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.

Preparation and characterization of ternary europium complex-doped thin sol-gel hybrid film by fluorescence spectroscopy

Ternary europium complex with dibenzoylmethane (DBM) and 1,10-phenanthroline (phen) was in-situ synthesized in thin SiO2/polyvinyl butyral (PVB) hybrid films by a two-step sol-gel process and characterized by;means of fluorescence spectroscopy. The luminescence spectra, fluorescence lifetimes and photostability were all investigated. The results showed that the hybrid films exhibited the characteristic emission bands of the Central rare earth Eu3+. In addition, Eu3+ presented longer fluorescence lifetime than in an ethanol solution and the complex had a higher photostability in the hybrid film than in the PVB film containing the corresponding pure complex.

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.

Fluorescence spectroscopy studies on the action of rare earths with bovine serum albumin in multimolecular system

In this paper. the interaction of tripositive terbium ions (Tb3+) with bovine serum albumin (BSA) has been investigated in the presence of some alkaline earth metals and citric acid (Cit) by using fluorescence spectroscopy. The results show that Tb-31. BSA and Cit can form ternary complex BSA . Tb-2. Cit(4) in mu =0.1 mol/l NaCl. pH6.3 hexamethylenetetramine buffer. Other tare earths are able to compete for the same binding site in BSA with Tb3+-. The sequence of con,petition is Eu3+>Pr3+>Yb3+>Gd3+>La3+>Ca2+ and Mg2+ cannot replace Tb bound to BSA.

STUDY ON RARE EARTH-HEDTA-SERINE TERNARY SYSTEM BY POTENTIOMETRY AND FLUORESCENCE SPECTROSCOPY

In this pare earth-HEDTA-serine (Ser) ternary system has been studied by potentiometric titration at 25 degrees C with an ionic strength of 0.15 mol.L-1 (NaCl). The excitation and fluorescence spectra of TbCl3, Tb-Ser, Tb-HEDTA and Tb-HEDTA-Ser have hem determined at room temperature. The stability constants of these ternary complexes have been obtained, It lieu been found that Tb-HEDTA-Ser ternary system exhibit characteriatic fluorescence spectrum of Tb3+ sensitized by two ligands,The results mean that the chemical hood between the control ion and the ligand in this ternary system is predominantly ionic in character,and the energy transfer from ligand to Tb8+ is performed by a kind of abort-range electron exchange action.

The surface sites of sulfated zirconia studied in situ by laser-induced fluorescence spectroscopy

The surface sites of sulfated zirconia were investigated in situ by laser-induced fluorescence spectroscopy using aniline as the probe molecule. Different from the cases for many other oxides, the aniline adsorbed on the unique active sites of sulfated zirconia at r.t. is changed into another species, which emits a characteristic fluorescence band at 422 nm. The results illustrate that the sulfate groups in sulfated zirconia are favorable for the generation of these unique active sites, which also rarely exist on pure zirconia composed of tetragonal and monoclinic phases but do not exist on pure zirconia composed of monoclinic phase. (C) 2004 Elsevier B.V. All rights reserved.

Kinetic study of DNA/DNA hybridization with electrochemical impedance spectroscopy

The hybridization of immobilized oligonucleotides probe strands with solution phase targets is the underlying principle of microarraybased techniques for the analysis of DNA variation. To study the kinetics of DNA/DNA hybridization, target DNA is often prior labeled with markers. A label-free method of electrochemical impedance spectra (EIS) for study the hybridization in process was reported. The Langmuir model was used to determine the association rate constant (K-on), the dissociation rate constant (K-off) and the affinity rate constant (K-A), for perfect matched DNA hybridization. The results show that, EIS is a successful technique possessing high effectivity and sensitivity to study DNA/DNA hybridization kinetics. This work can provide another view on EIS for the studying of DNA/DNA hybridization.

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.

Chiral metallo-supramolecular complexes selectively recognize human telomeric G-quadruplex DNA

Here, we report the first example that one enantiomer of a supramolecular cylinder can selectively stabilize human telomeric G-quadruplex DNA. The P-enantiomer of this cylinder has a strong preference for G-quadruplex over duplex DNA and, in the presence of sodium, can convert G-quadruplexes from an antiparallel to a hybrid structure. The compound's chiral selectivity and its ability to discriminate quadruplex DNA have been studied by DNA melting, circular dichroism, gel electrophoresis, fluorescence spectroscopy and S1 nuclease cleavage.

Comparative studies of AOT and NaDEHP by fluorescence technique and z-potential (ζ) measurements

The aggregation behaviors of two surfactants with the same hydrophobic tail, sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and sodium bis(2-ethylhexyl)phosphate (NaDEHP), have been investigated by the fluorescence technique and z-potential (ζ) measurements. Five fine peaks of the pyrene molecule fluorescence spectroscopy appear in the surfactant solution, and the micropolarity at which pyrene locates is monitored from the intensity ratio of the first (I1) and the third peak (I3). A wide peak around 475 nm, the emission spectra of the excimer of pyrene molecules, is observed in the NaDEHP solution, while this is not found for the AOT system. The value of I1/I3 decreases in a more limited concentration range for the AOT system than for NaDEHP, indicating that small aggregates can be more easily formed by NaDEHP molecules. The z-potential results for the aggregates formed by the two surfactants show that the interaction between AOT and PVP is stronger than that between NaDEHP and PVP.

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta(2) subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively. (c) 2006 Elsevier Inc. All rights reserved.

Comparison of fluorescence spectra of Yb : Y3Al5O12 and Yb : YALO(3) single crystals

Yb:Y3Al5O12 (Yb:YAG) single crystals with Yb doping concentration 0.5 at.%, 5 at.%, 15 at.%, 25 at.%, 50 at.%, 100 at.% and Yb:YAlO3 (Yb:YAP) single crystals with Yb doping concentration 0.5 at.%, 5 at.%, 15 at.%, 30 at.% were grown by the Czochralski process. The fluorescence spectra of these crystals and the effects of self-absorption on the shape of the fluorescence spectra were studied. Through comparing the fluorescence spectra of Yb:YAG and Yb:YAP, all results indicate that the effects of self-absorption on the fluorescence spectra of Yb:YAP are remarkably stronger than that of Yb:YAG at the same Yb concentration. (c) 2006 Elsevier B.V. All rights reserved.

Study on fluorescence emission and synchronous-scan fluorescence spectrea of Nitzschia hantzschiana solution with FE(III)

The characterization of the algal Nitzschia hantzschiana solution with (or without) Fe(III) was carried out using fluorescence emission and synchronous-scan spectroscopy. An emission peak (excited at 440 nm) was observed at 675 nm for Nitzschia hantzschiana solution. The effective characterization method used was synchronous-scan fluorescence spectroscopy (SFS). A wavelength difference (Delta lambda) of 90 nm was maintained between excitation and emission wavelengths. The peak was observed at about 236(ex) nm (326(em) nm) for synchronous fluorescence spectroscopy. Fe(III) was an effective quencher. The relationship between I-0/I (quenching efficiency) and c (concentration of Fe (III) added) was a linear correlation for the algal solution with Fe(III). Effects of pH on synchronous-scan fluorescence intensity were evident.

Investigations on the adsorption behavior of Neutral Red on mercaptoethane sulfonate protected gold nanoparticles

A detailed investigation on the adsorption behavior of Neutral Red (NR) molecules on mercaptoethane sulfonate-monolayer protected gold clusters (MES-MPCs) has been conducted by the spectroscopic method. It is found that cationic NR molecules are adsorbed on the negatively charged MPCs surfaces via electrostatic attractive forces. The absorption study shows that the optical properties of NR molecules are significantly influenced upon the adsorption. Based on the electrostatic adsorption nature and the excellent stability of MES-MPCs against the electrolytes, this association can be released by the addition of electrolyte salts, which can be monitored by both absorption and fluorescence spectroscopy. In addition, dication Ca2+ is found to be more effective in the release of NR than univalent Na+. Moreover, the MES-MPCs exert energy transfer quenching of NR fluorescence by both static and dynamic quenching. However, static quenching seems to be the dominating quenching mechanism. Furthermore, this energy transfer quenching exhibits strong dependence of Au core size, and 5.0 nm MPCs show stronger ability in quenching the NR fluorescence than that of 2.7 nm MPCs.

Possibility of lifetime measurement in a flowing medium with phase fluorometry

The possibility of lifetime measurement in a flowing medium with phase fluorometry is investigated theoretically. A 3-D time dependent partial differential equation of the number density of atoms (or molecules) in the upper level of the fluorescence transition is solved analytically, taking flow, diffusion, optical excitation, decay, Doppler shift, and thickness of the excitation light sheet into account. An analytical expression of the intensity of the fluorescence signal in the flowing medium is deduced. Conditions are given, in which the principle of lifetime measurement with phase fluorometry in the static sample cell can be used in a flowing medium.