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PubMed2007

Effect of wall shear rate on biofilm deposition and grazing in drinking water flow chambers.

The effect of four-wall shear rates (34.9, 74.8, 142.5, and 194.5 s(-1)) on bacterial deposition on glass slides in drinking water flow chambers was studied. Biofilm image acquisition was performed over a 50-day period. Bacterial accumulation and surface coverage curves were obtained. Microscopic observations allowed us to obtain information about the dynamics and spatial distribution of the biofilm. During the first stage of biofilm formation (210-518 h), bacterial accumulation was a function of the wall shear rate: the higher the wall shear rate, the faster the bacterial deposition (1.1 and 1.9 x 10(4) bacterial cells . cm(-2) for wall shear rates of 34.9 and 142.5 s(-1), respectively). A new similarity relationship characteristic of a non-dimensional time and function of the wall shear rate was proposed to describe initial bacterial deposition. After 50 days of exposure to drinking water, surface coverage was more or less identical under the entire wall shear rates (7.44 +/- 0.9%), suggesting that biofilm bacterial density cannot be controlled using hydrodynamics. However, the spatial distribution of the biofilm was clearly different. Under low wall shear rate, aggregates were composed of bacterial cells able to "vibrate" independently on the surface, whereas, under a high wall shear rate, aggregates were more cohesive. Therefore, susceptibility to the hydraulic discontinuities occurring in drinking water system may not be similar. In all the flow chambers, significant decreases in bacterial biomass (up to 77%) were associated with the presence of amoebae. This grazing preferentially targeted small, isolated cells.

Biofilms↗
PubMedDate not supplied

Peroxyoxalate chemiluminescence detection of condensates of malondialdehyde with thiobarbituric acids using a flow system.

The peroxyoxalate chemiluminescence(CL) detection method for the evaluation of the CL intensity of malondialdehyde(MDA) condensates with seven 2-thiobarbituric acid derivatives is described. The method consists of a flow injection technique together with a CL detection system using bis(2,4,6-trichlorophenyl) oxalate(TCPO) and hydrogen peroxide as chemiluminogenic reagents. Linear correlations between CL intensity and concentration are obtained for pmol levels of condensates. Among the condensates, 1,3-diethyl-2-thiobarbituric acid(DETBA)-MDA shows the largest CL intensity. High performance liquid chromatography (HPLC)/CL detection of DETBA-MDA and 1,3-diphenyl-2-thiobarbituric acid(DPTBA)-MDA using a mixture of TCPO and hydrogen peroxide in acetonitrile as a postcolumn reagent solution is also described. The detection limits for DETBA-MDA and DPTBA-MDA are 20 and 200 fmol, respectively, per 20 microL injection at a signal-to-noise ratio of 2. This HPLC/CL detection system was applied to the determination of MDA in rat brains by using DETBA as a fluorescent derivatizing reagent.

Animals↗
PubMedDate not supplied

A universal peroxyoxalate-chemiluminescence detection system for mobile phases of differing pH.

A universal peroxyoxalate-chemiluminescence detection system for high performance liquid chromatography, available for a variety of mobile phases, has been developed. The system consisted of a dual-head short-stroke pump and a chemiluminescence detector. The standard conditions using bis(2,4,6-trichlorophenyl) oxalate (TCPO) as aryl oxalate were as follows. The first postcolumn solution was the mixture of 0.5 M imidazole-nitric acid (pH 7.5) and acetonitrile (1:4, v/v). The second was acetonitrile containing TCPO-hydrogen peroxide. These two solutions were delivered by the two pump-heads. After the pH of the column eluate was adjusted to the optimum range (6.5-7.5) by the first postcolumn solution, the solution was mixed with the second postcolumn solution. After flowing through a reaction coil, the chemiluminescence of the mixture was monitored. Using this system, a high sensitivity (fmol level) was obtained for perylene as an analyte with mobile phases having different pH values (2.0-8.0). Polycyclic aromatic hydrocarbons became detectable to a high sensitivity even after the column separation using an acidic mobile phase. The detection sensitivity of nitrated pyrenes after on-line electrochemical reduction using an acidic mobile phase was also increased. This system might be available for other aryl oxalates by some modifications of the postcolumn solutions.

