Ultrabithorax (Ubx) is a Hox gene transcription factor regulating the growth of wings and limbs in Drosophila melanogaster. However, the protein can also be produced recombinantly, and can self-assemble to form a film at the air-water interface. Materials drawn from this film, including fibres and thin coatings, are elastic, bio- and cyto-compatible. Moreover, Ubx can be functionalised with other biomolecules to form protein fusions. In this work, the physical properties of Ubx fibres and electrospun mats containing Ubx were investigated. An electrical conductivity comparable to semiconductors was discovered in enhanced green fluorescent protein- Ubx (EGFP-Ubx) fibres. The photoluminescence properties of pure Ubx, EGFP-Ubx, and electrospun poly(ethylene oxide)/EGFP-Ubx complexes were compared and the fluorescence emission peaks were found at 420nm for poly(ethylene oxide), 442nm for Ubx, and 512nm for EGFP. Moreover, the effect of material production method on the fluorescence lifetime was investigated and revealed differences between self-assembled microfibres and electrospun mats with fibre diameters below 1 μm. Finally, Ubx fibres were functionalised with DNA aptamers, and E.coli binding was increased using three different aptamers compared to pure fibres. The increase was 15% increase for the P12 aptamer, 92% for the STC12 aptamer, and 167% for the Antibac2 aptamer. Developments in large-scale material formation could support the functional Ubx materials in becoming a platform material for biosensing and tissue regeneration.

Ultrabithorax (Ubx) is a Drosophila melanogaster transcription factor protein the the Bondos group discovered has the ability to form ordered materials in vitro. Ubx monomers are produced in E.coli and, following purification, are suspended in a buffer solution and where they do not aggregate in the volume of the solution when refrigerated. When allowed to rest at room temperature, the monomer self assembles at the air/water interface through nucleation, fibril formation and, eventually, film integration. The the self assembled film can then be pulled into a fibre with diameters in the range of 2???50 ?m or lifted off as a film with microscale thickness. These materials are highly elastic and maintain physical properties through cycles of drying and re-hydrating. Novel functions can be directly incorporated into Ubx-based materials via gene fusion to produce chimeric polypeptides capable of both self-assembly and the desired chemical reactivity. Unlike most protein-based materials, the gentle conditions under which Ubx self-assembles enable incorporation of active heterologous proteins. This talk will review recent work on the continued development of this unique materials system including mechanical properties enabled by dityrosine bonding between monomers, dynamics of surface film assembly, and advances in Ubxbased materials production. A key advantage of protein

This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically reduces the energy input required. The acetone extraction produced 10 fractions with dairy manure biochar-derived carbonaceous nanomaterials (DMB-CNs). The fraction with the carbonaceous nanomaterials, DMB-CN-E1, with highest fluorescence was selected for in-depth characterisation and for initial testing across a range of applications. DMB-CN-E1 was characterised using atomic force microscope, electrophoresis, and spectrophotometric methods. DMB-CN-E1 exhibited a lateral dimension between 11 and 28 nm, a negative charge, and excitation/emission maxima at 337/410 nm, respectively. The bioimaging potential of DMB-CN-E1 evidenced different locations and different interactions with the cellular models evaluated. DMB-CN-E1 was quenched by several heavy metal ions showing a future application of these materials in heavy metal ion detection and/or removal. The demonstrated capabilities in bioimaging and environmental sensing create the opportunity for generating added-value nanomaterials (NanoRefinery) from dairy manure biochar gasification and, thus, increasing the economic viability of gasification plants.

Optical spectroscopic imaging of biological systems has important applications in medical diagnosis, biochemistry, and image-guided surgery. Vibrational spectroscopy, such as Raman scattering, provides high chemical selectivity but is limited by weak signals and a large fluorescence background. Fluorescence imaging is often used by introducing specific dyes in biological systems to label different system parts and to increase the image contrast. However, the extrinsic fluorescence of the staining molecules often masks the intrinsic vibrational signals of biomolecules, which could also be simultaneously detected using the same excitation laser source. Therefore, fluorescence staining is often accompanied by the loss of other important complimentary information. For example, the high laser power often used for the rapid, high-quality imaging could lead to photo-induced suppression or bleaching of the fluorescence and Raman signals resulting in sample photodamage. Therefore, simultaneous imaging and photodamage analysis need to be performed in a controlled bioimaging experiment. Here we perform simultaneous spectroscopic bioimaging and photostability analysis of rhodamine 6G (R6G) stained red blood cells (RBCs) using both fluorescence and resonance Raman imaging in a single 532 nm laser excitation experiment. We develop a corresponding data processing algorithm which allows separation of the two spectroscopic signals. We control the relative intensity of the R6G and RBC signals by varying the excitation laser power and simultaneously monitor the photostability of RBCs. We observe no significant photodamage of RBCs through the absence of changes in the relative Raman peak intensities. Conversely, the R6G molecules show bleaching with the suppression of both the fluorescence and resonance Raman signals. Our approach may be generalized to other types of stained cells with the appropriate selection of fluorescent dyes and excitation sources.

