A Förster Resonance Energy Transfer (FRET)-Based Immune Assay for the Detection of Microcystin-LR in Drinking Water.

Cyanobacteria bloom is the term used to describe an abnormal and rapid growth of cyanobacteria in aquatic ecosystems such as lakes, rivers, and oceans as a consequence of anthropic factors, ecosystem degradation, or climate change. Cyanobacteria belonging to the genera Microcystis, Anabaena, Planktothrix, and Nostoc produce and release toxins called microcystins (MCs) into the water. MCs can have severe effects on human and animal health following their ingestion and inhalation. The MC structure is composed of a constant region (composed of five amino acid residues) and a variable region (composed of two amino acid residues). When the MC variable region is composed of arginine and leucine, it is named MC-LR. The most-common methods used to detect the presence of MC-LR in water are chromatographic-based methods (HPLC, LC/MS, GC/MS) and immunological-based methods (ELISA). In this work, we developed a new competitive Förster resonance energy transfer (FRET) assay to detect the presence of traces of MC-LR in water. Monoclonal antibody anti-MC-LR and MC-LR conjugated with bovine serum albumin (BSA) were labeled with the near-infrared fluorophores CF568 and CF647, respectively. Steady-state fluorescence measurements were performed to investigate the energy transfer process between anti-MC-LR 568 and MC-LR BSA 647 upon their interaction. Since the presence of unlabeled MC-LR competes with the labeled one, a lower efficiency of FRET process can be observed in the presence of an increasing amount of unlabeled MC-LR. The limit of detection (LoD) of the FRET assay is found to be 0.245 nM (0.245 µg/L). This value is lower than the provisional limit established by the World Health Organization (WHO) for quantifying the presence of MC-LR in drinking water.

Oriented Functionalization of Magnetic Beads with in Vivo Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples.

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 µg/L of MC.

Immunomodulatory effects of cyanobacterial toxin cylindrospermopsin on innate immune cells.

Cylindrospermopsin (CYN), a cyanobacterial toxin, is an important water pollutant with broad biological activity. It has been known mainly from tropical areas, but the area of occurrence of its producers is spreading to temperate climates. It can be found in high concentrations in the environment as well as in purified drinking waters. The aim of the study is to bring a basic information on the ability of CYN to interfere with mammalian innate immunity cells and thus increase the understanding of the immunomodulatory potency of CYN. This study investigated whether immune cells can be a target of CYN either alone or in combination with a model immunomodulatory agent, lipopolysaccharide (LPS). We examined the effects on cellular viability and inflammation signaling of CYN on murine macrophage-like RAW 264.7 cells. Macrophages were treated either with pure toxin (1 µM) or together with a known stimulator of immunologically active cells, bacterial or cyanobacterial LPS. CYN has had a significant effect on production on pro-inflammatory mediator tumor necrosis factor α (TNF-α) which correlates with its effect on reactive oxygen species (ROS) production. We found that CYN potentiated the effect of bacterial and cyanobacterial LPS that was documented by activation of inflammatory signaling pathways including mitogen-activated protein kinase p38 as well as consequent expression of inducible nitric oxide synthase (iNOS) and increased production of pro-inflammatory mediators such as nitric oxide (NO), TNF-α, interleukin-6 (IL-6). Our study brings one of the first information that contributes to the elucidation of immunomodulatory role of CYN in macrophages under normal and pro-inflammatory conditions.

High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay.

