Earticle

Genomics” based on the recent developments in high-throughput technologies such as automated DNA sequencer and DNA microarray opens the area of systemic approaches in bioscience and biotechnology (systems biology). Especially, almost inaccessible biological features are expected to be uncovered with these genomic analysis tools and eventually huge progress in chemical, pharmaceutical, and healthcare industries would be achieved. These methods are, however, not quite applicable to wide range of biological systems due to incorrect hybridization, insufficient resolution, image corruption (DNA microarray), and relatively low-throughput (automated DNA microarray). In order to avoid the significant errors caused by incorrect hybridization, we developed a novel DNA analysis method independent from hybridization using single-strand conformation polymorphism based on capillary electrophoresis (CE-SSCP). Since a number of genes can be separated in CE based on the folding changes dependent on sequences with high reproducibility, it is applicable to mRNA quantification, expression profiling, and mutation analysis with high precision and sensitivity.

Recently terrorist action poses the threat of bio-warfare using potential harmful bacterial spores. The anthrax attack of Oct 2001 in USA demonstrates the need for a rapid detection system for spores of Bacillus anthracis. In this work we have developed bacterial endospore detection technique based on the reaction dipicolinic acid (DPA) with terbium ion (TbCl3) and fabricated a prototype of spore detection system. The detector measures the fluorescence emission of DP A․TbCl3 in the Bacillus spores. The detection system consists of a air sampling device, band-pass filters and a photomultiplier tube array, which measures fluorescence intensity in the wavelength band from 550 nm to 565 nm. The DPA․TbCl3 complex has fluorescence emission band arround 560 nm, when it is excited by UV light in the excitation wavelength range around 280 nm. The experimental parameters investigated spores concentrated form air, amount of DPA release from Bacillus subtilis spores by dodecylamine solution. The limit of detection was 1000 CFU/ml.

Doxorubicin (DOX) is a highly valuable anticancer drug belonging to the anthracycline family, which is generated by a high G+C gram-positive soil bacterium, Streptomyces peucetius. It has been well documented that the biosynthesis of most antibiotics are tightly regulated at complicated genetic levels, thus leading to very low productivity in wild-type strains. To screen novel potential regulatory genes involved in antibiotic overproduction, wild type and overproducing strains such as actinorhodin (ACT)-producing S. lividans and DOX-producing S. peucetius were generated and compared via omics-driven strategies. Using 2-D gel electrophoresis and MALDI-TOF analyses between the wild type and the ACT-overproducing S. lividans, several proteomics-guided targets were identified. Comparative transcriptomes were also analyzed between the wild type and the DOX-overproducing S. peucetius via S. coelicolor cDNA microarrays. We found several potential regulatory genes involved in antibiotics-overproduction. To show the biological significance of the omics-driven potential target genes in Streptomyces species, these genes were functionally expressed and partially characterized both in S. coelicolor and S. peucetius. Further analysis revealed that expression of some of these genes inhibited antibiotics production. The identification and manipulation of these cryptic regulatory genes described herein may result in the maximization of the genetic productivity potentials in Streptomyces species.

Recently, hydroxylated products of daidzein and genistein are a growing scientific interest for their health-related qualities. Hydroxylated products have potent antioxidant properties that contribute to their cholesterol lowering effect, cardiovascular protection, antitumor effect, and anticarcinogenic properties Doenjang (Fermented Soybean paste) is a unique Korean traditional food fermented by many useful microorganisms including fungi and bacilli. Isoflavones have biological effects due to their potential beneficial properties for human health. We have been interested in a microbial metabolite of isoflavone in highly aged Doenjang. Recently, we isolated three o-dihydroxyisoflavones (ODI), 6-hydroxydaidzein (6-OHD), 8-hydroxydaidzein (8-OHD) and 3`-hydroxydaidzein (3`-OHD), and evaluated some biological activity. ODIs show predominant effect on antioxidation activity and melanogenesis inhibition. And we studied the formation of modified isoflavones from daidzein and genistein by the genus Streptomyces. Streptomyces avermitilis MA4680 could hydroxylate at ortho-specific position and methoxylate subsequently. The great potentiality in applications such as the production of hydroxylated isoflavones and drug metabolites or bioremediation will be motivated for researchers to find new paradigm.

