Earticle

Removal performance of the photocatalyst biofilter hybrid system for the removal of Styrene was evaluated to optimize operation conditions. First, to investigate the photocatalytic degradation of styrene, various nanocrystalline TiO2 thin films have been prepared by sol-gel method. And the optical fibers are employed as the light-transmitting guide with the immobilizing TiO2. Light source was used 500W high pressure mercury UV lamp. The effects of the inlet concentration of styrene, flow rate, relative humidity and the thickness of TiO2 thin films on the degradation of the styrene were examined. In addition, the effects of some metal ions that can be doped into the TiO2 thin films were considered in order to get higher photocatalytic degradation. Biofiltration involves the passage of a polluted air stream through a packed bed containing microorganisms immobilized within a biofilm attached to the bed-packing material. A biofilter system used in this study was packed with glass-type media with 5L of bed volume and inoculated with mixed microorganisms obtained from a sewage treatment plant. The biofilter systems were operated at an EBRT (Empty Bed Retention Time) of 20~30s under nitrogen deficient condition where nutrient solution was supplied only 1 L every 2 weeks. Toluene concentration of 10~20 ppmv corresponded to elimination capacity 12 ~ 60 g/m3 h. Air flow rate is 10L/min. GC (Gas Chromatography) and MS (Mass Spectroscopy) were used for analysis of the Toluene removal capacity and detection of Toluene degradation byproduct.

Alopecia is the medical description of the loss of hair from the head or body. Alopecia tends to be involuntary and unwelcome. Alopecia does not vitally influence one's life but patients who undergo alopecia appeal severe mental stress. The number of alopecia patients increases steadily. The current therapies used for treating alopecia have a number of limitations and to overcome such problems, many researchers have attempted to revive hair follicles by in vitro culturing hair follicle cells and implanting them in the treatment area. In this study, umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be isolated and expanded successfully from the Wharton's Jelly. Culture-expanded UC-MSCs formed aggregates similar to native dermal papilla (DP) in size and morphology under the special medium. Also the aggregates showed similar protein expression pattern of primary dermal papilla cells (DPCs). To confirm whether the aggregates could induce hair follicle instead of DP, they were transplanted with outer root sheath cells (ORSCs) in athymic nude mice's inner-dermis where cells underwent proliferation and differentiation. After 6 weeks of transplantation, we observed new hairs on the mice's scalp apparently. In conclusion, our results suggest that UC-MSC would be one of the cell sources for the treatment of alopecia in the future.

To support and enhance the in vitro differentiation and activity of cord stem cells(CSCs), the cell culture medium maybe supplemented with various proteins and factors in order to mimic the physiological environment in which cells optimally proliferate and differentiate. In this study, we evaluate the effects of mechanical tension on the differentiation and extracellular matrix (ECM) production of CSCs using a flexwell system that imposed cyclic mechanical tension at 0.03 Hz with 0, 5, and 10 % strains. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from CSCs during culture and increased GAG and elastin synthesis. And mechanical tension increased osteocalcin, osteopontin, and vimentin expression by RTPCR analysis. It is thought that the appropriate level of mechanical stretch served as a potent positive modulator of CSC differentioation.

Magnetic nanoparticles isolated from Magnetospirillum sp. AMB-1 can be applied to various therapeutic materials. However, the intracellular uptake pathway of magnetic nanoparticles has not been clearly elucidated yet. In this study, we investigated the intracellular uptake mechanism of magnetic nanoparticles. Magnetic nanoparticles were delivered to the endothelial progenitor cells (EPC) and their uptake pathways were investigated through various methods. Magnetic nanoparticle internalization into EPCs increased with incubation time in the presence of magnetic nanoparticles in the medium. Cellular uptake of magnetic nanoparticles was inhibited in the low temperature (4℃) and confocal study demonstrated that nanoparticles were internalized via endosome at least within 1 h after incubation. We also studied the exocytosis of magnetic nanoparticles by time-dependent manner. Our results demonstrated that the uptake process of magnetic nanoparticles was dynamic and the magnetic nanoparticles from AMB-1 could be internalized into the EPCs through energy-dependent endocytotic pathway.

