Earticle

Recently, surface plasmons of noble metallic materials (typically gold and silver) have been received much attention due to the phenomena are the bases of many standard tools for measuring adsorption of material onto planar metal surfaces (surface plasmon resonance (SPR)-based biosensors) or onto the surface of nanometer-sized metallic structures (localized surface plasmon resonance (LSPR)-based biosensors). For the planar surfaces, the SPR reflectivity measurements can be used to detect DNA, proteins or microorganisms by the changes in the local index of refraction upon adsorption of the target molecules to the metal surfaces. For the nanometer-sized particles, localized surface plasmon oscillations can give rise to the intense colors of solutions of plasmon resonance nanoparticles and/or very intense scattering. Nanoparticles of noble metals exhibit strong UV-Vis absorption bands that are not present in the bulk metal. Shifts in this resonance due to changes in the local index of refraction upon adsorption of target biomaterials to the nanoparticles can also be used to detect analytes such as DNA, proteins or other small molecules. In light of the useful and unique surface plasmon polaritons, many SPR-based biosensing platforms have been successfully carried out for detections of biomarker proteins and microbial spores in our lab. Moreover, LSPR of gold nanoparticles has also been being exploited to develop colorimetric biosensors and nanosensors for antigen-antibody biosensings. The strategy has several advantages, such as: non-invasive and non-labeling detection, high sensitivity, fast detection possibility, etc. More details will be thoroughly described in the presentation.

The immobilization of lipases, using ionic liquids (ILs) and multi-walled carbon nanotubes (MWNT) as additives to protect the inactivation of lipase during sol-gel process, was investigated. Among the lipase co-immobilized with ILs, the lipase showed the highest hydrolytic activity when the most hydrophilic IL, [C2mim][BF4], was used as an additive, while the highest stability of lipase was obtained by using the most hydrophobic IL, [C16mim][Tf2N]. Therefore, the binary mixtures of these ILs as additives were used to increase both activity and stability of immobilized lipase. The hydrolysis and esterification activities of lipase co-immobilized with the mixture of 1 : 1 were 10-fold and 14-fold greater than in silica gel without ILs, respectively. On the other hand, MWNT was used as an additive in the sol-gel process to immobilize lipase. The hydrolysis and esterification activities of lipase co-immobilized with MWNT were 6-fold and 5-fold greater than in silica gel without MWNT, respectively. After 7 times reuse of the lipase co-immobilized with 0.33% MWNT, the residual activity of lipase was 93%, while the immobilized lipase without MWNT showed no activity.

Oligosaccharides are used as therapeutic agents in infectious and inflammatory diseases, metabolic and cardiovascular disorders, transplantation, neutralization of toxins, and cancer immunotherapy. These are useful as bulking agents in diet foods and also act as pre-biotics. These activate the plant cell machinery leading to defence reactions or to plant developmental processes. They are also used as fertilizers. Gluconacetobacter hansenii PJK, a bacterial cellulose (BC) producing strain, can produce water-soluble oligosaccharides (WSOS) as a by-product from the chemically-defined medium. However, the yield of the WSOS from this medium was not enough for commercial applications. The current study deals with the optimization of medium for producing maximum amount of WSOS. The waste of beer fermentation broth was also tested as an alternative to the chemically-defined medium for the production of the WSOS. The structures of WSOS obtained were determined using various spectroanalytical techniques. Moreover, the various physical properties of the WSOS were determined to evaluate their potentials for commercial applications.

Silica-coated magnetite (Fe3O4) nanoparticles, synthesized by sol-gel method, were modified with iminodiacetic acid (IDA). The products with different particle sizes were obtained by controlling either sonication time or feed rate of tetraethyl orthosilicate（TEOS）during synthesis. A series of tests were then applied to characterize their physical properties. It was found that the particle size increased correspondingly with the increase of sonication time or feed rate of TEOS. Thermal gravimetry analysis suggested higher density coating of -OH groups on small-size silica particles, hence more IDA modified sites on their surfaces. The effects of particle size on adsorption ability were investigated by assessing the adsorption isotherm of bovine serum albumine to Cu2+-charged IDA-nanoparticles of varying sizes. With the increasing particle size, a higher maximum adsorption capacity and a higher dissociation constant was exhibited. This research provided an insight into synthesizing IDA-modified nanoparticles with controllable size and the resulting adsorption properties, thereby providing a simple method capable of addressing the various requirements in a wide range of applications.

