Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Toxigenic phytoplankton and concomitant toxicity in the mussel Choromytilus meridionalis off the west coast of South Africa

The variability of toxigenic phytoplankton and the consequent uptake and loss of toxins by the mussel Choromytilus meridionalis was investigated in the southern Benguela at the event scale (3–10 days) in response to the upwelling–downwelling cycle. Phytoplankton and mussel samples were collected daily (20 March–11 April 2007) from a mooring station (32.04°S; 18.26°E) located 3.5 km offshore of Lambert's Bay, within the St Helena Bay region. Rapid changes in phytoplankton assemblages incorporated three groups of toxigenic phytoplankton: (1) the dinoflagellate Alexandrium catenella; (2) several species of Dinophysis, including Dinophysis acuminata, Dinophysis fortii, Dinophysis hastata and Dinophysis rotundata; and (3) members of the diatom genus Pseudo-nitzschia. Analysis of phytoplankton concentrates by LC–MS/MS or LC-FD provided information on the toxin composition and calculated toxicity of each group. Several additional in vitro assays were used for the analysis of toxins in mussels (ELISA, RBA, MBA for PSP toxins; and ELISA for DSP toxins). Good correspondence was observed between methods except for the MBA, which provided significantly lower (approximately 2-fold) estimates of PSP toxins. PSP and DSP toxins both exceeded the regulatory limits in Choromytilis meridionalis, but ASP toxins were undetected. Differences were observed in the composition of both PSP and DSP toxins in C. meridionalis from that of the ingested dinoflagellates (PSP toxins showed an increase in STX, C1,2, and traces of dcSTX and GTX1,4 and a decrease in NEO; DSP toxins showed an increased in DTX1, and traces of PTX2sa, and a decrease in OA). The rate of loss of PSP toxins following dispersal of the A. catenella boom was 0.12 d⁻¹. Variation in the loss rates of different PSP toxins contributed to the change in toxin profile in C. meridionalis. Prediction of net toxicity in shellfish of the nearshore environment in the southern Benguela is limited due to rapid phytoplankton community changes, high variability in cellular toxicity, and the selective uptake and loss of toxins, and/or transformation of toxins.     

Effect of disease state on ionization during bioanalysis of MK-7009, a selective HCV NS3/NS4 protease inhibitor,in human plasma and human Tween-treated urine by high-performance liquid chromatography with tandem mass spectrometric detection

HPLC–MS/MS methods for the determination of a Hepatitis C NS3/NS4 protease inhibitor (MK-7009) in human plasma and Tween-treated urine were developed and validated over the concentration range 1–1000 ng/mL and 0.2–100 μg/mL respectively. A stable isotope labeled internal standard (ISTD), D₄-MK-7009, was employed. Analytes were chromatographed by reversed phase HPLC and quantified by an MS/MS system. Electrospray ionization in the positive mode was employed. Multiple reaction monitoring of the precursor to product ion pairs m/z 758.6 → 637.4 MK-7009 and m/z 762.5 → 637.4 ISTD was used for quantitation. Analyte and internal standard were extracted from 250 μL of plasma using an automated 96-well liquid–liquid extraction. Plasma pH adjustment prior to extraction minimized ionization suppression in plasma samples from patients with Hepatitis C. The urine method involved direct dilution in the 96-well format of 0.020 mL Tween-treated urine. These methods have supported several clinical studies. Incurred plasma sample reanalysis demonstrated adequate assay reproducibility and ruggedness.     

