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A millisecond infrared stopped-flow apparatus.

In this paper, we have described a stopped-flow apparatus that is capable of measuring infrared kinetics in the amide I' region of a protein's vibrational spectrum. The dead time of this setup, determined by the reducing reaction of 2,6-Dichlorophenolindophenol by L-ascorbic acid, is between 6 to 15 ms, depending on the flow rate. Therefore, this stopped-flow IR method provides a means of measuring infrared kinetics in a time window that is not easily accessible to other mixing-based IR techniques. Using this apparatus, we have studied the alkaline transition of cytrochrome c and have found that this conformational event proceeds in a biphasic manner. The characteristic time constants of these two phases were determined to be 68 +/- 20 ms and 624 +/- 37 ms, respectively.

Computer Systems↗

Development and application of an integrated system for monitoring ethanol content of fuels.

An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyses/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91-mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05-1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.

Alcohol Oxidoreductases↗

Fiberoptic biosensors based on chemiluminescent reactions.

The chemiluminescence of luminol in the presence of H2O2 has been exploited to develop fiberoptic biosensors associated with flow injection analysis systems. A chlorophenol sensor was developed based on the ability of certain halophenols to enhance the peroxidase-catalyzed luminol chemiluminescence. Horseradish peroxidase immobilized on a collagen membrane was used. Ten chlorophenols have been tested with this chemiluminescent-based sensor. The lower detection limit was obtained with 4-chloro-3-methylphenol and was equal to 0.01 microM. Electrochemiluminescent-based fiberoptic biosensors for glucose and lactate were also developed using glucose oxidase or lactate oxidase immobilized on polyamide membranes. In the presence of oxidase-generated H2O2, the light emission was triggered electrochemically by means of a glassy carbon electrode polarized at +425 mV vs a platinum pseudo-reference electrode. The detection limits for glucose and lactate were 150 and 60 pmol, respectively, and the dynamic ranges were linear from 150 pmol to 600 nmol and from 60 pmol to 60 nmol, respectively.

Biosensing Techniques↗

Rapid quantitative characterization of protein interactions by composition gradient static light scattering.

Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.

Aprotinin↗

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

Agrin↗

Relationship between concentration of citrate and ketone bodies in cow's milk.

The authors' hypothesis is that the members of the tricarboxylic acid cycle (TCA cycle) such as citrate decrease in association with increased ketone body formation. To prove this hypothesis the connection between ketone bodies and citrate formation of milk was studied. A fluorimetric method was used to determine citrate and a headspace sampling gas chromatographic (GC) method was developed for determination of ketone bodies. Under real conditions of milk sampling, transport and storage, preserved milk samples of 119 clinically healthy dairy cows obtained in the 48 hours after milking were investigated. A low level of acetoacetate (ACAC) was found in all samples. This fact can be explained by the spontaneous decarboxylation of acetoacetate during sample storage (previously decarboxylised acetoacetate = pdACAC) and, consequently, the majority of the amount of acetoacetate in the samples (AC + pdACAC) appeared in the measured acetone concentrations. Based on the measured acetone concentration of milk samples two groups were formed retrospectively: HA (high-acetone) group (n = 41) with an AC + pdACAC concentration of > 0.4 mmol/l and a LA (low-acetone) group (n = 78) with an AC + pdACAC level of < or = 0.4 mmol/l. In the milk of cows of Group HA a positive correlation (r = +0.623) and linear connection between acetone (AC + pdACAC) and beta-hydroxybutyrate (BOHB) levels was found [BOHB = 2.491 + 0.586 x (pdAC + ACAC)]. Furthermore, in this group a negative correlation between citrate and BOHB and AC + pdACAC was also established (r = -0.579). Focusing on the results of this group the authors found a significant drop of AC + pdACAC and citrate during the metabolically critical first 1-4 weeks of lactation. For this reason they suggest that simple, easy, automated methods (i.e. flow injection analysis, Fourier transformation infrared analysis) should be introduced for the simultaneous determination of acetone and citrate concentration in milk to make the evaluation of the energy status of high-producing dairy cows easier and more certain.

Animals↗

Preconcentration of trace aluminum from environmental water samples with 5'-chloro-5-dodecyl-2,4,2'-trihydroxyazobenzene impregnated macroporous resin.