Protein-based gene delivery including histone proteins has recently emerged as a powerful technique for non-viral DNA transfer. Histones are DNA-binding proteins that function in DNA packaging and protection. Several studies have examined the use of full-length histone H1-mediated gene transfer, and a few studies have investigated the use of C-terminal histone H1 fragments as gene-transfer materials. Previously, we cloned a novel histone H1 cDNA from the goldfish Carassius auratus and found that a recombinant histone H1 C-terminal short peptide (H1C) of 61 amino acids has comparable DNA binding and protection functions as full-length histone H1. In the present work, we successfully expressed and purified soluble recombinant H1C in an Escherichia coli expression system using a hexahistidine tag fusion strategy and providing tRNAs for rare codons. We confirmed its DNA-binding ability and found that this H1C peptide had similar or higher transfection efficiency in mammalian cells than the widely used agent lipofectamine. Therefore, we suggest that this novel goldfish-derived recombinant histone H1 C-terminal short peptide could be used as a peptide-based gene-transfer mediator.

A cell-once-through (COT) perfusion concept was applied to a dual bioreactor system coupled to a Centritech Lab II Centrifuge for culture of recombinant Chinese hamster ovary (rCHO) cells for monoclonal antibody production. In this new culture mode, i.e., the COT perfusion process, total spent medium was transferred the centrifuge and a fixed percentage was removed. Accordingly, a significant reduction of the cell-passage frequency to the centrifuge led to minimization of cell damage caused by mechanical shear stress. Although the suppressive effects of low culture temperature on cell growth led to a loss of stability in a long-term COT perfusion culture system, the average antibody concentration at 33°C was 157.8 mg/L, approximately 2.4-fold higher than that at 37°C. By the use of a fortified medium at 37°C, rCHO cells were maintained at high density above 1.2 × 107 cells/mL, and antibody was produced continuously in a range of 260 to 280 mg/L in a stable long-term COT perfusion culture. The proposed new culture mode, the COT perfusion approach, guarantees the recovery of rCHO cells damaged by lowered temperature or high lactate and ammonium concentration.

Angelica gigas is a common traditional medicinal plant that has been used for more than several thousand years in Korea. The commonly used Angelica roots showed pharmacological properties with their chemical compositions. The main chemical constituents of Angelica roots are ferulic acid, ligustilide, angelicide, brefeldin A, butylidenephthalide, butyphthalide, succinic acid, nicotinic acid, uracil, and adenine(2). Main constituents associated with the pharmacological activities of Angelica roots showed anticancer activity(1), inhibitor of platelet aggregation, antiasthmatic and spasmolytic activities(2). The best alternation for increasing production is elicitation. We activated all secondary metabolisms by the addition of biotic elicitors to suspended cultured cells and compared secondary metabolites between control (unelicited) cells and elicited cells. Elicitor-treated cells showed higher productivity of coumarin and coumarin derivatives than untreated cells. Also Elicitor-treated cells produced secondary metabolites which are not shown in unelicited cell by opening other secondary metabolism. We investigated major relationship between profiles of metabolite production and expression level of phenylalanine ammonia-lyase (PAL).

