Characterization of protein capacity of nanocation exchanger particles as filling material for functional magnetic beads for bioseparation purposes

In this study we describe the synthesis and characterization of nanocation exchanger particles (NCEX) as the functional filling material for magnetic beads. Polystyrene NCEX particles were synthesized from styrene via a mini-emulsion polymerization. The coupling of cation exchanger groups was done with chlorosulfuric acid after the polymerization reaction. The NCEX particles have an average diameter of 160-260 nm. Their ion exchange capacity amounts up to 4.58 mval/g. In an adsorption experiment it was possible to adsorb 192 mg lysozyme/g NCEX. Depending on the equilibrium concentration of lysozyme in the bulk solution 70-85% of the attached protein was desorbed. NCEX particles were used to produce magnetic beads with cation exchanger properties. Therefore an innovative production process for the synthesis of magnetic beads from different single components was used. The produced magnetic beads contained 40 wt % NCEX material and showed an ion exchanger capacity of 2 mequiv/g. It was possible to adsorb 75 mg lysozyme/g magnetic beads with a maximum recovery rate of 95%.     

Preparation and preliminary evaluation of a biotin-targeted, lectin-targeted dendrimer-based probe for dual-modality magnetic resonance and fluorescence imaging

A novel approach for the preparation of a biotinylated dendrimer-based MRI agent 5 is described, in which a unique disulfide bond in the core of the Gd(III)-1B4M-DTPA chelated G2 PAMAM dendrimer was reduced and then attached to a maleimide-functionalized biotin. The new MRI agent 5 features a well-defined dendron structure and a unique biotin functionality. Immobilization of up to four copies of biotinylated dendrimer 5 to fluorescently labeled avidin yields a supramolecular avidin-biotin-dendrimer-Gd(III) complex. Validation of the complex in mice bearing ovarian cancer tumors demonstrates that the avidin-biotin-dendrimer targeting system efficiently targets and delivers sufficient amounts of chelated Gd(III) and fluorophores (e.g., Rhodamine green) to ovarian tumors to produce visible changes in the tumors by both MRI and optical imaging, respectively. Thus, the avidin-biotin-dendrimer complex may be used as a tumor-targeted probe for dual-modality magnetic resonance and fluorescence imaging.     

Extraction of DNA from soil using nanoparticles by magnetic bioseparation

Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica‐magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR‐ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.     

Preparative agarose gel electrophoresis for the purification of small organelles and particles

A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically "downstream," well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellular structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of Mr 142,000, and an ovoid species (70 X 35 nm) whose protein composition is dominated by a polypeptide of Mr 104,000.     

Electric birefringence of polytetrafluoroethylene particles in agarose gels

Electric birefringence studies of strongly elongated, rod-like particles of polytetrafluoroethylene (PTFE) in agarose gels show that the negative effect observed by semi-diluted aqueous suspensions at low frequencies and at low electric field strengths (the so called "anomaly') disappears. The absolute value of the low frequency effect increases 3-4 times and the amplitude of modulation decreases faster compared to that of the suspensions. This together with decreased decay relaxation times in gels make the possibilty that the PTFE particles orientation in gels is not due to dipolar but to electrophoretic orientation mechanism quite probable. Similar change in the orientation mechanism could be expected also for suspensions of higher concentrations. The further elucidation of the orientation mechanism using fractions with lower polydispersity, broader ranges of experimental conditions (particle concentration, ionic strength and composition, electric field strengths, frequencies, etc.) could be interest for several fields: colloid electro-optics and especially that of concentrated colloids, pulsed field gel electrophoresis of DNA (and especially its sinusoidal biased field variant) and of nucleoprotein complexes and for the gel research.     

Macroporous gel particles as robust macroporous matrices for cell immobilization

A new design of robust matrices for cell immobilization is described. Macroporous gels (MGs) with immobilized microbial cells were prepared at subzero temperatures and were formed inside a plastic core (so-called, protective housing). Due to the protective housing the macroporous gel particles with immobilized cells can be used in well-stirred bioreactors. High retained activity of yeast (77-92%) and Escherichia coli (50-91%) cells immobilized in MGs after drying and storage in the dried state was due to the high structural stability and heterogeneous porous structure of the MGs.     