5'-Chloro-5-dodecyl-2,4,2'-trihydroxyazobenzene (CDTA) impregnated XAD-4 resin (CDTA resin) was prepared, and applied to the preconcentration of trace aluminum from environmental water samples with satisfactory results. Aluminum was measured by flow-injection analysis (FIA) with fluorescence detection by using lumogallion after the preconcentration of aluminum with CDTA resin. Fe3+ and other coexisting substances had no interference on the determination of aluminum by the present method. Chemical speciation of trace aluminum in environmental water was carried out by using the CDTA resin after the separation of aluminum species with ionic exchangers without changing the pH. About 50% of water-soluble aluminum in Lake Biwa was found to be aluminum organic species (anion species).

Journal Article↗

Native fluorescence flow-through optosensor for the fast determination of diphenhydramine in pharmaceuticals.

A single, rapid flow-through optosensor spectrofluorometric system is proposed for the determination of diphenhydramine in different pharmaceutical preparations. This sensor was developed in conjunction with a monochannel flow-injection analysis system with fluorometric solid-phase transduction. Diphenhydramine was directly injected into a carrier stream of ethanol/water, 50% (v:v), and transitorily retained on a sorption gel Sephadex G-15 placed in the detection area into the cell. The determination was carried out without any derivatization reaction by directly measuring the intrinsic fluorescence of the analyte and using the peak height as an analytical signal. The chemical and instrumental variables were optimized, and the influence of some foreign substances that can be found in typical pharmaceutical samples containing diphenhydramine was also investigated. Diphenhydramine could be determined in the concentration ranges of 0.5 - 8 microg ml(-1) and 0.1 - 1.2 microg ml(-1) with detection limits of 0.088 and 0.019 microg ml(-1) at sampling rates of 30 and 19 h(-1) for 200 and 800 microl of the sample volume, respectively.

Journal Article↗

Permanganate-based chemiluminescence analysis of ferulic acid using flow injection.

A simple, sensitive and rapid flow-injection chemiluminescence method has been developed for the determination of ferulic acid based on the chemiluminescence reaction of ferulic acid with potassium permanganate in a nitric acid medium. A strong chemiluminescence signal was observed when ferulic acid was injected into an acidic potassium permanganate solution in a flow-cell. The present method allowed the determination of ferulic acid in the concentration range of 6.0 x 10(-6) to 2.0 x 10(-4) mol l(-1); the detection limit (3sigma) for ferulic acid was 9.6 x 10(-8) mol l(-1). The relative standard deviation was 1.0% for 11 replicate analyses of 2.0 x 10(-4) mol l(-1) ferulic acid. The proposed method was applied to the determination of ferulic acid in real samples with satisfactory results. Moreover, the reaction mechanism of the chemiluminescence system was primarily considered.

Coumaric Acids↗

Liquid core waveguide spectrophotometry for the sensitive determination of nitrite in river water samples.

A flow injection analysis system was built with a liquid core waveguide spectrophotometric detector using an 80 cm Teflon AF-1600 capillary tube (2,2-bistrifluoromethyl-4,5-difluoro-1,3-dioxole/tetrafluoroethylene). The system was applied to determine nitrite ion in river water samples. The lower limit of detection for nitrite was 2.1 nmol dm(-3) (0.1 ng dm(-3) as NO(2)(-)) and the relative standard deviation of measurements was typically 0.56% (n = 5) at 0.21 micromol dm(-3).

Journal Article↗

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion.

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.

Cell Culture Techniques↗

Development of a coliforms monitoring system using an enzymatic fluorescence method.

A coliforms monitoring system in treated effluent of a wastewater treatment plant has been developed. In order to achieve rapid monitoring within 1 hour, an enzymatic fluorescence method without a culturing process was introduced to this system. It converts the increase rate of fluorescence intensity as enzymatic activity into the number of coliforms instead of converting fluorescence intensity itself. A flow injection analysis is used in this system for automatic measurement. Moreover, it is equipped with the pre-filtering unit to remove the interfering substances in the suspended solids causing deterioration in measurement precision. The good relationship (correlation coefficient of 0.90) between the obtained values using this system and the analysed values using the conventional direct counting method was observed in a test at an existing wastewater treatment plant.

Colony Count, Microbial↗

Insights to false positive total cyanide measurements in wastewater plant effluents.