Chromatography, High Pressure Liquid↗
PubMed2003

The benefit of the retrofitting of a conventional LC system to micro LC: a practical evaluation in the field of bioanalysis with fluorimetric detection.

The interests in liquid micro-chromatography (higher column efficiencies, increase in sensitivity) are now well established. The enhancement of fluorimetric response induced by the reduction of the inner diameter of columns (4.6, 3.0, 1.0 and 0.3 mm respectively) coupled with adapted detection cells to control the loss of efficiency (8 micro L for the two first columns and 100 nL for the two smaller ones) has been studied in the bioanalytical field, using the plasma determination of native fluorescent antibacterial agents: fluoroquinolones. Ten-fold enhancement of the signal can easily be obtained when substituting a 0.3 mm i.d. column and 100 nL detection cell for a 4.6 mm i.d. column, and 8 micro L detection cell. In addition to inner diameter reduction, the detection cell geometry appears to be an essential parameter to obtain the best enhancement of the recorded signal. Hence, the enhancement of signal with micro-chromatography with fluorimetric detection appears to be a compromise between column inner diameter and flow cell volume reduction.

Chromatography, Liquid↗
PubMed2004

On-line identification of trans-resveratrol in red wine using a sweeping technique combined with capillary electrophoresis/77 K fluorescence spectroscopy.

The feasibility of combining the techniques of on-line concentration and capillary electrophoresis/low-temperature fluorescence spectroscopy (CE/LTFS) for the detection and identification of trans-resveratrol in red wine at 77 K is demonstrated for the first time. This technique, involving sweeping-micellar electrokinetic chromatography (sweeping-MEKC), was used for the initial on-line concentration and separation, after which a cryogenic molecular fluorescence experiment was performed at 77 K. In comparison with normal-MEKC mode, a approximately 1500-fold improvement in detection sensitivity could be obtained when the sweeping-MEKC was applied. The proposed method permits not only the separation and detection of trans-resveratrol from red wine extracts but also ensures that the on-line spectrum is readily distinguishable and can be unambiguously assigned at 77 K.

Anticarcinogenic Agents↗
PubMed2004

The combination of flow injection with electrophoresis using capillaries and chips.

The combined flow injection capillary electrophoresis (FI-CE) system that integrates the essential favorable merits of FI and CE, can significantly expand the application scope of CE by exploring the various on-line sample pretreatments and preconcentration of FI. The principle behind this technique, some innovative designs of the split-flow interface, as well as novel applications to a variety of analytical problems, are reviewed and discussed. Some salient features and unique advantages of this technique are outlined.

Calibration↗
PubMed2006

The fabrication and evaluation of inline coupling of microdialysis with capillary electrophoresis and its application in the determination of blood glucose.

A novel method of inline coupling of microdialysis (MD) with CE has been investigated. A polysulfone MD membrane was in situ prepared at the inlet end of the separation capillary, which endued the capillary with an additional function of clean-up sampling and made the pretreatment of complex matrices simple. The properties of the membrane and its influence on electrophoresis were evaluated. The membrane cut off macromolecules and particles efficiently and persistently, and changing the components of membrane-casting solution could control the molecular weight cutoff of the membrane. Using the capillary having the MD membrane in electrophoresis, column efficiency with little reduction was achieved. The mixture of chlorpheniramine (CPA) and BSA was used to verify the analytical characteristics. Only the peak of CPA appears in the electropherograms and the RSDs of migration times and peak areas of CPA were 0.8 and 4.6%, respectively. The method was applied for the determination of glucose in human blood using an electrochemical detector. Whole blood containing glucose and macromolecules was electrokinetically injected directly into the capillary without pretreatment, and the concentration of glucose in human blood was 5.5 mmol/L. Equilibrium dialysis injection is also investigated in this paper. The results show that the method may be further used for in vivo sampling to monitor the endogenous or exogenous small molecules and their metabolites.

Blood Glucose↗
PubMed2006

Etched bare fused-silica capillaries for online preconcentration of amino acids in CE.

