• Title/Summary/Keyword: Immobilization method

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Development of Bioluminescence Immunoassay Using Photoprotein, Aequorin and Site-directed Immobilization

  • Shim, Yu-Nee;Rhee, In-sook
    • Bulletin of the Korean Chemical Society
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    • v.24 no.1
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    • pp.70-74
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    • 2003
  • The heterogeneous bioluminescence immunoassay for digoxin was developed using photoprotein, native aequorin as a label and the site-directed immobilization technique based on avidin/biotin interaction. Aequorin is a bioluminescence protein, originally isolated from the jellyfish Aequoria Victoria and an attractive label in analytical applications because of sensitive detection due to virtually no background bioluminescent signal. Digoxin is a cardioactive drug, and its therapeutic level in serum is at low concentration with very narrow therapeutic index. The aequorin-digoxigenin conjugates were synthesized by the N-hydroxysuccinimide ester method and characterized in terms of bioluminescent residual activity. The resulting dose-response curve shows that the detection limit is $1.0\;{\times}\;10^{-10}\;M$ and a dynamic range is three orders of magnitude, which was obtained by $1.0\;{times}\;10^{-10}\;M$ conjugate and 0.9 μg/mL anti-digoxin antibody. Three structurally similar molecules to digoxin were examined for their cross-reactivity. None of these three compounds showed any crossreactivity with digoxin antibody employed in this study. Standard amounts of digoxin corresponding to the therapeutic range were spiked into the each serum solution. Study of the serum matrix effect indicated that correlation coefficient shows good agreement between luminescence light intensity between in buffer and in serum.

Enhanced ${\varepsilon}$-Poly-$_L$-lysine Production from Streptomyces ahygroscopicus by a Combination of Cell Immobilization and In Situ Adsorption

  • Liu, Shengrong;Wu, Qingping;Zhang, Jumei;Mo, Shuping;Yang, Xiaojuan;Xiao, Chun
    • Journal of Microbiology and Biotechnology
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    • v.22 no.9
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    • pp.1218-1223
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    • 2012
  • ${\varepsilon}$-Poly-$_L$-lysine (${\varepsilon}$-PL), produced by Streptomyces or Kitasatospora strains, is a homo-poly-amino acid of $_L$-lysine, which is used as a safe food preservative. The present study investigates the combined use of cell immobilization and in situ adsorption (ISA) to produce ${\varepsilon}$-PL in shaken flasks. Loofah sponge-immobilized Streptomyces ahygroscopicus GIM8 produced slightly more ${\varepsilon}$-PL than those immobilized on synthetic sponge, and sugarcane bagasse. Moreover, loofah sponge supported the maximum biomass. Hence, loofah sponge was chosen for cell immobilization. Meanwhile, the ion-exchange resin D152 was employed for ISA. The loofah sponge-immobilized cells produced $0.54{\pm}0.1g/l$ ${\varepsilon}$-PL, which significantly increased to $3.64{\pm}0.32g/l$ after combining with ISA through the addition of resin bags. The free cells with ISA using the dispersed resin yielded $2.73{\pm}0.26g/l$ of ${\varepsilon}$-PL, an increase from $0.82{\pm}0.08g/l$. These data illustrate that the proposed combination method improved production most significantly compared with either immobilization or ISA only. Moreover, the immobilized cells could be repeatedly used and an ${\varepsilon}$-PL total amount of $8.05{\pm}0.84g/l$ was obtained. The proposed combination method offers promising perspectives for ${\varepsilon}$-PL production.