Magnetic nanoparticles isolated from Magnetospirillum sp. AMB-1 can be applied to various therapeutic materials. However, the intracellular uptake pathway of magnetic nanoparticles has not been clearly elucidated yet. In this study, we investigated the intracellular uptake mechanism of magnetic nanoparticles. Magnetic nanoparticles were delivered to the endothelial progenitor cells (EPC) and their uptake pathways were investigated through various methods. Magnetic nanoparticle internalization into EPCs increased with incubation time in the presence of magnetic nanoparticles in the medium. Cellular uptake of magnetic nanoparticles was inhibited in the low temperature (4℃) and confocal study demonstrated that nanoparticles were internalized via endosome at least within 1 h after incubation. We also studied the exocytosis of magnetic nanoparticles by time-dependent manner. Our results demonstrated that the uptake process of magnetic nanoparticles was dynamic and the magnetic nanoparticles from AMB-1 could be internalized into the EPCs through energy-dependent endocytotic pathway.

Transgenic rice, Oryza sativa L., cells were cultured to produce human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a fusion protein used for the treatment of rheumatoid arthritis. This plant cell culture system with the signal peptide for the secretion of target protein into the extracellular medium and the inducible RAmy3D promoter was used for the production of the recombinant therapeutic protein in order to overcome the constraint of low production level.1) However, the RAmy3D promoter system has a drawback in decreasing cell viability because the promoter is induced only by sugar starvation. The cell death stimulated the release of protease, which degraded target protein.2) In order to solve these instability problems of the product after secretion, in situ product recovery was performed by using a modified flask.3) The modified flask was specially designed with glass filter to make medium exchange easy and successful. The medium exchange provided an improved culture condition for the cells because proteases could be removed from the culture environment by dilution with new medium after exchange. Contrary to the expectation, it was found that the production level of hCTLA4Ig was not increased despite of the better maintenance of cell mass than those of control. These results show that in situ product recovery using a modified flask could not enhance the production of hCTLA4Ig in transgenic rice cell culture system.

Transgenic rice, Oryza sativa L., cells were cultured to produce human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), a fusion protein used for the treatment of rheumatoid arthritis. This plant cell culture system with the signal peptide for the secretion of target protein into the extracellular medium and the inducible RAmy3D promoter was used for the production of the recombinant therapeutic protein in order to overcome the constraint of low production level.1) However, the RAmy3D promoter system has a drawback in decreasing cell viability because the promoter is induced only by sugar starvation. The cell death stimulated the release of protease, which degraded target protein.2) In order to solve these instability problems of the product after secretion, in situ product recovery was performed by using a modified flask.3) The modified flask was specially designed with glass filter to make medium exchange easy and successful. The medium exchange provided an improved culture condition for the cells because proteases could be removed from the culture environment by dilution with new medium after exchange. Contrary to the expectation, it was found that the production level of hCTLA4Ig was not increased despite of the better maintenance of cell mass than those of control. These results show that in situ product recovery using a modified flask could not enhance the production of hCTLA4Ig in transgenic rice cell culture system.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious infection of swine, which is classified by the Office International des Epizooties (OIE) as a list of a disease. Envelope glycoprotein E2 is the major neutralizing antigen for CSFV infection. In this study, we describe the expression of recombined CSFV E2 gene by tobacco chloroplast transformation. The E2 gene was cloned into chloroplast targeting vector for high level expression. The recombinant vector was transformed to tobacco plant by particle bombardment method. Genomic DNA PCR analysis confirmed stable homologous recombination of the E2 gene into the chloroplast genome. And Northern blot analysis confirmed the expression level of mRNA.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious infection of swine, which is classified by the Office International des Epizooties (OIE) as a list of a disease. Envelope glycoprotein E2 is the major neutralizing antigen for CSFV infection. In this study, we describe the expression of recombined CSFV E2 gene by tobacco chloroplast transformation. The E2 gene was cloned into chloroplast targeting vector for high level expression. The recombinant vector was transformed to tobacco plant by particle bombardment method. Genomic DNA PCR analysis confirmed stable homologous recombination of the E2 gene into the chloroplast genome. And Northern blot analysis confirmed the expression level of mRNA.

