IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α.

The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-γ to activate bactericidal mechanisms in infected macrophages. Although IFN-γ-independent CD4 T cell based control of M. tuberculosis infection has been demonstrated in vivo it is unclear whether CD4 T cells are capable of directly activating macrophages to control infection in the absence of IFN-γ. We developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) to investigate mechanisms of CD4 dependent control of infection. We found that even in the absence of IFN-γ signaling, CD4 T cells drive macrophage activation, M1 polarization, and control of infection. This IFN-γ-independent control of infection requires activation of the transcription factor HIF-1α and a shift to aerobic glycolysis in infected macrophages. While HIF-1α activation following IFN-γ stimulation requires nitric oxide, HIF-1α-mediated control in the absence of IFN-γ is nitric oxide-independent, indicating that distinct pathways can activate HIF-1α during infection. We show that CD4 T cell-derived GM-CSF is required for IFN-γ-independent control in BMDMs, but that recombinant GM-CSF is insufficient to control infection in BMDMs or alveolar macrophages and does not rescue the absence of control by GM-CSF-deficient T cells. In contrast, recombinant GM-CSF controls infection in peritoneal macrophages, induces lipid droplet biogenesis, and also requires HIF-1α for control. These results advance our understanding of CD4 T cell-mediated immunity to M. tuberculosis, reveal important differences in immune activation of distinct macrophage types, and outline a novel mechanism for the activation of HIF-1α. We establish a previously unknown functional link between GM-CSF and HIF-1α and provide evidence that CD4 T cell-derived GM-CSF is a potent bactericidal effector.

New Method to Probe the Surface Properties of Polymer Thin Films by Two-Dimensional (2D) Inverse Gas Chromatography (iGC).

Polymer-based functional surface coatings are extensively used in advanced technologies, including optics, energy, and environmental applications. Surface thermodynamic properties profoundly impact the molecular interactions that control interfacial behaviors, such as adhesion and wettability, which in turn dictate coating processes and performance. Conventionally, contact angle measurements are used to assess the surface energy of polymer films and coatings, where the wettability of a surface is assessed using probe fluids (liquid drops). However, contact angle measurement oftentimes can be nontrivial due to the roughness or chemical heterogeneity of the solid surface, as well as the potential for the liquid drop to swell or even dissolve the material being measured. Alternatively, inverse gas chromatography (iGC) is a versatile technique to measure surface thermodynamics and Lewis acid-base properties while also providing environmental control such as temperature and humidity. Despite these benefits, the application of iGC has been limited to powders or fibers, while the direct measurement of supported thin films or coatings is still a nascent area of research. This creates a challenge when using iGC as a comprehensive platform for measuring the physicochemical properties of solid surfaces. Here, we demonstrate how to effectively use iGC to characterize the surface energy of supported polymer thin films by using a two-dimensional (2D) film holder and modifying operational controls, such as the concentration range of the injected gas probe molecules. This enables the precise control of surface coverage required for analyzing samples having minimal surface area, such as thin films. Poly(methyl methacrylate) (PMMA) was employed as a benchmark to determine suitable iGC parameters and to validate our approach on polymer thin films. The seminal work presented here expands the capability of state-of-the-art iGC to embrace supported thin films (2D iGC) that could either be smooth or display texture/roughness (patterned films) as well as coatings with heterogeneous chemical/structural composition.

Polyvinyl alcohol-montmorillonite composites for water purification: Analysis of clay mineral cation exchange and composite particle synthesis.

Municipal and residential water purification rely heavily on activated carbon (AC), but regeneration of AC is costly and cannot be performed at the point-of-use. Clay minerals (CMs) comprise a class of naturally abundant materials with known capacities for analyte adsorbance. However, the gel-forming properties of CMs in aqueous suspension pose problems for these materials being used in water-purification. In this study, we have taken three main steps to optimize the use of CMs in these applications. First, we produced several variants of montmorillonite CMs to evaluate the effect of interstitial cation hydrophobicity on the ability of the CM to uptake chargecarrying organic pollutants. These variants include CMs with the following cations: sodium, hexyl(triphenyl) phosphonium, hexyadecyl(triphenyl)phosphonium, and hexyl(tributyl)phosphonium. Second, we synthesized polymer-clay mineral composite films composed of polyvinyl alcohol (PVA), crosslinked in the presence of a CM variant. These films were evaluated for their ability to uptake malachite green (MG). Finally, we developed a one-pot synthetic method for the generation of polymer-clay particles for use in a continuous column process. We synthesized polymer-clay mineral particles using the highest performing CM (based on the film experiments) and evaluated the equilibrium capacity and kinetics of MG uptake from solution.

