Biocompatible fluorescent nanocrystals for immunolabeling of membrane proteins and cells

A methodology for simple convenient preparation of bright, negatively or positively charged, water-soluble CdSe/ZnS core/shell nanocrystals (NCs) and their stabilization in aqueous solution is described. Single NCs can be detected using a standard epifluorescent microscope, ensuring a detection limit of one molecule coupled with an NC. NCs solubilized in water by DL-Cys were stabilized, to avoid aggregation, by poly(allylamine) and conjugated with polyclonal anti-mouse antibodies (Abs). NC-Abs conjugates were tested in dot-blots and exhibited retention of binding capacity within several nanograms of antigen detected. We further demonstrated the advantages of NC-Abs conjugates in the immunofluorescent detection and three-dimensional (3D) confocal analysis of p-glycoprotein (p-gp), one of the main mediators of the MDR phenotype, overexpressed in the membrane of MCF7r breast adenocarcinoma cells. Immunolabeling of p-gp with NC-Abs conjugates was 4200-, 2600-, and 420-fold more resistant to photobleaching than its labeling with fluorescein isothiocyanate-Abs, R-phycoerythrin-Abs, and AlexaFluor488-Abs, respectively. The labeling of p-gp with NC-Abs conjugates was highly specific, and the data were used for confocal reconstruction of 3D images of the p-gp distribution in the MCF7r cell membrane. Finally, we demonstrated the applicability of NC-Abs conjugates obtained by the method described to specific detection of antigens in paraffin-embedded formaldehyde-fixed cancer tissue specimens, using immunostaining of cytokeratin in skin basal carcinoma as an example. We conclude that the NC-Abs conjugates may serve as easy-to-do, highly sensitive, photostable labels for immunofluorescent analysis, immunohistochemical detection, and 3D confocal studies of membrane proteins and cells.     

Injectable polymeric microspheres with X-ray visibility. Preparation,properties, and potential utility as new traceable bulking agents

The copolymer of methyl methacrylate (MMA) and 2-[2',3',5'-triiodobenzoyl]oxoethyl methacrylate (1), ratio 3:1 (mass:mass), was prepared via a free-radical polymerization in bulk. The copolymer (M(w) = 97.8 kD and M(n) = 41.5 kD) was dissolved in chloroform and subsequently transformed into beads with a diameter in the micrometer range, using a solvent evaporation technique. The resulting microbeads were characterized by different techniques, including NMR spectroscopy, differential scanning calorimetry, gel permeation chromatography, and scanning electron microscopy. The latter technique was used as the basis for statistical analysis of the bead size. Typically, an average diameter of 96 microm and a standard deviation of 21 microm were obtained. The beads were also subjected to some preliminary tests regarding cytotoxicity. The copolymer of MMA and 1 contains covalently bound iodine. Therefore, the material is intrinsically radiopaque, i.e., capable of absorbing X-radiation while no contrast additive is needed. Our interest in these microspheres stems primarily from their possible utility as injectable and afterward traceable (radiopaque) bulking agents, e.g., for use in urology for the treatment of female stress incontinence due to sphincter deficiency. As a first test into this direction, a sample of the microbeads was mixed with ethylene glycol, and the resulting suspension was studied with respect to injectability and radiopacity. The results suggest that the radiopaque microbeads may provide access to improved bulking agents. Further modification of the surface may be necessary in order to suppress the migratory aptitude of the radiopaque polymeric microspheres in vivo.     

A novel method of encapsulating and cultivating adherent mammalian cells within collagen microcarriers

A novel method of preparing collagen microcarriers was developed and used to entrap adherent cells for cell culturing. This new technique involved seeding of cells in micro gel beads comprised of collagen fibrils dispersed in alginate. The gel beads were washed with phosphate buffered saline (PBS) to remove alginate and the resulting microspheres, about 300-500 microm in diameter, contained evenly distributed collagen fibrils which provided a 3D biomimetic environment for cell growth. The applicability of this microencapsulating system was demonstrated by its ability to support the growth of C2C12 myoblast cells. When seeded and cultured within the 3D collagen microcarriers, the population of C2C12 cells entrapped within the microcarriers increased by 1.5 folds in 7 days after inoculation. This encapsulation technique is potentially useful for culturing cells and especially useful for adherent cells that require a 3D fibrillar collagen environment.     

