Jiho Lee

High throughput detection and selective enrichment of histidine-tagged enzymes with Ni-doped magnetic mesoporous silica

This work reports a simple and facile method to prepare novel magnetic mesoporous silica (MMS) materials with high magnetic strength for the convenient and high throughput detection of histidine-tagged enzymes with Ni-doped surfaces. These materials are designed by the incorporation of high-abundance and homogeneously dispersed iron nanoparticles within the mesopores by thermal hydrogen reduction after the incorporation of ferrous ions and demonstrated the selective enrichment and high-throughput recognition of His-tagged enzymes with multi-point anchoring by selective conjugation between the His-tag and Ni ions. Selective His-tagged enzyme enrichment efficiency was compared with nickel-based MMS materials, such as Ni2+-MMS and Ni-MMS, and nickel ion doped silica-coated magnetic nanoparticles (Ni2+-MNPs). The efficiency was calculated to be 100 ± 1.93%, 70.94 ± 1.95%, and 37.03 ± 5.93% for Ni2+-MMS, Ni-MMS, and Ni2+-MNPs, respectively. This method enables a high-throughput and advanced systematic approach for the separation and immobilization of proteins which cover a broad spectrum of polyhistidine-tagged proteins.

Amino acid side chain-like surface modification on magnetic nanoparticles for highly efficient separation of mixed proteins

This work reports on the realization of specific functionalized silica-coated magnetic nanoparticles (Si-MNPs) for effective protein separation through surface modification with various amino acid side chain-like functional groups such as thiol (-SH), disulfide (-S-S-), carbon chain (-C(n)), carboxyl (-COOH), amine (-NH(2)), and aldehyde (-CHO). This study also suggests an improved and convenient method for the synthesis of functionalized Si-MNPs by hydrolysis condensation with silan-coupling agents. The protein adsorption effects in a coexistent mixed state are explored using various proteins, which have different isoelectric point (pI) values and molecular weights, in order to elucidate the binding performance of different proteins one solution. The adsorption efficiency of bovine serum albumin (BSA; 66 kDa; pI=4.65) and lysozyme (LYZ; 14.3 kDa; pI=11) is 70-100% with various amino acid side chain-like functional groups. However, the adsorption efficiency of a mixed protein solution of BSA and LYZ was different. Although the relatively bulky BSA molecule displayed 50% and 20% adsorption corresponding to pH 4.65, and pH 11, respectively, the smaller LYZ provided almost 100% adsorption at both pH 4.65 and pH 11.

Hydrophobic partitioning approach to efficient protein separation with magnetic nanoparticles.

A novel approach to the hydrophobic partitioning effect on efficient separation of protein such as BSA was demonstrated by the modification of hydrophobic pockets on the surface of silica-coated magnetic nanoparticles with various alkyl groups at various pH levels. The separation efficiency is strongly reflected and can be attained by controlling the size of the hydrophobic pocket and other factors such as the alkyl chain length, the salt concentration, and the pH levels. Furthermore, the adsorption constant (k(ad)) was calculated for the hydrophobic partitioning interaction between BSA and the hydrophobic pocket size at various pH levels.

Rapid pathogen detection with bacterial-assembled magnetic mesoporous silica

We report rapid and accurate pathogen detection by coupling with high efficiency magnetic separation of pathogen by Ni(2+)-heterogeneous magnetic mesoporous silica (Ni-HMMS) and real time-polymerase chain reaction (RT-PCR) technique. Ni-HMMS was developed with a significant incorporation of Fe particles within the silica mesopores by programmed thermal hydrogen reaction and functionalized with Ni(2+) ion on the surface by the wet impregnation process. High abundant Ni(2+) ions on the Ni-HMMS surface were able to assemble with cell wall component protein NikA (nickel-binding membrane protein), which contains several pathogenic bacteria including Escherichia coli O157:H7. NikA protein expression experiment showed the outstanding separation rate of the nikA gene-overexpressed E. coli (pSY-Nik) when comparing with wild-type E. coli (44.5 ± 13%) or not over-expressed E. coli (pSY-Nik) (53.2 ± 2.7%). Moreover, Ni-HMMS showed lower obstacle effect by large reaction volume (10 mL) than spherical core/shell-type silica magnetic nanoparticles functionalized with Ni(2+) (ca. 40 nm-diameters). Finally, the Ni-HMMS was successfully assessed to separate pathogenic E. coli O157:H7 and applied to direct and rapid RT-PCR to quantitative detection at ultralow concentration (1 Log10 cfu mL(-1)) in the real samples (milk and Staphylococcus aureus culture broth) without bacterial amplification and DNA extraction step.

Rapid and selective separation for mixed proteins with thiol functionalized magnetic nanoparticles

Thiol group functionalized silica-coated magnetic nanoparticles (Si-MNPs@SH) were synthesized for rapid and selective magnetic field-based separation of mixed proteins. The highest adsorption efficiencies of binary proteins, bovine serum albumin (BSA; 66 kDa; pI = 4.65) and lysozyme (LYZ; 14.3 kDa; pI = 11) were shown at the pH values corresponding to their own pI in the single-component protein. In the mixed protein, however, the adsorption performance of BSA and LYZ by Si-MNPs@SH was governed not only by pH but also by the molecular weight of each protein in the mixed protein.

Nanoporous Silicified Phospholipids and Application to Controlled Glycolic Acid Release

This work demonstrates the synthesis and characterization of novel nanoporous silicified phospholipid bilayers assembled inorganic powders. The materials are obtained by silicification process with silica precursor at the hydrophilic region of phospholipid bilayers. This process involves the co-assembly of a chemically active phospholipids bilayer within the ordered porosity of a silica matrix and holds promise as a novel application for controlled drug release or drug containers with a high level of specificity and throughput. The controlled release application of the synthesized materials was achieved to glycolic acid, and obtained a zero-order release pattern due to the nanoporosity.

Histidine-tagged enzyme conjugated heterogeneous magnetic mesoporous silica for high efficient biodegradation of catechol

In this study, we present a novel method for mesoporous silicas which are functionalized with Fe and Ni ion to support His-tagged proteins. Mesoporous silicas were combined with iron (II) chloride tetrahydrate for initiating magnetizing process, followed by reduction in H2 media. The nickel (II) chloride hexahydrate was introduced into pore and surface of heterogeneous magnetic mesoporous silicas (HMMS) by wet impregnation process. The resulting materials were reduced in a flowing of H2 at 500 °C for 2h, thereby producing Ni particles on the surface of the HMMS. The Ni-HMMS was characterized by various state-of-the-art techniques, such as WAXS, FE-SEM, TEM, SQUID and nitrogen sorption. Ni-HMMS showed high adsorption level of His-tagged protein from Escherichia coli lysate. In addition, the purified His6-CatA was observed high enzyme activity. It suggests that Ni-MMS system provides contribution for maintain stable protein structure as well as simplified protein purification process.

The origin of coarse contrast modulation in In0.525Ga0.475As layer on InP grown by MBE at 310 °C

Abstract In the TEM plane view observation of phase separated microstructures in several regions of In 0.525 Ga 0.475 As layer grown at 310 °C by MBE on InP substrate, fine modulation is found in all the regions examined, but coarse modulation is found only in thick sections of TEM thin foils. These results strongly suggest that the coarse modulation in our sample is an artifact of thin foils which appears as a result of accommodation of the biaxial stress associated with the fine modulation.