Current characterization methods for cellulose nanomaterials.

A new family of materials comprised of cellulose, cellulose nanomaterials (CNMs), having properties and functionalities distinct from molecular cellulose and wood pulp, is being developed for applications that were once thought impossible for cellulosic materials. Commercialization, paralleled by research in this field, is fueled by the unique combination of characteristics, such as high on-axis stiffness, sustainability, scalability, and mechanical reinforcement of a wide variety of materials, leading to their utility across a broad spectrum of high-performance material applications. However, with this exponential growth in interest/activity, the development of measurement protocols necessary for consistent, reliable and accurate materials characterization has been outpaced. These protocols, developed in the broader research community, are critical for the advancement in understanding, process optimization, and utilization of CNMs in materials development. This review establishes detailed best practices, methods and techniques for characterizing CNM particle morphology, surface chemistry, surface charge, purity, crystallinity, rheological properties, mechanical properties, and toxicity for two distinct forms of CNMs: cellulose nanocrystals and cellulose nanofibrils.

Effect of cellulose nanocrystals on crystallization kinetics of polycaprolactone as probed by Rheo-Raman.

The development of biocompatible polymer nano-composites that enhance mechanical properties while maintaining thermoplastic processability is a longstanding goal in sustainable materials. When the matrix is semi-crystalline, the nanoparticles may induce significant changes to crystallization kinetics and morphology due to their ability to act as nucleating agents. To fully model this behavior in a process line, an understanding of the relationship between crystallinity and modulus is required. Here, we introduce a scalable model system consisting of surface-compatibilized cellulose nanocrystals (CNC) dispersed into poly(ε-caprolactone) (PCL) and study the effects of nanoparticle concentration on isothermal crystallization kinetics. The dispersion is accomplished by exchange of the Na+ of sulfated cellulose nanocrystals by tetra-butyl ammonium cations (Bu4N+) followed by melt mixing via twin-screw extrusion. Crystallization kinetics are measured through the recently developed rheo-Raman instrument which extracts the relationship between the growth of the transient mechanical modulus and that of crystallinity. With extrusion and increasing CNC content, we find the expected enhancement of crystallization rate, but we moreover find a significant change in the relative kinetics of increase in modulus versus crystallinity. We analyze this via generalized effective medium theory which allows computation of a critical percolation threshold ξ c and discuss the results in terms of a change in nucleation density and a change in the anisotropy of crystallization.

Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis.

The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis-infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

Visualizing and quantifying cell phenotype using soft X-ray tomography.

Soft X-ray tomography (SXT) is an imaging technique capable of characterizing and quantifying the structural phenotype of cells. In particular, SXT is used to visualize the internal architecture of fully hydrated, intact eukaryotic and prokaryotic cells at high spatial resolution (50 nm or better). Image contrast in SXT is derived from the biochemical composition of the cell, and obtained without the need to use potentially damaging contrast-enhancing agents, such as heavy metals. The cells are simply cryopreserved prior to imaging, and are therefore imaged in a near-native state. As a complement to structural imaging by SXT, the same specimen can now be imaged by correlated cryo-light microscopy. By combining data from these two modalities specific molecules can be localized directly within the framework of a high-resolution, three-dimensional reconstruction of the cell. This combination of data types allows sophisticated analyses to be carried out on the impact of environmental and/or genetic factors on cell phenotypes.

Concurrent zero-dimensional and one-dimensional biomineralization of gold from a solution of Au3+ and bovine serum albumin.

A technique was developed for preparing a novel material that consists of gold nanoparticles trapped within a fiber of unfolded proteins. These fibers are made in an aqueous solution that contains HAuCl4 and the protein, bovine serum albumin (BSA). By changing the ratio of gold to BSA in solution, two different types of outcomes are observed. At lower gold to BSA ratios (30-120), a purple solution results after heating the mixture at 80 °C for 4 h. At higher gold to BSA ratios (130-170), a clear solution containing purple fibers results after heating the mixture at 80 °C for 4 h. UV-Vis spectroscopy and light scattering techniques show growth in nanocolloid size as gold to BSA ratio rises above 100. Data indicate that, for the higher gold to BSA ratios, the gold is sequestered within the solid material. The material mass, visible by eye, appears to be an aggregation of smaller individual fibers. Scanning electron microscopy and transmission electron microscopy indicate that these fibers are primarily one-dimensional aggregates, which can display some branching, and can be as narrow as 400 nm in size. The likely mechanism for the synthesis of the novel material is discussed.

