Earticle

Great interests have been emerged in the development of bioelectronic devices which performed functions realized by current silicon based semiconductor devices because of their compatibilities for organic-inorganic interconnections or networks. Therefore the purpose of this study is to develop artificial molecular memory devices by biomimetic approach using metalloproteins. we fabricated a biomemory device that includes a recombinant azurin with a cysteine residue modified by site-directed mutagenesis technique [1]. Functional azurin proteins developed in this study were covalently-immobilized on four different Au electrodes patterned on a single Si substrate. The redox property of Azurin molecules on individual electrodes was measured by cyclic voltammetry (CV) experiments, respectively. Using open circuit potential amperometry (OCPA) method the memory function of the fabricated biodevice was confirmed. Upon applying particular oxidation voltage to Au electrode causes that particular protein layer becomes oxidized and stores positive charge which is represented as memory write (bit of “1”) and rest of the protein molecules on other electrodes are in its original state (bit of “0”). The memory read and erase functions are achieved by the application of open circuit and reduction potentials respectively. Applying oxidation potentials in different combinations bit of information can be stored in to the azurin molecules. Finally, based on the measurements of the charging currents of the fabricated biomemeory device, it is likely to be the key in the development of molecular/semiconductor electronic devices.

Surface modification and optimization is a principal part in developing a high sensitivity and high capacity protein sensor as an advanced immunoassay. To increase and improve the binding density of biomolecules on the surface matrices with biomolecules, dendrimer generation 4.5, which was terminated with carboxyl group (PAMAM-COOH), was layered on amine functionalized glass by covalent bonding. The surface properties were analyzed by ellipsometer and scanning electron microscopy to optimize the dispersion of the polymer and the chemistry for immobilization of proteins. IgG whole molecules (H+L) and Fc cleaved antibodies containing variable region were tested as protein-binding agents on the dendrimers and unbound free amines on the surfaces were capped by BSA or small PEG derivatives. This study demonstrates high-sensitivity by following attachment of covalent bond formation as compared with blank glass supports. Moreover, dendrimer-driven increased binding capacity was demonstrated with fragmented antibodies by reducing steric hindrance/resistance among the polymer and biomolecules and by inhibiting non-specific binding and undesired direct coupling between carboxylic acids of Antibody heavy chains and amines on the glass surfaces.

Hyaluronic acid (HA) and hyaluronidase have been implicated in cancers. The level of HA and hyaluronidase in cancer cells is higher than those in normal cells. The exact measurement of HA concentration and hyaluronidase activity, therefore, can be a potential diagnostic tool to detect cancers. The conventional methods to determine the amount of HA and hyaluronidase activity are time-consuming and requiring expensive instruments. In this study, a novel method to measure HA concentration and hyaluronidase activity was developed on the basis of converging bio- and nano-materials. Briefly, HA was conjugated to gold nanoparticles (AuNPs) to employ the color-changing property of AuNPs depending on interparticle distance. Thus, the AuNPs with amino surface (amino-AuNPs) easily formed AuNPs-HA complex through ionic interaction upon simple mixing the two component solutions. The formation of AuNPs-HA complex resulted in a color change of the solution depending on the HA concentration. The color change was easily detected by naked eyes or by UV/VIS spectrophotometer for more precise detection. In addition, the aggregation of amino-AuNPs caused by HA was also detected by transmission electron microscope (TEM). This phenomenon was also applied to determine the hyaluronidase activity. The details will be presented in the conference.

We report a micropump-actuated microfluidic platform based on pneumatically-controlled valves implementing molecular logic gates1 of fluorescent chemosensors which shows different intensities responding to different pH and ion conditions. The polydimethylsiloxane (PDMS) microfluidic device was fabricated by multilayer soft lithography2. As results, INHIBIT, a half adder, and negative INHIBIT logic gates have been achieved. These results suggest that our molecular logic gate platform can be considered as an important progress towards a molecular computing system in the future.

