Development of a membrane-based immunofiltration assay for the detection of T-2 toxin

An improved analytical device capable of performing simultaneous immunofiltration-based immunoassay on 30 samples in the presence of reference standards has been developed. The device consists of a rectangular membrane with 36 antibody spotted zones, one end of which was attached to a semirigid polyethylene card. A piece of wetted filter paper between the membrane and the polyethylene card absorbs the added reagent. The assay is a competitive one using T-2 toxin-horseradish peroxidase (T-2 toxin-HRP) as the labeled analyte and 4-chloro-1 naphthol (4CN) as the substrate. Signal amplification was done by the Super-CARD signal amplification method. Semiquantitative results were obtained by visual comparison of the color intensity of a sample spot with those of reference standards. Densitometric analysis was used for quantitation. The method allows rapid and easy determination of T-2 toxin in wheat and poultry feed with detection limits of 12.5 and 25 microg x kg(-)(1), respectively, with accuracy and precision. Matrix interference was eliminated by appropriate dilution of sample extracts with assay buffer. The detection sensitivity in ELISA was 10-fold higher than that in the membrane-based method. Noninfected samples were spiked with T-2 toxin at several concentrations and analyzed by the present method and rapid ELISA. Mean recoveries by both methods were between 80 and 108%. The correlation between the two methods was excellent (R(2) = 0.99).     

In vitro and in vivo evaluation of paralytic shellfish poisoning toxin potency and the influence of the pH of extraction

Paralytic shellfish poisoning (PSP) is one of the most severe forms of food poisoning. The toxins responsible for this poisoning are natural compounds, which cause the arrest of action potential propagation by binding to voltage-gated Na+ channels. Several standards for PSP toxins are nowadays commercially available; however, there is not accessible data on the biological activity of the toxins present on this standards and their in vivo toxicity. We have developed an in vitro quantification method for PSP toxins using cultured neurons and compared the potency of the commercial PSP toxin standards in this system with their relative toxicity by mouse bioassay. The in vitro potencies of the PSP toxin standards were saxitoxin (STX) > decarbamoylsaxitoxin (dcSTX) = neosaxitoxin (NeoSTX) > gonyautoxins 1, 4 (GTX1,4) > decarbamoylneosaxitoxin (dcNeoSTX) > gonyautoxins 2, 3 (GTX2,3) > decarbamoylgonyautoxins 2, 3 (dcGTX2,3) > gonyautoxin 5 (GTX5). The data in vitro correlated well with the toxicity values obtained by mouse bioassay. Using this in vitro model we also provide the first data evaluating the potencies of PSP toxins after extraction in acidic pHs, indicating that the toxicity of the sample increases in acidic conditions. This observation correlated well with the chemical transformations undergone by contaminated samples treated in several acidic conditions as corroborated by high-performance liquid chromatography (HPLC) detection of the toxins. Therefore, a variation of 2 units in the pH during PSP extraction may lead to large discrepancies regarding sample lethality during official PSP control in different countries. The results presented here constitute the first comprehensive and revised data on the potency of PSP toxins in vitro and their in vivo toxicity.     

Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces

There is a particular need in protein analysis and purification for specific, functional, and generic methods of protein immobilization on solid supports. Here we describe a double-hexahistidine (His6) tag sequence, comprising two hexahistidines separated by an 11-amino acid spacer, which shows at least 1 order of magnitude stronger binding to Ni-NTA-modified surfaces than a conventional single-His6 tag or two single-His6 tags at N- and C-termini. Using, as a model, tagged versions of green fluorescent protein (GFP), stable and tight binding of the double-His6 tag/Ni-NTA interaction was demonstrated by competitive elution from Ni-NTA agarose beads, surface plasmon resonance on a Ni-NTA chip, and ELISA in Ni-NTA microwell plates. Protein purification by Ni-NTA chromatography was improved by a 6-8-fold increase in imidazole concentration required for elution, while the dissociation rate of double-His6 GFP from Ni-NTA chips in SPR (BIAcore) was 10 times slower than for single-His6-tagged proteins. ELISA assays and protein microarrays constructed with double-His6 GFP demonstrated greater detection sensitivity with anti-His antibodies and Ni-NTA conjugates. Moreover, the double-His6 tag could serve simultaneously both for protein immobilization and for detection on surfaces. The double-His6 peptide has the potential to be a universal tag for protein immobilization and detection on arrays and single-step purification of proteins from crude mixtures.     

