Fluorescent europium-modified polymer nanoparticles for rapid and sensitive anthrax sensors

Novel fluorescent polyacrylonitrile nanoparticles were synthesized by microemulsion polymerization and Schiff base modification. By further modification with europium, the polyacrylonitrile nanoparticles could be used as a highly sensitive and rapid sensor for Bacillus anthracis spore detection in aqueous solution. The europium-modified polyacrylonitrile nanoparticles were readily combined with dipicolinic acid as a unique biomarker of B. anthracis, leading to high fluorescence emission. These nanoparticles enabled ratiometric detection without instrument-specific calibration due to the internal fluorescence reference. Additionally, the europium-modified polyacrylonitrile nanoparticle sensors exhibited a remarkable limit of detection (10pM) for dipicolinic acid and outstanding selectivity (160×) over aromatic ligands in aqueous solution. The ultrafine nanoparticle sensor showed a high capability for detecting anthrax due to the increased surface area-to-volume ratio and enhanced dispersibility.     

In Vivo Imaging of Far-red Fluorescent Proteins after DNA Electrotransfer to Muscle Tissue

DNA electrotransfer to muscle tissue yields long-term, high levels of gene expression; showing great promise for future gene therapy. We want to characterize the novel far-red fluorescent protein Katushka as a marker for gene expression using time domain fluorescence in vivo imaging. Highly efficient transgenic expression was observed after DNA electrotransfer with 100-fold increase in fluorescent intensity. The fluorescent signal peaked 1 week after transfection and returned to background level within 4 weeks. Katushka expression was not as stable as GFP expression, which was detectable for 8 weeks. Depth and 3D analysis proved that the expression was located in the target muscle. In vivo bio-imaging using the novel Katushka fluorescent protein enables excellent evaluation of the transfection efficacy, and spatial distribution, but lacks long-term stability.     

Serum amyloid A 2.2 refolds into a octameric oligomer that slowly converts to a more stable hexamer

Serum amyloid A (SAA) is an inflammatory protein predominantly bound to high-density lipoprotein in plasma and presumed to play various biological and pathological roles. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a marginally stable hexamer at 4–20 °C, but becomes an intrinsically disordered protein at 37 °C. Here we show that when urea-denatured SAA2.2 is dialyzed into buffer (pH 8.0, 4 °C), it refolds mostly into an octameric species. The octamer transitions to the hexameric structure upon incubation from days to weeks at 4 °C, depending on the SAA2.2 concentration. Thermal denaturation of the octamer and hexamer monitored by circular dichroism showed that the octamer is ∼10 °C less stable, with a denaturation mid point of ∼22 °C. Thus, SAA2.2 becomes kinetically trapped by refolding into a less stable, but more kinetically accessible octameric species. The ability of SAA2.2 to form different oligomeric species in vitro along with its marginal stability, suggest that the structure of SAA might be modulated in vivo to form different biologically relevant species.     

Monitoring of the Cytoskeleton-Dependent Intracellular Trafficking of Fluorescent Iron Oxide Nanoparticles by Nanoparticle Pulse-Chase Experiments in C6 Glioma Cells

Iron oxide nanoparticles (IONPs) are used for various biomedical and therapeutic approaches. To investigate the uptake and the intracellular trafficking of IONPs in neural cells we have performed nanoparticle pulse-chase experiments to visualize the internalization and the fate of fluorescent IONPs in C6 glioma cells and astrocyte cultures. Already a short exposure to IONPs for 10 min at 4 °C (nanoparticle pulse) allowed binding of substantial amounts of nanoparticles to the cells, while internalization of IONPs into the cell was prevented. The uptake of bound IONPs and the intracellular trafficking was started by increasing the temperature to 37 °C (chase period). While hardly any cellular fluorescence nor any iron staining was detectable directly after the nanoparticle pulse, dotted cellular fluorescence and iron patterns appeared already within a few minutes after start of the chase incubation and became intensified in the perinuclear region during further incubation for up to 90 min. Longer chase incubations resulted in separation of the fluorescent coat from the core of the internalized IONPs. Disruption of actin filaments in C6 cells strongly impaired the internalization of IONPs, whereas destabilization of microtubules traped IONP-containing vesicles to the plasma membrane. In conclusion, nanoparticle pulse-chase experiments allowed to synchronize the cellular uptake of fluorescent IONPs and to identify for C6 cells an actin-dependent early and a microtubule-dependent later process in the intracellular trafficking of fluorescent IONPs.     

