Staphylococcus aureus Detection in Milk Using a Thickness Shear Mode Acoustic Aptasensor with an Antifouling Probe Linker.

Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow's milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor's antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTTCOOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) quartz crystals, the sensitivity of the sensor's antifouling in milk improved by about 82-96%. The excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow's milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.

Dithiothreitol-Lucigenin Chemiluminescent System for Ultrasensitive Dithiothreitol and Superoxide Dismutase Detection.

1,4-Dithiothreitol (DTT), a highly water-soluble and well-known reducing agent for preservation and regeneration of sulfhydryl groups in biomedical applications, has been developed as an efficient and stable coreactant of lucigenin for the first time. DTT efficiently reacts with lucigenin to generate intense chemiluminescence (CL), eliminating the need for external catalysts to facilitate the lucigenin CL. The DTT-lucigenin CL is approximately 15-fold more intense when compared with the lucigenin-H2O2 classical system. Superoxide dismutase (SOD) remarkably quenches the DTT-lucigenin CL. Based on this phenomenon, a newly developed CL approach for the determination of SOD was proposed with a linear range of 0.01-1.5 µg/mL and a limit of detection of 2.2 ng/mL. Various factors affecting the CL emission of the DTT-lucigenin probe were studied and optimized. Plausible mechanistic pathways for the CL coreaction of lucigenin with DTT were proposed and fully discussed. Our proposed method not only has the merit of being selective toward the target analytes but also eliminates the need for the complex synthesis of luminescent probes and facilitates the sensitive detection of SOD in human serum and cosmetics SOD raw material with satisfactory recoveries.

Two-photon imaging of 1,4-dithiothreitol (DTT) by a red-emissive fluorescent probe in living cells, tissues and animals.

1,4-Dithiothreitol (DTT) is an important small-molecular reducing agent and has extensive applications in biochemistry, peptide/protein chemistry and clinical medicine. The development of effective methods for monitoring DTT is of great importance for its safe use and studying its toxicity to human. In this work, we present a two-photon red-emissive probe for the imaging of DTT in living cells, tissues and animals. The probe employed a two-photon red-emissive xanthene dye as the fluorophore and selected 2,4-dinitrophenylate as the novel recognition site for DTT. In response to DTT, the probe displayed excellent sensitivity and selectivity. The probe was successfully applied to the two-photon imaging of DTT in living cells, and the imaging of DTT in living tissues and animals.

A new xanthene-based two-photon fluorescent probe for the imaging of 1,4-dithiothreitol (DTT) in living cells.

1,4-Dithiothreitol (DTT) has wide applications in cell biology and biochemistry. Development of effective methods for monitoring DTT in biological systems is important for the safe handling and study of toxicity to humans. Herein, we describe a two-photon fluorescence probe (Rh-DTT) to detect DTT in living systems for the first time. Rh-DTT showed high selectivity and sensitivity to DTT. Rh-DTT can be successfully used for the two-photon imaging of DTT in living cells, and also can detect DTT in living tissues and mice.

Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.

Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

Spatial Variation and Land Use Regression Modeling of the Oxidative Potential of Fine Particles.

