Laser-time-resolved fluorescence spectroscopy in immunoassays

This paper described a laser-excited time-resolved fluoroimmunoassay set. It made lanthanide ion to couple the anhydrde of diethylenetriaminepentaacetic acid (DTPAA) for labeling antibodies. The experiment used polystyrene tap coated with HCV antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A nitrogen laser beam was used to excite the Eu3+ chelates and after 60 ys delay time,the emission fluorescence was measured. Background fluorescence of short lifetimes caused by serum components and Raman scattering can be eliminated by set the delay rime. In the system condition, fluorescent spectra and fluorescent lifetimes of Eu3+ beta-naphthoyltrifluroacetone (NTA) chelates were measured. The fluorescent lifetime value is 650 mu s. The maximum emssion wavelength is 613 nm. The linear range of europium ion concentration is 1 x 10(-7)- 1 x 10(-11) g.mL(-1) and the detection limit is 1 x 10(-13) g.mL(-1). The relative standard deviation of determination ( n = 12) for samples at 0.01 ng.mL(-1) magnitude is 6.4%. Laser-TRFIA was also found to be suitable for diagnosis of HCV. The sensitvity and specificity were comparable to enzyme immunoassay. The result was obtained with laser-TRFIA for 29 human correlated well with enzyme immunoassay.

Synthesis, 2D NMR, electrochemistry and luminescence of ruthenium(II) complexes with 2,2 '-bipyridine and 5-(omega-bromoalkylamido)-1,10-phenanthroline

The efficient synthesis of 5-(5-bromovaleramido)-1,10-phenanthroline, 5-(6-bromohexanamido)-1,10-phenanthroline, and 5-(11-bromoundecanamido)-1,10-phenanthroline are described, which reacted with cis-Ru(bpy)(2)Cl-2. 2H(2)O and sodium hexafluorophosphate to form Ru(bpy)(2)[phen-NHCO(CH2)(n)Br](PF6)(2) (n = 4, 5 or 10; phen = 1,10-phenanthroline). The intricate H-1 NMR spectra at low field of these complexes were completely assigned in virtue of H-1-H-1 COSY technique. Cyclic voltammetry was used to study electrochemical behaviours of these complexes, and their luminescent properties were investigated with fluorescent spectra.

Synthesis and properties of three tris(1,10-phenanthroline) ruthenium (II) derivatives

Three new asymmetrical ruthenium (II) complexes: [Ru(phen)(2){phen-NHCO(CH2)(4)Br}](PF6)(2), [Ru(phen)(2){phen-NHCO(CH2)(5)Br}](PF6)(2) and [Ru(phen)(2){phen-NHCO(CH2)(10)Br}](PF6)(2) were synthesized, which were confirmed by the technique of FT-IR, H-1 NMR and ESI-MS. The electrochemical and fluorescent properties of three Ru (II) complexes were investigated with cyclic voltammetry and fluorometry.

A new series of Ru(II)polypyridine surfactants: Synthesis, 2D NMR, fluorescence, electrochemistry and electrochemiluminescence

A new series of Ru(II) polypyridine surfactants, Ru(bpy)(2)[phenNH CO(CH2)(n)CH3](PF6)(2), where n is 10, 12, 14 or 16, has been prepared and characterized. The H-1-NMR spectra of these new Ru(II) complexes were attributed and discussed by the H-1-H-1 COSY method. The comparative analysis of IR spectra of these complexes and their responding free ligands has shown that there are intensive d --> pi* feedback interactions between central Ru(II) and their ligands. Their fluorescent, electrochemical and electrochemiluminescent behaviours are also reported.

Synthesis and spectroscopic studies of endohedral metallofullerenes Tb@C-2n

Endohedral metallofullerenes Tb@C-2n were synthesized and extracted with high-yield by K-H carbon-are evaporation and an effective pyridine extraction technique at high-temperature high-pressure. Laser-desorption-ionization time-of-flight (LD-TOF) mass spectrometry, X-ray photoelectron spectroscopy (XPS), solid-state fluorescent emission spectroscopy and gas phase derivation reaction with the self-chemical ionization mass spectrometric ion system of vinyl acetate were employed for studying the electronic structures, fluorescent properties and gas phase reactivities of metallofullerenes Tb@C-2n. The experimental results suggest that endohedral metallofullerenes Tb@C-2n would have the approximate structures of Tb3+@C-2n(3-) similar to other metallofullerenes, good fluorescent emission properties and active reactivities in gas phase ion-molecular reactions.

Blue-light emitting diodes and flat display based on poly(p-phenylenevinylene) copolymers

Copolymers containing alternating flexible aliphatic blocks and rigid poly(p-phenylenevinylene) (PPV) blocks were synthesized and characterized. It was found that the fluorescent intensity increases with increasing length of the flexible blocks. Bright blue-light emitting diodes were fabricated using PPV copolymers as electroluminescent layers. The devices show 190 cd/m(2) light-emitting brightness at 460 nm and 15 V rum-on voltage. The effects of oxadiazole derivative PBD and tris(8-hydroxyquinoline) aluminum Alq(3) electron-transporting layers on the luminance and stability of the devices are discussed.

