Synthesis, oxygen radical absorbance capacity, and tyrosinase inhibitory activity of glycosides of resveratrol, pterostilbene, and pinostilbene.

The stilbene compound resveratrol was glycosylated to give its 4'-O-ß-D-glucoside as the major product in addition to its 3-O-ß-D-glucoside by a plant glucosyltransferase from Phytolacca americana expressed in recombinant Escherichia coli. This enzyme transformed pterostilbene to its 4'-O-ß-D-glucoside, and converted pinostilbene to its 4'-O-ß-D-glucoside as a major product and its 3-O-ß-D-glucoside as a minor product. An analysis of antioxidant capacity showed that the above stilbene glycosides had lower oxygen radical absorbance capacity (ORAC) values than those of the corresponding stilbene aglycones. The 3-O-ß-D-glucoside of resveratrol showed the highest ORAC value among the stilbene glycosides tested, and pinostilbene had the highest value among the stilbene compounds. The tyrosinase inhibitory activities of the stilbene aglycones were improved by glycosylation; the stilbene glycosides had higher activities than the stilbene aglycones. Resveratrol 3-O-ß-D-glucoside had the highest tyrosinase inhibitory activity among the stilbene compounds tested.

Electrochemical biosensors for biocontaminant detection consisting of carbon nanotubes, platinum nanoparticles, dendrimers, and enzymes.

The presented approach provides the advanced development of effective, rapid, and versatile electrochemical sensors for a small amount of analytes on potential, cheap, and disposable printed chips. The electrocatalytic activity of this biosensor revealed the feasible detection of hydrogen peroxide at low potential (~0.09 V) and the detection of a biocontaminant inhibitor (organophosphorus pesticide) in a wide range of concentrations. This efficiency comes from the chemical immobilization of catalysts (Pt nanoparticles) and electron transfer-enlarging materials (carbon nanotubes) on an electrode. Especially, dendrimers raise the stable conjugation of enzymes (acetylcholinesterase/choline oxidase/peroxidase) as well as nanoparticles and carbon nanotubes on an electrode.

Detection of influenza virus using a lateral flow immunoassay for amplified DNA by a microfluidic RT-PCR chip.

Influenza virus RNA was amplified by a continuous-flow polydimethylsiloxane microfluidic RT-PCR chip within 15-20 min. The amplified influenza virus RNA was observed with the naked eye, as the red color at the test line, using a lateral flow immunoassay within 1 min.

Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat.

In this paper, the semi-real time electrochemical monitoring method using a screen-printed electrode, which employs reverse transcription loop-mediated isothermal amplification (RT-LAMP) for influenza virus RNA, is presented. The amplified DNA combined with methylene blue (MB), which was used as an electroactive DNA intercalator, and the electrochemical signal was monitored using square wave voltammetry in the presence of RT-LAMP reagent components. MB molecules binding to amplified DNA caused the reduction of the peak current due to the slow diffusion of MB-amplified DNA complex to the electrode surface. We successfully monitored the amplification process of DNA on the basis of RT-LAMP by measuring and analyzing the electrochemical signal of MB with only one screen-printed electrode that connected with a USB powered portable potentiostat. The peak height of the current was related to the extent of amplification of DNA and the amount of input RNA. Since laborious probe immobilization is not required and both the amplification and the monitoring are possible in a single tube, our method does not suffer from potential cross-contamination. Furthermore, our method provides a new rote for the development of electrochemical hand held biosensors.

A rapid gel electrophoretic chip for serum cholesterol determination.

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.

Rapid detection for primary screening of influenza A virus: microfluidic RT-PCR chip and electrochemical DNA sensor.

Rapid and definitive diagnosis is critical to the prevention of the spread of endemic human pathogenic viruses. Detection of variant specific genes by reverse transcription polymerase chain reaction (RT-PCR) has become a routine diagnostic test for accurate subtyping of RNA viruses, such as influenza. In this paper, we demonstrate the use of a continuous-flow polydimethylsiloxane (PDMS) microfluidic RT-PCR chip and disposable electrical printed (DEP) chips for rapid amplification and sensing of new influenza (AH1pdm) virus of swine-origin. The RT-PCR chip consisted of four zones: RT reaction zone, initial denaturation zone, thermal cycle zone for PCR (2-step PCR) and pressurizing-channel zone for preventing air-bubble formation. In order to measure electrochemical signals, methylene blue (MB), an electro-active DNA intercalator, was added to the RT-PCR mixture. The RT-PCR was completed within 15 min which was the total flow-through time from the inlet to the outlet, and the reduction signals from amplifications could be detected quickly on the DEP chip. The MB reduction current on the DEP chip with the amplicon significantly reduced compared to non-amplified controls. This microfluidic platform for rapid RT-PCR and the DEP chip for quick electrochemical sensing are suitable for integration, and have the potential to be a portable system for diagnostic tests.

