Earticle

Gene delivery received attention as a novel method for gene therapy. Especially non-viral delivery is safe and has low risk of provoking immune responses. But it has low gene delivery efficiency. To enhance it, this study focuses on the non-viral vector that consists of novel peptide which derived from virus. The peptide is an innovative material, and it is applied with polyethylenimine and DNA to increase the transfection efficiency. The uniqueness of the peptide is its derivation from adeno-associated virus r3.45 which was obtained by directed evolution toward rat neural stem cell. It has been proved to be a great tool to infect human neural stem cells (hNSCs). Hence, with the peptide, the positive effect in the non-viral delivery can be anticipated. In the experiment, the complex consists of PEI, DNA, and the peptide is used. The ternary complex was confirmed successfully by gel retardation. In the case of transfection into several cell lines, complex with ternary complex shows higher efficiency than binary complex. In conclusion, this specific peptide takes an important role in enhancing transfection. For the further study, application into the hNSCs will be researched.

Human neural stem cells (hNSCs) with the capacity of differentiating into transplantable neurons can provide a novel therapeutics for the treatment of neurodegenerative disease and neuronal injury. Recent studies have demonstrated that mechanical and physical properties of the culture substrate including stiffness and surface roughness control hNSC differentiation. Particularly, the surface topography at nanoscale level may significantly affect hNSC differentiation. Here, we report the effect of nanotopographical stimulation on hNSC differentiation. The polyurethane acrylate (PUA) nanopatterned surface was grafted with titanium (Ti) for potential electrical stimulation by initiated chemical vapor deposition technique. Human NSCs cultured on Ti-coated PUA groove nanopatterned surface exhibited the morphology with alignment and elongation along with the axis of patterned surfaces, as indicated by F-actin cytoskeleton (phalloidin) staining. The hNSCs on Ti-coated PUA flat surfaces without nanopatterning did not exhibit such aligned and elongated cell morphology. More importantly, neuronal differentiation of hNSCs was significantly enhanced by culturing them onto the nanopatterned groove surface, compared to the flat surface, as confirmed by immunocytochemistry and quantitative real-time polymerase chain reaction assay. In the future, we will consider electrical stimulation of Ti-grafted nanopatterned surface because electrical signals may further promote neuronal differentiation of hNSCs.

It is necessary and important to ferment Saururus chinensis(SC)massively with the effective specific microorganism for the short term fermenting period. Useful bacteria were identified and isolated from SC extract produced by the conventional fermentation process. It contains the highest number of bacillus species. Although many previous studies on SC were performed, most of them were based on the non-fermented SC. There was scarcely any study on the comparitive evaluation of fermented SC and non-fermented SC. This study is the first case, especially on the Bacillus subtilis 168-fermented SC. Optimum temperature of fermented Saururus chinensis extract(FSC) was 40°C and optimum time was 34 hour. The purpose of this study was to investigate the effect of FSC and non-fermented Saururus chinensis extract(NFSC). TLC and HPLC showed the increase of rutin and quercetin due to the fermentation. Furthermore, FSC had more effect than NFSC in free radical scavenging activity, anti-autooxidative activity and cell viability of prostate cancer cell in vitro. The high scavenging abilities of DPPH, OH-, O2 - were observed. Also FSC showed the inhibition effect on NO rate. This anti-inflammatory activities were further supported by the inhibition of iNOS and COX-2 expression. These results showed that decrease in unit activity of iNOS and COX-2 were principally due to inhibition of iNOS and COX-2 protein expression, which accounts for the decreased accumulation of nitrite in cells treated with FSC and NFSC. These results demonstrated that FSC could be useful as an anti-inflammatory and anti-oxidative functional ingredient.

Saururus chinensis is a traditional Korean herb for curing liver diseases. So this study was performed to investigate anti-inflammatory and anti-oxidative improving effects of Saururus chinensis fermented with Bacillus licheniformis sp. SMs-07 extract(SCFE) in the liver and sera of LPS-treated rats. SCFE, Saururus chinensis extract(SCE) and rutin were orally administered into experimental groups for 30 days, while the normal and control groups were administered with water. On day 31, rats were given 5 mg/kg of LPS(the normal group was given a vehicle). LPS-treated groups markedly increased the levels of AST and ALT in sera and MDA in the liver homogenates and mitochondrial fractions, but decreased the levels of SOD, CAT and GPx in liver homogenates and mitochondrial fractions. SCFE and SCE groups decreased AST, ALT and MDA, but they increased SOD, CAT and GPx. SCFE had better activities than SCE. It could be concluded that the fermentation played the important role in the improvement of anti-oxidation and anti-inflammation.