BACKGROUND: Oxidative potential (OP) has been suggested to be a more health-relevant metric than particulate matter (PM) mass. Land use regression (LUR) models can estimate long-term exposure to air pollution in epidemiological studies, but few have been developed for OP. OBJECTIVES: We aimed to characterize the spatial contrasts of two OP methods and to develop and evaluate LUR models to assess long-term exposure to the OP of PM2.5. METHODS: Three 2-week PM2.5 samples were collected at 10 regional background, 12 urban background, and 18 street sites spread over the Netherlands/Belgium in 1 year and analyzed for OP using electron spin resonance (OP(ESR)) and dithiothreitol (OP(DTT)). LUR models were developed using temporally adjusted annual averages and a range of land-use and traffic-related GIS variables. RESULTS: Street/urban background site ratio was 1.2 for OP(DTT) and 1.4 for OP(ESR), whereas regional/urban background ratio was 0.8 for both. OP(ESR) correlated moderately with OP(DTT) (R2 = 0.35). The LUR models included estimated regional background OP, local traffic, and large-scale urbanity with explained variance (R2) of 0.60 for OP(DTT) and 0.67 for OP(ESR). OP(DTT) and OP(ESR) model predictions were moderately correlated (R2 = 0.44). OP model predictions were moderately to highly correlated with predictions from a previously published PM2.5 model (R2 = 0.37-0.52), and highly correlated with predictions from previously published models of traffic components (R2 > 0.50). CONCLUSION: LUR models explained a large fraction of the spatial variation of the two OP metrics. The moderate correlations among the predictions of OP(DTT), OP(ESR), and PM2.5 models offer the potential to investigate which metric is the strongest predictor of health effects. CITATION: Yang A, Wang M, Eeftens M, Beelen R, Dons E, Leseman DL, Brunekreef B, Cassee FR, Janssen NA, Hoek G. 2015. Spatial variation and land use regression modeling of the oxidative potential of fine particles. Environ Health Perspect 123:1187-1192; http://dx.doi.org/10.1289/ehp.1408916.

Associations between three specific a-cellular measures of the oxidative potential of particulate matter and markers of acute airway and nasal inflammation in healthy volunteers.

INTRODUCTION: We evaluated associations between three a-cellular measures of the oxidative potential (OP) of particulate matter (PM) and acute health effects. METHODS: We exposed 31 volunteers for 5 h to ambient air pollution at five locations: an underground train station, two traffic sites, a farm and an urban background site. Each volunteer visited at least three sites. We conducted health measurements before exposure, 2 h after exposure and the next morning. We measured air pollution on site and characterised the OP of PM2.5 and PM10 using three a-cellular assays; dithiotreitol (OP(DTT)), electron spin resonance (OP(ESR)) and ascorbic acid depletion (OP(AA)). RESULTS: In single-pollutant models, all measures of OP were significantly associated with increases in fractional exhaled nitric oxide and increases in interleukin-6 in nasal lavage 2 h after exposure. These OP associations remained significant after adjustment for co-pollutants when only the four outdoor sites were included, but lost significance when measurements at the underground site were included. Other health end points including lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. CONCLUSIONS: We found significant associations between three a-cellular measures of OP of PM and markers of airway and nasal inflammation. However, consistency of these effects in two-pollutant models depended on how measurements at the underground site were considered. Lung function and vascular inflammatory and coagulation parameters in blood were not consistently associated with OP. Our study, therefore, provides limited support for a role of OP in predicting acute health effects of PM in healthy young adults.

A sensitive and selective detection method for thiol compounds using novel fluorescence probe.

In this work, a sensitive and selective detection method based on fluorescence resonance energy transfer (FRET) was developed for analyzing thiol compounds by using a novel fluorescent probe. The new fluorescent probe contains a disulfide bond which selectively reacts with nucleophilic thiolate through the thiol-disulfide exchange reaction. An obvious fluorescence recovery can be observed upon addition of the thiol compound in the fluorescent probe solution due to the thiol-disulfide exchange reaction and the destruction of FRET. This novel probe was successfully used to determine dithiothreitol (DTT), glutathione (GSH) and cysteine (Cys). The limits of detection (LOD) were 2.0µM for DTT, 0.6µM for GSH, and 0.8µM for Cys. This new detection method was further investigated in the analysis of compound amino acid injection.

Seasonal and spatial variation in dithiothreitol (DTT) activity of quasi-ultrafine particles in the Los Angeles Basin and its association with chemical species.