Our lab have tried to asymmetrically synthesize chiral amine, (s)-MBA with w-TA from Vibrio fluvialis JS17 using alanine and acetophenone as amine donor and amine acceptor respectively. But low equilibrium constant and product inhibition prevent from synthesizing highly concentrated (s)-MBA. The goals of this experiment is to analyze the problem of alanine, acetophenone reaction system and to find out the way to asymmetrically synthesize chiral amine compounds with high-yield. We succeed to get purified PLP-Enzyme and PMP-Enzyme. The typical absorption peak of PLP-enzyme and PMP-enzyme is 420nm, 340nm in UV-visible spectra respectively Doing half reaction between purified PLP-enzyme and ala, we can observe how absorption maxima is converted to counterpart. And, Doing half reaction between purified PMP-enzyme and acetophenone, the same as above. With these data we can find out the limit of alanine as amine donor and the limit of acetophenone as amine acceptor. Moreover, we can find out the way to synthesize divers chiral amine compounds with high-yield to circumvent low equilibrium problem. According the combination of relative activity of amine donor, amine acceptor, conjugated amine donor and conjugated amine acceptor, we classified 4 type reactions. To synthesize chiral amine compounds with high-yield, we conclude that we have to consider the relative activity of amine donor, amine acceptor, conjugated amine donor and conjugated amine acceptor

The reconstruction of rabbit corneal epithelium was performed through differentiation culture method using self-manufactured amniotic membrane (AM) supporter and lyophilized amniotic membrane (LAM). To evaluate the reconstructed corneal epithelium, we stained the reconstructed tissue sections against CK3, PCNA, and examined TEM. Tilting dynamic culture was better than static culture in terms of reconstructed corneal cell layers and proliferating activity for reconstructing corneal epithelium on LAM. Blinking dynamic culture, comparing with static culture, showed similar properties with rabbit cornea in stratification and differentiation of corneal epithelial cells under histological examination. The corneal epithelium was successfully reconstructed by the use of LAM and self-manufactured AM supporter by blinking dynamic culture. The reconstructed corneal epithelium in this experimental study is considered to be a good in vitro model in autograft and allograft for treating the patients with severely damaged corneal surface and allograft for treating damaged skin and mucosa.

L-homophenylalanine is a building block of Angiotensin-Converting Enzyme(ACE) inhibitor and 2-phenylethanol is a most widely used aroma compound with rose-like odor. L-homophenylalanine and 2-phenylethanol were synthesized simultaneously from 2-oxo-4-butanoic acid and L-phenylalanine by coupling reaction of aminotransferase and the Ehrlich pathway using a recombinant E.coli. By adding on carbonyl reductase cofactor regeneration system, high yield L-homophenylalanine and 2-phenylethanol synthesis reaction is available. To select carbonyl reductase, the activities of six carbonyl reductases from Saccharomyces cerevisiae were assayed. Among those candidates, YGL157w showed the highest activity on phenylacetaldehyde. Phenylpyruvate decarboxylase was recruited from the Ehrlich pathway of Saccharomyces cerevisiae, which is the metabolic pathway of L-phenylalanine. In addition, regeneration of an expensive cofactor NADPH is achieved using Glucose dehydrogenase from Bacillus subtilis. Despite of the low kcat value of decarboxylase, 39.2mM of 2-phenylethanol was produced via whole cell reaction although we have used 1mM of NADPH.

To alleviate oxidative stress generated during naphthalene metabolism, overexpression of antioxidant enzymes [Fpr (ferredoxin-NADP+ reductase) and SOD (superoxide dismutase)] was conducted in naphthalene-degrading bacterium Pseudomonas sp. strain As1. The fpr and sodA gene was placed under the transcriptional control of either constitutive lac promoter or native promoter. Both high performance liquid chromatography (HPLC) analysis and growth rate have shown that the recombinant cells effectively degraded naphthalene compared to wild type. All recombinant cells, except a strain As1(sodA), where sodA gene was up regulated by the lac promoter, exhibit more resistance to a superoxide generating agent, paraquat. Growth of As1(sodA) was inhibited by paraquat and its growth defect were significantly overcome by alkyl hydroperoxide reductase overproduction. Taken together, the data demonstrated that overproduction of these two genes contribute to oxidative tolerance during naphthalene degradation, however, more than necessary SOD, which may provoke generation of hydrogen peroxide, results in cell death.

