Glucose biosensor based on nanohybrid material of gold nanoparticles and glucose oxidase on a bioplatform

A simple and relatively cheap glucose biosensor based on a combination of gold nanoparticles (Au NPs) and glucose oxidase (GO(x) ) immobilized on a bioplatform eggshell membrane was established. Scanning electron microscopy showed successful immobilization of Au NPs/GO(x) on the eggshell membrane. The effects of pH, phosphate buffer concentration, and temperature on the glucose biosensor were studied in detail. The biosensor shows a linear response at a glucose concentration range of 5-525 μM. The detection limit of the biosensor is 2.5 μM (S/N = 3). The biosensor exhibits good repeatability with RSD = 3.6% (n = 6), good operational stability with over 300 measurements and long-term storage stability with a shelf life of at least 6 months. The response time is less than 60 s. The glucose level in commercial food samples has been successfully determined. The proposed work shows potential to develop cost-effective biosensors for biotechnological, biomedical and industrial use.     

Development and evaluation of a sensitive,Diffusive Gradients in Thin-Films (DGT) method for determining microcystin-LR concentrations in freshwater and seawater

A Diffusive Gradients in Thin-Films (DGT) passive sampling technique was developed for microcystin-LR (MC-LR), one of the most common and toxic microcystins. Three types of resins (HP20, SP700, and XAD18) were evaluated for MC-LR uptake kinetics, capacities, and extraction efficiencies and simple procedures were developed for determining MC-LR concentration in binding disc extracts by Adda-ELISA (U.S. EPA Method 546). The XAD18-DGT/Adda-ELISA method had a 7-d deployment time detection limit of ≈0.05 μg/L and capacity of >250 μg/L of MC-LR in water samples which encompass U.S. EPA and WHO advisory concentrations for drinking and recreational waters. The XAD18-DGT/Adda-ELISA method determined time-averaged MC-LR concentrations in waters with wide ranging pH (4.9–8.3) and ionic strength (0.04–0.8 M) under well-stirred and quiescent conditions with 90–101% accuracy. In addition to high sensitivity and accuracy, the method is simple, inexpensive, and applicable for determining MC-LR and related MCs concentrations in waterbodies with wide ranging chemical characteristics and hydrodynamic conditions.     

A new type sandwich immunoassay for microcystin: production of monoclonal antibodies specific to the immune complex formed by microcystin and an anti-microcystin monoclonal antibody.

To develop an ultrasensitive immunoassay for microcystins (MCs), a group of heptapeptide hepatotoxins produced by cyanobacteria, we produced monoclonal antibodies (MAbs) which specifically recognize the immune complex (IC) formed by an anti-MC MAb (MC MAb) and MCs. The use of the anti-IC MAb (IC MAb) as the secondary antibody made it possible to develop a sandwich type immunoassay, which is theoretically superior to the widely used competitive immunoassay in sensitivity as well as accuracy. A MC MAb mixed with microcystin-LR (MCLR) to form the IC was immunized to mice. Three IC MAbs were obtained, all of which specifically reacted with the IC, but almost never reacted to MC MAb or MCLR in enzyme-linked immunosorbent assays (ELISAs). Binding kinetics study of one of the IC MAbs, 3F7, by a BIAcore biosensor technique revealed that 3F7 IC MAb could associate with free MC MAb as well as the IC, but the binding to free MC MAb was much more easily dissociated than that to the IC, thus resulting in about 300-fold higher affinity of 3F7 for the IC than for MC MAb alone (1.8 x 10(9) M(-1) and 4.6 x 10(6) M(-1) for the IC and MC MAb, respectively). Finally, 3F7 IC MAb was shown to react with the IC formed by the addition of MCLR to MC MAb-coated plates in a dose-dependent manner. Therefore, a new type sandwich immunoassay, anti-immune complex ELISA (IC ELISA) for MCs, was indeed established. The detection limit of the IC ELISA was 2 pg of MCLR ml(-1) (50 fg per assay), making it the most sensitive of all the methods for detecting MCs reported to date.     

An amperometric biosensor based on laccase immobilized onto Fe(3)O(4)NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe(3)O(4)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, detection limit were 0.1-10μM (lower concentration range) and 10-500μM (higher concentration range), and 0.03μM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.     