White Spot Syndrome Virus (WSSV) occurs worldwide and causes up to 100% mortality within 7-10 days after the infection in commercial shrimp farm, resulting in large economic losses to the industry 1). Besides the economic impact of the disease, the natural aquatic ecology is also threatened as a wide host range of WSSV among crustacean 2). WSSV contains at least five major structural protein and the VP28 is a major component of its envelope protein. Of the viral structural protein, the envelope proteins usually play very important roles in virus infection and assembly. So the aim of this study is creating VP28 polyclonal antiserum and determining its WSSV neutralization ability for development of vaccines against WSSV. The VP28 ORF was successfully cloned in pCold I vector and expressed in E.coli BL21 . The purified VP28 protein was injected 3 times into a rabbit to produce VP28 polyclonal antibody. The polyclonal antibody was confirmed by ELISA and the antiserum was prepared for the neutralization. To study the role of VP28 in WSSV infection in Penaeus chinensis shrimp, an in vivo neutralization test was performed. The result of this experiment showed that WSSV infection was neutralizated by the VP28 polyclonal antiserum and that VP28 is involved in the infection process of WSSV. In further work, VP28 recombinant protein will be used for the production of vaccine against WSSV.

Protein patterning is crucial for the development of protein chip and biosensors. The most important point of this technique is how to bind both protein and surface. A lot of studies were reported about this, for example protein immobilization on aminosilane or poly-L-lysine functionalized surface. However theses conventional methods have problems that strong binding by covalent bonding cause deformation of three dimensional structures of protein and by electrostatic force by poly-l-lysine mono-layered surface have a weak binding force for effective patterning. In this study, we have introduced the development of protein micro-patterning on polyelectrolyte multilayer thin films(PEM) by consecutive layer-by-layer adsorption of polyelectrolyte via self-assembly technique. The property of PEM surface provided periodic oscillations in contact angle, which indicated the multilayer were correctly terminated by polycation and polyanion, respectively. Protein was immobilized on PEM by electrostatic interaction. As a model system, the printing of the fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA: 1 μg/ml) was performed. It showed good linearity over a range from 100 ng/ml to 255g/ml, indicating the feasibility of a quantitative measurement of concentration of target proteins in sample. The protein patterning fabricated on PEM provides higher signal intensity than patterning by conventional method. This novel method is well suited for immobilizing many types of biological molecules so that a wide variety of micro patterning formats can be developed.

Up to date, Silver nanopartilces (NPs) are the second largest products after carbon NP (Nanotubes & Fullerenes). There are many potential benefits of nanotechnology, but its hazards and risks are poorly understood. We have conducted an isolation of ESTs from male medaka liver and characterized unique 575 cDNAs cloned after sequencing and redundancy check of more than 2000 cDNAs obtained in randomly generated ESTs. Gene expression analysis with 575 cDNAs from specific tissues (gill, intestine, liver) were performed in exposure of Silver NPs (20 nm) to medaka using DNA microarray. A high dose (25 ppb) and a low dose (1 ppb) of the NPs were tested at various exposure time points. As the results, whether the NPs were uptake into the fish through gill or/and intestine they were accumulated in livers as the highest numbers of genes up or down regulated at any time point. For further understanding toxic mode causing by Silver NPs, its expression profile was compared to profiles of Gold colloid and Silver nitrate exposed to medaka. This study gives us additional knowledge of the environmental and ecological implications associated with nanotechnology.