Biosynthesis of Stable Polyshaped Gold Nanoparticles from Microwave-Exposed Aqueous Extracellular Anti-malignant Guava (Psidium guajava) Leaf Extract

Addition of microwave-exposed aqueous extracellular anti-malignant guava (Psidium guajava) leaf extract to the aqueous gold chloride solution yielded stable polyshaped gold nanoparticles of high composition. Microwave-assisted route selected for the preparation of aqueous guava leaf extract was to suppress the enzymatic action. The formation of nanoparticles was understood from the UV–visible and X-ray diffraction studies. The size and shape analysis was done using field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. Zeta potential experiment shows that the bio-functionalized gold nanoparticles colloidal solution obtained as above will maintain its stability even after 30 weeks of storage. It is observed that the flavonoids which are separated during microwave heating of extracellular solution of the guava leaves are responsible for the biosynthesis of gold nanoparticles.     

Etoposide-loaded nanoparticles made from glyceride lipids: Formulation,characterization, in vitro drug release,and stability evaluation

The aim of the study was to prepare etoposide-loaded nanoparticles with glyceride lipids and then characterize and evaluate the in vitro steric stability and drug release characteristics and stability. The nanoparticles were prepared by melt emulsification and homogenization followed by spray drying of nanodispersion. Spray drying created powder nanoparticles with excellent redispersibility and a minimal increase in particle size (20–40 nm). Experimental variables, such as homogenization pressure, number of homogenization cycles, and surfactant concentration, showed a profound influence on the particle size and distribution. Spray drying of Poloxamer 407-stabilized nanodispersion lead to the formation of matrix-like structures surrounding the nanoparticles, resulting in particle growth. The in vitro steric stability test revealed that the lipid nanoparticles stabilized by sodium tauroglycocholate exhibit excellent steric stability compared with Poloxamer 407. All 3 glyceride nanoparticle formulations exhibited sustained release characteristics, and the release pattern followed the Higuchi equation. The spray-dried lipid nanoparticles stored in black polypropylene containers exhibited excellent long-term stability at 25°C and room light conditions. Such stable lipid nanoparticles with in vitro steric stability can be a beneficial delivery system for intravenous administration as long circulating carriers for controlled and targeted drug delivery. Published: September 30, 2005     

Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication

Waxy maize starch in an aqueous sulfuric acid solution (3.16 M, 14.7% solids) was hydrolyzed for 2–6 days, either isothermally at 40 °C or 4 °C, or at cycled temperatures of 4 and 40 °C (1 day each). The starch hydrolyzates were recovered as precipitates after centrifuging the dispersion (10,000 rpm, 10 min). The yield of starch hydrolyzates depended on the hydrolysis temperature and time, which varied from 6.8% to 78%. The starch hydrolyzed at 40 °C or 4/40 °C exhibited increased crystallinity determined by X-ray diffraction analysis, but melted in broader temperature range (from 60 °C to 110 °C). However, the starch hydrolyzed at 4 °C displayed the crystallinity and melting endotherm similar to those of native starch. The starch hydrolyzates recovered by centrifugation were re-dispersed in water (15% solids), and the dispersion was treated by an ultrasonic treatment (60% amplitude, 3 min). The ultrasonication effectively fragmented the starch hydrolyzates to nanoparticles. The hydrolyzates obtained after 6 days of hydrolysis were more resistant to the ultrasonication than those after 2 or 4 days, regardless of hydrolysis temperatures. The starch nanoparticles could be prepared with high yield (78%) and crystallinity by 4 °C hydrolysis for 6 days followed by ultrasonication. Scanning electron microscopy revealed that the starch nanoparticles had globular shapes with diameters ranging from 50 to 90 nm.     

Fluidity properties of isolated chloroplast thylakoid lipids

Chloroplast thylakoid lipids have been isolated free of photosynthetic pigments using a combination of high performance liquid and thin layer chromatography. The hydrophobic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) has been incorporated into aqueous dispersions of the isolated lipids in order to investigate dynamic and structural properties of the resulting bilayer membranes. Time dependent fluorescence anisotropy decays have been measured and analysed assuming the wobbling-in-cone model (Kinosita et al., Biophys J 20 (1977) 289–305). The DPH fluorescence lifetimes and the static and dynamic fluorescence anisotropy decay parameters for the probe in a total lipid mixture or in pure digalactosyldiacylglycerol (DGDG), changed in a predictable way with increasing temperature (10°–36°C). For a given temperature, it was found that the total lipid mixture was in general less ordered and showed greater dynamic motion as judged from DPH fluorescence anisotropy and compared with the pure DGDG system, although at 36°C differences in dynamic parameters were less evident. Overall the results obtained emphasize the highly fluid nature of thylakoid membrane lipids and give a basis for investigating how intrinsic proteins modify structural and dynamic properties of the in vivo membrane.     