Glucose biosensor based on Au nanoparticles-conductive polyaniline nanocomposite

In this paper, we report a novel glucose biosensor based on composite of Au nanoparticles (NPs)-conductive polyaniline (PANI) nanofibers. Immobilized with glucose oxidase (GOx) and Nafion on the surface of nanocomposite, a sensitive and selective biosensor for glucose was successfully developed by electrochemical oxidation of H2O2. The glucose biosensor shows a linear calibration curve over the range from 1.0x10(-6) to 8.0x10(-4) mol/L, with a slope and detection limit (S/N=3) of 2.3 mA/M and 5.0x10(-7) M, respectively. In addition, the glucose biosensor system indicates excellent reproducibility (less than 5% R.S.D.) and good operational stability (over 2 weeks).     

Graphene Multilayer Supported Gold Nanoparticles for Efficient Electrocatalysts Toward Methanol Oxidation

This study reports a simple method of integrating electroactive gold nanoparticles (Au NPs) with graphene oxide (GO) nanosheet support by layer‐by‐layer (LbL) assembly for the creation of 3‐dimensional electrocatalytic thin films that are active toward methanol oxidation. This approach involves the alternating assembly of two oppositely charged suspensions of Au NPs with GO nanosheets based on electrostatic interactions. The GO nanosheets not only serve as structural components of the multilayer thin film, but also potentially improve the utilization and dispersion of Au NPs by taking advantages of the high catalytic surface area and the electronic conduction of graphene nanosheets. Furthermore, it is found that the electrocatalytic activity of the multilayer thin films of Au NPs with graphene nanosheet is highly tunable with respect to the number of bilayers and thermal treatment, benefiting from the advantageous features of LbL assembly. Because of the highly versatile and tunable properties of LbL assembled thin films coupled with electrocatalytic NPs, we anticipate that the general concept presented here will offer new types of electroactive catalysts for direct methanol fuel cells.     

Real-time label-free immunoassay of interferon-gamma and prostate-specific antigen using a Fiber-Optic Localized Surface Plasmon Resonance sensor

A Fiber-Optic Localized Surface Plasmon Resonance (FO LSPR) sensor was fabricated using spherical gold nanoparticles (Au NPs) on a flattened end-face of the optical fiber. The Au NPs were easily synthesized by the Turkevich method and were immobilized on the end-face of the optical fiber by using a self-assembled monolayer (SAM). In order to examine the possibility of its application as a biosensor for label-free immunoassays, the fabricated FO LSPR sensor was used for the detection of the antibody-antigen reaction of interferon-gamma (IFN-γ) and the limit of detection (LOD) was approximately 2pg/ml. Herein, The antibodies and bovine serum albumins (BSAs) were immobilized on the Au NPs by physisorption. Also, the FO LSPR sensor was used for the detection of a prostate-specific antigen (PSA) and the LOD was 1pg/ml below. The fabricated FO LSPR sensor can be used for real-time label-free immunoassay having fast detection time, high resolution and sensitivity. In addition, the proposed sensor platform has the advantages of low cost, simple optical setup, remote sensing, simple fabrication, real-time detection, low sample volume, and potential application to in-vivo detection systems.     

Electrochemical DNA biosensor for screening of chlorinated benzene pollutants

In this work, an electrochemical DNA biosensor based on double-stranded DNA modified Au electrode (dsDNA/Au) was proposed for the rapid screening and detection of chlorinated benzenes pollutants, in which redox-active methylene blue (MB) was used to amplify the interaction between dsDNA and the target analyte. Using hexachlorobenzene (HCB) as a model analyte of chlorinated benzenes, the biosensor demonstrated a linear response with the logarithm of HCB concentrations from 100 pmol L(-1) to 100 nmol L(-1). The obtained detection limit was 30 pmol L(-1), which was remarkably superior to other biosensors. The interaction mechanism of the biosensor with HCB was proposed based on systematical characterization by cyclic voltammetry (CV), differential pulse voltammetry (DPV), UV-vis spectrometry and electrochemical quartz crystal microbalance (EQCM). Further studies revealed that the biosensor could screen chlorinated benzenes in the presence of 100 fold amount of other co-existing chemicals (ethyl acetate and sodium oxalate, etc.), and the response signal of the biosensors for different chlorinated benzenes was correlative to their respective toxicity. The proposed biosensor proved to be a promising "alarm" tool for rapid screening of chlorinated benzenes in real water samples.     

