Development of analytical techniques for the analysis of submicron particles and protein aggregates

The goal of the research presented in this dissertation is to develop analytical techniques for the analysis of heterogeneous mixtures of submicron particles and protein aggregates. In Chapter 2, a simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis (CE) to take advantage of laser-induced fluorescence (LIF) detection. This technique uses laser ablation with a Nd:YAG laser to create gaps of 14.0 ¡Ó 2.2 ƒÝm. These structures have been used for reagent addition for postcolumn derivatization with LIF detection and have been tested for the separation of proteins and amino acids. In Chapter 3, laser-induced backside wet etching (LIBWE) has been adapted to improve the gap construction technique described in Chapter 2. A capillary filled with a solvent or a dye solution was cut by laser ablation. Gap size was reduced up to 56% in comparison to air cut gaps using only 22% of the laser pulse energy used in Chapter 2. The self focusing ability of the solvents tested due to nonlinear refractive index has been shown to play a role in the LIBWE process. Gaps created in Chapters 2 and 3 could be used to label individual protein aggregates and submicron particles. Separation and detection of individual submicron polystyrene spheres (110-992 nm) using CE with laser light scattering detection at 90o has been demonstrated in Chapter 4. Particles as small as 110 nm in diameter were detected individually using this method, but 57 nm particles could not be detected individually. Detection efficiencies ranging from 38 to 75% were determined for polystyrene spheres of different sizes. The instrument developed in Chapter 4 has been modified to collect scattered light at two different angles (20o and 90o) and fluorescence (90o) simultaneously. The ability of the new system to separate and detect individual 943 nm fluorescent particles was demonstrated. The smallest diameter particle that could be detected at 20o and 90o by scattering was 80 nm. The ability of the system to separate and detect individual rod-shaped biological particles (tobacco mosaic virus) was investigated