Sensors.

With recent advances in nanotechnology, development of nanomaterial bioconjugates is growing exponentially towards eventual translation into biomolecular recognition layers on surfaces. Label-free monitoring of biorecognition events is also key-technology and provides a promising platform, which is simple, cost-effective, and requires no external modification to biomolecules. In this review, we describe the application of nanomaterials, mainly metal nanoparticles, and specific applications of carbon nanotubes (CNTs) based label-free approaches.

Cell separation by an aqueous two-phase system in a microfluidic device.

We generated an aqueous two-phase laminar flow in a microfluidic chip and used the system to isolate leukocyte and erythrocyte cells from whole blood cells. The microfluidic system reduced the effect of gravity in the aqueous two-phase system (ATPS). Poly(ethylene glycol) (PEG) and dextran (Dex) solutions were used as the two phases, and the independent flow rates of the solutions were both 2 microL/min. When hydrophobic and hydrophilic polystyrene beads were introduced into the microfluidic device, the hydrophilic beads moved to the Dex layer and the hydrophobic beads to the interface between the two phases. In the case of living cells, Jurkat cells and erythrocytes moved more efficiently to the PEG and Dex layers, respectively, than they move in a conventional ATPS. When whole blood cells were inserted into the microfluidic chip, leukocytes could be separated from erythrocytes because erythrocytes moved to the Dex layer while leukocytes remained outside of this layer in the microfluidic system. The reported microfluidic chip for the whole blood cell separation can effectively be integrated into a Micro Total Analysis System designed for cell-based clinical, forensic, and environmental analyses.

Immunochromatographic assay using gold nanoparticles for measuring salivary secretory IgA in dogs as a stress marker.

The concentration of salivary secretory immunoglobulin A (sIgA) is a well-known stress marker for humans. The concentration of salivary sIgA in dogs has also been reported as a useful stress marker. In addition, salivary sIgA in dogs has been used to determine the adaptive ability of dogs for further training. There are conventional procedures based on enzyme-linked immunosorbent assay (ELISA) for measuring salivary sIgA in dogs. However, ELISA requires long assay time, complicated operations and is costly. In the present study, we developed an immunochromatographic assay for measuring salivary sIgA in dogs using a dilution buffer containing a non-ionic surfactant. We determined 2500-fold dilution as the optimum condition for dog saliva using a phosphate buffer (50 mM, pH 7.2) containing non-ionic surfactant (3 wt% Tween 20). The results obtained from the saliva samples of three dogs using immunochromatographic assay were compared with those obtained from ELISA. It was found that the immunochromatographic assay is applicable to judge the change in salivary sIgA in each dog. The immunochromatographic assay for salivary sIgA in dogs is a promising tool, which should soon become commercially available for predicting a dog's psychological condition and estimating adaptive ability for training as guide or police dogs.

Mechanism of destruction of microtubule structures by 4-hydroxy-2-nonenal.

A major end product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is an electrophilic alkenal and produces Michael adducts with cellular proteins. It is known that exposure of cultured cells to HNE causes rapid disappearance of microtubule networks. In this study we addressed the mechanism. Immunochemical studies revealed that HNE preferentially modified alpha-tubulin in rat primary neuronal cells, PC12 cells, and rat fibroblast cell line 3Y1 cells. This was morphologically associated with the disappearance of microtubule structures in those cells. In a purified rat brain microtubule fraction, HNE modified unpolymerized tubulin and impaired its polymerizability, with a concomitant increase in insolubilized tubulin. Nevertheless, HNE had a marginal effect on the stability of pre-polymerized microtubules. These results suggest that disruption of microtubule assembly as a result of HNE modification of unpolymerized tubulin, rather than destruction of assembled microtubules, is responsible for the disappearance of microtubule structures in cells exposed to HNE.

An electrochemical on-field sensor system for the detection of compost maturity.

A maturity sensor system was developed, based on the combination of three electrically measured parameters, pH, NH(4)(+) concentration, and phosphatase activity in the water extracts of compost samples. One of these parameters, the apparent phosphatase activity in crude test solutions was determined using screen-printed carbon strips (SPCSs) coated with alpha-naphthyl phosphate (alpha-NP) in Nafion film. The phosphatase activity was monitored in connection with differential pulse voltammetry (DPV) with an aliquot (30 microL) of the test solution on SPCS. The phosphatase activity sensor was validated using alkaline phosphatase (ALP) in Tris-HCl buffer (pH 8.0) and acid phosphatase (ACP) in citric acid buffer (pH 5.0). The activity of the spiked enzymes in the water extract of the compost sample could be confirmed with the change of corresponding oxidation peak current signal of the product, alpha-naphthol. The water extracts of compost samples (n=24) collected in various composting days were applied to our compost maturity sensor system, and the conventional germination tests. Using multiple regression analysis, the germination index (GI) was expressed by the multi-linear regression equation consisting of pH, NH(4)(+) concentration, and the phosphatase activity. The calculated GI from the regression equation had a good correlation with the measured GI of the corresponding composts (r=0.873). As a result, we have determined an equation for the determination of the compost stability using our portable sensor system rapidly at the composting site.

