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1.
Macromol Biosci ; : e2000049, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32253822

RESUMO

Cationic polymers exhibit high cytotoxicity via strong interaction with cell membranes. To reduce cell membrane damage, a hydrophilic polymer is introduced to the cationic nanoparticle surface. The hydrophilic polymer coating of cationic nanoparticles resulted in a nearly neutral nanoparticle. These particles are applied to mouse fibroblast (3T3) and human cervical adenocarcinoma (Hela) cells. Interestingly, nanoparticles with a long cationic segment decrease cell activity regardless of cell type, while those with a short segment only affect 3T3 cell activity at lower concentrations less than 500 µg mL-1 . Most nanoparticles are located inside 3T3 cells but on the cell membrane of Hela cells. The short cationic nanoparticle shows negligible cell membrane damage despite its high accumulation on Hela cell membranes. Cell activity changed by hydrophilic polymer-coated cationic nanoparticles is caused by incorporated nanoparticle accumulation in the cells, not cell membrane damage. To suppress the cytotoxicity from the cationic polymer, cationic nanoparticle needs to completely cover with hydrophilic polymer so as not to exhibit the cationic effect and applies to cell with low concentrations to reduce the nonselective cytotoxicity from the cationic polymer.

2.
Biosens Bioelectron ; 151: 111969, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999579

RESUMO

We report a peptide-based sensor that involves a multivalent interaction with L-ascorbate 6-phosphate lactonase (UlaG), a protein marker of Streptococcus pneumonia. By integrating the antifouling feature of the sensor, we significantly improved the signal-to-noise ratio of UlaG detection. The antifouling surface was fabricated via electrodeposition using an equivalent mixture of 4-amino-N,N,N-trimethylanilinium and 4-aminobenzenesulfonate. This antifouling layer not only effectively reduces the non-specific adsorption on the biosensor but also decreases the charge transfer resistance (Rct) of the screen-printed carbon electrode. The aniline-modified S7 peptide, an UlaG-binding peptide, was pre-synthesized and further electrochemically modified to bind onto the antifouling layer. Bio-electrochemical analysis confirms that the antifouling S7-peptide sensor binds strongly to the UlaG with a dissociation constant (Kd) = 0.5 nM. This strong interaction can be attributed to a multivalent interaction between the biosensor and the heximeric form of UlaG. To demonstrate the potential for clinical application, further detection of Streptococcus pneumonia from 50 to 5×104 CFU/mL were successfully performed in 25% human serum.

3.
Chembiochem ; 20(2): 237-240, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30239102

RESUMO

Many circulating cancer-related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans-particularly heparan sulfate-at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF-1 and FGF-2. Taking note of the typical pose of l-iduronic acid, we conceived of per-O-sulfonated analogues of such disaccharides, and devised a single-step procedure for per-O-sulfonation of unprotected sugars with concomitant 1,6-anhydro bridge formation to achieve such compounds through direct use of SO3 ⋅Et3 N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF-1 and FGF-2 mostly at the sub-micromolar level, although the tetrasaccharide analogue achieved low-nanomolar binding with FGF-2.


Assuntos
Fatores de Crescimento de Fibroblastos/química , Heparitina Sulfato/química , Açúcares/química , Configuração de Carboidratos
4.
Sensors (Basel) ; 19(1)2018 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-30577674

RESUMO

Boronic acids (BAs) provide strong potential in orientation immobilization of antibody and the modification method is crucial for efficiency optimization. A highly effective method has been developed for rapid antibody immobilization on gold electrodes through the electrodeposition of a BA⁻containing linker in this study. Aniline-based BA forms a condense layer while antibody could automatically immobilize on the surface of the electrode. Compare to traditional self-assembled monolayer method, the electrodeposition process dramatically reduces the modification time from days to seconds. It also enhances the immobilized efficiency from 95 to 408 (ng/cm²) with a strong preference being exhibited for shorter aniline-based linkers.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais , Ácidos Borônicos/química , Técnicas de Microbalança de Cristal de Quartzo , Adsorção , Compostos de Anilina/química , Anticorpos Imobilizados/imunologia , Eletrodos , Ouro/química , Humanos
5.
Nanomaterials (Basel) ; 8(8)2018 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127303