Glyconanoparticles for the colorimetric detection of cholera toxin

Cholera continues to represent a major threat to human health, particularly in developing countries. Death can be readily avoided when medical treatment is rapidly administered. In order to provide a means of detecting the bacterially secreted toxin, we have developed a simple, yet rapid, bioassay for the cholera toxin. The colorimetric bioassay is based on a specifically synthesized lactose derivative that is self-assembled onto gold nanoparticles of 16 nm diameter. In solution the lactose-stabilized nanoparticles are red in color due to the intense surface plasmon absorption band centered at 524 nm. Cholera toxin (added as the B-subunit) (CTB) binds to the lactose derivative and induces aggregation of the nanoparticles. Upon aggregation, the surface plasmon absorption band broadens and red shifts such that the nanoparticle solution appears a deep purple color. The selectivity of the bioassay stems from the thiolated lactose derivative that mimics the GM(1) ganglioside--the receptor to which cholera toxin binds in the small intestine. Consequently, added metal ions, anions, and a protein, at relevant concentrations, do not induce nonspecific aggregation of the nanoparticles. The simple color change of the bioassay provides a selective means to detect and quantify the cholera toxin within 10 min. The theoretical limit of detection of the bioassay was determined to be 54 nM (3 microg/mL) for CTB. The stability of the lactose-stabilized nanoparticles was established by freeze-drying and then resuspending the particles in water and subsequently measuring CTB in biologically relevant electrolyte solutions. This colorimetric bioassay provides a new tool for the direct measurement of cholera toxin.     

Colorimetric detection of nucleic acid signature of shiga toxin producing Escherichia coli using gold nanoparticles

Enterohemorrhagic E. coil (EHEC) serotype O157:H7 is one of the major pathogens, responsible for the severe disease outbreaks. EHEC causes diseases in humans through production of shiga-like toxin leading to bloody diarrhea. The toxin is encoded by stx2 gene in E. coli. The current methodology for detection of EHEC relies on fluorogenic-substrate based culture media or nucleic acid amplification based Real-Time Polymerase Chain Reaction assays that are either time consuming or need expensive instrumentation. In this study, the optical properties of gold nanoparticles (GNPs) have been exploited for detection of nucleic acid of Escherichia coli O157:H7. The stx2 gene representing EHEC signature has been targeted using the gold nanoparticle probes. Gold nanoparticles (GNPs) of 20 +/- 0.2 nm were synthesised by citrate reduction method and characterised by spectroscopy and transmission electron microscopy. The GNPs were functionalised with 19 and 22 bp of thiolated single stranded DNA complementary to target highly conserved 149 bp region of stx2 gene. Transmission Electron Microscopy revealed the hybridization, aggregation and reduction in the interparticle distances of the GNP probes in the presence of target DNA. The aggregation and the spectral shift in the plasmon band observed with 10(6) copies of target DNA indicates feasibility of a simple and quick colorimetric 'spot and read' test in contrast to amplification based detection methods.     

Ganglioside-liposome immunoassay for the ultrasensitive detection of cholera toxin

An extremely sensitive bioassay has been developed for cholera toxin (CT) detection, using ganglioside-incorporated liposomes. Cholera is a diarrheal disease, often associated with water or seafood contamination. Ganglioside GM1 was used to prepare the liposomes by spontaneous insertion into the phospholipid bilayer. CT recognition and signal generation is based on the strong and specific interaction between GM1 and CT. In a sandwich immunoassay, CT was detected as a colored band on the nitrocellulose membrane strip, where CT bound to GM1-liposomes can be captured by immobilized antibodies. The intensity of the band could be visually estimated or measured by densitometry, using computer software. The limit of detection (LOD) of CT in the assay system was found to be 10 fg/mL which is equivalent to 8 zmol in the 70-microL sample. The assay was also tested with water samples spiked with CT, providing a LOD of 0.1-30 pg/mL, which is much better than previously reported limits of detection from other assays. The assay could be completed within 20 min. These results demonstrate that the bioassay developed for CT is rapid and ultrasensitive, suggesting the possibility for detecting CT, simply and reliably, in field screening.     

基于神经网络的信息融合算法研究及其在入侵检测中的应用

目前，网络入侵技术越来越先进，许多黑客都具备反检测的能力，他们会有针对性地模仿被入侵系统的正常用户行为；或将自己的入侵时间拉长，使敏感操作分布于很长的时间周期中；还可能通过多台主机联手攻破被入侵系统．对于伪装性入侵行为与正常用户行为来说，仅靠一个传感器的报告提供的信息来识别已经相当困难，必须通过多传感器信息融合的方法来提高对入侵的识别率，降低误警率．应用基于神经网络的主观Bayes方法，经实验，效果良好．     

Selective photoelectrochemical detection of DNA with high-affinity metallointercalator and tin oxide nanoparticle electrode

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.     