Probing the mycobacterial trehalome with bioorthogonal chemistry.

Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.

SARS-CoV-2 down-regulates ACE2 through lysosomal degradation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes its Spike (S) glycoprotein to bind to the angiotensin-converting enzyme 2 (ACE2) receptor for cellular entry. ACE2 is a critical negative regulator of the renin-angiotensin system and plays a protective role in preventing tissue injury. Expression of ACE2 has been shown to decrease upon infection by SARS-CoV. However, whether SARS-CoV-2 down-regulates ACE2 and the underlying mechanism and biological impact of this down-regulation have not been well defined. Here we show that the SARS-CoV-2 infection down-regulates ACE2 in vivo in an animal model, and in cultured cells in vitro, by inducing clathrin- and AP2-dependent endocytosis, leading to its degradation in the lysosome. SARS-CoV-2 S-treated cells and ACE2 knockdown cells exhibit similar alterations in downstream gene expression, with a pattern indicative of activated cytokine signaling that is associated with respiratory distress and inflammatory diseases often observed in COVID-19 patients. Finally, we have identified a soluble ACE2 fragment with a stronger binding to SARS-CoV-2 S that can efficiently block ACE2 down-regulation and viral infection. Thus, our study suggests that ACE2 down-regulation represents an important mechanism underlying SARS-CoV-2-associated pathology, and blocking this process could be a promising therapeutic strategy.

SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-ß signaling.

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of vascular leak are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to induce barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-ß signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-ß signaling axis are required for S-mediated barrier dysfunction. Notably, we show that SARS-CoV-2 infection caused leak in vivo, which was reduced by inhibiting integrins. Our findings offer mechanistic insight into SARS-CoV-2-triggered vascular leak, providing a starting point for development of therapies targeting COVID-19.

Genome-wide bidirectional CRISPR screens identify mucins as host factors modulating SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a range of symptoms in infected individuals, from mild respiratory illness to acute respiratory distress syndrome. A systematic understanding of host factors influencing viral infection is critical to elucidate SARS-CoV-2-host interactions and the progression of Coronavirus disease 2019 (COVID-19). Here, we conducted genome-wide CRISPR knockout and activation screens in human lung epithelial cells with endogenous expression of the SARS-CoV-2 entry factors ACE2 and TMPRSS2. We uncovered proviral and antiviral factors across highly interconnected host pathways, including clathrin transport, inflammatory signaling, cell-cycle regulation, and transcriptional and epigenetic regulation. We further identified mucins, a family of high molecular weight glycoproteins, as a prominent viral restriction network that inhibits SARS-CoV-2 infection in vitro and in murine models. These mucins also inhibit infection of diverse respiratory viruses. This functional landscape of SARS-CoV-2 host factors provides a physiologically relevant starting point for new host-directed therapeutics and highlights airway mucins as a host defense mechanism.

Influence of cellulose nanocrystals (CNC) on permeation through intestinal monolayer and mucus model in vitro.

Cellulose nanocrystals (CNC) as a novel ingredient in foods and pharmaceuticals still lacks the safety and functionality information. We aimed to assess the absorption of CNC in small intestine and the effect on cell viability. In the second part, the impact of CNC on substance permeation through mucus layer, including the potential functionality in improving high blood cholesterol, was tested. No noticeable amount of CNC was found to penetrate through differentiated Caco-2 monolayer and in vitro mucus layer, and CNC had low toxicity on Caco-2 cell viability up to 10 mg/mL. CNC at 2 % (w/w) may affect the permeability of the mucus layer and larger molecules are more easily influenced. CNC may also alleviate hypercholesteremia by increasing viscosity of digesta, adsorbing cholesterol, and decreasing bile acids permeation. The results suggest CNC may not penetrate the small intestinal lining and may be used as a functional supplement.

