A new and sensitive catalytic resonance scattering spectral assay for the detection of laccase using guaiacol as substrate

In pH 4.0 succinic acid-sodium hydroxide buffer solution, laccase catalyzed the oxidization of guaiacol substrate to form red particles, which exhibited a strong resonance scattering (RS) peak at 590 nm. Under the chosen conditions, as the laccase increased, the RS intensity (ΔI) increased linearly. The ΔI was proportional to laccase activity in the range of 0.10-1.2 U/mL, with a regression equation of ΔI = 734.0 U(laccase) - 9.7, and a detection limit of 0.05 U/mL. This RS method was applied to the detection of laccase activity in real samples, and the results were agreement with those from spectrophotometry.     

CH2 domain orientation of human immunoglobulin G in solution: Structural comparison of glycosylated and aglycosylated Fc regions using small-angle X-ray scattering

The N-linked glycan in immunoglobulin G is critical for the stability and function of the crystallizable fragment (Fc) region. Alteration of these protein properties upon the removal of the N-linked glycan has often been explained by the alteration of the C_H2 domain orientation in the Fc region. To confirm this hypothesis, we examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution. Conformational characteristics of the C_H2 domain orientation were validated by comparison with SAXS profiles theoretically calculated from multiple crystal structures of the Fc region with different C_H2 domain orientations. The reduced chi-square values from the fitting analyses of gFc and aFc associated with the degree of openness or closure of each crystal structure, as determined from the first principal component that partially governed the variation of the C_H2 domain orientation extracted by a singular value decomposition analysis. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a “semi-closed” C_H2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the C_H2 domain orientation in solution. These findings will guide the development of methodology for the production of highly refined functional Fc variants.     

A simple and sensitive resonance Rayleigh scattering method for determination of As(III) using aptamer‐modified nanogold as a probe

A simple and selective aptamer (ssDNA)‐modified nanogold probe (AussDNA) was prepared for the determination of trace As(III) in HEPES buffer solution (pH 8.2) containing 0.05 mol/L NaCl. The method coupled the aptamer reaction of AussDNA–As(III) and the resonance Rayleigh scattering (RRS) of nanogold aggregations at 278 nm. When the As(III) concentration increased, the RRS intensity at 278 nm increased to form more nanogold aggregation and a stable As(III)–ssDNA complex. Under selected conditions, the increased RRS intensity (ΔI) was linear to the concentration of As(III) in the range 3.8–230.4 ng/mL, with a detection limit of 1.9 ng/mL. This RRS method was applied to detect As(III) in water samples, with simplicity, sensitivity and selectivity. Copyright © 2013 John Wiley & Sons, Ltd.     

A sensitive and selective immuno‐nanogold resonance‐scattering spectral method for the determination of trace penicillin G

A sensitive and selective immuno‐nanogold resonance scattering spectral assay was developed for the determination of trace hapten penicillin G, based on the resonance scattering (RS) effect of the nanogold at 560 nm, and the nanogold‐labelled immunoreaction took place in pH 5.4 phosphate citric acid buffer solutions and in the presence of polythylene glycol (PEG). The nanogold‐labelled immunocomplex formed more and more with addition of penicillin G. The enhanced RS intensity at 560 nm ΔI_RS was linear to the penicillin G concentration in the range 7.5–1700 ng/mL, with a detection limit of 0.78 ng/mL. The results indicate that the immunonanogold‐labelled RS spectral assay has a high specificity and sensitivity for quantitative determination of penicillin G in raw milk samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Catalytic effect of ReAu nanoalloy on the Te particle reaction and its application to resonance scattering spectral assay of CA125