Olfactory receptors (ORs) are the largest member of the G-protein-coupled receptors which mediated the early phase of the olfactory perception in discriminating among thousands of odorant molecules. However, very few ORs have been characterized from human origin. Consequently, the specificity and functional properties of individual OR still remains poorly understood, especially in humans. In this study, human olfactory receptor (hOR) 1-60 was expressed on cell surface of human embryonic kidney (HEK)-293 cells as a fusion protein with rho-tag to its N-terminus for plasma membrane targeting. The interactions between HEK-293 cells expressing hOR1-60 and various odorants elicited the change of Ca2+ ions and the change of Ca2+ ions was detected using ratiometric Ca2+ assay. Using Ca2+ assay, we found that hOR1-60 proteins are activated by esters. These findings will facilitate understanding about combinatorial code of hOR at the molecular level and our approach could be used as a tool for characterizing of other orphan hOR proteins.

It is very critical to develop the surface plasmon resonance (SPR) sensor in order to achieve a severe acute respiratory syndrome (SARS) coronavirus sensor. This work is specifically focused on the development of biosensor system to obtain recombinant proteins with the gold-specific affinity. The strategy is based on the principle that the gold binding polypeptide (GBP) as a fusion partner specifically binds onto the gold surface for immobilizing proteins. The SARS coronavirus envelope was employed as a model protein for the specific immobilization of GBP-fusion protein. The fusion proteins were successfully immobilized on the gold surface and examined by SPR analysis. We also analyzed the immobilization efficiency of intracellular protein on the micro-patterned gold surface. Finally, the GBP-fusion method immobilized proteins onto the gold surface without surface modification and in a bioactive form, which is suitable for studying interactions of protein-protein, and other biomolecules [This work was supported in part by MIC & IITA through IT Leading R&D Support Project.].

Pathogenic bacteria are one of the etiological agent causing infectious diseases, which is serious disease leading up to even death. In this reason, identification of pathogenic bacteria is very important in clinical diagnostics. Recently, DNA microarray-based technology is receiving much attention as a powerful tool for pathogen detection [1]. In this study, we suggest diagnostic microarray for pathogenic bacteria, which selected based on high prevalence and/or difficulty of cultivation. The probes were designed bioinformatic analysis using the sequence of 23S ribosomal DNA (rDNA) and 16S-23S rDNA intergenic spacer region. Our diagnostic microarray showed high sensitivity and specificity. The results of this study suggest that diagnostic DNA microarray facilitates more rapid and accurate identification of clinically predominant pathogenic bacteria causing infectious diseases. [This work was supported by Medigenes Co., Ltd.]

Detection method of Salmonellar typhimurium by binding with the ligands and its binding kinetics were conducted using the Surface Plasmon Resonance (SPR) biosensor. SptreetaTM was used as a SPR biosensor. Two kinds of ligands, such as phage P22 and antibody (rabbit anti Salmonellar typhimurium, polyclonal type), were employed with different concentration. The limit of detecting concentration for the Salmonellar typhimurium was evaluated in relation with the concentration of the ligands. The association rate constants and anffinities between Salmonellar typhimurium and the ligands were measured using the kinetic model by curve-fitting to the SPR sensorgrams

The chemotaxis of C.elegans has been conventionally studied by analyzing their movements toward attractant gradient on agar plates. Since the gradient was generated by diffusion of the attractant through porous agar, its concentration profile is ever changing. Thus, it needs to build consistent chemical gradient to perform accurate chemotaxis. Herein, we describe a PDMS (polydimethyl siloxane) microfluidic device which consists of a gradient-generating portion [1] and array of posts. Posts help worms swimming in water by enhancing their motility via mechanosensational reflex upon their bumping to the posts. Using this device, we were able to observe C. elegans swimming up the attractant gradient or down the repellent gradient. Moreover the device with linear gradient provides a sensitive platform to investigate chemo-response.