Biodegradation of triclosan by a wastewater microorganism

Available online xxx Keywords: Triclosan Biodegradation Sphingopyxis strain KCY1 Wastewater Meta-cleavage a b s t r a c t Triclosan, a synthetic antimicrobial agent, has been considered as an emerging environmental contaminant. Here we reported a triclosan-degrading wastewater bacterial isolate, Sphingopyxis strain KCY1, capable of dechlorinating triclosan with a stoichiometric release of chloride. The stain can degrade diphenyl ether but not 2,4,4 0 -tribromodiphenyl ether and 2,2 0 ,4,4 0 -tetrabromodiphenyl ether, despite all these three compounds are structurally similar to triclosan. While strain KCY1 was unable to grow on triclosan and catechol, it could grow with glucose, sodium succinate, sodium acetate, and phenol. When grown with complex nutrient medium containing a trace amount of triclosan (as low as 5 mg/L), the strain could retain its degradation ability toward triclosan. The maximum-specific triclosan degradation rate (q m ) and the half-velocity constant (K m ) are 0.13 mg-triclosan/mgprotein/day and 2.8 mg-triclosan/L, respectively. As triclosan degradation progressed, five metabolites were identified and these metabolites continue to transform into non-chlorinated end products, which was supported by a sharp drop in androgenic potential. The activity of catechol 2,3-dioxygenase in the cell extract was detected. No triclosan degradation was observed in the presence of 3-fluorocatechol, an inhibitor of meta-cleavage enzyme, suggesting that triclosan degradation proceed via meta-cleavage pathway. Based on all the observations, a degradation pathway for triclosan by strain KCY1 was proposed. ª 2012 Published by Elsevier Ltd. Introduction Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a common synthetic antimicrobial agent that has been incorporated into more than 700 different industrial and personal care products. These products, including deodorants, soaps, toothpastes, and various plastic products, contain 0.1e0.3% triclosan Biodegradation of triclosan in the environment and wastewater has recently become an interesting research topic Please cite this article in press as: Lee, D. In this study, we report isolation and characterization of a wastewater triclosan-degrading bacterium, Sphingopyxis strain KCY1. This isolate showed complete dechlorination of triclosan based on stoichiometric release of chloride. We also determined triclosan degradation kinetics, proposed possible degradation pathway for triclosan, and assessed the potential significance of this isolate to triclosan biodegradation in wastewater. 2. Material and methods Chemicals Triclosan Isolation and identification of triclosan-degrading bacteria A triclosan-degrading consortium, originally inoculated with activated sludge, was used as a source for the isolation of triclosan-degrading bacteria. Briefly, a loopful of the consortium was streaked onto nitrate mineral salts (NMS)-triclosan (5 mg/L) agar plates that were incubated at 30 C (Chu and Alvarez 2.3. Determination of degradation ability toward triclosan and compounds structurally similar to triclosan The isolate was initially grown in 20% R2A medium with 5 mg/ L triclosan for three days. The cell suspension was harvested by centrifugation and the pellet was washed with 50 mM phosphate-buffered saline and then resuspended in fresh NMS medium for experimental use. The degradation tests were conducted in 250 mL flasks containing the resting cell suspension and 5 mg/L of triclosan. The flasks were incubated on a rotary shaker at 150 rpm and at 30 C, and liquid samples were collected over time for triclosan measurements. A subset of collected liquid samples was used for BLYES and BLYAS assays. Liquid samples collected from degradation experiments were also used to measure concentrations of chloride. A parallel set of experiments was conducted to determine whether the isolate could degrade compounds that are structurally similar to triclosan. Three compounds, diphenyl . The degradation experiments were conducted similarly as described above, except using resting cell suspension (OD 600 ¼ 0.6e0.8) and each of these three compounds. Resistance to other antimicrobial agents Experiments were performed to determine whether strain KCY1 could resist to three common antimicrobial agents: kanamycin, trimethoprim and ampicillin (See SI for experimental details). Determination of Monod kinetic parameters for triclosan degradation Monod degradation kinetic model q ¼ q m $S=K s þ S, was used to describe triclosan degradation by strain KCY1. The kinetic experiments were conducted in a series of 40 mL EPA glass vials containing resting cells of strain KCY1 (OD 600 ¼ 0.4) and triclosan (ranging from 0.3 to 5 mg/L) in NMS medium. The vials were incubated on a rotary shaker at 150 rpm and at 30 C for 3 h, and then used for triclosan and protein measurements. The incubation duration (3 h) was determined in the laboratory where initial degradation rates remained linear. Experimental data obtained from kinetic tests were plotted as specific triclosan degradation rates (q, mass of substrate/mass of cell protein/time) against triclosan concentrations (S, mass/ volume). The maximum specific triclosan degradation rate (q m , mass of triclosan/mass of cell protein/time) and the halfvelocity constant (K m , mass of triclosan/volume) were determined by curve fitting using Sigmaplot 8.0 (SPSS Inc.) as w a t e r r e s e a r c h x x x ( 2 0 1 2 ) 1 e9 previously described Determination of degradation/utilization ability toward other organics Experiments were performed to determine whether the isolate could grow on three common macro-organics in wastewater: glucose (300 mg/L), sodium acetate (175 mg/L), and sodium succinate (300 mg/L) (Roh and Chu, 2010). These compounds were selected for the experiments because glucose is a common carbohydrate, and sodium succinate and sodium acetate are components present inside the tricarboxylic acid cycle (TCA cycle). Cell growth expressed as optical density (OD 600 ), protein contents, and volatile suspension solids, was monitored over time. Doubling times were determined from the exponential growth phase curves. Autoclavekilled cells were used as negative controls. Effect of complex nutrients on triclosan degradation Cells grown in a complex nutrient medium without prior exposure to triclosan were tested for their ability to retain its biodegradation of triclosan. Experiments were conducted as follows. Strain KCY1 was grown in 