We describe here ligation-based strategy to detect mutations in BRCA1 utilizing zip-code microarray technology. In our first approach, PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then used as templates in a subsequent ligation reaction using two ligation primers that flanked the mutation site. The primary allele-specific primer is designed to contain respective zip-code at its 5/ end while the secondary primer is modified by biotin at its 3/ end. Then the nick between two ligation primers was sealed in presence of DNA ligase. The ligation products were then hybridized on the zip-code microarray followed by staining with streptavidine-cy3 to generate fluorescent signal. Using this strategy we successfully genotyped selected Korean-specificmutation sites of BRCA1 with a wild type and two heterozygote mutant samples. Furthermore, we also demonstrated that ligase chain reaction using unamplified genomic DNA as direct templates is enough to generate sufficient signals for correct genotyping in a multiplexed manner, verifying first that PCR is not essential for this microarray-based strategy.

Efficient and safe nonviral gene delivery systems are a prerequisite for the clinical application of therapeutic genes. In this study, we report an enhancement of the transfection efficiency of plasmid DNA, via the use of positively charged colloidal gold nanoparticles (PGN). Plasmid DNA encoding for murine interleukin-2 was complexed with PGN at a variety of ratios. The delivery of pVAXmIL-2 into C2C12 cells was dependent on the complexation ratios between PGN and plasmid DNA, presented the highest delivery at a ratio of 2400:1. After complexation with DNA, PGN showed significantly higher cellular delivery and transfection efficiency than did the polyethylenimines (PEI) of different molecular weights. PGN resulted in a cellular delivery of pVAXmIL-2 6.3-fold higher than PEI25K. The PGN/DNA complex resulted in 3.2- and 2.1-fold higher murine IL-2 protein expression than PEI25K/DNA and PEI2K/DNA complexes. Following i.m. administration, PGN/DNA complex showed more than 4 orders of magnitude higher expression levels as compared to naked DNA. The results of this study suggest that the PGN/DNA complexes may harbor the potential for efficient and safe gene delivery vehicles

Serum stability of cationic liposomes is a prerequisite for in vivo application of therapeutic genes. In this study, we report an enhancement of the serum stability and delivery efficiency of siRNA via use of newly synthesized cationic lipid-containing liposomes. Two cholesterol derivatives, cholesteryloxypropan-1-amine (COPA) and cholesteryl-2-aminoethylcarbamate (CAEC) were synthesized. Cationic liposomes were composed of DOPE and COPA or CAEC. Lipofectamine 2000 and CAEC-containing liposomes showed significantly reduced cellular uptake of siRNA after delivery in serum-containing media as compared to serum-free media. In contrast, COPA-based liposomes revealed enhanced cellular uptake of siRNA in the presence of serum. The oncogene survivin-specific siRNA delivered via COPA-based liposomes reduced the mRNA expression levels of the target gene in Hepa 1-6 cells. The cytotoxicity of COPA-based liposomes was comparable to that of Lipofectamine 2000. These results suggest that the newly synthesized cholesterol derivative COPA-based liposomes could be further developed as a serum-stable non-viral delivery system of siRNA.

A protein chip was developed for melanogenesis inhibitor screening. MITF (Microphthalmia-associated transcription factor) is a key regulatory transcriptional factor of the pigmentation-related genes such as tyrosinase and MC1R, and binds to specific sequence (CATGTG) in E-box DNA within the promoter of tyrosinase and MC1R in the melanocytes.1), 2) We produced MITF as a MBP (maltose binding protein) fusion protein in E. coli and purified by affinity column and immobilized on β-cyclodextrin coated glass plate. Binding of MITF to its target DNA, E-box oligomer, was monitored by surface plasmon resonance (SPR) and SPR imaging (SPRi). Unlike SPR and SPRi which employed unlabeled DNA oligomer, we used Cy3-labeled DNA in fluorescent imaging.3) In this method, fluorescent intensity was proportional to the DNA concentration up to 20μM. Among these detection methods, fluorescence method was the most reliable.4) Kinetics of DNA binding with MITF showed Langmuir isotherm and its kinetic constants were determined. To determine if interaction between MITF and E-box is sequence specific, we modified E-box DNA (CATGTG → CAGCTG) and monitored fluorescent intensity. These results indicated the potentials of MITF-E-box DNA chip as a screening tool for depigmenting agent in the cosmetic industry.