Many publicly owned treatment works in North America are exceeding permitted limits for total cyanide in their wastewater treatment effluents. A recently introduced rapid, segmented, flow-injection analysis procedure using UV digestion and amperometric detection of the membrane-separated cyanide was used to investigate the various scenarios by which elevated cyanide levels might be present in wastewater treatment plant effluent. A number of significant interferences can produce false positive bias during sample analysis with the traditional acid distillation technique, but are minimized or absent with the new analytical method. However, increased levels of cyanide were found in some chlorinated wastewaters compared to the levels before chlorination, suggesting a fast reaction mechanism associated with the disinfectant and some precursor in the wastewater. In particular, the contact of chlorine with nitrite in the presence of a carbon precursor appears to contribute to cyanide formation during wastewater treatment and sample handling. This paper explores the scenarios under which cyanide can form during wastewater treatment as well as those in which a false bias for total cyanide can be obtained during sample processing and provides guidance for appropriate sample handling, screening, and processing to ensure valid analytical results.

Chemistry Techniques, Analytical↗

Rapid detection of Salmonella typhimurium in chicken carcass wash water using an immunoelectrochemical method.

An immunoelectrochemical method coupled with immunomagnetic separation was developed for rapid detection of Salmonella Typhimurium in chicken carcass wash water. Samples of chicken carcass wash water were inoculated with Salmonella Typhimurium at different cell numbers. Possible nonspecified inhibitors in the wash water were minimized by filtration and centrifugation. An approximately 9.4% loss of Salmonella cells was found after filtration (P < 0.01). The samples were mixed with anti-Salmonella-coated magnetic beads (ASCMB) and alkaline phosphatase-labeled anti-Salmonella (APLAS) to form ASCMB-Salmonella-APLAS conjugates. The conjugates were separated from the solution using a magnetic separator and then incubated with phenylphosphate substrate to produce phenol. The number of Salmonella was determined by measuring the phenol concentration using an amperometric tyrosinase carbon paste electrode in a flow injection analysis system. Under optimized parameters (1 mM MgCl2, 0.2 microg/ml APLAS, and 1 mM phenylphosphate in pH 7.0 Tris buffer solution), Salmonella Typhimurium in chicken carcass wash water could be identified and enumerated within 2.5 h with a detection limit of 5 x 10(3) CFU/ml. A linear relationship on a log-log scale was found between Salmonella cell number and the peak current ratio for Salmonella concentrations ranging from 10(3) to 10(7) CFU/ml (R2 = 0.963). The peak currents of multibacteria samples, containing Salmonella Typhimurium, Listeria monocytogenes, and Campylobacter jejuni, were not significantly different from Salmonella-only samples (P > 0.01).

Animals↗

Electrochemical immunoassay: an ultrasensitive method.

Hydrodynamic electrochemical techniques such as liquid chromatography and flow injection analysis with electrochemical detection are very effective for the rapid determination of the enzyme-generated product in enzyme immunoassays. The authors have used this detection method in various assay formats using both alkaline phosphatase and glucose-6-phosphate dehydrogenase as labels. Assays for digoxin will be used illustratively. Recently, the authors have used 70 mL microcapillary hematocrit tubes as the immunoassay reaction vessel and alkaline phosphatase as the labeling enzyme. The assay, complete in 30 min, had a detection limit of 5,6 x 10 -20 moles of IgG in serum. The linear range was four orders of magnitude. This low detection limit is due to a combination of the favorable geometry of the reaction vessel and the suppression of nonspecific adsorption by the addition of ion-pairing blocking agents. Even lower detectable amounts should be achievable with smaller reaction vessels. The capability for detecting such small amounts of analyte is potentially useful for the analysis of extremely small samples such as single cells and blood samples from premature infants.

Alkaline Phosphatase↗

High-throughput quantification of lysophosphatidylcholine by electrospray ionization tandem mass spectrometry.