A living cell can be properly described as an electrochemical dynamic system [1]. Many electrochemical methods have been used in studying intact living cells. We use the cyclic voltammetric (CV) and Potentiometric Stripping Analysis (PSA) methods to study the viability of Hela cells and the effect of anticancer drugs. HeLa cells were grown on gold patterned on silicon substrate; they wereused as a working electrode. Counter test was performed to confirm the results obtained from CV. The voltammetric behaviors of HeLa cells showed a quasi-reversible process and the peak current showed a linear relationship with cell number. PSA signal was used to detect the cell growth with time; as the culture time increases the area under the peak increase gradually reaches to amaximum value and then decreases sharply. The initial increase of PSA signal with the culture time is due to the proliferation of cells. In cell culture, nutrients are important in maintaining cell viability when the cells are cultured continuously in the CO2 incubator for longer time without any fresh nutrient replenished, the viability of cells can undoubtedly be affected. So the decrease of PSA signal in is related to the decrease of cell viability. Using Hydroxyurea and Cyclophosphamide as anticancer drugs, we observed reduction in peak current by increasing in their concentration. The electrochemical experiments on the cells also were verified in comparison with counter assay test. These results indicate that the voltammetric and PSA methods can be used tostudy the viability of Hela cells and the effect of anticancer drugs. Acknowledgments : This research was supported by Seoul R&BD Program(10816) and by the Nano/Bio science & Technology Program (M10536090001-05N3609-00110) of the Ministry of Science and Technology (MOST) and by the Korea government(MOST) (2006-05374).

The surface plasmon resonance (SPR) imaging technique has been used to characterize thin organic and biopolymer films at metal interfaces in a spatially resolved manner. Because this approach enables the measurement of interactions between unlabeled biological molecules and surface-bound species in an array format, SPR imaging is thought to be well-suited not only for DNA and protein microarrays but also for small-molecule microarrays. The biosensor for detection of small molecules or diagnosis markers had been required high sensitivity. One of the methods for enhancing sensitivity was surface modification to provide bio-adaptable space. In this study, we modified the surface of bare gold chip by using our NHS-SAM for enhancing of detection sensitivity. We immobilized the antibody on NHS surface by covalent bonding. And the target antigen were immobilized on modified surface using microarrayer. We observed images of these spots and compared with image of immobilized proteins on 2D surface. These results demonstrate that SPR imaging is well suited to study biomolecular interactions.

Alzheimer's disease(AD) is a neurodegenerative dementia characterized by memory loss and cognitive impairment. The sensitive and selective detection method is required in order to early definitive diagnosis of AD. Beta-amyloid is very attractive as a potential biomarker because of its natural association with the amyloid plaque. This study was based on measuring the beta-amyloid protein concentration and Scanning Tunneling Microscopy(STM) was studied for ultrasensitive feature measurement. STM is versatile technique to apply biosensor for beta-amyloid, because STM provides a topography and current of the atomic arrangement of a biosurface using gold nanoparticle. Gold nanoparticle immobilized on the surface has different electrical properties, it can be analyzed within nanoscale using STM. In this study, gold(Au) nanoparticle-antibody conjugate was applied and STM current change of biosurface can be utilized as quantitative analysis. Experimental results show that the proposed measurement technique can be applied to the nanoscale investigation with respect to a variety of biosensor for diagnosis, drug discovery and on-site monitoring of Alzheimer's Disease. Acknowledgments : This research was supported by the Nano/Bio science & Technology Program (M10536090001-05N3609-00110) of the Ministry of Science and Technology (MOST), by the Korea goverment (MOST) (2006-05374), and by the Korea Research Foundation Grant funded by the Korean Government(MOEHRD) (KRF-2006-005-J02301)

