The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.
24 Related JoVE Articles!
PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS
Institutions: Barrow Neurological Institute.
Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g.
poly(vinyl alcohol), PVA) or solvents (e.g.
Chemistry, Issue 82, Nanoparticles, Microparticles, PLGA, TPGS, drug delivery, scanning electron microscopy, emulsion, polymers
Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2
, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3
. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5
. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6
. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions
Institutions: Pacific Northwest National Laboratory.
Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ
characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3
(bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ
time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ
Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ
IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.
Chemistry, Issue 88, soft landing, mass selected ions, electrospray, secondary ion mass spectrometry, infrared spectroscopy, organometallic, catalysis
Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures
Institutions: University of Houston.
The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3
, drug delivery4
, improved hydrocarbon recovery5-7
, and flowable electrodes for energy storage8
. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9
. Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10
. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37
. In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.
Chemistry, Issue 87, confocal microscopy, particle tracking, colloids, suspensions, confinement, gelation, microfluidics, image correlation, dynamics, suspension flow
Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds
Institutions: Wayne State University.
This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.
Bioengineering, Issue 90, Electrospinning, Hyaluronic Acid, PLGA, Microspheres, Controlled Release, Neural Tissue Engineering, Directed Cell Migration
Preparation and Use of Photocatalytically Active Segmented Ag|ZnO and Coaxial TiO2-Ag Nanowires Made by Templated Electrodeposition
Institutions: University of Twente.
Photocatalytically active nanostructures require a large specific surface area with the presence of many catalytically active sites for the oxidation and reduction half reactions, and fast electron (hole) diffusion and charge separation. Nanowires present suitable architectures to meet these requirements. Axially segmented Ag|ZnO and radially segmented (coaxial) TiO2
-Ag nanowires with a diameter of 200 nm and a length of 6-20 µm were made by templated electrodeposition within the pores of polycarbonate track-etched (PCTE) or anodized aluminum oxide (AAO) membranes, respectively. In the photocatalytic experiments, the ZnO and TiO2
phases acted as photoanodes, and Ag as cathode. No external circuit is needed to connect both electrodes, which is a key advantage over conventional photo-electrochemical cells. For making segmented Ag|ZnO nanowires, the Ag salt electrolyte was replaced after formation of the Ag segment to form a ZnO segment attached to the Ag segment. For making coaxial TiO2
-Ag nanowires, a TiO2
gel was first formed by the electrochemically induced sol-gel method. Drying and thermal annealing of the as-formed TiO2
gel resulted in the formation of crystalline TiO2
nanotubes. A subsequent Ag electrodeposition step inside the TiO2
nanotubes resulted in formation of coaxial TiO2
-Ag nanowires. Due to the combination of an n
-type semiconductor (ZnO or TiO2
) and a metal (Ag) within the same nanowire, a Schottky barrier was created at the interface between the phases. To demonstrate the photocatalytic activity of these nanowires, the Ag|ZnO nanowires were used in a photocatalytic experiment in which H2
gas was detected upon UV illumination of the nanowires dispersed in a methanol/water mixture. After 17 min of illumination, approximately 0.2 vol% H2
gas was detected from a suspension of ~0.1 g of Ag|ZnO nanowires in a 50 ml 80 vol% aqueous methanol solution.
Physics, Issue 87, Multicomponent nanowires, electrochemistry, sol-gel processes, photocatalysis, photochemistry, H2 evolution
Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy
Institutions: Freie Universität Berlin.
Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102
repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4
, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 µg, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.
Biophysics, Issue 88, bacteriorhodopsin, channelrhodopsin, attenuated total reflection, proton transfer, protein dynamics, infrared spectroscopy, time-resolved spectroscopy, step-scan, membrane proteins, singular value decomposition
Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins
Institutions: George Mason University, Ceres Nanosciences.
Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.
Bioengineering, Issue 90, biomarker, hydrogel, low abundance, mass spectrometry, nanoparticle, plasma, protein, urine
Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique
Institutions: Appalacian State University, Appalachian State University, Carolinas Medical Center NorthEast.
The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergström needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergström needle consists of an outer cannula with a small opening (‘window’) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90° and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.