Nanofibrous mat fabricated by electrospinning has three-dimensional structure and mimics the extracellular matrix. Electrospun nanofibrous mat can be used as superior scaffolds. Astrocytes are one of the cells made up of central nervous system (CNS) and play an important role in structural support and nutrient supply to surrounding nerve cells in healthy CNS. When injury occurs in the CNS, astrocytes react to injury and play an important role in injured CNS. Polycaprolactone (PCL) has been generally applied to biomaterial application. Because it is one of biodegradable polymers and has good biocompatibility. In this study, PCL electrospun nanofiber was fabricated and rat primary astrocytes were cultured on PCL-coated surface and PCL nanofiber. Then, in order to analyze the cell mass and activity in each environment, crystal violet and MTT assay were tested. Cell mass and activity increased in PCL nanofiber. As result of the research, we suggest that PCL nanofiber can be used as superior cell scaffold to PCL-coated surface.

Phosphodiesterases (PDEs) are involved in many cellular signal transductions mediated by cAMP. In addition, PDEs are involved in phosphorylation when the precursors of soluble lysosomal enzymes transport into the lysosomes. To certify the relation between lysosomal activity and PDEs, Bovine Aortic Endothelial cells (BAECs) transfected with 200, 300 and 400 pmol antisense of PDE4A, B, C and D. Through cytochemical analysis of lysosomes with Lyso-Tracker, we demonstrated that the number of and fluorescent intensity of lysosomes in BAECs, and the endothelial nitric oxide synthase (eNOS), PDE4A, B, C, D and cathepsin B were quantitatively analyzed by western blotting. We found that the number of and fluorescent intensity of lysosomes decreased only the case of PDE4C knock-out, but was not changed by other PDE series. In addition, protein expression of PDE4A, B, C, and D were decreased by transfection but eNOS and cathepsin B were reduced by transfection of PDE4C antisense. Therefore, our results suggest that only PDE4C would affect the activity of lysosomal enzymes in cells.

Cell therapy has the potential for providing new way of clinical treatments. Mesenchymal stem cells (MSCs) are important tools for cell therapy because of their high proliferation and differentiation ability as well as immunosuppressive properties. MSCs are commonly cultured as monolayers, but there are indications that they lose cell-specific properties with multiplication in vitro. In addition, it was reported that the culture of MSCs as a spheroid form in a short period improved their therapeutic potential by increased expression of genes such as CXCR4 to promote adhesion to endothelial cells or IL-24 that has tumor suppressing properties. In this study, we developed a new three-dimensional (3D) culture system for periosteum-derived progenitor cells (PDPCs) which are one of diverse MSCs. The spheroid formation of PDPCs was induced by using a rotation platform. The spheres was found to maintain self-renewal ability. After 3D culture, the spheres expressed MSCs-specific surface markers and stem-cell-associated genes. In conclusion, an economical culture system could be developed. This system can be utilized for the scale-up 3D culture of the PDPCs.

To become stem cell therapy available as a product, efficient industrial cultivation method is required. From this point of view, threedimensional (3D) culture is inevitable. Mesenchymal stem cells (MSCs) are attractive cell sources because they do not have any ethical problem and are safe from tumor formation. In previous studies, we accomplished the induction of periosteum-derived progenitor cells (PDPCs), known as a kind of MSCs, to insulin-producing cells (IPCs) in two-dimensional (2D) culture. In addition, we applied our induction method to 3D culture system using ultra-low attachment plates with different cell densities. In this study, we investigated the viabilities of the inducted cells on diverse cultivation methods. Time course viabilities were measured by WST-1[2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2Htetrazolium] assay. Furthermore, we detected the functionalities of the cells by Enzyme-linked immunosorbent assay (ELISA) to measure the expression levels of insulin. In conclusion, it was found that PDPCs formed compact cellular spheroids and remained viable in 3D induction culture. It was also found that 3D culture was more effective than 2D culture for the differentiation of PDPCs to IPCs.