An online preconcentration method based on electrostatic interaction between the analytes and inner surface of the capillary column was developed for the determination of zwitterionic analytes such as amino acids in CE coupled with a DAD. The amino acids possessed positive charges when they were dissolved in an acidic solvent. When they were injected into the column, they were attracted by the negatively charged inner surface of the fused-silica capillary column. An etched column was used to increase the area of the capillary's inner surface and, consequently increase the electrostatic interaction between the amino acids and the inner surface of the capillary column. It was found that when the sample was injected at 10 psi for 1 min and the pH value of the sample was 4, the amount of amino acids attracted to the inner surface of the capillary was maximum. Under these optimized experimental conditions, the detection sensitivity of CE-DAD was enhanced by 5200, 2800, and 3100 times for asparagine, tryptophan and phenylalanine, respectively, compared with normal CE separation. The method provided good reproducibility in terms of both migration time and peak height. It can be successfully used for the preconcentration zwitterion.

Amino Acids↗
PubMed2006

Numerical model for DNA loading in microdevices: stacking and autogating effects.

Many electrophoresis-based DNA sequencing and genotyping microdevices rely on field-driven effects to load and preconcentrate the sample. A quantitative model is developed for a broad class of electrophoresis-based microfabricated sample injectors. Quantitative predictions of DNA preconcentration are compared with experimental data and are shown to qualitatively reproduce the detailed time-evolving sample distribution in the injector. The model provides practical guidance on device and protocol design, in order to optimize this critical aspect of microfluidic devices.

DNA↗
PubMed2006

Online acid barrage stacking anti-salt injection for capillary electrophoresis of 9-fluorenylmethylchloroformate-derivatized amino acids in high ionic strength solutions by UV detection.

An acid barrage stacking (ABS) method has been shown to be feasible for online anti-salt injection in CE of 9-fluorenylmethyl chloroformate (FMOC)-labeled amino acids (AAs) detected by common UV absorption. The operation was performed on normal polar CE by sucking in an extra plug of acid following a sample zone, serving as a selective acid barrage to block the backward migration of weak anionic analytes due to a sudden mobility reduction via acid-base reaction which does not affect strong co-ions such as Cl(-) to penetrate the barrage freely. By CE-UV of FMOC-AAs in various NaCl solutions, the effectiveness of ABS was firmly validated, able to stand up to 500 mM NaCl and to stack analytes by 10(3)-fold calculated from the UV detection limits, that is 0.01 microM for ABS and 10 microM for non-stacking injection. The method was also validated by determining trace Glu and Asp in real samples of rat brain microdialysate, rat serum and human saliva. The intraday RSDs were 0.33-4.9% for migration time and 1.8-9.6% for peak area. The recoveries measured by spiking technique were 82-115% for Glu and 86-116% for Asp. Working equations were obtained by plotting peak height vs. concentration at 0.1-50 microM, with correlation coefficients of >0.999. The contents of Glu and Asp were thus found at 0.26-0.83 microM and 0.24-0.64 microM respectively, in rat brain microdialyste; 37-40 microM and 8.4-10 microM, respectively, in rat serum; and 3.5-5.8 microM and 1.0-4.1 microM, respectively in human saliva. They were consistent with the data from other methods.

Amino Acids↗
PubMed2007

Combination of flow injection with electrophoresis using capillaries and chips.

The technique of combined flow injection CE (FI-CE) integrates the essential favorable merits of FI and CE and can significantly expand the application of CE by utilizing the various on-line sample pretreatments and preconcentration of FI. The basic principles, instrumental developments, and applications of the FI-CE system from 2004 to 2006 are reviewed. The recent developments and applications of FI-CE are outlined.

Calibration↗
PubMed1993

An automatic dehydrogenase-based flow-injection system: application for the continuous determination of glucose and lactate in mammalian cell-cultures.

