Polymer Matrix-Based Piezoelectric Composite for Structural Health Monitoring

Structural health monitoring (SHM) refers to the procedure of assessing the structure conditions continuously so it is an alternative to conventional nondestructive evaluation (NDE) techniques [1]. With the growing developments in sensor technology acoustic emission (AE) technology has been attracting attention in SHM applications. AE are characterized by waves produced by the sudden internal stress redistribution caused by the changes in the internal structure, such as fatigue, crack growth, corrosion, etc. Piezoelectric materials such as Lead Zirconate Titanate (PZT) ceramic have been widely used as sensor due to its high electromechanical coupling factor and piezoelectric d coefficients. Because of the poor mechanical characteristic and the lack in the formability of the ceramic, polymer matrix-based piezoelectric composites have been studied in the last decade in order to obtain better properties in comparison with a single phase material. In this study a composite film made of polyurethane (PU) and PZT ceramic particles partially recovered with polyaniline (PAni) was characterized and used as sensor for AE detection. Preliminary results indicate that the presence of a semiconductor polymer (PAni) recovering the ceramic particles, make the poling process easier and less time consuming. Also, it is possible to observe that there is a great potential to use such type of composite as sensor for structure health monitoring.

Analysing the fate of nanopesticides in soil and the applicability of regulatory protocols using a polymer-based nanoformulation of atrazine

For the first time, regulatory protocols defined in the OECD guidelines were applied to determine the fate properties of a nanopesticide in two agricultural soils with contrasting characteristics. The nanoformulation studied had no effect on the degradation kinetics of atrazine indicating that (1) the release of atrazine from the polymer nanocarriers occurred rapidly relative to the degradation kinetics (half-lives 36-53 days) and/or that (2) atrazine associated with the nanocarriers was subject to biotic or abiotic degradation. Sorption coefficients, derived from a batch and a centrifugation technique at a realistic soil-to-solution ratio, were higher for the nanoformulated atrazine than for the pure active ingredient. Results indicate that the nanoformulation had an effect on the fate of atrazine. However, since the protocols applied were designed to assess solutes, conclusions about the transport of atrazine loaded onto the nanocarriers should be made extremely cautiously. The centrifugation method applied over time (here over 7 days) appears to be a useful tool to indirectly assess the durability of nanopesticides under realistic soil-to-solution ratios and estimate the period of time during which an influence on the fate of the active ingredient may be expected. More detailed investigations into the bioavailability and durability of nanopesticides are necessary and will require the development of novel methods suitable to address both the "nano" and "organic" characteristics of polymer-based nanopesticides.

Fecal collection methods for the determination of protein digestibility in bullfrogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apparent protein and energy digestibility and amino acid availability of corn and co-products in extruded diets for nile tilapia, oreochromis niloticus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Increased cyclooxygenase-2 and vascular endothelial growth factor protein expression is associated with canine prostatic carcionogenesis process

Background: COX-2 is one of the most important prostaglandin involved in urologic cancer and seems to be associated with tumor progression, invasion, and metastasis. In addition, several effects have been reported for VEGF, including inducing angiogenesis, promoting cell migration, and inhibiting apoptosis. COX2 and VEGF up-regulation have been reported in human prostate cancer. Due to the importance of canine natural model for prostate cancer, the aim of this study was to evaluate COX-2 and VEGF protein expression in canine carcinogenic process. Material and Methods: Seventy-four prostatic tissues from dogs were selected to be evaluated for protein expression by immunohistochemistry (IHC), including: 10 normal prostatic tissues, 20 benign prostatic hyperplasias (BPH), 25 proliferative inflammatory atrophies (PIA) and 20 prostatic carcinomas (PCa). COX-2 and VEGF were detected using the monoclonal antibody CX-294 (1:50 dilution, Dako Cytomation and sc-53463 (1:100 dilution, Santa Cruz), respectively. The immunolabelling was performed by a polymer method (Histofine, Nichirei Biosciences). All reaction included negative controls by omitting the primary antibody. The percentage of C-MYC, E-cadherin, and p63- positive cells per lesion was evaluated according to Prowatke et al. (2007). The samples were scored separately according to staining intensity and graded semi-quantitatively as negative, weakly positive (1), moderately positive, and strongly positive. The score was done in one 400 magnification field, considering only the lesion, since this was done in a TMA core of 1 mm. For statistical analyses, the immunostaining classifications were reduced to two categories: negative and positive. The negative category included negative and weakly positive staining. Chi-square or Fisher exact test was used to determine the association between the categorical variables. Results: The COX-2 protein expression was elevated in the cytoplasm of the canine PCa and PIA compared to normal prostate (p=0.002). VEGF protein expression was increased in 94.75% of the PCa and 100% of the PIA compared with to normal prostate (p = 0.001). No difference was found when compared normal prostate with BPH. Conclusions: This study has demonstrated that the carcinogenesis of canine prostatic tissue may be related to gain of COX-2 and VEGF protein expression.

