Home About us Contact
|
Home About us Contact | ||
|
|
||
Sensitive Detection (sensitive + detection)
Kinds of Sensitive Detection Terms modified by Sensitive Detection Selected AbstractsSynthesis of Carbon Nanofibers for Mediatorless Sensitive Detection of NADHELECTROANALYSIS, Issue 15 2008Yang Liu Abstract Highly sensitive amperometric detection of dihydronicotinamide adenine dinucleotide (NADH) by using novel synthesized carbon nanofibers (CNFs) without addition of any mediator has been proposed. The CNFs were prepared by combination of electrospinning technique with thermal treatment method and were applied without any oxidation pretreatment to construct the electrochemical sensor. In amperometric detection of NADH, a linear range up to 11.45,,M with a low detection limit of 20,nM was obtained with the CNF-modified carbon paste electrode (CNF-CPE). Good selectivity was exhibited for the simultaneous detection of NADH and its common interferent of ascorbic acid (AA) by differential pulse voltammogram. The attractive electrochemical performance and the versatile preparation process of the CNF-CPE made it a promising candidate for designing effective NADH sensor. [source] Signal-On Electrochemiluminescence Biosensors Based on CdS,Carbon Nanotube Nanocomposite for the Sensitive Detection of Choline and AcetylcholineADVANCED FUNCTIONAL MATERIALS, Issue 9 2009Xiao-Fei Wang Abstract This work describes for the first time signal-on electrochemiluminescence (ECL) enzyme biosensors based on cadmium sulfide nanocrystals (CdS NCs) formed in situ on the surface of multi-walled carbon nanotubes (MWCNTs). The MWCNT,CdS can react with H2O2 to generate strong and stable ECL emission in neutral solution. Compared with pure CdS NCs, the MWCNT,CdS can enhance the ECL intensity by 5.3-fold and move the onset ECL potential more positively for about 400,mV, which reduces H2O2 decomposition at the electrode surface and increases detection sensitivity of H2O2. Furthermore, the ECL intensity is less influenced by the presence of oxygen in solution. Benefiting from these properties, signal-on enzyme-based biosensors are fabricated by cross-linking choline oxidase and/or acetylcholine esterase with glutaraldehyde on MWCNT,CdS modified electrodes for detection of choline and acetylcholine. The resulting ECL biosensors show wide linear ranges from 1.7 to 332,µM and 3.3 to 216,µM with lower detection limit of 0.8 and 1.7,µM for choline and acetylcholine, respectively. The common interferents such as ascorbic acid and uric acid in electrochemical enzyme-based biosensors do not interfere with the ECL detection of choline and acetylcholine. Furthermore, both ECL biosensors possess satisfying reproducibility and acceptable stability. [source] Innentitelbild: Sensitive Detection of Native Proteins Using Extractive Electrospray Ionization Mass Spectrometry (Angew. Chem.ANGEWANDTE CHEMIE, Issue 17 201017/2010) Die Charakterisierung nativer Proteine gelingt durch extrahierende Elektrosprayionisations-Massenspektrometrie (EESI-MS). Wie H. Chen und Mitarbeiter in ihrer Zuschrift auf S.,3117,ff. ausführen, werden native Proteine bei der EESI mit Ladungen belegt, ohne dass ein starkes elektrisches Feld benötigt wird. Das Verfahren erleichtert massenspektrometrische Studien, auch an biologischen Rohproben, und vermeidet signifikante Konformationsänderungen und Aktivitätsverluste. [source] Sensitive Detection of Native Proteins Using Extractive Electrospray Ionization Mass Spectrometry,ANGEWANDTE CHEMIE, Issue 17 2010Huanwen Chen Prof. ,Sanfte" Ionisation: Bei der EESI werden Ladungen auf native Proteine übertragen, ohne sie einem starken elektrischen Feld auszusetzen. So lassen sich Proteine in nichtaufbereiteten biologischen Proben massenspektrometrisch charakterisieren, ohne dass bedeutende Konformationsänderungen oder Aktivitätsverluste eintreten. Die EESI-MS bietet sich für die Hochdurchsatzanalyse kleinster Proteinmengen unter nativen Bedingungen an. [source] Determination of ethyl glucuronide in human serum by hyphenation of capillary isotachophoresis and zone electrophoresisELECTROPHORESIS, Issue 8 2008Michaela Novįkovį Abstract The determination of ethyl glucuronide (EtG), a marker of recent alcohol consumption, in human serum by hyphenation of capillary ITP (CITP) and CZE is reported. For CITP step, 1×10,2,M hydrochloric acid adjusted with ,-aminocaproic acid (EACA) to pH,4.4 was used as the leading electrolyte, and 1×10,2,M nicotinic acid with EACA, pH,4.4, was used as the terminating electrolyte (TE). All electrolytes contained 0.2% hydroxypropylcellulose to suppress electroosmosis. In CITP, EtG was separated from fast serum macrocomponents chloride, phosphate, lactate, and acetate. Zones of microcomponents