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.     

Preparation and evaluation of new bifunctional chelating agents: a preliminary report

A scheme has been designed to synthesize a homologous series of new bifunctional chelating agents (BFCAs), which may increase the thermodynamic stability of metal chelates and conjugate at the specific sites on the monoclonal antibody molecule (MoAb) permitting us to analyze the structure-activity relationships of the series of compounds. Four such compounds have been prepared and chacterized by FT-i.r. and NMR spectroscopy. One of them has been used to label an antibody with ¹¹¹In, the stability and distribution of which has been examined in tumor-bearing mice and compared to that of the ¹¹¹In-MoAb prepared using cyclic anhydride of DTPA. Enhanced tumor/blood ratios (9 vs 6.5), tumour to muscle ratios (7 vs 3), and decreased liver uptake (4 vs 12%) have been obtained.     

Preparation of crosslinked macroporous PVA foam carrier for immobilization of microorganisms

In this work, a new and economical way to prepare macroporous poly(vinyl alcohol) (PVA) foam was explored by adding calcium carbonate as a pore-forming agent and using epichlorhydrin as a chemical crosslinking agent to improve foam stability. The mixture for foam formation has been optimized to obtain macroporous PVA foam carriers with uniform apertures, narrow distribution of pore sizes, and good elasticity. The crosslinked PVA foam (CPVAF) carrier demonstrated better chemical and thermal stability, as well as larger specific surface area and diffusion coefficients than the traditional PVA (TPVA) carrier. Nitrifying bacteria were used to test the suitability of CPVAF and TPVA carriers for immobilized microorganisms. CPVAF carriers supported higher biomass density and microbial activity than TPVA carriers. At the same biomass density, the higher nitrification rate of CPVAF carriers was attributed to excellent mass transfer of the substrate (and oxygen) between the bulk solution and the immobilized microorganisms.     

On-chip manipulation and detection of magnetic particles for functional biosensors

We demonstrate the real-time on-chip detection and manipulation of single 1 microm superparamagnetic particles in solution, with the aim to develop a biosensor that can give information on biological function. Our chip-based sensor consists of micro-fabricated current wires and giant magneto resistance (GMR) sensors. The current wires serve to apply force on the particles as well as to magnetize the particles for on-chip detection. The sensitivity profile of the sensor was reconstructed by simultaneously measuring the sensor signal and the position of an individual particle crossing the sensor. A single-dipole model reproduces the measured sensitivity curve for a 1 microm bead. For a 2.8 microm bead the model shows deviations, which we attribute to the fact that the particle size becomes comparable to the sensor width. In the range between 1 and 10 particles, we observed a linear relationship between the number of beads and the sensor signal. The real-time detection and manipulation of individual particles opens the possibility to perform on-chip high-parallel single-particle assays.     

Preparation of agar and agarose from the red algae Ahnfeltia tobuchiensis

A simple and inexpensive procedure of agar and agarose production from the red alga Ahnfeltia tobuchiensis was developed, which needs no additional chromatographic purification.     

Hinge-thiol coupling of monoclonal antibody to silanized iron oxide particles and evaluation of magnetic cell depletion

