Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.
23 Related JoVE Articles!
Concurrent Quantitative Conductivity and Mechanical Properties Measurements of Organic Photovoltaic Materials using AFM
Institutions: Argonne National Laboratory, University of Chicago.
Organic photovoltaic (OPV) materials are inherently inhomogeneous at the nanometer scale. Nanoscale inhomogeneity of OPV materials affects performance of photovoltaic devices. Thus, understanding of spatial variations in composition as well as electrical properties of OPV materials is of paramount importance for moving PV technology forward.1,2
In this paper, we describe a protocol for quantitative measurements of electrical and mechanical properties of OPV materials with sub-100 nm resolution. Currently, materials properties measurements performed using commercially available AFM-based techniques (PeakForce, conductive AFM) generally provide only qualitative information. The values for resistance as well as Young's modulus measured using our method on the prototypical ITO/PEDOT:PSS/P3HT:PC61
BM system correspond well with literature data. The P3HT:PC61
BM blend separates onto PC61
BM-rich and P3HT-rich domains. Mechanical properties of PC61
BM-rich and P3HT-rich domains are different, which allows for domain attribution on the surface of the film. Importantly, combining mechanical and electrical data allows for correlation of the domain structure on the surface of the film with electrical properties variation measured through the thickness of the film.
Materials Science, Issue 71, Nanotechnology, Mechanical Engineering, Electrical Engineering, Computer Science, Physics, electrical transport properties in solids, condensed matter physics, thin films (theory, deposition and growth), conductivity (solid state), AFM, atomic force microscopy, electrical properties, mechanical properties, organic photovoltaics, microengineering, photovoltaics
A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration
Institutions: University of New South Wales .
Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1
Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2
Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3
Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5
Without this 'activation' process, SurgiLux films are readily removed.6-9
SurgiLux has been tested both in vitro
and in vivo
on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10
Bioengineering, Issue 68, Chitosan, Infra-red Laser, Indocyanine Green, Biomaterial, SurgiLux, Surgical Adhesive
In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa
Institutions: CNRS & Université Paris Descartes.
There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa
in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.
Biochemistry, Issue 84, membrane protein, transport, antibiotic resistance, liposomes, proton gradient, bacteriorhodopsin
The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism
Institutions: University of Montréal, McGill University, University of Minnesota.
Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33
. To help improve this understanding, proton magnetic resonance spectroscopy (1
H-MRS) can be used as it allows the in vivo
quantification of brain metabolites such as γ-aminobutyric acid (GABA) and glutamate in a region-specific manner 41
. In fact, a recent study demonstrated that 1
H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34
. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1
H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31
. Methodological factors to consider and possible modifications to the protocol are also discussed.
Neuroscience, Issue 93, proton magnetic resonance spectroscopy, transcranial direct current stimulation, primary motor cortex, GABA, glutamate, stroke
Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants
Institutions: The University of Akron, Saint Vincent Saint Mary's High School.
This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.
low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo
purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.
Bioengineering, Issue 82, hydrogels, microgels, polyethylene glycol, molecuar weight, photopolymerized precipitation reaction, polymers, polydispersity index
A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)
Institutions: Institut Pasteur, Stanford School of Medicine, Institut d'Imagerie Biomédicale, Vanderbilt School of Medicine, The Walter & Eliza Hall Institute of Medical Research, Institut Pasteur, Institut Pasteur.
Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro
and in vivo
. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the Förster radius, commonly less than 14 nm, FUEL can occur at distances up to µm or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.
Bioengineering, Issue 87, Biochemical Phenomena, Biochemical Processes, Energy Transfer, Fluorescence Resonance Energy Transfer (FRET), FUEL, BRET, CRET, Förster, bioluminescence, In vivo
Synthesis of an Intein-mediated Artificial Protein Hydrogel
Institutions: Texas A&M University, College Station, Texas A&M University, College Station.
We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans
-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans
-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 °C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a "docking station peptide" into the hydrogel building block enables convenient incorporation of "docking protein"-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.
Bioengineering, Issue 83, split-intein, self-assembly, shear-thinning, enzyme, immobilization, organic synthesis
Waste Water Derived Electroactive Microbial Biofilms: Growth, Maintenance, and Basic Characterization
Institutions: UFZ - Helmholtz-Centre for Environmental Research.