Mesenchymal stem cells are multipotent cells capable of differentiating into various mesenchymal tissues, such as bone, cartilage, fat, tendon and muscle. In order to explore there usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. The expansion of MSCs strongly depends on the culture conditions. In order to explore usages in medical applications, the ex vivo expansion of MSCs to sufficient cell numbers is necessary. The expansion of MSCs strongly depends on the culture conditions like pH, temperature, oxygen, osmolarity, basal medium, serum and other supplements. Carbon dioxide in the gas phase dissolves in the medium, establishes equilibrium with HCO3 − ions, and lowers the pH. The concentration of CO2 is in equilibrium with the sodium bicarbonate in the medium. We focused on effect of gas phase CO2 in the culture of mesenchymal stem cells. Human cord blood MSC, canine bone marrow MSC and canine cord blood MSCs were cultured in 5% CO2 and 10% CO2 condition. Media such as DMEM, KSFM and CDM 1.0 were used. As result cells cultured in DMEM shown batter growth in 10% CO2 and cells cultured in KSFM or SJ-CDM1.0 shown batter growth in 5% CO2 condition. We conclude that effect of gas phase CO2 depends on culture medium.

Insect cell lines have been used extensively for the production of numerous functional eukaryotic proteins. However, insect cell systems have limited commercial use due to a lack of complex glycosylation. Drosophila S2 cells have been developed as a plasmid-based and nonlytic expression system using recombinant baculovirus, infects into insect cells and does not require any selection markers. A gene encoding human erythropoietin (hEPO) or human 90K (h90K) fused with a hexa-histidine (His6) tag under the control of the Drosophila originated metallothionein promoter (pMT) was stably transfected into Drosophila S2 cells. In the preliminary studies, we found that expressed proteins had simple N-glycan structures instead of complex structures. We thought that this result caused by N-acetylglucosaminidase (GlcNAcase) through enzyme assay experiment. Therefore, this research will propose that suppression of GlcNAcase and multiple expression of N-glycosyltransferases using recombinant baculovirus strategy.

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), an immunosuppressive agent, is a glycoprotein and it was expressed in transgenic rice cell suspension cultures. As a production system, transgenic plant cells have several advantages including low risk of potential contamination with animal pathogens, economical large-scale production, and capacity to carry out most posttranslational modifications (PTMs). Among the PTMs, glycosylation is essential for proteins stability, folding and physiological activity.1, 2) However, there are differences in N-glycosylation between the glycoproteins produced in mammalian and plant cells. N-linked glycan structures contain penultimate galactose and terminal sialic acid residues in mammalian glycoproteins. In the case of plants, in vivo half-life of protein is decreased because of N-linked glycans with plant specific terminal β1, 3-galactose residues.3) To prolong the half-life of plant-made proteins, transgenic rice cells were transformed with human galactosyltransferase gene under the control of the RAmy3D promoter. Consequently, produced target proteins could have glycan structures with not only β1, 3-galactose but also β1, 4-galactose residues at the terminus. In this study, target proteins with terminal β1, 4-galactose oligosaccharides were purified by using Ricinus communis agglutinin (RCA120) lectins that binds to specific β1, 4-glactose structures. Through the purification process by RCA120 affinity chromatography, it was confirmed that N-linked glycan structures of hCTLA4Ig was modified similar to that of mammalian glycoproteins in transformed rice cells.

Dengue virus (DENV) spread by female mosquitoes, specifically the Aedes aegypti species is an infectious viral disease endemic to tropical areas worldwide. Currently four serotype (DEN1-4) of DENV exist. To date, it is estimated that two-fifths of the world’s population are at risk for being exposed to at least one serotype of DEN1-4(WHO,2002). No dengue vaccine is available yet. Recently, however, attenuated candidate vaccine viruses have been developed. This research was also being conducted to develop recombinant edible vaccine expressed in rice cell system. E glycoprotein has a major role in host cell attachment and entry and is recognized as the primary antigen that induces protective immunity. The binary expression vector, pMYN81, containing E glycoprotein gene was constructed, which was designed with RAmy3D promoter which is well known as a strong promoter under sugar starvation. Putative transformed calli were selected by genomic DNA PCR analysis after introducing into the nucleus of the rice cell by gene gun. The expression levels of mRNA and E glycoprotein were confirmed using Northern blot analysis and Western blot analysis.

