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Results for “Flow Injection Analysis”

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PubMed2004

Theoretical and experimental analysis of analyte transport in a fiber-optic, protein C immuno-biosensor.

Protein C (PC) is an important anticoagulant in human blood plasma, and early diagnosis of PC deficiency is critical for preventing dangerous thromboembolic complications. A fiber-optic PC immuno-biosensor has been under development in our research group for real-time PC-deficiency diagnosis. The sensor has demonstrated a good sensitivity and specificity for quantifying PC in buffered solutions. However, for plasma samples, with a limited sample reaction time, the sensor produced only 30% of the signal intensity of PC in buffer. The high plasma viscosity (1.9 cP) was speculated as the major reason for signal intensity reduction. In this investigation, the sensing performance of the fiber-optic PC biosensor is systematically characterized in terms of physical and chemical properties of the sample media. Theoretical and experimental analyses indicate that the reduced diffusion rate of PC molecules in viscous samples caused the sensing system to be more mass-transfer-limited. Convective flow of sample/reagent solutions during immunoreactions can increase the rate of the analyte mass transport from the bulk solution to the sensor surface, with reaction kinetics changing from mass-transfer-limited to reaction-limited as flow velocity increases. It was shown that PC sensor performance was significantly improved for plasma samples with convection. The effect of the flow velocity and incubation times for samples and reagents on the sensor performance was also systematically analyzed to optimize the assay protocol for PC sensing. Currently, a 6-cm-long immuno-biosensor is capable of quantifying PC in plasma (1 mL) in the heterozygous PC deficiency range (0.5 to 2.5 microg/mL) within 5 minutes, at an average signal-to-noise ratio of 50.

Biosensing Techniques↗
PubMed2007

A microfluidic platform for sequential ligand labeling and cell binding analysis.

Developing biochemical and cell biological assay for screening biomolecules, evaluating their characteristics in biological processes, and determining their pharmacological effects represents a key technology in biomedical research. A PDMS-based integrated microfluidic platform was fabricated and tested for facilitating the labeling of ligand on the nanogram scale and sequential cell binding analysis in a manner that saves both time and reagents. Within this microfluidic platform, ligand labeling, cell immobolization, and optical analysis are performed in a miniaturized, continuous and semi-automated manner. This microfluidic device for ligand labeling and cell analysis is composed of two functional modules: (i) a circular reaction loop for fluorophore-labeling of the ligand and (ii) four parallel-oriented incubation chambers for immobilization of cells, binding of ligand to different cell populations, and optical evaluation of interactions between the labeled ligand and its cell targets. Epidermal growth factor (EGF) as the ligand and different cell lines with various levels of EGF receptor expression have been utilized to test the feasiblity of this microfluidic platform. When compared to studies with traditional Petri dish handling of cells and tissues, or even microwell analyses, experiments with the microfluidic platform described here are much less time consuming, conserve reagents, and are programmable, which makes these platforms a very promising new tool for biological studies.

Animals↗
PubMed2004

Improving the monitoring of methanol concentration during high cell density fermentation of Pichia pastoris.

The Pichia pastoris expression system is widely used for the production of recombinant proteins. A simple and efficient experimental set-up allowing on-line monitoring of the methanol concentration during the fermentation of P. pastoris based on the detection of the methanol vapor concentration in the exhaust air from fermenter by a tin dioxide (SnO2) semiconductor sensor is described. An experimental procedure to allow precise calibration of the system and to reduce methanol sensor's interferences (>95% reduction) are also presented and discussed. Accuracy and measurement error were estimated about 0.05 g x l(-1) and 6%, respectively. The efficient monitoring of methanol will help to advanced control of recombinant protein production and process optimization.

Bioreactors↗
PubMed2005

Szilard's dream.

Explore the source record for details and available documents.

Bioreactors↗
PubMed2005

A microfluidic chemostat for experiments with bacterial and yeast cells.

Bacteria and yeast frequently exist as populations capable of reaching extremely high cell densities. With conventional culturing techniques, however, cell proliferation and ultimate density are limited by depletion of nutrients and accumulation of metabolites in the medium. Here we describe design and operation of microfabricated elastomer chips, in which chemostatic conditions are maintained for bacterial and yeast colonies growing in an array of shallow microscopic chambers. Walls of the chambers are impassable for the cells, but allow diffusion of chemicals. Thus, the chemical contents of the chambers are maintained virtually identical to those of the nearby channels with continuous flowthrough of a dynamically defined medium. We demonstrate growth of cell cultures to densely packed ensembles that proceeds exponentially in a temperature-dependent fashion, and we use the devices to monitor colony growth from a single cell and to analyze the cell response to an exogenously added autoinducer.

Bioreactors↗
PubMed2006

Absorption detection of enzymatic reaction using optical microfluidics based intermittent flow microreactor system.

The advantages of integrating microfluidics into photonics-based biosensing for fabricating microreactor type lab-on-a-chip devices carries a lot of advantages, such as smaller sample volume handling, controlled drug delivery and high throughput diagnosis, which is useful for in situ medical diagnosis and point-of-care (POC) testing. A hybrid integrated optical microfluidic system has been developed for the study of single molecules and enzymatic reactions. The method of optical absorption has been employed for biosensing and the feasibility of absorption-based detection on the microfluidic platform has been demonstrated using horseradish peroxidase and hydrogen peroxide, as an example. The results show that the device is useful for the analysis of both the individual chemical specimen and also the study of chemical and biological reaction between two reacting species. The hybrid integration of microfluidics and optical ensembles thus forms the basis for developing the microreactor type lab-on-a-chip device, which would have several important applications in the area of nanobiotechnology.

