His-tagged protein immobilization on cationic ferrite magnetic nanoparticles

Magnetic nanoparticles have been applied in various fields because of their interesting magnetic properties. Immobilization on magnetic nanoparticles is a very important step in functionalizing them. We examined protein immobilization efficiency using interactions between his-tagged enhanced green fluorescence protein and affordable cationic ferrite magnetic nanoparticles for the first time. Four types of ferrite magnetic nanoparticles were verified: cobalt iron oxide, copper iron oxide, nickel iron oxide, and iron (III) oxide as negative controls. Among the four ferrite magnetic nanoparticles, copper ferrite magnetic nanoparticle was confirmed to have the highest immobilization efficiency at 3.0 mg proteins per gram ferrite magnetic nanoparticle and 78% of total enhanced green fluorescence protein. In addition, the maximum binding efficiency was determined for copper ferrite magnetic nanoparticle. Consequently, this newly verified his-tag-immobilizing capacity of copper ferrite magnetic nanoparticle could provide a facile, capable, and promising strategy for immobilizing his-tagged proteins or peptides with high purity for biosensors, magnetic separation, or diagnostics.     

Facile and high-efficient immobilization of histidine-tagged multimeric protein G on magnetic nanoparticles

This work reports the high-efficient and one-step immobilization of multimeric protein G on magnetic nanoparticles. The histidine-tagged (His-tag) recombinant multimeric protein G was overexpressed in Escherichia coli BL21 by the repeated linking of protein G monomers with a flexible linker. High-efficient immobilization on magnetic nanoparticles was demonstrated by two different preparation methods through the amino-silane and chloro-silane functionalization on silica-coated magnetic nanoparticles. Three kinds of multimeric protein G such as His-tag monomer, dimer, and trimer were tested for immobilization efficiency. For these tests, bicinchoninic acid (BCA) assay was employed to determine the amount of immobilized His-tag multimeric protein G. The result showed that the immobilization efficiency of the His-tag multimeric protein G of the monomer, dimer, and trimer was increased with the use of chloro-silane-functionalized magnetic nanoparticles in the range of 98% to 99%, rather than the use of amino-silane-functionalized magnetic nanoparticles in the range of 55% to 77%, respectively.     

Simple synthesis of magnetic mesoporous FeNi/carbon composites with a large capacity for the immobilization of biomolecules

An easy co-impregnation method has been developed to synthesize magnetic mesoporous FeNi alloy/carbon composites with an ordered hexagonal structure. Furfuryl alcohol and metal nitrates were used as the carbon and magnetic particle precursors and were impregnated into the silica template in one step using a simple ethanol solution of furfuryl alcohol and metal nitrates. Compared to the co-casting route with two-step impregnation, the co-impregnation makes the synthesis simpler and eliminates the difficult second impregnation step. The mesoporous FeNi/carbon composites obtained have large surface areas, large pore volumes and a bimodal pore size distribution. The FeNi alloy nanoparticles were well dispersed in the mesoporous carbon walls. The composites exhibit excellent superparamagnetic behavior and the saturation magnetization strength can be adjusted from 3.4 to 5.7 emu/g by increasing the content of alloy. Such bimodal mesoporous composites show a large immobilization capacity for the biomolecules of cytochrome c (up to 732 mg/g) and lysozyme (up to 790 mg/g).     

Protease immobilization onto porous chitosan beads

Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L -arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized proteases on the porous chitosan beads are estimated. The apparent K_m values were larger for immobilized proteases than for the free ones, while V_m values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

Lipoprotein lipase immobilization onto porous polyvinyl alcohol beads

Water-insoluble proteases were prepared by immobilizing lipoprotein lipase (LPL) onto the surface of porous polyvinyl alcohol (PVA) beads by covalent fixation. The relative activity of the immobilized proteases was found to remain high toward small ester substrates, p-nitrophenyl laurate (pNPL). The relative activity of the immobilized LPL by cyanogen bromide (CNBr) method decreased gradually with the decreasing surface concentration of the immobilized LPL on the porous PVA beads. On the contrary, the immobilized LPL by hexamethylene diisocyante (HMDI) method gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave higher relative activity (RA) than that by the CNBr method. The Michaelis constant, K_m, and the maximum reaction velocity, V_m, were estimated for the free and the immobilized LPL. The apparent K_m was larger for the immobilized LPL than for the free one, and V_m was smaller for the immobilized LPL. The pH, thermal, and storage stabilities of the immobilized LPL were higher than those of the free ones. The initial enzymic activity of the immobilized LPL maintained almost unchanged without any leakage and inactivation of LPL when the batch enzymic reaction was performed repeatedly, indicating excellent durability of the immobilized LPL. © 1993 John Wiley & Sons, Inc.     

