Fluorescent Polystyrene Microspheres Research Articles

The uptake and translocation of latex nanospheres and microspheres after oral administration to rats.

Non-ionic and carboxylated fluorescent polystyrene microspheres (100, 500 nm, 1 and 3 microns in diameter), were fed by gavage (2.5% w/v; 1.25 mg kg-1) daily for 10 days to female Sprague Dawley rats. Peyer's patches, villi, liver, lymph nodes and spleen of animals fed the non-ionic microspheres from 100 nm to 1 micron showed unequivocal evidence of uptake and translocation of the particles. Heart, kidney and lung showed no evidence of the presence of microspheres. Carboxylated microspheres were taken up to a lesser degree than the non-ionised particles. Experiments with 125I radiolabelled 100 nm and 1 micron particles showed a higher uptake of the smaller particles, which were concentrated in GI tissue and liver. Particles were not distributed randomly in the tissues, but were concentrated at the serosal side of the Peyer's patches and could be seen traversing the mesentery lymph vessels towards the lymph nodes. The results demonstrate a need to re-examine the possibilities of particulate oral delivery, as well as the potential toxicity of ingested particulates.

Roll-to-roll fabricated lab-on-a-chip devices

We present a high-volume fabrication technique for making polymer lab-on-a-chip devices. Microfluidic separation devices, relying on pinched flow fraction, are roll-to-roll fabricated in a cellulose acetate (CA) film at a volume of 360 devices h−1 for a cost of approximately 0.5 euro/device. The manufacturing process consists of two steps: (i) roll-to-roll thermal nanoimprint for patterning the microchannels into a CA film and (ii) roll-to-roll lamination for bonding another CA film onto the imprinted film to seal the microchannels. Reverse gravure coating is used to apply an adhesive polymer onto the CA lid film before roll-to-roll lamination in order to increase the bonding strength. The fabricated devices are compared with planar imprinted devices with regard to the cross-sectional profile of the imprinted channels and their separation functionality. The separation functionality is characterized using fluorescent polystyrene microspheres with diameters ranging from 0.5 to 5 µm.

Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium.

The objective of this study was to examine the uptake mechanisms of fluorescent polystyrene microspheres of various diameters and surface chemistry by two human cell lines derived from the respiratory epithelium, A549 and Calu-3. Briefly, A549 and Calu-3 cells were grown to confluence in 12-well cluster plates and the uptake of fluorescent microspheres by the cells was determined at various time points. The amount of microspheres internalized by the cells was determined by correcting for non-specific binding to the cell surface. The data showed that A549 cells appeared to have more phagocytic activity than Calu-3 cells. Albumin-coated microspheres as large as 3 microm diameter can be internalized by A549 cells. The amount of internalization by A549 cells observed for 0.5-microm diameter albumin-coated microspheres was approximately 10-times greater than that observed for 1-microm diameter spheres and approximately 100-times greater than values observed for 2- and 3-microm diameter beads. Transmission electron micrographs confirmed that the microspheres were internalized by the cells. Uptake experiments conducted with Calu-3 cells indicated that albumin-coated microspheres were neither bound nor internalized by the cells. The effect of microsphere surface chemistry on the uptake mechanism indicated that amidine microspheres were internalized more rapidly and to a greater extent by both A549 and Calu-3 cells than carboxylate microspheres and non-coated microspheres. This phenomenon is thought to be attributed to masking of the negative polystyrene core by the positive amidine functional group; this effect was less marked for the carboxylate microspheres. These results suggest that A549 and Calu-3 cells can internalize microspheres and that size and effective charge played an important role in the uptake process.

Removal of virus to protozoan sized particles in point-of-use ceramic water filters

The particle removal performance of point-of-use ceramic water filters (CWFs) was characterized in the size range of 0.02–100 μm using carboxylate-coated polystyrene fluorescent microspheres, natural particles and clay. Particles were spiked into dechlorinated tap water, and three successive water batches treated in each of six different CWFs. Particle removal generally increased with increasing size. The removal of virus-sized 0.02 and 0.1 μm spheres were highly variable between the six filters, ranging from 63 to 99.6%. For the 0.5 μm spheres removal was less variable and in the range of 95.1–99.6%, while for the 1, 2, 4.5, and 10 μm spheres removal was >99.6%. Recoating four of the CWFs with colloidal silver solution improved removal of the 0.02 μm spheres, but had no significant effects on the other particle sizes. Log removals of 1.8–3.2 were found for natural turbidity and spiked kaolin clay particles; however, particles as large as 95 μm were detected in filtered water.