including EtG that migrated between acetate and nicotinate were forwarded to the second capillary filled with a BGE identical with the TE. Conductivity detection was used in the CITP step. Sensitive detection in the CZE step was performed using indirect spectrophotometric detection at 254,nm. The assay is based on a 1:5 dilution of serum with deionized water and has a concentration LOD for EtG in diluted sample of 9.8×10,9,M. The method was used for the determination of EtG in sera of volunteers consuming alcohol. [source] Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniquesPLANT PATHOLOGY, Issue 4 2000P. M. Pradhanang The sensitivity and specificity of various methods were compared for routine detection of Ralstonia solanacearum in a sandy loam soil. Populations fewer than 102 CFU per g soil were detected by dilution plating on a modified semiselective medium (SMSA). In comparison, a tomato bioassay was shown consistently to detect populations at or greater than 7·5 × 105 CFU per g soil. An indirect enzyme-linked immunosorbent assay (ELISA) was as sensitive as the tomato bioassay, but detected as few as 104 CFU per g soil when the suspension was first incubated in SMSA broth prior to testing. Detection using a nested polymerase chain reaction (PCR) was equally as sensitive as that using culture on SMSA agar, but only when the infested soil sample was first enriched overnight in SMSA broth prior to the nested PCR. Longer incubation periods in SMSA broth also increased the sensitivity of pathogen detection using a conventional PCR method, permitting detection of as few as 102 CFU per g soil after 60 h enrichment in SMSA broth. When evaluated using naturally infected field soils in Nepal, isolation of R. solanacearum on SMSA was reliable only when pathogen populations were higher than those of saprophytic soilborne bacteria. As few as 5 × 102 CFU of R. solanacearum per g were recovered from naturally infested soil, whereas the sensitivity of indirect ELISA was 106 CFU g,1. [source] Sensitive detection of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry: use of alkylphosphonic acids as matrix additivesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2008Hiroki Kuyama Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been one of the most powerful tools for analyzing protein phosphorylation. However, it is frequently difficult to detect phosphopeptides with high sensitivity by MALDI-MS. In our investigation of matrix/matrix-additive substances for improving the phosphopeptide ion response in MALDI-MS, we found that the addition of low-concentration alkylphosphonic acid to the matrix/analyte solution significantly enhanced the signal of phosphopeptides. In this study, the combination of methanediphosphonic acid and 2,5-dihydroxybenzoic acid gave the best results. In addition to enhancing the signal of the phosphopeptides, alkylphosphonic acid almost completely eliminated the signals of sodium and potassium ion adducts. We report herein sensitive detection of phosphopeptides by MALDI-MS with the use of alkylphosphonic acids as matrix additives. Copyright © 2008 John Wiley & Sons, Ltd. [source] Voltammetric Detection of Lead(II) Using Amide-Cyclam- Functionalized Silica-Modified Carbon Paste ElectrodesELECTROANALYSIS, Issue 15 2009Stéphanie Goubert-Renaudin Abstract 2-(4,8,11-Triscarbamoylmethyl-1,4,8,11-tetraazacyclotetradec-1-yl)acetamide (TETAM) derivatives bearing 1, 2, or 4 silylated arms have been synthesized and grafted to the surface of silica gel and ordered mesoporous silica samples. The resulting organic-inorganic hybrids have been incorporated into carbon paste electrodes and applied to the preconcentration electroanalysis of Pb(II). The attractive recognition properties of these cyclam derivatives functionalized with amide pendent groups toward Pb(II) species and the highly porous structure of the adsorbents can be exploited for the selective and sensitive detection of the target analyte. Various parameters affecting the preconcentration and detection steps have been discussed with respect to the composition and pH of both accumulation and detection media, the nature of the adsorbent (number of silylated groups linking the macrocycle to silica, texture of materials), the accumulation time, and the presence of interfering cations. Under optimal conditions and for 2,min accumulation at open-circuit, the voltammetric response increased linearly with the Pb(II) concentration in a range extending from 2×10,7 to 10,5,M, while a longer accumulation time of 15,min afforded a linear calibration curve between 10,8 and 10,7,M with a detection limit of 2.7×10,9,M which is well below the European regulatory limit of lead in consumption water. [source] Nanotube