Development and application of thermo-sensitive immunomicrospheres for antibody purification

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum. (c) 1994 John Wiley & Sons, Inc.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

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.     

Immunological agglutination kinetics of latex particles with covalently immobilized antigens

Hen egg-white lysozyme (HEL), ovalbumin and bovine serum albumin (BSA) were covalently immobilized onto styrene/methacrylic acid [P(St/MAA)] copolymer latex particles by the carbodiimide method. The initial rates of the immunological agglutination of these particles initiated by the addition of antibodies were quantified by the absorbance changes at a wavelength of 680 nm. The sensitivity of the immunological agglutination of the particles with covalently immobilized antigens was higher than that with physically adsorbed ones. The immunological agglutination kinetics showed a similar tendency irrespective of antigen-antibody systems. That is, the initial agglutination rates (i) increased with increasing immobilized amount of antigens, (ii) were largest in the ionic strength range of 0.02 to 0.05 at pH 7 and (iii) decreased with increasing pH. These results indicate that the electrostatic interactions of particle-particle and particle-antibody are main factors which control the immunological agglutination. On the other hand, the sensitivity of the immunological agglutination increased with increasing molecular size of antigens.     

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.     

Purification of recombinant enhanced green fluorescent protein expressed in Escherichia coli with new immobilized metal ion affinity magnetic absorbents

A new immobilized metal ion affinity (IMA) adsorbent containing superparamagnetic nanoparticles and coated with hydrophilic resins are proposed here to improve the purification of His-tagged proteins. The magnetic chelating resin was prepared by radical polymerization of magnetite (Fe3O4), styrene, divinyl benzene (DVB) and glycidyl methacrylate-iminodiacetic acid (GMA-IDA) in ethanol/water medium. IDA is immobilized on magnetite as a ligand and pre-charged Cu2+, Zn2+ and Ni2+ as metal ions. To identify the GMA-IDA magnetic particles easily, we named these particles MPGI. The MPGI adsorbent was used to test their suitability for the direct recovery of an intracellular, polyhistidine-tagged protein, enhanced green fluorescent protein [EGFP-(His)(6)], from Escherichia coli lysates in a single step. Parameters influencing the purification efficiencies such as pH, ionic strength and imidazole concentration were optimized to achieve improved separation. The optimal selectively was observed in binding buffer (0.2M NaCl, 0.02M imidazole), washing buffer (0.4M NaCl, 0.03 M imidazole) and elution buffer (0.50M imidazole). The Cu2+-charged MPGI adsorbent had the highest yield and purification factor at 70.4% and 12.3, respectively. The calculated isotherm parameters (Q(m)=53.5 mg/g, K(d)=5.84 mg/mL and Q(m)/K(d)=9.2 mL/g) indicated that the MPGI adsorbent could be used as a suitable adsorbent for EGFP from an aqueous solution.     

Hydrolysis of starchy materials by repeated use of an amylase immobilized on a novel thermo-responsive polymer

A copolymer of methacrylic acid (MAA) and N-isopropyl acrylamide (NIPAM) was used as a novel, reversibly soluble-insoluble support whose solubility changes depending on the temperature of the solution. Amylase (Dabiase K-27) immobilized covalently on the thermo-responsive polymer showed good solubility response: the immobilized enzyme (D-MN) was in a soluble state below 32°C, but insoluble above 42°C. D-MN in a soluble state has a high specific activity for the hydrolysis of soluble or uncooked starch. The solubility response of D-NM to changes in the temperature of the solution was more sensitive when 0.5% NaCl was added to a buffer solution (pH 4.5) with D-MN than in the buffer solution without NaCl. D-MN was used successively for repeated hydrolysis reactions of soluble and uncooked starches, in which D-MN was insolubilized either by changing the temperature of the reaction mixture from 30°C to 36°C with 0.5% NaCl or by adjusting the NaCl concentration of the reaction mixture from 0% to 1% at 30°C. In the repeated hydrolysis, glucose was produced successively from the soluble and uncooked starches, and D-MN could be repeatedly used after being recovered from the reaction product by centrifugation at the end of each batchwise hydrolysis.     

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.     