Au nanoparticles (AuNPs) have been widely used not only as optical labels or ‘weight” labels for the detections of biorecognition events but also an amplifier of surface plasmon resonance biosensors. The intrinsic property of gold nuclei composing of a group of Au atoms to catalyze the reduction of metal ions on the NPs and thereby to enlarge the metallic nanoparticles is employed in different biosensing paths. In a solution containing Au+ ions (e.g. HAuCl4) and the Au clusters, hydrated electrons which are reduced from oxidation of reducers (H2O2, sodium citrate, ascorbic acid, or NaBH4) will be used to reduce the Au+ ion leading to the deposition of Au+ to the Au0 (Au clusters). The reaction will be catalyzed continuously by the Au0 until the Au+ ions and hydrated electrons are exhausted. As a result, the AuNPs will be grown and their optical properties are also changed. If the AuNP nanoclusters are used as probes, the color change will be dependent on amount of analytes, thus give a quantitative monitoring of the analytes. In this study, we incorporate the use of magnetic beads with the nanocrystalline growth to quantify a target protein based on immunoreactions. Prostate specific antigen (PSA) is chosen as the target analyte because of its values in diagnosis of prostate cancer. A double-sandwiched immunoassay is performed by gold-tagged monoclonal PSA antibody-PSA antigen – magnetic bead-tagged polyclonal PSA antibody interactions. After the immunoreactions, the target analytes are preconcentrated and separated by the magnetic beads while the nanogrowth plays a role of colorimetric signal developer. The result shows that this is a very sensitive, robust and excellent strategy to detect biological interactions. PSA antigen is detected at femtomolar level with very high specificity under the presence of undesired proteins of crude samples. Furthermore, the method also shows great potential to detect other biological interactions. More details will be described in our presentation.

In this work, we first report self-assembly of insulin monomers into amyloid fibrils on a solid surface within microchannel by using microfluidics. To demonstrate amyloid formation and growth on a solid surface, we prepared NHS-activated glass and PDMS film embossed with a microchannel. According to the analysis with optical and fluorescence microscope, amyloid aggregates within microchannels were found to be increased with the duration of incubation due to the continuous flow of insulin monomers. We confirmed numerous amyloid fibrils grown inside microchannels through AFM analysis and the density of fibrils was much higher than those formed in other bulk systems. We observed the formation of spherulites showing “maltese cross” patterns inside the microchannel after 12 h. These results indicate that the microfluidic self-assembly into amyloid fibrils enables the low consumption of reagents, reduction in incubation time for amyloid formation, and a simultaneous detection. The microfluidic system for amyloid formation on the surface has a potential contribution in the field of study of the amyloidogenic peptides that are responsible for different neurodegenerative disease.

We present a simple and efficient method for specificity improvement of SNPs detection that combines PNA microarray with single-stranded DNA specific nuclease (S1 nuclease). When DNA is perfectly matched with PNA, the DNA/PNA duplexes are not digested by S1 nuclease as the PNA protects the DNA from the nuclease. However, DNA/PNA duplexes including one-base mismatch is efficiently hydrolyzed by the nuclease. This phenomenon was applied to the PNA microarray for single nucleotide polymorphisms (SNPs) detection. With Cy3-labeled target oligonucleotide hybridization to PNA microarray, non specific binding to mismatched PNA probe shows unexpected fluorescent intensity causing low specificity. Though, after treatment of S1 nuclease on PNA microarray, the intensity ratio between perfect matched and one-base mismatched case sharply increased. Therefore, we expect that combining PNA microarray with S1 nuclease will be a powerful tool in the development of accurate and high throughput SNPs detection.

An electrochemical method for glycated hemoglobin (HbA1c) analysis was developed. Gold electrodes was functionalized with boronic acid to prepare HbA1c sensitive surfaces. Boronic acid is known to form covalent bonds between its diol group and cis-diol group of the carbohydrate chain from glycoproteins. In order to modify boronic acid on electrode surface, a conjugation reaction of 3-formylphenyl boronic acid and cystamine were performed (Cys-FPBA). Glycated hemoglobin and glucose oxidase were immobilized onto the boronic acid modified electrode through its carbohydrate moiety under the competition reaction format. With increasing concentration of HbA1c from 2.5% to 15 % of total hemoglobin, a linear signal increase was registered. The limit of detection (LOD) is around 4%. Using this method, HbA1c could be analysed without pre-treatments and detection labels. It is not only expected to analyze HbA1c according to its concentration, but also a variety of glycoproteins that contain peripheral sugar groups could be applied.