Membrane Based BiosensorsELECTROANALYSIS, Issue 1-2 2004Punit Kohli Abstract We review highly sensitive detection based on electrochemical methods. These methods are based on monodisperse gold and alumina nanotubule membranes with inside diameter approaching molecular dimensions. The analyte species can be detected by measuring a change in trans-membrane current when the analyte is added to the nanotubule-based cell. The second method entails the use of a concentration change based on the nanotubule membrane. Biomemtic ion-gated channels micropore and nanotubule membrane sensors are also reviewed. These synthetic ion channels can be switched from an "off" state to an "on" state in response to an external chemical stimulus. Using these methods, we have achieved detection limits as low as 10,pM. Potential applications for these biosensors are in fields such as bioanalytical, biomedical, pharmaceutical and drug discovery. [source] Hydrodynamic Modulation Voltammetry with a Dual Disk Chopped Flow-Microjet Electrode (CF-MJE)ELECTROANALYSIS, Issue 18 2003Nafeesa Simjee Abstract A novel form of hydrodynamic modulation voltammetry (HMV) is described, based on the periodic variation of mass transport in a microjet electrode (MJE) system, in combination with phase-sensitive detection techniques. In the configuration developed, a jet of solution is fired from a nozzle that is aligned directly over the surface of a dual disk Pt-Pt ultramicroelectrode (UME). The potential at each electrode is controlled separately. A rotating blade, positioned between the nozzle and the UME probe, is used to periodically interrupt flow to the electrode surface, resulting in modulation of the overall mass transfer rate between two defined extremes. The use of a dual disk UME enables two transport-limited current signals to be recorded simultaneously, one for the analyte of interest, and the other for a ,reference species' (oxygen for the studies described herein). The latter current response corresponds to the variation in mass transport rate in the chopped flow (CF) arrangement and is used as the signal for phase sensitive detection of the analyte current. Studies of potassium hexachloroiridate (III) [IrCl] oxidation in aqueous solution are used to demonstrate the capabilities of the technique. HMV in the CF-MJE arrangement allows quantitative concentration measurements, down to at least 5×10,7,M. [source] High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresisELECTROPHORESIS, Issue 12 2008Yilin Li Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source] Simultaneous enantioseparation and sensitivity enhancement of basic drugs using large-volume sample stackingELECTROPHORESIS, Issue 19 2007Nerissa L. Deńola Abstract Simultaneous enantioseparation with sensitive detection of four basic drugs, namely methoxamine, metaproterenol, terbutaline and carvedilol, using a 20-,m ID capillary with native ,-CD as the chiral selector was demonstrated by the large-volume sample stacking method. The procedure included conventional sample loading either hydrodynamically or electrokinetically at longer injection times without polarity switching and EOF manipulation. In comparison to conventional injections, depending on the analyte, about several hundred- and a thousand-fold sensitivity enhancement was achieved with the hydrodynamic and the electrokinetic injections, respectively. The simple method developed was applied to the analysis of racemic analytes in serum samples and better recovery was achieved using hydrodynamic injection than electrokinetic injection. [source] Fast quantitative determination of diuretic drugs in tablets and human urine by microchip electrophoresis with native fluorescence detectionELECTROPHORESIS, Issue 16 2007Kamal Tolba Abstract Microchip electrophoresis (MCE) with native fluorescence detection has been applied for the fast quantitative analysis of pharmaceutical formulations. For this purpose, methods for fast separation and sensitive detection of the unlabeled diuretic drugs, amiloride, triamterene, bendroflumethiazide (BFMTZ), and bumetanide were developed. An epifluorescence setup was used enabling the coupling of different lasers into a commercial fluorescence microscope. The detection sensitivity of different excitation light sources was compared utilizing either a HeCd laser (,exc,=,325,nm), a frequency quadrupled Nd:YAG laser (,exc,=,266,nm), or a mercury lamp (,exc,=,330,380,nm). At optimal conditions using the HeCd laser, the drugs were separated within 15,s with LODs less than 1,,g/mL for the four compounds. A linear relationship between concentration and peak area was obtained in the concentration range of 0.05,20,,g/mL with a mean correlation coefficient of