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ABSTRACT: 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.
Nanoscale Research Letters 05/2012; 7(1):279. · 2.73 Impact Factor
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ABSTRACT: Here we present a sensitive DNA detection protocol using quantum dots (QDs) and magnetic beads (MBs) for large volume samples.
In this study, QDs, conjugated with streptavidin, were used to produce fluorescent signals while magnetic beads (MBs) were
used to isolate and concentrate the signals. The presence of target DNAs leads to the sandwich hybridization between the functionalized
QDs, the target DNAs and the MBs. In fact, the QDs-MBs complex, which is bound using the target DNA, can be isolated and then
concentrated. The binding of the QDs to the surface of the MBs was confirmed by confocal microscopy and Cd elemental analysis.
It was found that the fluorescent intensity was proportional to concentration of the target DNA, while the presence of non-complementary
DNA produced no significant fluorescent signal. In addition, the presence of low copies of target DNAs such as 0.5 pM in large
volume samples up to 40 mL was successfully detected by using a magnet-assisted concentration protocol which consequently
results in the enhancement of the sensitivity more than 100-fold.
Biotechnology and Bioprocess Engineering 04/2012; 11(5):449-454. · 1.28 Impact Factor
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ABSTRACT: This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)(3) linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO(2)-MNPs, Fe(3)O(4)@SiO(2)) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO(2)-MNPs was a time of 60 min and a concentration of 100 μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO(2)-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50 ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO(2)-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.
Biosensors & bioelectronics 07/2011; 28(1):146-51. · 5.43 Impact Factor
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Byoung Chan Kim,
Jinwoo Lee,
Wooyong Um,
Jaeyun Kim,
Jin Joo, Jin Hyung Lee,
Ja Hun Kwak,
Jae Hyun Kim,
Changha Lee,
Hongshin Lee,
R Shane Addleman,
Taeghwan Hyeon,
Man Bock Gu,
Jungbae Kim
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ABSTRACT: We have synthesized two different magnetic mesoporous materials that can be easily separated from aqueous solutions by applying a magnetic field. Synthesized magnetic mesoporous materials, Mag-SBA-15 (magnetic ordered mesoporous silica) and Mag-OMC (magnetic ordered mesoporous carbon), have a high loading capacity of contaminants due to high surface area of the supports and high magnetic activity due to the embedded iron oxide particles. Application of surface-modified Mag-SBA-15 was investigated for the collection of mercury from water. The mercury adsorption using Mag-SBA-15 was rapid during the initial contact time and reached a steady-state condition, with an uptake of approximately 97% after 7h. Application of Mag-OMC for collection of organics from water, using fluorescein as an easily trackable model analyte, was explored. The fluorescein was absorbed into Mag-OMC within minutes and the fluorescent intensity of solution was completely disappeared after an hour. In another application, Mag-SBA-15 was used as a host of tyrosinase, and employed as recyclable catalytic scaffolds for tyrosinase-catalyzed biodegradation of catechol. Crosslinked tyrosinase in Mag-SBA-15, prepared in a two step process of tyrosinase adsorption and crosslinking, was stable enough for catechol degradation with no serious loss of enzyme activity. Considering these results of cleaning up water from toxic inorganic and organic contaminants, magnetic mesoporous materials have a great potential to be employed for the removal of environmental contaminants and potentially for the application in large-scale wastewater treatment plants.
Journal of hazardous materials 06/2011; 192(3):1140-7. · 4.14 Impact Factor
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ABSTRACT: An enzyme-magnetic nanoparticle conjugate is prepared via conjugation of Ni(2+) ions onto the surface of magnetic nanoparticles to interact with a six histidine-tagged enzyme. The catalytic properties and enzyme rigidification of the conjugates are more stable at high concentrations of aromatic hydrocarbons.