A sensitive NADH and glucose biosensor tuned by visible light based on thionine bridged carbon nanotubes and gold nanoparticles multilayer

A NADH and glucose biosensor based on thionine cross-linked multiwalled carbon nanotubes (MWNTs) and Au nanoparticles (Au NPs) multilayer functionalized indium-doped tin oxide (ITO) electrode were presented in this paper. The effect of light irradiation on the enhancement of bioelectrocatalytic processes of the biocatalytic systems by the photovoltaic effect was investigated. This bioelectrode exhibited excellent catalytic activity of the oxidation towards dihydronicotinamide adenine dinucleotide (NADH). Most interesting, the performance of this NADH sensor could be tuned by the visible light. When the biosensor was performed in the dark, the anodic current increased linearly with NADH concentration over the range from 0.5 to 237 microM with detection limit 0.1 microM and sensitivity 17 nA microM(-1). The sensitivity became 115 nA microM(-1) with detection limit 0.05 microM with the light irradiation. Compared with the reaction in dark, the sensitivity increased around 7 folds while the detection limit decreased 2 folds. The glucose biosensor also exhibited the same behavior. The linear range was from 10 microM to 2.56 mM with the sensitivity of 7.8 microAmM(-1) and detection limit 5.0 microM in the dark. After the light irradiation, the linear range was from 1 microM to 3.25 mM with the sensitivity of 18.5 microA mM(-1) and detection limit 0.7 microM. It indicated a potential to provide an operational access to develop new kinds of photocontrolled dehydrogenase enzyme-based bioelectronics.     

Construction of an improved amperometric acrylamide biosensor based on hemoglobin immobilized onto carboxylated multi-walled carbon nanotubes/iron oxide nanoparticles/chitosan composite film

A method is described for construction of an improved amperometric acrylamide biosensor based on covalent immobilization of hemoglobin (Hb) onto nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT) and iron oxide nanoparticles (Fe₃O₄NPs) electrodeposited onto Au electrode through chitosan (CHIT) film. The Hb/cMWCNT-Fe₃O₄NP/CHIT/Au electrode was characterized by scanning electron microscopy, Fourier transform infra-red spectroscopy, electrochemical impedance spectroscopy, and differential pulse voltammetry at different stages of its construction. The biosensor was based on interaction between acrylamide and Hb, which led to decrease in the electroactivity of Hb, i.e., current generated during its reversible conversion [Fe(II)/Fe(III)]. The biosensor showed optimum response within 8 s at pH 5.0 and 30 °C. The linear working range for acrylamide was 3–90 nM, with a detection limit of 0.02 nM and sensitivity of 36.9 μA/nM/cm². The biosensor was evaluated and employed for determination of acrylamide in potato crisps.     

DNA Detection Based on Localized Surface Plasmon Resonance Spectroscopy of Ag@Au Biocomposite Nanoparticles

Noble metal nanoparticles (NPs) have attracted much attention due to their unique physical and chemical properties such as tunable surface plasmonics, high-efficiency electrochemical sensing, and enhanced fluorescence. We produced two biosensor chips consisting of Ag@Au bimetallic nanoparticles (BNPs) on a carbon thin film by simple RF-sputtering and RF-plasma-enhanced chemical vapor co-deposition. We deposited Au NPs with average size of 4 nm (Au₁ NPs) or 11 nm (Au₂ NPs) on a sensor chip consisting of Ag NPs with mean size of 15 nm, and we investigated the effect of shell size (Au NPs) on the chemical activities of the resulting Ag@Au₁ BNPs and Ag@Au₂ BNPs. We estimated the average size and morphology of Ag@Au BNPs by scanning electron microscopy (SEM) and atomic force microscopy (AFM) images. X-ray diffraction (XRD) patterns revealed that Ag NPs and Au NPs had face-centered cubic (FCC) structure. We studied aging of the biosensor chips consisting of Ag@Au BNPs by localized surface plasmon resonance (LSPR) spectroscopy for up to 3 months. UV–visible aging of the prepared samples indicated that Ag@Au₁ BNPs, which corresponded to Ag NPs covered with smaller Au NPs, were more chemically active than Ag@Au₂ BNPs. Furthermore, we evaluated changes in the LSPR absorption peaks of Ag@Au₁ BNPs and bare Ag NPs in the presence of a DNA primer decamer at fM concentrations, to find that Ag@Au₁ BNPs were more sensitive biosensor chips within a short response time as compared to bare Ag NPs.