Currently, there has been a great interest to generate patterns of biologically active molecules such as DNA, proteins, cells and spores on solid substrates for their applications in biosensors, biomaterials and tissue engineering. In this study, a simple lithographic process in conjunction with a novel biocompatible nonchemically amplified photoresist material was successfully used for cell patterning. A novel copolymer of DOBEMA and GMA, poly (DOBEMA0.84 - co - GMA0.16), the nonchemically amplified resist material was used in this study. GMA was incorporated in the polymer to avoid peeling off of the resist film from the glass substrate. UV light irradiation on selected regions of the nonchemically amplified resist film rendered the exposed regions hydrophilic by the formation of carboxylic groups. It was found that mouse fibroblast cells were preferentially aligned and proliferated on the UV light exposed regions of the nonchemically amplified resistfilm where carboxylic groups were present. The cell alignment on the exposed regions was maintained during cell roliferation. This simple strategy of generating carboxylic groups on the UV light exposed regions by the simplified lithographic process opens up the possibility of immobilizing various biomolecules such as DNA, proteins, and cells.[This work was supported by Medigenes Co., Ltd. and by the Brain Korea 21 Project. Further support through the LG Chemicals Chair Professorship is appreciated.]

Metabolic flux distributions are an ultimate cellular phenotype as an outcome of interplays among various cell components. They are condition-specific, so that they change drastically upon genetic/environmental perturbations. Their integral analysis obtained from various conditions could provide further insight into the science of metabolic fluxes. For this, we developed a framework that sequentially employs constraint-based flux analysis and Bayesian network analysis, which ultimately yields an integrated causal network of metabolic fluxes based on specified conditions. Constraint-based flux analysis is a simulation method of a stoichiometric model using various optimization techniques. Bayesian network is a probabilistic graphical model that reveals causal relationships among variables of interest and is useful for data integration. This framework enables integration of metabolic fluxes, which could supplement metabolic flux analysis. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (No. M10309020000-03B5002-00000). Further supports by LG Chem Chair Professorship, Microsoft and IBM SUR program are appreciated.]

The signal amplification technique of PNA-based electrochemical DNA sensor was developed utilizing enzymatic catalysis. Electrochemical detection of DNA hybridization on a PNA-modified electrode is based on the change of surface charge caused by the hybridization of negatively charged DNA molecules. Negatively charged mediator, ferrocenedicarboxylic acid, can not diffuse to the DNA hybridized electrode surface due to the charge repulsion with the hybridized DNA molecule while it can easily approach the neutral PNA-modified electrode surface without the hybridization. By employing glucose oxidase catalysis on this PNA-based electrochemical system, the oxidized mediator could be immediately reduced leading to greatly increased electrochemical signals. Using the enzymatic strategy, we successfully demonstrated its clinical utility by detecting one of the mutation sequences of breast cancer susceptibility gene BRCA1 at the sample concentration lower than 10-9 M. Furthermore, a single base-mismatched sample could be also discriminated from a perfectly matched sample.

Cell biosensor can be used to acquire functional information of the effect of a stimulus on a living system and detect various chemical compounds. An efficient strategy was developed here for immobilizing cells onto the gold surface by cell surface-expression of gold binding polypeptide (GBP). Additionally, core streptavidin (cSA) of Streptomyces avidinii was used as a model protein to demonstrate the effective immobilization of GBP-fusion proteins onto the gold surface. All specific binding of cells and other compounds to the gold surface were detected by surface plasmon resonance (SPR). As a series of data revealed, cells displaying GBP as well as GBP-fusion proteins bound to the gold surface stably and also, successful interaction between GBP-cSA pre-immobilized onto the gold surface and biotin-horse radish peroxidase (HRP) was observed. Mediated by GBP-fusion protein, whole cell biosensor system for biomolecular interaction studies could be obtained by effective immobilization of bioactive cells onto the gold surface without surface modification. [This work was supported by the KOSEF through the Center for Ultramicrochemical Process Systems, and by grant No. (R01-2006-000-11175-0) from the Basic Research Program of the Korea Science and Engineering Foundation.]