Humic acid assisted synthesis of silver nanoparticles and its application to herbicide detection

Silver nanoparticles have been synthesized by reduction of silver nitrate in the presence of humic acids (HA) which acted as capping agents. The HA protected nanoparticles were found to be sensitive to increasing concentrations of sulfurazon-ethyl herbicide in solution which induced a variation in color of the nanoparticles solution from yellow to purple. The effect of the humic acid concentration used in the nanoparticles synthesis was studied by varying the [Ag⁺:HA] ratio content from [1:1] to [1:100]. UV–Vis spectroscopy was used to monitor the extinction spectra of silver nanoparticles after the synthesis and in the herbicide sensing experiments. An average silver nanoparticles size of 5 nm was confirmed by transmission electron microscope (TEM). When exposed to increasing concentration of sulfurazon-ethyl (0, 100, 200, 300, 400, 500 ppm), the solution of nanoparticles was found to changes from yellow color to orange red and purple with increasing herbicide concentration.     

Development of a whole-cell-based biosensor for detecting histamine as a model toxin

A novel whole-cell potentiometric biosensor for screening of toxins has been developed. The constructed biosensor consists of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) attached to an ion-selective cellulose triacetate (CTA) membrane modified with a covalently attached RGD (arginine-glycine-aspartic acid) peptide sequence. When the HUVECs form a confluent monolayer, ion transport is almost completely inhibited, thereby reducing the response of the ion-selective electrode (ISE). When the monolayer is exposed to agents that increase its permeability (e.g., toxins), ions can diffuse through the membrane, and a potential response from the ISE is achieved. Histamine, a model toxin that increases the permeability of HUVEC monolayers, was used in this study. When the cell-based membranes are exposed to varying concentrations of histamine, the overall response increases with increasing histamine concentration. Thus, the measured potential is an indirect measurement of the histamine concentration. Further experiments were performed for a similar molecule, l-histidine, to test for selectivity. The cell permeability was unaffected by l-histidine, and the sensor response remained unchanged. This type of sensor should find multiple applications in medical, food, and environmental fields and in homeland security.     

A new competitive fluorescence assay for the detection of patulin toxin

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expansum, but it has also been extracted from rotten fruits, moldy feeds, and stored cheese. Human exposure to patulin can lead to serious health problems, and according to a long-term investigation in rats, the World Health Organization has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries. Conventional analytical detection methods involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. However, extensive protocols of sample cleanup are required prior to the analysis, and to accomplish it, expensive analytical instrumentation is necessary. An immunochemical analytical method, based on highly specific antigen-antibody interactions, would be desirable, offering several advantages compared to conventional techniques, i.e., low cost per sample, high selectivity, high sensitivity, and high throughput. In this paper, the synthesis of two new derivatives of patulin is described, along with their conjugation to the bovine serum albumin for the production of polyclonal antibodies. Finally, a fluorescence competitive immunoassay was developed for the on-line detection of patulin.     

Dioxin-binding pentapeptide for use in a high-sensitivity on-bead detection assay

The purpose of this study is to develop a dioxin detection method using a short peptide alternative to an immunoantibody. A full peptide library consisting of 2.5 million possible amino acid combinations was constructed by a solid-phase split synthesis approach using 19 natural amino acids. The peptide beads were subjected to a competitive binding assay between 2,3,7-trichlorodibenzo-p-dioxin and N-NBD-3-(3',4'-dichlorophenoxy)-1-propylamine (NBD-DCPPA) in a buffer containing 20% 1,4-dioxane. Two almost identical pentapeptides, FLDQI and FLDQV, that could bind dioxin were screened from the combinatorial library. NBD-DCPPA and the peptide synthesized on resin beads could be utilized to determine dioxin concentrations. The fluorescence intensity of the beads was measured using fluorescence microscopy to make a calibration curve for the dioxin concentrations. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TeCDD) could also detected in the presence of 30% 1,4-dioxane. To optimize the peptide sequence, a one-amino acid-substituted library was prepared using amino acids including nonnatural amino acids. The internal amino acids, LDQ, could not be substituted by any other amino acids. This result indicates that these three side chains are essential to recognize dioxins. The peptide C terminus substituted by phenylglycine showed a 10 times lower detection limit of 2,3,7,8-TeCDD of 150 pM (50 pg/mL) than the original sequence FLDQV. The cross reactivity of the dioxin binding peptides including the secondary derivatives was investigated. Some polycyclic aromatic hydrocarbons bound to the peptide beads, but nonchlorinated dibenzo-p-dioxin and PCB did not. From these results, we demonstrate the potential of short peptides as a practical sensor material targeting low molecular weight compounds such as dioxin.     

