Studies on interaction of bovine serum albumin with indo-1 by fluorescence spectroscopic method

The binding-site number was calculated by using fluorescence spectroscopic method with bovine serum albumin(BSA) and Indo-1 as protein and ligand models, respectively. The method for calculating binding-site number in BSA for Indo-1 was developed based on the relationships between the changes of Indo-1 fluorescence intensity and the analytical concentration of BSA. And the interaction of BSA with Indo-1 was investigated comprehensively by using fluorescence techniques as well as fluorescence resonance energy transfer, and the thermodynamic parameters were calculated according to the changes of enthalpy on temperature.,

Synthesis and Spectrum of Novel Pyrazoline Fluorescent Compounds

Pyrazoline derivatives have been used widely in dyeing industry as fluorescent whitening agents due to their excellent capability. According to Schellhammer theory of the relation between chemical structure and fluorescent quality, six new fluorescent compounds were designed and synthesized which contained the benzothiazole group in the I-pyrazoline, the indole group in the 3-pyrazoline and the derivatives of phenyl in the 5-pyrazoline. The structure of target compounds was confirmed by IR, H-1 NMR, MS and elementary analysis. The fluorescence spectra showed that these compounds had good fluorescence. They could absorb ultraviolet light at near 353 nm. The fluorescence maximum emission wavelengths were about 430-443 nm. It was a kind of promising fluorescence compounds. The largest fluorescence emission wavelength and the fluorescence intensity were related to the substituted group of the compounds. When the 6-Br group was introduced into benzothiazole, the fluorescence emission wavelength exhibited a blue shift, and the fluorescence intensity increased.

Surface plasmon resonance and electrochemistry for detection of small molecules using catalyzed deposition of metal ions on gold substrate

Small molecules are difficult to detect by conventional surface plasmon resonance (SPR) spectroscopy due to the fact that the changes in the refractive index resulted from the binding process of small biomolecules are quite small. Here, we report a simple and effective method to detect small biomolecule using SPR spectroscopy and electrochemistry by catalyzed deposition of metal ions on SPR gold film. As an example, the ascorbic acid-mediated deposition of Ag on gold film was monitored by in situ SPR spectrum. The deposition of Ag atom on gold film resulted in an obvious decrease of depth in SPR angular scan curves of reflectance intensity and minimum reflectivity angle. The depth change of the SPR reflectance intensity and minimum reflectivity angle curves mainly relied on the amount of Ag atom deposited on gold film that can be controlled by the concentration of ascorbic acid. By monitoring the deposition of Ag atom on gold film, ascorbic acid was detected in the concentration range of 2 x 10(-5) M to 1 x 10(-3) M. After each of detections, the SPR sensor surface was completely regenerated by a potential step that stripped off the Ag atom. Furthermore, the regeneration process of the sensor surface provides the feasibility for detecting the concentration of ascorbic acid by electrochemical method.

Investigation on preparation and fluorescence properties of oxy-fluoride glass co-doped with Er3+ and Yb3+

The glass sample based on the composition of 45PbF(2)-45GeO(2)-10WO(3) co-doped with Yb3+/Er3+ was prepared by the fusion method in two steps: melted at 950 degreesC for 20 similar to 25 min then annealed at 380 degreesC for 4 h. Through the V-prism it is found that the refractive index of host glass and the sample are 1.517 and 1.65 respectively. The transmittance was observed by using the ultraviolet-visible-infrared spectrometer in the wavelength range from 0.35 to 2.5mum. The transmittaitce of the host glass is beyond 73%. That of the sample is beyond 50% and there are characteristic absorption peaks of rare-earth ions. The emission spectrum was measured by using the Hitachi F-4500 fluorescent spectrometer pumped by 980 nm semiconductor laser. There are a strong emission peak at 530 nm and a weak peak at 650 nm.

The luminescence of Eu3+ ion in Ca2Al2SiO7

Ca2Al2SiO7:Eu3+ was prepared by the sol-gel method. Through the emission spectrum of Eu3+ ion, the fluorescence parameters such as Omega(i) (i = 2,4) and radiative transition probabilities of D-5(0)-F-7(j) were calculated. The Pb2+ ion with bigger radius has an effect on the fluorescence spectra of Eu3+ which can be explained by the structure of the matrix. Simultaneously, the energy transfers between mercury-like ions (Pb2+ and Bi3+) and Eu3+ ion were observed. The D-5(4) and D-5(2) energy levels of Eu3+ are the resonance ones for Pb2+ ion.

