Revealing the Kinetic Phase Behavior of Block Copolymer Complexes Using Solvent Vapor Absorption-Desorption Isotherms.

Controlling the self-assembled morphologies in block copolymers heavily depends on their molecular architecture and processing conditions. Solvent vapor annealing is a versatile processive pathway to obtain highly periodic self-assemblies from high chi (χ) block copolymers (BCPs) and supramolecular BCP complexes. Despite the importance of navigating the energy landscape, controlled solvent vapor annealing (SVA) has not been investigated in BCP complexes, partly due to its intricate multicomponent nature. We introduce characteristic absorption-desorption solvent vapor isotherms as an effective way to understand swelling behavior and follow the morphological evolution of the polystyrene-block-poly(4-vinylpyridine) block copolymer complexed with pentadecylphenol (PS-b-P4VP(PDP)). Using the sorption isotherms, we identify the glass transition points, polymer-solvent interaction parameters, and bulk modulus. These parameters indicate that complexation completely screens the polymer interchain interactions. Furthermore, we established that the sorption isotherm of the homopolymer blocks serves to deconvolute the intricacy of BCP complexes. We applied our findings by developing annealing pathways for grain coarsening while preventing macroscopic film dewetting under SVA. Here, grain coarsening obeyed a power law and the growth exponent revealed a kinetic transition point for rapid self-assembly. Overall, SVA-based sorption isotherms have emerged as a critical method for understanding and developing annealing pathways for BCP complexes.

New approach to electron microscopy imaging of gel nanocomposites in situ.

Characterization of Au-nanocomposites is routinely done with scattering techniques where the structure and ordering of nanoparticles can be analyzed. Imaging of Poloxamer gel-based Au-nanocomposites is usually limited to cryo-TEM imaging of cryo-microtomed thin sections of the specimen. While this approach is applicable for imaging of the individual nanoparticles and gauging their size distribution, it requires altering the state of the specimen and is prone to artifacts associated with preparation protocols. Use of Scanning Transmission Electron Microscopy (S/TEM) with fluid cell in situ provides an opportunity to analysis of these complex materials in their hydrated state with nanometer resolution, yet dispensing dense gel-based samples onto electron-transparent substrates remains challenging. We show that Poloxamer gel-based Au nanocomposites exhibiting thermoreversible behavior can be imaged in a fully hydrated state using a commercially available fluid cell holder, and we describe a specimen preparation method for depositing femtoliter amounts of gel-based nanocomposites directly onto the 50 nm-thick SiN window membranes. Ultimately, fluid cell S/TEM in situ imaging approach offers a pathway to visualization of individual nanoparticles within a thick gel media while maintaining the hydrated state of the carrier polymeric matrix.

Wind Energy Conversion by Plant-Inspired Designs.

In 2008 the U.S. Department of Energy set a target of 20% wind energy by 2030. To date, induction-based turbines form the mainstay of this effort, but turbines are noisy, perceived as unattractive, a potential hazard to bats and birds, and their height hampers deployment in residential settings. Several groups have proposed that artificial plants containing piezoelectric elements may harvest wind energy sufficient to contribute to a carbon-neutral energy economy. Here we measured energy conversion by cottonwood-inspired piezoelectric leaves, and by a "vertical flapping stalk"-the most efficient piezo-leaf previously reported. We emulated cottonwood for its unusually ordered, periodic flutter, properties conducive to piezo excitation. Integrated over 0°-90° (azimuthal) of incident airflow, cottonwood mimics outperformed the vertical flapping stalk, but they produced << daW per conceptualized tree. In contrast, a modest-sized cottonwood tree may dissipate ~ 80 W via leaf motion alone. A major limitation of piezo-transduction is charge generation, which scales with capacitance (area). We thus tested a rudimentary, cattail-inspired leaf with stacked elements wired in parallel. Power increased systematically with capacitance as expected, but extrapolation to acre-sized assemblages predicts << daW. Although our results suggest that present piezoelectric materials will not harvest mid-range power from botanic mimics of convenient size, recent developments in electrostriction and triboelectric systems may offer more fertile ground to further explore this concept.

High-Resolution Graphene Films for Electrochemical Sensing via Inkjet Maskless Lithography.