RESUMO

There are few reports on zero-field-cooled (ZFC) magnetization measurements for Fe@FeOx or FeOx particles synthesized by laser ablation in liquids (LAL) of Fe, and the minimum blocking temperature (TB) of 120 K reported so far is still much higher than those of their counterparts synthesized by chemical methods. In this work, the minimum blocking temperature was lowered to 52 K for 4⁻5 nm α-Fe2O3 particles synthesized by femtosecond laser ablation of Fe in acetone. The effective magnetic anisotropy energy density (Keff) is calculated to be 2.7⁻5.4 × 105 J/m³, further extending the Keff values for smaller hematite particles synthesized by different methods. Large amorphous-Fe@α-Fe2O3 and amorphous-Fe@C particles of 10⁻100 nm in diameter display a soft magnetic behavior with saturation magnetization (Ms) and coercivities (Hc) values of 72.5 emu/g and 160 Oe at 5 K and 61.9 emu/g and 70 Oe at 300 K, respectively, which mainly stem from the magnetism of amorphous Fe cores. Generally, the nanoparticles obtained by LAL are either amorphous or polycrystalline, seldom in a single-crystalline state. This work also demonstrates the possibility of synthesizing single-crystalline α-Fe2O3 hematite crystals of several nanometers with (104), (113), (116) or (214) crystallographic orientations, which were produced simultaneously with other products including carbon encapsulated amorphous Fe (a-Fe@C) and Fe@FeOx core-shell particles by LAL in one step. Finally, the formation mechanisms for these nanomaterials are proposed and the key factors in series events of LAL are discussed.

6.
ACS Omega ; 3(9): 10953-10966, 2018 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31459206

RESUMO

Laser ablation in liquids (LAL) offers a facile technique to develop a large variety of surfactant-free nanomaterials with high purity. However, due to the difficulty in the control of the particle synthesis process, the as-prepared nanomaterials always have a broad size distribution with a large polydispersity (σ). Surfactant-free properties can also cause problems with particle growth, which further increases the difficulty in size control of the colloids. Therefore, searching for strategies to simultaneously unify the sizes of colloids and inhibit particle growth has become significantly important for LAL-synthesized nanomaterials to be extensively used for biological, catalytic, and optical applications, in which fields particle size plays an important role. In this work, we present a facile way to simultaneously realize these two goals by ex situ SU-8 photoresist functionalization. Ag nanoparticles (NPs) synthesized by femtosecond laser ablation of silver in acetone at laser powers of 300 and 600 mW were used as starting materials. The synthesized Ag NPs have a broad size distribution between 1 and 200 nm with an average size of ca. 5.9 nm and σ of 127-207%. After ex situ SU-8 functionalization and 6 months storage, most particles larger than 10 nm become aggregates and precipitate, which makes the size distribution narrow with an average diameter of 4-5 nm and σ of 48-78%. The precipitation process is accompanied by the decrease in colloid mass from the initial ∼0.2 to 0.10-0.11 mg after ex situ SU-8 functionalization and 6 months colloid storage. Morphology analysis indicates that ex situ SU-8 functionalization inhibits the particle growth into polygonal nanocrystals. Radical polymerization of SU-8 on Ag NPs is considered to be the reason for both spontaneous size separation and growth inhibition phenomena. Benefiting from Ag NPs embedment and acetone dissolution, the glass-transition temperature of SU-8 photoresist increased from 314 to 331 °C according to thermogravimetric analysis. The universality of ex situ SU-8 functionalization-induced growth inhibition and size separation behaviors is further proved using the Au colloids generated by LAL in acetone. This work is expected to provide a new route for better size control of LAL-synthesized colloids via ex situ photoresist functionalization, although a half of colloidal mass is wasted due to radical polymerization-induced colloidal precipitation.

7.
Biotechnol Lett ; 39(3): 407-413, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27888365

RESUMO

OBJECTIVES: A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv). RESULTS: Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine. CONCLUSIONS: The recombinant scFv could detect Neisseria strains at 106 CFU/ml.