The immobilization of enzymes onto hydrolyzed polyethylene-g-co-2-HEMA

A study of the immobilization of trypsin and other enzymes onto hydrolyzed poly(2-hydroxyethyl methacrylate)-g-co-polyethylene using hydroxyl and carboxyl activating agents has been undertaken. Some emphasis was placed on the immobilized trypsin system which involved examination of the variation of (i) the extent of hydrolysis of the graft copolymer, (ii) the concentration of activating agent, and (iii) the temperature of coupling. With the trypsin system, an increase in carbodi-imide concentration gave an increase in the amount of protein immobilized but a marked decrease in the retention of enzymic activity. Comparison of the behavior of the free with the immobilized enzyme showed that satisfactory yields were obtained and the immobilized system has an extended pH profile and good stability and thus would have broad applicability. The kinetic factors were examined further, and the role of the graft copolymer chains in the immobilized system is discussed.     

Growing spherical polypyrrole nanoparticles onto the magnetic carbon aerogel for improving electrochemical performance for supercapacitors

Journal of Porous Materials - In this work, hierarchical porous magnetic activated carbon aerogels (aCA)/polypyrrole (PPy) composites were synthesized by a facile and cost effective in situ...     

Covalent immobilization of nucleic acid probes onto reactive synthetic polymers

Oligodeoxyribonucleotide (ODN)/polymer conjugates were obtained by covalent immobilization of various nucleic acid sequences to three different polymers. Two of them bore active ester groups for the covalent linkage via the formation of amide bonds and the third one featured aldehyde moieties for immobilization via reductive amination. The factors controlling the conjugation reaction were found to be the reactivities of the polymers and their abilities at forming hydrogen bonds with the ODNs to be tethered. It was found, as well, that the observed aggregation of the grafting reaction products was essentially due to hydrogen bonding with the nucleic bases of the tethered oligonucleotides. © 1997 John Wiley & Sons, Inc. J Appl Polym Sci 66: 233–242, 1997     

Theranostic magnetic nanoparticles

Early detection and treatment of disease is the most important component of a favorable prognosis. Biomedical researchers have thus invested tremendous effort in improving imaging techniques and treatment methods. Over the past decade, concepts and tools derived from nanotechnology have been applied to overcome the problems of conventional techniques for advanced diagnosis and therapy. In particular, advances in nanoparticle technology have created new paradigms for theranostics, which is defined as the combination of therapeutic and diagnostic agents within a single platform. In this Account, we examine the potential advantages and opportunities afforded by magnetic nanoparticles as platform materials for theranostics. We begin with a brief overview of relevant magnetic parameters, such as saturation magnetization, coercivity, and magnetocrystalline anisotropy. Understanding the interplay of these parameters is critical for optimizing magnetic characteristics needed for effective imaging and therapeutics, which include magnetic resonance imaging (MRI) relaxivity, heat emission, and attractive forces. We then discuss approaches to constructing an MRI nanoparticle contrast agent with high sensitivity. We further introduce a new design concept for a fault-free contrast agent, which is a T1 and T2 dual mode hybrid. Important capabilities of magnetic nanoparticles are the external controllability of magnetic heat generation and magnetic attractive forces for the transportation and movement of biological objects. We show that these functions can be utilized not only for therapeutic hyperthermia of cancer but also for controlled release of cancer drugs through the application of an external magnetic field. Additionally, the use of magnetic nanoparticles to drive mechanical forces is demonstrated to be useful for molecular-level cell signaling and for controlling the ultimate fate of the cell. Finally, we show that targeted imaging and therapy are made possible by attaching a variety of imaging and therapeutic components. These added components include therapeutic genes (small interfering RNA, or siRNA), cancer-specific ligands, and optical reporting dyes. The wide range of accessible features of magnetic nanoparticles underscores their potential as the most promising platform material available for theranostics.     