Protective immunity in rats by intranasal immunization with Streptococcus mutans glucan-binding protein D encapsulated into chitosan-coated poly(lactic-co-glycolic acid) microspheres

Recombinant Streptococcus mutans glucan-binding protein D (rGbpD) was incorporated into poly(lactic-co-glycolic acid) (PLGA) microspheres which then were surface-coated with chitosan. The microspheres, with a mean diameter of ca. 1.8 μm, were intranasally administered in rats. There were elevated salivary immunoglobulin A and serum immunoglobulin G antibody responses to rGbpD, as well as lower molar caries scores in immunized animals as compared to sham immunized ones. The chitosan-coated PLGA microspheres are thus potentially useful for antigen delivery in dental caries vaccination.     

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

Nanophased porous hydroxyapatite beads with particle diameters of 25 microm and 30 microm intended for use in protein and biomolecule separation are characterized with respect to chromatographic characteristics. These particles were produced from a hydroxyapatite gel by a controlled spray process yielding microspheres containing hydroxyapatite nanocrystals. By calcification of the microspheres, nanophased porous hydroxyapatite beads were obtained. As a reference material, ceramic hydroxyapatite Types I and II with a particle diameter of 40 microm was chosen. SEM pictures show that the surface of the nanophased hydroxyapatite is very rough compared to ceramic hydroxyapatite Types I and Type II. The calcium-to-phosphorous ratio of this nanophased hydroxyapatite is 1.6, which is slightly below the theoretical ratio of 1.67 of pure hydroxyapatite. The porosity is greater than 60%. An IgG binding capacity of 60.7 mg/ml for Bio-Rad Type I and 36.0 mg/ml for Type II, 42.0 mg/ml for the nanophased material with 25 microm and 19.7 mg/ml for the nanophased material with 30 microm were observed. The nanophased material with 30 microm had the lowest mass transfer resistancy as indicated by the dependency of the dynamic binding capacity on velocity. It is assumed that the mass transport properties are characterized by a low particle diffusion resistancy or by slight intraparticle convection. The material also showed high selectivity for IgG. When culture supernatant with 5% FCS containing 3 mg/ml was loaded, pure IgG could be eluted by linear gradient with increasing sodium phosphate concentration. This nanophased material comprises a novel stationary phase for IgG separation.     

Polymer support for exonucleolytic sequencing

Different kinds of particles were investigated for their potential use as supports for exonucleolytic sequence analysis. Composite beads composed of an unreactive polystyrene "core" and a "shell" of functionalized silica nanoparticles were found to best fulfill the various prerequisites. The biotin/streptavidin system was used for attachment of DNA to composite beads of 6 microm diameter. Applying M13 ssDNA in extremely high dilution (approximately 1 molecule versus 100 beads) with internal fluorescent labels, only a small fraction of beads was found to be associated with fluorescent entities, which likely correspond to a very small number of bound DNA molecules per particle. For better selection and transfer of DNA-containing beads into microstructures for exonuclease degradation the loading experiments were repeated with composite beads of 2.3 microm diameter. In this case a covalent bond was formed between carboxylate-functionalized beads and amino-terminated oligonucleotides, which were detected through external labelling with fluorescent nanoparticles interacting with biotinylated segments of the complementary strand.     

Structural aspects of the zona pellucida of in vitro-produced bovine embryos: a scanning electron and confocal laser scanning microscopic study

Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.     