Protein disulfide isomerase (PDI), an ER-resident molecular chaperone, forms and isomerizes disulfide bonds1. Calnexin (Cnx), also an ER-resident protein interacts with glycoproteins promoting proper folding. The Ca2+ binding property of Cnx links it to apoptosis as Ca2+ signal dissemination from ER generates and mediates apoptosis cascade from the mitochondria2. This study attempts to investigate the effect of PDI and Cnx expression level on specific productivity (q) and apoptosis of rCHO cells producing thrombopoietin (TPO), antibody (Ab) and TNFR-Fc. Doxycycline-regulated PDI and Cnx expression system in Tet-Off rCHO cells was established. It was found that the q of TPO (qTPO) was unaffected but that of antibody producing cells was increased by 15-27% due to the PDI expression level. The cells with down regulated Cnx showed an increase in the viability by ～20% throughout the batch culture. Western blot analyses showed a delay in apoptosis. The effect of overexpression of chaperones on q is dependent on the target protein and the chaperone concerned and that calnexin deficiency confers resistance to apoptosis induced by NaBu.

Extracellular production of recombinant proteins in E. coli has received considerable attention due to its significant advantages over cytoplasmic or periplasmic production. However, efficient secretion of recombinant proteins into the culture medium of E. coli remains a challenge due to the intrinsic limitations of the secretion machinery. Here we report a systematic proteome-based approach for high-level extracellular production of recombinant proteins. First, the extracellular proteome of an E. coli B strain was analyzed to identify motifs as potential fusion partners. Next, we expressed each open reading frame of the selected motifs and determined the protein profiles of the culture medium. The highest secreting motif was used as the carrier protein to produce several "passenger proteins" in the culture medium. Those model passenger proteins show a wide versatility with respect to the proteins' length and origin. In addition, the polypeptides accumulated in the medium at high concentrations ranging from 15 to 500 mg/L. These findings demonstrate a proteome-based approach for high-level production of recombinant proteins in the culture medium of E. coli.

This study describes micropatterning of proteins on the surface of 3-dimensional hydrogel microstructures. PEG-based hydrogel microstructures were fabricated on a glass substrate using a PDMS replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica. BSA, a model protein, was covalently immobilized to the surface of the hydrogel microstructure via 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker, which has a phenyl azide group and a protein-binding group on either end. The immobilization of BSA on the hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase was immobilized on the surface and HRP was encapsulated.

The library number and peptide structures are the most important aspects to find out the substrates of a protease. We introduced mRNA-display to screening for protease substrates and immobilized cDNA/mRNA-peptide library on acylated hexa-lysine bead by transglutaminase (TG) activity.1,2 TG generates the covalent bond by its specific peptide sequence containing glutamine. We identified the reacted substrates for 1×10-5 unit of thrombin, V8 protease, and caspase3. Screening of protease substrates supplied critical information for its specificity. We confirmed the proteolytic cleavage site of thrombin and V8 protease and founded novel peptides reacted by caspase3.

We tried to evaluate genotoxicity. Three different mutagens, mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG) and Nalidixic acid (NDA) were used to evaluate their toxicogenomic impacts on Escherichia coli. Different doses such as, sublethal, LC20 and LC50 and four testing time points, 5 min, 25 min, 45 min and 65 min were used. Commonly expressed genes as potential genotoxicity biomarkers on E. coli were obtained. By analyzing time-course expression data, distinct impacts of each chemical were pivotallized based on SOS resulted genes. And depending on time course expression, we can figure out different chemical effects based on SOS related genes. Clustering analysis of the DNA microarray gene expression data resulted in the presentation and visualization of functional classification and chemical-gene network.

The binding interaction of Salmonella typhimurium was studied with bacteriophage (P22) ligand and antibody ligand. A locally constructed fluidics system was used to deliver solutions to the compact, three-channel SPRTEETATM sensor. The binding interactions between Salmonella typhimurium and bacteriophage (P22) in addition to antibody were measured using surface plasmon spectrum by monitoring a variation of the Response Unit (RU) values. The association and dissociation rate constants of Salmonella typhimurium with bacteriophage (P22) and antibody were determined from a kinetic model applied curve-fitting method. The affinities between Salmonella typhimurium with bacteriophage (P22) and antibody were also considered with their kinetic parameters. The affinity appeared in the binding of Salmonella typhimurium with bacteriophage (P22) ligand.1) Salmonella typhimurium also exhibited significant adhesive properties antibody ligand.