Solution-phase printing of nanomaterial-based graphene inks are rapidly gaining interest for fabrication of flexible electronics. However, scalable manufacturing techniques for high-resolution printed graphene circuits are still lacking. Here, we report a patterning technique [i.e., inkjet maskless lithography (IML)] to form high-resolution, flexible, graphene films (line widths down to 20 µm) that significantly exceed the current inkjet printing resolution of graphene (line widths ∼60 µm). IML uses an inkjet printed polymer lacquer as a sacrificial pattern, viscous spin-coated graphene, and a subsequent graphene lift-off to pattern films without the need for prefabricated stencils, templates, or cleanroom technology (e.g., photolithography). Laser annealing is employed to increase conductivity on thermally sensitive, flexible substrates [polyethylene terephthalate (PET)]. Laser annealing and subsequent platinum nanoparticle deposition substantially increases the electroactive nature of graphene as illustrated by electrochemical hydrogen peroxide (H2O2) sensing [rapid response (5 s), broad linear sensing range (0.1-550 µm), high sensitivity (0.21 µM/µA), and low detection limit (0.21 µM)]. Moreover, high-resolution, complex graphene circuits [i.e., interdigitated electrodes (IDE) with varying finger width and spacing] were created with IML and characterized via potassium chloride (KCl) electrochemical impedance spectroscopy (EIS). Results indicated that sensitivity directly correlates to electrode feature size as the IDE with the smallest finger width and spacing (50 and 50 µm) displayed the largest response to changes in KCl concentration (∼21 kΩ). These results indicate that the developed IML patterning technique is well-suited for rapid, solution-phase graphene film prototyping on flexible substrates for numerous applications including electrochemical sensing.

Rapid and Label-Free Detection of Interferon Gamma via an Electrochemical Aptasensor Comprising a Ternary Surface Monolayer on a Gold Interdigitated Electrode Array.

A label-free electrochemical impedance spectroscopy (EIS) aptasensor for rapid detection (<35 min) of interferon-gamma (IFN-γ) was fabricated by immobilizing a RNA aptamer capture probe (ACP), selective to IFN-γ, on a gold interdigitated electrode array (Au IDE). The ACP was modified with a thiol group at the 5' terminal end and subsequently co-immobilized with 1,6-hexanedithiol (HDT) and 6-mercapto-1-hexanolphosphate (MCH) to the gold surface through thiol-gold interactions. This ACP/HDT-MCH ternary surface monolayer facilitates efficient hybridization with IFN-γ and displays high resistance to nonspecific adsorption of nontarget proteins [i.e., fetal bovine serum (FBS) and bovine serum albumin (BSA)]. The Au IDE functionalized with ACP/HDT-MCH was able to measure IFN-γ in actual FBS solution with a linear sensing range from 22.22 pM to 0.11 nM (1-5 ng/mL) and a detection limit of 11.56 pM. The ability to rapidly sense IFN-γ within this sensing range makes the developed electrochemical platform conducive toward in-field disease detection of a variety of diseases including paratuberculosis (i.e., Johne's Disease). Furthermore, experimental results were numerically validated with an equivalent circuit model that elucidated the effects of the sensing process and the influence of the immobilized ternary monolayer on signal output. This is the first time that ternary surface monolayers have been used to selectively capture/detect IFN-γ on Au IDEs.

Randomized, controlled trial evaluating the effect of multi-strain probiotic on the mucosal microbiota in canine idiopathic inflammatory bowel disease.

The intestinal microbiota is increasingly linked to the pathogenesis of idiopathic inflammatory bowel disease (IBD) in dogs. While studies have reported alterations in fecal (luminal) microbial populations, only limited information is available about the mucosal microbiota of IBD dogs at diagnosis and following medical therapy. Our aim was to characterize the mucosal microbiota and determine the clinical, microbiological, and mucosal homeostatic effects of probiotic treatment in dogs with IBD. Thirty four IBD dogs were randomized to receive standard therapy (ST = diet + prednisone) with or without probiotic. Tissue sections from endoscopic biopsies were evaluated by fluorescence in situ hybridization (FISH) on a quantifiable basis. Disease activity and changes in mucosal microbiota and tight junction protein (TJP) expression were assessed before and after 8 weeks of IBD therapy. ST and ST/probiotic therapy modulated the number of mucosal bacteria of IBD dogs in a similar fashion. Both treatments increased the numbers of total bacteria and individual species residing within adherent mucus, with ST therapy increasing Bifidobacterium spp. and ST/probiotic therapy increasing Lactobacillus spp (P < 0.05 for both), respectively. Both treatments were associated with rapid clinical remission but not improvement in histopathologic inflammation. Probiotic therapy was associated with upregulated (P < 0.05) expression of TJPs E-cadherin, occludin, and zonulin versus ST. The probiotic effect on mucosal bacteria is similar to that of IBD dogs receiving ST. IBD dogs fed probiotic had increased TJP expression suggesting that probiotic may have beneficial effects on mucosal homeostasis.

Helicobacter bilis Infection Alters Mucosal Bacteria and Modulates Colitis Development in Defined Microbiota Mice.