Here we describe a highly specific, robust and rapid new method for labeling of cell surface proteins with fluorescent probes. The method uses the trans-splicing reaction of Nostoc punctiforme (Npu) DnaE split-intein. Split-intein based protein trans-splicing reaction has been used for site-specific labeling of proteins in vitro for various applications. The trans-splicing reaction is self-processing reaction and can be used for introduction of various synthetic probes to target proteins without a large linker proteins. In this report, we have utilized split-intein based protein trans-splicing reaction for site specific labeling of cell surface proteins. We have prepared a model cell surface protein fused to N-terminal fragment of Npu DnaE Intein and C-terminal fragment of Npu DnaE Intein fused to a fluorescent probe. The model protein was expressed on the cell surface and the proteins were labeled with synthetic fluorescent probes through specific interaction between Nand C-fragment of inteins. This labeling reaction was performed within 30 mins using low concentration of fluorescent molecules (4 uM). No external energy was required for the labeling reaction. This improved labeling method will provide a useful tool for studying functions and mobility of proteins in live cells.

Three-dimensional (3D) cell culture systems have been fabricated using a magnetic force [1], scaffolds [2], and biocompatible gels [3]. In this report, fibroin microcapsules approximately 500 nm in diameter were fabricated to entrap iron oxide nanoparticles (Fe3O4) and introduced to 3T3 cells cultured in vitro. Magnetized cells were levitated using a neodymium magnet placed on the top of the culture plate containing trypsin-treated 3T3 cell (6.2X105 cells) and further incubated for three days with daily media change. Disk-shaped dendroid cell clumps were formed up to 2400 mm2, which were analyzed using confocal laser scanning microscope (CLSM) and flow cytometry to evaluate cell numbers, shape and viability. The CLSM im ages of fluorescence stained cells indicate that the 3D cell clumps were concave and full of cells. Flow cytometry shows that the morphology of cells in the 3D cell clumps was significantly influenced. Viability of the cell clumps decreased with the increasing culture period but was not influenced by the increasing amount of microcapsules. The results indicate that mass transfer limitation is critical to maintain viability of 3D cell culture.

The terminal sialylation of N-linked glycan is most important factor to determine the in vivo clearance rate of therapeutic glycoproteins. In this study, we introduced the sialuria-mutated rat GNE/MNK to increase the intracellular CMP-sialic acid by genetic engineering. From in vitro activity assays of various sialuria-mutated rat UDP-GlcNAc2-epimerases, rat GNE/MNK-R263L-R266Q mutant sustained the highest activity among the five mutants, and feedback inhibition was not detected. To improve the sialylation path way of CHO cells, Chinese hamster CMP-SAT, and human α2,3-ST were transfected simultaneously with sialuria-mutated rat GNE/MNK into the rhEPO-producing CHO cells. The intracellular CMP-sialic acid pool of engineered CHO cells was significantly increased up to 10.7 folds compared to control. The sialic acid contents of produced rhEPO were significantly increased up to 43%. The asialo-and monosialylated N-glycans were decreased by half compared to control, and the tetra-sialylated N-glycans were increased up to 32.0%. In this study, we found the effective sialuria-mutated rat GNE/MNK to increase the intracellular CMP-sialic acid pool by genetic engineering. And, the new CHO cell lines were constructed which could produce the more sialylated therapeutic glycoproteins by overexpression of sialuriamutated GNE/MNK, CMP-SAT, and α2,3-ST.

Plant-derived peptones have been utilized to improve cell growth and product formation in plant cell cultures over 10 years. Supplementation of defined medium with plant-derived peptone was found to enhance cell growth and the production of human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) significantly in transgenic rice cell suspension cultures. Production of hCTLA4Ig was induced by sugar starvation. Nitrogen (N) and carbone (C) sources have been known as major factors in plant cell cultures. Therefore, it was necessary to investigate the effects of C and N sources for the improvement of protein production. In addition, optimization of the concentrations of C and N sources is required. As a carbon source, 0 (control), 3, 5% (w/v) of sucrose was added in 100-mL Erlenmeyer flasks with 1% plant-based peptone. In case of adding 5% sucrose, the maximum dry cell weight and hCTLA4Ig production were higher than those of control culture. In conclusion, it was found that the determination of optimal ratio between C and N sources are important for the production of recombinant protein in transgenic rice cultures.