Microcystin-LR (MC-LR) is one of common high-toxic biotoxins produced by cyanobacteria in waterbody. A high sensitive and convenient detection method is necessary for monitoring for MC-LR. To establish a high sensitive indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) based on single chain variable fragment (scFv) for detecting MC-LR, 16 positive anti-MC-LR phage scFv particles were screened out from a MC-LR-immunized mouse phage scFv library, which was successfully constructed with the capacity of 8.67 × 107 CFU/mL. The most positive anti-MC-LR phage scFv (MscFv7) was successfully expressed in Escherichia coli (E.coli) HB2151. The molecular weight (M.W.) of expressed protein was about 30 kDa, and the concentration of purified protein was 512.6 µg/mL analyzed by SDS-PAGE and protein quantitative respectively. The IC-ELISA based on MscFv7-scFv for MC-LR shows a half-maximum inhibition (IC50) of 0.471 µg/L and a limit of detection (LOD) of 0.044 µg/L, which is below the maximum residue limit standard (MRLs) of 1.0 µg/L in drinking water. The MscFv7-scFv has a strong cross-recognition for MC-RR and MC-YR with cross-reactivity (CRs) of 93.1% and 85.9%, respectively, but weak for MC-LW with that of 9.7%, even non-recognition for MC-WR, MC-LF and MC-LY. The recovery rates of IC-ELISA to detect MC-LR spiked in different cleanliness of water samples were 81.2-106.3% with CVs of 2.62-10.22% at intra-assay and inter-assay. The results showed that we obtained a high sensitive anti-MC-LR scFv, and the established IC-ELISA based on MscFv7-scFv should be promising for ultrasensitive monitoring MC-LR, MC-RR and MC-YR in water samples.

Understanding microcystin-LR antibody binding interactions using in silico docking and in vitro mutagenesis.

Microcystins (MCs) are a group of highly potent cyanotoxins that are becoming more widely distributed due to increased global temperatures and climate change. Microcystin-leucine-arginine (MC-LR) is the most potent and most common variant, with a guideline limit of 1 µg/l in drinking water. We previously developed a novel avian single-chain fragment variable (scFv), designated 2G1, for use in an optical-planar waveguide detection system for microcystin determination. This current work investigates interactions between 2G1 and MC-LR at the molecular level through modelling with an avian antibody template and molecular docking by AutoDock Vina to identify key amino acid (AA) residues involved. These potential AA interactions were investigated in vitro by targeted mutagenesis, specifically, by alanine scanning mutations. Glutamic acid (E) was found to play a critical role in the 2G1-MC-LR binding interaction, with the heavy chain glutamic acid (E) 102 (H-E102) forming direct bonds with the arginine (R) residue of MC-LR. In addition, alanine mutation of light chain residue aspartic acid 57 (L-D57) led to an improvement in antigen-binding observed using enzyme-linked immunosorbent assay (ELISA), and was confirmed by surface plasmon resonance (SPR). This work will contribute to improving the binding of recombinant anti-MC-LR to its antigen and aid in the development of a higher sensitivity harmful algal toxin diagnostic.

Microcystin-LR Enrichment from Freshwater by a Recombinant Plant-derived Antibody Using Sol-Gel-Glass Immunoextraction.

Eutrophication of water bodies can promote cyanobacterial (blue-green algae) blooms, which has become a source of increasing concern for both recreational and drinking water use. Many bacterial species can produce toxins that pose threats to wildlife, domestic animals and humans. Microcystin-leucine-arginine (MC-LR) is the most frequent and most toxic microcystin congener. For the first time, lab-scale investigations were performed to test the application of a recombinant plant-derived anti-MC-LR antibody immobilized on an immunoaffinity support material to selectively extract the toxin from spiked freshwater samples. As a comparison, its hybridoma-derived counterpart (murine monoclonal antibody) was evaluated. The antibody-doped material was prepared via an optimized sol-gel process; its stability and binding efficiency of MC-LR in spiked freshwater samples were thoroughly tested using the ELISA and orthogonal LC-MS methods. For removal, two column-based procedures with sequential or continuous cyclic sample addition and a suspension mode (moving adsorbent) were tested. Noteworthy the results obtained with a crude antibody fraction were fully compatible with the highly purified preparation. This study paves the way for further investigation being focused on novel applications of plant-derived anti-MC-LR antibodies in bioremediation to selectively deplete the toxin from freshwater: a green and promising technology without secondary pollution.