Chemical Communications 05/2011; 47(36):9989-91. · 6.17 Impact Factor
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ABSTRACT: Over the years, nanostructures have been developed to enable to support enzyme usability to obtain highly selective and efficient biocatalysts for catalyzing processes under various conditions. This review summarizes recent developments in the nanostructures for enzyme supporters, typically those formed with various inorganic materials. To improve enzyme attachment, the surface of nanomaterials is properly modified to express specific functional groups. Various materials and nanostructures can be applied to improve both enzyme activity and stability. The merits of the incorporation of enzymes in inorganic nanomaterials and unprecedented opportunities for enhanced enzyme properties are discussed. Finally, the limitations encountered with nanomaterial-based enzyme immobilization are discussed together with the future prospects of such systems.
BMB reports 02/2011; 44(2):77-86. · 1.72 Impact Factor
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ABSTRACT: In Corynebacterium glutamicum, the ArgR protein, a transcriptional repressor, affects the expression level of the argB gene through binding to its promoter region. The argB promoter region (positions -77 to -25) has been found by in vitro electrophoretic mobility shift assay (EMSA) results and in silico analysis to be important for the DNA binding of ArgR. Proline supplementation prevented the DNA binding of ArgR to the argB promoter region and triggered an increase of the argB mRNA level. Additional mutational analyses of the argB promoter region found nucleotides critical for ArgR binding (G located at position -58, C at position -55, and A at position -41 of the argB promoter) in that region. Another transcriptional repressor, FarR, was also demonstrated to bind to the argB promoter region. This binding was delimited to positions -57 to -77 on the argB promoter. FarR has only one putative binding domain located at positions -57 to -77, but this region exactly overlapped with the binding region located from positions -55 to -77 for the binding of ArgR within the argB promoter; thus, if ArgR bound with the argB promoter first, the binding of FarR was not observed in this region. However, if FarR bound to the binding domain located at positions -57 to -77 first, ArgR could bind other binding sites located at positions -49 to -25 within the argB promoter. Finally, this study suggests that ArgR can affect FarR binding to the argB promoter region, as protein binding is dominated by the protein most able to do so.
Applied and environmental microbiology 02/2011; 77(3):711-8. · 3.69 Impact Factor
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ABSTRACT: This study shows the optimization of biotin labeling to antibodies using mouse IgG. Several parameters of the biotin labeling, including the molar ratio of biotin to antibody, the coupling time and the dialysis time, were studied to optimum conditions. The biotin-tagged mouse IgGs were immobilized on avidin-coated PMMA (Polymethyl Methacrylate) plates via a biotin-avidin linkage. The immobilization of the IgG to the chip was quantified using goat anti-mouse IgG bound fluorescent beads. It was found that the binding of the fluorescent beads saturated when a 10-fold or higher molar ratio of biotin to antibody was used. In biotin coupling time tests, sixty minutes was sufficient for the capture probes to bind to the surface. However, the results from the dialysis experiments showed no difference, indicating that 2 hours was sufficient to remove any unbound biotin. Finally, to prove the universality of this protocol using mouse antibodies, the optimum conditions were successfully applied in sandwich immunoassays designed to detect troponin I (TnI) and N-terminal probrain natriuretic peptide (NT-proBNP).
Journal of immunological methods 10/2010; 362(1-2):38-42. · 2.35 Impact Factor
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ABSTRACT: 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.
Analytical Biochemistry 10/2010; 405(1):135-7. · 3.00 Impact Factor
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ABSTRACT: The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the
ground for the next step in the evolution of both disciplines – the whole cell array. In the present chapter, we highlight
the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering
of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning
on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by
the reporter cell arrays, some of the transduction methodologies for reading these signals, and the mathematical approaches
proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the
future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation
into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for
multiplex signal analysis, and – most importantly – enhanced long term maintenance of viability and activity on the fabricated
biochips.