In-vitro sensing of biomechanical forces in live cells by a whispering gallery mode biosensor

Direct measurement of the biomechanical stress induced by a live cell during endocytosis is reported. Fluorescent dye-doped polystyrene microspheres were used as microscopic remote optical sensors applying whispering gallery modes (WGMs) as transducer mechanism. Monitoring of the WGMs throughout the incorporation of the microsphere into the cell enabled the determination of the deformation experienced by the microsphere, characterized by both a broadening and a blue shift of the resonances, and consequently the stress induced by the cell. The results reveal an unexpectedly high stress with a magnitude of up to five times that of the passive cortical tension, which can be only explained by a so far undetermined active stress component induced by the cytoskeletal machinery during particle incorporation. The method is adaptable to the study of any other kind of phagocyte and thus provides a novel research tool of high interest for cell biology.

Murine B-1 B lymphocytes have active phagocytic and microbicidal abilities (134.31)

Abstract It has long been recognized that B cells lack phagocytic capabilities. Until recently, B cells from teleost fish were shown to have potent phagocytic abilities for particle antigens; however, whether mammalian B cells are able to accomplish phagocytosis is still not clear. Here we demonstrated that mouse peritoneal cavity (PerC) B-1 B cells, which are thought to be the counterpart of the teleost fish blood B cells, had in vivo and in vitro phagocytic activities to Staphylococcus aureus and polystyrene fluorescent microspheres. Ingestion of particles by B cells led to activation of degradative pathways, resulting in phagolysosome formation and intracellular killing of ingested microbes. About 10% of PerC B cells, mostly CD11b+ B-1 B cells, showed phagocytic activities, and there was no apparent difference between B-1a and B-1b cells in the capabilities for phagocytotisis. Spleen B cells showed a very low degree of phagocytosis. Our results revealed for the first time that mammalian B-1 B cells have phagocytic capacities, which may contribute to their role in innate and adaptive immunity.

Preparation of polystyrene fluorescent microspheres based on some fluorescent labels

Five fluorescent labels were synthesized and used in the preparation of polystyrene fluorescent microspheres by the dispersion copolymerization and absorption method. Spectral properties of copolymerization fluorescent microspheres in tetrahydrofuran indicated these individual characteristics of labels should be maintained in the fluorescent microspheres. The differences of the fluorescent spectra between five fluorescent microspheres and their corresponding parent labels in ethanol have been investigated. These fluorescent microspheres were characterized by environmental scanning electron microscopy, laser scanning confocal microscopy and fluorescence spectrophotometry. They exhibited good dispersion and stable and high fluorescence intensity. Furthermore, copolymerization fluorescent microspheres were functionalized with amino groups. This means that a method for the preparation of copolymerization fluorescent microspheres was developed as a platform for the generation of functional fluorescent microspheres for diverse applications.

Preparation of fluorescent polystyrene microspheres by gradual solvent evaporation method

Highly cross-linked polystyrene beads of 9.2 μm were synthesized by seed polymerization with styrene as monomer and divinylbenzene as cross linker. Other sized monodisperse PS microspheres were also prepared by varying seed particle diameter and proportion of swelling agents. Furthermore, the polystyrene beads were stained by gradual solvent evaporation method using dyes such as rhodamine 101 and acridine orange. Gradual solvent evaporation method facilitates a high concentration of fluorescent dyes on beads. This is the key to obtain fluorescent beads with high intensity. The results showed that the fabricated fluorescent microspheres could be excited to various wavelengths (such as yellow, green, red and scarlet). Our synthesized microspheres offer high fluorescence emission efficiency compared to commercial fluorescent microspheres in the mean time have other properties in common.

