Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.     

Development of binding assays in microfabricated picoliter vials: an assay for biotin

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.     

Nanomechanical assay to investigate the selectivity of binding interactions between volatile benzene derivatives

Understanding the interactions between aromatic gas molecules and various simple aromatic receptor molecules is important in developing selective receptors for volatile organic compounds (VOCs). Here, five benzene thiols with different functional end groups were used to investigate the weak binding of aromatic vapors such as dinitrotolouene (DNT) and toluene. A multiplexed microcantilever array in conjunction with a very low concentration vapor generation system was developed to study multiple receptor-target interactions simultaneously. Differential nanomechanical responses of such devices provided insight into the influence of various chemical and structural features of such molecules.     

Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.     

Chemiluminescent reaction of fluorescent organic compounds with KHSO5 using cobalt(II) as catalyst and its first application to molecular imprinting

The decomposition of peroxomonosulfate (HSO5-) has been investigated by chemiluminescence (CL). A weak CL was observed during mixing the HSO5- solution with the Co2+ solution in unbuffered conditions. An appropriate amount of fluorescent organic compounds (FOCs), such as dansyl amino acids and pyrene, was added to the KHSO5/Co2+ solution, a strong CL was recorded. A possible CL mechanism, based on studies of the fluorescence, CL, and UV-visible spectra and comparison of Co3+ oxidation ability with the SO4.- radical ion, was discussed. The CL from HSO5-/Co2+ is the emission of singlet oxygen produced from the catalytic decomposition of HSO5-. It was suggested that the decomposition of HSO5- in aqueous solution with Co2+ proceeds via one-electron transfer to yield SO4.- radical ion. The FOC was attacked by SO4.- radical ion and oxidized to decompose into small molecules. During this proceeding, CL emission was given out. The present CL system has been developed as a flow injection analysis for FOCs. The detection limits (S/N = 3) were in the concentration range 10(-9)-10(-7) M for FOCs. Oxidation decomposition and CL emission of the analytes have been used in the molecular imprinting recognition. As an initial attempt, dansyl-L-phenylalanine was used as a template molecule and methacrylic acid and 2-vinylpyridine were used as functional monomers. The network copolymer imprinted with dansyl-L-phenylalanine exhibits an affinity for the template molecule. When the flowing streams of HSO5- and Co2+ solutions mixing through the molecularly imprinted polymer particles filled the flow cell, the template molecule, dansyl-L-phenylalanine reacted with the HSO5-/Co2+ solution and CL was emitted. The dansyl-L-phenylalanine was decomposed during the CL process, and the cavities of a defined shape and an arrangement of functional groups complementary to the template in the polymer were left for the next sample analysis.     

Detection of Cryptosporidium parvum using oligonucleotide-tagged liposomes in a competitive assay format

To meet the technical challenge of accurately and rapidly detecting Cryptosporidium parvum oocysts in environmental water, the authors developed a single-use visual-strip assay. The first step in the overall assay procedure involves extracting C. parvum's mRNA coding for heat-shock protein hsp70, followed by amplification using nucleic acid sequence-based amplification (NASBA) methodology as described previously (Baeumner, A. J.; Humiston, M.; Montagna, R. A.; Durst, R. A. Anal. Chem., in press). Subsequently, generated amplicons are hybridized with dye-entrapping liposomes bearing DNA oligonucleotides (reporter probes) and biotin on their surface. The liposome-amplicon complex is then allowed to migrate upward on a nitrocellulose membrane strip. On the nitrocellulose strip, antisense-reporter probes are immobilized in a capture zone and antibiotin antibodies are immobilized in a second zone above the capture zone. Depending on the presence or absence of amplicon in the sample, the liposomes will bind to the capture zone, or they will be caught via their biotin tag in the second zone. Visual detection or gray-scale densitometry allows the quantification of liposomes that are present in either zone. The detection limit of the assay was determined to be 80 fmol amplicon/test. High accuracy and an internal assay control is established using this competitive format, because the presence or absence of liposomes can be quantified in the two capture zones.     

非线性光学材料聚二乙炔及其衍生物的制备及应用

非线性光学材料聚二乙炔及其衍生物的研究已引起国内外的广泛关注。本文总结了近年来聚二乙炔及其衍生物的合成方法 ,综述了聚二乙炔及其生物在非线性光学方面的研究进展 ,并且对聚二乙炔及其衍生物在非线性光学领域的进一步应用提出了展望。     

Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol

Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 μM and V = 1.98 μM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.     

MDA及其衍生物的制备和应用进展

本文简要介绍了MDA的制备及应用 ,并就其在作为固化剂或交联剂时存在的问题 ,介绍了几种可作为替代品的MDA衍生物及其制备和应用情况     

Liquid-phase binding assay of alpha-fetoprotein using DNA-coupled antibody and capillary chip electrophoresis

An immunoassay using DNA-coupled antibody for bound/free separation in a liquid-phase binding assay format is described. Anti-alpha-fetoprotein monoclonal antibody was conjugated with DNA, mixed with alpha-fetoprotein (AFP), and incubated, and then 1 muL of the mixture was applied to capillary electrophoresis on a microchip. The DNA molecule of the antibody-DNA conjugate and the DNA-conjugated immune complex peak were detectable fluorophotometrically using intercalator dye within 90 s, whereas the Alexa-labeled antibody was detected as a broad and slower migrating peak. The electrophoretic mobility of the immune complex could be optimized for resolution and sharpness by changing the length of the DNA coupled to the antibody. The detection limit of AFP was approximately 300 pM in a sample. This immunoassay method utilizing a liquid-phase binding assay format is simple and convenient for antigen measurements on microchips.     