The aim of this study is to develop a rapid diagnostic DNA chip to detect most common βigh3 mutations which cause corneal dystrophies (CDs). Recent studies have shown that LASIK can worsen the CDs, and thus initial screening of βigh3 gene mutation is urgently needed before LASIK. Direct sequencing of the exons 4 and 12 of the relevant gene showed that CDs could be mostly divided into homozygous Avellino CD and heterozygous Avellino CD, Heterozygous Lattice Type I, heterozygous Reis-Buckers CD and heterozygous Granular CD. Suitable primer and probe sets were designed to examine the exons 4 and 12 of the βigh3 gene in order to identify mutant and wild type alleles. DNA chip diagnosing these CDs was successfully developed. Mutations were then identified by signals of the probes immobilized on DNA chip developed in this study. Ninety eight participants volunteered for this test. Each DNA sample from peripheral blood was analyzed by DNA sequencing and DNA chip, and then compared with each result. We concluded that this rapid genotyping using DNA chip allowed successful detection of CDs with 100% sensitivity and specificity. We thus conclude that mutational analysis of exons 4 and 12 of the βigh3 gene, which is the mutational hot spot in CDs, can be successfully performed with DNA chip technology providing enough information for the diagnosis of CDs. [This work was supported by Medigenes Co., Ltd. and by the Brain Korea 21 Project. Further support through the LG Chemicals Chair Professorship is appreciated.]

We report real-time interactive manipulation of microparticles using optoelectronic tweezers (OET) on liquid crystal display (LCD). This report demonstrates an interactive control program for OET-based microparticle manipulation. The OET has become a remarkable technique for parallel manipulation of live cells and microparticles using image-driven dielectrophoresis (DEP). Due to its programmability, the automatic parallel manipulation of microparticles using computer softwares such as Microsoft PowerPoint has been possible.1,2) However, the conventional softwares are not compatible with the interactive particle manipulation. Therefore, here we develop a new control program which offers a useful user interface for the real-time interactive OET. We can manipulate the particles in OET device using LCD images controlled by keyboard and mouse, as seeing a microscopic view of OET in real-time. Our Flash-based control program is successfully demonstrated to the interactive manipulation of polystyrene beads with 75 ㎛ diameter. The microparticles trapped by ones inside a virtual DEP cage are formed a single particle array within only a few minutes. This interactive OET system can be utilized to several biological and medical applications including cell patterning and bead-based protein assays.

Electrochemical microchip was fabricated by assembling a surface-functionalized poly(dimethylsiloxane) (PDMS) microchannel with an interdigitated array (IDA) gold electrode to sensitively and rapidly detect human cardiac troponin I (cTnI). Using a confocal microscope, a significant fluorescence detected within a FITC-labeled immunoglobulin G (instead of antibodies) bound to the PDMS channel indicated the feasibility of the direct binding of antibodies onto the PDMS channel. To suppress electrode fouling, anti-cTnI was immobilized onto the PDMS channel on which protein G layer had been generated by silanization, and then the PDMS channel was assembled with an IDA chip. The detection experiments were performed with successive injection of cTnI, alkaline phosphatase-labeled anti-cTnI, and p-aminophenylphosphate. Then, cyclic voltammograms were obtained by the oxidation peak current proportionally to the concentration of enzymatic product, p-aminophenol. The optimal packing density of anti-cTnI on the surface of PDMS channel was determined at the anti-cTnI concentration of 30 µg/ml. These demonstrate that the proper orientation and best packing density of antibody as well as no electrode fouling contributed to the low detection limit (148 pg/ml) of cTnI within 8 min.