Prediction of crude protein digestibility of animal by-product meals for dogs by the protein solubility in pepsin method

Animal by-product meals have large variability in crude protein (CP) content and digestibility. In vivo digestibility procedures are precise but laborious, and in vitro methods could be an alternative to evaluate and classify these ingredients. The present study reports prediction equations to estimate the CP digestibility of meat and bone meal (MBM) and poultry by-product meal (PM) using the protein solubility in pepsin method (PSP). Total tract CP digestibility of eight MBM and eight PM samples was determined in dogs by the substitution method. A basal diet was formulated for dog maintenance, and sixteen diets were produced by mixing 70 % of the basal diet and 30 % of each tested meal. Six dogs per diet were used to determine ingredient digestibility. In addition, PSP of the MBM and PM samples was determined using three pepsin concentrations: 0·02, 0·002 and 0·0002 %. The CP content of MBM and PM ranged from 39 to 46 % and 57 to 69 %, respectively, and their mean CP digestibility by dogs was 76 (2·4) and 85 (2·6) %, respectively. The pepsin concentration with higher Pearson correlation coefficients with the in vivo results were 0·0002 % for MBM (r 0·380; P = 0·008) and 0·02 % for PM (r 0·482; P = 0·005). The relationship between the in vivo and in vitro results was better explained by the following equations: CP digestibility of MBM = 61·7 + 0·2644 × PSP at 0·0002 % (P = 0·008; R (2) 0·126); and CP digestibility of PM = 54·1 + 0·3833 × PSP at 0·02 % (P = 0·005; R (2) 0·216). Although significant, the coefficients of determination were low, indicating that the models were weak and need to be used with caution.

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two-phase polymer systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A New Fluorinated Polymer Having Two Connected Rings in the Main Chain: Synthesis and Characterization of Fluorinated Poly(1,3-cyclohexadiene)

The high electronegativity and small size of the fluorine atom and the high stability of C-F bonds impart interesting properties and applications to fluorine containing polymers. The unique properties of fluoropolymers include high thermal stability, improved chemical resistance, low surface energies, low coefficients of friction, and low dielectric constants. Applications of fluorinated polymers include use as noncorrosive materials, polymer processing aids, chemically resistant and antifouling coatings, as well as interlayer dielectrics. Fluorine-containing polymers can be directly synthesized via polymerization of fluorine-containing monomers or by post-polymerization modification. The latter method can be used to attach fluorinated species, such as perfluoroalkyl groups, onto conventional polymer chains, thereby imparting properties of fluorine-containing polymers into conventional polymers and widening their range of potential applications.

A stable liquid-liquid extraction system for clavulanic acid using polymer-based aqueous two-phase systems