A year-long sampling campaign of quasi-ultrafine particles (dp < 0.25 µm) was conducted at 10 distinct sites representing source, urban and/or near-freeway, rural receptor and desert locations across the Los Angeles air basin. Redox activity of the PM samples was measured by means of the Dithiothreitol (DTT) assay and detailed chemical analysis was performed to measure the concentrations of chemical species. DTT activity per unit air volume and unit PM mass (expressed in nmol min(-1) m(-3) and nmol/min/µg PM, respectively) showed similar trends across sites and seasons. DTT activity was generally higher during cold seasons (winter and fall) compared to warm seasons (summer and spring). Noticeable peaks were observed at urban near-freeway locations representing "source" sites impacted by fresh traffic emissions. Regression analysis indicated strong association (R > 0.7) between the DTT activity and the concentrations of carbonaceous species (OC, EC, WSOC and WIOC) across all seasons and strong winter-time correlations with organic tracers of primary vehicular emissions including polycyclic aromatic hydrocarbons (PAHs), alkanes, hopanes and steranes. Strong correlations were also observed, particularly during winter, between DTT activity and transition metals (e.g., Cr, Mn, V, Fe, Cu, Cd and Zn), which share similar vehicular sources with primary organics. A multivariate linear regression analysis indicated that the variability in DTT activity is best explained by the variability in concentrations of WSOC, WIOC, EC and hopanes. Combined contributions from these species explained 88% of the DTT activity. The appearance of WSOC as a typical tracer of secondary organic aerosol, along with EC, WIOC and hopanes, all markers of emissions from primary combustion sources, emphasizes the contributions of both primary and secondary sources to the overall oxidative potential of quasi-ultrafine particles. Supplemental materials are available for this article. Go to the publisher's online edition of the Journal of Environmental Science and Health, Part A, to view the supplemental file.

Microfluidic paper-based analytical device for aerosol oxidative activity.

Human exposure to particulate matter (PM) air pollution has been linked with respiratory, cardiovascular, and neurodegenerative diseases, in addition to various cancers. Consistent among all of these associations is the hypothesis that PM induces inflammation and oxidative stress in the affected tissue. Consequently, a variety of assays have been developed to quantify the oxidative activity of PM as a means to characterize its ability to induced oxidative stress. The vast majority of these assays rely on high-volume, fixed-location sampling methods due to limitations in assay sensitivity and detection limit. As a result, our understanding of how personal exposure contributes to the intake of oxidative air pollution is limited. To further this understanding, we present a microfluidic paper-based analytical device (µPAD) for measuring PM oxidative activity on filters collected by personal sampling. The µPAD is inexpensive to fabricate and provides fast and sensitive analysis of aerosol oxidative activity. The oxidative activity measurement is based on the dithiothreitol assay (DTT assay), uses colorimetric detection, and can be completed in the field within 30 min following sample collection. The µPAD assay was validated against the traditional DTT assay using 13 extracted aerosol samples including urban aerosols, biomass burning PM, cigarette smoke, and incense smoke. The results showed no significant differences in DTT consumption rate measured by the two methods. To demonstrate the utility of the approach, personal samples were collected to estimate human exposures to PM from indoor air, outdoor air on a clean day, and outdoor air on a wildfire-impacted day in Fort Collins, CO. Filter samples collected on the wildfire day gave the highest oxidative activity on a mass normalized basis, whereas typical ambient background air showed the lowest oxidative activity.

Speciation and determination of thiols in biological samples using high performance liquid chromatography-inductively coupled plasma-mass spectrometry and high performance liquid chromatography-Orbitrap MS.

An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quadrupole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103 fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 µmol g(-1) and 0.61 to 1.32 µmol g(-1), respectively.

A highly selective ratiometric fluorescent probe for 1,4-dithiothreitol (DTT) detection.

A highly selective ratiometric fluorescent probe, which contains an aminonaphthalimide fluorophore and a self-immolative spacer for 1,4-dithiothreitol (DTT) detection was designed and synthesized. The probe displays a 66 nm red-shift of fluorescence emission and the color changes from colorless to jade-green upon reaction with DTT. These properties are mechanistically ascribed to the strong reducing capability of DTT.

The role of covalent dimerization on the physical and chemical stability of the EC1 domain of human E-cadherin.