Determination of azathioprine and its related substances by capillary zone electrophoresis and its application to pharmaceutical dosage forms assay

The development of a stability-indicating capillary zone electrophoresis (CZE) method for the determination of the drug azathioprine (AZA) and its related substances in bulk and dosage forms is described. Theophylline was used as an internal standard to improve quantitative results. The method was fully validated in terms of repeatability (n = 10, RSD for migration time and peak area ratio were 0.15% and 0.60%, respectively), reproducibility (n = 5, RSD of peak area ratio was 0.84%), linearity at two ranges of the azathioprine concentration, limits of detection (LOD) and quantitation (LOQ), and robustness. The method was applied for determination of the drug in bulk and a commercial tablet dosage form (recovery 98.3-101.3%) and in powder for injection (recovery 98.7-100.6%). The method was fast and reliable for the analysis of AZA and its related substances in bulk and dosage forms.     

Assay principle for modulators of protein-protein interactions and its application to non-ATP-competitive ligands targeting protein kinase A

Targeting sites that modulate protein-protein interactions represents an ongoing challenge for drug discovery. We have devised an assay principle, named ligand-regulated competition (LiReC), in an effort to find non-ATP competitive small-molecule regulators for type Ialpha cAMP-dependent Protein kinase (PKA-Ialpha), a protein complex that is implicated in disease. Our assay based on the LiReC principle utilizes a competitive fluorescent peptide probe to assess the integrity of the PKA-Ialpha complex upon introduction of an allosteric ligand. The developed fluorescence polarization method screens for small molecules that specifically protect (antagonists) or conversely activate (agonists) this protein complex. In high-throughput format, various cyclic nucleotide-derived agonists and antagonists are successfully detected with high precision. Furthermore, assay performance (Z'-factors above 0.7) far exceeds the minimum requirement for small-molecule screening. To identify compounds that operate through novel modes of action, our method shields the ATP-binding site and purposely excludes ATP-competitive ligands. These proof-of-principle experiments highlight the potential of the LiReC technique and suggest its application to other protein complexes, thereby providing a novel approach to identify and characterize modulators (small molecules, proteins, peptides, or nucleic acids) of protein-protein systems.     

Molecular control of quantum-dot internal electric field and its application to CdSe-based solar cells

Inorganic nanocrystals are attractive materials for solar-cell applications. However, the performance of such devices is often limited by an insufficient alignment of energy levels in the nanocrystals. Here, we report that by attaching two different molecules to a single quantum dot or nanocrystal one can induce electric fields large enough to significantly alter the electronic and optoelectronic properties of the quantum dot. This electric field is created within the nanocrystals owing to a mixture of amine- and thiol-anchor-group ligands. Examining the steady state as well as temporal evolution of the optical properties and the nuclear magnetic resonances of the nanocrystals we found that the first excitonic peak shifts as a function of the capping-layer composition. We also demonstrate that the use of a mixed-ligand-induced electric field markedly enhances the charge generation efficiency in layer-by-layer CdSe-nanocrystal-based solar cells, thus improving the overall cell efficiency.     

Element-free Galerkin method using directed graph and its application to creep problems

The element-free Galerkin method (EFGM) is one of the meshless methods proposed by Belytschko et al. Since node-element connectivities used in the finite element method (FEM) are not needed in the EFGM, the EFGM is expected to be applied to many problems of the continuum mechanics and to be utilized for a tool in a CAE system instead of the FEM. However the EFGM requires more CPU time to search nodes of the MLSM than the FEM. In this paper, the method of the directed graph and the Delaunay triangulation are respectively used for searching nodes and the division of the integral domain respectively. These techniques are useful for saving the CPU time and the simplification of the analysis for the EFGM. Furthermore, the EFGM has not been applied to nonlinear problems such as creep problems under elevated temperature. In this paper, the EFGM using the method of the directed graph and the Delaunay triangulation is applied to several creep problems. The CPU times for the analyses are reduced by the proposed EFGM. The results obtained from the EFGM analyses agree well with those of the FEM.     

Matching IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay and its clinical application for tracing tumor-associated glycosphingolipids in hepatocellular and pancreatic cancer

Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.     

Bioanalytical method development and its application to pharmacokinetics studies on Simvastatin in the presence of piperine and two of its synthetic derivatives

Piperine has been widely used as a bioenhancer. Simvastatin belongs to a group of medicines known as statins. It acts by inhibiting HMG CoA reductase and acts primarily as a hypolipidemic agent. In this study some derivatives of Piperine were synthesized. They were studied for their bioenhencing effect (10?mg kg^?1) and this effect was compared with that of Piperine. The pharmacokinectic profile of Simvastatin alone and in combination with Piperine and Piperine derivatives were investigated by validated HPLC method as per USFDA guidelines. It was seen that the two synthesized derivatives of Piperine significantly improved the bioavailability of Simvastatin in Wistar rats. The 5-(benzo) [1,3]dioxol-5-yl)-N-(pyridin-4-yl)penta-2,4-dienamide was better amongst the synthesized in increasing the bioavailability of Simvastatin in Wistar rat.     

Identification of binding interactions between myeloperoxidase and its antibody using SERS

Nano-Micro Letters - Surface Enhanced Raman Spectroscopy (SERS) is a widely used spectroscopic method that can dramatically increase the sensitivity of Raman spectroscopy and has demonstrated...