The partitioning of Clavulanic Acid (CA) in a novel inexpensive and stable aqueous two-phase system (ATPS) composed by poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA) has been studied. The aqueous two-phase systems are formed by mixing both polymers with a salt (NaCl or Na2SO4) and an aqueous solution of CA. The stability of CA on the presence of both polymers was investigated and it was observed that these polymers do not degrade the biomolecule. The effect of PEG-molecular size, polymer concentrations on the commercial CA partitioning has been studied, at 25 degrees C. The data showed that commercial CA was preferentially partitioned for the PEG-rich phase with a partition coefficient (K-CA) between 1 and 12 in the PEG/NaPA aqueous two phase systems supplemented with NaCl and Na2SO4. The partition to the PEG phase was increased in the systems with high polymer concentrations. Furthermore, Na2SO4 caused higher CA preference for the PEG-phase than NaCl. The systems having a composition with 10 wt.% of PEG4000, 20 wt.% of NaPA8000 and 6 wt.% of Na2SO4 were selected as the optimal ones in terms of recovery of CA from fermented broth of Streptomyces clavuligerus. The partitioning results (K-CA = 9.15 +/- 1.06) are competitive with commercial extraction methods of CA (K-CA = 11.91 +/- 2.08) which emphasizes that the system PEG/NaPA/Na2SO4 can be used as a new process to CA purification/concentration from fermented broth. (C) 2012 Elsevier B.V. All rights reserved.

Systematic studies of protein immobilization by surface plasmon field-enhanced fluorescence spectroscopy

The research interest of this study is to investigate surface immobilization strategies for proteins and other biomolecules by the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) technique. The recrystallization features of the S-layer proteins and the possibility of combining the S-layer lattice arrays with other functional molecules make this protein a prime candidate for supramolecular architectures. The recrystallization behavior on gold or on the secondary cell wall polymer (SCWP) was recorded by SPR. The optical thicknesses and surface densities for different protein layers were calculated. In DNA hybridization tests performed in order to discriminate different mismatches, recombinant S-layer-streptavidin fusion protein matrices showed their potential for new microarrays. Moreover, SCWPs coated gold chips, covered with a controlled and oriented assembly of S-layer fusion proteins, represent an even more sensitive fluorescence testing platform. Additionally, S-layer fusion proteins as the matrix for LHCII immobilization strongly demonstrate superiority over routine approaches, proving the possibility of utilizing them as a new strategy for biomolecular coupling. In the study of the SPFS hCG immunoassay, the biophysical and immunological characteristics of this glycoprotein hormone were presented first. After the investigation of the effect of the biotin thiol dilution on the coupling efficiently, the interfacial binding model including the appropriate binary SAM structure and the versatile streptavidin-biotin interaction was chosen as the basic supramolecular architecture for the fabrication of a SPFS-based immunoassay. Next, the affinity characteristics between different antibodies and hCG were measured via an equilibrium binding analysis, which is the first example for the titration of such a high affinity interaction by SPFS. The results agree very well with the constants derived from the literature. Finally, a sandwich assay and a competitive assay were selected as templates for SPFS-based hCG detection, and an excellent LOD of 0.15 mIU/ml was attained via the “one step” sandwich method. Such high sensitivity not only fulfills clinical requirements, but is also better than most other biosensors. Fully understanding how LHCII complexes transfer the sunlight energy directionally and efficiently to the reaction center is potentially useful for constructing biomimetic devices as solar cells. After the introduction of the structural and the spectroscopic features of LHCII, different surface immobilization strategies of LHCII were summarized next. Among them the strategy based on the His-tag and the immobilized metal (ion) affinity chromatography (IMAC) technique were of great interest and resulted in different kinds of home-fabricated His-tag chelating chips. Their substantial protein coupling capacity, maintenance of high biological activity and a remarkably repeatable binding ability on the same chip after regeneration was demonstrated. Moreover, different parameters related to the stability of surface coupled reconstituted complexes, including sucrose, detergent, lipid, oligomerization, temperature and circulation rate, were evaluated in order to standardize the most effective immobilization conditions. In addition, partial lipid bilayers obtained from LHCII contained proteo-liposomes fusion on the surface were observed by the QCM technique. Finally, the inter-complex energy transfer between neighboring LHCIIs on a gold protected silver surface by excitation with a blue laser (λ = 473nm) was recorded for the first time, and the factors influencing the energy transfer efficiency were evaluated.