Up-conversion spectrum properties of the oxy-fluoride glass co-doped with Er3+ and Yb3+

Up-conversion of 45PbF(2)-45GeO(2)-10WO(3) oxy-fluoride glasses co-doped with Yb3+ and Er3+ ions were prepared by fusion method through melting at 1223 K and then annealing at 653 K for 4 h. Transmittance of the undoped host glass was beyond 73% in a range of 0.6-2.5 mu m and the co-doped glasses still provided good transmittance beyond 50%. Refractive indices of the host and co-doped glasses were 1.517 and 1.650, respectively. Blue, green and red fluorescence spectra were observed in a range of 400-700 nm under 980 nm diode laser excitation. Up-conversion spectra at about 410, 518, 530and 650 nm were assigned to the 4f electron transitions of H-2(9/2) -> I-4(15)/(2), H-2(15/2) -> I-4(15/2) S-4(3/2) -> I-4(15/2) and F-4(9/2) -> I-4(15/2) of Er3+ ion, respectively. The mechanism of energy transfer between Yb3+ and Er3+ ions in the glass was analyzed. Raman shift shows the non-radiative relaxation of the glass sample is low.

Probing UPD-induced surface atomic rearrangement of polycrystalline gold nanofilms with surface plasmon resonance spectroscopy and cyclic voltammetry

Silver underpotential deposition (UPD)-induced surface atomic rearrangement of polycrystalline gold nanofilms was probed with use of surface plasmon resonance spectroscopy (SPRs) as a novel probe tool in combination with cyclic voltammetry. Interestingly, upon repetitive electrochemical UPD and stripping of Ag, the surface structure of the resulting bare Au film is rearranged due to strong adatom-substrate interactions, which causes a large angle shift of SPR R-theta curves, in a good linear relationship with the number of UPDs, to a lower SPR angle. The n, K values of the surfacial Au monolayers before and after the repetitive Ag UPD and stripping for 27 times are found to be 0.133, 3.60 and 0.565, 9.39, respectively, corresponding to the huge shift of 1.61degrees to the left of the SPR minima. Cyclic voltammetry experiments in 0.10 M H2SO4 are carried out before and after the UPD treatment to examine the quality of the whole electrode surface and confirmed this change. To correlate the angle change in SPRs with the profile change in the cyclic voltammogram, the UPD treatment was also performed on a Au(111) textured thin film. It was therefore confirmed that the resonance position of the SPR spectrum is very sensitive to the surface crystallographic orientation of the bare Au substrates. Some surface atomic rearrangement can cause a pronounced SPR angle shift.

Synthesis and characterization of a time-resolved fluorescence analysis chelate reagent-BCPDA

In this paper, we report on a solid phase time-resolved fluorescence immunoassay chelate reagent-4,7-bis(chlorosulfophenyl)1, 10-phenanthroline-2,9-dicarboxylic acid (BCPDA), which is suitable as a fluorescent labeling agent. The five step synthesis product of BCPDA was presented for improving the purity of the product based on the three step synthesis product. The approach involves chlorization, hydrolyzing the ester, preparing disodium, carboxylate to diacid, sulfonation. The yield of five step product is 99 %, 45 %, 94 %, 95 %, 80 % respectively. The structure and purity of product was characterized by the melting point, IR,H-1 NMR, UV spectrum, element analysis, and proved to be consistent with the structure predictal.

Studies on the interaction of porphyrin with antibody with fluorescent spectrum and UV-Vis differential spectra

After meso-tetra (alpha, alpha, alpha, alpha-O-phenylacetyl benzene)porphyrin combined with McAb 1F2, there was a significant hyperchromic effect, indicating that the combination of porphyrin and antibody is rigid and compact, aromatic amino acids exist at the combining sites of antigen in antibody. These aromatic amino acids are Trys and Trps, but the numbers of Trp are more than that found for Trys. The stochiometric ratio of porphyrin to 1F2 is 1:1, the disassociation constant was determined as(2.084+/-0.216) x 10(-10) mol/L by a method of fluorescence quenching, showing that both have a high affinity.

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described, REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i,e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M+. ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation. Copyright (C) 2000 John Wiley & Sons, Ltd.