Polyethylene glycol grafting and attachment of encapsulated magnetic iron oxide silica nanoparticles onto chlorosilanized single-wall carbon nanotubes

It is demonstrated that functionalization of single-wall carbon nanotubes (SWCNTs) can be carried out using simple chlorosilane chemistry, which allows coupling of the SWCNTs with external chemical species. As a result, organic polymers and magnetically iron oxide-in-silica nanoparticles are specifically bonded onto SWCNTs. It is also found that the SWCNTs functionalized with long PEG chains greatly enhance dispersion of the carbon structure in water. Furthermore, the SWCNT-COO-Si(Fe₃O₄@SiO₂) can be quickly separated and redispersed from their aqueous colloid with the application of external magnetic field.     

Magnetically separable bimodal mesoporous carbons with a large capacity for the immobilization of biomolecules

A method for the fabrication of carbon-based mesoporous magnetic composites with a large capacity for the adsorption/immobilization of biomolecules is presented. The composites consist of iron oxide spinel nanoparticles inserted into the pores of templated unimodal or bimodal mesoporous carbons. The deposition of the magnetic iron oxide nanoparticles was carried out following two synthetic routes: (1) the direct incorporation of nanoparticles into the pores of the templated carbons and (2) the insertion of nanoparticles into the mesopores of the carbon-silica composite followed by the selective removal of silica framework. The carbon-iron oxide magnetic composites prepared according to route 2 were found to have better textural properties (larger BET surface areas and pore volumes) and significantly higher capacity for the adsorption of hemoglobin and immobilization of lysozyme. The amounts of hemoglobin or lysozyme adsorbed/immobilized by these materials were 176 mg hemoglobin g⁻¹ support and 131 mg lysozyme g⁻¹ support using route 1 and 430 mg hemoglobin g⁻¹ support and 322 mg lysozyme g⁻¹ support by route 2. Furthermore, we have demonstrated that, when no inorganic nanoparticles are deposited, the bimodal mesoporous carbon shows exceptionally a large immobilization capacity for hemoglobin (830 mg g⁻¹ support) and lysozyme (510 mg g⁻¹).     

功能化磁性纳米颗粒固定化脂肪酶研究

用葡萄糖酸对Fe3O4磁性纳米颗粒表面进行修饰,然后用水溶性碳化二亚胺（EDC）作偶联剂,对脂肪酶进行固定化。考察了偶联剂浓度、给酶量和反应时间对脂肪酶固定化过程的影响。结果表明,制备功能化磁性颗粒固定化酶的最佳条件为：偶联剂浓度为12．5mg／mL磷酸缓冲液（PBS）,给酶量为2．5mg／mLPBS,反应时间为24h。固定化脂肪酶表现出优异的热稳定性,60℃时酶活为游离酶的6倍。重复使用10次后,酶促活力依然保持80％以上。     

A comparative study on antibody immobilization strategies onto solid surface

Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering real-to-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels) had the following order: secondary antibody-mediated immobilization>protein A/G-mediated immobilization>direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies, which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.     

The sorption of acid dye onto chitosan nanoparticles

The behavior of chitosan nanoparticles as an adsorbent to remove Acid Green 27 (AG27), an acid dye, from an aqueous solution has been investigated with nanochitosan (particle size = 180 nm; degree of deacetylation = 74%). The dye concentration at equilibrium (Q_e, mg/g) was calculated using the weight of the nanoparticles in the mixed solution (Q_es) and the weight of chitosan in the nanoparticles (Q_ep). The experimental isotherm data were analyzed using the Langmuir equation for each chitosan sample; the Langmuir monolayer adsorption capacity (Q₀) was calculated with Q_es and Q_ep and the results were 1051.8 mg/g and 2103.6 mg/g, respectively, which were significantly higher than that of the micron-sized chitosan.     

Grafting of hyperbranched aromatic polyamide onto silica nanoparticles

Hyperbranched aromatic polyamide grafted nano-silica particles were successfully prepared via “one-pot” melt polycondensation. The particles were firstly treated with a silane coupling agent to introduce amine groups as the growth points, and then grafting of the hyperbranched polymers started from the modified surface. The percentage grafting can be as high as 32.8%. The key reaction conditions, including feeding ratio, reaction temperature and time, were carefully investigated to determine a set of optimal grafting parameters. It was found that the significant steric hindrance between the hyperbranched polymers during graft polymerization played a key role in controlling molecular structure of the grafted polymers, while adsorption of grafting monomer onto SiO₂ particles prior to the reaction facilitated higher degree of branching and apparent molecular weight of ungrafted polymers.     