100% R2A (fully nutrientrich) medium without or with (5 or 500 mg/L) triclosan and transferred to its respective growth medium every two days. After four consecutive transfers, the cells were harvested as described above for degradation tests. The degradation experiments were conducted in glass vials containing 5 mg/L triclosan and the resting cells in NMS medium. Bioluminescent androgenic/estrogenic screening assays To evaluate androgenic and estrogenic potential of triclosan degradation metabolites and end products, the bioluminescent androgenic and estrogenic screening (BLYES and BLYAS) assays were performed as described previously Determination of enzymes responsible for triclosan degradation The isolate was screened for the presence of catechol 2,3-dioxygenase and/or catechol 1,2-dioxygenase using a spectrophotometric method as described previously . In addition, triclosan degradation via meta-cleavage pathway was tested by adding 3-fluorocatechol (50 mg/L) or in the absence of it. 3-Fluorocatechol is an inhibitor of catechol 2,3-dioxygenase that catalyzes meta-cleavage reactions (Bartels et al., 1984; Chemical analysis Chloride concentrations were measured using a DX-80 Ion Chromatography (IC) system (Dionex, Sunnyvale, CA) equipped with an IonPac AS14A-5 mm analytical column (3  150 mm). Triclosan, tri-BDE, tetra-BDE, and DE concentrations and degradation metabolites were determined using a GC (Agilent 6890)/MS (Agilent 5973) equipped with DB-5 column. In addition, to detect possible degradation metabolites, LC/MS analysis was performed using a Surveyor HPLC system (ThermoFinnigan, San Jose, CA) interfaced with quadruple ion trap mass spectrometer (LCQ-DECA; ThermoFinnigan). Detailed description of chemical analysis is available in SI. 3. Results and discussion 3.1. Identification of a triclosan-degrading microorganism, strain KCY1 Among three presumptive triclosan-degrading colonies, one isolate (yellow-mucoid), designated strain KCY1, showed the ability to degrade triclosan in NMS medium. Strain KCY1 is a short, rod-shaped (0.5 mm  1.7 mm) Gram-negative bacterium with a flagellum a t e r r e s e a r c h x x x ( 2 0 1 2 ) 1 e9 Characteristics of strain KCY1 Since strain KCY1 can grow rapidly on R2A agar, it is expected that the strain can also grow on glucose, sodium succinate, and sodium acetate Degradation of triclosan by strain KCY1 As shown in 3.4. Androgenic and estrogenic potential of triclosan degradation metabolites and end products BYLES and BLYAS assays were used to evaluate estrogenic and androgenic potential of triclosan degradation metabolites and end products. Triclosan itself triggered weak androgenic activity in the BLYAS assay, but not estrogenic activity in the BLYES assay in this study. sensitivities between the yeast cells and human breast cancer cells. As shown in Since triclosan is a chlorinated organic compound, the decrease of androgenic potential over time might correlate to the extent of dechlorination during the triclosan degradation. The decline of androgenic potential could be explained by the decrease in initial triclosan concentration and the transformation into less-chlorinated metabolites that were detected in this study before day 1 (see identification of metabolites below). Between day 1 and day 2, the reduction rate of androgenic activity became slower than the triclosan degradation rate, suggesting that (i) other androgenic metabolites might be produced during this period or (ii) androgenic metabolites might be transformed at a slower rate than the triclosan degradation rate. Interestingly, the androgenic response (w40% of original response) was observed despite that triclosan was no longer detected after day 2. This indicated a slow transformation of triclosan metabolites with androgenicity, like 2,4-dichlorophenol. Previous studies have reported that 2,4-dichloropehnol exhibited in-vivo androgenic activity in zebrafish embryos (Sawle et al., 2010) and in human prostate cancer cells Factors affecting triclosan degradation As wastewater contains a wide range of complex organics that are readily available for microbial growth, it is important to know whether strain KCY1 would retain its ability to degrade triclosan after it has grown on complex nutrients. To determine the effects of nutrients on triclosan biodegradation by strain KCY1, the strain was initially grown in nutrient-rich medium (100% R2A) without triclosan for 8 days (4 consecutive transfers every 2-day). After grown on nutrient-rich medium without triclosan, strain KCY1 lost its degradation ability toward triclosan Degradation kinetic parameters for triclosan The results of triclosan degradation tests were used to develop the relationship between specific triclosan degradation rate (q) and triclosan concentrations 3.7. Degradation ability toward compounds which are structurally similar to triclosan Since two BDEs (tri-and tetra-BDEs) and DE are structurally similar to triclosan and strain KCY1 can degrade triclosan, we hypothesized that strain KCY1 could degrade these compounds as well. Strain KCY1 was able to degrade approximately 78% of DE (1 g/L) within 5 days, but unable to use DE as a sole carbon source (data not shown). Although earlier it was reported that CeBr and CeCl bonds are at least equally viable for enzymatic reaction (Dos Santos et al., 1999), strain KCY1 was unable to degrade tri-BDE and tetra-BDE. The inability of strain KCY1 to degrade these two BDEs could be due to a combination of various factors, including the difference in electronwithdrawing effects that would result from the difference between the halogen species (Cl vs Br) and the absence of hydroxyl group in both BDEs that could contribute to selectivity of the enzymes. The reason why strain KCY1 can degrade DE but not tri-and tetra-BDEs was unclear in this study. Enzymes involved in triclosan biodegradation Another set of experiments was conducted to examine whether 3-flurocatechol, a meta-cleavage inhibitor, would affect triclosan biodegradation. As shown in Degradation metabolites and possible degradation pathway for triclosan During the triclosan biodegradation, five metabolites were identified. These metabolites are monohydroxy-triclosan, 6-chloro-3-(2,4-dichlorophenoxy)-4-hydroxycyclohexa-3,5-diene-1,2-dione, 3-chloro-4-(5,7-dichloro-3-oxo-2,3-dihydrobenzo[1,4]dioxin-2-yl)-2-oxobut-3-enal, 3,5-dichloro-4,6-dihydroxycyclohexa-3,5-diene-1,2-dione, and 2,4-dichlorophenol ( Here we proposed a biodegradation pathway for triclosan by strain KCY

Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNAPyl Pairs in the Escherichia coli BL21(DE3) Cell Strain

Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl) pairs and cross recognition between nonsense codons and various tRNA(Pyl) anticodons in the Escherichia coli BL21(DE3) cell strain are reported. tRNA(CUA)(Pyl) is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N(ε)-tert-butoxycarbonyl-L-lysine (BocK). Similar to tRNA(CUA)(Pyl), tRNA(UUA)(Pyl) is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA)(Pyl) is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA)(Pyl) pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA)(Pyl) pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU)(Pyl) fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli

Mineralization of Acephate, a Recalcitrant Organophosphate Insecticide Is Initiated by a Pseudomonad in Environmental Samples

An aerobic bacterium capable of breaking down the pesticide acephate (O,S-dimethyl acetyl phosphoramidothioic acid) was isolated from activated sludge collected from a pesticide manufacturing facility. A phylogenetic tree based on the 16 S rRNA gene sequence determined that the isolate lies within the Pseudomonads. The isolate was able to grow in the presence of acephate at concentrations up to 80 mM, with maximum growth at 40 mM. HPLC and LC-MS/MS analysis of spent medium from growth experiments and a resting cell assay detected the accumulation of methamidophos and acetate, suggesting initial hydrolysis of the amide linkage found between these two moieties. As expected, the rapid decline in acephate was coincident with the accumulation of methamidophos. Methamidophos concentrations were maintained over a period of days, without evidence of further metabolism or cell growth by the cultures. Considering this limitation, strains such as described in this work can promote the first step of acephate mineralization in soil microbial communities

Desorption electrospray ionization of aerosol particles

We have applied desorption electrospray ionization to aerosol particles. Ions were formed from aerosols by merging suspended dry particles with an electrospray of solvent in a modified ion trap mass spectrometer. Dry aerosol particles were generated using a fluidized bed powder disperser and directed toward the inlet of the mass spectrometer. A nanospray source was used to create a spray of solvent droplets directed at the inlet and at a right angle with respect to the aerosol. Ions generated by the interaction of the particles and electrospray were transferred into the ion trap mass spectrometer. Using this method, pure samples of caffeine and erythromycin A were analyzed. In addition, commonly available food and drug powders including instant cocoa powder, artificial sweetener and ibuprofen were analyzed. Copyright © 2007 John Wiley & Sons, Ltd