Uptake of paralytic shellfish poisoning and spirolide toxins by paddle crabs (Ovalipes catharus) via a bivalve vector

The uptake of paralytic shellfish poisoning (PSP) toxins and spirolides by the paddle crab (Ovalipes catharus) was investigated in two laboratory feeding trials using Greenshell? mussels (Perna canaliculus), which had been fed toxic strains of either Alexandrium catenella or A. ostenfeldii, as a vector. Toxin uptake by crabs occurred in both feeding trials and was limited to the visceral tissue; no toxins were detected in the body meat or the gills. The first trial utilized a strain of A. catenella that had high total PSP toxin content, 442.3 ± 91.6 fmol/cell, that was dominated by low toxicity N-sulfocarbamoyl toxins resulting in a low cellular toxicity, 5.5 ± 1.6 pg STXequiv./cell. In this trial, toxin accumulation in the crabs was highly variable and ranged from 3.8 to 221.5 μg STXequiv./100 g, with 3/4 of the crabs exceeding the regulatory limit of 80 μg STXequiv./100 g. Eight days after feeding on toxic mussels the crabs still retained high levels of toxin suggesting that depuration rates in this species may be slow. In the second feeding trial, the A. ostenfeldii strain fed to mussels produced low levels of both PSP toxins (52.0 ± 19.5 fmol/cell; 1.4 ± 0.3 pg STXequiv./cell) and spirolides (1.8 pg/cell) and, as a result, the concentration transferred to crabs via the mussels was very low-PSP toxins ranged from 2.5 to 6.8 μg STXequiv./100 g and spirolides from 6 to 7 μg/kg. The results of our study demonstrate that paddle crabs are capable of acquiring both PSP toxins and spirolides and suggest that this may occur in the wild during a toxic shellfish event. It also highlights the need to remove the viscera before consumption.     

First development and characterisation of polyclonal and monoclonal antibodies to the emerging fresh water toxin cylindrospermopsin

As increasing incidences in the occurrence of cylindrospermopsin (CYN) appear, in addition to further research on its toxicological nature, improved rapid methods to detect this toxin are required. Antibody based assays are renowned for their ability to provide rapid, portable, simple to use tests. As yet however there are no publications outlining how an antibody to CYN can be produced. A range of chemical approaches was investigated to synthesise CYN immunogens for antibody production but failed to generate a response. Finally, a modified Mannich reaction for immunogen synthesis was employed to couple the toxin to two carrier proteins. Both protein conjugates were successfully used to raise both polyclonal and monoclonal antibodies of high sensitivity to CYN. These antibodies were characterised employing competitive indirect ELISA and an optical biosensor assay. By ELISA the sensitivity achieved ranged from 27 to 131 pg/mL and by SPR 4.4 to 11.1 ng/mL thus demonstrating that the selection of immunoassay platform is important for the detection level required by the end user for their application. Low cross-reactivity to the much less toxic metabolite deoxyCYN was observed. This is the first reported production of antibodies to this toxin.     

Estimating the contribution of N-sulfocarbamoyl paralytic shellfish toxin analogs GTX6 and C3 + 4 to the toxicity of mussels (Mytilus galloprovincialis) over a bloom of Gymnodinium catenatum

Gymnodinium catenatum, a dinoflagellate species with a global distribution, is known to produce paralytic shellfish poisoning (PSP) toxins. The profile of toxins of G. catenatum is commonly dominated by sulfocarbamoyl analogs including the C3 + 4 and GTX6, which to date has no commercial certified reference materials necessary for their quantification via chemical methods, such as liquid chromatography. The aim of this study was to assess the presence of C3 + 4 and GTX6 and their contribution to shellfish toxicity. C3 + 4 and GTX6 were indirectly quantified via pre-column oxidation liquid chromatography with fluorescence detection after hydrolysis conversion into their carbamate analogs. Analyses were carried out in mussel samples collected over a bloom of G. catenatum (>63 × 10³ cells l⁻¹) in Aveiro lagoon, NW Portuguese coast. Concentration levels of sulfocarbamoyl toxin analogs were two orders of magnitude higher than decarbamoyl toxins, which were in turn one order of magnitude higher than carbamoyl toxins. Among the sulfocarbamoyl toxins, C1 + 2 were clearly the dominant compounds, followed by C3 + 4 and GTX6. The least abundant sulfocarbamoyl toxin was GTX5. The most important compounds in terms of contribution for sample toxicity were C1 + 2, which justified 26% of the PSP toxicity. The lesser abundant dcSTX constitutes the second most important compound with similar % of toxicity to C1 + 2, C3 + 4 and GTX6 were responsible for approximately 11% and 13%, respectively. The median of the sum of C3 + 4 and GTX6 was 27%. These levels reached a maximum of 60% as was determined for the sample collected closest to the G. catenatum bloom. This study highlights the importance of these low potency PSP toxin analogs to shellfish toxicity. Hydrolysis conversion of C3 + 4 and GTX6 is recommended for determination of PSP toxicity when LC detection methods are used for PSP testing in samples exposed to G. catenatum.     