This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRefinery process transforms biochar into advanced nanomaterials using depolymerisation/chemical oxidation and purification of nanomaterials using solvent extraction. Dairy manure biochar-derived carbonaceous nanomaterials (DMB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were utilised as probes for the evaluation of their fluorescent properties and the detection of heavy metal ions. The BCN fluorescence quenching and fluorescence recovery was tested with lead (Pb2+), nickel (Ni2+), copper (Cu2+) and mercury (Hg2+). Principal component analysis (PCA) and discriminant analysis were used to differentiate among heavy metal ions in water samples. The BCN from different feedstocks had different characteristics and produced different interactions with heavy metal ions. DMB-CN had the highest quenching for Hg2+ and Ni2+ while SSB-CN and RSB-CN responded best to Cu2+ and Pb2+, respectively. The fluorescence quenching was modelled using linear and empirical functions. PCA and discriminant analysis used the quenching measurements to differentiate heavy metal ions in aqueous system. A key result was that the discriminant analysis had a 100% accuracy to detect Pb2+, 66% for Ni2+ and Cu2+, and 33% for Hg2+. This study has shown that biochar-derived carbonaceous nanomaterials could be used in heavy metal ions sensing applications. This is the first step in the development of a fast and accurate method for the detection of heavy metal ions in waters using environmentally friendly BCN.

This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy metal ion sensors in aqueous systems. The experimental phase included the transformation of MAB into microalgae biochar-derived carbon dots (MAB-Cdots), MAB-Cdot characterisation and the evaluation of the MAB-Cdots as transducers for the detection of four heavy metal ions (Pb-2 (broken vertical bar), Cu-2 (broken vertical bar), Cd-2 (broken vertical bar), and Ni-2 (broken vertical bar)). MAB-Cdot fluorescence was stable over a wide range of pH and resistant to photo-bleaching, making them suitable as fluorescence probes. The MAB-Cdot fluorescence was quenched by all of the metal ions and displayed different quenching levels. Depending upon the ions involved, MAB-Cdots were used to detect the presence of heavy metal ions from concentrations of 0.012 mu M up to 2mM by measuring the reduction in fluorescence intensity. Neutral and slightly alkaline pHs were optimal for Cu2+ Ni2+ and Pb2+ heavy metal quenching. To quantify the concentration of the heavy metal ions, linear and logarithmic functions were used to model the MAB-Cdot fluorescence quenching. The sensing mechanism was determined to be reversible and purely collisional with some fluorophores less accessible than the others. This work demonstrated the ability to produce Cdots from microalgae biochar, examined their application as a transducer for detecting heavy metal ions in aqueous systems and paves the way for novel sensing systems using MAB-Cdots. (C) 2018 Elsevier B.V. All rights reserved.

Three types of biochar (microalgae, rice straw and sorghum straw) from biomass thermal conversion production were tested for producing biochar-derived carbonaceous nanomaterials (BCN). BCN were obtained after using chemical depolymerisation and solvent extraction, NanoRefinery process. Microalgae biochar-derived carbonaceous nanomaterials (MAB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were characterised using spectroscopic and microscopic techniques. This characterisation evidenced significant differences among the three BCN with MAB-CN exhibiting greater structural differences compared to RSB-CN and SSB-CN. Biocompatibility, cellular uptake, and cellular localisation were evaluated using three yeast species, Saccharomyces cerevisiae, Candida albicans, and Yarrowia lipolytica. While all BCN were biocompatible, the degree of biocompatibility for each species was dependent on pH, BCN concentration and BCN type. Additionally, BCN were evaluated as transducers for the detection of 12 heavy metal ions. MAB-CN, RSB-CN, and SSB-CN had different responses to the 12 heavy metal ions. The SSB-CN/Cu (II) and the MAB-CN/Zn (II) combinations evidenced selectivity over the other metal ions with these combinations having limits of detection of 0.0125 mu M and 9 mu M, respectively. The results from this research pave the way for BCN novel applications for bioimaging and heavy metal ions sensing probes.