The development of porcine cholera marker vaccine is needed for the maintenance of porcine cholera free region. Insect cells are used as host cells for the expression of marker vaccine by baculovirus vector system. Insect cell lines are able to grow suspension culture and easy to scale up. The use of FBS has several disadvantages and is undesirable in large scale insect cell cultures media. Therefore, high density insect cell culture in serum free medium is prerequisite to produce a marker vaccine with high productivity and safety. Therefore, it is important to select the optimum serum free medium for host cell line. In this work, two insect cell lines, Hi5 originated from the eggs of Tricoplusiu ni and Sf9 derived from the ovary of Spodopteru frugiperdu, were investigated for their ability to grow in a serum-containing medium and in serum free media in erlenmeyer flask. An inoculum concentration of 5 x 105 cells/ml results in high growth rates in both Sf9 and Hi 5 cell line. The best medium for the cell growth was sf900Ⅲ SFM and Ex-cell 405 SFM in sf9 and Hi 5cell, respectively. The optimum serum-free media were selected in terms of cell density, and the selected serum free media showed higher cell growth than the medium containing FBS.

Genitourinaty infections diseases are known to occur frequently and some of them have high possibility to pass into chronic diseases. However, current treatments are carried out without broad and exact screening of infected pathogens and antibiotic resistances, causing overdose of multiple antibiotics. For the efficient diagnosis and treatment, we developed a DNA chip for the simultaneous detection of multiple pathogens causing genitourinary infectious diseases. In this study, we selected 14 pathogens which are abundantly found in Koreans, and known to cause major infections. Through full consideration of their sequence, we settled the condition for multiplex PCR, and the aldehyde glass surface by our novel method, using gold nanoparticles. By using this method, we could increase the immobilization efficiency, and at the same time, it was able to control the quality of the manufactured DNA chip by silver enhancement. The target samples were fluorescently labeled by using Cy3 labeled primers. Then, the PCR products were applied to the manufactured DNA chip at the optimized hybridization condition. As a result, we could get the correct signals of the single infection and the multiple infections simultaneously. The developed chip is suitable for the point-of-care testing due to its efficiency, convenience, and the eligible cost of money and time.

Recently, a successful outcome of solid device such as high integrated memory, optoelectronic device and biochip is in the development of nanosclae lithographic technique. In other words, nanoscale controlling is key point to improve these devices. Compare with electrical beam lithography, optics lithography, and NIL, Nanoimprint lithography (NIL) is most effective to make nanostructure because of its merits. NIL can provide inexpensive and easier method to generate large area of nanoscale patterns. Especially ultraviolet-nanoimprint lithography (UV-NIL) is extensively used among nanoimprint lithography method because of efficiency in view of cost and time. By using this UV-NIL technique, we have successfully fabricated 100nm wide silicon nanowire patterns on the Silicon on insulator (SOI) wafer. The fabricated nanowire patterns were distinguished by FE-SEM and the characteristic of nanowire conductance were measured by semiconductor parameter analyzer. Consequently, these nanopatterned SOI substrates would be able to use as bioplatform for nanoscale biochips. Acknowledgments: This research was supported by Nuclear R&D program through the Korea Science and Engineering Foundation (KOSEF) funded by the Ministry of Education, Science and Technology (MEST) of Korea (Grant No. M20706010003-08M0601-00310), and by the Ministry of Knowledge Economy (MKE) and Korea Industrial Technology Foundation (KOTEF) through the Human Resource Training Project for Strategic Technology and by The Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST) (2006-05374)

Bioengineering and modification of antibody as a capture molecule is very important to increase sensitivity and specificity of the biosensor. As a detecting molecule to diagnose Legionella infectious disease, PAL (peptidoglycanassociated lipoprotein) specific antibody was immunoaffinity purified from rabbit polyclonal anti-PAL IgG by immobilizing PAL onto CNBr-activated sepharose. For the immunoassay, intact antibody was modified into F(ab’)2 and either intact antibody (H+L) or F(ab’)2 was layered on carboxyl terminated PAMAM dendrimer (generation 4.5), which was immobilized onto an amine functionalized polystyrene surface. Subsequently, soluble antigen of Legionella pneumophila serogroup 1 or Legionella anisa diluted with sodium phosphate buffer or patient’s plasma was applied respectively and detected by HRPconjugated immunoaffinity purified rabbit polyclonal anti-PAL. As a result, each antibody on dendrimer presented better binding activity than the antibody on blank surface. Moreover, F(ab’)2 on dendrimer showed higher binding capacity than intact antibody on dendrimer against soluble Legionella antigens diluted in plasma. This study demonstrated modified antibody immobilized on dendrimer amplified the signal by increasing binding capacity/sensitivity and minimizing steric hindrance. Furthermore, this sensor format demonstrated it could be applied as a protein biosensor in clinical samples. (The authors gratefully acknowledge supplying materials from the laboratory of Dr. Minja Kim, Division of Infectious Disease, Department of Internal Medicine, College of Medicine, Korea University.)