Currently, there has been a great interest to generate patterns of biologically active molecules such as DNA, proteins, cells and spores on solid substrates for their applications in biosensors, biomaterials and tissue engineering. In this study, a simple lithographic process in conjunction with a novel biocompatible nonchemically amplified photoresist material was successfully used for cell patterning. A novel copolymer of DOBEMA and GMA, poly (DOBEMA0.84 - co - GMA0.16), the nonchemically amplified resist material was used in this study. GMA was incorporated in the polymer to avoid peeling off of the resist film from the glass substrate. UV light irradiation on selected regions of the nonchemically amplified resist film rendered the exposed regions hydrophilic by the formation of carboxylic groups. It was found that mouse fibroblast cells were preferentially aligned and proliferated on the UV light exposed regions of the nonchemically amplified resistfilm where carboxylic groups were present. The cell alignment on the exposed regions was maintained during cell proliferation. This simple strategy of generating carboxylic groups on the UV light exposed regions by the simplified lithographic process opens up the possibility of immobilizing various biomolecules such as DNA, proteins, and cells.[This work was supported by Medigenes Co., Ltd. and by the Brain Korea 21 Project. Further support through the LG Chemicals Chair Professorship is appreciated.]

Protein adsorption on single-walled nanotube field effect transistors (FETs) leads to appreciable changes in the electrical conductance of the devices, a phenomenon that can be exploited for label free detection of biomolecules with a high potential for miniaturization. This work presents an elucidation of the electronic biosensing in immune reaction with a newly developed microarray of nanotube "micromat" sensors. First, anti-human IgG was immobilized by 1-pyrenebutanoic acid succinimidyl ester on carbon nanotube surface1). And then, the introduction of human IgG at various concentrations caused a decrease in the source-drain current. Thus, because the CNT-FET biosensor can detect immune reaction, it is able to use for various disease diagnosis.

본 연구에서는 미생물 발효공정의 모니터링을 위해서 졸겔을 이용한 pH와 용존산 소 검출막을 개발하였다. 졸겔은 물리, 화학적 안정성이 우수하여 다양한 분야의 센 서에 적용이 가능하다. 특히, GPTMS와 MTMS 그리고 TEOS의 실란계 화합물로 제조한 졸겔은 폴리에스테르 필름에 스핀코팅시 열경화 건조를 하기 때문에 건조시 간이 매우 짧은 이점을 가진다. 제조한 pH와 용존산소 검출막은 pH와 용존산소량에 따라 형광세기를 측정한 결과, 응답속도가 빠르며 재현성이 우수하였다.

A thin film oxygen and pH sensing membrane were fabricated by entrapping Ru(Ⅱ) complex and 6-aminofluorescein in the sol-gel matrix. This sol-gel matrix was prepared by the silane compounds of precursors, tetraethyl-orthosilicate(TEOS), methyl-trimethoxy silane(MTMS) and 3-glycidoxypropyl-trimethoxysilane(GPTMS), in ethanol solution. The high sensitivity and fast response time make these sensing membranes suitable for a variety of applications, including environmental monitoring and cell cultivations.

One of the serious problems concerning water in the environment is its pollution with naturally occurring toxins presumably produced by cyantobacteria (blue-green algae) that produce algae blooms. The cyanobacteria produce various types of toxins. One of them is microcystin, a hepatotoxin. Microcystins from cyanobacteria are released when the cells are lysed, die and break open. The incidents of wild and domestic animal poisoning and human health problems attributed to exposure to microcystins have been well documented. So for health risks assessment from the microcystins, the rapid system to detect and monitor microcystins is required. Quantum-dot nanocrystals have high fluorescence efficiency, lack of photobleaching, and long fluorescence lifetimes. Here we biocojugated quantum-dot 525 (Invitrogen) to the monoclonal microcystin antibody to detect the microcystins extracted from cultured Microcystin aeruginosa (NIER 10038). When quantum-dot 525 antibody conjugates interact with the microcystins, the fluorescence emission spectra and intensities changes were observed using a fluorescence spectrophotometer.