     

A sensitive SERS quantitative analysis method for Ni2+ by the dimethylglyoxime reaction regulating a graphene oxide nanoribbon catalytic gold nanoreaction

The nanogold reaction between HAuCl₄ and trisodium citrate (TCA) proceeded very slowly at 60°C in a water bath. The as‐prepared graphene oxide nanoribbons (GONRs) exhibited strong catalysis during the reaction to form gold nanoparticles (Au NPs) and appeared as a strong surface‐enhanced Raman scattering (SERS) peak at 1616 cm^?1 in the presence of the molecular probe Victoria blue 4R (VB4r). With increase in GONR concentration, the SERS peak increased due to increased formation of Au NPs. Upon addition of dimethylglyoxime (DMG) ligand, which was adsorbed onto the GONR surface to inhibit GONR catalysis, the SERS peak decreased. When Ni²⁺ was added, a coordination reaction between DMG and Ni²⁺ took place to form stable complexes of [Ni (DMG)₂]²⁺ and the release of free GONR catalyst that resulted in the SERS peak increasing linearly. A SERS quantitative analysis method for Ni²⁺ was therefore established, with a linear range of 0.07–2.8 μM, and a detection limit of 0.036 μM Ni²⁺.     

An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide

In this article, a supersandwich-type electrochemical biosensor for sequence-specific DNA detection is described. In design, single-strand DNA labeled with methylene blue (MB) was used as signal probe, and auxiliary probe was designed to hybridize with two different regions of signal probe. The biosensor construction contained three steps: (i) capture DNA labeled with thiol was immobilized on the surface of gold nanoparticles decorated reduced graphene oxide (Au NPs/rGO); (ii) the sandwich structure formation contained “capture–target–signal probe”; and (iii) auxiliary probe was introduced to produce long concatamers containing signal molecule MB. Differential pulse voltammetry (DPV) was used to monitor the DNA hybridization event using peak current changes of MB in phosphate-buffered saline (PBS) containing 1.0 M NaClO₄. Under optimal conditions, the peak currents of MB were linear with the logarithm of the concentration of target DNA in the range of 0.1 μM to 0.1 fM with a detection limit of 35 aM (signal/noise = 3). In addition, this biosensor exhibited good selectivity even for single-base mismatched target DNA detection.     

Bioaccumulation of Microcystins in Lettuce

The contamination of lettuce (Lactuca sativa L.) by water‐borne crude extracts of the cyanobacterium microcystin‐producing Microcystis aeruginosa (Kützing) Kützing was investigated. The aim of the study was to determine whether bioaccumulation of microcystins occurs in lettuce foliar tissue when sprayed with solutions containing microcystins at concentrations observed in aquatic systems (0.62 to 12.5 μg · L^?1). Microcystins were found in lettuce foliar tissues (8.31 to 177.8 μg per Kg of fresh weight) at all concentrations of crude extracts. Spraying with water containing microcystins and cyanobacteria may contaminate lettuce at levels higher than the daily intake of microcystins recommended by the World Health Organization (WHO), underscoring the need to monitor such food exposure pathways by public authorities.     

Depth profiles of cyanobacterial hepatotoxins (microcystins) in three Turkish freshwater lakes

The Turkish freshwater lakes, Sapanca, Iznik and Taskisi (Calticak) have been enriched with nutrients from agriculture and domestic sources for many years. A major bloom of cyanobacteria (blue-green algae) in Lake Sapanca was recorded in May 1997, closely followed by a fish kill. Investigations were subsequently made on the cyanobacteria and water quality of the lakes, including analysis for cyanobacterial hepatotoxins (microcystins) in the filtered particulate fraction. Samples, taken from the beginning of May to end of August 1998, were analysed for microcystins by high–performance liquid chromatography with photodiode array detection (HPLC-PDA), protein phosphatase inhibition assay (PPIA) and an enzyme-linked immunosorbent assay (ELISA). No microcystins were detected in the water column in Lake Sapanca above 10 m, but toxins were found in filtered cyanobacterial samples from 20 m depth at a concentration of 3.65 μg l^?1 microcystin–LR equivalents. Ninety percent of the microcystin pool detected in L. Sapanca was found between depths of 15 and 25 m. The principal microcystin detected by HPLC-PDA was similar to microcystin–RR. Two unidentified microcystin variants were found in Lake Taskisi surface samples at a concentration of 2.43 μg l^?1 microcystin–LR equivalents in the filtered cyanobacterial cell fraction. Although 10 water samples (10 × 5 l) were taken from Lake Iznik (surface to 20 m, 5 m intervals), no microcystins were detected by HPLC-PDA (limit of detection 10 ng). The depth at which microcystins were detected in L. Sapanca coincided with the draw-off depth for the drinking water supply for the city of Sakarya     

