this study, imidazolium-rich COFs (IMCOFs) with well-defined active sites and characteristic three-dimensional assembly structures were readily prepared, and their electronic structures were tuned by Co incorporation to elicit bifunctional electrocatalytic activities for the ORR and OER. The Co nanoparticle-incorporated spherical IMCOF-derived electrocatalyst (CoNP-s-IMCOF) exhibited lower overpotentials for the ORR and OER compared with the atomic Co-incorporated planar IMCOF-derived electrocatalyst (Co-p-IMCOF). Computational simulations revealed that the imidazole carbon sites of CoNP-s-IMCOF rather than the triazine carbons were the active sites for the ORR and OER, and its p-band center downshifted via charge transfer, facilitating the chemisorption of oxygen intermediates during the reactions. A Zn-air battery with CoNP-s-IMCOF exhibited a small voltage gap of 1.3 V with excellent durability for 935 cycles. This approach for control over the three-dimensional assembly and electronic structures of IMCOFs can be extended to the development of diverse catalytic nanomaterials for applications of interest.

Graphene oxide film guided skeletal muscle differentiation.

Engineered muscle tissues can be used for the regeneration or substitution of irreversibly damaged or diseased muscles. Recently, graphene oxide (GO) has been shown to improve the adsorption of biomolecules through its biocompatibility and intrinsic π-π interactions. The possibility of producing various GO modifications may also provide additional functionality as substrates for cell culture. In particular, substrates fabricated from pristine GO have been shown to improve cellular functions and influence stem cell differentiation. In this study, we fabricated tunable GO substrates with various physical and chemical properties and demonstrated the ability of the substrate to support myogenic differentiation. Higher cellular adhesion affinity with unique microfilament anchorage was observed for GO substrates with increased GO concentrations. In addition, amino acid (AA)-conjugated GO (GO-AA) substrates were fabricated to modify GO chemical properties and study the effects of chemically modified GO substrates on myogenic differentiation. Our findings demonstrate that minor tuning of GO significantly influences myogenic differentiation.

Milk Protein-Derived Antioxidant Tetrapeptides as Potential Hypopigmenting Agents.

Excessive accumulation of melanin can cause skin pigmentation disorders, which may be accompanied by significant psychological stress. Although many natural and synthetic products have been developed for the regulation of melanogenesis biochemistry, the management of unwanted skin pigmentation remains challenging. Herein, we investigated the potential hypopigmenting properties of peptide sequences that originated from milk proteins such as ĸ-casein and ß-lactoglobulin. These proteins are known to inhibit melanogenesis and their hydrolysates are reported as antioxidant peptides. We synthesize tetrapeptide fragments of the milk protein hydrolysates and investigate the amino acids that are essential for designing peptides with tyrosinase inhibitory and antioxidant activities. We found that the peptide methionine-histidine-isoleucine-arginine amide sufficiently inhibits mushroom tyrosinase activity, shows potent antioxidant activity and effectively impedes melanogenesis in cultured melanocytes via cooperative biological activities. Our findings demonstrate the potential utility of the bioactive tetrapeptide from milk proteins as a chemical alternative to hypopigmenting agents.

Real-time detection of wound-induced H2O2 signalling waves in plants with optical nanosensors.

Decoding wound signalling in plants is critical for understanding various aspects of plant sciences, from pest resistance to secondary metabolite and phytohormone biosynthesis. The plant defence responses are known to primarily involve NADPH-oxidase-mediated H2O2 and Ca2+ signalling pathways, which propagate across long distances through the plant vasculature and tissues. Using non-destructive optical nanosensors, we find that the H2O2 concentration profile post-wounding follows a logistic waveform for six plant species: lettuce (Lactuca sativa), arugula (Eruca sativa), spinach (Spinacia oleracea), strawberry blite (Blitum capitatum), sorrel (Rumex acetosa) and Arabidopsis thaliana, ranked in order of wave speed from 0.44 to 3.10 cm min-1. The H2O2 wave tracks the concomitant surface potential wave measured electrochemically. We show that the plant RbohD glutamate-receptor-like channels (GLR3.3 and GLR3.6) are all critical to the propagation of the wound-induced H2O2 wave. Our findings highlight the utility of a new type of nanosensor probe that is species-independent and capable of real-time, spatial and temporal biochemical measurements in plants.

