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.

Study on charge transfer reactions at multilayers of polyoxometalates clusters and poly(allylamine hydrochloride) (Grotthuss-018)

Multilayers of anionic phosphotungstic acid (PTA) clusters and positively charged protonated poly(allylamine hydrochloride) (PAH) were assembled by layer-by-layer self-assembled method on Au electrode modified by 3-mercaptopropionic acid (3-MPA). The effect of the charge of the surface of the multilayer assembly on the kinetics of the charge transfer reaction was studied by using the redox probes [Fe(CN)(6)](3-)/(4-) [Ru(NH3)(6)](2+/3+). The cyclic voltammetry experiments showed that the peak currents and peak-to-peak potential differences changed after assembling different layers on the electrode surface indicating that the charge of the surface has a significant effect on the kinetics of the studied charge transfer reactions. These reactions were studied in more detail by electrochemical impedance spectroscopy. When [Fe(CN)(6)](3-/-) was used as the redox label, multilayers that terminated with negatively charged PTA showed a high charge transfer resistance but multilayers that terminated with positively charged PAH showed lower charge transfer resistance. With [Ru(NH3)(6)](2+/3+) as the redox label, the charge transfer resistance at multilayers that terminated with positively charged PAH was much higher than at the multilayer terminated by the negatively charged PTA.

Europium ion labeling anti-HBs

A microsecond time-resolved laser fluorescence spectroscopic analysis set was developed, A chelate-cyclic anhydride of diethylenetrimin pentaacetic acid anhydride (DTPAA) was synthesized. An anti-HBs antibody was purified, A EU3+ -DTPAA-anti-HBs label was prepared by two step procedure. We described the optimal condtion with EU3+ as marker and DTPAA as chelate bounding to antibody molecule. Labeling parameters such as solvent pH, protein and chelate molar ratio, reaction time, separation method were discussed in detail.

Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor

The biosensor based on surface plasmon resonance(SPR) technology is a very useful tool to study the interaction between biomolecles. The main advantages of this technique is to "visualize" macromolecular interactions directly in real time, and in a label-free mode rather than indirect methods like enzyme-linked immunosorbent assays (ELISAs). We immobilize human serum albumin (HSA) to the carboxymethyldextran-modified sensor chip surface covalently to detect the activity of anti-HSA in serum, and regenerate the surface with .1 mol/L phosphoric acid. The results show that SPR biosensor can detect the activity of anti-HSA in real-time quickly and the sensor chip can be used over 100 cycles.

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.

Nanostructured hybrid organic-inorganic lanthanide complex films produced in situ via a sol-gel approach

Communication: Nanostructural hybrid organic-inorganic lanthanide complex films were prepared in situ by use of a novel sol-gel precursor containing pendant triethoxy-silyl and carboxyl groups (see Figure). The resulting transparent and crack-free films gave rise to strong red or green emission, even at low lanthanide ion concentration. Phase separation and lanthanide ion aggregation were controlled at the nanoscale.

Self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine as an immobilization matrix for the construction of a tyrosinase-based amperometric biosensor

A tyrosinase-based amperometric biosensor using a self-gelatinizable graft copolymer of poly(vinyl alcohol) with 4-vinylpyridine (PVA-g-PVP) as an immobilization matrix was constructed. The 4-vinylpyridine component of PVA-g-PVP enhances the adherence to a glassy carbon electrode surface. The content of 4-vinylpyridine in this immobilization matrix plays a key role in retaining the activity of tyrosinase. A simple, milder method was adopted by simply syringing the copolymer-tyrosinase aqueous solution on to the electrode surface and allowing water to evaporate at 4 degrees C in a refrigerator. Several parameters, including copolymer composition; pH, applied potential and enzyme membrane composition, ware optimized. The enzyme membrane composition can be varied to obtain higher sensitivity or a wider linear detection range. The biosensor was used for the determination of phenol, p-cresol and catechol. The biosensor exhibited excellent reproducibility, stability and sensitive response and can be used in flow injection analysis. The biosensor showed an extended linear range in hydrophilic organic solvents and it can be used in monitoring organic reaction processes. The analytical performance demonstrated this immobilization matrix is suitable for the immobilization of tyrosinase.