BACKGROUND: Lysophosphatidylcholine (LPC) has been suggested to play a functional role in various diseases, including atherosclerosis, diabetes, and cancer mediated by LPC-specific G-protein-coupled receptors. Initial studies provided evidence for a potential use of LPC as diagnostic maker. However, existing methodologies are of limited value for a systematic evaluation of LPC species concentrations because of complicated, time-consuming procedures. We describe a methodology based on electrospray ionization tandem mass spectrometry (ESI-MS/MS) applicable for high-throughput LPC quantification. METHODS: Crude lipid extracts of EDTA-plasma samples were used for direct flow injection analysis. LPC 13:0 and LPC 19:0 were added as internal standards, and the ESI-MS/MS was operated in the parent-scan mode for m/z 184. Quantification was achieved by standard addition. Data processing was highly automated by use of the mass spectrometer software and self-programmed Excel macros. RESULTS: The calibrators LPC 16:0, LPC 18:0, and LPC 22:0 showed a linear response independent of sample dilution and plasma cholesterol concentration for both internal standards. The within-run imprecision (CV) was 3% for the major and 12% for the minor species, whereas the total imprecision was approximately 12% for the major and 25% for the minor species. The detection limit was <1 micromol/L. CONCLUSION: The developed ESI-MS/MS methodology with an analysis time of 2 min/sample, simple sample preparation, and automated data analysis allows high-throughput quantification of distinct LPC species from plasma samples, which could be a valuable tool for the evaluation of LPC as diagnostic marker.

Calibration↗

Comparison of different advanced oxidation process to reduce toxicity and mineralisation of tannery wastewater.

Many organic compounds contained in wastewater are resistant to conventional chemical and/or biological treatment. Because of this reason different degradation techniques are studied as an alternative to biological and classical physico-chemical processes. Advanced Oxidation Processes (AOPs) probably have developed to become the best options in the near future. AOP while making use of different reaction systems, are all characterised by the same chemical feature: production of OH radicals (*OH). The versatility of AOPs is also enhanced by the fact that they offer different possibilities for OH radical production, thus allowing them to conform to specific treatment requirements. The main problem with AOPs is their high cost. The application of solar technologies to these processes could help to diminish that problem by reducing the energy consumption required for generating UV radiation. In this work, different AOPs (O3, TiO2/UV, Fenton and H2O2/UV) were examined to treat tannery wastewater or as a pre-treatment step for improving the biodegradation of tannery wastewater, at different pH and dosage of the chemicals. Under certain circumstances retardation in biodegradation and/or an increase in toxicity may be observed within these treatment steps. Two different bioassays (Daphnia magna and Vibrio fischeri) have been used for testing the progress of toxicity during the treatment. In parallel other objectives were to analyse and identify organic compounds present in the untreated wastewater and arising degradation products in AOP treated wastewater samples. For this purpose substance specific techniques, e.g., gas chromatography-mass spectrometry (GC-MS) in positive electron impact (El(+)) mode and atmospheric pressure ionisation (API) in combination with flow injection analysis (FIA) or liquid chromatography-mass and tandem mass spectrometry (LC-MS or LC-MS-MS) were performed.

Aliivibrio fischeri↗

Antioxidant activities of novel alpha-lipoic acid derivatives: N-(6, 8-dimercaptooctanoyl)-2-aminoethanesulfonate- and N-(6, 8-dimercaptooctanoyl)-L-aspartate-zinc complex.

Two new compounds, sodium N-(6, '8-dimercaptooctanoyl)-2-amino ethanesulfonate- and sodium N-(6, 8-dimercaptooctanoyl)-L-aspartate - zinc complex were synthesized from alpha-lipoyl-2-aminoethanesulfonate and alpha-lipoyl-L- aspartate by reduction of zinc/acetic acid respectively. These alpha-lipoyl-amino acids were obtained by a coupling of alpha-lipoic acid and 2-aminoethanesulfonate or L-aspartate, using a mixed anhydride method. Scavenging activities of these derivatives against hydroxyl radicals (*OH) was demonstrated directly using electron spin resonance (ESR) spectrometry with spin trapping. Otherwise an apparent superoxide anion radical (O2*-) scavenging effect of these derivatives may be due to the inhibition of 02*- generation system, i.e., xanthine oxidase. Scavenging activities of these compounds against nitric oxide radicals (NO*), and peroxynitrite (ONOO-) were estimated by the flow injection analysis using the Griess reagent and by a fluorescence spectrometry using dihydrorhodamine 123 respectively. Meanwhile, these derivatives showed protective effects against lipid peroxidation and protein carbonyl formation. Scavenging activities against NO* and ONOO-, and inhibitory effects on protein carbonyl formation of these derivatives were much stronger than these of alpha-lipoic acid itself.

Alkanesulfonates↗