In this work, a novel microfluidic device was prepared for the enzyme-based bioassay. Microfluidic device had two serial chambers connected by microchannels, reaction chamber and detection chamber. The reaction chamber was filled with glass beads with immobilized enzymes via APTES. Key feature of first chamber was filters on both sides, which was used to retain the beads with D of 40-70 µm in the flowing system. In the second chamber, PEG hydrogel microarray was fabricated via photolithography, which could immobilize proteins or fluorescent dyes for the optical analysis of the reaction. As a model, various concentration of glucose was detected in the system. In this system, first chamber was filled with GOX-immobilized beads, and second chamber with HRP-entrapped hydrogel microarray. When solution of glucose and Amplex Red flowed into channels, glucose reacted with GOX on the beads to produce H2O2, which moved to the second chamber and diffused into hydrogel array with HRP. Next, Amplex Red reacts with H2O2 in the presence of HRP inside array to produce fluorescent resorufin. By measuring the fluorescence of resorufin, glucose concentration ranging from 0.1–50 mM could be detected.

Quantitative analysis of DNA microarray is one of important issues in mRNA expression profiling and estimation for infectious doses of pathogens1). Here, we newly introduced an artificial standard probe strategy for quantitative data analysis of pathogen detection oligonucleotide chip as a model system improved from our previous report2). The artificial standard probe was designed to have below 85% similarity with any bacteria in order not to occur non-specific hybridizations with other targets. Based on fluorescence intensities of artificial standard spots, we successfully demonstrated that raw fluorescence intensities of specific probe spots could be corrected to have quantitative correlations with target concentrations. Therefore, our suggested rtificial standard probe strategy may be used for correction of chip-to-chip variation and making possible quantitative analysis of one-color oligonucleotide microarray experiment.

Phthalates and their esters (PE’s) are industrially important chemicals used for the manufacture of plastics, synthetic fibers, cosmetics etc. These phthalate compounds enter into the environment through effluents and aerosols. One of the most threatening and toxic behaviors of PE’s is that they mimic human endocrine hormone/s. There is no information available on the other toxicity aspects of these PE’s apart from their endocrine disrupting action. Here, by the use of recombinant bacteria specific for a known type of toxicity as signature, it was possible to identify that the PE’s also cause serious membrane damage along with moderate oxidative and protein damages. Authentic chemical compounds as possible candidates of PE’s breakdown products also showed similar toxicity behavior as that of their microbial mediated degradation byproducts.(1, 2)

RGD peptide sequence is an effective cell recognition motif and used to enhance the cell adhesion on desired solid material for cell immobilization. We have synthesized CRGD, CRGD-MAP (Multiple-Armed Peptide), RGD-MAP-C and evaluated their comparative efficacy for cell immobilization. Each peptide was assembled on gold surface and investigated by AFM technique in contact mode. The viability of immobilized animal cells was examined by MTT assay. Our results showed that RGD-MAP-C in comparison to others was the most effective proliferation of cells on the gold surface. The goal of this present work is integration to the nano-pattern cell chip bioplatform for biomedical assays or can provide valuable insights into cell biology and design of biomaterials.

We propose a mediator-free glucose biosensor which use immobilized glucose oxidase on tin oxide. Direct electron transfer is possible, because the tin oxide shows redox properties similar to those of the mediators. The preparation method for tin oxide and glucose oxidase-modified tin oxide is also simpler than the existing methods. Tin oxide provides an efficient surface for the immobilization of large amounts of enzymes, due primarily to the porosity of the surface. Glucose oxidase was immobilized on tin oxide via the entrapment method, using nafion. The suggested method provides a simple process for enzyme electrode fabrication. Glucose oxidase immobilized on tin oxide prepared in accordance with this method has a relatively large current response, as compared to those of other glucose biosensors. The sensitivity of the biosensor was 39A/mM, and a linear response was observed between 0mM to 3mM glucose. The Michaelis-Menten constant was determined to be lower than in electrodes prepared via the methods in current use. Also, this biosensor shows good reproducibility and stability.