Purification of fusion proteins using affinity microspheres in aqueous two-phase systems

Affinity microspheres were prepared by immobilizing human -globulin (HGb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H; average diameter 0.33 m], which were prepared by emulsifier-free emulsion polymerization. HGB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced. Correspondence to: A. Kondo     

Magnetic characterizations of nickel hyperaccumulating plants (Planchonella oxyhedra and Rinorea bengalensis) from Halmahera,Indonesia

Magnetic minerals, such as magnetite and hematite, have been reported to be present, in particular, leaves as biogenic particles. The magnetic minerals and properties of Ni hyperaccumulators have not previously been reported in the literature. This study aimed to characterize the magnetic properties of two Ni hyperaccumulating plant species, R. bengalensis and P. oxyhedra, which grow in an ultramafic region on Halmahera Island, Indonesia. For comparison, similar characterization was carried out on two non-hyperaccumulating plant species which grow in the same region. Concentrations of Ni, Fe, and Mn in the leaves of the hyperaccumulating plants were measured using atomic absorption spectroscopy (AAS) and their magnetic properties were characterized using measurements of magnetic susceptibility, low temperature magnetic susceptibility, and hysteresis curves. The results show that, compared to the non-hyperaccumulating plants, the Ni hyperaccumulating plants have higher concentrations of Ni and similar concentration of Fe. The magnetic susceptibilities of hyperaccumulating plants are positive, and those of non-hyperaccumulating plants are negative. This suggests that the abundance of Ni, rather than Fe, may control the magnetic properties of Ni hyperaccumulating plants. This probable connection between Ni concentration and plant magnetic properties could be advantageous for identifying hyperaccumulators, and should, therefore, be explored further.     

The aggregation behaviour of protein-coated particles: a light scattering study

The different mechanisms involved in the aggregation of spherical latex particles coated with bovine serum albumin (BSA) have been studied using static and dynamic light scattering. These techniques assess the fractal dimension of the aggregates and their mean hydrodynamic radius. Particles with different degrees of surface coverage have been prepared. The net charge of the covered particles has been modified by varying the pH of the aqueous phase. The aggregation rate was measured and used to determine the importance of the different aggregation mechanisms that are responsible for these types of flocculation processes. At low and intermediate degrees of surface coverage, bridging flocculation is the principal aggregation mechanism irrespective of the electrical state of the protein-particle complexes. At high degree of surface coverage, however, weak flocculation is important only when the BSA molecules are at their isoelectric point.     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

Flocculation performance of a cationic biopolymer derived from a cellulosic source in mild aqueous solution

The flocculation behavior of cationic, quaternary ammonium groups containing cellulosic biopolymers, CDACs, synthesized by cationizing dialdehyde cellulose in mild aqueous solution was studied in a kaolin suspension. In particular, the role of CDAC dosage and solution pH, NaCl concentration, and temperature were clarified. In addition, the initial apparent charge densities (CDs), particle sizes, ζ-potential, and stability of CDs were determined. CDACs possessed a high flocculation activity in neutral and acidic solutions, but a significant decrease was observed in alkaline solutions (pH >9). This was also seen as a decline in the apparent CD and particle size of the CDACs in alkaline conditions. The measurements also indicated that the apparent CD decreased to a constant level of 3 mmol/g in aqueous solutions. However, no notable decrease in flocculation performance was obtained after several days of storage. Moreover, the variation of NaCl concentration and temperature did not affect the flocculation activity.     

A novel protein tightly bound to bacterial magnetic particles in Magnetospirillum magneticum strain AMB-1

Magnetic bacteria synthesize magnetite crystals with species-dependent morphologies. The molecular mechanisms that control nano-sized magnetite crystal formation and the generation of diverse morphologies are not well understood. From the analysis of magnetite crystal-associated proteins, several low molecular mass proteins tightly bound to bacterial magnetite were obtained from Magnetospirillum magneticum strain AMB-1. These proteins showed common features in their amino acid sequences, which contain hydrophobic N-terminal and hydrophilic C-terminal regions. The C-terminal regions in Mms5, Mms6, Mms7, and Mms13 contain dense carboxyl and hydroxyl groups that bind iron ions. Nano-sized magnetic particles similar to those in magnetic bacteria were prepared by chemical synthesis of magnetite in the presence of the acidic protein Mms6. These proteins may be directly involved in biological magnetite crystal formation in magnetic bacteria.     