We have developed a biosensing strategy for glycated protein in red blood cells. Glycated hemoglobin (HbA1c) is recognized as an important target molecule to improve diabetes control and treatment. HbA1C is made by nonenzymatic reaction between glucose and the N-terminal valine of the β-chain of hemoglobin. We selected ferrocene boronic acid (FcBA) as an electrochemical signaling molecule. Boronic acid has an ability to form a covalent bond between its diol group and the cis-diol group from the carbohydrate chain on the surface of HbA1c. FcBA was conjugated with HbA1c, and the conjugate was collected and purified by precipitated with zinc chloride. After the solution-phase step, we used cyclic voltammetry for the analytical signal registration. Detection range obtained for HbA1c was 5∼20% (HbA1c/total Hb), which covers the clinical reference range. From this simple method, we were able to obtain clinically meaningful sensitivity as well as detection range. This study can be implemented in the diagnostic biosensor and would be applicable to other glycoproteins having analytical needs.

An application of a polymer microfluidic chip for immuno-analysis is described. The microfluidic device was designed to achieve sample replacement by natural capillary force only, which would be suitable for point-of-care-testing. Complete and automatic replacement of the sample in the reaction chamber with another one makes the chip able to mimic affinity chromatography and immunoassay processes. The microfluidic chip was made using polymer replication techniques, which were suitable for fast and cheap fabrication. Dinitrophenyl (DNP) and anti-DNP antibody coordination was employed on the chip for fluorescence analysis. Fluorescein tagged anti-DNP was successfully observed by a fluorescence microscope after the completion of the entire flow sequence. A multiplex sensing was accomplished by adding biotin/streptavidin coordination to the system. DNP and biotin conjugated beads were placed in the reaction chamber in an ordered fashion and biospecific bindings of anti-DNP antibody and streptavidin were observed at their expected sites. A ratiometric analysis was carried out with different concentration ratios of anti-DNP/streptavidin.

LAL (Limulus amebocyte lysate) test is used to detect bacterial endotoxin or LPS (lipopolysaccharide), a membrane component, of gram negative bacteria. There are 3 methods available for conventional testing: gel clotting, KTA (kinetic turbidimetric assay) and KCA (kinetic chromogenic assay). These require long incubation and expensive reagents. And LOC (lab-on-a-chip) is one of the solutions. We evaluated the assays' feasibility in terms of reducing assay time and volume. Among the 3, gel clotting was excluded because its result was too rough to be quantitative. The volume of LAL and CSE (control standard endotoxin) mixture was from 50 to 200 μl, and its final concentration was from 0.05 to 5 EU/ml. The test was triplicated. 96-well plate reader read the absorbance of a well every 3 min during 60±1 min at 340 nm for KTA, and 405 nm for KCA with 37±1°C. Absorbance per light path length of each reaction volume was calculated. Absorbance controlled regression analysis showed the coefficient of determination above 0.98. These data suggested the assay time was shortened by 1/2 hr than the conventional methods and the LOC could distinguish various concentration of samples.

BSA is normally used as a blocking agent in microarrays. However, it will cause the immobilized peptides on a chip to become slightly interactive with macromolecules such as enzymes and antibodies. We developed a recombinant protein-fused substrate (PFS) which can be constantly produced at a reasonable quantity with a small outlay. Using the mep45 gene encoding the 45 kDa major envelope protein (Mep45) of Selenomonas ruminantium, we cloned a protein-fused substrate (AAKIQASFRGHMARKK-Mep45) for protein kinase C (PKC). In the present study, we describe a suitable strategy for the detection of phosphorylation of a PFS catalysed by PKC by using a powerful radioisotope detection technique in protein microarrays. Further, it can be a useful tool for a high throughput screening and for biological and medical research.