We develop a micro analysis and detection system for replicase of the hepatitis C virus. The fabricated analysis system consists of a bead based microchip and a computer based controller. A microheater, a micro temperature sensor and a microreator for bead packing is integrated in one microchip to realize several sequences for the capture and the elution of replicase. In this study, we used the PEG-grafted PS bead on which RNA aptamers are immobilized and performed thermal denaturation for releasing target molecules, and analyzed by MALDI-TOF MS. We performed several experiments to optimize conditions. As a result, we could acquire several matched peaks (26 % sequence coverage) of HCV replicase using the following condition: at room temperature for 20 min of binding time and at 85℃ for 30s of thermal denaturation time.

Protein microarrays are studied as a rapid analysis of multiple samples, and a critical point for an optical detection signal is ligand density on a chip surface[1]. In this study, to evaluate the activity of the adsorbed ligand proteins on the hydrophilic or hydrophobic surface. We immobilized ligand proteins on different membranes: NC (nitrocellulose) membrane as a hydrophilic surface and PVDF (polyvinylidenedifluoride) membrane as a hydrophobic surface. We used rabbit IgG as the ligand for direct ELISA, and anti-rabbit IgG antibody as the ligand for sandwich ELISA. The adsorption of anti-rabbit IgG antibody to the membrane required 12 hr at 4℃. In sandwich ELISA, anti-rabbit IgG antibody with HRP (horseradish peroxidase) could specifically bind to rabbit IgG antibody. We obtained the results of ligand antibody adsorption and their activity and specificity toward antigen binding on each surface for comparison. In the case of NC membrane, diffusion of a spot was shown faster and wide. Resulting in a lower surface density than PVDF membrane. In conclusion, the optimal surface density was saturated in 10 uL volume and concentration of 2.5 ug/mL of anti-rabbit IgG antibody. Additionally, we apply this experiment to glass surface, too. We compared conjugation with adsorption of immobilized protein on the glass surface. In the case of glass surface, conjugation of proteins was essential process. Because adsorption of proteins was washed out after washing buffer[2]. So we have to use conjugation method of ligand proteins for microarray chip. And we confirmed the possibility of a new microarray system.

The filed of molecular electronics has seen an outburst of interest particularly motivated by the possibility of developing and characterizing the individual molecules to perform functions in electronic circuitry as performed by semiconductor devices[1-3]. Towards this goal, we have developed a molecular memory device that includes novel immobilization method for ferredoxin on gold surface. A recombinant protein with cysteine residue by using Polymerase Chain Reaction (PCR) was designed and then directly immobilized on Au surface without any chemical linker. The immobilization of the functionalized protein is confirmed by Surface Plasmon Resonance (SPR) and its surface morphology is analyzed by Scanning Tunneling Microscopy (STM). The memory function of self assembled ferredoxin was investigated by its redox properties of metalloprotein using Cyclic voltammetry, Chronoamperometry and Open Circuit Potential Amperometry (OCPA).The approaches outlined here can be applied for the development of the molecular memory device

Here we describe a useful method for the site-directed immobilization of proteins with a DNA-binding domain (DNA-BD) on the cognate DNA-coated gold surface for surface plasmon resonance (SPR) imaging analyses. In order to assess the performance of this procedure, we utilized two DNA-BDs, yeast GAL4 DNA-BD, and bacterial LexA DNA-BD. After the immobilization of the cognate double-stranded DNAs (dsDNAs) to a gold chip surface with a monolayer of poly(L-lysine) for sequence-specific DNA-protein interaction, purified recombinant GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP fusion proteins were applied to a dsDNA-spotted gold chip, and were subsequently analyzed using an SPR imaging system. Consequently, the recombinant DNA-binding proteins, GAL4 DNA-BD:EGFP and LexA DNA-BD:RFP, were shown to bind selectively to their cognate DNA sequences on the gold chip. Collectively, our results revealed that sequence-specific dsDNA microarray approach might prove useful in performing the site-directed immobilization of DNA-binding proteins onto a gold thin film in a parallel format.