around 0.996 for all analytes. The method was successfully applied to the analysis of the respective drugs in commercial formulations and in human urine without interference from other constituents. These data show that MCE has a great potential for reliable drug analysis. [source] Optimization of the electrokinetic supercharging preconcentration for high-sensitivity microchip gel electrophoresis on a cross-geometry microchipELECTROPHORESIS, Issue 14 2004Zhongqi Xu Abstract We developed a novel on-line preconcentration procedure for microchip gel electrophoresis (MCGE), which enables application of electrokinetic supercharging (EKS) for highly sensitive detection of DNA fragments on a cross-geometry microchip. In comparison with conventional pinched injection using the cross microchip, the present approach allows loading a much larger amount of the sample by taking advantage of a newly developed operational mode. In order to obtain high preconcentration effect and prevent splitting of an enriched sample into subchannels, i.e., off the detector range, effects of the voltage applied on the reservoirs and the time of isotachophoretic preconcentration were examined. The optimal balance between the voltage and time was found for a high-sensitivity analysis of DNA fragments. After experimental optimization the detection limit of a 150 bp fragment was as low as 0.22 mg/L (S/N = 3) that is 10 times better than using the conventional pinched injection. [source] Separation of twenty underivatized essential amino acids by capillary zone electrophoresis with contactless conductivity detectionELECTROPHORESIS, Issue 4 2003Pavel Coufal Abstract Twenty underivatized essential amino acids were separated using capillary zone electrophoresis and consequently detected with contactless conductivity detection (CCD). A simple acidic background electrolyte (BGE) containing 2.3 M acetic acid and 0.1% w/w hydroxyethylcellulose (HEC) allowed the electrophoretic separation and sensitive detection of all 20 essential amino acids in their underivatized cationic form. The addition of HEC to the BGE suppressed both, electroosmotic flow and analyte adsorption on the capillary surface resulting in an excellent migration time reproducibility and a very good analyte peak symmetry. Additionally, the HEC addition significantly reduced the noise and long-term fluctuations of the CCD baseline. The optimized electrophoretic separation method together with the CCD was proved to be a powerful technique for determination of amino acid profiles in various natural samples, like beer, yeast, urine, saliva, and herb extracts. [source] Application of real-time PCR for quantitative detection of Campylobacter jejuni in poultry, milk and environmental waterFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2003Chengbo Yang Abstract Campylobacter jejuni is a leading human food-borne pathogen. The rapid and sensitive detection of C. jejuni is necessary for the maintenance of a safe food/water supply. In this article, we present a real-time polymerase chain reaction (PCR) assay for quantitative detection of C. jejuni in naturally contaminated poultry, milk and environmental samples without an enrichment step. The whole assay can be completed in 60 min with a detection limit of approximately 1 CFU. The standard curve correlation coefficient for the threshold cycle versus the copy number of initial C. jejuni cells was 0.988. To test the PCR system, a set of 300 frozen chicken meat samples, 300 milk samples and 300 water samples were screened for the presence of C. jejuni. 30.6% (92/300) of chicken meat samples, 27.3% (82/300) of milk samples, and 13.6% (41/300) of water samples tested positive for C. jejuni. This result indicated that the real-time PCR assay provides a specific, sensitive and rapid method for quantitative detection of C. jejuni. Moreover, it is concluded that retail chicken meat, raw milk and environmental water are commonly contaminated with C. jejuni and could serve as a potential risk for consumers in eastern China, especially if proper hygienic and cooking conditions are not maintained. [source] Trichlorofluoroethene: A reactive tracer for evaluating reductive dechlorination in large-diameter permeable columnsGROUND WATER MONITORING & REMEDIATION, Issue 2 2005Jennifer A. Field Trichlorofluoroethene (TCFE) was used as a reactive tracer to determine the in situ rate of reductive dechlorination in treatment zones impacted by three large-diameter permeable columns (LDPCs) that were installed at a trichloroethene (TCE),contaminated site. The LDPCs were part of a pilot study to evaluate the effectiveness of hydrogen, lactate, and zero-valent iron for remediating TCE-contaminated ground water. The rate of TCFE