The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (jmax
) and the coulombic efficiency (CE
) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e
. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e
. turnover conditions, and in the absence of the substrate, i.e.
nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (Ep
), peak current density (jp
), formal potential (Ef
) and peak separation (ΔEp
). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.
Environmental Sciences, Issue 82, Electrochemistry, Microbial fuel cell, microbial bioelectrochemical system, cyclic voltammetry, electroactive bacteria, microbial bioelectrochemistry, bioelectrocatalysis
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
Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells
Institutions: Georgia Institute of Technology , Georgia Institute of Technology .
Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7
. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ
conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2
. Among the relatively few in situ
surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12
. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ
Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14
("coking") and sulfur poisoning11, 15
and the manner in which surface modifications stave off this degradation16
. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22
. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2
S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ
Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.
Materials Science, Issue 67, Chemistry, Electrical Engineering, Physics, electrochemistry, catalysts (chemical), spectroscopic chemical analysis (application), microscopes, Fuel cell, Raman, AFM, SOFC, Surface, Electrode
Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry
Institutions: University of Richmond, University of Richmond.
Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3
In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4
and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1
H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa
azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c
) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8
Bioengineering, Issue 56, Monolayer protected clusters, film assemblies, protein monolayer electrochemistry, azurin, self-assembled monolayers
Molecular Beam Mass Spectrometry With Tunable Vacuum Ultraviolet (VUV) Synchrotron Radiation
Institutions: Lawrence Berkeley National Laboratory.
Tunable soft ionization coupled to mass spectroscopy is a powerful method to investigate isolated molecules, complexes and clusters and their spectroscopy and dynamics1-4
. Fundamental studies of photoionization processes of biomolecules provide information about the electronic structure of these systems. Furthermore determinations of ionization energies and other properties of biomolecules in the gas phase are not trivial, and these experiments provide a platform to generate these data. We have developed a thermal vaporization technique coupled with supersonic molecular beams that provides a gentle way to transport these species into the gas phase. Judicious combination of source gas and temperature allows for formation of dimers and higher clusters of the DNA bases. The focus of this particular work is on the effects of non-covalent interactions, i.e.
, hydrogen bonding, stacking, and electrostatic interactions, on the ionization energies and proton transfer of individual biomolecules, their complexes and upon micro-hydration by water1, 5-9
We have performed experimental and theoretical characterization of the photoionization dynamics of gas-phase uracil and 1,3-dimethyluracil dimers using molecular beams coupled with synchrotron radiation at the Chemical Dynamics Beamline10
located at the Advanced Light Source and the experimental details are visualized here. This allowed us to observe the proton transfer in 1,3-dimethyluracil dimers, a system with pi stacking geometry and with no hydrogen bonds1
. Molecular beams provide a very convenient and efficient way to isolate the sample of interest from environmental perturbations which in return allows accurate comparison with electronic structure calculations11, 12
. By tuning the photon energy from the synchrotron, a photoionization efficiency (PIE) curve can be plotted which informs us about the cationic electronic states. These values can then be compared to theoretical models and calculations and in turn, explain in detail the electronic structure and dynamics of the investigated species 1, 3
Physics, Issue 68, mass spectroscopy (application), physical chemistry, radiation chemistry, molecular beams, molecular physics, molecular structure, photon interactions with atoms and molecules, Molecular beam, mass spectrometry, vacuum ultraviolet, synchrotron radiation, proton transfer, DNA bases, clusters
Fabrication of Nano-engineered Transparent Conducting Oxides by Pulsed Laser Deposition
Institutions: Politecnico di Milano, Instituto Italiano di Tecnologia.
Nanosecond Pulsed Laser Deposition (PLD) in the presence of a background gas allows the deposition of metal oxides with tunable morphology, structure, density and stoichiometry by a proper control of the plasma plume expansion dynamics. Such versatility can be exploited to produce nanostructured films from compact and dense to nanoporous characterized by a hierarchical assembly of nano-sized clusters. In particular we describe the detailed methodology to fabricate two types of Al-doped ZnO (AZO) films as transparent electrodes in photovoltaic devices: 1) at low O2
pressure, compact films with electrical conductivity and optical transparency close to the state of the art transparent conducting oxides (TCO) can be deposited at room temperature, to be compatible with thermally sensitive materials such as polymers used in organic photovoltaics (OPVs); 2) highly light scattering hierarchical structures resembling a forest of nano-trees are produced at higher pressures. Such structures show high Haze factor (>80%) and may be exploited to enhance the light trapping capability. The method here described for AZO films can be applied to other metal oxides relevant for technological applications such as TiO2
Materials Science, Issue 72, Physics, Nanotechnology, Nanoengineering, Oxides, thin films, thin film theory, deposition and growth, Pulsed laser Deposition (PLD), Transparent conducting oxides (TCO), Hierarchically organized Nanostructured oxides, Al doped ZnO (AZO) films, enhanced light scattering capability, gases, deposition, nanoporus, nanoparticles, Van der Pauw, scanning electron microscopy, SEM
Development of a 3D Graphene Electrode Dielectrophoretic Device
Institutions: Michigan Technological University, Michigan Technological University, XG Sciences, Inc..