Low protein yield has been known as a main obstacle in transgenic plant cell cultures for the production of recombinant protein. Production of a fusion protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig), was induced by sugar starvation using rice alpha-amylase 3D (RAmy3D) promoter in transgenic rice cell suspension cultures. Due to the sugar depletion, RAmy3D promoter causes cell death during the production phase. Under this condition, the rice cells can be more sensitive to hydrodynamic stress. In flask cultures, hydrodynamic stress can be changed by the parameters such as the size of flasks, agitation rate and working volume. In this study, hCTLA4Ig productivity was compared between the cultures in different flask sizes (0.1, 0.25, 0.5, 1, 2 and 5 L). In larger flasks, significant decrease of hCTLA4Ig level was observed and this decrease was thought to be originated from the changes in hydrodynamic stress. To confirm the effect of hydrodynamic stress on hCTLA4Ig production in flask cultivation, both agitation rate and working volume were changed in 1-L flask. It was confirmed that hydrodynamic stress could lower the production of hCTLA4Ig as well as cell viability. In conclusion, hydrodynamic stress should be controlled to improve the hCTLA4Ig production during the scale-up process.

Chinese hamster ovary (CHO) cells are one of the predominant systems for the production of recombinant proteins among eukaryotic expression systems. Recently, various strategies have been studied on increasing specific productivity in CHO cell cultures. Under nutrient deprivation, cells are unable to proliferate because they are subjected to two types of programmed cell death (apoptosis and autophagy) or necrosis in batch culture. Anti-apoptotic agents have been known to inhibit cell death in mammalians cell cultures and extensively applied in recombinant CHO cell cultures. To investigate the effects of anti-apoptotic agents on the production of recombinant albumin-erythropoietin (Alb-EPO) fusion protein, various chemicals were added into CHO cell cultures in exponential growth phase. Among the agents tested, sericin was found to be the most effective. Compared to control cultures without sericin, the culture with 1 mg/mL sericin enhanced the cell viability by 12%. In conclusion, it was found that the addition of adequate anti-apoptotic agent could increase the viability of CHO cells and accordingly enhance the production of recombinant Alb-EPO fusion protein.

A proof-of-concept research was reported on analysis of antigen-antibody realization based on resonant Rayleigh scattering response of single Au nanoparticles in an imaging chamber. As benefited by a traditional dark-field microscope and a spectrograph, individual Au nanoparticles (20 nm) were observed with high signal to-noise ratio and they were effectively utilized to monitor changes in refractive index induced by specific binding of the adsorbates. Using PSA antigen as a model, a LSPR λmax shift of about 2.85 nm was recorded for a molecular binding corresponding to 0.1 pg/ml of the protein biomarker. This result successfully explain a non-labeling detection system for proteins as well as thousands of different chemical or biological species, and it possesses a superb potential as a sensitive, on-chip and multiplexing detection.

In the past decade, many analysis methods of DNA mismatch detection have been proposed for analyzing SNPs (single nucleotide polymorphisms), which are one-base DNA sequence variations that are responsible for various inherited diseases and drug resistance. Nevertheless, ligase-based assay is more proper methods in terms of various applications then other SNP analysis methods. However, DNA ligase-based methods for mismatch detection showed unavoidable limitation in cytosine/thymine (C/T)-type SNP analyses. Theoretically, ligation product should be detected for guanine:cytosine (G:C) match and not for guanine:thymine (G:T) mismatch. But, C/T type SNP analysis is not distinguished clearly due to false-positive induced by hydrogen bondings between G:T. In this study, DNA oligomers containing modified nucleobases (5-Nitroindole, dSpacer, Iso-dG, and Iso-dC) at the end were employed as new ligation fragments for DNA-ligase-based SNP analysis. When such modified-end DNA ligation fragments are used, false-positive result for G:T mismatch was not found both using T4 DNA ligase and Thermostable DNA ligase (Tth DNA ligase). This ligation method using modified-end DNA ligation fragment has potential for the accurate analysis of C/T-type SNPs.

Inflammation is known for complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a protective reaction by the organism to remove the injurious stimuli and to initiate the healing process. In other view-point, long lasting of inflammation destroys tissue and organism that cause disease. Therefore, it is recognized important issue that detection of inflammation on living cell in the initial stage. Previous study, we found out bradykinin 1 receptor is one of the proper biomarker for cell inflammation. In this study, we set up chip-based cell-inflammation imaging system through investigation of bradykinin 1 receptor and modified bradykinin peptide. The channel for live cell imaging which made of PDMS wall and quartz panel shows great image difference between normal cells and inflammated cells. The image analyze system by peptide probe would be an important basis for developing cell-inflammation analysis system for efficient high-throughput screening of anti-inflammation drug.

