Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Flow injection determination of dopamine based on inhibited electrochemiluminescence of luminol

A flow injection method has been developed for the determination of dopamine based on its inhibition of the electrochemiluminescence of luminol. This method is simple and sensitive for dopamine detection. Under the selected experimental conditions, the decreased electrochemiluminescent intensity is linear with dopamine concentration in the range of 5.0 x 10(-8)-1.0 x 10(-5) mol/L with a detection limit of 30 nmol/L. The relative standard deviation of eleven determinations is 1.9% for 1.0 x 10(-6) mol/L dopamine. The proposed method has been applied to the detection of dopamine in pharmaceutical injections with satisfactory results.

A novel flow through optical fiber biosensor for glucose based on luminol electrochemiluminescence

A novel flow injection optical fiber biosensor for glucose based on luminol electrochemiluminescence (ECL) is presented. The sol-gel method is introduced to immobilize glucose oxidase (GOD) on the surface of a glassy carbon electrode. After optimization of the working conditions, glucose could be quantitated in the concentration ranges between 50 muM and 10 mM with a detection limit of around 26 muM. Signal reproducibility was about 3.62% relative standard deviation for 11 replicated measurements of 0.1 mM glucose. The ECL biosensor also showed good selectivity and operational stability. The proposed method can be applied to determination of glucose in soft drink samples.

Electrochemiluminescent determination of glucose with a sol-gel derived ceramic-carbon composite electrode as a renewable optical fiber biosensor

A sol-gel derived ceramic-carbon composite electrode is used for fabrication of a new type of optical fiber biosensor based on luminol electrochemiluminescence (ECL). The electrode consists of graphite powder impregnated with glucose oxidase in a silicate network. In this configuration, the immobilized enzyme oxidizes glucose to liberate hydrogen peroxide and graphite powder provides percolation conductivity for triggering the ECL between luminol and the liberated hydrogen peroxide. Both of the reactions occur simultaneously on the surface of the composite electrode, thereby the response of the biosensor is very fast. The peak intensity was achieved within only 20 s after glucose injection. In addition, the electrode could be renewed by a simple mechanical polishing step in case of contamination or fouling. The linear range extends from 0.01 to 10 mM for glucose and the detection limit is about 8.16 muM. The renewal repeatability and stability of the biosensor are also investigated in detail.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Rapid quantification of total microcystins in cyanobacterial samples by periodate-permanganate oxidation and reversed-phase liquid chromatography

Microcystins (MCs) comprise a family of more than 80 related cyclic hepatotoxic heptapeptides. Oxidation of MCs causes cleavage of the chemically unique C-20 beta-amino acid (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) amino to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB), which has been exploited to enable analysis of the entire family. In the present study, the reaction conditions (e.g. concentration of the reactants. temperature and pH) used in the production of MMPB by oxidation of cyanobacterial samples with permanganate-periodate were optimized through a series of well-controlled batch experiments. The oxidation product (MMPB) was then directly analyzed by high-performance liquid chromatography with diode array detection. The results of this study provided insight into the influence of reaction conditions on the yield of MMPB. Specifically, the optimal conditions, including a high dose of permanganate (>= 50 mM) in saturated periodate solution at ambient temperature under alkaline conditions (pH similar to 9) over 1-4 h were proposed, as indicated by a MMPB yield of greater than 85%. The technique developed here was applied to determine the total concentration of MCs in cyanobacterial bloom samples, and indicated that the MMPB technique was a highly sensitive and accurate method of quantifying total MCs. Additionally, these results will aid in development of a highly effective analytical method for detection of MMPB as an oxidation product for evaluation of total MCs in a wide range of environmental sample matrices, including natural waters, soils (sediments) and animal tissues. (C) 2009 Elsevier B.V. All rights reserved.

A novel chemiluminescent immunoassay for microcystin (MC) detection based on gold nanoparticles label and its application to MC analysis in aquatic environmental samples

A novel chemiluminescent immunoassay method based on gold nanoparticles was developed for the detection of microcystins (MCs). The immunoassay included three main steps: indirect competitive immunoreaction, oxidative dissolution of gold nanoparticles, and indirect determination for MCs with Au3+-catalysed luminol chemiluminesent system. The method has a wide working range (0.05-10 mu g L-1, r(2) = 0.9914), the limit of detection was determined to be 0.024 mu g L-1, which is much lower than the World Health Organization's proposed guidelines (1 mu g L-1) for drinking-water. The proposed method was applied to MC analysis in natural water and fish tissue samples, and most results in the proposed method were in agreement with the conventional indirect competitive enzyme-linked immunosorbent assay method, which indicated that the new chemiluminescent immunoassay was sensitive, reliable, and suitable for MC analysis in natural water and fish tissue samples.