Assuntos
Anticorpos Antibacterianos/imunologia , Neisseria/imunologia , Engenharia de Proteínas/métodos , Especificidade de Anticorpos/imunologia , Eletroforese em Gel de Poliacrilamida , Ouro/química , Nanopartículas/química , Anticorpos de Cadeia Única/metabolismo , Especificidade da Espécie
8.
Appl Microbiol Biotechnol ; 100(19): 8411-24, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27198725

RESUMO

Glycosyltransferase-1 from Bacillus cereus (BcGT1) catalyzes a reaction that transfers a glucosyl moiety to flavonoids, such as quercetin, kaempferol, and myricetin. The enzymatic glucosidation shows a broad substrate specificity when the reaction is catalyzed by wild-type BcGT1. Preliminary assays demonstrated that the F240A mutant significantly improves the regioselectivity of enzymatic glucosidation toward quercetin. To unveil and further to control the catalytic function of BcGT1, mutation of F240 to other amino acids, such as C, E, G, R, Y, W, and K, was performed. Among these mutants, F240A, F240G, F240R, and F240K greatly altered the regioselectivity. The quercetin-3-O-glucoside, instead of quercetin-7-O-glucoside as for the wild-type enzyme, was obtained as the major product. Among these mutants, F240R showed nearly 100 % product specificity but only retained 25 % catalytic efficiency of wild-type enzyme. From an inspection of the protein structure, we found two other amino acids, F132 and F138, together with F240, are likely to form a hydrophobic binding region, which is sufficiently spacious to accommodate substrates with varied aromatic moieties. Through the replacement of a phenylalanine by a tyrosine residue in the substrate-binding region, the mutants may be able to fix the orientation of flavonoids, presumably through the formation of a hydrogen bond between substrates and mutants. Multiple mutants-F240R_F132Y, F240R_F138Y, and F240R_F132Y_F138Y-were thus constructed for further investigation. The multiple points of mutants not only maintained the high product specificity but also significantly improved the catalytic efficiency, relative to F240R. The same product specificity was obtained when kaempferol and myricetin were used as a substrate.


Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Bacillus cereus/enzimologia , Flavonoides/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Glicosilação , Glicosiltransferases/química , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformação Proteica , Especificidade por Substrato
10.
PLoS One ; 11(5): e0155905, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27214294

RESUMO

Streptococcus pneumoniae, a penicillin-sensitive bacterium, is recognized as a major cause of pneumonia and is treated clinically with penicillin-based antibiotics. The rapid increase in resistance to penicillin and other antibiotics affects 450 million people globally and results in 4 million deaths every year. To unveil the mechanism of resistance of S. pneumoniae is thus an important issue to treat streptococcal disease that might consequently save millions of lives around the world. In this work, we isolated a streptococci-conserved L-ascorbate 6-phosphate lactonase, from S. pneumoniae ATCC 49136. This protein reveals a metallo-ß-lactamase activity in vitro, which is able to deactivate an ampicillin-based antibiotic by hydrolyzing the amide bond of the ß-lactam ring. The Michaelis parameter (Km) = 25 µM and turnover number (kcat) = 2 s(-1) were obtained when nitrocefin was utilized as an optically measurable substrate. Through confocal images and western blot analyses with a specific antibody, the indigenous protein was recognized in S. pneumoniae ATCC 49136. The protein-overexpressed S. pneumonia exhibits a high ampicillin-tolerance ability in vivo. In contrast, the protein-knockout S. pneumonia reveals the ampicillin-sensitive feature relative to the wild type strain. Based on these results, we propose that this protein is a membrane-associated metallo-ß-lactamase (MBL) involved in the antibiotic-resistant property of S. pneumoniae.


Assuntos
Resistência a Ampicilina , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Técnicas de Inativação de Genes , Hidrólise , Streptococcus pneumoniae/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo
11.
ACS Appl Mater Interfaces ; 8(22): 13714-23, 2016 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-27198164

RESUMO

Fluorescent silicon quantum dots (SiQDs) have shown a great potential as antiphotobleaching, nontoxic and biodegradable labels for various in vitro and in vivo applications. However, fabricating SiQDs with high water-solubility and high photoluminescence quantum yield (PLQY) remains a challenge. Furthermore, for targeted imaging, their surface chemistry has to be capable of conjugating to antibodies, as well as sufficiently antifouling. Herein, antibody-conjugated SiQD nanoparticles (SiQD-NPs) with antifouling coatings composed of bovine serum albumin (BSA) and polyethylene glycol (PEG) are demonstrated for immunostaining on live cancer cells. The monodisperse SiQD-NPs of diameter about 130 nm are synthesized by a novel top-down method, including electrochemical etching, photochemical hydrosilylation, high energy ball milling, and "selective-etching" in HNO3 and HF. Subsequently, the BSA and PEG are covalently grafted on to the SiQD-NP surface through presynthesized chemical linkers, resulting in a stable, hydrophilic, and antifouling organic capping layer with isothiocyanates as the terminal functional groups for facile conjugation to the antibodies. The in vitro cell viability assay reveals that the BSA-coated SiQD-NPs had exceptional biocompatibility, with minimal cytotoxicity at concentration up to 1600 µg mL(-1). Under 365 nm excitation, the SiQD-NP colloid emits bright reddish photoluminescence with PLQY = 45-55% in organic solvent and 5-10% in aqueous buffer. Finally, through confocal fluorescent imaging and flow cytometry analysis, the anti-HER2 conjugated SiQD-NPs show obvious specific binding to the HER2-overexpressing SKOV3 cells and negligible nonspecific binding to the HER2-nonexpressing CHO cells. Under similar experimental conditions, the immunofluorescence results obtained with the SiQD-NPs are comparable to those using conventional fluorescein isothiocyanate (FITC).


