Application of methyl parathion hydrolase (MPH) as a labeling enzyme

Methyl parathion hydrolase (MPH) is an enzyme that catalyzes the degradation of methyl parathion, generating a yellow product with specific absorption at 405 nm. The application of MPH as a new labeling enzyme was illustrated in this study. The key advantages of using MPH as a labeling enzyme are as follows: (1) unlike alkaline phosphatase (AP), horseradish peroxidase (HRP), and glucose oxidase (GOD), MPH is rarely found in animal cells, and it therefore produces less background noise; (2) its active form in solution is the monomer, with a molecular weight of 37 kDa; (3) its turnover number is 114.70 +/- 13.19 s(-1), which is sufficiently high to yield a significant signal for sensitive detection; and (4) its 3D structure is known and its C-terminal that is exposed to the surface can be easily subjected to the construction of genetic engineering monocloning antibody-enzyme fusion for enzyme-linked immunosorbent assay (ELISA). To demonstrate its utility, MPH was ligated to an single-chain variable fragment (scFv), known as A1E, against a white spot syndrome virus (WSSV) with the insertion of a [-(Gly-Ser)(5)-] linker peptide. The resulting fusion protein MPH-A1E possessed both the binding specificity of the scFv segment and the catalytic activity of the MPH segment. When MPH-A1E was used as an ELISA reagent, 25 ng purified WSSV was detected; this was similar to the detection sensitivity obtained using A1E scFv and the HRP/Anti-E Tag Conjugate protocol. The fusion protein also recognized the WSSV in 1 mu L hemolymph from an infected shrimp and differentiated it from a healthy shrimp.

tilt menu: using the 3d orientation information of pen devices to extend the selection capability of pen-based user interfaces

We present a new technique called‘Tilt Menu’ for better extending selection capabilities of pen-based interfaces.The Tilt Menu is implemented by using 3D orientation information of pen devices while performing selection tasks.The Tilt Menu has the potential to aid traditional onehanded techniques as it simultaneously generates the secondary input (e.g., a command or parameter selection) while drawing/interacting with a pen tip without having to use the second hand or another device. We conduct two experiments to explore the performance of the Tilt Menu. In the first experiment, we analyze the effect of parameters of the Tilt Menu, such as the menu size and orientation of the item, on its usability. Results of the first experiment suggest some design guidelines for the Tilt Menu. In the second experiment, the Tilt Menu is compared to two types of techniques while performing connect-the-dot tasks using freeform drawing mechanism. Results of the second experiment show that the Tilt Menu perform better in comparison to the Tool Palette, and is as good as the Toolglass.

Development of functionalized terbium fluorescent nanoparticles for antibody labeling and time-resolved fluoroimmunoassay application

Silica-based functionalized terbium fluorescent nanoparticles were prepared, characterized and developed as a fluorescence probe for antibody labeling and time-resolved fluoroimmunoassay. The nanoparticles were prepared in a water-in-oil (W/O) microemulsion containing a strongly fluorescent Tb3+ chelate. N,N.N-1,N-1-12,6-bis(3'-aminomethyl-1'-pyrazolyl)phenylpyridine] tetrakis(acetate)-Tb3+ (BPTA-Tb3+), Triton X-100, octanol, and cyclohexane by controlling copolymerization of tetraethyl orthosilicate (TEOS) and 3-[2-(2- aminoethylamino)-ethylamino]propyl-trimethoxysilane (AEPS) with ammonia water. The characterizations by transmission electron microscopy and fluorometric quantum methods show that the nanoparticles are spherical and uniform in size, 45 +/- 3 nm in diameter, strongly fluorescent with fluorescence yield of 10% and a long fluorescence lifetime of 2.0 ms. The amino groups directly introduced to the nanoparticle's surface by using AEPS in the preparation made the surface modification and bioconjugation of the nanoparticles easier. The nanoparticle-labeled anti-human alpha-fetoprotein antibody was prepared and used for time-resolved fluoroimmunoassay of (x-fetoprotein (AFP) in human serum samples. The assay response is linear from 0.10 ng ml(-1) to about 100 ng ml(-1) with the detection limit of 0.10 ng ml(-1). The coefficient variations (CVs) of the method are less than 9.0%. and the recoveries are in the range of 84-98% for human serum sample measurements. (C) 2004 Elsevier B.V. All rights reserved.