Multiple label-free detection of antigen-antibody reaction using localized surface plasmon resonance-based core-shell structured nanoparticle layer nanochip.

In this research, a localized surface plasmon resonance (LSPR)-based bioanalysis method for developing multiarray optical nanochip suitable for screening bimolecular interactions is described. LSPR-based label-free monitoring enables to solve the problems of conventional methods that require large sample volumes and time-consuming labeling procedures. We developed a multiarray LSPR-based nanochip for the label-free detection of proteins. The multiarray format was constructed by a core-shell-structured nanoparticle layer, which provided 300 nanospots on the sensing surface. Antibodies were immobilized onto the nanospots using their interaction with Protein A. The concentrations of antigens were determined from the peak absorption intensity of the LSPR spectra. We demonstrated the capability of the array measurement using immunoglobulins (IgA, IgD, IgG, IgM), C-reactive protein, and fibrinogen. The detection limit of our label-free method was 100 pg/mL. Our nanochip is readily transferable to monitor the interactions of other biomolecules, such as whole cells or receptors, with a massively parallel detection capability in a highly miniaturized package. We anticipate that the direct label-free optical immunoassay of proteins reported here will revolutionize clinical diagnosis and accelerate the development of hand-held and user-friendly point-of-care devices.

Label-free electrochemical immunoassay for the detection of human chorionic gonadotropin hormone.

Here we report on a new and rapid immunoassay for the label-free voltammetric detection of human chorionic gonadotropin hormone (hCG) in urine. Monitoring the changes in the current signals of antibodies (Abs) before and after the binding of the antigen (Ag) provides the basis for an immunoassay that is simple, rapid, and cost-effective. Since hCG is found at highly elevated levels in pregnant female urine with the range of 30,000-200,000 mIU/mL (approximately 30-200 nM) by 8-10 weeks into pregnancy, its label-free electrochemical detection was achieved by using our method. The coverage of the electrode surface with the Ab and the incubation time with the target Ag were optimized for the detection of hCG. The limit of detection of our method was calculated to be 15 pM (n = 3, approximately 15 mIU/mL) in synthetic hCG samples and 20 pM (n = 3, approximately 20 mIU/mL) in human urine. The electrochemical results for the detection of hCG in the urine samples were in agreement with the results obtained using a reference system, enzyme-linked immunosorbent assay. Further research about the intrinsic electroactivity of Abs and their target molecules would surely provide new and sensitive screening assays, as well as extensive data regarding their interaction mechanisms.

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles.

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.

Resin-based micropipette tip for immunochromatographic assays in urine samples.

A novel bioanalysis system based on immunochromatography was developed in connection with a nitrocellulose resin modified micropipette tip, such as ZipTip. The sandwich-type immunoassay was applied to our bioanalysis system. The simple handling of the micropipette enabled us to increase the sample volume and detect low concentrations of target antigens in urine samples. In addition, the washing procedure could also be performed easily to reduce the background signal levels. For analytical evaluations, the color intensity was captured by a flatbed scanner, and processed by a software. We have achieved the detection of human chorionic gonadotropin (hCG) and prostate-specific antigen (PSA). The detection limit of hCG was 0.5 ng/ml (0.05 ng/tip), which is comparable to that of other conventional immunochromatographic systems. Moreover, the detection of PSA was greatly improved over the existing systems with the application of different sample volumes, such as 1 ng/ml (0.2 ng/tip) in a 200 microl sample volume, and 1 ng/ml (0.3 ng/tip) in 300 microl sample volume. Our bioanalysis system is a promising candidate for application to point-of-care tests with its simple handling and high sensitivity.

Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The attachment of long single-strands on GCE surface caused the accumulation of [Co(NH3)6]3+ and a high current response. Here, we report a versatile method that would allow for simple and rapid analysis of nucleic acids in combination with PNA-mediated PCR and asymmetric PCR techniques by using an electrochemical genosensor.

Label-free detection of peptide nucleic acid-DNA hybridization using localized surface plasmon resonance based optical biosensor.