Assuntos
Nanopartículas , Pontos Quânticos , Coloração e Rotulagem/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Nanopartículas/toxicidade , Polietilenoglicóis/química , Soroalbumina Bovina/química , Silício
12.
Appl Microbiol Biotechnol ; 100(10): 4459-71, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26795959

RESUMO

Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-ß-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are ß-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.


Assuntos
Bacillus cereus/enzimologia , Proteínas de Bactérias/metabolismo , Glucosídeos/metabolismo , Glicosiltransferases/metabolismo , Bacillus cereus/genética , Proteínas de Bactérias/genética , Compostos de Benzil/metabolismo , Cumarínicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Estradiol/metabolismo , Glicosiltransferases/genética , Espectroscopia de Ressonância Magnética , Purinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Uridina Difosfato Glucose/metabolismo
13.
Chem Commun (Camb) ; 51(74): 14080-3, 2015 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-26251847

RESUMO

We report a novel 'fluorescent dopamine' that possesses essential features of natural dopamine. Our method is simple and is readily extended to monoamine neurotransmitters such as L-norepinephrine, serotonin and GABA, providing a more practical approach. Because of its compatibility with sensitive fluorescent measurements, we envisage that our approach will have a broad range of applications in neural research.


Assuntos
Dopamina/metabolismo , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Imagem Molecular/métodos , Neurotransmissores/metabolismo , Transmissão Sináptica , Animais , Transporte Biológico , Células CHO , Cricetulus , Fluorometria/métodos , Células PC12 , Ratos
14.
Protein Expr Purif ; 115: 132-40, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26166179

RESUMO

ß-Xylosidases catalyze the breakdown of ß-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 ß-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5' end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70°C and at pH 4.0-4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the ß-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 ß-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry.


Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes/metabolismo , Xilosidases/metabolismo , Clonagem Molecular , Códon/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Hidrólise , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Xilanos , Xilosidases/química , Xilosidases/genética , Xilosidases/isolamento & purificação
15.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 9): 1228-31, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25195897

RESUMO

Glycosyltransferases (GTs), which are distributed widely in various organisms, including bacteria, fungi, plants and animals, play a role in synthesizing biological compounds. Glycosyltransferase-1 from Bacillus cereus (BcGT-1), which is capable of transferring glucose to small molecules such as kaempferol and quercetin, has been identified as a member of the family 1 glycosyltransferases which utilize uridine diphosphate glucose (UDP-glucose) as the sugar donor. BcGT-1 (molecular mass 45.5 kDa) has been overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. According to X-ray diffraction of BcGT-1 crystals to 2.10 Šresolution, the crystal belonged to space group P1, with unit-cell parameters a = 54.56, b = 84.81, c = 100.12 Å, α = 78.36, ß = 84.66, γ = 84.84°. Preliminary analysis indicates the presence of four BcGT-1 molecules in the asymmetric unit with a solvent content of 50.27%.


Assuntos
Bacillus cereus/enzimologia , Glicosiltransferases/química , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida
16.
Biosens Bioelectron ; 60: 101-11, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-24787124

RESUMO

Silicon nanowire field effect transistors (SiNW-FETs) have shown great promise as biosensors in highly sensitive, selective, real-time and label-free measurements. While applications of SiNW-FETs for detection of biological species have been described in several publications, less attention has been devoted to summarize the conjugating methods involved in linking organic bio-receptors with the inorganic transducer and the strategies of improving the sensitivity of devices. This article attempts to focus on summarizing the various organic immobilization approaches and discussing various sensitivity improving strategies, that include (I) reducing non-specific binding, (II) alignment of the probes, (III) enhancing signals by charge reporter, (IV) novel architecture structures, and (V) sensing in the sub-threshold regime.


