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A method of fluorescent nanoparticle-based indirect immunofluorescence assay using either fluorescence microscopy or flow cytometry for the rapid detection of pathogenic Escherichia coli O157:H7 was developed. The dye-doped silica nanoparticles (NPs) were synthesized using W/O microemulsion methods with the combination of 3-aminopropyltriethoxysilane (APTES) and fluorescein isothiocyanate (FITC) and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers and the surface coverage of Protein A on NPs was determined by the Bradford method. Rabbit anti- E. Coli O157:H7 antibody was used as primary antibody to recognize E. coli O157:H7 and then antibody binding protein (Protein A) labeled with FITC-doped silica NPs (FSiNPs) was used to generate fluorescent signal. With this method, E. Coli O157:H7 in buffer and bacterial mixture was detected. In addition, E. coli O157:H7 in several spiked background beef samples were measured with satisfactory results. Therefore, the FSiNPs are applicable in signal-amplified bioassay of pathogens due to their excellent capabilities such as brighter fluorescence and higher photostability than the direct use of conventional fluorescent dyes. [ABSTRACT FROM AUTHOR]

Microextrusion is becoming increasingly important for the manufacturing of microcomponents. However, this reduction in scale to a microlevel means that the influence of friction and the need for suitable lubrication are greatly increased. This study therefore looks at the use of a low-friction and highly wear resistant Graphit-ic coating on the mold-forming section of a microextrusion mold, this coating being applied by a closed-field unbalanced magnetron sputter ion plating technique. A microcup of CuZn33 brass alloy was then extruded, with a wall thickness of 0.45 mm, outside diameter of 2.9 mm, and an internal diameter of 2 mm. The experimental results in which extrusion uses the mold coating with Graphit-ic film are compared against the experimental results in which extrusion uses the mold uncoating with Graphit-ic film. This showed that the load was decreased a lot and the self-lubricating solid coating facilitates a smooth extrusion process. As the extrusion rate was quite high, smoothed particle hydrodynamics method simulations of the extrusion process were conducted, these being then compared with the experimental results. These result showed that the SPH simulation can be applied to show the deformation of materials and predict the load trend. [ABSTRACT FROM AUTHOR]

We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation. [ABSTRACT FROM AUTHOR]

Abstract: In this work, we have prepared three types of aptamer-conjugated Rubpy-doped silica nanoparticles for Human breast carcinoma MCF-7 cells labeling. Probe A is prepared through covalent conjugation between amine-labeled MUC-1 aptamer and carboxyl-modified Rubpy-doped NPs (NPs-aptamer). Probe B is prepared based on the interaction between biotin-labeled MUC-1 aptamer and avidin-conjugated Rubpy-doped NPs (NPs-avidin-biotin-aptamer). For Probe C, there is a PEG with flexible long chain as the bridge between avidin and the NPs (NPs-PEG-avidin-biotin-aptamer). In addition, we further investigate the practical number of MUC-1 aptamers on an NP of each probe using hoechst33258 dye. The binding efficiency of MUC-1 aptamer on the three types of probes as follows: Probe A < Probe B < Probe C. In addition, microscopic fluorescence imaging shows that Probe C containing the PEG molecules can be effectively applied for the recognition of MUC-1 protein in human breast carcinoma MCF-7 cells thus demonstrates that the PEG with flexible long chain as the bridge between the aptamer and NP can greatly enhances the freedom of MUC-1 aptamer. Compared with common organic dyes, the dye-doped silica nanoparticles serve as a stable bioprobe because of their facile conjugation with the desirable biomolecules, and have exhibited great potential in bioanalysis. [Copyright &y& Elsevier]