Construction of an immunized rabbit phage display antibody library for screening microcystin-LR high sensitive single-chain antibody.

Microcystin-LR (MC-LR) is one of the most common biotoxin that pollutes water and agricultural products. The study aims to obtain the high sensitive anti-MC-LR single-chain antibody (scFv) for detecting MC-LR. Here, a MC-LR-immunized rabbit phage display scFv library with its capacity of 3.26×109CFU/mL was constructed and used for anti-MC-LR phage scFv particles screening. After four rounds of biopanning, 18 positives were isolated and identified successfully. The most positive scFv (RscFv3) was expressed in Escherichia coli HB2151 and purified with a concentration of 796.7µg/mL, and the purified anti-MC-LR polyclonal antibodies (PAbs) were 3.6mg/mL. The PAbs and scFv based indirect competitive enzyme linked immunosorbent assay (IC-ELISAs) were established for MC-LR and its analogues, and the results showed they all had high sensitive for MC-LR with detection limits of 0.03 and 0.05µg/L, and had strong cross-reactivity for MC-RR, MC-WR and MC-YR, respectively. The average recovery rate was 91.9% with coefficient of variation <6.8% for scFv-based IC-ELISA to detect MC-LR spiked in water samples, which met the requirement of indoor testing. The present results indicate that we have obtained a high sensitive anti-MC-LR scFv by the MC-LR-immunized phage library construction and screening, and the scFv-based IC-ELISA can be used for monitoring MC-LR in water samples.

Enhancing recombinant antibody performance by optimally engineering its format.

Antibody-based sensors are now widely used in therapeutics, diagnostics, and in environmental monitoring. Recombinant antibodies are becoming integral parts of such devices due to their reported high affinities, their capacity for engineering to achieve highly defined performance characteristics and the fact that their production can be optimized to a significant degree. To aid as a model for the identification of important analyte binding residues within the antibody sub-structure and elucidate the docking characteristics of small molecules such as metabolites, illicit drugs, biotherapeutics (proteins, peptides and nucleic acids) or toxins towards the antibody, herein we report the binding of the harmful cyanobacterial-toxin, microcystin-leucine-arginine (MC-LR) to a single chain fragment variable (scFv) antibody fragment. Analysis of the binding of MC-LR to this scFv was used to identify key residues of interest and to show how 'freely-available' and 'easily-accessible' computer-based webservers can be utilized to initiate an investigation into the binding characteristics of interacting molecules. In this study, a detailed investigation of the sub-structure of the anti-MC-LR (scFv) was carried out and antibody/small-molecule binding interactions were analyzed. The profile elucidated using computational analysis revealed amino acids of importance in the complementarity determining region light chain region 3 (CDRL3) and framework region 3 (FR3) of the heavy chain. Important amino acid residues within CDRL3 and FR3 were mutated in vitro and sensitivity and binding profiles were examined. It was identified that phenylalanine (F) at position 91 and aspartate (D) at position 92 of the light chain region, and arginine (R) at position 66 in framework region 3 (FR3) of the heavy chain were nvolved in binding. The introduction of an auxiliary antibody domain to the variable heavy and variable light (scFv) to ascertain its influence on stability and binding was also examined. The strategy adopted provided a deeper knowledge of scFv sub-structure and identified the regions and amino acids essential to antibody/small-molecule binding.

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC50) were 0.87, 1.17 and 1.47µg/L, their detection limits (IC10) were 0.06, 0.08 and 0.12µg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs.

Addressed immobilization of biofunctionalized diatoms on electrodes by gold electrodeposition.