Recently, many commercial electronic noses have been developed and are being applied to such areas as food, beverage, agriculture, health, industry and environment. But the electronic noses have limitation; less sensitivity, high cost, large equipment and so on. Biosensor using olfactory receptor can overcome these limitation having high sensitivity and specificity. In previous study, quartz crystal microbalance (QCM) was used to check olfactory receptor protein ODR10 interact with diacetyl. Such system could be applied to test the interaction of human olfactory receptor and specific odorants. For this experiment, Human olfactory receptor OR52D1 interacting with ethyl isobutyrate is chosen. OR52D1 was expressed in E.coli as a fusion protein with GST tag and the expression was confirmed through western blot using anti-GST antibody. Crude membrane extract of the expressed protein was coated on the surface of gold, and the interaction of the olfactory receptor and specific odorant was examined. OR52D1 interacted with isobutyrate stronger than other odorants is confirmed by QCM.

Spore-forming bacterium Bacillus anthracis is cause of anthrax which is an acute infectious disease. Anthrax most commonly occurs in mammal such cattle, sheep, goats, camels and antelopes, but can also occur in humans when they are exposed to infected animals or tissue from infected animals. The goal of this research is to propose new experimental ideas that may serve detection method of B. subtilis which is similar with B. anthracis by surface plasmon resonance (SPR). Surface construction provided by R. A. Vega et al. previously was used. After immobilization of poly- and monoclonal antibodies with metal ions on gold thin film, response of a layer of antibody in the presence of microbes is recorded through the shift of SPR angle. In this report, passivation layer of thiolated poly-ethylene glycol (PEG-SH) is suggested to improve sensitivity and to get optimum antibody orientation. Relatively uniform immobilization of antibody structures in active states leads to abundant possibility of efficient measurement of B. subtilis.

Here we report in vitro selection of a novel ribozyme that catalyzes the 5'-nucleotidyl transfer reaction forming the 2'–5' phosphodiester bond. This ribozyme was underrepresented in the pool enriched for the 5'-triphosphate-dependent activities in incorporating ATP-γS, and thus it could have been undiscovered previously using similar selection strategies. The originally selected ribozyme consisting of 109-nucleotide (nt) was miniaturized to 45-nt M4 ribozyme via a series of mutation studies, and based on this mini-ribozyme a trans-acting system was constructed. One of the most challenging tasks in our study was to determine the chemistry occurring at the 5'-ppp site. We utilized various analytical methods including MALDI-TOF analysis of the product generated by the trans-acting system and elucidated the chemistry to be 3'→5' mononucleotide extension forming the 2'–5' phosphodiester bond. Interestingly, M4 ribozyme promiscuously accepts a variety of purine nucleotides bearing 5'-mono-, di- and triphosphates as substrates. This remarkable ability of M4 ribozyme would lead us to the development of a new tool for the 5'-modification of RNAs with unique chemical groups.

The ability to organize multiple biomolecules on micro/nanometer scale pattern is crucial for the development of biological integrated devices such as DNA/protein chip. In this study, a gold pattern on silver substrate was prepared. A novel and ultrasensitive detection method of prostate specific antigen (PSA) by scanning tunneling microscopy (STM) was developed on the micro gold pattern. PSA is a marker for prostate cancer and it has been identified as a potential marker for breast cancer in women. The detection method is based on the change of tunneling current given by the gold nanoparticle dispersed on micro gold pattern insulated with biomaterial. Biomolecular thin film on the pattern and the Au nanoparticle-antibody conjugate were fabricated in an oriented manner with the antibody fragments. As a result, the frequency of current peaks was generated in accordance with the surface density of the dispersed Au nanoparticle on pattern. And, the change of the power spectrum was observed in accordance with the concentration of PSA molecule. The lowest detection limit of the assay system for PSA was 33fM (10pg/mL).