Use of particulate surrogates for assessing microbial mobility in subsurface ecosystems

Mass fluxes from the ground surface can play a vital role in influencing groundwater ecosystems. Rates of delivery may influence intact ecosystem composition, while fluxes of substances associated with anthropogenic activity may critically alter the functioning of associated microbial assemblages. Field-based tracing experiments offer a valuable means of understanding mass transport rates and mechanisms, particularly in complex heterogeneous epikarst systems overlying vulnerable fissured aquifers. A short-term tracer experiment monitoring solute and particle tracer concentrations after they passed through a 10-m-thick sequence of limestone, capped by a thin soil, revealed rapid travel times and variable attenuation rates for the substances employed. Results demonstrated that particle tracers have shorter average travel times and can reach the subsurface in higher concentrations and over shorter times than non-reactive solutes. High recovery rates for the bacterial tracer Ralstonia eutropha H16 contrasted strongly with those of similarly sized fluorescent polystyrene microspheres, highlighting the importance of physico-chemical surface characteristics of particle tracers. Complementary laboratory batch experiments examined the role played by organic and inorganic soil/rock surfaces on particle tracer attenuation. Findings suggest that biofilms may significantly promote transport of particulate material below ground, i.e., the delivery of allochthonous microorganisms to karst groundwater.

Efficient Visible-Light-Induced Photocatalytic Activity of a 3D-Ordered Titania Hybrid Photocatalyst with a Core/Shell Structure of Dye-Containing Polymer/Titania

Photostable 3D-ordered titania hybrid photocatalyst with the core/shell structure of dye-containing polymer/titania was prepared by layer-by-layer coating of adsorption and hydrolysis of Ti(OBu)4 on fluorescent polystyrene microspheres (YG), in which a sensitized dye of fluorescein isothiocyanate with a strong absorption in the visible light region of 400−500 nm is fixed. Photocatalytic tests show that the nanostructured titania hybrid photocatalyst exhibited efficient and stable photocatalytic activity for the photodegradation of crystal violet (CV) under visible light irradiation. The titania hybrid photocatalyst was characterized by UV−visible spectroscopy, SEM, TEM, XRD, photoluminescence (PL), time-resolved fluorescence, and electrochemical measurement. The characterization results show that the dye in YG spheres acts as a sensitizer for titania. By absorbing a visible photon it is promoted to an excited electronic state YG*, from which an electron can be injected into the conduction band of titania ...

A microfluidic approach to assembling ordered microsphere arrays

Hydrodynamic flow through an array of channels has been utilized to assemble microspheres on a flat surface. The channels, about 6 µm in lateral size, were etched through a 60 µm thick silicon wafer using deep reactive ion etching (DRIE). Droplets containing 6–8 µm fluorescent polystyrene microspheres were placed on the top side of the horizontally-oriented silicon wafer, while the bottom side was connected to a syringe that draws the fluid through the channels. In this way the microspheres are guided and secured at the inlets of the channels, and remain in place when the suction ceases. This technique, which combines favorable features such as high throughput, high resolution rate and reusability, can be a powerful platform for a new generation of protein microarrays. Antigens can be bound to the microspheres as ‘targets’, which can then be exposed to different fluorescence-tagged antibodies so that their binding can be confirmed. This system can also be used to study the functional roles of gene fragments and their relations to human diseases. The high throughput feature will make it possible to screen a large number of DNA fragments and identify the genetic basis of various diseases effectively.

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.

Visualization of membrane cleaning using confocal laser scanning microscopy

Fouling of membranes in water treatment processes can be caused by the precipitation of inorganic salts, the accumulation of organic substances like polysaccharides, the deposition of organic and inorganic particles as well as the formation of microbial biofilms. The aim of this study was the reproducible generation of membrane deposits and their subsequent visualization by confocal laser scanning microscopy (CLSM). Model foulants were bacterial cells ( Pseudomonas fluorescens) labeled with the DNA-binding dyes SYTO 62 or 4′,6-diamidino-2-phenylindole, the synthetic clay laponite RD stained with the fluorescent dye rhodamine 6G, fluorescent carboxylate-modified polystyrene microspheres as well as the polysaccharide dextran labeled with fluorescein isothiocyanate. Mixtures of fluorescently labeled foulants were filtered through a polyethersulfone ultrafiltration membrane. CLSM in combination with image analysis allowed the three-dimensional reconstruction of the membrane deposits and the localization of each single foulant within the membrane deposits. This approach was applied to the visualization of cleaning effects on fouled membranes that had been treated by ultrasound in such a way that only partial removal of the deposits was achieved. By means of CLSM, uncleaned, weakly cleaned and completely cleaned areas could be detected on the membrane surface, so that the extent of ultrasound-induced membrane cleaning could be localized. In conclusion, CLSM in combination with image analysis proved suitable to visualize the three-dimensional distribution of fluorescently labeled foulants in multi-layered deposits and to evaluate the efficiency of cleaning measures for deposit removal.