Synchronous fluorescence spectra of myoglobin

The synchronous fluorescence spectra of myoglobin were studies for the first time. The fluorescence peals observed in the spectra were assigned, When the wavelength interval (Delta lambda) is 80 nm, the main peak at 335 nm is originated from the tryptophan residues in the myoglobin molecule. When Delta lambda is 20 nn, the peak at 308 nm is mainly due to the tyrosine residues in the myoglobin molecule and in a small part due to the tryptophan residues. Two peaks at 322 and 596 nm were observed in the spectrum of myoglobin for Delta lambda = 40 nm. The peak at 322 nm is due to both tyrosine and tryptophan residues. The peak at 596 nm is attributed to the heme group in the myoglobin molecule.

Three-dimensional fluorescence characteristics of dissolved organic matter produced by Prorocentrum donghaiense Lu

Filtration and cross-flow ultrafiltration techniques were used to separate culture media of Prorocentrum donghaiense at the exponential growth, stationary and decline stages into < 0.45 mu m filtrate, 100 kDa-0.45 mu m, 10-100 kDa and 1-10 kDa retentate and < 1 kDa ultrafiltrate fractions. The fluorescence. properties of different molecular weights of dissolved organic matter (DOM) were measured by excitation-emission matrix spectra. Protein-like and humic-like fluorophores were observed in the DOM produced by P. donghaiense. The central positions of protein-like fluorophores showed a red shift with prolonged growth duration, shifting from tyrosine-like properties at the exponential growth stage to tryptophan-like properties at the stationary and decline stages. The excitation wavelengths of protein-like fluorophores exhibited some change in the exponential growth and stationary stages with increased molecular size, but showed little change in the decline stage. However, the emission wavelengths in the decline stage exhibited a blue shift. Very distinct C type and A type peaks in humic-like fluorophores were observed. With a prolonged culture time, the intensities of both of the peaks became strong and the excitation wavelengths of peak A showed a red shift, while the A:C ratios fell. More than 94% of fluorescent DOM was in the lower than 1 kDa molecular weight fraction.

Homogeneous time-resolved fluoroimmunoassay of bensulfuron-methyl by using terbium fluorescence energy transfer

A sensitive homogenous time-resolved fluoroimmunoassay (TR-FIA) method for bensulfuron-methyl (BSM) based on fluorescence resonance energy transfer (FRET) from a Tb3+ fluorescent chelate with N,N,N',N'-[2,6-bis(3'-aminomethyl-1'-pyrazoly)-4-phenylpyridine] tetrakis(acetic acid) (BPTA-Tb3+) to organic dye, Cy3 or Cy3.5 has been developed. New method combined the use of BPTA-Tb3+ labeled streptavidin, Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody and biotinylated BSM-BSA conjugate (BSA is bovine serum albumin) for competitive-type immunoassay. After BPTA-Tb3+ labeled streptavidin was reacted with a competitive immune reaction solution containing biotinylated BSM-BSA, BSM sample and Cy3 or Cy3.5 labeled anti-BSM monoclonal antibody, the sensitized and long-lived emission of Cy3 or Cy3.5 derived from FRET was measured, and thus the concentration of BSM in sample was calculated. The present method has the advantages of rapidity, simplicity and high sensitivity since the B/F (bound reagent/free reagent) separation steps and the solid-phase carrier are not necessary. The method gives the detection limit of 2.10 ng ml(-1). The coefficient variations of the method are less than 1.5% and the recoveries are in the range of 95-105% for BSM water sample measurement. (C) 2001 Elsevier Science B.V. All rights reserved.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Time-Resolved Synchronous Fluorescence for Biomedical Diagnosis.

This article presents our most recent advances in synchronous fluorescence (SF) methodology for biomedical diagnostics. The SF method is characterized by simultaneously scanning both the excitation and emission wavelengths while keeping a constant wavelength interval between them. Compared to conventional fluorescence spectroscopy, the SF method simplifies the emission spectrum while enabling greater selectivity, and has been successfully used to detect subtle differences in the fluorescence emission signatures of biochemical species in cells and tissues. The SF method can be used in imaging to analyze dysplastic cells in vitro and tissue in vivo. Based on the SF method, here we demonstrate the feasibility of a time-resolved synchronous fluorescence (TRSF) method, which incorporates the intrinsic fluorescent decay characteristics of the fluorophores. Our prototype TRSF system has clearly shown its advantage in spectro-temporal separation of the fluorophores that were otherwise difficult to spectrally separate in SF spectroscopy. We envision that our previously-tested SF imaging and the newly-developed TRSF methods will combine their proven diagnostic potentials in cancer diagnosis to further improve the efficacy of SF-based biomedical diagnostics.