Iron oxide particles of average size 0.5-1.5 microns, covered by a silane coat carrying amino groups (Bio-Mag, Advanced Magnetics, Boston), were derivatized by reaction with N-[(gamma-maleimidobutyryl)oxy]-succinimide (GMBS), N-hydroxysuccinimidyl iodoacetate (NHIA), 2-iminothiolane (2-It), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The derivatized particles were suitable for the reaction with sulfhydryl groups and subsequently coated with monoclonal antibodies (MoAbs) of different classes and isotypes (IgM, IgG1, IgG2a, IgG2b, IgG3) as well as polyclonal rabbit anti-mouse IgG (RAM). The antibodies were reduced by dithiothreitol (DTT) and covalently conjugated to the BioMag derivatives via liberated sulfhydryls of the hinge region. The observed conjugation ratios, expressed as protein/iron (micrograms/mg), could be reproducibly varied for optimization. These ratios were dependent on the type and amount of antibody offered for coupling to the derivatized particles, decreasing as follows: polyclonal = IgM greater than IgG2b greater than IgG2a = IgG3 greater IgG1. The conjugation ratios were also dependent on the type and amount of the spacer used to derivatize the BioMag particles, decreasing as follows: GMBS greater than NHIA greater than 2-It greater than SPDP. The magnetically responsive magnetite-antibody conjugates ("magneto-beads"), carrying MoAb BMA 081 (anti-CD8; IgG2a), MoAb BB10 (anti-CD10/CALLA; IgG2b), MoAb VIL-A1 (anti-CD10; IgM), and polyclonal RAM, coupled similarly via 3.6 mumol of GMBS spacer per mg of Fe, were further investigated with respect to a depletion effect on specific cell subsets. The rates of cell depletion were found to be strongly dependent on the individual characteristics of the antibody used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.     

Preparation of chitosan particles suitable for enzyme immobilization

Macro-, micro- and nanosized chitosan particles suitable as immobilization carriers were prepared by precipitation, emulsion cross-linking and ionic gelation methods, respectively. Effects of particle preparation parameters on particle size were investigated. Activities of β-galactosidase covalently attached to differently sized particles have been evaluated and compared. The highest activity was shown by the biocatalyst immobilized on nanoparticles obtained by means of the ionotropic gelation method with sodium sulphate as gelation agent. β-Galactosidase fixed on macro- and microspheres exhibited excellent storage stability in aqueous solution, with no more than 5% loss of activity after 3 weeks storage at 4 °C and pH 7.0.     

Novel detection system for biomolecules using nano-sized bacterial magnetic particles and magnetic force microscopy

A system for streptavidin detection using biotin conjugated to nano-sized bacterial magnetic particles (BMPs) has been developed. BMPs, isolated from magnetic bacteria, were used as magnetic markers for magnetic force microscopy (MFM) imaging. The magnetic signal was obtained from a single particle using MFM without application of an external magnetic field. The number of biotin conjugated BMPs (biotin-BMPs) bound to streptavidin immobilized on the glass slides increased with streptavidin concentrations up to 100 pg/ml. The minimum streptavidin detection limit using this technique is 1 pg/ml, which is 100 times more sensitive than a conventional fluorescent detection system. This is the first report using single domain nano-sized magnetic particles as magnetic markers for biosensing. This assay system can be used for immunoassay and DNA detection with high sensitivities.     

Preparation and evaluation of decellularized porcine carotid arteries cross-linked by genipin: the preliminary results

Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.     

Preparation and preliminary biological evaluation of a 177Lu labeled sanazole derivative for possible use in targeting tumor hypoxia

The preparation of a polyazamacrocyclic-nitrotriazole conjugate for radiolabeling with the therapeutic radioisotope viz. (177)Lu is described. The nitroimidazole used for the present study is [N-2'(carboxyethyl)-2-(3'-nitro-1'-triazolyl)acetamide], the carboxylic acid derivative of sanazole, which possesses an optimal combination of desired properties such as, selective toxicity for hypoxic cells, lowered lipophilicity resulting in lowered neurotoxicity. The bifunctional chelating agent is a DOTA derivative viz. 1,4,7,10-tetraaza-1-(4'-aminobenzylacetamido)-cyclododecane-4,7,10- triacetic acid (p-amino-DOTA-anilide). (177)Lu was produced in adequate specific activity (110TBq/g) and high radionuclidic purity (approximately 100%) by irradiating enriched (60.6% (176)Lu) Lu(2)O(3) target and used for radiolabeling of the sanazole-BFCA conjugate. approximately 98% Complexation yield was achieved under optimized conditions. The complex has been characterized by paper chromatography and HPLC studies. Bioevaluation studies in Swiss mice bearing fibrosarcoma tumors revealed moderate tumor uptake (0.88%/g at 1h post-injection) with favorable tumor to blood (4.00 at 1h post-injection) and tumor to muscle (4.63 at 1h post-injection) ratios.     

Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.