β-Cyclodextrin polymer nanoparticles as carriers for doxorubicin and artemisinin: a spectroscopic and photophysical study

The association of doxorubicin (DOX) and artemisinin (ART) to a β-CyD-epichlorohydrin crosslinked polymer (pβ-CyD), organized in nanoparticles of ca. 15 nm size, was investigated in neutral aqueous medium by circular dichroism (CD), UV-vis absorption and fluorescence. The stability constants and the absolute CD spectra of the drug complexes were determined by global analysis of multiwavelength data from spectroscopic titrations. The polymer pβ-CyD proved able to disrupt the DOX dimer when the latter is the predominant form of DOX in solution. The spectroscopic and photophysical properties of the complexes evidenced an alcohol-like environment for ART and an improved inherent emission ability for DOX in the nanoparticle frame.     

Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts

Leaf extracts of two plants, Magnolia kobus and Diopyros kaki, were used for ecofriendly extracellular synthesis of metallic gold nanoparticles. Stable gold nanoparticles were formed by treating an aqueous HAuCl₄ solution using the plant leaf extracts as reducing agents. UV–visible spectroscopy was used for quantification of gold nanoparticle synthesis. Only a few minutes were required for >90% conversion to gold nanoparticles at a reaction temperature of 95 °C, suggesting reaction rates higher or comparable to those of nanoparticle synthesis by chemical methods. The synthesized gold nanoparticles were characterized with inductively coupled plasma spectrometry (ICP), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and particle analysis using a particle analyzer. SEM and TEM images showed that a mixture of plate (triangles, pentagons, and hexagons) and spherical structures (size, 5–300 nm) were formed at lower temperatures and leaf broth concentrations, while smaller spherical shapes were obtained at higher temperatures and leaf broth concentrations.     

A fluorescent substrate for the continuous assay of phosphatidylinositol-specific phospholipase C: synthesis and application of 2-naphthyl myo-inositol-1-phosphate.

A fluorescent water-soluble substrate for phosphatidylinositol-specific phospholipase C was synthesized. The diacylglycerol moiety of the natural substrate, phosphatidylinositol, was replaced by the fluorescent moiety, 2-naphthol, resulting in the synthetic substrate, racemic 2-naphthyl myo-inositol-1-phosphate. The synthetic substrate provided a continuous fluorometric assay for the phosphatidylinositol-specific phospholipase C from Bacillus cereus. Initial rates of the cleavage of the 2-naphthyl substrate by the phospholipase measured by fluorometry were linear with time and the amount of enzyme added. The specific enzyme activity at pH 8.5 and 25 degrees C was about 0.04 mumol/min mg protein at an initial substrate concentration of 0.8 mM. 31P NMR experiments suggest that, as with phosphatidylinositol itself, cleavage of the fluorescent substrate proceeds in two steps via a myo-inositol-1,2-cyclic phosphate intermediate, and that only the D-isomer is a substrate for the B. cereus phospholipase. The synthetic substrate was stable during long-term storage as a solid in the dark at -20 degrees C. It was also stable for several weeks when stored in the dark frozen in aqueous solution near neutral pH.     

Differently Environment Stable Bio-Silver Nanoparticles: Study on Their Optical Enhancing and Antibacterial Properties

Generally, limited research is extended in studying stability and applicational properties of silver nanoparticles (Ag NPs) synthesized by adopting ‘green chemistry’ protocol. In this work, we report on the synthesis of stable Ag NPs using plant-derived materials such as leaf extract of Neem (Azadirachta indica) and biopolymer pectin from apple peel. In addition, the applicational properties of Ag NPs such as surface-enhanced Raman scattering (SERS) and antibacterial efficiencies were also investigated. As-synthesized nanoparticles (NPs) were characterized using various instrumentation techniques. Both the plant materials (leaf extract and biopolymer) favored the synthesis of well-defined NPs capped with biomaterials. The NPs were spherical in shape with an average particle size between 14-27 nm. These bio-NPs exhibited colloidal stability in most of the suspended solutions such as water, electrolyte solutions (NaCl; NaNO₃), biological solution (bovine serum albumin), and in different pH solutions (pH 7; 9) for a reasonable time period of 120 hrs. Both the bio-NPs were observed to be SERS active through displaying intrinsic SERS signals of the Raman probe molecule (Nile blue A). The NPs were effective against the Escherichia coli bacterium when tested in nutrient broth and agar medium. Scanning and high-resolution transmission electron microscopy (SEM and HRTEM) images confirmed cellular membrane damage of nanoparticle treated E. coli cells. These environmental friendly template Ag NPs can be used as an antimicrobial agent and also for SERS based analytical applications.     

Fluorescence-based temperature control for polymerase chain reaction

The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95 °C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3 min and 45 s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29 ± 0.17 and 0.96 ± 0.26 °C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.     

Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.     