Carbon nanotubes (CNTs) have stimulated intense research activities, leading to remarkable scientific and technological advances in nanostructured materials. Although CNTs provide extraordinary mechanical, electrical, and thermal properties for numerous potential applications, it is difficult to process them due to a major drawback, i.e. poor dispersibility. Recently, the physical gels consisting of ionic liquids (ILs) and CNTs were fabricated by using imidazolium-ion-based ILs as new dispersants of CNTs. The CNT/IL bucky gel-modified electrodes are important in terms of understanding electron transfer of biomolecules. We here report the immobilization of organophosphorus hydrolase (OPH) on the multi-walled carbon nanotube (MWNT)/IL bucky gel electrode for the biosensors. The MWNT/IL bucky gels were formed by grinding the mixture of MWNTs and 1-butyl-3-methylimidazolium tetrafluoroborate (BMimBF4) with an agate mortar. After the immobilization of MWNT/IL gel, MWNT/IL/OPH electrode was obtained by immersing the modified GC electrode into OPH. The OPH immobilized on the CNT/IL bucky gel electrode surface was confirmed by SEM, EDX and UV-vis spectroscopy analysis. The interactions between ILs and OPHs were evaluated by FT-IR spectroscopy. The electrochemical properties of MWNT, MWNT/IL, MWNT/OPH, and MWNT/IL/OPH electrode were characterized by AC impedance and cyclic voltammetry. MWNT/IL/OPH electrode with effective stability and electrocatalytic performance provides a useful platform for the development of environmental biosensors [This work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through IITA].

Nano structure has been introduced to fabricate cell based biochip for the adhesion of an epithelial cell in various applications because cells are adhered by nano-scaled adhesive protein in the cell membrane. In this study, we fabricated to silica nano structure on the PDMS surface. Silica pillars were well arrayed on PDMS (pillar diameter: 80nm, gap: 30nm). The length of silica pillar was controlled to optimize the hydrophobicity of the surface. The adhesion quality of cell on nano structured surface was investigated in aspects of cell morphology, cell adhesion force and viability using AFM (Atomic Force Microscopy), bright-field microscopy and confocal microscopy. Acknowledgments: This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10755160002-07N5516-00210).

A sensitive protease assaying method is key tool for elucidating protease function, and developing therapeutics because of implications in the pathogenesis of diverse disease such as viral and bacterial infections and cancer and neurodegenerative diseases. Also it has been reported that secreted amount of proteases are related to cancers. Activity of proteases is assayed by measuring amount of digested substrates. Due to feasibility for developing high throughput assaying formats, synthetic short peptides are preferred. For developing fast and sensitive protease assaying methods, also nanoparticles such as quantum dots, gold nanoparticles and magnetic nanoparticles have been utilized. The reported methods show relatively low detection limits. However still these techniques require long assaying times(more than 12 hours) for acquiring enough detecting signals. Therefore, I propose a more rapid and sensitive way to assay activity of protease by incorporating a signal amplification method with gold nanoparticle and RNase H. More detail working principle and detection limit of our protease assaying method will be presented

Basic biological and medical research continues to provide the building blocks necessary to develop solutions to human health problems and to preserve and repair the natural environment. In general, kinases and phosphatases are of considerable interest to researchers involved in a drug discovery and development, because of their role in a wide variety of diseases. In this study, we investigated the feasibility of applying a RI detection technique to a protein kinase assay for the development of a diagnostic biochip. We cloned a protein-fused substrate for protein kinases using an elevating protein. Results of the study indicate that the protein-fused substrate can be used effectively as a substrate for detecting the activity of protein kinases and it can be a useful tool for a high throughput screening and for studying enzyme-substrate interactions.