Assembly and characterization of supramolecular architectures for biosensor applications

The research has included the efforts in designing, assembling and structurally and functionally characterizing supramolecular biofunctional architectures for optical biosensing applications. In the first part of the study, a class of interfaces based on the biotin-NeutrAvidin binding matrix for the quantitative control of enzyme surface coverage and activity was developed. Genetically modified ß-lactamase was chosen as a model enzyme and attached to five different types of NeutrAvidin-functionalized chip surfaces through a biotinylated spacer. All matrices are suitable for achieving a controlled enzyme surface density. Data obtained by SPR are in excellent agreement with those derived from optical waveguide measurements. Among the various protein-binding strategies investigated in this study, it was found that stiffness and order between alkanethiol-based SAMs and PEGylated surfaces are very important. Matrix D based on a Nb2O5 coating showed a satisfactory regeneration possibility. The surface-immobilized enzymes were found to be stable and sufficiently active enough for a catalytic activity assay. Many factors, such as the steric crowding effect of surface-attached enzymes, the electrostatic interaction between the negatively charged substrate (Nitrocefin) and the polycationic PLL-g-PEG/PEG-Biotin polymer, mass transport effect, and enzyme orientation, are shown to influence the kinetic parameters of catalytic analysis. Furthermore, a home-built Surface Plasmon Resonance Spectrometer of SPR and a commercial miniature Fiber Optic Absorbance Spectrometer (FOAS), served as a combination set-up for affinity and catalytic biosensor, respectively. The parallel measurements offer the opportunity of on-line activity detection of surface attached enzymes. The immobilized enzyme does not have to be in contact with the catalytic biosensor. The SPR chip can easily be cleaned and used for recycling. Additionally, with regard to the application of FOAS, the integrated SPR technique allows for the quantitative control of the surface density of the enzyme, which is highly relevant for the enzymatic activity. Finally, the miniaturized portable FOAS devices can easily be combined as an add-on device with many other in situ interfacial detection techniques, such as optical waveguide lightmode spectroscopy (OWLS), the quartz crystal microbalance (QCM) measurements, or impedance spectroscopy (IS). Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) allows for an absolute determination of intrinsic rate constants describing the true parameters that control interfacial hybridization. Thus it also allows for a study of the difference of the surface coupling influences between OMCVD gold particles and planar metal films presented in the second part. The multilayer growth process was found to proceed similarly to the way it occurs on planar metal substrates. In contrast to planar bulk metal surfaces, metal colloids exhibit a narrow UV-vis absorption band. This absorption band is observed if the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as the localized surface plasmon resonance (LSPR). LSPR excitation results in extremely large molar extinction coefficients, which are due to a combination of both absorption and scattering. When considering metal-enhanced fluorescence we expect the absorption to cause quenching and the scattering to cause enhancement. Our further study will focus on the developing of a detection platform with larger gold particles, which will display a dominant scattering component and enhance the fluorescence signal. Furthermore, the results of sequence-specific detection of DNA hybridization based on OMCVD gold particles provide an excellent application potential for this kind of cheap, simple, and mild preparation protocol applied in this gold fabrication method. In the final chapter, SPFS was used for the in-depth characterizations of the conformational changes of commercial carboxymethyl dextran (CMD) substrate induced by pH and ionic strength variations were studied using surface plasmon resonance spectroscopy. The pH response of CMD is due to the changes in the electrostatics of the system between its protonated and deprotonated forms, while the ionic strength response is attributed from the charge screening effect of the cations that shield the charge of the carboxyl groups and prevent an efficient electrostatic repulsion. Additional studies were performed using SPFS with the aim of fluorophore labeling the carboxymethyl groups. CMD matrices showed typical pH and ionic strength responses, such as high pH and low ionic strength swelling. Furthermore, the effects of the surface charge and the crosslink density of the CMD matrix on the extent of stimuli responses were investigated. The swelling/collapse ratio decreased with decreasing surface concentration of the carboxyl groups and increasing crosslink density. The study of the CMD responses to external and internal variables will provide valuable background information for practical applications.