Rapid detection of food-borne bacterial pathogens is desperately needed in clinical, food safety and. environmental field. In this study, we developed a rational method to evaluate the diagnostic ability by using a fluorescence labeled colony-PCR (FLC-PCR) that is possible for simultaneous detection of 16S ribosomal DNA of multiple bacterial species. The diagnostic ability of the Fluorescent (Cyanine 5) labeled colony polymerase chain reaction (FLC-PCR) based DNA microarray method was evaluated. The PCR frgments were carried out successfully, and these genes were differentiated by the heterologous region of their PCR products in same region of bacterial 16s rDNA sequence. The 8 species-specific capture probes (Eco2, Eco3, Lisn2, Lis3, Clo3, StaA, Vib1, and Vc1, 5'-Amine labelled) were designed from 16S rDNA genes encoding food borne bacterial genomic DNA of different six pathogens (Escherichia coli O157:H7, Vibrio cholera, Vibrio parahaemolyticus, Listeria monocytogenes, Clostridium perfringens, and Staphylococcus aureus). The FLC-PCR frgments (302 bp) of 6 pathogens were hybridized 1 hour (60℃) to immobilize speciesspecific and universal probes on microarray. The fragment sizes of PCR products were identical to the sizes calculated from nucleotide sequences of 16s rRNA deposited in EMBL, GenBank, and/or DDBJ databases, and the sequences were also identical. In conclusions, the method and newly designed primers applied in this work can differentiate the Pathogen’s 16s rRNA sequence very fast (< 3 hours) and effectively, without liquid cultivation and purified DNA.

Autonomous bottom-up fabrication based on molecular recognition1 is a conceptually attractive and potentially powerful approach to engineering structures and devices at the molecular scale. DNA is a versatile construction material that can be programmed to self-assemble into nanoscale structures based on specific complementarity between bases.2-4 Here, our research effort aims at developing bio-inspired nanomaterials capable of detecting single nucleotide polymorphism (SNP). About 0.1% single nucleotide variation is observed in human population, leading to challenges in achieving effective medical treatment of different individuals. Considering the importance of SNP in individual drug efficacy, hugh research efforts have been focused on coming up with fast and convenient SNP detection methods. We developed a novel SNP biosensor material made of DNA-programmed protein nanostructures of which disassembly of the whole structure is triggered by a target DNA oligonucleotide carrying a specific sequence. We designed DNA sequences incorporated in the sensor materials in a way that even a single base mutation in the target DNA (e.g. a single mutation in a 24-mer DNA oligonucleotide) effectively prevents the protein nanostructures from being disassembled. We could detect SNP by monitoring structural changes in the protein nanostructure. The SNP biosensor material is promising for fast identification of SNP in genes of interest, which enables patient-tailored drug theraphy.

This study describes formation of Poly(ethylene glycol) (PEG) hydrogel nanofibers via electrospinning, which can be used as a scaffold for 3-dimensional cell culture system. Currently, most of in vitro cell-based assays for drug screening and toxicity evaluation were performed using cells adhered on the 2-dimensional substrates. However, in a 2-dimensional system, cells are in an unnatural environment; I.e., in tissue they exist in a 3-dimensional hydrogel nanofiber matrix consisting of proteins and polysaccharides (i.e., the extracellular matrix). As a result, the response of these cells to drug candidates may be very different than that of the same cells in their native tissue. To overcome thes problems and mimic the native tissue, PEG hydrogel nanofiber was prepared by electrospinning PEG precursor solution containing poly(ethylene oxide) (PEO) and thermal or photo-initiators. Without PEO, electrospinning of PEG solution produced microsphere instead of fiber due to low viscosity. Addition of high molecular weight of PEO made it possible to create nanofiber and resultant nanofiber was crosslinked thermally or photochemically to prevent dissolution of nanofiber in aqueous environment. Formation of PEG hydrogel nanofiber was confirmed using SEM and other characterization including water content and mechanical strength were carried out varying the molecular weight of PEG.