VOLTAMMETRY OF CYTOCHROME-C ENTRAPPED IN HYDROGEL MEMBRANE ON GRAPHITE ELECTRODE

The voltammetric behavior of cytochrome c entrapped in hydrogel membranes at paraffin wax-impregnated spectroscopic graphite electrodes (WISGE) was studied in this paper. A pair of well-defined peaks appeared at +70 mV (vs. Ag/AgCl). Beside these two peaks, another pair of peaks emerged at around +225 mV. Further investigations suggested that at least three states of cytochrome c existed in the membranes due to the special structure of the hydrogel. The native conformation of cytochrome c molecules was stabilized by the hydrophilic environment that was formed by the hydroxyl structure of the membranes and facilitated the cytochrome c electron transfer reaction at +70 mV. The molecules directly adsorbed on the surface of the graphite electrode were responsible for the redox peaks at around +225 mV. Whether the adsorption peaks were detectable or not was related to the thickness of membranes and the pre-retaining time before the formation of membranes.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by C-14 label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20 : 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-C-14] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG C-14-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, C-14-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of C-14-labeled fatty acids between sn-1 and sn-2 positions might be related to 20 : 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDC.

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (Pseudosciaena crocea) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1: 100 with Ringer's solution and UV irradiated with doses ranging from 0-150 J cm (-2). The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm(-2), after fertilization, motility was < 10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm -2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 208 degrees C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures ( 28 degrees C, 38 degrees C or 48 degrees C) and five shock durations ( 4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 +/- 2.99%) were obtained at 38 degrees C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.

Intracellular copper/zinc superoxide dismutase from bay scallop Argopecten irradians: Its gene structure, mRNA expression and recombinant protein

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Two unique and highly compartmentalized bay scallop Argopecten irradians superoxide dismutases: MnSOD and ecCuZnSOD, have been molecularly characterized in our previous study. To complete characterize the SOD family in A. irradians, a novel intracellular copper/zinc SOD from the A. irradians (Ai-icCuZnSOD) was obtained and characterized. The full-length cDNA of Ai-icCuZnSOD was 1047 bp with a 459 bp open reading frame encoding 152 amino acids. The genomic length of the Ai-icCuZnSOD gene was about 4279 bp containing 4 exons and 3 introns. The promoter region containing many putative transcription factor binding sites were analyzed. Furthermore, quantitative reverse transcriptase real-time PCR (qRT-PCR) analysis indicated that the highest expression of the Ai-icCuZnSOD was detected in gill and the expression profiles in hemocytes of bay scallops challenged with bacteria Vibrio anguillarum and lipopolysaccharide (LPS) were different. The result presented an increased expression after injection with LPS whereas no significant changes were observed after V. anguillarum injection. A fusion protein containing Ai-icCuZnSOD was produced in vitro. The rAi-icCuZnSOD is a stable enzyme, retaining more than 80% of its activity between 10 and 60 degrees C and keeping above 88% of its activity at pH values between 5.8 and 9. Ai-icCuZnSOD is more stable under alkaline than acidic conditions. Crown Copyright (C) 2009 Published by Elsevier Ltd. All rights reserved.

Feeding behaviour of yaks on spring, transitional, summer and winter pasture in the alpine region of the Qinghai-Tibetan plateau

Of the present estimated world population of 14.2 million yaks, approximately 13.3 million occur within Chinese territories (Food and Agriculture Organization of the United Nations, 2003). Although there is an extensive bibliography covering the species, few studies have been conducted in the area of foraging behaviour. The present study was conducted at pasture during the spring, transitional, summer and winter seasons to determine the daily temporal patterns of grazing and ruminating behaviour by yaks. During each study period, two 24 h recordings were undertaken with each of six mature dairy yaks. One study period was conducted on each of the transitional, summer and winter pastures, whereas, due to the considerable changes occurring in the morphology of the spring pasture, three separate studies were completed during March, April and May. During the second of these studies (April), the effect of level of concentrate supplementation on grazing and ruminating behaviour was also examined. Behaviour recordings were made using solid-state behaviour recorders. Short-term intake rates (IR, g min(-1)) were calculated by weighing yaks before and after approximately 1 h of grazing, retaining the faeces and urine excreted and applying a correction for insensible weight loss. Yaks spent less time grazing during the dry season (the early period on the spring pasture) compared with the later green swards (the later period on the spring pasture, the transitional pasture and the summer pasture) (P < 0.05). When the forage quality improved, but there was still insufficient mass (the later period on the spring pasture), the yaks extended their grazing time at the expense of other activities. During the early periods on the spring pasture, the short-term IR by yaks was up to 53 g DM min(-1), significantly higher than at other times (P < 0.05). The level of concentrate offered had little or no effect on grazing or ruminating time. The total eating time of the yaks offered 0.5 or 1.0 kg concentrate was 2.9 and 4.5 h day(-1) respectively, significantly lower than unsupplemented yaks (6.8 h) (P < 0.05). In general, yaks can regulate their foraging behaviour according to the changes of sward conditions in order to achieve optimal grazing strategies. (C) 2007 Published by Elsevier B.V.