Iron oxide/carbon black composite (Fe2O3/CB) electrode was prepared and used for NAD(P)H oxidation under very low overpotential (+0.00V vs. Ag/AgCl) without employment of mediators or any chemical treatment. Based on the capability for oxidation of NAD(P)H by Fe2O3/CB electrode, we developed an amperometric biosensor for the detection of NAD(P)H and ethanol using NAD+-dependent alcohol dehydrogenase. Not only the method of sensor construction is very simple, but also it shows good sensing performances such as high sensitivity, stability, and broad detection range. The sensitivity of this system is 2.54 μA mM-1 and the limit of detection (S/N=3) is 10 μM. The linear range was observed between 10 μM and 1000 μM of NADH (R2=0.993), which is preferable to previous studies. Moreover, when integrating the developed sensor system into dehydrogenasebased sensor system, it also showed good sensing performance. Details will be presented.

T lymphocyte adhesion to adhesion molecules on endothelium is important in immune function, cancer metastasis and inflammation. This cell-cell binding is controlled via cell adhesion molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) of endothelial cells and adhesive properties of T-cell which is greatly modified in several diseased conditions. Thus, therapeutic agents that down-regulate leukocyte-endothelial interactions have been focused to markedly regulate the progression of inflammatory responses in a number of in vivo models. Here, we present a protein based microfluidic system that mimics an inflammatory site which occur the binding of leucocytes to endothelium. This system can easily monitor the binding leucocytes to adhesion molecules under various physiological conditions, such as, shear stress and the density of adhesion molecules. We fabricate protein based microfluidic system by immobilization of cell adhesion molecules (ICAM-1, VCAM-1, E-selectin) on the glass surface. The T cell binding to cell adhesion molecules were affected by the height of channel, density of adhesion molecules and shear stress. The maximum binding was observed at 2 dyne/cm2 of shear stress and 20 ug/ml of the density of adhesion molecule in the case of ICAM-1. This system is easily integrated with SPR as detection method to monitor the dynamics of reactions in real time. Furthermore, this device is useful as a biosensor for clinical diagnostics and high throughput drug screening.

We present DNA templated silver nano-wires linking nanogap electrodes for electrical detection of DNA hybridization. The nanogap was fabricated using anisotropic silicon etching on SOI wafer substrate (bottom gap size was about 60nm). Nine single-stranded DNA oligomers were designed to form 80 nm length double-stranded DNA connecting nanogap electrodes. Current changes between nanogap electrodes upon this DNA bridge formation, however, were rather small. For current signal amplification, silver nano-wires were grown on the DNA bridges through silver ion deposition and enhancing procedures. The current signal was significantly increased by this silver nano-wire formation.

Microarrays can be used to screen thousands of binding events in a parallel and high throughput fashion, and are of major importance in the detection of a disease and drug discovery. The use of radioisotope (RI) is conventionally regarded as one of the most sensitive detection methods. However, it takes a long time to implement a data analysis for a signal intensity. A glass chip is exposed to an X-ray film or imaging plate and then developed or analyzed by a bioimage analyzer. Radioactive labeling is mainly performed using different RI’s such as 33P and 32P into ATP for a phosphorylation. We reported here on the degree of incorporation of 33P and 32P into a substrate measured by a radio-TLC. In the examined substrate concentration range, the signal intensity was continually increased up to a high concentration of E. coli malic-kemptide fusion protein as determined by the radio-TLC. The use of this detection method facilitates a rapid data analysis with a high sensitivity.

Protein immobilization is a key step for the development of protein biosensor. In recent years, people have been seeking various immobilization ways aiming at satisfying a number of standards for excellent performance of a biosensor, among which, gold binding polypeptide (GBP) based immobilization began to attract people’s attention. Here, we established a universal detecting platform utilizing the GBP, protein A (SpA) and protein G (SpG). Kinds of GBP-fusion protein, such as GBP-SpA, SpA-GBP, GBP-SpG and SpG-GBP, were prepared via recombinant DNA technology. Surface plasmon resonance (SPR) was applied to investigate the affinity of these proteins with the gold surface as well as with immunoglobins. Furthermore, electrochemical impedance spectroscopy was analyzed for the detection optimization of hepatitis B viral surface antigen [Our work was supported in part by the IT Leading R&D Support Project from the Ministry of Knowledge Economy through IITA.].