The activation of caspase-3 plays an important role in the apoptotic process. In this study, we describe a novel method by which caspase-3-dependent proteolytic cleavage can be monitored, using a surface plasmon resonance (SPR) imaging protein chip system. To the best of our knowledge, this is the first report regarding the SPR imaging-based monitoring of caspase-3 activation. In order to evaluate the performance of this protocol, we constructed a chimeric caspase-3 substrate (GST:DEVD:EGFP) comprised of glutathione-S transferase (GST) and enhanced green fluorescent protein (EGFP) with a specialized linker peptide harboring the caspase-3 cleavage sequence, DEVD. Using this reporter, we assessed the cleavage of the artificial caspase-3 substrate in response to caspase-3 using an SPR imaging sensor. The purified GST:DEVD:EGFP protein was initially immobilized onto a glutathionylated gold chip surface, and subsequently analyzed using an SPR imaging system. As a result, caspase-3 activation predicated on the proteolytic properties inherent to substrate specificity could be monitored via an SPR imaging system with a detection performance similar to that achievable by the conventional method, including fluorometric assays. Collectively, our data showed that SPR imaging protein chip system can be effectively utilized to monitor the proteolytic cleavage in caspase-3, thereby potentially enabling the detection of other intracellular protease activation via the alteration of the protease recognition site in the linker peptides.

Here we describe an n-type ion sensitive field effect transistor (n-type ISFET) biosensor, which is designed to directly monitor the surface charge of structurally altered maltose binding protein (MBP) upon stimulation with maltose. To our best knowledge, this study reports the first application of FET biosensor for the monitoring of conformationally changed protein. In this work, silicon dioxide (SiO2) has been used as the a material of the ISFET device, and Pt reference electrode employed for detecting the variations of surface charge in MBP. Histidine-tagged MBP was immobilized onto a NTA-Ni(II)-coated sensing layer (SiO2), subsequently treated with maltose to induce conformational changes in MBP. As a result, a significant decrease in drain current of ISFET device has been clearly observed in response to maltose, but not glucose as a control, indicating that substrate-specific conformational properties of target protein could be successfully monitored using ISFET biosensor. Collectively, our data strongly demonstrated that ISFET biosensor provides a high fidelity system for the detection of maltose-induced structural alterations in MBP by analyzing the changes in drain current based on the charge-dependent capacitance measurement.

The apoptotic caspases have been classified according to their substrate specificities, as the optimal tetrapeptide recognition motifs for various caspases have been determined by the positional scanning substrate combinatorial library technology. Here we chose to focus on two proteolytic recognition motifs, DEVD and IETD, due to their wide use in cell-based apoptosis assay. Although, DEVE and IETD have been considered selective for caspase-3 and -8, respectively, the proteolytic cleavage of these substrates does not display absolute specificity for a particular caspase. Thus, this work for the non-specificity of the substrates was achieved with caspase-3 via evaluating the difference in proteolytic cleavage between DEVD- and IETD-based substrates. To design a rigorous protocol for this aim, we developed the DEVD- and IETD-based substrate, which are referred to as the GST:DEVD:EGFP and GST:IETD:EGFP, respectively. Using these substrates, the efficiency of caspase-3-induced proteolytic cleavages could be compared via the mass-based (Western blotting and SPR imaging), and fluorescence-based (on-chip visualization and bead assay) methods. Consequently, the GST:IETD:EGFP was partially cleaved in response to caspase-3, whereas GST:DEVD:EGFP completely proteolysed, indicating that GST:DEVD:EGFP is a better substrate than GST:IETD:EGFP for caspase-3. Therefore, our results revealed that both GST:DEVD:EGFP and GST:IETD:EGFP substrates proved cleavable to caspase-3, although the GST:DEVD:EGFP substrate is more appropriate than the GST:IETD:EGFP substrate in favor of the proteolytic cleavage for caspase-3, which may potentially help to develop more rigorous caspase assay system for the measurement of caspase-3 activation during apoptosis.