DNA-Ag nanoclusters as fluorescence probe for turn-on aptamer sensor of small molecules

As promising substitutes for organic dyes and quantum dots, few-atom fluorescent silver nanoclusters (Ag NCs) have recently gained much attention in a wide range from cellular imaging to chemical/biological detection applications owing to their ultrasmall size (<2 nm), excellent photostability, good biocompatibility and water solubility. Herein, we design an aptamer, guanine-rich (G-rich) DNA and Ag NCs nanocomplex to investigate its ability for the detection of small molecules. The design contains two DNA strands which are both chimeric conjugates of the DNA aptamer sequence fragment and G-rich sequence fragment. Using cocaine as a model molecule, the two DNA strands are in free state if there is no cocaine present, and the formed Ag NCs through the reduction of Ag(+) by NaBH(4) show weak fluorescence emission. In the presence of cocaine, however, the two aptamer fragments bind cocaine, which in turn puts the two G-rich sequence fragments in proximity and the fluorescent intensity of DNA-Ag NCs enhances greatly. As a result, DNA-Ag NCs are demonstrated as a novel, cost-effective and turn-on fluorescent probe for the analysis of cocaine, with a detection limit of 0.1 μM. Besides, successful detection of adenosine triphosphate (ATP) with detection limit of 0.2 μM demonstrates its potential to be a general method.     

High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

Quantitative single particle tracking of NGF-receptor complexes: transport is bidirectional but biased by longer retrograde run lengths

The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054+/-0.020 microm/s. Individual runs had a mean velocity of approximately 0.15 microm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites.     

Analysis of antigen-specific antibodies and their isotypes in experimental malaria.

BACKGROUND: Measuring antibody production in response to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi -quantitative analysis of antibody production in response to malaria infection. METHODS: We used microspheres as solid supports on which to capture and analyze circulating antibodies. Antigen immobilized on beads captured antigen-specific antibodies for semi- quantitative analysis using fluorescent secondary antibodies. Anti-immunoglobulin antibodies on beads captured specific antibody isotypes for affinity estimation using fluorescent antigen. RESULTS: Antigen-mediated capture of plasma antibodies enables determination of antigen-specific antibody "titer," a semi-quantitative parameter describing a convolution of antibody abundance and avidity, as well as parameters describing numbers of antibodies bound/bead at saturation and the plasma concentration-dependent approach to saturation. Results were identical in single-plex and multiplex assays, and in qualitative agreement with similar parameters derived from ELISA-based assays. Isotype-specific antibody-mediated capture of plasma antibodies allowed the estimation of the affinity of antibody for antigen. CONCLUSION: Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in speed, sample size, and quantification over standard ELISA-based titer methods.     

利用悬液芯片系统建立一种高通量检测牛奶中头孢氨苄和莱克多巴胺残留的方法

目的:旨在应用基于荧光编码微球技术的悬液芯片系统建立一种方便、稳定性好及高通量的检测牛奶中头孢氨苄和莱克多巴胺残留的免疫检测方法。方法:利用碳二亚胺法将抗生素合成抗原与表面具有羧基的聚苯乙烯微球通过酰胺键偶联成捕获抗原。利用捕获抗原、抗生素的单克隆抗体及抗生素标准品构建竞争性免疫检测体系。荧光标记的羊抗小鼠的IgG作为荧光探针标记与捕获抗原结合的单克隆抗体得到悬液芯片系统的检测物。悬液芯片系统的检测器由两束特殊的激光构成,能够检测荧光探针荧光强度的同时分辨不同型号的微球以实现高通量检测的目的。结果:通过对头孢氨苄和莱克多巴胺合成抗原包被微球的条件进行优化得到包被100μl的微球所需两者合成抗原的量分别是8.4μg 和 87.73μg;实验结果表明抗生素的单克隆抗体特异性良好;在牛奶中,该方法对头孢氨苄和莱克多巴胺的检测限(LOD)分别是20.59 ng/ml 和23.51 ng/ml, 标准添加回收率在70%~110%之间。     

Single-bead-based immunofluorescence assay for snake venom detection

In this communication, we reported a rapid and sensitive immunofluorescence method for the detection of snake venom by using microscale polystyrene beads as platform combined with semiconductor quantum dots (Qdots) as fluorescence label. Briefly, control rabbit IgG or capture antibody for venom was covalently immobilized onto the microspheres (surface activated with carboxyl group, dyed with different color) to form the control or capture beads. When incubated with the testing samples, the venom binds to the specific capture beads to form the complex through antibody-antigen interaction. Then, the second antibody conjugated Qdot was added, which targeted the Qdot to bind to the capture bead/antigen complex. The complex can be directly observed under a UV microscope. The system was applied to the testing of Naja kaouthia venom. Fluorescent microscopic images of QD-labeled capture beads demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of capture beads, indicating that the conjugated antibody molecules remained active and were able to recognize their specific target in solution. The detection limit of this method was 5-10 ng/mL. The detection could be completed within 3 h.     