Measuring the Accessible Surface Area within the Nanoparticle Corona Using Molecular Probe Adsorption.

The corona phase-the adsorbed layer of polymer, surfactant, or stabilizer molecules around a nanoparticle-is typically utilized to disperse nanoparticles into a solution or solid phase. However, this phase also controls molecular access to the nanoparticle surface, a property important for catalytic activity and sensor applications. Unfortunately, few methods can directly probe the structure of this corona phase, which is subcategorized as either a hard, immobile corona or a soft, transient corona in exchange with components in the bulk solution. In this work, we introduce a molecular probe adsorption (MPA) method for measuring the accessible nanoparticle surface area using a titration of a quenchable fluorescent molecule. For example, riboflavin is utilized to measure the surface area of gold nanoparticle standards, as well as corona phases on dispersed single-walled carbon nanotubes and graphene sheets. A material balance on the titration yields certain surface coverage parameters, including the ratio of the surface area to dissociation constant of the fluorophore, q/KD, as well as KD itself. Uncertainty, precision, and the correlation of these parameters across different experimental systems, preparations, and modalities are all discussed. Using MPA across a series of corona phases, we find that the Gibbs free energy of probe binding scales inversely with the cube root of surface area, q. In this way, MPA is the only technique to date capable of discerning critical structure-property relationships for such nanoparticle surface phases. Hence, MPA is a rapid quantitative technique that should prove useful for elucidating corona structure for nanoparticles across different systems.

Chloroplast-selective gene delivery and expression in planta using chitosan-complexed single-walled carbon nanotube carriers.

Plant genetic engineering is an important tool used in current efforts in crop improvement, pharmaceutical product biosynthesis and sustainable agriculture. However, conventional genetic engineering techniques target the nuclear genome, prompting concerns about the proliferation of foreign genes to weedy relatives. Chloroplast transformation does not have this limitation, since the plastid genome is maternally inherited in most plants, motivating the need for organelle-specific and selective nanocarriers. Here, we rationally designed chitosan-complexed single-walled carbon nanotubes, utilizing the lipid exchange envelope penetration mechanism. The single-walled carbon nanotubes selectively deliver plasmid DNA to chloroplasts of different plant species without external biolistic or chemical aid. We demonstrate chloroplast-targeted transgene delivery and transient expression in mature Eruca sativa, Nasturtium officinale, Nicotiana tabacum and Spinacia oleracea plants and in isolated Arabidopsis thaliana mesophyll protoplasts. This nanoparticle-mediated chloroplast transgene delivery tool provides practical advantages over current delivery techniques as a potential transformation method for mature plants to benefit plant bioengineering and biological studies.

Polymethacrylamide and Carbon Composites that Grow, Strengthen, and Self-Repair using Ambient Carbon Dioxide Fixation.

Plants accumulate solid carbon mass and self-repair using atmospheric CO2 fixation from photosynthesis. Synthetic materials capable of mimicking this property can significantly reduce the energy needed to transport and repair construction materials. Here, a gel matrix containing aminopropyl methacrylamide (APMA), glucose oxidase (GOx), and nanoceria-stabilized extracted chloroplasts that is able to grow, strengthen, and self-repair using carbon fixation is demonstrated. Glucose produced from the embedded chloroplasts is converted to gluconolactone (GL) via GOx, polymerizing with APMA to form a continuously expanding and strengthening polymethacrylamide. The extracted spinach chloroplasts exhibit enhanced stability and produce 12 µg GL mg-1 Chl h-1 after optimization of the temporal illumination conditions and the glucose efflux rate, with the insertion of chemoprotective nanoceria inside the chloroplasts. This system achieves an average growth rate of 60 µm3 h-1 per chloroplast under ambient CO2 and illumination over 18 h, thickening with a shear modulus of 3 kPa. This material can demonstrate self-repair using the exported glucose from chloroplasts and chemical crosslinking through the fissures. These results point to a new class of materials capable of using atmospheric CO2 fixation as a regeneration source, finding utility as self-healing coatings, construction materials, and fabrics.