The optimization of high performance liquid chromatographic method for analysis of trace microcystins in natural water bodies

The ratio of methanol., water and trifluoroacetic acid ( TFA) was regulated to change the polarity and the pH of the rinse solution and the eluent, so as to improve the high performance liquid chromatography HPLC) detection method for trace microcystines (MCs) in natural water bodies. The results showed that 40 % similar to 45 % methanol-water solution containing 0. 1 % TFA could get good effects on the rinse of impurity, and 70% methanol-water solution containing 0. 1% TFA could elute all the MCs in solid phase extraction ( SPE) cartridge ( C-18), In this way. it is suggested that, in analysis of environmental samples with high concentration of impurity, impurity should be washed with 40% similar to 45% methanol-water solution containing 0. 1% TFA, and MCs should be eluted with 70% similar to 100% methanol-water solution containing 0. 1% TFA.

Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China)

The species-specific production of extracellular phosphatases in phytoplankton of a subtropical polymictic take was investigated from March to May 2004. Phosphatase activity was detected directly at the site of enzyme action using the enzyme-labelled fluorescence (ELF) technique. Size fractionation of bulk phosphatase activity (PA), concentrations of soluble reactive phosphorus (SRP), chlorophyll a, and phytoplankton composition were determined in parallel. Phosphatase-positive cells were present in every phytoplankton sample; labelled cells were detected in 33 algal taxa, including many chlorophytes, dinoflagellates and some diatoms, but never among cyanobacteria. We recorded an unusual dinoflagellate bloom (Peridiniopsis sp.), of which similar to 25% of the cells were phosphatase-positive. Several populations were partly phosphatase-positive whenever present, while some other species never showed any activity. The production of extracellular phosphatases was not primarily regulated by ambient P concentrations; algae produced these enzymes even if SRP concentrations were high. Moreover, heterotrophic nanoflagellates most probably contributed to the pool of particle-bound PA in some samples.

Investigation of PL properties of C-doped SiO2/Si samples after high energy Pb ion irradiation

Amorphous SiO2 thin films with about 400-500 nm in thickness were thermally grown on single crystalline silicon. These SiO2/Si samples were firstly implanted at room temperature (RT) with 100 keV carbon ions to 2.0 x 10(17),5.0 X 10(17) or 1.2 x 10(18) ions/cm(2), then irradiated at RT by 853 MeV Pb ions to 5.0 x 10(11), 1.0 X.10(12) 2.0 x 10(12) or 5.0 x 10(12) ions/cm(2), respectively. The variation of photoluminescence (PL) properties of these samples was analyzed at RT using a fluorescent spectroscopy. The obtained results showed that Pb-ion irradiations led to significant changes of the PL properties of the carbon ion implanted SiO2 films. For examples, 5.0 x 10(12) Pb-ions/cm(2) irradiation produced huge blue and green light-emitters in 2.0 x 10(17) C-ions/cm(2) implanted samples, which resulted in the appearance of two intense PL peaks at about 2.64 and 2.19 eV. For 5.0 x 10(17) carbon-ions/cm(2) implanted samples, 2.0 x 10(12) Pb-ions/cm(2) irradiation could induce the formation of a strong and wide violet band at about 2.90 eV, whereas 5.0 x 10(12) Pb-ionS/cm(2) irradiation could,create double peaks of light emissions at about 2.23 and 2.83 eV. There is no observable PL peak in the 1.2 x 10(18) carbon-ions/cm(2) implanted samples whether it was irradiated with Pb ions or not. All these results implied that special light emitters could be achieved by using proper ion implantation and irradiation conditions, and it will be very useful for the synthesis of new type Of SiO2-based light-emission materials.

Photo-sensitized oxidation in natural water via .OH radicals

A method is presented for determining production and consumption rates of .OH radicals produced photochemically in natural surface waters. It is based on the determination of the kinetics by which the concentration of a specified trace compound decreases during irradiation. In samples from Lake Greifensee (Switzerland) low production rates for .OH limit its possible effects. In addition, fast consumptions by the natural dissolved organic solutes and by the bicarbonate protect organic micropollutants from oxidation by .OH. Neither direct nor indirect H2O2 photolysis was a significant source of .OH in the lakewater studied lacking iron, whereas nitrate photolysis could have been a source. Comparison with reaction kinetic formulations allows generalizations for other types of waters.