Abstract: Progress in biomedical imaging depends on the development of bioprobes with a high sensitivity and stability. Fluorescent silica nanoparticles (NPs) covalent conjugation of avidin has been proposed for cancer cells imaging by fluorescence microscopy. Uniform silica NPs were prepared using water-in-oil (W/O) microemulsion methods and primary amine groups were introduced onto the surface of the NPs by condensation of tetraethyl orthosilicate (TEOS). Optically stable organic dyes, tris(2,2''-bipyridyl) dichlororuthenium(II) hexahydrate (Rubpy), were doped inside the silica NPs. The amine functions were transferred to carboxyl groups coupled with a linker elongation. Avidin was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified by titration with biotin(5-fluorescein) conjugate to 1.25 biotin binding sites/100nm2. We used biotinylated antibody and cell recognition by fluorescence microscopy imaging technique. The lung carcinoma cells were identified easily with high efficiency using these antibody-coated NPs. By comparison with fluorescein isothiocyanate (FITC), dye-doped silica NPs display dramatically increased stability of fluorescence as well as photostability, as compared to the common organic dye, when under continuous irradiation. [Copyright &y& Elsevier]

Abstract: In this work, we have developed a simple and sensitive method for ATP detection using silica nanoparticles (NPs) as the platform and hoechst33258 as the signal reporter. The ATP-binding aptamers hybridize with the probe DNA (DNAp) immobilized NPs to form the aptamer/DNAp duplex on the NPs surface. The conformational change of the aptamer leads to the decrease of the aptamer/DNAp duplex on the NPs due to the ATP-binding aptamer switches its structure from the aptamer/DNAp duplex to the aptamer/target complex in the presence of ATP. ATP detection can be easily realized by separating the silica nanoparticles and adding the hoechst33258 of intercalating to aptamer/DNAp (dsDNA). Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between ATP aptamer and ATP. The K d was estimated to be ∼1mM from 0 to 4mM and a liner response was observed from 0 to 0.2mM with a detection limit of ∼20μM. Compared with other methods, the carboxyl-modified silica nanoparticles (∼60nm) prepared by the reverse microemulsion method can serve as a stable and sensitive sensor platform because of their smaller size and facile conjugation with amine-containing molecules. In addition, the high sensitivity and selectivity of hoechst33258 was employed for the ssDNA and dsDNA determination, which takes advantage of the label-free aptamer and lower cost. [Copyright &y& Elsevier]

A new method for real-time detection of carcinoembryonic antigen (CEA) in human serum with high sensitivity and selectivity using surface plasmon resonance (SPR) biosensor was developed. Two kinds of antibodies were used to recognize CEA at different epitopes with high affinity and specificity. Gold nanoparticles (GNPs) modified with streptavidin (SA) were used to further enhance signal specifically via biotin–streptavidin interaction. The binding capacity of the streptavidin-modified gold nanoparticles (SA–GNPs) for ligand biotin was quantified by titration with biotin (5-fluorescein) conjugate to be 10.54 biotin binding sites per 100 nm 2 . The developed GNPs enhanced sandwich SPR biosensor successfully fulfilled the sensitive detection of CEA in the range of 1–60 ng/mL with a detection limit of 1.0 ng/mL. Compared to the direct assay format, sandwich format without GNPs and SA–GNPs enhanced sandwich format led to 4.2-fold and 13.8-fold in the sensitivity, respectively. This sensor also showed good selectivity for CEA in the interference study. The results demonstrated that the proposed method could provide a high sensitivity and selectivity in the detection of CEA and offer a promising alternative for cancer biomarker than traditional clinical examinations. [ABSTRACT FROM AUTHOR]

Abstract: The effect of methylene blue/TiO2 nanocomposites on Staphylococcus aureus was evaluated by a LKB-2277 Bioactivity Monitor at 37.0°C. By analyzing the thermogenic power–time curves, kinetic parameters such as the growth rate constant (k), the maximum heat power of growth phase (P m), generation time (t G) and the inhibitory ratio (I) were determined. The k and P m decrease while t G and I increases with increasing concentrations of the methylene blue/TiO2 nanocomposites added into the microbe suspension. Both k and I have linear correlations with the concentration of the methylene blue/TiO2 nanocomposites. The effect of the methylene blue sensitized-TiO2 nanocomposites on the cell damage of S. aureus was investigated by microscopic method. [Copyright &y& Elsevier]