Alterations in ocular blood flow have been implicated in mechanisms that lead to vision loss in patients with various ocular disorders such as diabetic retinopathy, glaucoma, and age-related macular degeneration. Assessment of retinal and choroidal blood flow is also a window to evaluate systemic diseases that affect microvasculature. Quantification and qualification of the blood flow in the retina and choroid help us understand pathophysiology, stratify disease risk, and monitor disease progression in these disorders. Multiple methods are used by researchers for assessment of blood flow, but a gold standard is lacking. We review commonly used methods, both invasive and noninvasive, for evaluation of blood flow, including intravital microscopy, laser Doppler velocimetry, laser Doppler flowmetry, laser interferometry, confocal scanning laser Doppler flowmetry, laser speckle flowgraphy, Doppler optical coherence tomography, blue-field entoptic simulation, retinal vessel caliber assessment, optical coherence tomography angiography, retinal function imaging, color Doppler imaging, and scanning laser ophthalmoscope angiogram. As technology evolves, better evaluation of blood flow in various ocular and systemic diseases will likely bring new perspectives into clinical practice and translate to better diagnosis and treatment.

Understanding the delivered cellular dose of nanoparticles is imperative in nanomedicine and nanosafety, yet is known to be extremely complex because of multiple interactions between nanoparticles, their environment, and the cells. Here, we use 3-D reconstruction of agglomerates preserved by cryogenic snapshot sampling and imaged by electron microscopy to quantify the "bioavailable dose" that is presented at the cell surface and formed by the process of individual nanoparticle sequestration into agglomerates in the exposure media. Critically, using 20 and 40 nm carboxylated polystyrene-latex and 16 and 85 nm silicon dioxide nanoparticles, we show that abrupt, dose-dependent "tipping points" in agglomeration state can arise, subsequently affecting cellular delivery and increasing toxicity. These changes are triggered by shifts in the ratio of the total nanoparticle surface area to biomolecule abundance, with the switch to a highly agglomerated state effectively changing the test article midassay, challenging the dose-response paradigm for nanosafety experiments. By characterizing nanoparticle numbers per agglomerate, we show these tipping points can lead to the formation of extreme agglomeration states whereby 90% of an administered dose is contained and delivered to the cells by just the top 2% of the largest agglomerates. We thus demonstrate precise definition, description, and comparison of the nanoparticle dose formed in different experimental environments and show that this description is critical to understanding cellular delivery and toxicity. We further empirically "stress-test" the commonly used dynamic light scattering approach, establishing its limitations to present an analysis strategy that significantly improves the usefulness of this popular nanoparticle characterization technique.

Erythrocyte abundance, mobility, and carrying capacity make them attractive as a platform for blood analyte sensing as well as for drug delivery. Sensor-loaded erythrocytes, dubbed erythrosensors, could be reinfused into the bloodstream, excited noninvasively through the skin, and used to provide measurement of analyte levels in the bloodstream. Several techniques to load erythrocytes, thus creating carrier erythrocytes, exist. However, their cellular characteristics remain largely unstudied. Changes in cellular characteristics lead to removal from the bloodstream. We hypothesize that erythrosensors need to maintain native erythrocytes’ (NEs) characteristics to serve as a long-term sensing platform. Here, we investigate two loading techniques and the properties of the resulting erythrosensors. For loading, hypotonic dilution requires a hypotonic solution while electroporation relies on electrical pulses to perforate the erythrocyte membrane. We analyze the resulting erythrosensor signal, size, morphology, and hemoglobin content. Although the resulting erythrosensors exhibit morphological changes, their size was comparable with NEs. The hypotonic dilution technique was found to load erythrosensors much more efficiently than electroporation, and the sensors were loaded throughout the volume of the erythrosensors. Finally, both techniques resulted in significant loss of hemoglobin. This study points to the need for continued development of loading techniques that better preserve NE characteristics.

Cross-system comparisons of drug delivery vectors are essential to ensure optimal design. An in-vitro experimental protocol is presented that separates the role of the delivery vector from that of its cargo in determining the cell response, thus allowing quantitative comparison of different systems. The technique is validated through benchmarking of the dose–response of human fibroblast cells exposed to the cationic molecule, polyethylene imine (PEI); delivered as a free molecule and as a cargo on the surface of CdSe nanoparticles and Silica microparticles. The exposure metrics are converted to a delivered dose with the transport properties of the different scale systems characterized by a delivery time, τ. The benchmarking highlights an agglomeration of the free PEI molecules into micron sized clusters and identifies the metric determining cell death as the total number of PEI molecules presented to cells, determined by the delivery vector dose and the surface density of the cargo.