The objective of this work was to evaluate the solution stability of the EC1 domain of E-cadherin under various conditions. The EC1 domain was incubated at various temperatures (4, 37, and 70 degrees C) and pH values (3.0, 7.0, and 9.0). At pH 9.0 and 37 or 70 degrees C, a significant loss of EC1 was observed due to precipitation and a hydrolysis reaction. The degradation was suppressed upon addition of dithiothreitol (DTT), suggesting that the formation of EC1 dimer facilitated the EC1 degradation. At 4 degrees C and various pH values, the EC1 secondary and tertiary showed changes upon incubation up to 28 days, and DTT prevented any structural changes upon 28 days of incubation. Molecular dynamics simulations indicated that the dimer of EC1 has higher mobility than does the monomer; this higher mobility of the EC1 dimer may contribute to instability of the EC1 domain.

Determination of dithiothreitol in complex protein mixtures by HPLC-MS.

Dithiothreitol (DTT) and other reducing agents are typically used in refolding processes of recombinant human proteins during their purification from inclusion bodies. Due to its toxicity, it is essential to monitor the clearance of DTT throughout the analytical flow from the refolding phase to the final formulated product. Here we report a direct, simple, and fast liquid chromatography method using UV and tandem mass spectrometry (MS/MS) detection for DTT evaluation in complex protein mixtures. In aqueous solution DTT exists as an equilibrium mixture of the oxidized and the reduced form (H(2)DTT --> DTT(ox)) and the quantitation tools should therefore be applicable to both forms in a single step or in multiple steps. Oxidation of DTT with aqueous copper(II) nitrate trihydrate solution was introduced to determine a single oxidized compound, i. e. DTT(ox). Proteins and other components of high molecular masses were separated from DTT(ox) by ultrafiltration. Consequently, efficient separation of the DTT(ox )from other flow-through mixture components (sugars, polymers, salts, protein stabilizers) was achieved on an Atlantis dC(18) column. After chromatographic separation, DTT(ox) was selectively identified by UV absorbance at 285 nm or by selected reaction monitoring, measuring signal transition between m/z 151 --> 105. The method was validated in terms of specificity, accuracy, precision, linearity, and limit of quantification and detection. A reversed-phase HPLC separation method with atmospheric pressure chemical ionization and MS/MS detection in negative ion mode is highlighted as a viable alternative to currently existing quantitation methods involving DTT derivatization and HPLC fluorescence detection. The described approach offers simple, straightforward, selective, and high-throughput DTT quantitation in protein mixtures.

Detection and analysis of the cyanobacterial peptide hepatotoxins microcystin and nodularin using SELDI-TOF mass spectrometry.

Surface-enhanced laser desorption ionization mass spectrometry (SELDI-TOFMS) was used to develop a new and useful method for determination and identification of the cyanobacterial (blue-green algae) toxins: microcystin and nodularin. The technique, combining chromatography and MS, enables microcystin/nodularin capture, purification, analysis, and processing from complex biological mixtures directly onto a hydrophobic chip. Factors affecting ion intensities, including matrix concentration and laser intensity, were investigated to optimize sensitivity of the method. Microcystins and nodularin were analyzed for femtomolar sensitivity (about 2.5 pg microcystin-LR in 2 microl water). Samples of blood sera and liver tissue were spiked with microcystin-LR and analyzed. The detection limit was 1 ng in 2 microl blood sera solution. Reactions of microcystins by compounds containing mercaptan groups, such as dithiothreitol, aminoethanethiol and protein phosphatase 1, were examined on the chip by mass spectrometry. Formation of the microcystin-dithiothreitol conjugate was used to confirm the target compounds. The MS/MS data obtained showed the presence of the microcystin conjugate. The reaction position of the toxin with target compound was confirmed by a series of MS/MS fragment ions. The protein profile of microcystins reacting with protein phosphatase 1 was also obtained from the SELDI-TOF mass spectra.