Olfactory receptors, G protein-coupled receptor (GPCR), are integral membrane proteins composed of seven transmembrane spanning domains. These are not easy to be overexpressed, solubilized and purified because of their structures and strong hydrophobicity. Purification of olfactory receptor, where only a few studies have been done, is critical step for developing protein-based olfactory biosensor. In this work, human olfactory receptor, OR2AG1, was overexpressed in Escherichia coli. Electrophoresis of the overexpressed protein, visualized with Coomassie Blue staining and by immunoblotting with glutathione S-transferase (GST) antibody, revealed bands of 53, 118 and 160KDa, which were identified as the monomeric, dimeric and trimeric forms of the receptor proteins, respectively. The protein was difficult to solubilize with many detergents and only N-lauroyl sarcosine was found to be suitable for efficient solubilization. Human olfactory receptor, OR2AG1, was purified and solubilized by N-Lauroyl Sarcosine after treating membrane fraction with Triton X-100. Purified protein can be used as the primary transducer of olfactory biosensor after reconstitution in phospholipid vesicles.

Surface Plasmon resonance (SPR)-based sensors have been used to detect the binding etween interaction molecules. In this study, the SPR technology was applied to the cell-based measurement of odorant molecules. Human embryonic kidney (HEK)-293 cells were used as a heterologous cell system. An olfactory receptor protein of rats, I7, was expressed on the surface of HEK-293 cells. For targeting and detecting the protein, rho-tag import sequence was fused with the I7 protein. The interactions between HEK-293 cells expressing I7 and octanal; its specific odorant, elicited the change of Ca2+ ions within the cells and hence the change of Ca2+ ions was detected using SPR. This was confirmed by measuring the cytosolic Ca2+ ions using fluorescent calcium indicator. The SPR response induced by octanal in HEK-293 cells expressing I7 was dependent on the dose of octanal. These SPR responses were regarded as a result of the intracellular signaling triggered by the binding of odorant molecules to the olfactoryreceptors.

To detect bacterial pathogens, 16s rRNA was analyzed using a surface plasmon resonance (SPR) biosensor with a signal amplification technology. The 16s rRNA has been used as a genetic marker for identifying organism, and can be analyzed directly without PCR amplification due to relatively high copy number [1]. However, the direct detection of 16s rRNA shows sensitivity limitation compared with PCR-based assays. In this study, a signal enhancing method for high sensitive direct detection of bacterial 16S rRNA using peptide nucleic acid (PNA) probes was developed. The amount of hybridization was monitored by the SPR biosensor, which enables detection of molecular interactions on surface in response to changes in the index of refraction [2]. To amplify the interaction of PNA and 16s rRNA, surface-modified nanoparticles were designed to interact with 16s rRNA selectively. Using this amplification strategy, detection of various pathogens such as E. coli O157, Clostridium perfringens, Staphylococcus aureus was possible.

DNA computing is a computing paradigm as information storage media and information processing operators. Especially, information processing of DNA computing has emerged for gene expression pattern analysis and disease diagnosis. Recently, we developed a theoretical model for pattern classification, biomolecular perceptron. In the model, each pattern is a solution of various nucleotide strands. The patterns of the DNA species are summed with corresponding weight factors, where the result is used for the pattern’s classification. The calculation of this weighted summation is performed via competitive hybridization reactions between input molecules and differentially labeled probe molecules (molecular beacon), and the calculation is detected as fluorescence signals. Here, we demonstrate the experimental feasibility of pattern classification. We perform disease type discrimination using the subsequence of the acute leukemia marker genes. The results show the possibility of our method as a pattern classification tool and diagnosis tool using gene expression. Because of the absence of information conversion steps, diagnosis process can be simplified.