ReAu nanoparticles with a molar ratio of 2:8 Re and Te nanoparticles were prepared by NaBH₄ reduction. In HCl medium at 65°C, ultratrace Re, Te and ReAu bimetallic nanoparticles strongly catalyzed the slow reaction between Sn(II) and Te(VI) to form Te particles, which exhibited the strongest resonance scattering (RS) peak at 782 nm. As the amount of nanocatalyst increased, the RS intensity at 782 nm (I_{782 nm}) increased linearly, and the increase in intensity ΔI_{782 nm} was linear to the ReAu, Re and Te concentrations in the ranges 0.07–9.0, 0.01–4.5 and 30–1200 nm , respectively. As a model, a ReAu immunonanoprobe catalytic Te–particle resonance scattering spectral (RSS) method was established for detection of CA125, using ReAu nanoparticle labeling CA125 antibody (CA125Ab) to obtain an immunonanoprobe (ReAuCA125Ab) for CA125. In pH 7.6 citric acid–Na₂HPO₄ buffer solution, ReAuCA125Ab aggregated nonspecifically. Upon addition of CA125, the immunonanoprobe reacted with it to form ReAuCA125Ab–CA125 dispersive immunocomplex in the solution. After the centrifugation, the supernatant containing the immunocomplex was used to catalyze the reaction of Te(VI)–Sn(II) to produce the Te particles that resulted in the I_{782 nm} increasing. The ΔI_{782 nm} was linear to CA125 concentration (C_CA125) in the range 0.1–240 mU/mL. The regression equation, correlation coefficient and detection limit were ΔI_{782 nm} = 1.61 C_CA125 + 1.5, 0.9978 and 0.02 mU/mL, respectively. The proposed method was applied to detect CA125 in serum samples, with satisfactory results. Copyright © 2010 John Wiley & Sons, Ltd.     

A new and highly sensitive resonance Rayleigh scattering assay for lysozyme using aptamer–nanogold as a probe

Gold nanoparticles (GN), 10 nm in size, were modified by using lysozyme aptamer (Apt) to obtain a stable Apt–GN probe in pH 8.05 Tris/HCl buffer solutions containing 0.04 mol/L NaCl. Upon addition of lysozyme (LYS), it reacted with the Apt of the probe to form a very stable Apt–LYS complex and to release GNs, which aggregated to form large clusters with a resonance Rayleigh scattering (RRS) peak at 368 nm. The enhanced peak intensity, ΔI, was linear to the LYS concentration in the range 0.2–5.2 nmol/L, with a detection limit of 0.05 nmol/L. The influence of foreign substance was tested, and the results showed that this RRS method has high selectivity. This Apt–GN RRS method was applied to the analysis of LYS in a real sample, with satisfactory results. Copyright © 2014 John Wiley & Sons, Ltd.     

Mercaptoheterocyclic ligands grafted on a poly(ethylene vinyl alcohol) membrane for the purification of immunoglobulin G in a salt independent thiophilic chromatography

In this study, we attempted a limited combinatorial approach for designing affinity ligands based on mercaptoheterocyclic components. The template, divinyl sulfone structure (DVS), which was grafted on poly(ethylene vinyl alcohol) (PEVA) hollow fiber membrane, has served for the tethering of different heterocyclic compounds as pyridine, imidazole, purine and pyrimidine rings. Their ability to adsorb specifically IgG in a salt independent manner out of pure IgG solution, mixture of IgG/albumin and human plasma was demonstrated. Mercapto methyl imidazole (MMI) has shown the best adsorption of IgG in terms of binding capacity. No subclass discrimination was observed on all tested ligands except for mercapto methyl pyrimidine where the major IgG subclass adsorbed was IgG3. MMI gave an IgG binding capacity of 100 microg/cm2 of hollow fiber membrane surface area.     

A substrate-unspecified glycosylation reaction promoted by copper(II) trifluoromethanesulfonate in benzotrifluoride

A glycosylation reaction induced by copper(II) trifluoromethanesulfonate is described. Using benzotrifluoride as the reaction solvent, five kinds of glycosyl donors, a glucosyl chloride, a fluoride, a trichloroacetimidate, a 1-O-acetyl compound, and a lactol were activated to give the corresponding glucosides.     

A mixed-valence Cu(I)-Cu(II) and metal-metal bond containing coordination polymer obtained from an in situ oxidation reaction route

A new mixed-valence copper coordination polymer with copper-copper metal bonds in a two-dimensional network was generated from an in situ oxidation reaction route under hydrothermal conditions. The synthesis of this coordination polymer demonstrated that the novel compounds that may not be accessible using the known methods could be synthesized via an oxidation reaction route. The reaction conditions are mild enough to keep the building blocks intact during the oxidation and self-assembly process under hydrothermal conditions.     