Erythrocytes, or red blood cells, transport oxygen to and carbon dioxide from the body's tissues and organs. Red blood cell mechanical properties are altered in a number of diseases such as sickle cell anaemia and malaria. Additionally, mechanically modified red blood cell ghosts are being considered as a long-term, biocompatible carrier for drug delivery and for blood analyte sensing. Brillouin spectroscopy enables viscoelastic characterization of samples at the microscale. In this report, Brillouin spectroscopy is applied to characterize the mechanical properties of red blood cells and red blood cell ghosts.

Keys to successful treatment of disease include early diagnosis and timely treatment. It is hypothesized that early clotting events may contribute to a pro-thrombotic state that exacerbates atherothrombotic vascular disease. Brillouin spectroscopy involves inelastic coupling of light with phonons and enables viscoelastic characterization of samples at the microscale. In this work, we apply Brillouin spectroscopy to a model fibrinogen-thrombin clotting system with the goal of measuring clotting dynamics at the microscale and providing characterization that is not possible with standard rheometric techniques. Here, the clotting dynamics of the model clotting system are measured at various fibrinogen and thrombin concentrations.

The layer-by-layer modification of ≈5nm mercaptocarboxylic acid stabilized gold nanoparticles was studied in an effort to illustrate effective means to overcome practical issues in handling and performing surface modification of such extremely small materials. To accomplish this, each layer deposition cycle was separated into a multi-step process wherein solution pH was controlled in two distinct phases of polyelectrolyte adsorption and centrifugation. Additionally, a solvent precipitation step was introduced to make processing more amenable by concentrating the sample and exchanging solution pH before ultracentrifugation. The pH-dependent assembly on gold nanoparticles was assessed after each layer deposition cycle by monitoring the plasmon peak absorbance location, surface charge, and the percentage of nanoparticles recovered. The selection of solution pH during the adsorption phase was found to be a critical parameter to enhance particle recovery and maximize surface charge when coating with weak polyelectrolytes. One bilayer was deposited with a high yield and the modified particles exhibited enhanced colloidal stability across a broad pH range and increased ionic strength. These findings support the adoption of this multi-step processing approach as an effective and generalizable approach to improve stability of high surface curvature particles.

Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background-free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular- and subcellular-level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA-based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.

The extensive use of nanomaterials in new products for the building envelope is expanding rapidly and continues to influence materials science and environmental health. However, there is still uncertainly around the safety of these novel materials to humans and the environment; therefore, more life cycle data is needed. One analytical technique to gather information critical to the development of safe nanomaterials is to characterize the physicochemical properties of the nano-enabled product along various stages of the product life cycle. This research investigates two different products fortified with nanoscale titania relevant to the building and construction industry: (1) a white paint and (2) a lacquer formulation. Three brands of commercially available titanium dioxide (TiO2) nanoparticles were incorporated into labformulated paint and lacquer mixtures. These fortified products were subjected to simulated degradation processes (a.k.a. “wear-and-tear” scenarios) to mimic the real world. Unincorporated TiO2, as well as TiO2 incorporated “wornand- torn” powders, were processed and analyzed for physical, chemical, and toxicological characteristics. Electron microscopy confirmed the presence of nanoscale TiO2 particles in the painted and lacquered matrices for all three nanoparticle-types. Cell viability and reactive oxygen species (ROS) generation was measured in immortalized human lung epithelial cells (A549). Results from bioassays showed that ROS generation between unincorporated TiO2 particles and TiO2 incorporated paint powders showed little difference, but, the TiO2 incorporated lacquered powders demonstrated significantly lower ROS generation and cell death. Thus, the polymer-based lacquer mitigated tissue damage. Our results show that using a life cycle approach may help provide data – and by extension, an understanding - of nanomaterial properties in the context of developed consumer products.

The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self‐assembles in vitro into biocompatible materials that are remarkably extensible and strong. Here, it is demonstrated that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials autofluoresce blue, a characteristic of dityrosine, and bind dityrosine‐specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx are identified by site‐directed mutagenesis followed by measurements of fiber fluorescence intensity. One bond is located between the N‐terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. This is the first identification of specific residues that participate in dityrosine bonds in protein‐based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine‐containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self‐assembling proteins to strengthen the corresponding materials.