Spatial distribution of toxic Alexandrium tamiyavanichii (Dinophyceae) in the southeastern South China Sea-Sulu Sea: A molecular-based assessment using real-time quantitative PCR (qPCR) assay

In this study, a quantitative real-time PCR (qPCR) assay targeting the second internal transcribed spacer (ITS2) of the nuclear-encoded ribosomal RNA gene (rDNA) was developed for Alexandrium tamiyavanichii, a harmful tropical marine dinoflagellate. This species is of concern because it produces toxins that cause paralytic shellfish poisoning (PSP). The qPCR assay employed hydrolysis probe technology and showed high specificity, with a detection limit of 10² gene copies (less than one cell equivalent). Using this assay, the spatial distribution of A. tamiyavanichii was assessed, for the first time, in the southeastern South China Sea and the Sulu Sea. Plankton samples were collected from 71 stations during a scientific cruise from the Research Vessel Sonne as part of the joint EU project on Stratosphere ozone: Halogens in a Varying Atmosphere (SHIVA), conducted in November 2011. The highest cell densities were detected offshore of Kuching, southern Borneo (150 cells l⁻¹) and exceeded the threshold level of 20–40 cells l⁻¹ where the bioaccumulation of PSP toxins by shellfish is of concern. The distribution of A. tamiyavanichii was patchy horizontally with the highest cell concentrations found mainly offshore of southern Borneo, and a heterogeneous vertical distribution was observed above the pycnocline. The A. tamiyavanichii qPCR assay proved its applicability, specificity and sensitivity, and provides an alternative implementation tool for harmful microalgae monitoring programs.     

The toxicity and intraspecific variability of Alexandrium andersonii Balech

The toxicity of Alexandrium andersonii Balech is unclear and its intraspecific variability has yet to be studied. To address these gaps in our knowledge, in the present work five strains of A. andersonii from four different localities were characterized. The results showed that despite genetic homogeneity in the 5.8-ITS (internal transcribed spacer) and large subunit (LSU) regions and similar growth rates, strains originating from different locations varied with respect to cell size, the ratios of certain pigments, and their growth patterns. Cultures of the strains grown at 20 °C were analyzed for toxicity using four different methodologies. The two officially established methods, mouse bioassay and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and post-column reaction analysis of PSP toxins, failed to show the toxicity of any strain. Strains grown at 14 °C were also negative for PSP toxins by HPLC-FLD. However, strains grown at 20 °C exhibited both a response characteristic of the presence of toxin-inhibiting voltage-gated sodium channels, as demonstrated in a neuroblastoma neuro-2a cell-based assay, as well as hemolytic activity in a sheep red blood cell assay.     