DNA-directed immobilization of horseradish peroxidase onto porous SiO2optical transducers

ABSTRACT: Multifunctional porous Si nanostructure is designed to optically monitor enzymatic activity of Horseradish Peroxidase. First, an oxidized PSi optical nanostructure, a Fabry-Perot thin film, is synthesized and is used as the optical transducer element. Immobilization of the enzyme onto the nanostructure is performed through DNA-Directed Immobilization. Preliminary studies demonstrate high enzymatic activity levels of the immobilized Horseradish Peroxidase, while maintaining its specificity. The catalytic activity of the enzymes immobilized within the porous nanostructure is monitored in real time by reflective interferometric Fourier transform spectroscopy. We show that we can easily regenerate the surface for consecutive biosensing analysis by mild de-hybridization conditions.     

BSA adsorption onto magnetic polyvinylbutyral microbeads

Bovine serum albumin (BSA) adsorption onto novel and feasible magnetic polyvinylbutyral‐based microbeads was investigated. The microbeads were made of Mowital® B30HH, a commercial product, in the range 125–250 μm by a modified solvent evaporation technique. Magnetite particles were embedded in the polymer structure for favorable magnetic properties, 4.80 emu/g microbeads of saturation magnetization at 6000 Gauss magnetic field. Glutaraldehyde (GA) was used as a bonding agent to increase stability and as a ligand for protein adsorption. The amount of adsorbed BSA was optimized by changing the medium pH and the initial concentrations of GA and BSA. Dynamic adsorption data of batch runs fitted best to Langmiur kinetics. The parameters q_max, k^′_f and k^′_r of the model were estimated through nonlinear regression analysis as 138 mg BSA/g adsorbent, 0.058 ml/(mg BSA · min) and 0.002 min⁻¹, respectively, for magnetic microbeads at pH 5.0 and 25°C. The adsorbed BSA was eluted successfully at pH 8.0 and 25°C. Possibly due to surface roughness and magnetic properties, q_max was found higher than the other adsorbents reported in the literature. The results denote that these microbeads can be an alternative protein adsorbent due to high adsorption capacity and rate, as well as remarkable separation characteristics. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 707–715, 2001     

Grafting polystyrene onto silica nanoparticles via RAFT polymerization

Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to graft polystyrene (PS) onto silica nanoparticles. A novel route was used to prepare the RAFT agent, 2-butyric acid dithiobenzoate (BDB) by substitution of dithiobenzoate magnesium bromide with sodium 2-bromobutyrate under alkali condition in aqueous solution. Epoxy groups were covalently attached to silica nanoparticles by condensation reaction of 3-glycidyloxypropyltrimethoxysilane (GPS) with the hydroxyl on the silica particle surface. RAFT agent functionalized nanoparticles were produced by ring-open reaction of the epoxy group with the carboxyl group of BDB. Then, PS chains with controlled molecular weights and narrow polydispersities (less than 1.1) were grown from the RAFT agent anchored nanoparticle surface. FT-IR, transmission electron microscopy (TEM) and thermogravimetric analysis (TGA) results showed that PS chains grew from silica particles by surface RAFT polymerization.     

Bisacryloylpiperazine as vinylating agent for enzyme immobilization by graft-copolymerization onto polysaccharides

A method of enzyme immobilization by graft-copolymerization onto polysaccharides is reported. Bisacryloylpiperazine has been used as a vinylating reagent and the reaction product with several enzymes (HRP, GOD, Am, ChT, Cel) was copolymerized onto different matrices (cellulose, Sepharose, Sephadex, starch). Immobilization parameters which influence the copolymer activity have been studied for the insolubilization of horseradish peroxidase onto cellulose. These parameters are pH, time, and temperature of bisacryloylpiperazine enzyme activation reaction. Under the best immobilization conditions copolymer activity linearly depends on enzyme concentration. Enzyme coupling efficiency depends on the type of enzyme and it ranges from 7 to 20%. The most important characteristics of these immobilized enzyme systems were tested and compared with those of similar systems obtained by glycidylmethacrylate enzyme activation (stability in continuous washing, kinetic characteristics, and storage, thermal, and operational stability). Immobilized enzyme graft copolymers have kinetic behaviour very close to that of the free enzymes. Diffusion is not seriously limited because immobilization reaction does not alter the enzymatic activity. By means of bisacryloylpiperazine it was possible to immobilize chymotrypsin with better results than those previously obtained, particularly coupling efficiency and long term continuous working.