Improved HPLC methodology for monitoring thiopurine metabolites in patients on thiopurine therapy.

OBJECTIVE: Standardization of thiopurine metabolite testing is currently lacking. This paper presents in-depth methodological analysis and optimization of two currently available HPLC procedures (Lennard-Singleton [J. Chromatogr. 583 (1992) 83] and Dervieux-Boulieu [Clin. Chem. 44 (1998) 551]) to improve precision, turn-around time, ruggedness, and cost effectiveness. DESIGN AND METHODS: Reversed-phase chromatography with UV detection was performed on a Waters HPLC system. The two protocols were improved with regards to internal standardization (IS), chromatographic conditions, as well as reagent preparation, storage, and use. 6-Thioguanine nucleotides (6-TGNs) were analyzed by our optimized techniques in samples from patients on thiopurine therapy (n = 24) and the results were compared. RESULTS: 6-Mercaptopurine (6-MP) was an ideal internal standard in either procedure. Isocratic elution with 5% acetonitrile (ACN) in 20 mmol/l phosphate buffer pH 2.5 allowed for minimal background interference in both protocols. 6-Thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine (6-MMP) eluted at around 4, 5, and 6 min, respectively. Dithiothreitol (DTT) was critical only during the acid hydrolysis step. Less mercury-containing waste was generated in the Lennard-Singleton procedure. With our optimized protocols recovery of 6-TGNs was on average 1.38-fold higher in the Dervieux-Boulieu method over a range of 10-678 pmol/8 x 10(8) RBC and no interfering peaks hindered analysis. Specific extraction of thiopurines before their analysis as per Lennard-Singleton procedure may be redundant. CONCLUSIONS: We improved the quality and cost effectiveness of two known procedures for thiopurine metabolite assay. Through common chromatographic conditions and internal standardization, future comparison studies are now facilitated a great deal. The less tedious Dervieux-Boulieu procedure for routine thiopurine metabolite testing is warranted.

[Determination of L-cysteine in enzymatic reaction mixture with pre-column derivatization by high performance liquid chromatography].

A new sensitive high performance liquid chromatographic method for the determination of L-cysteine in an enzymatic reaction mixture using ultra violet spectrometric detection was developed. The sample reacted with 5,5'-dithio-bis-nitrobenzoic acid (DTNB) and was analyzed on a Shimadzu VP-ODS column at room temperature, using gradient elution with detection at 330 nm. The L-cysteine chromatographic peak was determined in comparison with derivatives of 2-mercapto ethanol and dithiothreitol. The linear range was 5-950 micromol/L. The recoveries were 99.7%-100.5% and the relative standard deviations (RSDs) were less than 1.3%. The detection limit was 0.8 micromol/L. The method is simple and accurate.

Monitoring of dithiothreitol clearance by means of micellar electrokinetic chromatography.

A new method for specific determination of dithiothreitol (DTT) using micellar electrokinetic chromatography and on-column reaction with reactive disulfide-2,2'-dipyridyldisulfide is described. DTT in this reaction is quantitatively transformed into a mixed disulfide concomitantly with formation of equimolar amount of the 2-thiopyridone that is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of DTT is thus estimated indirectly from the result of 2-thiopyridone determination. The linear detection range for concentration versus peak area for the assay is from 0.05 to 2.5 mM (correlation coefficient 0.993) with a detection limit of 2.5 microM. The inter-day reproducibility of the peak area was 1.35% and the inter-day reproducibility of the migration time 0.56%. The method can be applied for DTT monitoring both in chemical and biological systems.

Thiol and disulfide determination by free zone capillary electrophoresis.

A rapid, sensitive and simple method for the determination of reduced and oxidized glutathione, cysteine, cystine, cysteamine, cystamine and their respective mixed disulfides is described. The compounds were separated and identified in a single step by capillary zone electrophoresis. The method was used to follow the thiol-disulfide interconversion and to measure glutathione levels in lens extracts.

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.