Valiolamine is an important chemical intermediate for the synthesis of voglibose, antidiabetic medicine and has been also isolated from the fermentation broth of Streptomyces hygroscopicus subsp.. Nowadays, intensive effort has been made to produce valiolamine by the fermentation of Streptomyces cells using metabolic engineering tool. Quantitative analysis of valiolamine is crucial for quality control of voglibose production process or monitoring the fermentation process to produce valiolamine. Valiolamine has no significant UV absorption and thus derivatization by chromophoric reagents can increase the detection sensitivity. This study was aimed to develop a rapid, accurate and precise HPLC method for UV detection of valiolamine following pre-column derivatization. The performance of several derivatization methods were evaluated for valiolamine analysis. The electrospray ionization-mass (ESI-MS) analysis was performed to identify the new peak after derivatization. The experimental conditions for derivatization and separation of valiolamine derivatives were optimized to increase sensitivity.

Currently, there are many reports in the literature regarding technological methods for paclitaxel purification. However, there have been few reports on the separation of paclitaxel using a micellar system. This method is based on the transfer of paclitaxel within the crude extract to an aqueous surfactant solution as a micelle, allowing the use of organic solvents to be used for the removal of lipids and non-polar impurities. In this study, we optimized the important process parameters of micellar extraction to obtain a high purity and yield of paclitaxel in a pre-purification step. The optimal surfactant (N-cetylpyridinium chloride, CPC) concentration, initial crude paclitaxel concentration, organic solvents (methyl t-butyl ether/hexane) ratio, extraction temperature, and extraction time were 7.5% (w/v), 16.4 mg/mL, 1.5/1 (v/v), 25°C, and 30 min, respectively. The crude extracts from the liquid-liquid extracts were efficiently pre-purified by micellar extraction. Overall, the use of micellar extraction in the pre-purification process allowed for rapid and efficient separation of paclitaxel from interfering compounds, and dramatically increased the yield and purity of the crude paclitaxel for subsequent purification steps.

Magnetic particles (MPs) carrying an immobilized affinity ligand have been successfully used in the purification and separation of several biomolecules from biological mixtures such as crude cell lysates, whole blood, etc [1]. In these techniques, there is no need for a prior step such as centrifugation and filtration for solids removal from the suspensions [2]. So the advantages include separation efficiency, simplicity, low cost, and easy scale-up ability and automation. Hepatitis B surface antigen (HBsAg) and anti-HBsAg antibody were the model ligand and capturing molecules for this study. Anti-HBsAg antibody is used for treatment of Hepatitis B virus (HBV) and detection of HBsAg in the serum as a immunosensor [3]. 255 umol of HBsAg was immobilized by covalently linking onto amine-coated 1 mg of MPs. Molar ratio of immobilized HBsAg to bound anti-HBsAg antibody was 0.58, and these HBsAg-immobilized MPs can be used to purify anti-HBsAg antibody from mixtures including impurities. This method is expected to be highly cost-effective as a single-step purification of various antibodies or proteins.

The use of L-carbohydrates and their corresponding nucleosides in medicinal application has greatly increased. For example L-ribose has been much in demand as the starting material for curing hepatitis B. SMB was invented by UOP in the early 1960s, and the SMB utilized the movement of ports or columns to mimic a counter-current movement of liquid and solid phase. The classical closed-loop 4-zone SMB approach reduces solvent consumption, but there can be occurred a risk of cross-contamination or mechanical demage in lines due to high pressure. In the open-loop 3-zone SMB, the recycle line of eluent be removed and the SMB consumes more solvent. However it can reduce the risk of cross-contamination or mechanical demage compared to the 4-zone SMB. In this study, we carried out simulations of the separation of L-ribose and L-arabinose in 3-zone SMB for the operating points in the so-called m2-m3 plane from triangle theory using Aspen chromatography.