Sensitive determination of estriol-16-glucuronide using surface plasmon resonance sensing

For the quantitative evaluation of low levels of an estriol metabolite of estriol (estriol-16-glucuronide (E3-16G)) in liquid media, we developed a simple and highly sensitive immunoassay using a surface plasmon resonance (SPR) biosensor which did not require any time-consuming sample pretreatment steps. E3-16G was conjugated to ovalbumin (OVA) through an oligoethylene glycol (OEG) linker to form protein conjugates (E3-16G-OEG-OVA), which were then immobilized on a carboxymethyl dextran-coated sensor chip via amine coupling to develop inhibition immunoassays. A limit of detection (LOD) of 76 pg/mL was achieved using a rabbit anti-sheep primary antibody as a binding agent. The detection limit was further improved by using synthesized gold colloids (15 nm) as high mass labels conjugated to the primary antibody. In this Au nanoparticle-enhanced assay, the concentration of E3-16G in aqueous samples could be determined in 7.5 min at a level as low as 14 pg/mL. In addition, the high stability of the E3-16G-OEG-OVA surface gave no obvious drop in antibody-binding capability after more than 1000 binding/regeneration cycles which significantly lowered the research cost.     

Improved screening of microcystin genes and toxins in blue-green algal dietary supplements with PCR and a surface plasmon resonance biosensor

Microcystins (MCs) comprise a group of cyclic heptapeptide toxins that share a common backbone and have two variable l-amino acids that yield at least 21 known analogs of varying potency. These hepatotoxins and potential tumor promoters are produced by certain cyanobacteria, including Microcystis aeruginosa. The cyanobacterium M. aeruginosa blooms in freshwater lakes and can potentially co-occur with other species such as Aphanizomenon flos-aquae, which is targeted and harvested for the production of dietary supplements known as blue-green algae (BGA). BGA supplements are currently marketed in the U.S. and internationally as a product that may elevate mood, increase energy, and alleviate attention deficit hyperactivity disorder. However, the potential for BGA dietary supplements to be contaminated with MCs is of concern, and there are currently no validated methods for detection of MCs in these products. This research focused on establishing screening methods for toxic Microcystis and MCs in BGA supplements. A DNA-based method employing polymerase chain reaction (PCR) was used as a prescreening tool to evaluate the dietary supplements and to detect the presence of toxin genes (i.e., presence of toxic Microcystis). A rapid, sensitive surface plasmon resonance (SPR) biosensor, directed towards recognition of all MC forms, was also developed and validated. This improved SPR biosensor incorporates a commercial Adda-group antibody (Ab) that has the capacity for broader recognition of MCs than previously developed sensors for BGA supplements that rely solely on an arginine-reactive Ab and can quantitate MC levels down to 0.24 ng/mL (equivalent to 0.24 μg per gram of BGA supplement) in less than 10 min. Such a rapid, quantitative screening method may allow for further surveillance of BGA products to assist risk assessment efforts, establishment of regulatory guidance levels, and response to potential consumer complaints related to BGA products. The PCR technique and SPR biosensor may be used in concert as prescreening and screening tools, respectively or individually, thereby limiting the number of samples that must be evaluated with confirmatory methods.     

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C₁₈ column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis^®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.     

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

BackgroundClinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases.MethodsAntibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones.ResultsCross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R² = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice.ConclusionWe describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia.     

A novel electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

The emergence of Dinophysis acuminata blooms and DSP toxins in shellfish in New York waters