A concept for the development of an automatic flow-injection analyzer with integrated dehydrogenase columns and its application in the control of industrial processes is presented. The system is based upon a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer and values for switching on the one hand between sample and standard solutions and on the other hand between different enzyme columns. A Microsoft Windows 3.x application 'WIN-FIA' controls interactively the whole system and can be easily adapted to a specific solution of an analytical problem. As an example, the flow-injection system was used for the continuous determination of glucose and lactate, using glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) as indicator enzymes, in a mammalian cell-culture fermentation process. The resulting concentration values are in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays.

Animals↗
PubMedDate not supplied

Kinetic study of site directed and randomly immobilized his-tag alkaline phosphatase by flow injection chemiluminescence.

We immobilized his-tag alkaline phosphatase (ALP) randomly and with the desirable orientation (site directed) to compare the effects of the enzyme activity on the beads. The chemiluminescence was employed to increase the sensitivity of enzyme labelled assays. Flow injection was also carried out for the detection of chemical and biological molecules in flow solutions. The Vmax of randomly immobilized his-tag ALP was 1.2 and the Vmax of site directed immobilized his-tag ALP was 1.5. In other words, the activity of site directed immobilized his-tag ALP was about 1.3-folds increased. The detection limit was detected to be 6 x 10(-6) M for the flow injection system.

Alkaline Phosphatase↗
PubMed2006

Simultaneous determination of eight biologically active thiol compounds using gradient elution-liquid chromatography with Coul-Array detection.

The most active form of sulfur in biomolecules is the thiol group, present in a number of biologically active compounds. Here we present a comprehensive study of thiol analysis using flow injection analysis/HPLC with electrochemical detection. The effect of different potentials of working electrodes, of organic solvent contents in the mobile phase, and of isocratic and gradient elution on simultaneous determination of thiol compounds (cysteine, cystine, N-acetylcysteine, homocysteine, reduced and oxidised glutathione, desglycinephytochelatin, and phytochelatins) are described and discussed. These thiol compounds were well separated and detected under optimised HPLC-electrochemical detection conditions (mobile phase: 80 mM trifluoroacetic acid and methanol with a gradient profile starting at 97:3 (TFA:methanol), kept constant for the first 8 min, then decreasing to 85:15 during one minute, kept constant for 8 min, and finally increasing linearly up to 97:3 from 17 to 18 min; the flow rate was 0.8 mL/min, column and detector temperature 25 degrees C, and the electrode potential 900 mV). We were able to determine tens of femtomoles (3 S/N) of the thiols per injection (5 microL), except for phytochelatin5 whose detection limit was 2.1 pmole. This technique was consequently used for simultaneous determination of compounds of interest in biological samples (maize tissue and human blood serum).

Chromatography, Liquid↗
PubMedDate not supplied

Determination of cardamonin using a chemiluminescent flow-injection method.

A sensitive and selective flow injection chemiluminescence method for the determination of cardamonin over the range 1.0 x 10(-8) to 8.0 x 10(-6) g/mL is described. The method is based on the enhancement by cardamonin of the chemiluminescence of the reaction between cerium (IV) and rhodamine 6G in sulphuric acid medium. The optimised flow injection procedure yielded a detection limit for cardamonin of 8.8 x 10(-9) g/mL, whilst the relative standard deviations of intraday and inter-day precision were below 2.5%. The method has the advantages of high sensitivity and a wide linear range. It was successfully applied to the determination of cardamonin in Alpinia katsumadai Hayata. The mechanism of the chemiluminescence reaction is proposed.

Cerium↗
PubMed1993

NMR and protein folding: equilibrium and stopped-flow studies.

NMR studies are now unraveling the structure of intermediates of protein folding using hydrogen-deuterium exchange methodologies. These studies provide information about the time dependence of formation of secondary structure. They require the ability to assign specific resonances in the NMR spectra to specific amide protons of a protein followed by experiments involving competition between folding and exchange reactions. Another approach is to use 19F-substituted amino acids to follow changes in side-chain environment upon folding. Current techniques of molecular biology allow assignments of 19F resonances to specific amino acids by site-directed mutagenesis. It is possible to follow changes and to analyze results from 19F spectra in real time using a stopped-flow device incorporated into the NMR spectrometer.