Color-tuned FRET polystyrene microspheres by single wavelength excitation

Fluorescent monodisperse polystyrene microspheres were prepared by two-stage dispersion polymerization, which successfully covalently labeled microspheres with two dyes without disturbing the final particle size and size distribution. By varying the dye concentrations, microspheres show tuned colors with different fluorescent intensity under a single wavelength excitation. Fluorescence resonance energy transfer (FRET) between two labeled dyes was proved to contribute to the emission of the longer-wavelength dye at a shorter-wavelength excitation. There is no dye leakage for microspheres because of the covalent incorporation of dye molecules. The microsphere matrix provides good protection of dye molecules and blocks the influence of media outside on the fluorescence of microspheres. Single microsphere shows intense fluorescence due to a large number of encapsulated dye molecules. These uniform barcoding fluorescent microspheres have potential application in multiplexed bioanalysis.

A new single nucleotide polymorphism genotyping method based on gap ligase chain reaction and a microsphere detection assay

The establishment of new methods to detect human genetic variations, such as single nucleotide polymorphisms, is of importance in hereditary disease diagnosis and pharmacogenomics. Several single nucleotide polymorphism genotyping technologies have been presented in recent years. However, techniques which would allow accurate, fast and cheap allelic determination and multiple single nucleotide polymorphism detection in parallel are still in great need. Here, we present a new genotyping technique based on gap ligase chain reaction and a fluorescent polystyrene microsphere measurement platform. We chose the human polymorphism rs3730386 as our candidate to establish this method. Probes for gap ligase chain reaction were designed to recognize the target alleles sensitively and specifically and to produce templates for amplifying the correspondent target fragments used in the following hybridization. The genotypes were finally determined by a hybridization process based on the Luminex fluorescent polystyrene microspheres measurement platform. This method was successfully applied for the detection of selected single nucleotide polymorphisms with high sensitivity and specificity. The genotypes were validated by DNA sequencing. The new genotyping method benefited from the high sensitivity and specificity of gap ligase chain reaction and the detection platform of Luminex. The method allows multiplex analysis of single nucleotide polymorphism, because 100 types of microspheres are available from Luminex.

Development of a fluorescent-microsphere immunoassay for detection of antibodies specific to equine arteritis virus and comparison with the virus neutralization test.

The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia coli were covalently bound to fluorescent polystyrene microspheres and analyzed with the Luminex xMap 100 instrument. Of the eight recombinant proteins, the highest concordance with the virus neutralization test (VNT) results was obtained with the partial GP5(55-98) protein. The MIA was validated by testing a total of 2,500 equine serum samples previously characterized by the VNT. With the use of an optimal median fluorescence intensity cutoff value of 992, the sensitivity and specificity of the assay were 92.6% and 92.9%, respectively. The GP5(55-98) MIA and VNT outcomes correlated significantly (r = 0.84; P < 0.0001). Although the GP5(55-98) MIA is less sensitive than the standard VNT, it has the potential to provide a rapid, convenient, and more economical test for screening equine sera for the presence of antibodies to EAV, with the VNT then being used as a confirmatory assay.