Micromechanical Compatibility between Cells and Scaffolds Directs the Phenotypic Transition of Stem Cells.

This study experimentally substantiates that the micromechanical compatibility between cell and substrate is essential for cells to achieve energetically favorable mechanotransduction that directs phenotypic transitions. The argument for this compatibility is based on a thermodynamic model that suggests that the response of cells to their substrate mechanical environment is a consequence of the interchange between forms of energy governing the cell-substrate interaction. Experimental validation for the model has been carried out by investigating the osteogenic differentiation of dental follicle stem cells (DFSCs) seeded on electrospun fibrous scaffolds. Electrospinning of blends containing polycaprolactone (PCL) and silk fibroin (SF) with varying composition of cellulose nanocrystals (CNCs) resulted in three-dimensional (3D) fibrous scaffolds with bimodal distribution of fiber diameter, which provides both macroscopically stiff and microscopically compliant scaffolds for cells without affecting the surface chemical functionality of scaffolds. Atomic force microscopy (AFM) with a colloidal probe and single-cell force spectroscopy were used to characterize cell stiffness and scaffold stiffness on the cellular level, as well as cell-scaffold adhesive interaction (chemical functionality). This study has successfully varied scaffold mechanical properties without affecting their surface chemistry. In vitro tests indicate that the micromechanical compatibility between cells and scaffolds has been significantly correlated with mechanosensitive gene expression markers and osteogenic differentiation markers of DFSCs. The agreement between experimental observations and the thermodynamic model affirms that the cellular response to the mechanical environment, though biological in nature, follows the laws of the energy interchange to achieve its self-regulating behavior. More importantly, this study provides systematic evidence, through extensive and rigorous experimental studies, for the first time that rationalizes that micromechanical compatibility is indeed important to the efficacy of regenerative medicine.

Fluorescently Labeled Cellulose Nanofibers for Environmental Health and Safety Studies.

An optimal methodology for locating and tracking cellulose nanofibers (CNFs) in vitro and in vivo is crucial to evaluate the environmental health and safety properties of these nanomaterials. Here, we report the use of a new boron-dipyrromethene (BODIPY) reactive fluorescent probe, meso-DichlorotriazineEthyl BODIPY (mDTEB), tailor-made for labeling CNFs used in simulated or in vivo ingestion exposure studies. Time-correlated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (FLIM) was used to confirm covalent attachment and purity of mDTEB-labeled CNFs. The photoluminescence properties of mDTEB-labeled CNFs, characterized using fluorescence spectroscopy, include excellent stability over a wide pH range (pH2 to pH10) and high quantum yield, which provides detection at low (µM) concentrations. FLIM analysis also showed that lignin-like impurities present on the CNF reduce the fluorescence of the mDTEB-labeled CNF, via quenching. Therefore, the chemical composition and the methods of CNF production affect subsequent studies. An in vitro triculture, small intestinal, epithelial model was used to assess the toxicity of ingested mDTEB-labeled CNFs. Zebrafish (Danio rerio) were used to assess in vivo environmental toxicity studies. No cytotoxicity was observed for CNFs, or mDTEB-labeled CNFs, either in the triculture cells or in the zebrafish embryos.

Screening a Library of FDA-Approved and Bioactive Compounds for Antiviral Activity against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has emerged as a major global health threat. The COVID-19 pandemic has resulted in over 168 million cases and 3.4 million deaths to date, while the number of cases continues to rise. With limited therapeutic options, the identification of safe and effective therapeutics is urgently needed. The repurposing of known clinical compounds holds the potential for rapid identification of drugs effective against SARS-CoV-2. Here, we utilized a library of FDA-approved and well-studied preclinical and clinical compounds to screen for antivirals against SARS-CoV-2 in human pulmonary epithelial cells. We identified 13 compounds that exhibit potent antiviral activity across multiple orthogonal assays. Hits include known antivirals, compounds with anti-inflammatory activity, and compounds targeting host pathways such as kinases and proteases critical for SARS-CoV-2 replication. We identified seven compounds not previously reported to have activity against SARS-CoV-2, including B02, a human RAD51 inhibitor. We further demonstrated that B02 exhibits synergy with remdesivir, the only antiviral approved by the FDA to treat COVID-19, highlighting the potential for combination therapy. Taken together, our comparative compound screening strategy highlights the potential of drug repurposing screens to identify novel starting points for development of effective antiviral mono- or combination therapies to treat COVID-19.