STUDIES ON STATES OF CYTOCHROME-C MOLECULES IN AQUOUS SOLUTIONS USING SYNCHRONOUS FLUORESCENCE SPECTROSCOPY

The states of cytochrome C molecules in aquous solution were studied with synchronous fluorescence spectroscopy, It was found that the synchronous fluorescent spectra of cytochrome C were contributed by tyrosine and tryptophan residues separately at Delta lambda = 20 nm and Delta lambda = 80 nm, The peak position in synchronous fluorescent spectra of tyrosine residues in cytochrome C molecule does not change with its concentration, but that of tryptophan residue changes with its concentration, Only one peak at 340.0 nm was observed in the dilute solution of cytochrome C, With increasing the concentration of cytochrome C, a new peak at 304. 0 nm appeared. The peak at 340.0 nm disappeared and only one peak at 304.0 nm was observed at a higher concentration of cytochrome C, It may originate from the change of aggregation states of cytochrome C molecules and it was considered that the peak at 340.0 nm was attributed to the monomer and peak at 304.0 nm was due to the dimmer or oligomers. When urea was added into cytochrome C solution in which both monomer and dimmer or oligomers exist, cytochrome C molecules do not denature in the range of the specific concentrations of urea. The concentration of monomer of cytochrome C molecules increased and that of aggregation slates decreased by adding urea, Therefore, the synchronous fluorescence spectroscopy can be used to identify monomer and aggregation states of cytochrome C molecules.

SYNTHESIS OF THE CLINOPYROXENES JOIN CAMGSI2O6-NAALSI2O6 FOR JEWELRY

Clinopyroxenes of the join CaMgSi2O6(diopside)-NaAlSi2O6 (jadeite) were synthesized in the temperature range 800-1900 degrees C and under varying pressure, 10-55 kbar. The stability regions of various compositions of diopside-jadeite have been established experimentally using different compositions of glass materials: stoichiometric composition NaAlSi2O6, 0.1CaMgSi(2)O(6)-0.9NaAlSi(2)O(6), 0.2CaMgSi(2)O(6)0.8NaAlSi(2)O(6), 0.3CaMgSi(2)O(6)-0.7NaAlSi(2)O(6), and 0.4CaMgSi(2)O(6)-0.6NaAlSi(2)O(6). Unit cell parameters of synthetic clinopyroxenes with the above compositions were determined. The physical properties, such as hardness, toughness, density, and refractive index, etc., were also measured. The results show that synthetic clinopyroxenes have the same properties as the natural one. The gem quality of diopside-jadeite clinopyroxenes was achieved by synthesised on the basis of the above experiments. Various colouring agents, such as Cr2O3, Co2O3, NiO2, Fe2O3, TiO2, MnO, CuO, and their combinations, FeO-Cr2O3, etc., were added to obtain the different colours of gem. In addition, small amounts of the rare-earth oxides, such as CeO2, Nd2O3, Sm2O3, Dy2O3, Eu2O3, Er2O3, Pr6O11, Lu2O3 and CuO-Eu2O3, Co2O3Nd2O3, etc., were also added to produce fluorescent clinopyroxenes for jewellery.

SYNTHESIS OF K5LALI2F10, K5GDLI2F10, K5YLI2F10 AND LUMINESCENCE OF CE-3+

The compounds, K_5LnLi_2F_(10)(Ln=La, Gd, Y) were synthesized by solid-state reaction in argon atmosphere. Powder X-ray diffraction patterns showed that K_5LnLi_2F_(10) is isostructural with K_5NdLi_2F_(10)(KNLF) except K_5YLi_2F_(10). The cell parameters and volumes of K_5LnLi_2F_(10)(Ln=La, Ce, Gd) were calculated. They decrease regularly with radii of La~(3+), Ce~(3+) and Gd~(3+). The excitation and fluorescent spactra of K_5Ce_xLn_(1-x) Li_2F_(10) were determined. It was found that the excitat...

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO2 and HCO3-, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full-length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5' untranslated region (UTR) of 177 bp, a 3' UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274-amino-acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the beta-CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real-time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4-fold higher and 10-fold higher, respectively.

Molecular cloning, characterization and mRNA expression of peroxiredoxin in Zhikong scallop Chlamys farreri

Peroxiredoxin V (PRX V) is known as an atypical 2-cysteine peroxiredoxin that protects the organisms against various oxidative stresses and functions in signal transduction. The cDNA of a PRX V gene (designated as CfPRX) was cloned from scallop Chlamys farreri. The full-length sequence of CfPRX cDNA was of 2,179 bp with a 564 bp open reading frame encoding a peptide of 187 amino acids. Sequence comparison showed that CfPRX shared higher identities with PRX Vs than that with other isoforms of PRX, indicating CfPRX was a member of the PRX V family. Fluorescent real-time quantitative PCR analysis revealed the presence of CfPRX transcripts in gill filaments, adductor muscle, heart, gonad, kidney and hemocytes, and the stimulation of Listonella anguillarum significantly (P < 0.01) enhanced the mRNA expression of CfPRX in hemocyte. These results indicated that CfPRX was a constitutive and inducible acute-phase protein which was involved in the immune resistance to L. anguillarum stimulation.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli

Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g 1(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

Highly soluble and stable recombinant holo-phycocyanin alpha subunit expressed in Escherichia coli

C-phycocyanin (Cpc) is one of the phycobiliproteins with highly fluorescent and various pharmacological activities Holo-Cpc-alpha Subunit (holo-CpcA) expressed in Escherichia colt resulted in low yield and tended to aggregate after purification in this Study, we constructed a new plasmid coding holo-CpcA fused with hexahistidine and maltose-binding protein tag, which designated as HMCpcA. to Improve Its Solubility and stability without the Impairment of its spectra anti fluorescent properties HMCpcA was significantly more stable over time and a wider range of pH as compared to holo-CpcA. In addition. both the solubility and yields of HMCpcA increase significantly We here provided an example to demonstrate that MBP could also Improve the stability of the protein it fused while it has been reported as a soluble fusion partner before. This novel fluorescent protein will facilitate the large-scale production and be potentially applicable for the development Of fluorescent probes, as well as antioxidant agents (C) 2009 Elsevier B V. All rights reserved

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.