Diatoms are single cell microalgae with a silica shell (frustule), which possess a micro/nanoporous pattern of unparalleled diversity far beyond the possibilities of current micro- and nanofabrication techniques. To explore diatoms as natural three-dimensional nanostructured supports in sensing and biosensing devices, a simple, rapid and stable method to immobilize diatoms via gold electrodeposition is described. In this process, gold microstructures are formed, immobilizing diatoms by entrapment or crossing their nanopores. Varying the applied potential, time and HAuCl4 concentration, gold deposits of different morphologies and roughness are obtained, thereby determining the diatom immobilization process. Optical and scanning electron microscopy have been used to characterize diatom immobilization yields, the morphology of the gold microstructures, and the morphological integrity of diatoms. Cyclic voltammetry has been performed to characterize the gold deposits and to demonstrate the enhanced electrocatalytic activity of the gold-diatom electrodes. Electro-addressed immobilization of different diatoms on specific bands of interdigitated electrode arrays has been achieved, highlighting the potential application of diatoms for site-specific immobilization on microarrays. The feasibility to combine tailored immobilization with diatom biofunctionalization has also been demonstrated. Antibody-functionalized diatoms were immobilized on electrodes retaining their ability to detect its cognate antigen. The reported method exploits the natural three-dimensional nanostructures of diatoms together with their easy modification with biomolecules and the simplicity of gold electrodeposition to produce micro/nanostructured and highly electrocatalytic electrodes, providing low-cost and eco-friendly platforms and arrays with potential application in biosensing devices.

Cloning of scFv from hybridomas using a rational strategy: Application as a receptor to sensitive detection microcystin-LR in water.

Single chain variable fragment (scFv), containing of heavy and light chains (VH and VL) joined by a short peptide linker, has been used widely for immunodetection. Nevertheless, cloning functional variable genes is still a bottle neck for the scFv generation technology. Here, a rational strategy for cloning and selecting variable region genes from an anti-microcystin-LR hybridoma was devised, then the functional VH and VL genes were recloned and assembled to scFv using splicing overlap extension PCR. The resulting scFv gene was recombinantly expressed as a soluble scFv-alkaline phosphatase fusion protein (scFv-AP) by vector PLIP6/GN. Then an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for detection of microcystin-LR was developed. The half-maximum inhibition concentrations (IC50) and limits of detection (LODs, IC15) were 0.81 ± 0.04 µgL(-1) and 0.13 ± 0.03 µgL(-1), respectively. With the mean coefficient of variation lowing 8%, the mean recovery in intra-assay and inter-assay were 100.06% and 96.46%, The proposed strategy should be useful for generation scFv in a rapid and simple way.

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water.

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 µg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

An aptamer-based immunoassay in microchannels of a portable analyzer for detection of microcystin-leucine-arginine.

The rapid detection of microcystin-leucine-arginine (MC-LR), the most highly toxic among MCs, is significantly important to environmental and human health protection and prevention of MC-LR from being used as a bioweapon. Although aptamers offer higher affinity, specificity, and stability with MC-LR than antibodies in the immunodetection of MC-LR due to steric hindrance between two antibodies and limited epitopes of MC-LR for use in a sandwich immunoassay, no sandwich immunoassay using an aptmer has been developed for MC-LR detection. This study is aimed at developing an aptamer-antibody immunoassay (AAIA) to detect MC-LR using a portable analyzer. The aptamers were immobilized onto the glass surface of a microchamber to capture MC-LR. MC-LR and horseradish peroxidase (HRP)-labeled antibody were pulled into the microchamber to react with the immobilized aptamer. The chemiluminescence (CL) catalyzed by HRP was tested by a photodiode-based portable analyzer. MC-LR at 0.5-4.0 µg/L was detected quantitatively by the AAIA, with a CL signal sensitivity of 0.3 µg/L. The assay took less than 35 min for a single sample and demonstrated a high specificity, detecting only MC-LR, but not MC-LA, MC-YR, or nodularin-R. The recovery of two spiked real environmental samples calculated as 94.5-112.7%. Therefore, this AAIA was proved to be a rapid and simple method to detect MC-LR in the field by a single analyst.