[Ru(bpy)(2)(dcbpy)NHS] Labeling/Aptamer-Based Biosensor for the Detection of Lysozyme by Increasing Sensitivity with Gold Nanoparticle Amplification

A novel [Ru(bpy)(2) (dcbpy)NHS] labeling/aptamer-based biosensor combined with gold nanoparticle amplification for the determination of lysozyme with an electrochemiluminescence (ECL) method is presented. In this work, an aptamer, an ECL probe, gold nanoparticle amplification, and competition assay are the main protocols employed in ECL detection. With all the protocols used, an original biosensor coupled with an aptamer and [Ru(bpy)(2)(dcbpy)NHS] has been prepared. Its high selectivity and sensitivity are the main advantages over other traditional [Ru(bpy)(3)](2+) biosensors. The electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) characterization illustrate that this biosensor is fabricated successfully. Finally, the biosensor was applied to a displacement assay in different concentrations of lysozyme solution, and an ultrasensitive ECL signal was obtained. The ECL intensity decreased proportionally to the lysozyme concentration over the range 1.0 x 10-(13)-1.0 x 10(-8) mol L-1 with a detection limit of 1.0 x 10(-13) mol L-1.

Europium ion labeling anti-HBs

A microsecond time-resolved laser fluorescence spectroscopic analysis set was developed, A chelate-cyclic anhydride of diethylenetrimin pentaacetic acid anhydride (DTPAA) was synthesized. An anti-HBs antibody was purified, A EU3+ -DTPAA-anti-HBs label was prepared by two step procedure. We described the optimal condtion with EU3+ as marker and DTPAA as chelate bounding to antibody molecule. Labeling parameters such as solvent pH, protein and chelate molar ratio, reaction time, separation method were discussed in detail.

Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC

Since the discovery of multiple bioactivities for agarobiose oligomers, a quantitative method has been in great need to monitor the agarobiose oligomers. This report demonstrates that agarobiose oligomers can be separated with high resolution in HPLC after introducing a-naphthylamine into compounds. Agarobiose oligomers ranged from biose to decaose were isolated by Sephadex column. HPLC analysis indicated that each oliomer could be quantified with good linearity and a low detection limit of 0.1-4 mug/ml. The chromatographic profiles of agaro-oligosaccharides with different hydrolysis modes (hydrochloride, citric acid, solid acid, and hydroxyl radical degradation) showed that agarobiose could be obtained more than 57.8% using solid acid mediated hydrolysis, while hydrochloride acid could degrade agar into a series of agaro-oligosaccharides from biose to decaose. The yield of oligosaccharides was low if hydrolyzed by citric acid. The Fenton degradation can increase the speed of hydrolysis, but the product was complex. (C) 2004 Elsevier B.V. All rights reserved.

Topographic changes in nascent and early mesoderm in amphioxus embryos studied by Dil labeling and by in situ hybridization for a Brachyury gene

In amphioxus embryos, the nascent and early mesoderm (including chorda-mesoderm) was visualized by expression of a Brachyury gene (AmBra-2). A band of mesoderm is first detected encircling the earliest (vegetal plate stage) gastrula sub-equatorially. Soon thereafter, the vegetal plate invaginates. resulting in a cap-shaped gastrula with the mesoderm localized at the blastoporal lip and completely encircling the blastopore. As the gastrula stage progresses, DiI (a vital dye) labeling demonstrates that the entire mesoderm is internalized by a slight involution of the epiblast into the hypoblast all around the perimeter of the blastopore. Subsequently. during the early neurula stage, the internalized mesoderm undergoes anterior extension mid-dorsally (as notochord) and dorsolaterally (in paraxial regions when segments will later form). By the late neurula stage, AmBra-2 is no longer transcribed throughout the mesoderm as a whole; instead. expression is detectable only in the posterior mesoderm and in the notochord, but not in par axial mesoderm where definitive somites have formed.

LC-DAD-ESI-MS Characterization of Carbohydrates Using a New Labeling Reagent

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupling to liquid chromatography with electrospray ionization mass spectrometry for the detection of carbohydrates from the derivatized rape bee pollen samples is reported. Carbohydrates are derivatized to their bis-NMP-labeled derivatives. Derivatives showed an intense protonated molecular ion at m/z [M+H](+) in positive-ion detection mode. The mass-to-charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative analysis of carbohydrates. This characteristic fragment ion is from the cleavage of C2-C3 bond in carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose and glyceraldehydes and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids such as galacturonic acid and glucuronic acid (m = n - 2, n is carbon number of carbohydrate). No interferences for all aliphatic and aromatic aldehydes presented in natural environmental samples were observed due to the highly specific parent mass-to-charge ratio and the characteristic fragment ions. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed-phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected.