Stable and fluid ethylphosphocholine membranes in a poly(dimethylsiloxane) microsensor for toxin detection in flooded waters

Highly stable and fluid supported bilayer membranes were fabricated by fusion of positively charged ethylphosphocholine (DOPC+) vesicles into poly(dimethylsiloxane) (PDMS) microchannels for immunosensing of cholera toxin (CT) in flooded waters. Compared to phosphatidylcholine (PC) layers in the microchannels, DOPC+ membranes show exceptionally strong resistance to air-dry damage, as demonstrated by fluorescence recovery after photobleaching (FRAP) measurements and protein adsorption studies. In FRAP experiments, the mobile fraction of PC membranes was found to decrease by 10% upon drying/rehydration and the lateral diffusion coefficient decreased from 2.2 to 1.6 microm(2)/s, whereas the mobile fraction and diffusion coefficient for DOPC+ membranes remain virtually unchanged during this process. Characterization by confocal microscopy reveals that only 1% of the DOPC+ membrane in the microchannels was removed by the drying/rehydration process, as compared to 11% for PC. Protein adsorption trends indicate that the charge of DOPC+ membranes allows for tuning of solution conditions to enable the desired protein-membrane interaction to predominate at the interface. A flow-based immunoassay for bacterial toxin was developed with 5% GM1/DOPC+ membranes in PDMS channels, and a detection limit of 250 amol for CT was obtained from the calibration curves. The assay was successfully applied to detection of CT spiked in water samples from the Santa Ana River, with nearly identical response and sensitivity.     

Ground-based full-sky imaging polarimetry of rapidly changing skies and its use for polarimetric cloud detection

For elimination of the shortcomings of imaging polarimeters that take the necessary three pictures sequentially through linear-polarization filters, a three-lens, three-camera, full-sky imaging polarimeter was designed that takes the required pictures simultaneously. With this polarimeter, celestial polarization patterns can be measured even if rapid temporal changes occur in the sky: under cloudy sky conditions, or immediately after sunrise or prior to sunset. One of the possible applications of our polarimeter is the ground-based detection of clouds. With use of the additional information of the degree and the angle of polarization patterns of cloudy skies measured in the red (650 nm), green (550 nm), and blue (450 nm) spectral ranges, improved algorithms of radiometric cloud detection can be offered. We present a combined radiometric and polarimetric algorithm that performs the detection of clouds more efficiently and reliably as compared with an exclusively radiometric cloud-detection algorithm. The advantages and the limits of three-lens, three-camera, full-sky imaging polarimeters as well as the possibilities of improving our polarimetric cloud detection method are discussed briefly.     

Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology

Paralytic shellfish poisoning (PSP) toxin monitoring in shellfish is currently performed using the internationally accredited AOAC mouse bioassay. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. The feasibility of using a surface plasmon resonance optical biosensor to detect PSP toxins in shellfish tissue below regulatory levels was examined. Three different PSP toxin protein binders were investigated: a sodium channel receptor (SCR) preparation derived from rat brains, a monoclonal antibody (GT13-A) raised to gonyautoxin 2/3, and a rabbit polyclonal antibody (R895) raised to saxitoxin (STX). Inhibition assay formats were used throughout. Immobilization of STX to the biosensor chip surface was achieved via amino-coupling. Specific binding and inhibition of binding to this surface was achieved using all proteins tested. For STX calibration curves, 0-1000 ng/mL, IC50 values for each binder were as follows: SCR 8.11 ng/mL; GT13-A 5.77 ng/mL; and R895 1.56 ng/mL. Each binder demonstrated a different cross-reactivity profile against a range of STX analogues. R895 delivered a profile that was most likely to detect the widest range of PSP toxins at or below the internationally adopted regulatory limits.     