Assuntos
Técnicas Biossensoriais/instrumentação , Condutometria/instrumentação , Eletrodos , Nanotecnologia/instrumentação , Nanofios/química , Silício/química , Transistores Eletrônicos , Condutividade Elétrica , Desenho de Equipamento , Análise de Falha de Equipamento , Nanofios/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
17.
Chem Commun (Camb) ; 50(51): 6793-6, 2014 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-24836378

RESUMO

A new approach to immobilize zwitterionic molecules rapidly and highly efficiently on a gold surface applies aniline-based electrodeposition. The zwitterion-functionalized antifouling surface enables a decrease of the adsorption of non-specific proteins by 95% from fetal bovine serum (FBS, 10%).


Assuntos
Técnicas Biossensoriais , Galvanoplastia/métodos , Ouro/química , Proteínas/isolamento & purificação , Compostos de Anilina/química , Imunofluorescência , Indicadores e Reagentes , Soroalbumina Bovina/química
18.
Lab Chip ; 13(20): 4078-86, 2013 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-23966230

RESUMO

This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.


Assuntos
Técnicas de Cultura de Células/métodos , Proteínas da Matriz Extracelular , Lasers , Microtecnologia/métodos , Sobrevivência Celular , Vidro/química , Células HeLa , Humanos , Propriedades de Superfície , Fatores de Tempo , Água/química
19.
Carbohydr Polym ; 93(2): 615-21, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23499103

RESUMO

An entomopathogenic fungus, Paecilomyces lilacinus, was found to grow on chitosanase-detecting plates. Besides an endo-chitosanase, an exo-ß-D-glucosaminidase was purified by cation-exchange chromatography from this microorganism cultivated in M9 minimal media containing 0.5% chitosan as the sole carbon source. The molecular weight of the enzyme is 95kDa; the optimum pH and temperature for activity are 6.0 and 45°C, respectively. The purified exo-ß-D-GlcNase promotes the hydrolysis of 95% deacetylated chitosan from its non-reducing end and liberates 2-amino-2-deoxy-D-glucopyranose (GlcN) as the sole product; however, 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) was not detected when chitin was used as the substrate. The cleavage pattern confirmed using real-time mass spectrometry shows that exo-ß-D-glucosaminidase cleaves the glycosidic bonds between GlcN-GlcN and GlcN-GlcNAc but not between GlcNAc-GlcN or GlcNAc-GlcNAc. In the presence of a 10% solution of various alcohols, many alkyl-ß-D-glucosaminides were obtained, indicating that exo-ß-D-glucosaminidase is a retaining enzyme.


Assuntos
Proteínas Fúngicas/isolamento & purificação , Hexosaminidases/isolamento & purificação , Paecilomyces/enzimologia , Acetilação , Quitina/metabolismo , Quitosana/metabolismo , Meios de Cultura/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Indução Enzimática , Estabilidade Enzimática , Proteínas Fúngicas/biossíntese , Glicosilação , Hexosaminidases/biossíntese , Hidrólise , Micélio/enzimologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Estereoisomerismo , Especificidade por Substrato
20.
Chem Asian J ; 7(12): 2848-53, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23012064

RESUMO

An improved method for the synthesis of high-performance and water-soluble quantum dots (QDs) involving the encapsulation of mercaptosuccinic acid coated QDs (MSA-QDs) with poly(diallyldimethylammonium chloride) (PDDA) followed by their direct photoactivation with fluorescent radiation near 295 K to yield PDDA-coated QDs (PDDA-QDs) has been demonstrated. The quantum yield (QY) of the PDDA-QDs was significantly improved from 0.6 (QY of MSA-QDs) to 48%. By using this synthetic strategy, highly photoluminescent PDDA-QDs of varied size were readily prepared. The surface properties of PDDA-QDs and MSA-QDs were extensively characterized. The highly luminescent and positively charged PDDA-QDs serve as a useful and convenient tool for protein adsorption. With a Δ(5)-3-ketosteroid isomerase adsorbed PDDA-QD complex, the biorecognition of steroids was demonstrated through the application of fluorescent resonance energy transfer.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Substâncias Luminescentes/química , Pontos Quânticos , Esteroides/análise , Técnicas Biossensoriais/métodos , Enzimas Imobilizadas/metabolismo , Polietilenos/química , Compostos de Amônio Quaternário/química , Esteroide Isomerases/metabolismo , Tiomalatos/química
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