An organic-phase optical phenol biosensor coupling enzymatic oxidation with chemical reduction

This paper presents a recycle amplification optical biosensor to monitor phenol in hydrophobic organic solvents. Tyrosinase was first immobilised by entrapping it in a copolymer membrane of poly(vinyl alcohol)-hydroxyethyl carboxymethylcellulose doped with octadecylsilica particles. The biosensor was then constructed by co-mixing small particles of the immobilised tyrosinase with the adduct of L-ascorbic acid-poly(vinyl alcohol) (AsA-PVA) in conjunction with an optical oxygen transducer. The biosensor was characterised by its amplifying response to phenol, stable biocatalytic activity of entrapped-tyrosinase, free of interference from o-quinone polymerisation, and large water buffer capacity in hydrophobic organic solvents. The working range of the biosensor to phenol was 0.08-40 mmol dm(-3) in the flow mode. The response times (95%) of the biosensor were 4-7 min for phenol. The operational lifetime was more than 40 assays and the shelf lifetime of the biosensor was longer than 3 months. The biosensor has been successfully applied to quantify the phenol contents in some commercial ointment samples.     

Characterization of the O-4 phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipopolysaccharide using tandem mass spectrometry

The molecular structure of the wild strain of the lipopolysaccharide core of Aeromonas salmonicida, ssp salmonicida has been sequenced using tandem mass spectrometry. The core oligosaccharide was determined to contain an O-4 phosphorylated and O-5 substituted Kdo reducing group, and its structure is proposed as the follows: [structure: see text] After the core oligosaccharide of LPS was released from the lipid A portion by conventional treatment with 1% acetic acid, we demonstrated the existence of a homogeneous mixture composed mainly of the native core oligosaccharide containing the Kdo with its O-4 phosphate group intact, and a degraded core oligosaccharide mixture, which eliminated the O-4 phosphate group with extreme facility. The precise molecular structure and glycone sequence of the homogeneous mixture of phosphorylated and dephosphorylated core oligosaccharides was determined by electrospray ionization (ESI) mass spectrometry and tandem mass spectrometric analysis. CID-MS/MS of the homogeneous mixture of permethylated core oligosaccharides afforded a series of diagnostic product ions which confirmed the established sequence of the glycones to be determined. Matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry reconfirmed the molecular structure of the dephosphorylated homogeneous permethylated mixture of the core oligosaccharides containing the diastereomeric 4,8- and 4,7-anhydro-alpha-keto acids.     

DNA-nanotube artificial ion channels

There is considerable interest in developing chemical devices that mimic the function of biological ion channels. We recently described such a device, which consisted of a single conically shaped gold nanotube embedded within a polymeric membrane. This device mimicked one of the key functions of voltage-gated ion channels: the ability to strongly rectify the ionic current flowing through it. The data obtained were interpreted using a simple electrostatic model. While the details are still being debated, it is clear that ion-current-rectification in biological ion channels is more complicated and involves physical movement of an ionically charged portion of the channel in response to a change in the transmembrane potential. We report here artificial ion channels that rectify the ion current flowing through them via this "electromechanical" mechanism. These artificial channels are also based on conical gold nanotubes, but with the critical electromechanical response provided by single-stranded DNA molecules attached to the nanotube walls.     

Sequential DNA hybridisation assays by fast micromixing

The prospects of performing DNA hybridisation assays in a novel sequential scheme are explored in this article. It is based on recording the kinetics of hybridisation on a microfluidic device measuring only 10 by 5 mm. It contains a split channel system for fast mixing and a subsequent meandering channel to observe the evolution of the mixture by optical means. The problems of diffusion limitations in the laminar flow regime are overcome by reducing the average diffusion distance to a few micrometers only. DNA oligomers (20-mers) of different sequences were injected on the chip for mixing. The detection of hybridisation was based on the fluorescence of DNA-intercalating dyes. Two modes of operation were investigated. First, the samples were injected into the micromixing device at a high flow rate of 40 microl min(-1). When the sample passed through the actual micromixing unit, the flow rate was reduced to allow for measurement of fluorescence levels at various steady-state reaction times in the range of 2-15 s, as defined by the channel geometry. Using this continuous flow approach, photobleaching of fluorophores could be avoided. In a buffer containing 0.2 M NaCl, 2 base-pair mismatches could routinely be detected within 5-20 s. Single base-pair mismatches were successfully identified under low salt conditions. In the second mode, the flow was completely stopped and the evolution of the total fluorescence signal influenced by the hybridisation of oligomers and photobleaching was observed. Whereas the sequence-dependent effects remained unchanged, the assay times between the mixing of two oligomers and clear identification of their hybridisation properties could be reduced down to a maximum of 5-7 s, in some cases even below 1 s.     