A highly rapid one-step immunoassay biosensor to diagnosis various biomarkers was developed based on flow-through-hole (FTH) system. FTH system was usually applied to measure a wide range of target antigen concentrations and relatively small volume of samples in human serum within a few minutes. In the presence of antigen, the assay takes place in the holes contacting a porous Nitrocellulose membrane. The detection antibody-conjugated gold nanoparticle (antibody-AuNP) could bind to the capture antibody-immobilized membrane as a form of sandwich immunoassay, otherwise passed through hundreds of micro-size holes, due to the size of antibody-AuNP much smaller than the membrane pore size. Under optimal conditions, High-sensitivity C-reactive protein (hsCRP) from 10ngmL-1 to 10 μmL-1 was detected by using the FTH immunoassay system. Furthermore, the assay requires only a 2 minuets period in order to obtain sactisfactory results, which was comparable to the conventional LFA.

A sensitive, fluorescence based, label-free, immunoassay system, relying on emission from the dianion form of ochratoxin A (OTA2-) in the OTA/anti-OTA complex under weakly acidic conditions, was developed. Although free OTA exists in its monoanion form (OTA-) at pH 6.0, OTA in the OTA/anti-OTA complex is present in the OTA2- form predominantly at this same pH. This is caused by deprotonation of both the carboxylic acid group of a phenylalanine and the isocoumarin phenolic group in this substance. The results of the current effort show that the fluorescence intensities (λex = 390 nm, λem = 450 nm) of solutions increase with increasing concentrations of OTA at pH 6.0 owing to formation of the OTA/anti-OTA complex. With an optimized system, the fluorescence immunoassay can be used to detect OTA in a highly specific manner with a limit of detection (LOD) of 0.5 ng mL-1. In order to demonstrate a practical application of this assay, LODs for OTA in spiked red wine, white wine, and grape juice were determined and found to be 2.15, 1.23, and 2.06 ng mL-1, respectively. The new strategy, involving a simple sample preparation procedure, serves as a powerful tool for the rapid and sensitive quantitative determination of OTA in food and feed matrices.

Magnetic beads modified with DNA intercalating agents have been developed for simple separation of DNA in solution after PCR by Microwave reactor. It is widely reported that a microwave system could enhance the chemical and biological reactions. An uniform type of microwave system for biological and mild chemical reaction has been developed and applied for a synthetic and some enzymatic reactions. In this study, We modified the bare magnetic particle to 9-aminoacridine coated magnetic particle using conventional method and microwave assisted method. As a result, we confirm, when we reacted with microwave systems, that 9-aminoacridine coated magnetic was increased reaction efficiency in short time(<30min). We developed and confirmed the microwave system that increases the reaction efficiency of surface modification on magnetic particles and shortens the time for reaction.

In order to achieve highly sensitive biosensors and electrochemical sensors, we require specialized materials with superior biocompatibility, physical-chemical durableness and stability. The diamond thin film is an excellent candidate for such biosensors and electrochemical sensors since it has low background current, biocompatibility, wide potential window (3.0~3.5 V), and physical-chemical stability. Diamond thin film will be enables to the selective and quantitative detection of biomolecule in vivo such as DNA, proteins, and cell. In this work we culture the nerve cell (SH-SY5Y) on diamond thin film surface to evaluate the biocompatibility. We investigated the effect of diamond thin film by cytotoxicity and biocompatibility with cultured human nerve cells by using MTT, calcein AM and DAPI. Human nerve cell exhibit the 110% cell viability on microdiamond compared with cell culture dish by MTT assay. Fluorescence data with calcein AM and DAPI exhibit 100% human nerve cells were detected onto microdiamond thin film, which is similar to MTT data. It has demonstrated that human cells proliferated on microdiamond thin film with very high viability and microdiamond material had no toxicity to cultured SH-SY5Y cells. We conclude that microdiamond thin film is a biological compatible material for potential cell culture matrix and bio-medical applications.