Rational Design Principles for the Transport and Subcellular Distribution of Nanomaterials into Plant Protoplasts.

The ability to control the subcellular localization of nanoparticles within living plants offers unique advantages for targeted biomolecule delivery and enables important applications in plant bioengineering. However, the mechanism of nanoparticle transport past plant biological membranes is poorly understood. Here, a mechanistic study of nanoparticle cellular uptake into plant protoplasts is presented. An experimentally validated mathematical model of lipid exchange envelope penetration mechanism for protoplasts, which predicts that the subcellular distribution of nanoparticles in plant cells is dictated by the particle size and the magnitude of the zeta potential, is advanced. The mechanism is completely generic, describing nanoparticles ranging from quantum dots, gold and silica nanoparticles, nanoceria, and single-walled carbon nanotubes (SWNTs). In addition, the use of imaging flow cytometry to investigate the influence of protoplasts' morphological characteristics on nanoparticle uptake efficiency is demonstrated. Using DNA-wrapped SWNTs as model nanoparticles, it is found that glycerolipids, the predominant lipids in chloroplast membranes, exhibit stronger lipid-nanoparticle interaction than phospholipids, the major constituent in protoplast membrane. This work can guide the rational design of nanoparticles for targeted delivery into specific compartments within plant cells without the use of chemical or mechanical aid, potentially enabling various plant engineering applications.

Persistent drought monitoring using a microfluidic-printed electro-mechanical sensor of stomata in planta.

Stomatal function can be used effectively to monitor plant hydraulics, photosensitivity, and gas exchange. Current approaches to measure single stomatal aperture, such as mold casting or fluorometric techniques, do not allow real time or persistent monitoring of the stomatal function over timescales relevant for long term plant physiological processes, including vegetative growth and abiotic stress. Herein, we utilize a nanoparticle-based conducting ink that preserves stomatal function to print a highly stable, electrical conductometric sensor actuated by the stomata pore itself, repeatedly and reversibly for over 1 week. This stomatal electro-mechanical pore size sensor (SEMPSS) allows for real-time tracking of the latency of single stomatal opening and closing times in planta, which we show vary from 7.0 ± 0.5 to 25.0 ± 0.5 min for the former and from 53.0 ± 0.5 to 45.0 ± 0.5 min for the latter in Spathiphyllum wallisii. These values are shown to correlate with the soil water potential and the onset of the wilting response, in quantitative agreement with a dynamic mathematical model of stomatal function. A single stoma of Spathiphyllum wallisii is shown to distinguish between incident light intensities (up to 12 mW cm-2) with temporal latency slow as 7.0 ± 0.5 min. Over a seven day period, the latency in opening and closing times are stable throughout the plant diurnal cycle and increase gradually with the onset of drought. The monitoring of stomatal function over long term timescales at single stoma level will improve our understanding of plant physiological responses to environmental factors.

A Nanobionic Light-Emitting Plant.

The engineering of living plants for visible light emission and sustainable illumination is compelling because plants possess independent energy generation and storage mechanisms and autonomous self-repair. Herein, we demonstrate a plant nanobionic approach that enables exceptional luminosity and lifetime utilizing four chemically interacting nanoparticles, including firefly luciferase conjugated silica (SNP-Luc), d-luciferin releasing poly(lactic-co-glycolic acid) (PLGA-LH2), coenzyme A functionalized chitosan (CS-CoA) and semiconductor nanocrystal phosphors for longer wavelength modulation. An in vitro kinetic model incorporating the release rates of the nanoparticles is developed to maximize the chemiluminescent lifetimes to exceed 21.5 h. In watercress (Nasturtium officinale) and other species, the nanoparticles circumvent limitations such as luciferin toxicity above 400 µM and colocalization of enzymatic reactions near high adenosine triphosphate (ATP) production. Pressurized bath infusion of nanoparticles (PBIN) is introduced to deliver a mixture of nanoparticles to the entire living plant, well described using a nanofluidic mathematical model. We rationally design nanoparticle size and charge to control localization within distinct tissues compartments with 10 nm nanoparticles localizing within the leaf mesophyll and stomata guard cells, and those larger than 100 nm segregated in the leaf mesophyll. The results are mature watercress plants that emit greater than 1.44 × 1012 photons/sec or 50% of 1 µW commercial luminescent diodes and modulate "off" and "on" states by chemical addition of dehydroluciferin and coenzyme A, respectively. We show that CdSe nanocrystals can shift the chemiluminescent emission to 760 nm enabling near-infrared (nIR) signaling. These results advance the viability of nanobionic plants as self-powered photonics, direct and indirect light sources.