KeywordsWhole-cell arrays-Biosensors-Cell immobilization-Cell deposition-Toxicity testing-Gene expression-Bioreporters
07/2010: pages 85-108;
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ABSTRACT: In this study, recombinant plasmid was constructed to analyze the effect of hydrogen
production on the expression HupSL hydrogenase isolated from Rhodobacter sphaeroides in
Escherichia coli. Although most of recombinant HupSL hydrogenase was produced as
inclusion bodies the solubility of the protein increased significantly when the expression
temperature shifted from 37 �C to 30�C. Hydrogen production by expression of HupSL
hydrogenase from recombinant E. coli increased 20.9-fold compared to control E. coli and
218-fold compared to wild type R. sphaeroides under anaerobic dark condition. The results
demonstrate that HupSL hydrogenase, consisting of small and large subunits of hydrogenase
isolated from R. sphaeroides, increases hydrogen production in recombinant E. coli. In
addition conditions for enhancing the activity of HupSL hydrogenase in E. coli were
suggested and were used to increase bacterial hydrogen production.
International Journal of Hydrogen Energy 01/2010; 35:1112-1116. · 4.05 Impact Factor
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ABSTRACT: The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines - the whole cell array. In the present chapter, we highlight the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals, and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis, and - most importantly - enhanced long term maintenance of viability and activity on the fabricated biochips.
Advances in biochemical engineering/biotechnology 01/2010; 117:85-108. · 1.64 Impact Factor
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Small 07/2008; 4(6):746-50. · 8.35 Impact Factor
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ABSTRACT: The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines - the whole-cell array. In the present review, we highlight the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis and - most importantly - enhanced long-term maintenance of viability and activity on the fabricated biochips.
Microbial Biotechnology 03/2008; 1(2):137-48. · 2.53 Impact Factor
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ABSTRACT: The recombinant bioluminescent bacterium, DNT5, containing a nagR-nagAa::luxCDABE fusion, was tested in a multi-channel continuous monitoring system to evaluate its ability to detect benzoic acid derivatives. Seven chemicals, benzoic acid, salicylic acid, 2,5-dihydroxy benzoic acid, 3,5-dihydroxy benzoic acid, benzene, naphthalene and phenol, were used to characterize the responses of DNT5. This strain responded uniquely to each chemical, and these responses were then evaluated based upon the structures of each chemical. The greatest bioluminescent responses were to salicylic acid and benzoic acid, followed by 2,5-dihydroxy benzoic acid and 3,5-dihydroxy benzoic acid, but DNT5 was unresponsive when exposed to benzene, phenol and naphthalene, suggesting it has a strong preference for benzoic acid derivatives with few or no ring-substituted groups.
Journal of Biotechnology 10/2007; 131(3):330-4. · 3.05 Impact Factor
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ABSTRACT: This study shows the preparation and application of enzyme-nanofiber composites for long-term stable operation. The enzyme-nanofiber composite was prepared by coating an enzyme aggregate, the esterase from Rhizopus oryzae, on the surface of the nanofibers. After immobilization on the nanofiber, the apparent K ( m ) for the immobilized esterase was 1.48-fold higher than that of the free esterase, with values of 0.98 and 1.35 mM for the free and immobilized enzymes, respectively. It was found that enzyme-nanofiber was very stable, even when the fibers were shaken in glass vials, preserving 80% of the initial activity for 100 days. In addition, the enzyme-nanofiber composite was used repeatedly in 30 cycles of substrate hydrolysis and still remained active. Consequently, the esterase-nanofiber composite was employed within a continuous reactor system to evaluate its use in a long-term and stable continuous substrate hydrolysis reaction. It was found that the production of p-nitrophenol was stable for at least 400 h. This study demonstrates that the enzyme-nanofiber composite can be used in both repeated-batch mode and a continuous mode for a long-term stable operation.