Flow Injection Analysis↗
PubMed1997

Absence of a stable intermediate on the folding pathway of protein A.

The B-domain of protein A has one of the simplest protein topologies, a three-helix bundle. Its folding has been studied as a model for elementary steps in the folding of larger proteins. Earlier studies suggested that folding might occur by way of a helical hairpin intermediate. Equilibrium hydrogen exchange measurements indicate that the C-terminal helical hairpin could be a potential folding intermediate. Kinetic refolding experiments were performed using stopped-flow circular dichroism and NMR hydrogen-deuterium exchange pulse labeling. Folding of the entire molecule is essentially complete within the 6 ms dead time of the quench-flow apparatus, indicating that the intermediate, if formed, progresses rapidly to the final folded state. Site-directed mutagenesis of the isoleucine residue at position 16 was used to generate a variant protein containing tryptophan (the 116 W mutant). The formation of the putative folding intermediate was expected to be favored in this mutant at the expense of the native folded form, due to predicted unfavorable steric interactions of the bulky tryptophan side chain in the folded state. The 116 W mutant refolds completely within the dead time of a stopped-flow fluorescence experiment. No partly folded intermediate could be detected by either kinetic or equilibrium measurements. Studies of peptide fragments suggest that the protein A sequence has an intrinsic propensity to form a helix II/helix III hairpin. However, its stability appears to be marginal (of the order of 1/2 kT) and it could not be an obligatory intermediate on a defined folding pathway. These results explicitly demonstrate that the protein A B domain folds extremely rapidly by an apparent two-state mechanism without formation of stable partly folded intermediates. Similar mechanisms may also be involved in the rapid folding of subdomains of larger proteins to form the compact molten globule intermediates that often accumulate during the folding process.

Amino Acid Sequence↗
PubMed1995

Phe-46(CD4) orients the distal histidine for hydrogen bonding to bound ligands in sperm whale myoglobin.

The role of Phe-46(CD4) in modulating the functional properties of sperm whale myoglobin was investigated by replacing this residue with Leu, Ile, Val, Ala, Trp, Tyr, and Glu. This highly conserved amino acid almost makes direct contact with the distal histidine and has been postulated to affect ligand binding. The overall association rate constants for CO, O2, and NO binding were little affected by decreasing the size of residue 46 step-wise from Phe to Leu to Val to Ala. In contrast, the rates of CO, O2, and NO dissociation increased 4-, 10-, and 25-fold, respectively, for the same series of mutants, causing large decreases in the affinity of myoglobin for all three diatomic gases. The rates of autooxidation at 37 degrees C, pH 7.0 increased dramatically from approximately 0.1-0.3 h-1 for wild-type, Tyr-46, and Trp-46 myoglobins to 1.5, 5.2, 4.9, and 5.0 h-1 for the Leu-46, Ile-46, Val-46 and Ala-46 mutants, respectively. Rates of NO and O2 geminate recombination were measured using 35 ps and 9 ns laser excitation pulses. Decreasing the size of residue 46 causes significant decreases in the extent of both picosecond and nanosecond rebinding processes. High resolution structures of Leu-46 and Val-46 metmyoglobins, Val-46 CO-myoglobin, and Val-46 deoxymyoglobin were determined by X-ray crystallography. When Phe-46 is replaced by Val, the loss of internal packing volume is compensated by (1) contraction of the CD corner toward the core of the protein, (2) movement of the E-helix toward the mutation site, (3) greater exposure of the distal pocket to intruding solvent molecules, and (4) large disorder in the position of the side chain of the distal histidine (His-64). In wild-type myoglobin, the van der Waals contact between C zeta of Phe-46 and C beta of His-64 appears to restrict rotation of the imidazole side chain. Insertion of Val at position 46 relieves this steric restriction, allowing the imidazole side chain to rotate about the C alpha - C beta bond toward the surface of the globin and about the C beta - C gamma bond toward the space previously occupied by the native Phe-46 side chain. This movement disrupts hydrogen bonding with bound ligands, causing significant decreases in affinity, and opens the distal pocket to solvent water molecules, causing marked increases in the rate of autooxidation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