The development of label-free optical biosensors for DNA and other biomolecules has the potential to impact life sciences as well as screening in medical and environmental applications. In this report, we developed a localized surface plasmon resonance (LSPR) based label-free optical biosensor based on a gold-capped nanoparticle layer substrate immobilized with peptide nucleic acids (PNAs). PNA probe was designed to recognize the target DNA related to tumor necrosis factor. The nanoparticle layer was formed on a gold-deposited glass substrate by the surface modified silica nanoparticles using silane-coupling reagent. The optical properties of gold-capped nanoparticle layer substrate were characterized through monitoring the changes in the absorbance strength, as the thickness of the biomolecular layer increased with hybridization. The detection of PNA-DNA hybridization with target oligonucleotides and PCR-amplified real samples were performed with a limit of detection value of 0.677 pM target DNA. Selective discrimination against a single-base mismatch was also achieved. Our LSPR-based biosensor with the gold-capped nanoparticle layer substrate is applicable to the design of biosensors for monitoring of the interaction of other biomolecules, such as proteins, whole cells, or receptors with a massively parallel detection capability in a highly miniaturized package.

A rapid label-free electrochemical detection and kinetic study of Alzheimer's amyloid beta aggregation.

We present the first electrochemical detection, characterization, and kinetic study of the aggregation of Alzheimer's disease (AD) amyloid beta peptides (Abeta-40, Abeta-42) using three different voltammetric techniques at a glassy carbon electrode (GCE). This method is based on detecting changes in the oxidation signal of tyrosine (Tyr) residue. As the peptides aggregate, there are structure conformational changes, which affect the degree of exposure of Tyr to the molecular surface of the peptides. The results show significant differences in the aggregation process between the two peptides, and these correlate highly with established techniques. The method is rapid and label-free, and the principle can be universally applied to other protein aggregation studies related to diseases, such as Huntington's, Parkinson's, and Creutzfeldt Jacob (CJD). This method could also be explored in screening for the effectiveness of AD therapies.

On-chip micro-flow polystyrene bead-based immunoassay for quantitative detection of tacrolimus (FK506).

Tacrolimus (FK506) is a widely used immunosuppressant for preventing allograft rejection and the treatment of atopic dermatitis. FK506 necessitates therapeutic drug monitoring because of inter- and intrapatient variability and the lack of correlation between the administered dose and the blood concentration. Previous immunoassay-based methods required a relatively long assay time and troublesome liquid-handling procedures. In the present study, we aimed to establish a rapid monitoring method for FK506 determination by using a poly(dimethylsiloxane) (PDMS)-based microfluidic device. Polystyrene beads were coated with mouse anti-FK506 antibody and placed in the flow channel. As a competitive assay, sample solution was allowed to react in the flow channel. After the addition of the fluorogenic substrate, the fluorescent signal was observed under a microscope. As a result, the developed assay allowed a short detection time of approximately 15 min per each sample and a high sensitivity even by using only a single bead. The feasibility of performing a competitive assay using a PDMS-based antibody chip gives promising results over the existing immunoassay-based methods.

Reduction of alpha-galactosyl xenoantigen by expression of endo-beta-galactosidase C in pig endothelial cells.

BACKGROUND: Elimination of the Galalpha1-3Galbeta1-4GlcNAc (alphaGal) epitope has been considered to be essential for successful pig-to-human xenotransplantation but, unfortunately, has not been achieved. Endo-beta-galactosidase C (EndoGalC) is an endoglycosidase which cleaves the Galbeta1-4GlcNAc linkage in the alphaGal epitope and digests out the Galalpha1-3Gal disaccharide. Because of its potent activity in physiological pH conditions, EndoGalC can remove alphaGal epitopes expressed on the cell surface of pig erythrocytes and vascular endothelial cells almost completely. In vivo or ex vivo administration of EndoGalC successfully reduced alphaGal expression in pig kidneys to an undetectable level, but alphaGal epitopes soon reappeared. Gene expression of EndoGalC in pig cells was attempted to solve this problem. As the terminal alphaGal is transferred in the trans-Golgi network by alpha-1,3-galactosyltransferase (alpha1,3GT), colocalization of the EndoGalC gene with the alpha1,3GT gene was expected to be one of the most reliable ways to eliminate the alphaGal epitope. METHODS AND RESULTS: The sequence of pig alpha1,3GT, including the cytoplasmic tail, transmembrane domain and stem region, was ligated upstream of EndoGalC, and the conjugated gene was expressed in pig aortic endothelial cells and COS7 cells. Following the introduction of the gene, the alphaGal epitope on pig aortic endothelial cells was effectively reduced. Transfection studies in COS7 cells using EndoGalC combined with alpha1,3GT showed that the expressed EndoGalC was localized not only inside, but also outside, the cells. The expression of EndoGalC conjugated with a murine immunoglobulin (Igkappa)-chain signal sequence also showed a similar effect. CONCLUSIONS: These results suggest the effectiveness of gene transfer with EndoGalC into pig endothelial cells, and strongly encourage us to produce transgenic animals with the expressed enzyme.