Nanosensor Technology Applied to Living Plant Systems.

An understanding of plant biology is essential to solving many long-standing global challenges, including sustainable and secure food production and the generation of renewable fuel sources. Nanosensor platforms, sensors with a characteristic dimension that is nanometer in scale, have emerged as important tools for monitoring plant signaling pathways and metabolism that are nondestructive, minimally invasive, and capable of real-time analysis. This review outlines the recent advances in nanotechnology that enable these platforms, including the measurement of chemical fluxes even at the single-molecule level. Applications of nanosensors to plant biology are discussed in the context of nutrient management, disease assessment, food production, detection of DNA proteins, and the regulation of plant hormones. Current trends and future needs are discussed with respect to the emerging trends of precision agriculture, urban farming, and plant nanobionics.

Nitroaromatic detection and infrared communication from wild-type plants using plant nanobionics.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm-1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Proteolytic disassembly of peptide-mediated graphene oxide assemblies for turn-on fluorescence sensing of proteases.

Molecule-induced assembly of nanomaterials can alter their unique chemical and physical properties, which can be a promising approach for sensing. Herein, we demonstrate an optical 'turn-on' biosensor for the detection of matrix metalloproteinase-2 (MMP-2), fabricated by means of a peptide-induced assembly of fluorescent graphene oxide (GO). Functionalization of GO with a peptide substrate for MMP-2 bearing a thiol group leads to its self-assembly via disulfide bonding, accompanied by self-quenching of GO's strong fluorescence. This peptide-induced GO assembly is then disassembled by proteolytic cleavage in the presence of MMP-2, thereby restoring the level of self-quenched GO fluorescence. With this approach, we are able to detect MMP-2 and to investigate the kinetic parameters of MMP-2 activity. The GO-peptide assembly is successfully applied to the selective and sensitive detection of MMP-2 secreted by living cells, human hepatocytes HepG2, at a concentration of 2 ng mL(-1).

Lipid Exchange Envelope Penetration (LEEP) of Nanoparticles for Plant Engineering: A Universal Localization Mechanism.

Nanoparticles offer clear advantages for both passive and active penetration into biologically important membranes. However, the uptake and localization mechanism of nanoparticles within living plants, plant cells, and organelles has yet to be elucidated.1 Here, we examine the subcellular uptake and kinetic trapping of a wide range of nanoparticles for the first time, using the plant chloroplast as a model system, but validated in vivo in living plants. Confocal visible and near-infrared fluorescent microscopy and single particle tracking of gold-cysteine-AF405 (GNP-Cys-AF405), streptavidin-quantum dot (SA-QD), dextran and poly(acrylic acid) nanoceria, and various polymer-wrapped single-walled carbon nanotubes (SWCNTs), including lipid-PEG-SWCNT, chitosan-SWCNT and 30-base (dAdT) sequence of ssDNA (AT)15 wrapped SWCNTs (hereafter referred to as ss(AT)15-SWCNT), are used to demonstrate that particle size and the magnitude, but not the sign, of the zeta potential are key in determining whether a particle is spontaneously and kinetically trapped within the organelle, despite the negative zeta potential of the envelope. We develop a mathematical model of this lipid exchange envelope and penetration (LEEP) mechanism, which agrees well with observations of this size and zeta potential dependence. The theory predicts a critical particle size below which the mechanism fails at all zeta potentials, explaining why nanoparticles are critical for this process. LEEP constitutes a powerful particulate transport and localization mechanism for nanoparticles within the plant system.

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.