Medicine, Issue 91, percutaneous muscle biopsy, needle biopsy, suction-modified, metabolism, enzyme activity, mRNA, gene function, fiber type, histology, metabolomics, skeletal muscle function, humans
Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts
Institutions: Aix-Marseille Université, Aix-Marseille Université.
Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.
Neuroscience, Issue 91, metabolomics, brain tissue, rodents, neurochemistry, tissue extracts, NMR spectroscopy, quantitative metabolite analysis, cerebral metabolism, metabolic profile
Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish
Institutions: University of Ottawa, University of Ottawa, University of Ottawa.
Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals' access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal's body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal's body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.
Neuroscience, Issue 85, animal tracking, weakly electric fish, electric organ discharge, underwater infrared imaging, automated image tracking, sensory isolation chamber, exploratory behavior
Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape
Institutions: US Naval Research Laboratory, North Carolina State University and University of North Carolina at Chapel Hill.
A “sheath” fluid passing through a microfluidic channel at low Reynolds number can be directed around another “core” stream and used to dictate the shape as well as the diameter of a core stream. Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid. By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed. Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid. Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers. Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light. Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.
Bioengineering, Issue 83, hydrodynamic focusing, polymer fiber, biohybrid, microfabrication, sheath flow, click chemistry
Designing Silk-silk Protein Alloy Materials for Biomedical Applications
Institutions: Rowan University, Rowan University, Cooper Medical School of Rowan University, Rowan University.
Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi
) and domestic mulberry silk (Bombyx mori
) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.
Bioengineering, Issue 90, protein alloys, biomaterials, biomedical, silk blends, computational simulation, implantable electronic devices
Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles
Institutions: Technische Universität Braunschweig.
The filamentous fungus A. niger
is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most intriguing and often uncontrollable characteristics of this filamentous organism is its complex morphology. It ranges from dense spherical pellets to viscous mycelia (Figure 1
). Various process parameters and ingredients are known to influence fungal morphology 1
. Since optimal productivity correlates strongly with a specific morphological form, the fungal morphology often represents the bottleneck of productivity in industrial production.
A straight forward and elegant approach to precisely control morphological shape is the addition of inorganic insoluble micro particles (like hydrous magnesium silicate, aluminum oxide or titanium silicate oxide) to the culture medium contributing to increased enzyme production 2-6
. Since there is an obvious correlation between micro particle dependent morphology and enzyme production it is desirable to mathematically link productivity and morphological appearance. Therefore a quantitative precise and holistic morphological description is targeted.
Thus, we present a method to generate and characterize micro particle dependent morphological structures and to correlate fungal morphology with productivity (Figure 1
) which possibly contributes to a better understanding of the morphogenesis of filamentous microorganisms.
The recombinant strain A. niger
SKAn1015 is cultivated for 72 h in a 3 L stirred tank bioreactor. By addition of talc micro particles in concentrations of 1 g/L, 3 g/L and 10 g/L prior to inoculation a variety of morphological structures is reproducibly generated. Sterile samples are taken after 24, 48 and 72 hours for determination of growth progress and activity of the produced enzyme. The formed product is the high-value enzyme β-fructofuranosidase, an important biocatalyst for neo-sugar formation in food or pharmaceutical industry, which catalyzes among others the reaction of sucrose to glucose 7-9
. Therefore, the quantification of glucose after adding sucrose implies the amount of produced β-fructofuranosidase. Glucose quantification is made by a GOD/POD-Assay 10
, which is modified for high-throughput analysis in 96-well micro titer plates.
Fungal morphology after 72 hours is examined by microscope and characterized by digital image analysis. In doing so, particle shape factors for fungal macro morphology like Feret's diameter, projected area, perimeter, circularity, aspect ratio, roundness und solidity are calculated with the open source image processing program ImageJ. Relevant parameters are combined to a dimensionless Morphology number (Mn) 11
, which enables a comprehensive characterization of fungal morphology. The close correlation of the Morphology number and productivity are highlighted by mathematical regression.
Immunology, Issue 61, morphology engineering, Morphology number (Mn), filamentous fungi, fructofuranosidase, micro particles, image analysis
Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival
Institutions: Swedish University of Agricultural Sciences, Uppsala University.
Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida
KT2440 and A. chlorophenolicus
A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1
Microbiology, Issue 78, Cellular Biology, Molecular Biology, Biochemistry, Biophysics, Basic Protocols, Cell survival, sucrose, polysaccharides, cellulose, Ficoll, freeze-drying, Pseudomonas putida, Arthrobacter chlorophenolicus, cells, cell culture
Encapsulation and Permeability Characteristics of Plasma Polymerized Hollow Particles
Institutions: The Pennsylvania State University.
In this protocol, core-shell nanostructures are synthesized by plasma enhanced chemical vapor deposition. We produce an amorphous barrier by plasma polymerization of isopropanol on various solid substrates, including silica and potassium chloride. This versatile technique is used to treat nanoparticles and nanopowders with sizes ranging from 37 nm to 1 micron, by depositing films whose thickness can be anywhere from 1 nm to upwards of 100 nm. Dissolution of the core allows us to study the rate of permeation through the film. In these experiments, we determine the diffusion coefficient of KCl through the barrier film by coating KCL nanocrystals and subsequently monitoring the ionic conductivity of the coated particles suspended in water. The primary interest in this process is the encapsulation and delayed release of solutes. The thickness of the shell is one of the independent variables by which we control the rate of release. It has a strong effect on the rate of release, which increases from a six-hour release (shell thickness is 20 nm) to a long-term release over 30 days (shell thickness is 95 nm). The release profile shows a characteristic behavior: a fast release (35% of the final materials) during the first five minutes after the beginning of the dissolution, and a slower release till all of the core materials come out.
Physics, Issue 66, Chemical Engineering, Plasma Physics, Plasma coating, Core-shell structure, Hollow particles, Permeability, nanoparticles, nanopowders
Harvesting Solar Energy by Means of Charge-Separating Nanocrystals and Their Solids
Institutions: Bowling Green State University, Bowling Green State University, Bowling Green State University.
Conjoining different semiconductor materials in a single nano-composite provides synthetic means for the development of novel optoelectronic materials offering a superior control over the spatial distribution of charge carriers across material interfaces. As this study demonstrates, a combination of donor-acceptor nanocrystal (NC) domains in a single nanoparticle can lead to the realization of efficient photocatalytic1-5
materials, while a layered assembly of donor- and acceptor-like nanocrystals films gives rise to photovoltaic materials.
Initially the paper focuses on the synthesis of composite inorganic nanocrystals, comprising linearly stacked ZnSe, CdS, and Pt domains, which jointly promote photoinduced charge separation. These structures are used in aqueous solutions for the photocatalysis of water under solar radiation, resulting in the production of H2
gas. To enhance the photoinduced separation of charges, a nanorod morphology with a linear gradient originating from an intrinsic electric field is used5
. The inter-domain energetics are then optimized to drive photogenerated electrons toward the Pt catalytic site while expelling the holes to the surface of ZnSe domains for sacrificial regeneration (via methanol). Here we show that the only efficient way to produce hydrogen is to use electron-donating ligands to passivate the surface states by tuning the energy level alignment at the semiconductor-ligand interface. Stable and efficient reduction of water is allowed by these ligands due to the fact that they fill vacancies in the valence band of the semiconductor domain, preventing energetic holes from degrading it. Specifically, we show that the energy of the hole is transferred to the ligand moiety, leaving the semiconductor domain functional. This enables us to return the entire nanocrystal-ligand system to a functional state, when the ligands are degraded, by simply adding fresh ligands to the system4
To promote a photovoltaic charge separation, we use a composite two-layer solid of PbS and TiO2
films. In this configuration, photoinduced electrons are injected into TiO2
and are subsequently picked up by an FTO electrode, while holes are channeled to a Au electrode via PbS layer6
. To develop the latter we introduce a Semiconductor Matrix Encapsulated Nanocrystal Arrays
(SMENA) strategy, which allows bonding PbS NCs into the surrounding matrix of CdS semiconductor. As a result, fabricated solids exhibit excellent thermal stability, attributed to the heteroepitaxial structure of nanocrystal-matrix interfaces, and show compelling light-harvesting performance in prototype solar cells7
Physics, Issue 66, Materials Science, Chemical Engineering, Chemistry, Electrical Engineering, Photovoltaics, nanorods, dye-sensitized, solids, titanium dioxide, photocatalysis, quantum dots
Template Directed Synthesis of Plasmonic Gold Nanotubes with Tunable IR Absorbance
Institutions: University of Toronto.