A novel FRET-based optical fiber biosensor for rapid detection of Salmonella typhimurium

A biosensor that is portable and permits on-site analysis of samples would significantly reduce the large economical burden of food products recalls. A fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) was developed for fast detection of Salmonella typhimurium (S. typhimurium) in ground pork samples. Labeled antibody-protein G complexes were formed via the incubation of anti-Salmonella antibodies labeled with FRET donor fluorophores (Alexa Fluor 546) and protein G (PG) labeled with FRET acceptor fluorophores (Alexa Fluor 594). Utilizing silanization, the labeled antibodies-PG complexes were then immobilized on decladded, tapered silica fiber cores to form the evanescent wave-sensing region. The biosensors were tested in two different solutions: (1) PBS doped with S. typhimurium and (2) homogenized pork sample with S. typhimurium. The fiber probes tested in a S. typhimurium doped phosphate buffered solution demonstrated the feasibility of the biosensor for detecting S. typhimurium as well as determined the optimal packing density of the labeled antibody-PG complexes on the surface of fibers. The results showed that a packing density of 0.033 mg/ml produced the lowest limit of detection of 10(3)cells/ml with 8.2% change in fluorescence. The fiber probes placed in homogenized pork samples inoculated with S. typhimurium showed a limit of detection of 10(5)CFU/g with a 6.67% in fluorescence within a 5-min response time. These results showed that the FRET-based fiber optic biosensor can become a useful analytical tool for detection of S. typhimurium in real food samples.     

Rapid isolation of a single-chain antibody against the cyanobacterial toxin microcystin-LR by phage display and its use in the immunoaffinity concentration of microcystins from water

A na?ve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 microg liter(-1)) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 micro g of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.     

Current approaches to cyanotoxin risk assessment and risk management around the globe

Toxic cyanobacteria became more widely recognized as a potential health hazard in the 1990s, and in 1998 the World Health Organization (WHO) first published a provisional Guideline Value of 1 μg L⁻¹ for microcystin-LR in drinking-water. In this publication we compare risk assessment and risk management of toxic cyanobacteria in 17 countries across all five continents. We focus on the three main (oral) exposure vehicles to cyanotoxins: drinking-water, water related recreational and freshwater seafood. Most countries have implemented the provisional WHO Guideline Value, some as legally binding standard, to ensure the distribution of safe drinking-water with respect to microcystins. Regulation, however, also needs to address the possible presence of a wide range of other cyanotoxins and bioactive compounds, for which no guideline values can be derived due to insufficient toxicological data. The presence of microcystins (commonly expressed as microcystin-LR equivalents) may be used as proxy for overall guidance on risk management, but this simplification may miss certain risks, for instance from dissolved fractions of cylindrospermopsin and cyanobacterial neurotoxins. An alternative approach, often taken for risk assessment and management in recreational waters, is to regulate cyanobacterial presence – as cell numbers or biomass – rather than individual toxins. Here, many countries have implemented a two or three tier alert level system with incremental severity. These systems define the levels where responses are switched from Surveillance to Alert and finally to Action Mode and they specify the short-term actions that follow. Surface bloom formation is commonly judged to be a significant risk because of the elevated concentration of microcystins in a scum. Countries have based their derivations of legally binding standards, guideline values, maximally allowed concentrations (or limits named otherwise) on very similar scientific methodology, but underlying assumptions such as bloom duration, average body size and the amount of water consumed while swimming vary according to local circumstances. Furthermore, for toxins with incomplete toxicological data elements of expert judgment become more relevant and this also leads to a larger degree of variation between countries’ thresholds triggering certain actions. Cyanobacterial blooms and their cyanotoxin content are a highly variable phenomenon, largely depending on local conditions, and likely concentrations can be assessed and managed best if the specific conditions of the locality are known and their impact on bloom occurrence are understood. Risk Management Frameworks, such as for example the Water Safety Plan concept of the WHO and the ‘bathing water profile’ of the European Union are suggested to be effective approaches for preventing human exposure by managing toxic cyanobacteria from catchment to consumer for drinking water and at recreational sites.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.     

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter⁻¹) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples.