Gene/Protein Disease Symptom Drug Enzyme Compound
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A method was developed to prepare silk fibroin microspheres using lipid vesicles as templates to efficiently load protein drugs in active form for controlled release. The lipid was subsequently removed by methanol or sodium chloride treatments, resulting in silk microspheres consisting of beta-sheet structure and about 2 mum in diameter. NaCl treated microspheres had smoother surfaces compared to the methanol treatments based on SEM analysis, and both types of microspheres had a mixture of multilamellar and unilamellar structures. A model protein drug, horseradish peroxidase, was encapsulated in the microspheres. Freeze-thaw cycles during preparation led to higher loading of the peroxidase due to improved mixing between the silk and drug, while without this process the drug and silk remained in separate layers or domains in microspheres. This partitioning was determined with fluorescein-labeled silk and rhodamine-labeled dextran. Small molecules such as the enzyme substrate 3,3',5,5'-tetramethylbenzidine, Mw=240 Da, and its oxidized product freely diffused through the MeOH- and NaCl-processed silk microspheres so that enzyme loading and activity could be determined. Enzyme activity was retained during processing and in the final microspheres. The enzyme release profile depended on the NaCl-process used in microsphere preparation. The physically cross-linked beta-sheet structure of silk fibroin and the residual lipids in the microspheres played important roles in controlling enzyme release profiles. The silk microspheres have the potential for diverse applications where controlled protein release from biocompatible, mechanically tough, and slowly biodegradable carriers is desirable.
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PMID:Silk microspheres for encapsulation and controlled release. 1721 36

The simple and efficient method for preparing sensitive carbon nanotube/polysulfone/RIgG immunocomposite is described. The membrane of the modified disposable screen-printed electrochemical immunosensor is based on phase inversion method. Carbon nanotube/polysulfone membrane acts both as reservoir of immunological material and transducer while offering high surface area, high toughness and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a much higher sensitivity for those prepared with carbon nanotubes coupled with polysulfone (PSf). The membrane was characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), laser profilometer and by atomic force microscopy (AFM). The purity of the materials was evaluated by thermogravimetric analysis (TGA). The roughness value is doubled when MWCNTs are used instead of graphite into the PSf membranes and the incorporation of antibodies enhances the dispersion of the carbon with the polymeric membrane reducing the roughness in all cases. This biosensor was based on the competitive assay between free and labelled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG was incorporated into the polysulfone membrane by a phase inversion method. Horse radish peroxidase (HRP) enzyme was used as label and hydroquinone as mediator. The detection limit for competitive assay was determined to be 1.66 microg/ml. the linear range of anti-RIgG from 2 to 5 microg/ml and the C(50) was found at 3.56 microg/ml. The sensitivity is five times higher for MWCNT than for graphite electrodes, showing lower unspecific adsorption.
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PMID:Carbon nanotube/polysulfone screen-printed electrochemical immunosensor. 1756 Jan 2

Bombyx mori silk fibroin self-assembles on surfaces to form ultrathin nanoscale coatings based on our prior studies using layer-by-layer deposition techniques driven by hydrophobic interactions between silk fibroin protein molecules. In the present study, poly(lactic-co-glycolic acid) (PLGA) and alginate microspheres were used as substrates and coated with silk fibroin. The coatings were visualized by confocal laser scanning microscopy using fluorescein-labeled silk fibroin. On PLGA microspheres, the coating was approximately 1microm and discontinuous, reflecting the porous surface of these microspheres determined by SEM. In contrast, on alginate microspheres the coating was approximately 10microm thick and continuous. The silk fibroin penetrated into the alginate gel matrix. The silk coating on the PLGA microspheres delayed PLGA degradation. The silk coating on the alginate microspheres survived ethylenediamine tetraacetic acid (EDTA) treatment used to remove the Ca(2+)-cross-links in the alginate gels to solubilize the alginate. This suggests that alginate microspheres can be used as templates to form silk microcapsules. Horseradish peroxidase (HRP) and tetramethylrhodamine-conjugated bovine serum albumin (Rh-BSA) as model protein drugs were encapsulated in the PLGA and alginate microspheres with and without the silk fibroin coatings. Drug release was significantly retarded by the silk coatings when compared to uncoated microsphere controls, and was retarded further by methanol-treated silk coating when compared to silk water-based coatings on alginate microspheres. Silk coatings on PLGA and alginate microspheres provide mechanically stable shells as well as a diffusion barrier to the encapsulated protein drugs. This coating technique has potential for biosensor and drug delivery applications due to the aqueous process employed, the ability to control coating thickness and crystalline content, and the biocompatibility of the silk fibroin protein used in the process.
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PMID:Silk coatings on PLGA and alginate microspheres for protein delivery. 1758 88

The design, synthesis and properties of a new class of enzyme/DNA/inorganic nanobiomaterials are described here. DNA has been used to stabilize the enzymes intercalated in the galleries of the inorganic solid, alpha-Zr(iv) phosphate (alpha-Zr(HPO(4))(2).H(2)O, abbreviated as alpha-ZrP). Interestingly, the presence of DNA improved the activity and stability of the bound enzymes. Key studies leading to the current strategy are presented initially, and these are followed by more recent developments. Several enzymes and proteins, including horseradish peroxidase, lysozyme, glucose oxidase, chymotrypsin, bovine serum albumin, cytochrome c, met-hemoglobin and met-myoglobin are successfully intercalated in the galleries of alpha-ZrP, under benign ambient conditions (aqueous buffered solutions, at room temperature and neutral pH). These novel materials are characterized by XRD, SEM and TEM as well as by biochemical, calorimetric and spectroscopic methods. Spectroscopic studies (circular dichroism, CD), for example, indicated that co-intercalation of DNA improved the retention of bound enzyme structure. The activity was enhanced markedly (five-fold) when DNA is co-intercalated, when compared to the activity in the absence of DNA. Addition of DNA to the sample, after enzyme intercalation, did not make any improvements. Our hypothesis is that enzyme-DNA supramolecular complex binds to the solid and the unfavorable interactions between the enzyme and the solid are minimized. These novel nanobiocomposite materials provide a simple method for packaging DNA and aid in engineering more effective synthetic materials for gene/RNA-delivery and drug delivery applications.
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PMID:Novel enzyme/DNA/inorganic nanomaterials: a new generation of biocatalysts. 1804 9

Glycidyl methacrylate and N-vinyl-2-pyrrolidone (GMA-co-NVP) copolymers with various GMA:NVP ratios were synthesized by solution polymerization technique in toluene using 2,2'-azobisisobutyronitrile (AIBN) as free radical initiator and dip coated onto polypropylene strips. The copolymer composition in polymeric coatings was confirmed by proton NMR spectroscopy. Various techniques like FTIR, SEM and contact angle were used for surface characterization of the polymer coatings. These polymer coated strips were evaluated and standardized for their application in dot-ELISA in two steps. In first step, specificity, sensitivity and reproducibility of the assay on developed polymer coated strips was evaluated through a model system using rabbit anti-goat IgG, goat anti-rabbit IgG and goat anti-rabbit IgG HRP (horseradish peroxidase)-conjugate. Polymer coating with GMA-NVP mol% ratio of 78:22 was able to detect rabbit anti-goat IgG antibody at a concentration as low as 2 ng mL(-1) with 1% BSA as blocking agent using antispecies IgG peroxidase conjugate diluted 1500 times. In the second step, the sensitivity and specificity of the developed system was established with human blood and finally used to identify the source of mosquito blood meal which is an important parameter in epidemiological studies, particularly in determining the role of mosquito in malaria transmission. The time duration of standardized assay with developed polymer coated strips was cut down to one hour compared to the 3-4h required in usual dot-ELISA.
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PMID:Glycidyl methacrylate-co-N-vinyl-2-pyrrolidone coated polypropylene strips: synthesis, characterization and standardization for dot-enzyme linked immunosorbent assay. 1911 Jan 2

The entrapment of enzymes within biomimetic silica nanoparticles offers unique and simple immobilization protocols that merge the stability of proteins confined in solid phases with the high loading and reduced diffusion limitations inherent to nano-sized structures. Herein, we report on the biomimetic silica entrapment of chemically derivatized horseradish peroxidase for amperometric sensing applications. Scanning electron microscopy shows evidence of the formation of enzyme-modified nanospheres using poly(ethylenimine) as a template for silicic acid condensation. When these nanospheres are directly deposited on graphite electrodes, chemically modified anionic peroxidase shows direct electron transfer at 0 mV vs Ag|AgCl. Microgravimetric measurements as well as SEM images demonstrate that negatively charged peroxidase is also entrapped when silica precipitates at gold electrodes are modified with a self-assembled monolayer of poly(ethylenimine). Electrostatic interactions may play a crucial role for efficient enzyme entrapment and silica condensation at the PEI template monolayer. The in-situ biomimetically synthesized peroxidase nanospheres are catalytically active, enabling direct bioelectrocatalysis at 0 mV vs Ag|AgCl with long-term stability.
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PMID:Enzyme-modified nanoparticles using biomimetically synthesized silica. 1954 Jan 73

The topography and morphology of retinal ganglion cells (RGCs) in the eastern newt were studied. Cells were retrogradely labeled with tetramethylrhodamine-conjugated dextran amines or horseradish peroxidase and examined in retinal wholemounts. Their total number was 18,025 +/- 3,602 (mean +/- SEM). The spatial density of RGCs varied from 2,100 cells/mm(2) in the retinal periphery to 4,500 cells/mm(2) in the dorsotemporal retina. No prominent retinal specializations were found. The spatial resolution estimated from the spatial density of RGCs varied from 1.4 cycles per degree in the periphery to 1.95 cycles per degree in the region of the peak RGC density. A sample of 68 cells was camera lucida drawn and subjected to quantitative analysis. A total of 21 parameters related to RGC morphology and stratification in the retina were estimated. Partitionings obtained by using different clustering algorithms combined with automatic variable weighting and dimensionality reduction techniques were compared, and an effective solution was found by using silhouette analysis. A total of seven clusters were identified and associated with potential cell types. Kruskal-Wallis ANOVA-on-Ranks with post hoc Mann-Whitney U tests showed significant pairwise between-cluster differences in one or more of the clustering variables. The average silhouette values of the clusters were reasonably high, ranging from 0.52 to 0.79. Cells assigned to the same cluster displayed similar morphology and stratification in the retina. The advantages and limitations of the methodology adopted are discussed. The present classification is compared with known morphological and physiological RGC classifications in other salamanders.
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PMID:Retinal ganglion cells in the eastern newt Notophthalmus viridescens: topography, morphology, and diversity. 1967 5

Fe(3)O(4) magnetic nanoparticles (Fe(3)O(4) MNPs) with much improved peroxidase-like activity were successfully prepared through an advanced reverse co-precipitation method under the assistance of ultrasound irradiation. The characterizations with XRD, BET and SEM indicated that the ultrasound irradiation in the preparation induced the production of Fe(3)O(4) MNPs possessing smaller particle sizes (16.5nm), greater BET surface area (82.5m(2)g(-1)) and much higher dispersibility in water. The particle sizes, BET surface area, chemical composition and then catalytic property of the Fe(3)O(4) MNPs could be tailored by adjusting the initial concentration of ammonia water and the molar ratio of Fe(2+)/Fe(3+) during the preparation process. The H(2)O(2)-activating ability of Fe(3)O(4) MNPs was evaluated by using Rhodamine B (RhB) as a model compound of organic pollutants to be degraded. At pH 5.4 and temperature 40 degrees C, the sonochemically synthesized Fe(3)O(4) MNPs were observed to be able to activate H(2)O(2) and remove ca. 90% of RhB (0.02mmolL(-1)) in 60min with a apparent rate constant of 0.034min(-1) for the RhB degradation, being 12.6 folds of that (0.0027min(-1)) over the Fe(3)O(4) MNPs prepared via a conventional reverse co-precipitation method. The mechanisms of the peroxidase-like catalysis with Fe(3)O(4) MNPs were discussed to develop more efficient novel catalysts.
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PMID:Sono-assisted preparation of highly-efficient peroxidase-like Fe(3)O(4) magnetic nanoparticles for catalytic removal of organic pollutants with H(2)O(2). 1994 1

Blood is a waste product of the slaughter industry, while its main component hemoglobin (Hb) is a pseudo-peroxidase which is able to oxidize polycyclic aromatic hydrocarbons (PAHs) in the presence of H(2)O(2). In order to use Hb for wastewater treatment, we encapsulated it in silica-based matrix by modified "fish-in-net" approach. The as-synthesized catalysts were characterized by SEM, TEM, BET and solid-state UV-vis spectroscopy. It was found that Hb was partially homogeneously dispersed in microspheres and showed more stable peroxidase-like activity than free Hb. Moreover, it had substantially increased storage stability as well as pH stability. It was used as biocatalyst to remove aniline in aqueous solution and gave a reduction of 65% aniline removal, while 76% in the presence of additive PEG. No significant activity loss was observed after ten runs. These experimental results suggest that the resultant product was a promising biocatalyst for aromatic wastewater treatment.
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PMID:Hemoglobin immobilized with modified "fish-in-net" approach for the catalytic removal of aniline. 2248 38

Flaxseed (Linum usitatissimum L.) fibers were modified by oxidoreductive and cellulolytic enzymes. The lignin amount and intrinsic plant peroxidase activity was evaluated by histochemical and spectrophotometric assays. Peroxidase activity was not found from bast fibers. The flaxseed fibers were further separated and treated with laccase to conjugate the model compounds, that is, the hydrophobic gallate molecules on fiber surfaces. Laccase was able to slowly oxidize fiber-conjugated phenolics, but no fundamental changes in fiber cell surface structure or notable coupling of the applied hydrophobic gallate molecules onto the fibers occurred, as revealed by Fourier transform infrared spectroscopy. The reactivity of the mature fibers was further investigated using cellulolytic enzymes. Cellobiohydrolase (CBH) and endoglucanase (EG)-rich enzyme preparations were applied to reach a hydrolysis degree of 1-6% (of the fiber dry matter) using a standard enzyme dosage. The CBH mixture altered the fiber surface morphology distinctly, and SEM images illustrated fibers in which the cellulose fibrils seemed to be loosened and partially hydrolyzed. In contrast, the effect of the EG-rich preparation without CBH activity was notable on the fiber surface, polishing the surfaces. The cellulolytic treatments were potentially interesting for specific enzymatic modifications of flax fiber surfaces, whereas the approach to use oxidoreductive enzyme treatments on mature linseed fibers offered little potential, obviously due to the low lignin content of the fibers.
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PMID:Enzymatic modification of flaxseed fibers. 2309 92


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