The Effect of Cellulose Nanocrystal Coatings on the Glass Fiber-Epoxy Interphase.

This study focuses on understanding the effect of cellulose nanocrystals (CNCs) on glass fiber/epoxy interfacial interactions. The glass fibers (GF) were coated with solutions containing cellulose nanomaterial. The parameters that were investigated were the CNC surface chemistry, concentration, and dispersing medium, i.e., aqueous solution only versus emulsions. To determine the effect of the CNC coatings on the interfacial adhesion, specimens of a single GF in an epoxy matrix were prepared for GF coating by varying the coating formulations. The interfacial shear stress (IFSS) was determined by the single fiber fragmentation test (SFFT). Following the SFFT, the samples were investigated by cross-polarized microscopy in order to understand the fracture modes which are related to the nature of the interphase. According to the SFFT data and photoelastic fracture patterns, both the emulsion and aqueous coatings containing cellulose nanocrystals functionalized with methyl(triphenyl) phosphonium (CNCPh) improve the IFSS in comparison to coated GFs without CNCs.

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower ΔEP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

Dielectric Characterization of Confined Water in Chiral Cellulose Nanocrystal Films.

A known deterrent to the large-scale development and use of cellulose nanocrystals (CNCs) in composite materials is their affinity for moisture, which has a profound effect on dispersion, wetting, interfacial adhesion, matrix crystallization, water uptake, and hydrothermal stability. To quantify and control the hydration and confinement of absorbed water in CNCs, we studied sulfated-CNCs neutralized with sodium cations and CNCs functionalized with less hydrophilic methyl(triphenyl)phosphonium cations. Films were cast from water suspensions at 20 °C under controlled humidity and drying rate, yielding CNC materials with distinguishably different dielectric properties and cholesteric structures. By controlling the evaporation rate, we obtained self-assembled chiral CNC films with extended uniformity, having helical modulation length (nominal pitch) tunable from 1300 to 600 nm. SEM imaging and UV-vis-NIR total reflectance spectra revealed tighter and more uniform CNC packing in films cast at slow evaporation rates or having lower surface energy when modified with phosphonium. The dielectric constant was measured by a noncontact microwave cavity perturbation method and fitted to a classical mixing model employing randomly oriented ellipsoidal water inclusions. The dielectric constant of absorbed water was found to be significantly smaller than that for free liquid indicating a limited mobility due to binding with the CNC "matrix". In the case of hydrophilic Na-modified CNCs, a decreasing pitch led to greater anisotropy in the shape of moisture inclusions (ellipsoidal to platelet-like) and greater confinement. In contrast, the structure of hydrophobic phosphonium-modified CNC films was found to have reduced pitch, yet the shape of confined water remained predominantly spherical. These results provide a useful perspective on the current state of understanding of CNC-water interactions as well as on CNC self-assembly mechanisms. More broadly, we believe that our results are beneficial for the realization of CNC-based functional materials and composites.

Protein-templated gold nanoparticle synthesis: protein organization, controlled gold sequestration, and unexpected reaction products.

Emerging applications that exploit the properties of nanoparticles for biotechnology require that the nanoparticles be biocompatible or support biological recognition. These types of particles can be produced through syntheses that involve biologically relevant molecules (proteins or natural extracts, for example). Many of the protocols that rely on these molecules are performed without a clear understanding of the mechanism by which the materials are produced. We have investigated a previously described reaction in which gold nanoparticles are produced from the reaction of chloroauric acid and proteins in solution. We find that modifications to the starting conditions can alter the product from the expected solution-suspended colloids to a product where colloids are formed within a solid, fibrous protein structure. We have interrogated this synthesis, exploiting the change in products to better understand this reaction. We have evaluated the kinetics and products for 7 different proteins over a range of concentrations and temperatures. The key factor that controls the synthetic outcome (colloid or fiber) is the concentration of the protein relative to the gold concentration. We find that the observed fibrous structures are more likely to form at low protein concentrations and when hydrophilic proteins are used. An analysis of the reaction kinetics shows that AuNP formation occurs faster at lower protein (fiber-forming) concentrations than at higher protein (colloid-forming) concentrations. These results contradict traditional expectations for reaction kinetics and protein-fiber formation and are instructive of the manner in which proteins template gold nanoparticle production.