BACKGROUND: Helicobacter bilis infection of C3H/HeN mice harboring the altered Schaedler flora (ASF) triggers progressive immune responsiveness and the development of colitis. We sought to investigate temporal alterations in community structure of a defined (ASF-colonized) microbiota in normal and inflamed murine intestines and to correlate microbiota changes to histopathologic lesions. METHODS: The colonic mucosal microbiota of healthy mice and ASF mice colonized with H. bilis for 3, 6, or 12 weeks were investigated by fluorescence in situ hybridization targeting the 16S ribosomal RNA genes of total bacteria, group-specific organisms, and individual ASF bacterial species. Microbial profiling of ASF and H. bilis abundance was performed on cecal contents. RESULTS: Helicobacter bilis-colonized mice developed colitis associated with temporal changes in composition and spatial distribution of the mucosal microbiota. The number of total bacteria, ASF519, and helicobacter-positive bacteria were increased (P < 0.05), whereas ASF360/361-positive bacteria were decreased (P < 0.05) versus controls. Adherent biofilms in colitic mice were most often (P < 0.05) composed of total bacteria, ASF457, and H. bilis. Total numbers of ASF519 and H. bilis bacteria were positively correlated (P = 0.03, r = 0.39 and P < 0.0001, r = 0.73), and total numbers of ASF360/361 bacteria were negatively correlated (P = 0.003, r = -0.53) to histopathologic score. Differences in cecal abundance of ASF members were not observed. CONCLUSIONS: Altered community structure with murine colitis is characterized by distinct ASF bacteria that interact with the colonic mucosa, by formation of an isolating interlaced layer, by attachment, or by invasion, and this interaction is differentially expressed over time.

Alterations of the Ileal and Colonic Mucosal Microbiota in Canine Chronic Enteropathies.

BACKGROUND: The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon. AIM: To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls. METHODS: Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis. RESULTS: The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05). CONCLUSION: Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota.

Detection and quantification of protein biomarkers from fewer than 10 cells.

The use of antibody microarrays continues to grow rapidly due to the recent advances in proteomics and automation and the opportunity this combination creates for high throughput multiplexed analysis of protein biomarkers. However, a primary limitation of this technology is the lack of PCR-like amplification methods for proteins. Therefore, to realize the full potential of array-based protein biomarker screening it is necessary to construct assays that can detect and quantify protein biomarkers with very high sensitivity, in the femtomolar range, and from limited sample quantities. We describe here the construction of ultramicroarrays, combining the advantages of microarraying including multiplexing capabilities, higher throughput, and cost savings with the ability to screen very small sample volumes. Antibody ultramicroarrays for the detection of interleukin-6 and prostate-specific antigen (PSA), a widely used biomarker for prostate cancer screening, were constructed. These ultramicroarrays were found to have a high specificity and sensitivity with detection levels using purified proteins in the attomole range. Using these ultramicroarrays, we were able to detect PSA secreted from 100 LNCaP cells in 3 h and from just four LNCaP cells in 24 h. Cellular PSA could also be detected from the lysate of an average of just six cells. This strategy should enable proteomic analysis of materials that are available in very limited quantities such as those collected by laser capture microdissection, neonatal biopsy microspecimens, and forensic samples.

Functional protein nanoarrays for biomarker profiling.

The use of microarrays for parallel screening of nucleic acid profiles has become an industry standard. Similar efforts for screening protein-protein interactions are gaining momentum, however, they remain limited by the requirement for relatively large sample volumes. One strategy for overcoming this problem is to significantly decrease the size and consequently the sample volume of the protein interaction assay. We report here on our progress over the last two years in the construction of ultraminiaturized, functional protein capture assays. Each one micron spot in these array-based assays covers less than 1/1000(th) of the surface area of a conventional microarray spot while still maintaining enough antibodies to provide a useful dynamic range. These nanoarray assays can be read by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy. The size reduction realized by functional protein nanoarrays also creates opportunities for novel applications including highly multiplexed single cell analysis and integration with microfluidics and other "lab-on-a-chip" technologies.

Virus particle detection by solid phase immunocapture and atomic force microscopy.

A novel application of atomic force microscopy (AFM) in the rapid, label-free detection and identification of viruses is described. Multiplexed, miniaturized antibody domains were constructed using "ink-jet" protein arraying technology. The solid-phase affinity substrate termed the "ViriChip" was used in the immunocapture of bacteriophage fd, canine parvoviruses, and coxsackieviruses and analyzed by AFM. Immunocapture was found to be antibody-specific with a sensitivity of 10(8)pfu/ml in 30min. Virus binding was found to be linear for concentration between 10(8) and 10(10)pfu/ml and did not reach saturation through 4h.

Microfabricated quill-type surface patterning tools for the creation of biological micro/nano arrays.

Novel quill-type cantilever-based surface patterning tools (SPTs) were designed and constructed for use in controlled placement of femtoliter volumes of biological molecules on surfaces for biological applications. These tools were fabricated from silicon dioxide using microelectromechanical systems (MEMS) techniques. They featured a 1 microm split gap, fluidic transport microchannels and self-replenishing reservoirs. Experimental trials were performed using these tools on NanoArrayer molecular deposition instrumentation. Cy3-streptavidin was loaded as a biological sample and patterned on an amine-reactive dithiobis-succinimidyl undecanoate (DSU) monolayer on gold. Results showed these tools were capable of generating high quality biological arrays with routine spot sizes of 2-3 microm. The spot size could potentially achieve sub-micron dimensions if these SPT designs are reduced in size by more precise microfabrication techniques. The geometric designs of these tools facilitated sample replenishment from the local reservoir on the cantilever which allowed printing of large numbers of spots without sample reloading.