We developed a biomemory device with enhanced signal which consists of heterolayers of cysteine-modified azurin/gold nano particle (Az/GNP). The cysteine residues that were attached onto azurin proteins were introduced via site-directed mutagenesis method (SDM) and the purpose was to effectively immobilize the modified azurin onto gold surface. Then gold nanoparticles of 5nm size were adsorbed onto the self-assembled azurin layer. The atomic force microscopy (AFM) and surface plasmon resonance (SPR) were used to investigate the fabricated Az/GNP bilayer. Also, cyclic voltammetry (CV) was employed to confirm the amplified electrochemical signal. As a result, we obtained two redox potentials and with these parameters, using chronoamperometry (CA), function of biomemory device was verified. If foretold biomemory device could be developed in molecular size, it would be able to replace the existing silicon based memory devices in the near future.

In this study, E.coli was directly determined by conjugation of localized antibody fragment on a gold nanopattern modified Indium Tin Oxide (ITO) substrate without use of any labelling materials. Electrochemical method was used for the fabrication of gold nanopattern on ITO substrate and confirmed by Scanning Electron Microscope (SEM) and UV-vis Spectroscopy. Then, antibody against E.coli was immobilized on the gold nanopattern modified ITO substrate, and different concentrations of E.coli were applied to the system by antigen- antibody interaction. After all, Ecoli was detected by using SERS, the intensity was showing linear relationship with different concentration of E.coli. Consequently, this system could be applied to develop the biosensor for pathogen detection. Acknowledgments: This research was supported by the Original Technology Research Program for Brain Science through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (2009-0093907), by the Ministry of Knowledge Economy(MKE) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology, and by National Nuclear R&D Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0018194)

The discovery of numerous nanomaterials has added a new dimension to the rapid development of nanotechnology. The potential applications of nanomaterials are supposed to increase dramatically. Especially, carbon- based nanomaterials are considered to be one of the key elements in nanotechnology and biomedicine. Recently, graphene has much focal point for use in biomedicine due to its nanoscopic and electrical properties. Here, we investigate the cell culture conditions, which is exposed to graphene onto polyethylene terephthalate (PET) using human nerve cell, SH-SY5Y. In order to confirm the cell viability and morphology, we adopt the MTT assay and fluorescence microscopy images stained with DAPI and Calcein AM. Human nerve cell exhibit the 65% growth on coated with graphene compared with PET substrate by MTT assay after 3 days culturing. Fluorescence data with DAPI and Calcein AM exhibit 60% human nerve cells were detected onto graphene coated substrate, which is similar to MTT data. Our data strongly support that graphene can be applied to the fabrication of biological sensor, biomolecules patterning, and study of cell-surface interaction.

In this study, cell chip was fabricated with conductive composite film composed of RGD peptide and carbon nanotubes (CNTs) for improving conductivity of RGD peptide film on gold electrode. The topological characteristics of modified electrode surface were validated by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Human embryo kidney (HEK293T) cells were immobilized on fabricated cell chip to detect electrochemical characteristics by cyclic voltammetry. The electrochemical sensitivity of fabricated electrode evidently increase compared with bare gold and RGD film coated electrode. Thereafter, 2 kinds of nephrotoxic antibiotics were treated to HEK293T cells which were immobilized on modified electrode in cell chip. The electrochemical signal intensities linearly decreased with increase of antibiotics’ concentration. These results were confirmed by MTT assay. Our newly developed renal cell chip can be used in drug development field as label-free screening device to detect nephrotoxic effects of antibiotics on kidney. Acknowledgement: This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2012-0000163) and by the Nano/Bio Science &Technology Program (M10536090001-05N3609- 00110) of the Ministry of Education, Science, and Technology (MEST), and by Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology.

The expression of metallothionein in O.javanicus (OjaMT) increases as a result of heavy metal exposure. Measuring the level of OjaMT expression in O.javanicus can be a good monitoring method for heavy metal contamination of environment and their toxicity. Previously, western blotting and ELISA methods were used to monitor the level of OjaMT expression and these methods requires long detection time and gives high level of background. Also, it was rather difficult to achieve quantitative measurement of OjaMT. Here, we set-up an assay to quantitatively detect OjaMT using surface plasmon resonance (SPR) system. We have used OjaMT specific antibody for the SPR based label free detection of OjaMT. Also, we have constructed OjaMT specific scFv using the total RNA of antibody generating hybridoma cells using jumping PCR. The generated scFv’s exhibited specific binding activity to OjaMT in solution. ScFv’s were immobilized to gold substrate using a 3-Cys tag introduced during the cloning vector construction. The performance of two different probes were compared and the amount of OjaMT was measured quantitatively.