Synthesis,spectroscopic properties and protein labeling of water soluble 3,5-disubstituted boron dipyrromethenes

Sulfur-containing 3,5-disubstituted boron dipyrromethene (Bodipy^®) fluorescent probes with improved water solubility were synthesized. A dicarboxylic acid derivative that can be excited by the 543 nm HeNe laser line is very soluble in aqueous solution and retains high fluorescence quantum yield of the unionizable parent molecule. Conversion of the dicarboxylic acid to the succimidyl or sulfosuccinimidyl diester produces molecules capable of labeling proteins with a bright and stable fluorescence signal.     

Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles

Magnetic Fe₃O₄-chitosan nanoparticles are prepared by the coagulation of an aqueous solution of chitosan with Fe₃O₄ nanoparticles. The characterization of Fe₃O₄-chitosan is analyzed by FTIR, FESEM, and SQUID magnetometry. The Fe₃O₄-chitosan nanoparticles are used for the covalent immobilization of lipase from Candida rugosa using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The response surface methodology (RSM) was employed to search the optimal immobilization conditions and understand the significance of the factors affecting the immobilized lipase activity. Based on the ridge max analysis, the optimum immobilization conditions were immobilization time 2.14 h, pH 6.37, and enzyme/support ratio 0.73 (w/w); the highest activity obtained was 20 U/g Fe₃O₄-chitosan. After twenty repeated uses, the immobilized lipase retains over 83% of its original activity. The immobilized lipase shows better operational stability, including wider thermal and pH ranges, and remains stable after 13 days of storage at 25 °C.     

Some factors contributing to the survival of rapidly cooled plant cells

The stability of innocuous intracellularly frozen cells was found to be influenced remarkably by the surrounding solutions and the degree of prefreezing. In the cells suspended in isotonic or slightly hypertonic glucose solution, innocuous intracellular freezing readily occurred and these rapidly frozen cells remained stable even at −30 °C for 20 min, while cells prefrozen to −10 °C after suspending in water survived standing at −30 °C only for 5 sec at most. These facts suggest that when the innocuous intracellularly frozen cells are surrounded with concentrated sugar solutions, these cells remain far more stable than those surrounded with ice.     

G-quadruplex-forming oligonucleotide conjugated to magnetic nanoparticles: synthesis, characterization, and enzymatic stability assays

In the present work, we report the conjugation of superparamagnetic nanoparticles to a fluorescently labeled oligodeoxyribonucleotide (ODN) able to fold into stable unimolecular guanine quadruple helix under proper ion conditions by means of its thrombin-binding aptamer (TBA) sequence. The novel modified ODN, which contained a fluorescent dU(Py) unit at 3'-end and a 12-amino-dodecyl spacer (C(12)-NH(2)) at 5' terminus, was characterized by ESI-MS and optical spectroscopy (UV, CD, fluorescence), and analyzed by RP-HPLC chromatography and electrophoresis. From CD and fluorescence experiments, we verified that dU(Py) and C(12)-NH(2) incorporation does not interfere with the conformational stability of the G-quadruplex. Subsequently, the conjugation of the pyrene-labeled ODN with the magnetite particles was performed, and the ODN-conjugated nanoparticles were studied through optical spectroscopy (UV, CD, fluorescence) and by enzymatic and chemical assays. We found that the nanoparticles enhanced the stability of the TBA ODN to enzymatic degradation. Finally, we evaluated the amount of the TBA-conjugated nanoparticles immobilized on a magnetic separator in view of the potential use of the nanosystem for the magnetic capture of thrombin from complex mixtures.     

High-pressure SANS and fluorescence unfolding study of calmodulin

Apo-calmodulin, a small soluble mainly α protein, is a calcium-dependent protein activator. Calcium binding affects the calmodulin conformation but also its stability. Calcium free form unfolds between 40 and 80 °C, whereas the calcium-saturated form is stable up to temperatures as high as 100 °C, forbidding comparison of the thermal unfolding pathways of the two forms. Thus, this paper focuses especially on the conformation of pressure-induced unfolding states of both forms of calmodulin, by combining small-angle neutron scattering (SANS) with biophysical techniques such as tyrosines and ANS fluorescence. In contrast to heat denaturation (Gibrat et al., BBA, 2012), the pressure denaturation of calmodulin is reversible up to pressures of 3000 bar (300 MPa). A pressure-induced compact intermediate state has been found for the two calmodulin forms, but their unfolding pathways are different. A domain compaction and an increase of the ANS fluorescence of holo form have been evidenced. On the contrary, a domain dilatation and an ANS fluorescence decrease have been found for the apo form. The pressure induced an increase of the interdomain distance for both calmodulin forms, suggesting that the central linker of calmodulin is flexible in solution.