reductive dechlorination was determined for each LDPC by means of push-pull tests conducted in each treatment layer. In addition, the distribution of TCFE's lesser chlorinated transformation products was determined. The rates of TCFE reductive dechlorination ranged from 0.05/d to 0.20/d and corresponded to half-lives ranging from 3.5 to 13.9 d. cis -Dichlorofluoroethene was the dominant transformation product detected in all the tests, which is consistent with the findings from pilot tests conducted in the LDPCs prior to the TCFE push-pull tests. cis -Chlorofluoroethene (CFE) and 1,1-CFE also were detected and indicate the potential for vinyl chloride to form under all treatment regimes. Significant production of fluoroethene (FE), the analog of ethene, was observed for only one of the hydrogen treatments. Unambiguous and sensitive detection of the lesser chlorinated products, such as CFE and FE, is possible because TCFE and its transformation products are not found in the background ground water at contaminated sites. Good agreement between the rates and transformation product profiles for TCFE and TCE in both field and laboratory experiments indicates the suitability of TCFE as a surrogate for predicting the rates of TCE reductive dechlorination. [source] Highly Surface-roughened "Flower-like" Silver Nanoparticles for Extremely Sensitive Substrates of Surface-enhanced Raman ScatteringADVANCED MATERIALS, Issue 45 2009Hongyan Liang Abstract Surface-enhanced Raman scattering (SERS) is a new optical spectroscopic analysis technique with potential for highly sensitive detection of molecules. Recently, many efforts have been made to find SERS substrates with high sensitivity and reproducibility. In this Research News article, we provide a focused review on the synthesis of monodispersed silver particles with a novel, highly roughened, "flower-like" morphology by reducing silver nitrate with ascorbic acid in aqueous solutions. The nanometer-scale surface roughness of the particles can provide several hot spots on a single particle, which significantly increases SERS enhancement. The incident polarization-dependent SERS of individual particles is also studied. Although the different "hot spots" on a single particle can have a strong polarization dependency, the total Raman signals from an individual particle usually have no obvious polarization dependency. Moreover, these flower-like silver particles can be measured by SERS with high enhancement several times, which indicates the high stability of the hot spots. Hence, the flower-like silver particles here can serve as highly sensitive and reproducible SERS substrates. [source] Highly sensitive detection of the MGB1 transcript (mammaglobin) in the peripheral blood of breast cancer patientsINTERNATIONAL JOURNAL OF CANCER, Issue 4 2004Nuno Cerveira Abstract We describe a new one-step RT-PCR assay for the detection of the mammaglobin (MGB1) gene transcript in the peripheral blood of breast cancer patients. With this approach, the MGB1 transcript could be detected in the peripheral blood of 22 of 54 (41%) breast cancer patients prior to any therapy. This method, using specific primers for cDNA synthesis, proved to be more sensitive (10,6 to 10,11, usually 10,7) than previously reported methodologies. This increased sensitivity was achieved without compromising specificity, as the MGB1 transcript was not detected in 38 blood samples of healthy donors and in only 1 of 18 blood samples of patients presenting with hematologic malignancies. A positive correlation was seen between MGB1 positivity and breast cancer stage: 0/3 (0%) in stage 0, 3/13 (23%) in stage I, 6/17 (35%) in stage II, 5/10 (50%) in stage III, 8/11 (73%) in stage IV (p = 0.003). The prognostic and therapeutic implications of MGB1 positivity by one-step RT-PCR in the peripheral blood of breast cancer patients, especially in clinically localized disease (stages I and II), should be evaluated after long-term clinical follow-up of these patients. © 2003 Wiley-Liss, Inc. [source] A PCR-DGGE method for detection and identification of Campylobacter, Helicobacter, Arcobacter and related Epsilobacteria and its application to saliva samples from humans and domestic petsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2007R.F. Petersen Abstract Aims:, To develop a PCR-denaturing gradient gel electrophoresis (PCR-DGGE) method for the detection and identification of Campylobacter, Helicobacter and Arcobacter species (Epsilobacteria) in clinical samples and evaluate its efficacy on saliva samples from humans and domestic pets. Methods and Results:, A semi-nested PCR was developed to allow sensitive detection of all Epsilobacteria, with species separation undertaken by DGGE. A