Analysis of carbohydrates in a tibetan medicine using new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone, by HPLC with DAD detection and ESI-MS identification

A new labeling reagent, 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP), coupling with liquid chromatography (LC) with electrospray ionization mass spectrometry (ESI-MS) for the detection of carbohydrates from a famous Tibetan medicine is reported. Carbohydrates were derivatized to their bis-NMP-labeled derivatives. The method, in conjunction with a gradient elution, offered a baseline resolution of carbohydrate derivatives on a reversed phase Hypersil ODS-2 column. The carbohydrates such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose, and fucose could be successfully detected by UV and ESI-MS. Derivatives showed intense protonated molecular ion at m/z [M+H]+ in positive ion mode. The mass to charge ratios of characteristic fragment ions at m/z 473.0 could be used for the accurately qualitative identification of carbohydrates; this characteristic fragment ion was from the cleavage of C2-C3 bond in the carbohydrate chain giving the specific fragment ions at m/z [MH-CmH2m+1Om-H2O](+) for pentose, hexose, and glyceraldehydes, and at m/z [MH-CmH2m-1Om+1-H2O](+) for alduronic acids, such as galacturonic acid and glucuronic acid (m=n-2, n is carbon atom number of carbohydrate). Compared with the traditional 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent, currently synthesized NMP show the advantage of higher sensitivity to carbohydrate compounds with UV and ESI-MS detection.

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV)

A novel labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone (NMP) coupled with capillary electrophoresis (CE) with DAD detection for the determination of carbohydrates has been developed. The chromophore in the 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent is replaced by naphthyl functional group, which results in a reagent with very high molar absorptivity (epsilon(251nm) = 5.58 x 10(4) L mol(-1) cm(-1)). This pen-nits NMP-labeled carbohydrates to be detected with UV absorbance in standard 50-mu m-i.d. fused silica capillaries by zone electrophoresis. in this mode, nanomolar concentrations of detection limits are obtained. The method for the derivatization. of carbohydrates with NMP is simplified. The derivatization reaction is rapid and mild in the presence of ammonia catalyst without further transfer steps. Nine monosaccharide derivatives such as mannose, galacturonic acid, glucuronic acid, rhamnose, glucose, galactose, xylose, arabinose and fucose can successfully be detected in CE mode. Good reproducibility can be obtained with relative standard deviation (R.S.D.) values of the migration times and peak area, respectively, from 0.44 to 0.48 and from 3.2 to 4.8. Furthermore, the developed method has been successfully applied to the analysis of carbohydrates in the hydrolyzed rape bee pollen samples. (C) 2008 Published by Elsevier B.V.

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance, liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+ H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH2O-CO and CH2-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate, (FMOC-Cl) as labeling reagents. The ratios I-BCEC/I-BCEOC = 1.94-2.17 and I-BCEC/I-FMOC = 1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits calculated from 0.50 pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4 fmol. The relative standard deviations for within-day determination (n = 11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were < 3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of > 0.9996. (C) 2006 Elsevier B.V. All rights reserved.

A Label-Free Multisensing Immunosensor Based on Imaging Ellipsometry

An immunosensor based on imaging ellipsometry and its potential applications was demonstrated in this paper. It has been proven a fast, reliable, and convenient method to quantify the thickness distribution of protein layers or detect protein concentration in solution. Combined with a protein chip, the immunosensor was able to detect multiple analytes simultaneously without any labeling. Preliminary results demonstrated how this immunosensor could be used to monitor several independent biospecific binding processes in real-time and in situ conditions.

Micro-systems for Optical Protein-Chip

Protein-Chip as micro-assays for the determination of protein interaction, the analysis, the identification and the purification of proteins has large potential applications. The Optical Protein-Chip is able to detect the multi-interaction of proteins and multi-bio-activities of molecules directly and simultaneously with no labeling. The chip is a small matrix on solid substrate containing multi-micro-area prepared by microfabrication with photolithography or soft lithography for surface patterning, and processed with surface modification which includes the physical, chemical, and bio-chemical modifications, etc. The ligand immobilization, such as protein immobilization, especially the oriented immobilization with low steric hindrance and high bio-specific binding activity between ligand and receptor is used to form a sensing surface. Each area of the pattern is corresponding to only one bioactivity. The interval between the areas is non-bioactive and optically extinctive. The affinity between proteins is used to realize non-labeling microassays for the determination of protein identification and protein interaction. The sampling of the chip is non-disturbing, performed with imaging ellipsometry and image processing on a database of proteins.

The intensity distributions of collected signals in coherent anti-Stokes Raman scattering microscopy

Coherent anti-Stokes Raman scattering (CARS) microscopy with the combining of confocal and CARS techniques is a remarkable alternative for imaging chemical or biological specimens that neither fluoresce nor tolerate labeling. The CARS is a nonlinear optical process, the imaging properties of CARS microscopy will be very different from the conventional confocal microscopy. In this paper, we calculated the propagation of CARS signals by using the wave equation in medium and the slowly varying envelope approximation (SVEA), and find that the intensity angular distributions vary considerably with the different experimental configurations and the different specimen shapes. So the conventional description of microscopy (e.g.. the point spread function) will fail to descript the imaging properties of CARS microscopy. (c) 2004 Elsevier B.V. All rights reserved.