Surface-enhanced fluorescence immunosensor using Au nano-crosses for the detection of microcystin-LR.

A surface-enhanced fluorescence (SEF) immunosensor for the detection of microcystin-LR was developed using Au nano-crosses as fluorescence enhancement nanoparticles and cy5 as a fluorescence label molecule. The SEF effects of cy5 in the proximity of Au nanorods and gold nano-crosses was investigated by using Au nanorods or nano-crosses coated negative-charged glass surfaces. Fluorescence measurements indicated that SEF was influenced by the size, shape and distribution of the Au nanoparticles, with an appropriate spacer layer between the Au nanoparticles and the cy5. The enhancement factor was from 2.3- to 35-fold. Under optimal conditions, the SEF immunosensor exhibited a good linear response at microcystin-LR concentrations of 0.02-16 ng mL(-1) (R(2)=0.9981). The limit of detection was 0.007 ng mL(-1) with little adsorption of microcystin-RR, microcystin-LW, and microcystin-LF. High microcystin-LR recoveries were obtained from naturally contaminated fish samples. The SEF immunosensor allows the reliable detection of microcystin-LR in seafood, and has potential in simple, sensitive detection applications.

Multihapten approach leading to a sensitive ELISA with broad cross-reactivity to microcystins and nodularin.

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 µg/L, well below the WHO's maximum recommendation of 1 µg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

Two ScFv antibody libraries derived from identical VL-VH framework with different binding site designs display distinct binding profiles.

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.

Automated online optical biosensing system for continuous real-time determination of microcystin-LR with high sensitivity and specificity: early warning for cyanotoxin risk in drinking water sources.

The accelerated eutrophication of surface water sources and climate change have led to an annual occurrence of cyanobacterial blooms in many drinking water resources. To minimize the health risks to the public, cyanotoxin detection methods that are rapid, sensitive, real time, and high frequency must be established. In this study, an innovative automated online optical biosensing system (AOBS) was developed for the rapid detection and early warning of microcystin-LR (MC-LR), one of the most toxic cyanotoxins and most frequently detected in environmental water. In this system, the capturing molecular MC-LR-ovalbumin (MC-LR-OVA) was covalently immobilized onto a biochip surface. By an indirect competitive detection mode, samples containing different concentrations of MC-LR were premixed with a certain concentration of fluorescence-labeled anti-MC-LR-mAb, which binds to MC-LR with high specificity. Then, the sample mixture was pumped onto the biochip surface, and a higher concentration of MC-LR led to less fluorescence-labeled antibody bound onto the biochip surface and thus to lower fluorescence signal. The quantification of MC-LR ranges from 0.2 to 4 µg/L, with a detection limit determined as 0.09 µg/L. The high specificity and selectivity of the sensor were evaluated in terms of its response to a number of potentially interfering cyanotoxins. Potential interference of the environmental sample matrix was assessed by spiked samples, and the recovery of MC-LR ranged from 90 to 120% with relative standard deviation values <8%. The immunoassay performance of the AOBS was validated with respect to that of conventional high-performance liquid chromatography, and the correlation between methods agreed well (R(2) = 0.9762). This system has successfully been applied to long-term, continuous determination and early warning for MC-LR in Lake Tai from June 2011 to May 2012. Thus, the AOBS paves the way for a vital routine online analysis that satisfies the high demand for ensuring the safety of drinking water sources. The AOBS can also serve as early warning system for accidental or intentional water pollution.

Next generation planar waveguide detection of microcystins in freshwater and cyanobacterial extracts, utilising a novel lysis method for portable sample preparation and analysis.