Thermodynamics of aqueous biopolymer solutions and fractionation of Dextran

Flory-Huggins interaction parameters and thermal diffusion coefficients were measured for aqueous biopolymer solutions. Dextran (a water soluble polysaccharide) and bovine serum albumin (BSA, a water soluble protein) were used for this study. The former polymer is representative for chain macromolecules and the latter is for globular macromolecules. The interaction parameters for the systems water/dextran and water/BSA were determined as a function of composition by means of vapor pressure measurements, using a combination of headspace sampling and gas chromatography (HS-GC). A new theoretical approach, accounting for chain connectivity and conformational variability, describes the observed dependencies quantitatively for the system water/dextran and qualitatively for the system water/BSA. The phase diagrams of the ternary systems water/methanol/dextran and water/dextran/BSA were determined via cloud point measurements and modeled by means of the direct minimization of the Gibbs energy using the information on the binary subsystems as input parameters. The thermal diffusion of dextran was studied for aqueous solutions in the temperature range 15 < T < 55 oC. The effects of the addition of urea were also studied. In the absence of urea, the Soret coefficient ST changes its sign as T is varied; it is positive for T > 45.0 oC, but negative for T < 45.0 oC. The positive sign of ST means that the dextran molecules migrate towards the cold side of the fluid; this behavior is typical for polymer solutions. While a negative sign indicates the macromolecules move toward the hot side; this behavior has so far not been observed with any other binary aqueous polymer solutions. The addition of urea to the aqueous solution of dextran increases ST and reduces the inversion temperature. For 2 M urea, the change in the sign of ST is observed at T = 29.7 oC. At higher temperature ST is always positive in the studied temperature range. To rationalize these observations it is assumed that the addition of urea opens hydrogen bonds, similar to that induced by an increase in temperature. For a future extension of the thermodynamic studies to the effects of poly-dispersity, dextran was fractionated by means of a recently developed technique called Continuous Spin Fractionation (CSF). The solvent/precipitant/polymer system used for the thermodynamic studies served as the basis for the fractionation of dextran The starting polymer had a weight average molar mass Mw = 11.1 kg/mol and a molecular non-uniformity U= Mw / Mn -1= 1.0. Seventy grams of dextran were fractionated using water as the solvent and methanol as the precipitant. Five fractionation steps yielded four samples with Mw values between 4.36 and 18.2 kg/mol and U values ranging from 0.28 to 0.48.

Influence of spatial constraints in non-crystalline regions on the polymer dynamics in semi-crystalline polyethylene: a solid state NMR study

A broad variety of solid state NMR techniques were used to investigate the chain dynamics in several polyethylene (PE) samples, including ultrahigh molecular weight PEs (UHMW-PEs) and low molecular weight PEs (LMW-PEs). Via changing the processing history, i.e. melt/solution crystallization and drawing processes, these samples gain different morphologies, leading to different molecular dynamics. Due to the long chain nature, the molecular dynamics of polyethylene can be distinguished in local fluctuation and long range motion. With the help of NMR these different kinds of molecular dynamics can be monitored separately. In this work the local chain dynamics in non-crystalline regions of polyethylene samples was investigated via measuring 1H-13C heteronuclear dipolar coupling and 13C chemical shift anisotropy (CSA). By analyzing the motionally averaged 1H-13C heteronuclear dipolar coupling and 13C CSA, the information about the local anisotropy and geometry of motion was obtained. Taking advantage of the big difference of the 13C T1 relaxation time in crystalline and non-crystalline regions of PEs, the 1D 13C MAS exchange experiment was used to investigate the cooperative chain motion between these regions. The different chain organizations in non-crystalline regions were used to explain the relationship between the local fluctuation and the long range motion of the samples. In a simple manner the cooperative chain motion between crystalline and non-crystalline regions of PE results in the experimentally observed diffusive behavior of PE chain. The morphological influences on the diffusion motion have been discussed. The morphological factors include lamellar thickness, chain organization in non-crystalline regions and chain entanglements. Thermodynamics of the diffusion motion in melt and solution crystallized UHMW-PEs is discussed, revealing entropy-controlled features of the chain diffusion in PE. This thermodynamic consideration explains the counterintuitive relationship between the local fluctuation and the long range motion of the samples. Using the chain diffusion coefficient, the rates of jump motion in crystals of the melt crystallized PE have been calculated. A concept of "effective" jump motion has been proposed to explain the difference between the values derived from the chain diffusion coefficients and those in literatures. The observations of this thesis give a clear demonstration of the strong relationship between the sample morphology and chain dynamics. The sample morphologies governed by the processing history lead to different spatial constraints for the molecular chains, leading to different features of the local and long range chain dynamics. The knowledge of the morphological influence on the microscopic chain motion has many implications in our understanding of the alpha-relaxation process in PE and the related phenomena such as crystal thickening, drawability of PE, the easy creep of PE fiber, etc.