Determination of aliphatic amines from soil and wastewater of a paper mill by pre-column derivatization using HPLC and tandem mass spectrometry (HPLC-MS/MS)

A pre-column derivatization method for the sensitive determination of aliphatic amines using the labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) followed by HPLC with fluorescence detection and APCI/NIS identification in positive-ion mode has been developed. The chromophore of 2-(9-carbazole)-ethyl chloroformate (CEOC) reagent was replaced by the 1,2-benzo-3,4-dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent, BCEOC, that could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M + H](+) with APCI/MS in positive-ion mode. The collision induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 264.1, m/z 246.0 and m/z 218.1, corresponding to the cleavages of CH2CH2O-CO, CH2CH2-OCO, and N-CH2CH2O bonds. Studies on derivatization conditions demonstrated that excellent derivatization yields close to 100% were observed with a 3 to 4-fold molar reagent excess in acetonitrile solvent, in the presence of borate buffer (pH 9.0) at 40 degrees C for 10 min. In addition, the detection responses for BCEOC derivatives were compared with those obtained with CEOC and FMOC as labeling reagents. The ratios I-BCEOC/I-CEOC and I-BCEOC/I-FMOC were, respectively, 1.40-2.76 and 1.36-2.92 for fluorescence responses (here, I was the relative fluorescence intensity). Separation of the amine derivatives had been optimized on an Eclipse XDB-C-8 column. Detection limits calculated from an 0.10 pmol injection, at a signal-to-noise ratio of 3, were 18.65-38.82 fmol (injection volume 10 mu L for fluorescence detection. The relative standard deviations for intraday determination (n = 6) of standard amine derivatives (50 pmol) were 0.0063-0.037% for retention times and 3.36-6.93% for peak areas. The mean intra-and inter-assay precision for all amines were <5.4% and 5.8%, respectively. The recoveries of amines ranged from 96 to 113%. Excellent linear responses were observed with correlation coefficients of >0.9994. The established method provided a simple and highly sensitive technique for the quantitative analysis of trace amounts of aliphatic amines from biological and natural environmental samples.

Grazing behavior of lactating and non-lactating yaks in the summer season of the Qinghai-Tibetan plateau

The effect of the physiological states of lactating vs. non-lactating (dry) on grazing behavior and herbage intake by yaks was examined in the summer season in the Qinghai alpine area under continuous stocking management. Intake rates were estimated over periods of 1 h by weighing the animals before and after grazing, retaining the feces and urine excreted, and applying a correction for insensible weight loss (the 1-h weight changes of yaks when non-eating before or after the intake rate measurement). It is hypothesized that the lactating yaks should eat more and spend more time eating than nonlactating yaks, because they expend more energy. In our experiment, there were no differences in the effect of physiological state (lactating vs. dry) of yaks observed on the rate of insensible weight loss, intake rate, grazing jaw movement rate, bites per grazing jaw movement, or bite mass. The dry yaks tended to eat more and spend more time eating than lactating yaks, but not significantly so. Compared with the dry yaks, the lactating yaks had a significantly lower bite rate and bites per bolus.

Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance, liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+ H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH2O-CO and CH2-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate, (FMOC-Cl) as labeling reagents. The ratios I-BCEC/I-BCEOC = 1.94-2.17 and I-BCEC/I-FMOC = 1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C-8 column. Detection limits calculated from 0.50 pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4 fmol. The relative standard deviations for within-day determination (n = 11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were < 3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of > 0.9996. (C) 2006 Elsevier B.V. All rights reserved.