Infrared laser-assisted desorption electrospray ionization mass spectrometry

We have used an infrared laser for desorption of material and ionization by interaction with electrosprayed solvent. Infrared laser-assisted desorption electrospray ionization (IR LADESI) mass spectrometry was used for the direct analysis of water-containing samples under ambient conditions. An ion trap mass spectrometer was modified to include a pulsed Er:YAG laser at 2.94 μm wavelength coupled into a germanium oxide optical fiber for desorption at atmospheric pressure and a nanoelectrospray source for ionization. Analytes in aqueous solution were placed on a stainless steel target and irradiated with the pulsed IR laser. Material desorbed and ablated from the target was ionized by a continuous stream of charged droplets from the electrosprayed solvent. Peptide and protein samples analyzed using this method yield mass spectra similar to those obtained by conventional electrospray. Blood and urine were analyzed without sample pretreatment to demonstrate the capability of IR LADESI for direct analysis of biological fluids. Pharmaceutical products were also directly analyzed. Finally, the role of water as a matrix in the IR LADESI process is discussed. © The Royal Society of Chemistry

Laser ablation construction of on-column reagent addition devices for capillary electrophoresis

A simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis. This technique uses laser ablation with the second harmonic of a Nd:YAG laser (532 nm) at 13.5 mJ/pulse and a repetition rate of 15 Hz to create these gaps. A capillary is glued to a microscope slide and positioned at the focal point of a cylindrical lens using the focused beam from a laser pointer as a reference. Gaps of 14.0 ± 2.2 μm (n = 33) at the bore of the capillary are produced with a success rate of 94% by ablation with 400 pulses. This simple method of gap construction requires no micromanipulation under a microscope, hydrofluoric acid etching, or use of column fittings. These structures have been used for reagent addition for postcolumn derivatization with laser-induced fluorescence detection and have been tested for the separation of proteins and amino acids. Detection limits of 6 × 10-7 and 1 × 10-8 M have been obtained for glycine and tranferrin, respectively. Separation efficiencies obtained using these gap reactors range from 38 000 to 213 000 theoretical plates

Banca electrónica en el Ecuador

El sector bancario en el Ecuador realizó un avance tecnológico. Su conexión electrónica con el sistema financiero internacional promueve, a mediano plazo, cambios significativos en el sector. Mediante el enlace electrónico los bancos se adelantan a los gobiernos en la integración regional

Separation and detection of individual submicron particles by capillary electrophoresis with laser-light-scattering detection

Separation and detection of individual submicron polystyrene spheres using capillary electrophoresis with laser-light-scattering detection has been demonstrated. Electrophoretically separated particles were passed through a focused laser beam and light scattered from individual particles was collected at 90°. Each diameter of polystyrene spheres injected (from 110 to 992 nm) resulted in the observation of a well-defined migration window containing multiple peaks, each arising from the light scattered by an individual particle. The migration time window for individual particles of a particular size corresponded well to the migration time of a peak from a population of particles of the same size detected using a UV absorbance detector. The electrophoretic mobility and scattered light intensity were determined for each particle detected. The average scattered light intensity for each particle size was consistent with Mie scattering theory. Particles as small as 110 nm in diameter were detected individually using this method, but particles with a diameter of 57 nm could not be individually detected. The number of single particle scattering events was counted and compared to the theoretical number of particles injected electrokinetically, and the detection efficiency determined ranged from 38 to 57% for polystyrene spheres of different sizes. The laser-light-scattering detection method was directly compared to laser-induced fluorescence detection using fluorescent polystyrene microspheres. The number of particles detected individually by each method was in agreement. © The Royal Society of Chemistry