Applied Microbiology and Biotechnology 07/2007; 75(6):1301-7. · 3.42 Impact Factor
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ABSTRACT: An oxidative stress-specific bacterial cell array chip was fabricated and implemented in the analysis of various different chemicals. The chip consisted of twelve toxicity responsive strains that respond specifically to different oxidative toxicities such as the generation of the superoxide radical, except for strain EBMalK, which was included as a negative control. Each bioluminescent strain carried a fusion of a stress gene promoter (sodA, pqi-5, soxR, fumC, soxS, inaA, hmp, malK, katG, zwf, fpr or pgi) to the bacterial lux reporter genes. A total of nine chemicals were selected to exhibit the capabilities of this array when analyzing different oxidative toxicities. Each of the chemicals were categorized according to their structure and their ability to form radicals in vivo: (I) paraquat, an active radical producer, (II) structural analogs of paraquat that produce radicals, (III) chemicals that are distinct from paraquat but still produce radicals and (IV) chemicals having similar structures as paraquat but do not produce radicals. The results found that each strain was responsive to one or more of the compounds tested but, as a definitive factor, the responses from the chip were dependent upon the production of radicals, i.e., the strains were unresponsive to compounds that were similar in structure to paraquat but lacked the ability to generate radicals. The specificity of the strains used in the chip was also demonstrated by their ability to discriminate between the superoxide radical and hydrogen peroxide. Therefore, this cell array chip could be implemented in characterizing and understanding the toxic impacts of newly synthesized chemicals and drugs in terms of toxicity classification and the nature of oxidative damage experienced by cells.
Biosensors and Bioelectronics 05/2007; 22(9-10):2223-9. · 5.60 Impact Factor
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ABSTRACT: A bio-MEMS based cell-chip that can detect a specific toxicity was fabricated by patterning and immobilizing bioluminescent bacteria in a microfluidic chip. Since the emitted light intensity of bioluminescent bacteria changed in response to the presence of chemicals, the bacteria were used as the toxicity indicator in this study. A pattern of immobilized cells was successfully generated by photolithography, utilizing a water-soluble and negatively photosensitive polymer, PVA-SbQ (polyvinyl alcohol-styrylpyridinium) as an immobilization material. Using the recombinant Escherichia coli (E. coli) strain, GC2, which is sensitive to general toxicity, the following were investigated for the immobilization: an acceptable dose of long-wavelength UV light, the biocompatibility of the polymer, and the effect of the chip-environment. We found that 10 min of UV light exposure, the toxicity of polymer (SPP-H-13-bio), and the other chip-environment did not inhibit cell metabolism significantly for making a micro-cell-chip. Detection of a specific toxicity was demonstrated by simply immobilizing the bioluminescent bacteria, DK1, which increased bioluminescence in the presence of oxidative damage in the cells. An injection of hydrogen peroxide of 0.88 mM induced 10-fold increase in bioluminescent intensity confirming the capability of the chip for toxicity monitoring.
Biosensors and Bioelectronics 04/2007; 22(8):1586-92. · 5.60 Impact Factor
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ABSTRACT: In this study, a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity has been implemented to develop two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 microl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.
Biosensors and Bioelectronics 10/2005; 21(3):500-7. · 5.60 Impact Factor
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ABSTRACT: An integrated water toxicity monitoring system that uses recombinant bioluminescent bacteria was successfully developed for the continuous monitoring and classification of toxicities present in water. This system consists of four channels arranged horizontally inside of a cylinder, with each channel having two small bioreactors that are vertically connected to each other to maintain a separation of the culture reactor from test reactor. This system is easily handled and installed, making its application in the field a potential reality. As well, it performed stably and continuously due to the vertical separation of the culture reactor from the test reactor and a long term operation was also performed because of its small working volume, i.e., only 1 ml for the 1st bioreactor and 2 ml for the 2nd. During an operation with four strains, i.e., EBHJ2, DP1, DK1 and DPD2794, which are responsive to superoxide damage (EBHJ2 and DP1), hydrogen peroxide (DK1), and DNA damage (DPD2794), the O.D. and bioluminescence of the bacterial cultures inside the system were constant when no chemical was injected. However, with the addition of paraquat, hydrogen peroxide or mitomycin C, the bioluminescent responses of the strains were found to be dose-dependent to different concentrations of these chemicals.
Biosensors and Bioelectronics 04/2005; 20(9):1744-9. · 5.60 Impact Factor