Bat head contains soft magnetic particles: Evidence from magnetism

Recent behavioral observations have indicated that bats can sense the Earth's magnetic field. To unravel the magnetoreception mechanism, the present study has utilized magnetic measurements on three migratory species (Miniopterus fuliginosus, Chaerephon plicata, and Nyctalus plancyi) and three non‐migratory species (Hipposideros armiger, Myotis ricketti, and Rhinolophus ferrumequinum). Room temperature isothermal remanent magnetization acquisition and alternating‐field demagnetization showed that the bats' heads contain soft magnetic particles. Statistical analyses indicated that the saturation isothermal remanent magnetization of brains (SIRM_{1T_brain}) of migratory species is higher than those of non‐migratory species. Furthermore, the SIRM_{1T_brain} of migratory bats is greater than their SIRM_{1T_skull}. Low‐temperature magnetic measurements suggested that the magnetic particles are likely magnetite (Fe₃O₄). This new evidence supports the assumption that some bats use magnetite particles for sensing and orientation in the Earth's magnetic field. Bioelectromagnetics 31:499–503, 2010. © 2010 Wiley‐Liss, Inc.     

Magnetic and structural properties of magnetite in radular teeth of chiton Acanthochiton rubrolinestus

The teeth of the Polyplacophora Chiton Acanthochiton Rubrolinestus contain biomineralized magnetite crystallites whose biological functions in relation to structure and magnetic properties are not well understood. Here, using superconducting quantum interference device (SQUID) magnetometry, we find that the saturation magnetization (σ(s)) and the Verwey transition temperature (T(v)) of tooth particles are 78.4 emu/g and 105 K, respectively. These values are below those of the stoichiometric magnetite. An in situ examination of the structure of the magnetite-bearing region within an individual tooth using high-resolution transmission electron microscopy indicates magnetite microcrystals form electron dense polycrystalline sheets with typical lengths of about 800 nm and widths of about 150 nm. These polycrystalline sheets are arranged regularly along the longitudinal direction of the tooth cutting surface. In addition, the crystallites in polycrystalline sheets take on generally good crystallinity. The magnetic microstructures of in situ magnetic force microscopy demonstrate that the [111] easy direction of magnetite microcrystals are aligned along the length of the tooth, whereas the [111] direction is parallel to the thickness of the tooth. Both M?ssbauer spectra and magnetization versus temperature measurements under field cooled and zero-field cooled conditions do not detect superparamagnetic magnetite crystallites in the mature major lateral tooth particles of this chiton.     

Preparation of Fine Magnetic Particles and Application for Enzyme Immobilization

Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe²⁺ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe²⁺ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.     

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.     

Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp.

A marine photosynthetic bacterium (PS88), identified as Rhodovulum sp., with flocculating ability was isolated from the sea sediment mud of a shrimp cultivation farm in Thailand. This bacterium flocculated in glutamate/malate medium during aerobic dark or anaerobic light cultivation. The flocculating ability was enhanced with the increase of NaCl concentration to 6% (w/v). When PS88 was grown in glutamate/malate medium containing 3.5% NaCl, protein, RNA and DNA were produced exocellularly and there was flocculation. The yields of DNA, RNA and protein were 8.3, 62.5 and 48.5 mg/g dry cell, respectively. The flocculated cells were deflocculated by treatment with a nucleolytic enzyme such as RNase or DNase, while amylase, protease, trypsin, cellulase and pectinase had no deflocculating effect. These results suggest that the exocellular nucleic acids are active in flocculation. Received: 10 April 1998 / Received revision: 14 July 1998 / Accepted: 8 August 1998     

Multifunctional organic–inorganic composite luminescent nanospheres

A simple general strategy was successfully developed for the preparation of magnetic–luminescent multifunctional nanocomposites by incorporating fluorescent (pyrene) and magnetic (Fe₃O₄) components simultaneously into a poly(styrene‐co‐methacrylic acid) [poly(St‐co‐MAA)] copolymer matrix. The nanospheres so prepared were characterized using scanning electron microscopy (SEM), powder X‐ray diffraction (XRD) and Fourier transform infrared (FTIR) analysis. The prepared magnetic–fluorescent inorganic–organic nanocomposites have excellent magnetic and photoluminescent properties. They can be used in magnetic separation of trace amounts of sample, fluorescence detection and imaging applications, including magnetic resonance imaging (MRI) and fluorescence imaging. The fluorescence quenching of the nanospheres in the presence of different amounts of Cu²⁺ ions was also investigated. Under optimal experimental conditions, the relative fluorescence intensity of the composite nanosphere colloidal solution is proportional to the concentration of Cu²⁺ ions, which indicates that these multifunctional nanocomposites can be used for the magnetic separation and fluorescence detection of Cu²⁺ ions. Copyright © 2011 John Wiley & Sons, Ltd.