Effect of a Hydrophobic Phospholipid Lining of the Gastric Mucosa in Bioadhesion

The role of a model hydrophobic phospholipid simulating lining of the gastric mucosa, as to adhesion of polymers with different surface functional groups and surface hydrophobicities, was evaluated using an in vitro gastric mucus model. Front-faced fluorescence measurement was used to determine adhesion of fluorescent polystyrene microspheres with different surface functional groups. Contact angle measurements and sticking bubble technique were used to measure relative surface hydrophobicity of the polymers. Adhesion of fluorescent polystyrene microspheres using front-faced fluorescence measurement revealed the hydrophobic phospholipid lining of the in vitro gastric mucus model did not allow adhesion of microspheres with -COOH and -NH(2) functional groups, whereas it did allow adhesion of microspheres with hydrophobic attributes. In addition, in vitro adhesive force studies using diblock copolymers of polystyrene and polyacrylate showed that the in vitro adhesive force between the hydrophobic phospholipid lining of the in vitro gastric mucus model and the polymer increased when the surface hydrophobicity of the polymer increased. The hydrophobic phospholipid acts as an adhesion barrier to hydrophilic bioadhesive polymers and polymers with surface functional groups of carboxylic acid and amine. The hydrophobic phospholipid lining of the gastric mucosa should be taken into considerations for screening and designing of a new gastric bioadhesive polymer.

Micron-pore-sized metallic filter tube membranes for filtration of particulates and water purification

Robust filtering techniques capable of efficiently removing particulates and biological agents from water or air suffer from plugging, poor rejuvenation, low permeance, and high backpressure. Operational characteristics of pressure-driven separations are in part controlled by the membrane pore size, charge of particulates, transmembrane pressure and the requirement for sufficient water flux to overcome fouling. With long term use filters decline in permeance due to filter-cake plugging of pores, fouling, or filter deterioration. Though metallic filter tube development at ORNL has focused almost exclusively on gas separations, a small study examined the applicability of these membranes for tangential filtering of aqueous suspensions of bacterial-sized particles. A mixture of fluorescent polystyrene microspheres ranging in size from 0.5 to 6 μm in diameter simulated microorganisms in filtration studies. Compared to a commercial filter, the ORNL 0.6 μm filter averaged approximately 10-fold greater filtration efficiency of the small particles, several-fold greater permeance after considerable use and it returned to approximately 85% of the initial flow upon backflushing versus 30% for the commercial filter. After filtering several liters of the particle-containing suspension, the ORNL composite filter still exhibited greater than 50% of its initial permeance while the commercial filter had decreased to less than 20%. When considering a greater filtration efficiency, greater permeance per unit mass, greater percentage of rejuvenation upon backflushing (up to 3-fold), and likely greater performance with extended use, the ORNL 0.6 μm filters can potentially outperform the commercial filter by factors of 100–1000 fold.

Fluorescent polystyrene microspheres with large Stokes shift by fluorescence resonance energy transfer

We prepared fluorescent microspheres with notably large Stokes shift and long-wavelength fluorescence by applying fluorescence resonance energy transfer (FRET) between two common julolidine dyes. Short distance between dye molecules caused by high dye concentration results in efficient FRET in microspheres. However, adequate dye concentration and moderate molar ratio of the donor and acceptor should be chosen to avoid aggregation of dye molecules, which leads to the decrease of fluorescent intensity. Microrspheres with average distance between dye molecules of 1.94 nm and molar ratio of 3.08:1 realize highly efficient FRET with no fluorescence of donor and intense long-wavelength emission of acceptor. In addition, the applied solvent evaporation method for preparing microspheres provided better protection of dyes from ambient medium than traditional surface-labeled method. These results demonstrate the feasibility of applying FRET in microspheres to expand useful fluorescent probes, and reveal their potential application in bioassays field.

The use of CdTe quantum dot fluorescent microspheres in fluoro‐immunoassays and a microfluidic chip system

Polystyrene fluorescent microspheres prepared by deposition of CdTe quantum dots (QDs) are used in an immunoassay in this study. CdTe QDs/polyelectrolyte multilayers on the surface of polystyrene microspheres have been formed by layer-by-layer self-assembly via electrostatic interactions. As a model antigen, rabbit IgG has been bound to the outermost layer of the fluorescent microspheres. The immunoreaction between fluorescent microspheres/rabbit IgG and the corresponding antibody was confirmed by change of the fluorescence spectrum and competitive immunoassay. This approach allowed detection of the antigen (rabbit IgG) in the range 1-500 mg/L, based on the change in the fluorescence intensity of the reporter (fluorescent microspheres/rabbit IgG). A novel microfluidic chip device with a laser-induced fluorescence system was established and used for the detection of fluorescent microspheres in this study.