The design and fabrication of a novel 3D electrode microdevice using 50 µm thick graphene paper and 100 µm double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 µm x 0.7 cm x 2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 μm diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 μm polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.
Physics, Issue 88, graphene paper, dielectrophoresis, graphene electrodes, 3D laminated microdevice, polystyrene beads, cell diagnostics
Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation
Institutions: University of Rochester, University of Rochester, University of Rochester Medical Center.
One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young’s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.
primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;
H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.
Chemistry, Issue 80, Poly(ethylene glycol), peptides, polymerization, polymers, methacrylation, peptide functionalization, 1H-NMR, MALDI-ToF, hydrogels, macromer synthesis
Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation
Institutions: Virginia Tech, Virginia Tech, Illinois Institute of Technology- Moffett Campus, University of Guadalajara, Virginia Tech, Virginia Tech.
Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.
Plant Biology, Issue 88, nanocellulose, thin films, quartz crystal microbalance, layer-by-layer, LbL
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
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
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
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
Micropunching Lithography for Generating Micro- and Submicron-patterns on Polymer Substrates
Institutions: University of Texas at Arlington .
Conducting polymers have attracted great attention since the discovery of high conductivity in doped polyacetylene in 19771
. They offer the advantages of low weight, easy tailoring of properties and a wide spectrum of applications2,3
. Due to sensitivity of conducting polymers to environmental conditions (e.g., air, oxygen, moisture, high temperature and chemical solutions), lithographic techniques present significant technical challenges when working with these materials4
. For example, current photolithographic methods, such as ultra-violet (UV), are unsuitable for patterning the conducting polymers due to the involvement of wet and/or dry etching processes in these methods. In addition, current micro/nanosystems mainly have a planar form5,6
. One layer of structures is built on the top surfaces of another layer of fabricated features. Multiple layers of these structures are stacked together to form numerous devices on a common substrate. The sidewall surfaces of the microstructures have not been used in constructing devices. On the other hand, sidewall patterns could be used, for example, to build 3-D circuits, modify fluidic channels and direct horizontal growth of nanowires and nanotubes.
A macropunching method has been applied in the manufacturing industry to create macropatterns in a sheet metal for over a hundred years. Motivated by this approach, we have developed a micropunching lithography method (MPL) to overcome the obstacles of patterning conducting polymers and generating sidewall patterns. Like the macropunching method, the MPL also includes two operations (Fig. 1
): (i) cutting; and (ii) drawing. The "cutting" operation was applied to pattern three conducting polymers4
, polypyrrole (PPy), Poly(3,4-ethylenedioxythiophen)-poly(4-styrenesulphonate) (PEDOT) and polyaniline (PANI). It was also employed to create Al microstructures7
. The fabricated microstructures of conducting polymers have been used as humidity8
, and glucose sensors9
. Combined microstructures of Al and conducting polymers have been employed to fabricate capacitors and various heterojunctions9,10,11
. The "cutting" operation was also applied to generate submicron-patterns, such as 100- and 500-nm-wide PPy lines as well as 100-nm-wide Au wires. The "drawing" operation was employed for two applications: (i) produce Au sidewall patterns on high density polyethylene (HDPE) channels which could be used for building 3D microsystems12,13,14
, and (ii) fabricate polydimethylsiloxane (PDMS) micropillars on HDPE substrates to increase the contact angle of the channel15
Mechanical Engineering, Issue 65, Physics, micropunching lithography, conducting polymers, nanowires, sidewall patterns, microlines
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
Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation
Institutions: Karlsruhe Research Centre, University of Twente, Institute for Heavy Ion Research, Karlsruhe Research Centre, Karlsruhe Research Centre.
Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.
Cellular Biology, Issue 15, SMART, microthermoforming, microfabrication, scaffolds, polymer