Formation of organolithium hetero-aggregates [Li4Ar2(nBu)2] (Ar=C6H4CH(Me)NMe2-2) during the directed ortho-lithiation of [1-(dimethylamino)ethyl]benzene

(R)-[1-(Dimethylamino)ethyl]benzene reacts with nBuLi in a 1:1 molar ratio in pentane to quantitatively yield a unique hetero-aggregate (2 a) containing the lithiated arene, unreacted nBuLi, and the complexed parent arene in a 1:1:1 ratio. As a model compound, [Li(4)(C(6)H(4)CH(Me)NMe(2)-2)(2)(nBu)(2)] (2 b) was prepared from the quantitative redistribution reaction of the parent lithiated arene Li(C(6)H(4)CH(Me)NMe(2)-2) with nBuLi in a 1:1 molar ratio. The mono-Et(2)O adduct [Li(4)(C(6)H(4)CH(Me)NMe(2)-2)(2)(nBu)(2)(OEt(2))] (2 c) and the bis-Et(2)O adduct [Li(4)(C(6)H(4)CH(Me)NMe(2)-2)(2)(nBu)(2)(OEt(2))(2)] (2 d) were obtained by re-crystallization of 2 b from pentane/Et(2)O and pure Et(2)O, respectively. The single-crystal X-ray structure determinations of 2 b-d show that the overall structural motifs of all three derivatives are closely related. They are all tetranuclear Li aggregates in which the four Li atoms are arranged in an almost regular tetrahedron. These structures can be described as consisting of two linked dimeric units: one Li(2)Ar(2) dimer and a hypothetical Li(2)nBu(2) dimer. The stereochemical aspects of the chiral Li(2)Ar(2) fragment are discussed. The structures as observed in the solid state are apparently retained in solution as revealed by a combination of cryoscopy and (1)H, (13)C, and (6)Li NMR spectroscopy.     

Photodissociation of heme distal methionine in ferrous cytochrome C revealed by subpicosecond time-resolved resonance Raman spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.     

Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.     

Hybrid fluorocarbon-hydrocarbon CO2-philic surfactants. 2. formation and properties of water-in-CO2 microemulsions

Hybrid fluorocarbon-hydrocarbon (F-H) sulfate surfactants are shown to be efficient stabilizers in water-in-CO2 (w/c) microemulsions. The chain structure and F-H ratio affect the regions of P-T phase stability and aggregation structure in these w/c phases. High-pressure near-infrared spectroscopy and small-angle neutron scattering measurements of microemulsified water provide evidence for the stabilization of w/c microemulsion droplets. The relative lengths of the two chains were found to influence the favored aggregation structure: for symmetric chain surfactants (F8H8, F7H7) spherical reverse micelles are present, but for asymmetric chain surfactants (F7H4, F8H4) extended cylinder aggregates form. These changes in aggregation are consistent with different surfactant packing parameters owing to the controlled variations in molecular structure. Furthermore, the general order of w/c phase transition pressures (F8H8 < F7H7 and F8H4 < F7H4) is in line with estimations of surfactant fractional free volume, as proposed by Johnston et al. (J. Phys. Chem. B 2004, 108, 1962-1966). Studies of adsorption at the poly(dimethylsiloxane)-water interface are shown to be valuable for assessing the CO2-philicity of new surfactants. All in all, the symmetric F8H8 and F7H7 analogues are seen to be the most efficient compounds from this class for applications in CO2.     

Synthesis and conformational studies of palladium(II) complexes containing [PhP(O)NP(E)Ph]− (E=S or Se)

The ligands [Ph₂P(O)NP(E)Ph₂]⁻ (E=S I; E=Se II) can readily be complexed to a range of palladium(II) starting materials affording new six-membered Pd–O–P–N–P–E palladacycles. Hence ligand substitution reaction of the chloride complexes [PdCl₂(bipy)] (bipy=2,2′-bipyridine), [{Pd(μ-Cl)(L–L)}₂] (HL–L=C₉H₁₃N or C₁₂H₁₃N), [{Pd(μ-Cl)Cl(PMe₂Ph)}₂] or [PdCl₂(PR₃)₂] [PR₃=PPh₃; 2PR₃=Ph₂PCH₂CH₂PPh₂or cis-Ph₂PCH=CHPPh₂] with either I (or II) in thf or CH₃OH gave [Pd{Ph₂P(O)NP(E)Ph₂-O,E}(bipy)]PF₆, [Pd{Ph₂P(O)NP(E)Ph₂-O,E}(L–L)], [Pd{Ph₂P(O)NP(E)Ph₂-O,E}Cl(PMe₂Ph)] or [Pd{Ph₂P(O)NP(E)Ph₂-O,E} (PR₃)₂]PF₆ in good yields. All compounds described have been characterised by a combination of multinuclear NMR [31 P{1 H} and 1 H] and IR spectroscopy and microanalysis. The molecular structures of five complexes containing the selenium ligand II have been determined by single-crystal X-ray crystallography. Three different ring conformations were observed, a pseudo-butterfly, hinge and in the case of all three PR₃ complexes, pseudo-boat conformations. Within the Pd–O–P–N–P–Se rings there is evidence for π-electron delocalisation.