Diffusion in polymer systems studied by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique to determine the diffusion of fluorescence molecules in various environments. The technique is based on detecting and analyzing the fluctuation of fluorescence light emitted by fluorescence species diffusing through a small and fixed observation volume, formed by a laser focused into the sample. Because of its great potential and high versatility in addressing the diffusion and transport properties in complex systems, FCS has been successfully applied to a great variety of systems. In my thesis, I focused on the application of FCS to study the diffusion of fluorescence molecules in organic environments, especially in polymer melts. In order to examine our FCS setup and a developed measurement protocol, I first utilized FCS to measure tracer diffusion in polystyrene (PS) solutions, for which abundance data exist in the literature. I studied molecular and polymeric tracer diffusion in polystyrene solutions over a broad range of concentrations and different tracer and matrix molecular weights (Mw). Then FCS was further established to study tracer dynamics in polymer melts. In this part I investigated the diffusion of molecular tracers in linear flexible polymer melts [polydimethylsiloxane (PDMS), polyisoprene (PI)], a miscible polymer blend [PI and poly vinyl ethylene (PVE)], and star-shaped polymer [3-arm star polyisoprene (SPI)]. The effects of tracer sizes, polymer Mw, polymer types, and temperature on the diffusion coefficients of small tracers were discussed. The distinct topology of the host polymer, i.e. star polymer melt, revealed the notably different motion of the small tracer, as compared to its linear counterpart. Finally, I emphasized the advantage of the small observation volume which allowed FCS to investigate the tracer diffusions in heterogeneous systems; a swollen cross-linked PS bead and silica inverse opals, where high spatial resolution technique was required.

Development and applications of hollow fiber flow field-flow fractionation in the bioanalytical field. Studies of aggregation phenomena in complex protein samples

Recent advances in the fast growing area of therapeutic/diagnostic proteins and antibodies - novel and highly specific drugs - as well as the progress in the field of functional proteomics regarding the correlation between the aggregation of damaged proteins and (immuno) senescence or aging-related pathologies, underline the need for adequate analytical methods for the detection, separation, characterization and quantification of protein aggregates, regardless of the their origin or formation mechanism. Hollow fiber flow field-flow fractionation (HF5), the miniaturized version of FlowFFF and integral part of the Eclipse DUALTEC FFF separation system, was the focus of this research; this flow-based separation technique proved to be uniquely suited for the hydrodynamic size-based separation of proteins and protein aggregates in a very broad size and molecular weight (MW) range, often present at trace levels. HF5 has shown to be (a) highly selective in terms of protein diffusion coefficients, (b) versatile in terms of bio-compatible carrier solution choice, (c) able to preserve the biophysical properties/molecular conformation of the proteins/protein aggregates and (d) able to discriminate between different types of protein aggregates. Thanks to the miniaturization advantages and the online coupling with highly sensitive detection techniques (UV/Vis, intrinsic fluorescence and multi-angle light scattering), HF5 had very low detection/quantification limits for protein aggregates. Compared to size-exclusion chromatography (SEC), HF5 demonstrated superior selectivity and potential as orthogonal analytical method in the extended characterization assays, often required by therapeutic protein formulations. In addition, the developed HF5 methods have proven to be rapid, highly selective, sensitive and repeatable. HF5 was ideally suitable as first dimension of separation of aging-related protein aggregates from whole cell lysates (proteome pre-fractionation method) and, by HF5-(UV)-MALS online coupling, important biophysical information on the fractionated proteins and protein aggregates was gathered: size (rms radius and hydrodynamic radius), absolute MW and conformation.