We developed new strategy for the detection of mutations in breast cancer susceptibility gene BRCA on PNA zip-code microarray by Single strand specific (SSS) nuclease. SSS nucleases was reported that is working from different sources to cleave single base pair mismatches in heteroduplex DNA templates used for mutation and single nucleotide polymorphism analysis. We focused this property and then we combined to detect of BRCA mutation site using on PNA zip-code microarray by SSS nuclease. The PCR products were performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent SSS nuclease reaction using chimeric primers with biotin-labeled 3' complementarity to the specific mutation site and 5' complementarity to the respective PNA zip-code sequence on the microarray. Hybridization of the labeled primers to the PCR products and This primer was cleaved by sss nuclease at each wild site in the presence of biotin-labeled region and mutation site in the absent of biotin-labeled region, and the products were hybridized to the PNA zip-code microarray. We conclude that SSS nuclease assay on PNA zip-code microarray can be effectively used for high-throughput detection of mutation.

In the present research, a novel ultra-sensitive detection method of β-amyloid 1-40 was investigated by scanning tunneling microscopy (STM). It is known that, β-amyloid 1-40 is a most challenging marker for the early diagnosis of alzheimer disease. So, to achieve this, an ultra-sensitive detection system for β-amyloid 1-40 is required. In this study, novel ultra-sensitive of β-amyloid 1-40 was developed and STM was performed for it's investigation. In STM, the frequency of current peaks was measured in accordance with the surface density of the dispersed Au nanoparticle-antibody complex on the gold surface. Which was represented as periodogram with it's logarithmic regression curve, and the change of the power spectrum was observed in accordance with the concentration of β-amyloid 1-40 molecule. the lowest detection limit of the assay system for β-amyloid 1-40 was found as 1fg/ml.

A n-type field-effect-transistor (n-type FET) biosensor for detecting the protein-protein interaction on the sensing layer was fabricated. Silicon dioxide (SiO2) of the gate oxide was used as the layer to detect the protein. As a platform model, we have used C-reactive protein (CRP) which is a cardiac and inflammation marker. The reagents such as APTS, poly(ethylene glycol) iglycidyl ether were used to bind with amine portion of the anti-CRP. After binding the anti-CRP on the sensing layer, CRP was injected. The binding reaction of the anti-CRP and CRP was detected to the electrical signal by FET-type biosensor. The measurement was performed in phosphate buffer saline (PBS;pH 7.4) solution and Pt was used as the reference electrode. The response of the anti-CRP and CRP have caused the variation of the drain current of a FET-type biosensor. To verify the interactions between the anti-CRP and CRP on the sensing layer, surface plasmon resonance (SPR) measurement has been performed. We confirmed that FET-type biosensor can detect the characterization of the protein-protein binding reaction using the real-time system.

The strategy for electrochemical detection of DNA mutation was developed with SURVEYOR nuclease and synthesized signal enhancing material. 50 base-long capture probe having thiol group and biotin at 3’ and 5’-end respectively was immobilized on the gold electrode surface and mercaptohexanol was applied subsequently as a blocking reagent. Perfect matched target and single-base mismatched target was hybridized on the each electrode. SURVEYOR nuclease was added and it cleaved the single-base mismatched dsDNA. The cleaved fraction including biotin at 5’-end was removed during washing step. Glucose oxidase (GOX, signal generating enzyme)-avidin conjugate was reacted biotin at 5’-end of capture probe, while it could not react with single-base mismatched duplex. Additionally, biotin-modified G4 PAMAM (Polyamidoamime) dendrimer was added to increase density of the GOX on the electrode surface. Cyclic voltammetry was performed with electrolyte containing 0.1 mM ferrocenemethanol and 10 mM glucose. The higher portion of single-base mismatched target in whole sample, the lower peak current was observed. With this simple and fast electrochemical system, the possibility of mutation in unknown sample can be detected clearly.