database was constructed in BioNumerics using 145 strains covering 51 Campylobacter, Arcobacter and Helicobacter taxa; Nineteen distinct DGGE profile-groups were distinguished. This approach detected Epsilobacteria in all saliva samples collected from humans, cats and dogs, and identified Campylobacter concisus and/or Campylobacter gracilis in the human samples. The pet animal samples were taken from individuals with oral/dental diseases; PCR-DGGE identified up to four different species in each sample. The most common species detected included Wolinella succinogenes, Arcobacter butzleri and two hitherto uncultured campylobacters. The enteropathogen Campylobacter lari was also found. Conclusions:, PCR combined with DGGE is a useful tool for direct detection and preliminary identification of Epsilobacteria in the oral cavity of humans and small animals. Significance and Impact of the Study:, The PCR-DGGE method should allow determination of the true prevalence and diversity of Epsilobacteria in clinical and other samples. Contact with the oral cavity of domestic pets may represent a route of transmission for epsilobacterial enteric diseases. [source] Detection of cryptosporidia and Cryptosporidium parvum oocysts in environmental water samples by immunomagnetic separation,polymerase chain reactionJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2000S. Hallier-Soulier Cryptosporidium parvum has emerged as one of the most important new contaminants found in drinking water. Current protocols for the detection of cryptosporidia are time-consuming and rather inefficient. We recently described an immunomagnetic separation,polymerase chain reaction (IMS,PCR) assay permitting highly sensitive detection of C. parvum oocysts in drinking water samples. In this study, a second IMS,PCR assay to detect all cryptosporidial oocysts was developed, and both IMS,PCR assays were optimized on river water samples. A comparative study of the two IMS,PCR assays and the classical detection method based on an immunofluorescence assay (IFA) was carried out on 50 environmental samples. Whatever the type of water sample, the discrepancy in C. parvum detection between the IFA and IMS,PCR took the form of IFA-negative/IMS,PCR-positive results, and was caused mainly by the greater sensitivity of IMS,PCR as compared with IFA. Of the 50 water samples, only five tested positive for C. parvum using IMS,PCR, and could constitute a threat to human health. These results show that both IMS,PCR assays provide a rapid (1 d) and sensitive means of screening environmental water samples for the presence of cryptosporidia and C. parvum oocysts. [source] SPECIFIC DETECTION OF AMANITA PHALLOIDES MYCELIUM AND SPORES BY PCR AMPLIFICATION OF THE GPD (GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE) GENE FRAGMENTJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2000OWSKI, ROMAN KOT ABSTRACT Oligonucleotide primers designed to flank a 635 bp fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from Araanita muscaria were used to amplify the corresponding gpd fragment from Amanita phalloides. The A. phalloides PCR product was cloned, sequenced and found to be 70 - 77% similar to the known basidiomycetes gpd genes within the exon part and 25 - 52% within the intron part. Based on these data, species-specific amplification was achieved using a pair of oligonucleotide primers complementary to the A. phalloides gpd intron sequences. These primers allowed the amplification of the corresponding gpd fragment from the A. phalloides but not from various other basidiomycetes, ascomycetes and human matrices. PCR amplification of the A. phalloides DNA gave the predicted PCR product of 284 bp. The created PCR system is an efficient tool for the specific, rapid and sensitive detection of A. phalloides mycelium and spores. [source] Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed ProductsJOURNAL OF FOOD SCIENCE, Issue 1 2006Lihua Liu ABSTRACT: A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat-processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin-conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory-adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy-based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy-based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety. [source] Detection and validated quantification of nine herbal phenalkylamines and methcathinone in human blood plasma by LC-MS/MS with electrospray ionizationJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2007Jochen Beyer Abstract The herbal stimulants Ephedra species, Catha edulis (khat), and Lophophora williamsii (peyote) have been abused for a long time. In recent years, the herbal drug market has grown owing to publicity