The study details the development of a fully validated, rapid and portable sensor based method for the on-site analysis of microcystins in freshwater samples. The process employs a novel lysis method for the mechanical lysis of cyanobacterial cells, with glass beads and a handheld frother in only 10 min. The assay utilises an innovative planar waveguide device that, via an evanescent wave excites fluorescent probes, for amplification of signal in a competitive immunoassay, using an anti-microcystin monoclonal with cross-reactivity against the most common, and toxic variants. Validation of the assay showed the limit of detection (LOD) to be 0.78 ng mL(-1) and the CCß to be 1 ng mL(-1). Robustness of the assay was demonstrated by intra- and inter-assay testing. Intra-assay analysis had % C.V.s between 8 and 26% and recoveries between 73 and 101%, with inter-assay analysis demonstrating % C.V.s between 5 and 14% and recoveries between 78 and 91%. Comparison with LC-MS/MS showed a high correlation (R(2)=0.9954) between the calculated concentrations of 5 different Microcystis aeruginosa cultures for total microcystin content. Total microcystin content was ascertained by the individual measurement of free and cell-bound microcystins. Free microcystins can be measured to 1 ng mL(-1), and with a 10-fold concentration step in the intracellular microcystin protocol (which brings the sample within the range of the calibration curve), intracellular pools may be determined to 0.1 ng mL(-1). This allows the determination of microcystins at and below the World Health Organisation (WHO) guideline value of 1 µg L(-1). This sensor represents a major advancement in portable analysis capabilities and has the potential for numerous other applications.

Effect of cyanobacteria on immune function of crucian carp (Carassius auratus) via chronic exposure in diet.

Cyanobacterial blooms caused by water eutrophication have become a worldwide problem. Microcystins (MCs) released during cyanobacterial blooms exert toxicity on fish. Up to now, immunotoxicity of MCs on fish has been rarely reported. The present study investigated immune response of crucian carp (Carassius auratus) to cyanobacteria via chronic exposure in diet. Fish were fed with diets containing 20% (low dose group) and 40% (high dose group) of cyanobacteria lyophilized powder. After exposure of 30 d, a batch of assays was determined for assessing immunotoxicity of MCs. The head kidney and spleen indexes significantly increased in high dose group. Blood nitroblue tetrazolium activity in high dose group was nearly twice as much as that in control group with no cyanobacteria additive. Marked haemorrhage and hyperemia were observed in kidney and spleen in high dose group. The edematous mitochondria, deformation of the nucleus and compaction of chromatin occurred in lymphocytes of head kidney and spleen in both cyanobacteria groups. Lysozyme activity showed an obvious increase in low dose group but a sharp decrease in high dose group. Significant increase of macrophage bactericidal activity was detected in low dose group. The present findings indicate that via chronic diet exposure of different cyanobacteria levels, fish exhibit various immune responses. Fish immunity tends to proceed toward the direction of immunostimulative response at low MCs concentrations but toward the trend of immunosuppressive answer at high MCs concentrations.

Magnetic particle-based enzyme assays and immunoassays for microcystins: from colorimetric to electrochemical detection.

In this work, magnetic particles (MPs) are used as supports for the immobilization of biorecognition molecules for the detection of microcystins (MCs). In one approach, a recombinant protein phosphatase 1 (PP1) has been conjugated to MPs via coordination chemistry, and MC-LR detection has been based on the inhibition of the enzyme activity. In the other approach, a monoclonal antibody (mAb) against MC-LR has been conjugated to protein G-coated MPs, and a direct competitive enzyme-linked immunoparticle assay (ELIPA) has been then performed. Conjugation of biomolecules to MPs has been first checked, and after optimization, MC detection has been performed. The colorimetric PPIA with PP1-MP and the best ELIPA strategy have provided limits of detection (LOD) of 7.4 and 3.9 µg/L of MC-LR, respectively. The electrochemical ELIPA has decreased the LOD to 0.4 µg/L, value below the guideline recommended by the World Health Organisation (WHO). The approaches have been applied to the analysis of a cyanobacterial culture and a natural bloom, and MC equivalent contents have been compared to those obtained by conventional assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results have demonstrated the viability of the use of MPs as biomolecule immobilization supports in biotechnological tools for MCs monitoring.