Phenoxyl-copper(II) complexes: models for the active site of galactose oxidase

 The reaction of the macrocycles 1,4,7-tris (3,5-di-tert-butyl-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L¹H₃, or 1,4,7-tris(3-tert-butyl-5-methoxy-2-hydroxy-benzyl)-1,4,7-triazacyclononane, L²H₃, with Cu(ClO₄)₂·6H₂O in methanol (in the presence of Et₃N) affords the green complexes [Cu^II(L¹H)] (1), [Cu^II(L²H)]·CH₃OH (2) and (in the presence of HClO₄) [Cu^II(L¹H₂)](ClO₄) (3) and [Cu^II(L²H₂)] (ClO₄) (4). The Cu^II ions in these complexes are five-coordinate (square-base pyramidal), and each contains a dangling, uncoordinated pendent arm (phenol). Complexes 1 and 2 contain two equatorially coordinated phenolato ligands, whereas in 3 and 4 one of these is protonated, affording a coordinated phenol. Electrochemically, these complexes can be oxidized by one electron, generating the phenoxyl-copper(II) species [Cu^II(L¹H)]^+ ·, [Cu(L²H)]^+ ·, [Cu^II(L¹H₂)]^2+ ·, and [Cu^II(L²H₂)]^2+ ·, all of which are EPR-silent. These species are excellent models for the active form of the enzyme galactose oxidase (GO). Their spectroscopic features (UV-VIS, resonance Raman) are very similar to those reported for GO and unambiguously show that the complexes are phenoxyl-copper(II) rather than phenolato-copper(III) species. Received: 10 February 1997 / Accepted: 7 April 1997     

Probing inter-ligand excited state interaction in homo and heteroleptic ruthenium(II) polypyridyl complexes using selective deuteriation

The effect of deuteriation on the photophysical properties of two series of regioselectively deuteriated Ru(II) complexes ([Ru(bipy)_x(ph₂phen)_3−x]²⁺, where x = 0-3 and ph₂phen is 4,7-diphenyl-1,10-phenanthroline and [Ru(bipy)₂(dcbipy²⁻)], where H₂dcbipy is 4,4′-dicarboxy-2,2′-bipyridyl) is reported. Although overall, deuteriation results in an increase in emission lifetime for all complexes, the effect of substitution of hydrogen for deuterium shows strong regioselectivity both in terms of the ligand and the position on individual ligands that are exchanged.     

A simple and sensitive assay for evaluation of sediment toxicity using Lumbriculus variegatus (Müller)

The burrowing oligochaete Lumbriculus variegatus (Muller) was evaluated as a test organism for sediment bioassessment. As part of a battery of assays, the survival, growth, reproduction and burrowing behaviour of Lumbriculus in sediments from several areas of the North American Great Lakes were examined. Although not abundant, this species can be found in oligotrophic and mesotrophic environments. They are readily cultured and can be commercially purchased. Chronic tests indicated that Lumbriculus was as sensitive to contaminated sediments as Hyalella azteca, but less sensitive than the Holarctic amphipod Pontoporeia (Diporeia) hoyi. The growth of individuals or their reproduction by cloning can be measured after a two-week exposure at 20 °C.     

A dye-binding assay for measurement of the binding of Cu(II) to proteins

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to α-synuclein of ∼1 × 10⁹ M⁻¹ was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).     

A new and simple resonance Rayleigh scattering method for human serum albumin using graphite oxide as probe

Graphite oxide (GO) was prepared by the Hummer procedure, and can be dispersed to stable colloid solution by ultrasonic wave. The GO exhibited an absorption peak at 313 nm, and a resonance Rayleigh scattering (RRS) peak at 490 nm. In pH 4.6 HAc‐NaAc buffer solution, human serum albumin (HSA) combined with GO probe to form large HSA‐GO particles that caused the RRS peak increasing at 490 nm. The increased RRS intensity was linear to HSA concentration in the range 0.50–200 µg/mL. Thus, a new and simple RRS method was proposed for the determination of HSA in samples, with a recovery of 98.1–104%. Copyright © 2012 John Wiley & Sons, Ltd.     