The dynamics of Dinophysis acuminata and its associated diarrhetic shellfish poisoning (DSP) toxins, okadaic acid (OA) and dinophysistoxin-1 (DTX1) as well as pectenotoxins (PTXs), were investigated within plankton and shellfish in Northport Bay, NY, USA, over a four year period (2008–2011). Over the course of the study, Dinophysis bloom densities ranged from ~10⁴ to 10⁶ cells L⁻¹ and exceeded 10⁶ L⁻¹ in 2011 when levels of total OA, total DTX1, and PTX in the water column were 188, 86, and 2900 pg mL⁻¹, respectively, with the majority of the DSP toxins present as esters. These cell densities exceed – by two orders of magnitude – those previously reported within thousands of samples collected from NY waters from 1971 to 1986. The bloom species was positively identified as D. acuminata via scanning electron microscopy and genetic sequencing (cox1 gene). The cox1 gene sequence from the D. acuminata populations in Northport Bay was 100% identical to D. acuminata from Narragansett Bay, RI, USA and formed a strongly supported phylogenetic cluster (posterior probability = 1) that included D. acuminata and Dinophysis ovum from systems along the North Atlantic Ocean. Shellfish collected from Northport Bay during the 2011 bloom had DSP toxin levels (1245 ng g⁻¹ total OA congeners) far exceeding the USFDA action level (160 ng g⁻¹ total OA of shellfish tissue) representing the first such occurrence on the East Coast of the U.S. D. acuminata blooms co-occurred with paralytic shellfish poisoning (PSP) causing blooms of Alexandrium fundyense during late spring each year of the study. D. acuminata cell abundances were significantly correlated with levels of total phytoplankton biomass and Mesodinium spp., suggesting food web interactions may influence the dynamics of these blooms. Given that little is known regarding the combined effects of DSP and PSP toxins on human health and the concurrent accumulation and depuration of these toxins in shellfish, these blooms represent a novel managerial challenge.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

Activation of nylon net and its application to a biosensor for determination of glucose in human serum

A glucose biosensor using a glucose oxidase (GO_x)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O₂) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O₂ in oxidation of glucose by the membrane bound GO_x. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.     

Electrochemical detection of xanthine in fish meat by xanthine oxidase immobilized on carboxylated multiwalled carbon nanotubes/polyaniline composite film

A commercial xanthine oxidase (XOD) was immobilized covalently onto carboxylated multiwalled carbon nanotubes (c-MWCNT) and polyaniline (PANI) composite film electrodeposited on the surface of a Pt electrode, using N-ethyl-N′-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. A xanthine biosensor was fabricated using XOD/c-MWCNT/PANI/Pt electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 4 s at pH 7.0 and 35 °C, when polarized at 0.4 V. The optimized xanthine biosensor showed linear response range of 0.6–58 μM, with a detection limit of 0.6 μM (S/N = 3), and a correlation coefficient of 0.98. The biosensor was applied to determine xanthine in fish meat. The biosensor lost 50% of its initial activity after its 200 uses over a period of 100 days.     

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine,verticinone and isoverticine in rat plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC–LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 μm particle size (Agilent Zorbax StableBond-C₁₈, 4.6 mm × 50 mm, 1.8 μm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic–lipophilic balanced copolymer (2.1 mm × 20 mm, 25 μm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC–MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R > 0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.     

Design and synthesis of a photocleavable biotin-linker for the photoisolation of ligand–receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution

The ability of avidin (Avn) to form strong complex with biotin (Btn) is frequently used in the detection and isolation of biomolecules in biochemical, analytical, and medicinal research. The fact that the binding is nealy irreversible, however, constitutes a drawback in term of the isolation and purification of intact biomolecules. We recently found that 8-quinolinyl esters of aromatic or aliphatic sulfonic acids undergo photolysis when irradiated at 300–330 nm in aqueous solution at neutral pH. In this work, a biotin–dopamine (BD) conjugate containing a photocleavable 8-quinolinyl benzenesulfonate (QB) linker, BDQB, was designed and synthesized for use in the efficient recovery of dopamine–protein (e.g., antibody) complexes from an Avn–Btn system. The complexation of BDQB with a primary anti-dopamine antibody (anti-dopamine IgG₁ from mouse) on an Avn-coated plate was confirmed by an enzyme-linked immunosorbent assay (ELISA) utilizing a secondary antibody (anti-IgG₁ antibody) conjugated with horseradish peroxidase (HRP). Upon the photoirradiation (at 313 nm) of the BDQB–IgG₁ complex, the release of dopamine–IgG₁ complex was confirmed by ELISA. Characterization of the resulting photoreleased dopamine–anti-dopamine IgG₁ complex was performed by SDS–PAGE and Western blot.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.