Electrochemical Detection of $17{\beta}-estradiol$ by using DNA Aptamer Immobilized Nanowell Gold Electrodes

  • Kim, Yeon-Seok;Jung, Ho-Sup;Lee, Hea-Yeon;Kawai, Tomoji;Gu, Man-Bock
    • 한국생물공학회:학술대회논문집
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    • 2005.04a
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    • pp.88-92
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    • 2005
  • Aptamer is the single-stranded oligonucleotide which binds to various target molecules such as proteins, peptides, lipids and small organic molecules with high affinity and specificity. DNA aptamers specific for the $17{\beta}-estradiol$ were selected by SELEX (Systematic Evolution of Ligands by EXponential enrichment) process from a random DNA library. These DNA aptamers have a high affinity to $17{\beta}-estradiol$ as an endocrine disrupting chemical. Nanowell and $200{\mu}m$ gold electrode were used as substrate for DNA aptamer immobilization and electrochemical analysis. Especially, nanowell gold electrode was fabricated by e-beam lithography. The size of single nanowell is 130nm and 40,000 nanowells were deposited on one gold electrode. The immobilization method was based on the interaction between the biotinylated aptamer and streptavidin deposited on gold electrode previously. Immobilization procedure was optimized by surface plasma resonance (SPR) and electrochemical analysis. After the immobilization of DNA aptamer on streptavidin modified gold electrode, $17{\beta}-estradiol$ solution was treated on aptamer immobilized gold electrode. The current of gold electrode was decreased by the binding of $17{\beta}-estradiol$ to DNA aptamer immobilized on gold electrode. However, in negative control experiments of 1-aminoanthraquinone and 2-methoxynaphthalene, the current was rarely decreased. And more sensitive data was obtained from nanowell gold electrode comparing with $200{\mu}m$ gold electrode.

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The Effects of Gammaekdaejo-tang (Ganmaidazao-tang) on Rats Subjected to Stress by Immobilization (감맥대조탕(甘麥大棗湯)이 신체부동 스트레스 백서(白鼠)에 미치는 영향(影響))

  • Baek, Hyun;Kim, Jang-Hyun
    • The Journal of Korean Medicine
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    • v.28 no.3 s.71
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    • pp.183-196
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    • 2007
  • Objective : This study was conducted in order to find the effects of Gammaekdaejo-tang (Ganmaidazao-tang, GDT) by subjecting rats to immobilization stress, thereby inducing depression, anxiety and acquisition-retention defects. Method : Rats treated with normal saline, GDT 200mg/kg and GDT 400mg/kg were subjected to stress by immobilization. Afterwards, behavior changes were observed by elevated plus maze test, acquisition test and retention test in the Morris water maze. The results were obtained by immunohistochemically measuring stress hormone (corticosteroid) levels in the blood. Results and Conclusions : 1. The open arm test in the elevated plus maze showed that compared with the normal group, the time spent decreased in the control group and increased in the GDT 400mg/kg group. 2. The locomotor activity test in the elevated plus maze revealed that the control group showed significant activity decrease compared with the normal group but significant increase in the GDT 400mg/kg group. 3. The acquisition test in the Morris water maze showed that the acquisitive ability of the control group significantly deteriorated on the 3rd and 4th day compared with the normal group, but improved significantly in the GDT 200mg/kg and GDT 400mg/kg groups. 4. The retention test on the 7th day in the Morris water maze revealed that the retentive ability of the control group significantly deteriorated compared with the normal group, but the retentive ability of the GDT 400mg/kg group significantly improved. 5. The blood levels of corticosteroid in the control group increased significantly compared with the normal group but the levels of corticosterone in the blood of the GDT 400mg/kg group significantly decreased.