on the Internet. Some ingredients of these plants are also ingredients of cold remedies. The aim of the presented study is to develop a multianalyte procedure for detection and validated quantification of the phenalkylamines ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, methylephedrine, methylpseudoephedrine, cathinone, mescaline, synephrine (oxedrine), and methcathinone in plasma. After mixed-mode solid-phase extraction of 1 ml of plasma, the analytes were separated using a strong cation exchange separation column and gradient elution. They were detected using a Q-Trap LC-ESI-MS/MS system (MRM mode). Calibration curves were used for quantification using norephedrine- d3, ephedrine- d3, and mescaline- d9 as internal standards. The method was validated according to international guidelines. The assay was selective for the tested compounds. It was linear from 10 to 1000 ng/ml for all analytes. The recoveries were generally higher than 70%. Accuracy ranged from , 0.8 to 20.0%, repeatability from 2.5 to 12.3%, and intermediate precision from 4.6 to 20.0%. The lower limit of quantification was 10 ng/ml for all analytes. No instability was observed after repeated freezing and thawing or in processed samples. The applicability of the assay was tested by analysis of authentic plasma samples after ingestion of different cold medications containing ephedrine or pseudoephedrine, and after ingestion of an aqueous extract of Herba Ephedra. After ingestion of the cold medications, only the corresponding single alkaloids were detected in human plasma, whereas after ingestion of the herb extract, all six ephedrines contained in the plant were detected. The presented LC-MS/MS assay was found applicable for sensitive detection and accurate and precise quantification of all studied analytes in plasma. Copyright © 2006 John Wiley & Sons, Ltd. [source] Off-line combination of reversed-phase liquid chromatography and laser desorption/ionization time-of-flight mass spectrometry with seamless post-source decay fragment ion analysis for characterization of square-planar nickel(II) complexesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2006Pavel, ehulka Abstract Characterization of square-planar nickel(II) complexes of the Schiff base of (S)- N -benzylproline (2-benzoylphenyl)amide and various amino acids that are used as efficient ,-amino acids synthons was carried out using laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) in off-line combination with liquid chromatography. A mixture of four square-planar nickel(II) complexes was separated using reversed-phase liquid chromatography (RPLC) and the separated fractions from the chromatographic run were spotted on the metal target directly from the column outlet using a lab-made sample deposition device. The separated fractions were then analyzed by LDI-TOF MS. Seamless postsource decay (sPSD) fragment ion analysis was used for their structural characterization, which made possible the confirmation of expected chemical structures of the analyzed compounds. The off-line combination of the separation by RPLC and analysis by LDI-TOF MS allowed successful separation, sensitive detection and structure elucidation of the square-planar nickel(II) complexes. Copyright © 2006 John Wiley & Sons, Ltd. [source] Rapid and sensitive detection of mumps virus RNA directly from clinical samples by real-time PCRJOURNAL OF MEDICAL VIROLOGY, Issue 3 2005Kazue Uchida Abstract A rapid, sensitive, and specific assay to detect mumps virus RNA directly from clinical specimens using a real-time PCR assay was developed. The assay was capable of detecting five copies of standard plasmid containing cDNA from the mumps virus F gene. No cross-reactions were observed with other members of Paramyxoviridae, or with viruses or bacteria known to be meningitis pathogens. Seventy-three clinical samples consisting of throat swabs collected from patients with parotitis, and cerebrospinal fluid (CSF) collected from patients with aseptic meningitis, were examined with a real-time PCR assay developed by the authors, reverse-transcription nested-PCR (RT-n-PCR), and virus isolation using cell culture. Like the RT-n-PCR assay, the real-time PCR assay could detect mumps virus RNA in approximately 70% of both throat swabs and CSF samples, while, by tissue culture, mumps virus was isolated from only approximately 20% of CSF and 50% of throat swab samples. In addition, the real-time PCR assay could be developed easily into a quantitative assay for clinical specimens containing more than 1,800 copies of mumps virus RNA/ml by using serial dilutions of the standard plasmid. The results suggest that the real-time PCR assay is useful for identification of mumps virus infections, not only in typical cases, but also in suspected cases, which show only symptoms of meningitis or encephalitis. J. Med. Virol. 