Oriented antibody immobilization is very important in the development of immunological analytic devices. To immobilize antibodies on assay surfaces, most of all technologies rely on physical adsorption or covalent coupling of the protein. However, these methods often cause reduced antigen binding affinity due to random orientation, denaturation, and chemical modification of antibodies. To overcome this drawback, we suggested a novel strategy to immobilize an antibody on various sensor surfaces by fusion protein with Fc-binding peptide and mussel adhesive protein. Fc-binding peptide can afford oriented antibody immobilization on sensor surfaces by binding Fc region of immunoglobulin G. Mussel adhesive protein from Mytilus galloprovincialis can attach a variety of surfaces and therefore, the fusion protein can attach various sensor surfaces without loss of activity. Through SDS-PAGE analysis, we identified whether the fusion protein capture immunoglobulin G or not and the species specificity of the fusion protein. Quartz Crystal Microbalance (QCM) analysis indicated antibody immobilization by the fusion protein exhibited higher antigen binding capability than random antibody immobilization.

Olfactory receptors, belonging to G-protein coupled receptor family, are integral membrane proteins composed of seven transmembrane spanning domains. The olfactory receptor protein is thought to be an efficient primary transducer for the protein-based olfactory biosensor which has high selectivity for odorant detection. Since these proteins have complicated structures and strong hydrophobicity, it is important to optimize reconstitution and refolding condition. The human olfactory receptor was expressed at high-level in E. coli as insoluble form and pre-treated with Triton X-100 to remove impurities. The solubilized receptor proteins were reconstituted with artificial lipid membrane by mixing with an optimal detergent. The functionalization of reconstituted olfactory receptor protein in lipid membrane on sensor chip was observed by surface plasma resonance (SPR). The proteinbased olfactory bioseonsors using olfactory receptors reconstituted with lipid membranes might demonstrate highly selective odorant detection. This reconstitution method can be applied to other sensing devices such as QCM or FET for the development of highly sensitive olfactory biosensor.

It is known that the human olfactory system has remarkable sensitivity and specificity. Since olfactory system plays an important role in recognizing environmental conditions, kinds of olfactory research have been carried out due to its potential application. In recent years, a few significant studies regarding biotechnology-based olfactory sensors have been reported. However, these sensors still have limitations in sensitivity and specificity. In this work, human olfactory receptor (hOR) 2AG1-conjugated carboxylated polypyrrole nantubes (CPNTs) field effect transistor (FET) sensor was used to measure the binding of odorant molecules to specific receptor proteins. hOR2AG1 was expressed in Escherichia coli (E. coli) as a fusion protein with glutathione-S-transferase (GST)-tag to its N-terminus for efficient expression. Conductive CPNTs were synthesized by copolymerizing pyrrole monomers with intrinsically functionalized P3CA (pyrrole-3-carboxylic acid) monomers. CPNTs were immobilized onto an interdigitated microelectrode array (IDA) via covalent linkages. hOR2AG1 was then immobilized with CPNTs. As a result, the hOR2AG1-conjugated CPNT FET sensor showed highly specific response to a target odorant, amyl butyrate, and gave measurable signal up to concentrations as low as tens of femtomoles. The hOR-conjugated CPNT FET system offers a new direction of the highly sensitive and selective recognition of odorants, and could be expanded to allow the multiplexed detection of various odorants after a judicious optimization.

In this study we have developed optical progesterone sensing probe based on modified CdSe/ZnS quantum dot (QDs). Water-soluble quantum dots (MPA-QDs) were used to label anti-progesterone antibody (P4Ab), and the labeling process was characterized by column purification. The QDs obtained in organic solvent were modified with mercaptopropionic acid (MPA) and became water-soluble. These water-soluble QDs were linked to the progesterone antibody using the coupling reagents ethyl-3-(dimethyl aminopropyl) carbodiimide(EDC) and N-hydroxysuccinimide(NHS). The linking process was shown to be effective by ultra-filter centrifugation and column purification. After comparing the quantities of P4Ab and water-soluble QDs involved in the linking reaction via column purification, it was found that a molar P4Ab:QDs ratio of 250ng:0.8mg/ml resulted in most of the water-soluble QDs becoming covalently linked to the P4Ab. CdSe/ZnS quantum dots were synthesized and conjugated with progesterone antibody as progesterone optical sensing probe. The P4Ab conjugated CdSe/ZnS probe sensor has high sensitivity and rapid response time, that it can be used to assay the quality of progesterone.