Hexanal, an odorant generated by oxidation of unsaturated fatty acids, can be used as an indicator of food quality. In this work, we screened specific human olfactory receptors (hORs) which can selectively recognize hexanal. Diverse HEK-293 cells each transiently expressing one of the 43 selected hORs were produced. The hexanal-discriminating ability of hORs was confirmed using Fura-2/AM, a dye which illuminates when it binds to calcium ions flowed into the cells once the olfactory signaling occurs. Consequently, we identified that the ORL3687 can selectively recognize hexanal. The ORL3687 can be effectively used for the development of bioelectronic sensors to determine the quality of foods.

Each odorant has its own odor fingerprint, a relationship between specific olfactory receptors and odorants, and human use this to percept certain scents. In this work, the odor fingerprints of three odorants, 1-octen-3-ol, trimethylamine, and 3-methyl-1-butanol, were discovered using artificial olfactory cells that each express diverse olfactory receptors on their membrane. The activation of artificial olfactory cells was identified using cAMP response element (CRE)-directed reporter gene assay which produce green fluorescence after external stimulation. The CRE-directed reporter gene assay method was optimized and subsequently used to screen the odor fingerprints of three selected odorants. 1-octen-3-ol, trimethylamine, and 3-methyl-1-butanol are indicators of fungal contamination, seafood spoilage, and salmonella contamination respectively. The discovered odor fingerprints will be a foundation on the development of biosensors applicable to food safety.

For an early disease diagnosis, highly sensitive detection technology is necessary to analyze biomarkers quantitatively at low concentration. In case of prion which is a marker protein for mad cow disease, it is impossible to detect prion protein in serum using the conventional detection technology as the concentration is low. In this study, the orientation of antibodies is controlled using aptamer to improve sensitivity of a sensor. Antigens are analyzed by Real-time PCR measurements. +Targets to detect are Prostate specific antigen (PSA) and Escherichia coli O157:H7 (E.coli O157:H7). This system can be applied to disease diagnosis and environmental toxicity test. Two probes to capture antigens are magnetic microparticle (MMP) and gold nanoparticle (AuNP). Antibodies for antigens are immobilized on each particles. Using Fc-specific aptamer, antibodies are coupled with gold nanoparticle. An antigen is captured with magnetic probe and gold probe. After reaction, magnetic separation is carried out and washing steps are followed. This aptamer is sensitive to the change of pH. Gold nanopaticle coupled with aptamer is obtained by treatment of distilled water. Finally, antigens can be detected by Real-time PCR of aptamer. Acknowledgments: This research was a part of the project titled "Development of Sustainable Remediation Technology for Marine Contaminated Sediments" funded by the Ministry of Land, Transport and Maritime Affairs, Korea, This research was supported by by the R&D Program of MKE/KEIT(No. 10035578, Development of 2,3-butanediol and derivate production technology for C-Zero bio-platform industry), This work (research) is financially supported by the Ministry of Knowledge Economy(MKE) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology.

Lipopolysaccharide (LPS), also known as endotoxin, is a major component of the outer membrane of Gram-negative bacteria and triggers a fatal septic shock upon its internalization into mammalian cells.1 Fast and accurate detection of even small amount of LPS in therapeutic products and the blood purification is therefore of primary importance. Several LPS affinity binders have been reported by far but few of them have proved their efficacy in developing electrochemical sensors capable of selectively detecting LPS from crude biological liquors.2-4 In this study, we identified 10 different single stranded DNA aptamers (designated as B1 to B10) showing specific affinity to LPS with dissociation constants (Kd) in the nanomolar range using NECEEM-based non-SELEX method. Based on the sequence and secondary structure analysis of the LPS binding aptamers, an aptamer exhibiting the highest affinity to LPS (i.e. B2) was selected as a sensing probe to construct an impedance biosensor on a gold surface. The developed electrochemical aptasensor showed excellent sensitivity and specificity in the linear detection range from 0.01 to 1 ng/mL of LPS with significantly reduced detection time compared with the traditional Limulus Amoebocyte Lysate (LAL) assay.