75:470,474, 2005. © 2005 Wiley-Liss, Inc. [source] Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain ReactionJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009Xiaojie Wang Abstract Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst. Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst. In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants. [source] Use of peptide for selective and sensitive detection of an Anthrax biomarker via peptide recognition and surface-enhanced Raman scatteringJOURNAL OF RAMAN SPECTROSCOPY, Issue 2 2010Kyungtag Ryu Abstract A short 16-amino acid peptide has been used in place of an antibody to selectively detect the specific Anthrax biomarker, protective antigen (PA), using surface-enhanced Raman scattering (SERS). Peptides are more stable than antibodies under various biological conditions and are easily synthesized for a specific target. A peptide that has high affinity to PA was conjugated onto gold nanoparticles along with a Raman reporter and then incubated in various concentrations of PA. Parallel studies in which the peptide sequence was replaced with an antibody were performed to compare the performance of the two methodologies. Both the peptide and antibody functionalized nanoparticles were able to specifically detect PA concentrations down to 6.1 fM. These results demonstrate that these short, robust peptides can be used in the place of traditional antibodies to specifically recognize target biomarkers in the field for the potential diagnosis of disease. Copyright © 2010 John Wiley & Sons, Ltd. [source] Surface-enhanced resonance Raman scattering using pulsed and continuous-wave laser excitationJOURNAL OF RAMAN SPECTROSCOPY, Issue 6-7 2005Rachael E. Littleford Abstract Pulsed and continuous-wave (CW) lasers were compared as excitation sources for surface-enhanced resonance Raman scattering (SERRS). CW excitation provided SERRS spectra with a greater signal-to-noise ratio and more sensitive detection by a factor of ,50 compared with the high peak power, low repetition rate pulsed configuration used. The SERRS intensity using a pulsed laser produced a non-linear response with respect to changes in power of the laser. At powers of less than ,0.012 mW, the absolute intensity under the peaks of the CW and pulsed SERRS spectra converged, suggesting that lower peak power, high repetition rate systems may be more effective excitation sources for SERRS. Transmission electron microscopy of pulsed laser-irradiated silver particles showed significant sample damage and morphological changes. This problem was overcome with the use of a recirculating large-volume flow cell system, providing a fresh sample for each measurement. A picosecond-resolved time delay experiment found that SERRS intensity decreased by ,60% when exposed to a 400 nm pump pulse and probed with a 529 nm pulse. As the time delay between pump and probe increased the system recovered gradually to ,60% of the original SERRS intensity after 50 ps, where it remained constant. This suggests that the surface bonding between the silver and the dye is significantly perturbed, with some nanoscale diffusion occurring of the dye away from the metal surface. Hence chemical enhancement is temporarily prevented and electromagnetic enhancement is reduced as a function of 1/r3 as the dye moves away from the surface. Additionally, transient heating of the colloidal particles caused by the pulsed laser may also lead to plasmon shifts and changes in absorption intensity. Other factors such as surface annealing or decomposition of the silver particle or dye due the extreme temperature conditions may account for the permanent loss in SERRS intensity. Copyright © 2005 John Wiley & Sons, Ltd. [source] Quantification of D -Asp and D -Glu in rat brain and human cerebrospinal fluid by microchip electrophoresisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 17 2009Yong Huang Abstract A microchip electrophoresis (MCE) method with LIF detection was presented for quantification of D -aspartic acid (D -Asp) and D -glutamate (D -Glu) in biological samples. D -Asp and D -Glu were determined after precolumn derivatization with FITC. The chiral separation was performed on a glass/PDMS hybrid microfluidic chip using ,-CD as chiral selector in the running buffer. High sensitive detection was obtained by the LIF detection. The LODs (S/N = 3) for D -Asp and D -Glu were 6.0×10,8 and 4.0×10,8 M, respectively. Using this method, the levels of D -Asp and D -Glu in rat brain and human cerebrospinal fluid (CSF) were determined. [source] | ||||||||||||||||||||||||||||||||||