Building a cascade detector and its applications in automatic target detection

A hierarchical classifier (cascade) is proposed for target detection. In building an optimal cascade we considered three heuristics: (1) use of a frontier-following approximation, (2) controlling error rates, and (3) weighting. Simulations of synthetic data with various underlying distributions were carried out. We found that a weighting heuristic is optimal in terms of both computational complexity and error rates. We initiate a systematic comparison of several potential heuristics that can be utilized in building a hierarchical model. A range of discussions regarding the implications and the promises of cascade architecture as well as of techniques that can be integrated into this framework is provided. The optimum heuristic--weighting algorithms--was applied to an IR data set. It was found that these algorithms outperform some state-of-the-art approaches that utilize the same type of simple classifier.     

Synthesis of silver nanoparticles by oleylamine-oleic acid reduction and its use in making nanocable by coaxial electrospinning

In the present study, silver nanoparticles were produced by hydrazine hydrate and oleylamine/oleic acid systems in order to investigate the effects of reducing agents with different strengths on the reduction mechanism. Particle size and size distribution of silver particles produced by slow reducing system were studied in detail by using transmission electron microscopy (TEM), X-Ray Diffraction (XRD), photon correlation spectroscopy (PCS), and surface plasmon resonance spectroscopy (SPR). Finally, reduction mechanism by oleylamine and oleic acid system was clarified and particles with average diameter of 2.7 nm were produced. Nano-sized particles were then placed at the center of the polymer fibers by coaxial electrospinning and nanocable like structures were produced. SEM and TEM were used for the characterization of these cables.     

Ultrasensitive detection of cancer biomarkers in the clinic by use of a nanostructured microfluidic array

Multiplexed biomarker protein detection holds unrealized promise for clinical cancer diagnostics due to lack of suitable measurement devices and lack of rigorously validated protein panels. Here we report an ultrasensitive electrochemical microfluidic array optimized to measure a four-protein panel of biomarker proteins, and we validate the protein panel for accurate oral cancer diagnostics. Unprecedented ultralow detection into the 5-50 fg·mL(-1) range was achieved for simultaneous measurement of proteins interleukin 6 (IL-6), IL-8, vascular endothelial growth factor (VEGF), and VEGF-C in diluted serum. The immunoarray achieves high sensitivity in 50 min assays by using off-line protein capture by magnetic beads carrying 400,000 enzyme labels and ~100,000 antibodies. After capture of the proteins and washing to inhibit nonspecific binding, the beads are magnetically separated and injected into the array for selective capture by antibodies on eight nanostructured sensors. Good correlations with enzyme-linked immunosorbent assays (ELISA) for protein determinations in conditioned cancer cell media confirmed the accuracy of this approach. Normalized means of the four protein levels in 78 oral cancer patient serum samples and 49 controls gave clinical sensitivity of 89% and specificity of 98% for oral cancer detection, demonstrating high diagnostic utility. The low-cost, easily fabricated immunoarray provides a rapid serum test for diagnosis and personalized therapy of oral cancer. The device is readily adaptable to clinical diagnostics of other cancers.     

Development of a cryogenic detection concept for GNO

The Gallium-Neutrino-Experiment at GNO measures solar neutrinos radiochemically via the reaction ⁷¹Ga(ν,e)⁷¹Ge. In order to reduce statistical and systematic uncertainties in detecting the decay of ⁷¹Ge to ⁷¹Ga, a favourable way could be the use of high-resolution cryogenic detectors instead of the presently integrated miniaturized proportional counters. After the successful development of a highly efficient 4π-detector, optimization of thermal Ge-deposition and decoupling of deposition and detection, present activity concentrates on low background considerations, enhancement of external and internal shielding of the cryostat and a long-term run with artificially activated ⁷¹Ge to prove the feasibility of ‘cryoGNO’ in our Underground Laboratory in Garching.     

SWCNT-DNA修饰电极的制备及其电化学检测性能

利用单壁碳纳米管(SWCNT)与单链DNA(ssDNA)自组装生成单根离散的sWCNT-DNA复合物,将其吸附在玻炭(GC)电极表面形成一层SWCNTT-DNA薄膜,构建SWCNT-DNA修饰玻炭电极.循环伏安测试表明,与裸玻炭电极和未分散SWCNT修饰的玻炭电极相比,该电极的响应峰电流明显增大,而且在一定范围内对不同浓度的铁氰化钾有一个线性响应,表现出良好的灵敏度和稳定性.ssDNA通过缠绕在SWCNT外壁使其离散,可提高电极的有效表面积,加快Fe(CN)3-6/Fe(CN)4-6-氧化还原反应的电子传递,使之表现出良好的电化学检测性能.