Behavior of human immunoglobulin G adsorption onto immobilized Cu(II) affinity hollow‐fiber membranes

Iminodiacetic acid (IDA) and tris(2‐aminoethyl)amine (TREN) chelating ligands were immobilized on poly(ethylene vinyl alcohol) (PEVA) hollow‐fiber membranes after activation with epichlorohydrin or butanediol diglycidyl ether (bisoxirane). The affinity membranes complexed with Cu(II) were evaluated for adsorption of human immunoglobulin G (IgG). The effects of matrix activation and buffer system on adsorption of IgG were studied. Isotherms of batch IgG adsorption onto finely cut membranes showed that neither of the chelates, IDA‐Cu(II) or TREN‐Cu(II), had a Langmuirean behavior with negative cooperativity for IgG binding. A comparison of equilibrium and dynamic maximum capacities showed that the dynamic capacity for a mini‐cartridge in a cross‐flow filtration mode (52.5 and 298.4 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively) was somewhat higher than the equilibrium capacity (9.2 and 73.3 mg g^?1 dry weight for PEVA‐TREN‐Cu(II) and PEVA‐IDA‐Cu(II), respectively). When mini‐cartridges were used, the dynamic adsorption capacity of IDA‐Cu(II) was the same for both mini‐cartridge and agarose gel. Copyright © 2013 John Wiley & Sons, Ltd.     

Identification of aqueous pollen extracts using surface enhanced Raman scattering (SERS) and pattern recognition methods

Aqueous pollen extracts of varying taxonomic relations were analyzed with surface enhanced Raman scattering (SERS) by using gold nanoparticles in aqueous suspensions as SERS substrate. This enables a selective vibrational characterization of the pollen water soluble fraction (mostly cellular components) devoid of the spectral contributions from the insoluble sporopollenin outer layer. The spectra of the pollen extracts are species‐specific, and the chemical fingerprints can be exploited to achieve a classification that can distinguish between different species of the same genus. In the simple experimental procedure, several thousands of spectra per species are generated. Using an artificial neural network (ANN), it is demonstrated that analysis of the intrinsic biochemical information of the pollen cells in the SERS data enables the identification of pollen from different plant species at high accuracy. The ANN extracts the taxonomically‐relevant information from the data in spite of high intra‐species spectral variation caused by signal fluctuations and preparation specifics. The results show that SERS can be used for the reliable characterization and identification of pollen samples. They have implications for improved investigation of pollen physiology and for allergy warning.

     

An integrated system of pyrene and rhodamine-6G for selective colorimetric and fluorometric sensing of mercury(II)

A pyrene and rhodamine-6G functionalized simple chemosensor L is studied toward sensing of metal ions in solution extensively. L shows selective color change from colorless to pink in the presence of Hg²⁺ in acetonitrile and the UV-Vis study shows peak at 525 nm with a ε value of 5.2 × 10⁴ M⁻¹ cm⁻¹ due to selective ring opening of rhodamine spirolactam moiety. The selective sensing of Hg²⁺ by L in the presence of other metal ions and reversible nature of “OFF-ON-OFF” functionality of L by Hg²⁺ and EDTA, respectively, are also established. The fluorescence study of L in the presence of Hg²⁺ shows emission at 550 nm when excited at 525 nm (ring opened rhodamine wavelength) or 340 nm (pyrene wavelength) in dry CH₃CN. Thus L acts as a selective colorimetric and fluorometric probe (dual probe) for the Hg²⁺ in solution. Metal ion sensing ability of L is also carried out in water as well as in aqueous Hepes buffer. These studies suggest that the fluorescence output of L in presence of Hg²⁺ in aqueous environment is apparently due to the generation of acid upon addition of Hg²⁺ salt in water.     

Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H(2)O(2) in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.