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Synthesis and Characterization of Magnetic Nanoparticles and Its Application in Lipase Immobilization

  • Xu, Jiakun;Ju, Caixia;Sheng, Jun;Wang, Fang;Zhang, Quan;Sun, Guolong;Sun, Mi
    • Bulletin of the Korean Chemical Society
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    • v.34 no.8
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    • pp.2408-2412
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    • 2013
  • We demonstrate herein the synthesis and modification of magnetic nanoparticles and its use in the immobilization of the lipase. Magnetic $Fe_3O_4$ nanoparticles (MNPs) were prepared by simple co-precipitation method in aqueous medium and then subsequently modified with tetraethyl orthosilicate (TEOS) and 3-aminopropyl triethylenesilane (APTES). Silanization magnetic nanoparticles (SMNP) and amino magnetic nanomicrosphere (AMNP) were synthesized successfully. The morphology, structure, magnetic property and chemical composition of the synthetic MNP and its derivatives were characterized using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) analysis, X-ray diffraction, superconducting quantum interference device (SQUID) and thermogravimetric analyses (TGA). All of these three nanoparticles exhibited good crystallization performance, apparent superparamagnetism, and the saturation magnetization of MNP, SMNP, AMNP were 47.9 emu/g, 33.0 emu/g and 19.5 emu/g, respectively. The amino content was 5.66%. The AMNP was used to immobilize lipase, and the maximum adsorption capacity of the protein was 26.3 mg/g. The maximum maintained activity (88 percent) was achieved while the amount of immobilized lipase was 23.7 mg $g^{-1}$. Immobilization of enzyme on the magnetic nanoparticles can facilitate the isolation of reaction products from reaction mixture and thus lowers the cost of enzyme application.

Picomolar Scale Determination of Carbohydrates Covalently Immobilized on Activated Beads Using Hydroxyl Functionality

  • Yu, Jae-Hoon;Chun, Sung-Min;Park, Ho-Koon;Park, Yong-Keun;Jeong, Sun-Joo
    • BMB Reports
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    • v.32 no.1
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    • pp.98-102
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    • 1999
  • Since carbohydrates are major mediators in cell-to-cell adhesion and communication, the development of specific and strong binders against them could generate promising therapeutics. As the first step towards that goal, sugar molecules have to be immobilized to be used as an affinity matrix. The amino functionality in sugar is the most active nucleophile for the immobilization, if the amino group is available. An alternative and general method is to use the hydroxyl group as a direct nucleophile, but the quantitation of immobilized hydroxyl groups is not easily done. To overcome this limitation, we have developed a method to immobilize various isomers of monosaccharides with p-nitrophenyl groups to the beads by using their hydroxyl groups. It was found that the amount of immobilized sugar was independent of the structure of the sugar, but was dependent on the number of hydroxyl groups. We also developed a sensitive method to quantify the amount of immobilized sugar at the picomolar scale by utilizing commercially available glycosidases to release a sensitive reporter molecule, p-nitrophenol, and detect it by HPLC. This new technique would allow a facile quantitation method for immobilized sugar molecules, which could be used as the affinity matrix to develop strong binders against biologically important sugars.

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Comparison Study of Open Reduction and Closed Reduction in Treatment of Mandibular Subcondylar Fractures (하악골 과두하부골절 정복술에서 관혈적 정복술과 비관혈적 정복술의 비교 연구)

  • Jang, Ju Yun;Kang, Dong Hee
    • Archives of Craniofacial Surgery
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    • v.9 no.2
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    • pp.51-54
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    • 2008
  • Purpose: The choice of open versus closed reduction for mandibular subcondylar fracture is a debatable issue. To evaluate the advantage of open approach to closed method with IMF(intermaxillary fixation), we conducted a retrospective study to compare the outcomes of each method. Methods: From 2002 to 2006, 29 patients with mandibular subcondylar fractures were treated by open or closed reduction. 17 patients were treated by open reduction and 12 patients by closed reduction and IMF. Each group was assessed for duration of mandibular immobilization, incidences of buccal palsy, malocclusion, TMJ(temporomandibular joint) pain, and deviation of the mandible on mouth opening. Results: All cases showed accurate reduction in anatomical position, no significant displacement and no deviation on mouth opening during the follow-up period. IMF period is statistically shorter in open reduction (p<0.05). Differences in incidence of other complications were not significant statistically. Conclusion: As there are significant independent morbidities associated with IMF which requires postoperative rehabilitation, prolonged temporomandibular immobilization should not be overlooked. Some patients with poor compliances will not tolerate IMF in nonsurgical treatment. In the aspect of patient's convenience and early recovery by short IMF period, open reduction would be recommended as a better treatment method.