A nearly parallel array of pores can be produced by anodizing aluminum foils in acidic environments1, 2
. Applications of anodic aluminum oxide (AAO) membranes have been under development since the 1990's and have become a common method to template the synthesis of high aspect ratio nanostructures, mostly by electrochemical growth or pore-wetting. Recently, these membranes have become commercially available in a wide range of pore sizes and densities, leading to an extensive library of functional nanostructures being synthesized from AAO membranes. These include composite nanorods, nanowires and nanotubes made of metals, inorganic materials or polymers 3-10
. Nanoporous membranes have been used to synthesize nanoparticle and nanotube arrays that perform well as refractive index sensors, plasmonic biosensors, or surface enhanced Raman spectroscopy (SERS) substrates 11-16
, as well as a wide range of other fields such as photo-thermal heating 17
, permselective transport 18, 19
, catalysis 20
, microfluidics 21
, and electrochemical sensing 22, 23
. Here, we report a novel procedure to prepare gold nanotubes in AAO membranes. Hollow nanostructures have potential application in plasmonic and SERS sensing, and we anticipate these gold nanotubes will allow for high sensitivity and strong plasmon signals, arising from decreased material dampening 15
Chemistry, Issue 74, Chemical Engineering, Materials Science, Physics, Nanotechnology, Chemistry and Materials (General), Composite Materials, Inorganic, Organic and Physical Chemistry, Metals and Metallic Materials, Gold, nanotubes, anodic aluminum oxide templates, surface plasmon resonance, sensing, refractive index, template directed synthesis, nano
Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry
Institutions: Georgia Institute of Technology.
Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo
. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.
Bioengineering, Issue 77, Molecular Biology, Biomedical Engineering, Chemistry, Biochemistry, Biophysics, Physics, Cellular Biology, Molecular Imaging, Mass Spectrometry, MS, MSI, Desorption electrospray ionization, DESI, Ambient mass spectrometry, tissue, sectioning, biomarker, imaging
Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques
Institutions: Lawrence Berkeley National Laboratory, University of Illinois at Chicago, Stanford Synchrotron Radiation Lightsource, Haldor Topsøe A/S, PolyPlus Battery Company.
Intercalation compounds such as transition metal oxides or phosphates are the most commonly used electrode materials in Li-ion and Na-ion batteries. During insertion or removal of alkali metal ions, the redox states of transition metals in the compounds change and structural transformations such as phase transitions and/or lattice parameter increases or decreases occur. These behaviors in turn determine important characteristics of the batteries such as the potential profiles, rate capabilities, and cycle lives. The extremely bright and tunable x-rays produced by synchrotron radiation allow rapid acquisition of high-resolution data that provide information about these processes. Transformations in the bulk materials, such as phase transitions, can be directly observed using X-ray diffraction (XRD), while X-ray absorption spectroscopy (XAS) gives information about the local electronic and geometric structures (e.g.
changes in redox states and bond lengths). In situ
experiments carried out on operating cells are particularly useful because they allow direct correlation between the electrochemical and structural properties of the materials. These experiments are time-consuming and can be challenging to design due to the reactivity and air-sensitivity of the alkali metal anodes used in the half-cell configurations, and/or the possibility of signal interference from other cell components and hardware. For these reasons, it is appropriate to carry out ex situ
on electrodes harvested from partially charged or cycled cells) in some cases. Here, we present detailed protocols for the preparation of both ex situ
and in situ
samples for experiments involving synchrotron radiation and demonstrate how these experiments are done.
Physics, Issue 81, X-Ray Absorption Spectroscopy, X-Ray Diffraction, inorganic chemistry, electric batteries (applications), energy storage, Electrode materials, Li-ion battery, Na-ion battery, X-ray Absorption Spectroscopy (XAS), in situ X-ray diffraction (XRD)
A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent
Institutions: University of Maine, Orono, University of Maine, Orono.
Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g.
by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5
. Mast cell granule component and the allergic mediator β-hexosaminidase, which is released linearly in tandem with histamine from mast cells6
, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1
. Originally published by Naal et al.1
, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11
, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2
. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using ε280
= 4,200 L/M/cm)12
. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.
Immunology, Issue 81, mast cell, basophil, degranulation, RBL-2H3, triclosan, irgasan, antibacterial, β-hexosaminidase, allergy, Asthma, toxicants, ionophore, antigen, fluorescence, microplate, UV-Vis
Ultrahigh Density Array of Vertically Aligned Small-molecular Organic Nanowires on Arbitrary Substrates
Institutions: University of Alberta.
In recent years π-conjugated organic semiconductors have emerged as the active material in a number of diverse applications including large-area, low-cost displays, photovoltaics, printable and flexible electronics and organic spin valves. Organics allow (a) low-cost, low-temperature processing and (b) molecular-level design of electronic, optical and spin transport characteristics. Such features are not readily available for mainstream inorganic semiconductors, which have enabled organics to carve a niche in the silicon-dominated electronics market. The first generation of organic-based devices has focused on thin film geometries, grown by physical vapor deposition or solution processing. However, it has been realized that organic nanostructures
can be used to enhance performance of above-mentioned applications and significant effort has been invested in exploring methods for organic nanostructure fabrication.
A particularly interesting class of organic nanostructures is the one in which vertically oriented organic nanowires, nanorods or nanotubes are organized in a well-regimented, high-density array
. Such structures are highly versatile and are ideal morphological architectures for various applications such as chemical sensors, split-dipole nanoantennas, photovoltaic devices with radially heterostructured "core-shell" nanowires, and memory devices with a cross-point geometry. Such architecture is generally realized by a template-directed approach. In the past this method has been used to grow metal and inorganic semiconductor nanowire arrays. More recently π-conjugated polymer nanowires have been grown within nanoporous templates. However, these approaches have had limited success in growing nanowires of technologically important π-conjugated small molecular weight organics
, such as tris-8-hydroxyquinoline aluminum (Alq3
), rubrene and methanofullerenes, which are commonly used in diverse areas including organic displays, photovoltaics, thin film transistors and spintronics.
Recently we have been able to address the above-mentioned issue by employing a novel "centrifugation-assisted" approach. This method therefore broadens the spectrum of organic materials that can be patterned in a vertically ordered nanowire array. Due to the technological importance of Alq3
, rubrene and methanofullerenes, our method can be used to explore how the nanostructuring of these materials affects the performance of aforementioned organic devices. The purpose of this article is to describe the technical details of the above-mentioned protocol, demonstrate how this process can be extended to grow small-molecular organic nanowires on arbitrary substrates
and finally, to discuss the critical steps, limitations, possible modifications, trouble-shooting and future applications.
Physics, Issue 76, Electrical Engineering, Chemistry, Chemical Engineering, Nanotechnology, nanodevices (electronic), semiconductor devices, solid state devices, thin films (theory, deposition and growth), crystal growth (general), Organic semiconductors, small molecular organics, organic nanowires, nanorods and nanotubes, bottom-up nanofabrication, electrochemical self-assembly, anodic aluminum oxide (AAO), template-assisted synthesis of nanostructures, Raman spectrum, field emission scanning electron microscopy, FESEM
Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals
Institutions: UC Berkeley, UC Berkeley, UC Berkeley, Lawrence Berkeley National Laboratory, University of Chicago, Argonne National Laboratory.
We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.
Chemistry, Issue 82, nanostructures, synthesis, nanocrystals, seeded rods, tetrapods, nanoheterostructures
Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters
Institutions: University of Texas at Austin, University of Texas M.D. Anderson Cancer Center.
Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture and detection of circulating tumor cells (CTCs), and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.
Chemistry, Issue 90, nanoparticles, plasmonic, magnetic, nanocomposites, magnetic trapping, circulating tumor cells, dark-field imaging