A new rod-shaped virus from parasitic wasp Diachasmimorpha longicaudata (Hymenoptera: Braconidae)

A new rod-shaped nucleocapsids (NCs) was found inadvertently in Diachasmimorpha longicaudata accessory gland filaments (AGFs). The NCs were 30 nm in diameter and nearly 900 nm in length. They replicated in a small cell type of the AGFs in D. longicaudata, and following oviposition, invaded and proliferated in the hemocytes of a parasitized host Bactrocera dorsalis Hendel. This finding of a completely new virus in the AGF indicate that different geographical populations (subspecies) of D. longicaudata may carry different sybionts. This is the first report showing that the same wasp species, but from a different geographical populations, can carry an entirely different virus.     

Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique

Neutron activation is an accurate analytic method in which trace quantities of isotopes of interest in a sample are activated and the emitted radiation is measured with high-resolution detection equipment. This study demonstrates the application of neutron activation for the measurement of myocardial perfusion using stable isotopically labeled microspheres. Stable labeled and standard radiolabeled microspheres (15 microm) were coinjected in an in vivo rabbit model of myocardial ischemia and reperfusion. Radiolabeled microspheres were detected with a standard gamma-well counter, and stable labeled microspheres were detected with a high-resolution Ge detection after neutron activation of the myocardial and reference blood samples. Regional myocardial blood flow was calculated from the deposition of radiolabeled and stable labeled microspheres. Both sets of microspheres gave similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation (r = 0.95-0.99). Neutron activation is capable of detecting a single microsphere in an intact myocardial sample while providing simultaneous quantitative measurements of multiple isotope labels. This high sensitivity and capability for measuring perfusion in intact tissue are advantages over other techniques, such as optical detection of microspheres. Neutron activation also can provide an effective method for reducing the production of low-level radioactive waste generated from biomedical research. Further applications of neutron activation offer the potential for measuring other stable labeled compounds, such as fatty acids and growth factors, in conjunction with microsphere measured flow, providing the capability for simultaneous measurement of regional metabolism and perfusion.     

High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.     

Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection

Sensitive and specific electrochemiluminescence (ECL) assays were used to detect Clostridium botulinum neurotoxins serotypes A, B, E, and F in undiluted human serum, undiluted human urine, assay buffer, and selected food matrices (whole milk, apple juice, ground beef, pastry, and raw eggs). These novel assays used paramagnetic bead-based electrochemiluminescent technology in which biotinylated serotype-specific antibodies were bound to streptavidin-coated paramagnetic beads. The beads acted as the solid support and captured analyte from solution. Electrochemiluminescent detection relied on the use of ruthenium chelate-labeled anti-serotype antibodies and analysis with a BioVeris M-Series M1R analyzer. The sensitivities of the assays in clinically relevant matrices were 50 pg/ml for serotypes A and E, 100 pg/ml for serotype B, and 400 pg/ml for serotype F. The detection limits in selected food matrices ranged from 50 pg/ml for serotype A to 50 to 100 pg/ml for serotypes B, E, and F. The antibodies used for capture and detection exhibited no cross-reactivity when tested with the other serotypes. When purified native toxin was compared with toxins complexed to neurotoxin-associated proteins, no significant differences in assay response were noted for serotypes A, B, and F. Interestingly, the native form of serotype E exhibited reduced signal and limit of detection compared with the complexed form of the protein. We suspect that this difference may be due to trypsin activation of this particular serotype. The assays described in this article demonstrate limits of detection similar in range to the gold standard mouse bioassay, but with greatly reduced time to data. These rapid sensitive assays may have potential use in clinical settings, research studies, and screening of food products for botulinum toxins.     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.