Melting and draining tests on glass waste form for the immobilization of Cs, Sr, and rare-earth nuclides using a cold-crucible induction melting system

  • Choi, Jung-Hoon;Lee, Byeonggwan;Lee, Ki-Rak;Kang, Hyun Woo;Eom, Hyeon Jin;Park, Hwan-Seo
    • Nuclear Engineering and Technology
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    • v.54 no.4
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    • pp.1206-1212
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    • 2022
  • Cold-crucible induction melting (CCIM) technology has been intensively studied as an advanced vitrification process for the immobilization of highly radioactive waste. This technology uses high-frequency induction to melt a glass matrix and waste, while the outer surface of the crucible is water-cooled, resulting in the formation of a frozen glass layer (skull). In this study, for the fabrication of borosilicate glass waste form, CCIM operation test with 60 kg of glass per batch was conducted using surrogate wastes composed of Cs, Sr, and Nd as a representative of highly radioactive nuclides generated during spent nuclear fuel management. A 60 kg-scale glass waste form was successfully fabricated through melting and draining processes using a CCIM system, and its physicochemical properties were analyzed. In particular, to enhance the controllability and reliability of the draining process, an air-cooling drain control method that can control draining through air-cooling near drain holes was developed, and its validity for draining control was verified. The method can offer controllability on various draining processes, such as molten salt or molten metal draining processes, and can be applied to a process requiring high throughput draining.

SrAl2Si2O8 ceramic matrices for 90Sr immobilization obtained via spark plasma sintering-reactive synthesis

  • Papynov, E.K.;Belov, A.A.;Shichalin, O.O.;Buravlev, I. Yu;Azon, S.A.;Golub, A.V.;Gerasimenko, A.V.;Parotkina, Yu. А.;Zavjalov, A.P.;Tananaev, I.G.;Sergienko, V.I.
    • Nuclear Engineering and Technology
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    • v.53 no.7
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    • pp.2289-2294
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    • 2021
  • In the present study, an original spark plasma sintering-reactive synthesis (SPS-RS) method for minerallike ceramic materials based on SrAl2Si2O8 feldspar-like skeleton structure was used for the first time, promising solid-state matrices for reliable immobilization of high-energy 90Sr. The method is based on the "in-situ" reaction of a mixture of SrO, Al2O3 and SiO2 oxides when heated by a unipolar pulsed current under compacting pressure. The phase and elemental composition structure were studied. The dynamics of the consolidation of the reaction mixture of oxides was studied in the range of 900-1200 ℃. The study found the temperature of the high-speed (minutes) SPS-RS formation of single-phase SrAl2Si2O8 composition ceramic in the absence of intermediate reaction products with a relative density of up to 99.2% and compressive strength up to 145 MPa and a strontium leaching rate of 10-4g/cm2·day.

Immobilization of Microorganisms - Part 1. Preparation of Immobilized Lactobacillus bulgaricus - (미생물(微生物) 고정화(圖定化)에 관한 연구(硏究) - 제1보(第1報). Lactobacillus 균(菌)의 고정화조건(圖定化條件) -)

  • Lee, Kang-Heup
    • Applied Biological Chemistry
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    • v.24 no.2
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    • pp.149-152
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    • 1981
  • The immobilization of Lactobacillus bulgaricus was investigated by various method, e.g. by use of polyacrylamide gel and Al-, Ca-, Fe- or Mg-alginate beads, and the most active immobilized cells were obtained by entrapment in a Ca-alginate beads. These immobilized microbial cells, when introduced into 4.5% lactose solution and whey solution showed maximum relative activity of 28% or lactose solution and 18% for whey solution as measured against the native microbial reference standard (100).

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