Absorption↗
PubMed2006

Accumulation of E. Coli bacteria in mini-channel flow.

The objective of this research is to design and optimize a mini/micro-channel based surface-accumulator of E. coli bacteria to be detected by acoustic wave biosensors. A computational approach has been carried out using the state of the art software, CFD-ACE with water as bacteria bearing fluid. E. coli bacteria have been modeled as random discrete particles tracked by solving the Lagrangian equations. The design challenges are to achieve low shear force (pico-N), high concentration at accumulation, and high enough Reynolds number to avoid bacteria swimming. A range of low Reynolds number (Re) has been considered along with the effects of particle boundary interactions, gravity, Saffman lift, etc. More than two orders of magnitude higher concentration at the accumulation than the inlet concentration, and lower shear force of less than pico-N have been achieved in the optimized designs.

Cell Aggregation↗
PubMed2003

Flow-injection chemiluminescence detection for studying protein binding of terbutaline sulfate with on-line microdialysis sampling.

The binding of terbutaline sulfate to bovine serum albumin was studied in vitro using the technique of microdialysis sampling combined with flow-injection chemiluminescence analysis (FIA-CL). In the presence of formaldehyde, terbutaline sulfate can be oxidized by KMnO(4) to produce high chemiluminescence emission in sulfate acid media. The concentration of terbutaline sulfate is proportional with the CL intensity in the range of 1 x 10(-7)-2 x 10(-5) mol l(-1) with a detection limit of 3 x 10(-8) mol l(-1). The drug and protein were mixed in different molar ratios in 0.067 mol l(-1) phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water bath. The microdialysis probe was utilized to sample the mixed solution at a perfusion rate of 5 microl min(-1) and the dialytic efficiency of terbutaline sulfate under the experimental conditions was 26.3%. The data obtained by proposed microdialysis flow-injection chemiluminescence method was analyzed with Scrathard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scrathard analysis were 4.11 x 10(4) l mol(-1) and 1.06, respectively. The proposed system proved that FIA-CL coupled with on-line microdialysis sampling is a simple and reliable technique for the study of drug-protein interaction.

Animals↗
PubMed2002

Field preconcentration of cadmium from seawater by using a minicolumn packed with Amberlite XAD-4/4-(2-pyridylazo) resorcinol and its flow-injection-flame atomic absorption spectrometric determination at the ng L(-1) Level.

A flow injection analysis-flame atomic absorption spectrometric method for the determination of cadmium in seawater was developed with the aim of yielding a sensitive assay with a low detection limit. The method employs a field flow preconcentration technique involving a minicolumn containing Amberlite XAD-4 impregnated with the complexing agent 4-(2-pyridylazo) resorcinol. A Plackett-Burman 2(7)x3/32 design for seven factors (sample pH, sample flow rate, eluent volume, eluent concentration, eluent flow rate, ethanol percentage in the eluent and minicolumn diameter) was carried out in order to find the significant variables affecting the field continuous preconcentration system (FCPS) and the flow injection elution manifold for cadmium determination in seawater samples by flame atomic absorption spectrometry. Cadmium can be preconcentrated with an enrichment factor of 1053 for a sample volume of 200 mL and a preconcentration time of 57 min. In these experimental conditions, the method provides a linear relationship between absorbance and cadmium concentration in the range from 22-1900 ng L(-1), with a detection limit (3SD) of 6 ng L(-1). The precision (expressed as relative standard deviation) for eleven independent determinations reached values of 8.9-0.8% in cadmium solutions of 50-700 ng L(-1). Analysis of certified reference materials (SLEW-3 and NASS-5) showed good agreement with the certified value. This procedure was applied to the determination of cadmium in seawater from Galicia (Spain).

Cadmium↗
PubMed2005

Optimizing an online SPE-HPLC method for analysis of (R)-[11C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide [(R)-[11C]PK11195] and its metabolites in humans.

(R)-[11C]PK11195 is used as a positron emission tomography tracer for activated microglia in several neurological disorders. Quantification of specific binding requires a metabolite-corrected plasma input function. In this study, a high-performance liquid chromatography (HPLC) procedure with online solid phase extraction was modified for analyzing (R)-[11C]PK11195 plasma samples, yielding total sample recoveries of more than 98%. When applied to human studies, the use of two HPLC systems enabled analysis of up to seven plasma samples under regular conditions. Online radioactivity detection was compared with offline sample measurements of HPLC profiles. Offline measurements provided the most reliable results especially for late plasma samples. In 10 patients, an average decrease of parent compound from 94.6% at 2.5 min to 45.2% at 1 h after administration was observed.

Amides↗
PubMed2004

Chloride-selective membrane electrodes and optodes based on an indium(III) porphyrin for the determination of chloride in a sequential injection analysis system.

Two quasi-independent methods for potentiometric and optical determination of chloride were simultaneously implemented in a flow system, providing real-time assessment of the quality of results. A potentiometric and an optical polymeric membrane doped with the same indium(III) octaethyl-porphyrin were used as sensor ionophore. The working mechanism and the analytical characteristics of these porphyrin-based sensors with respect to dynamic range, selectivity, repeatability and lifetime are discussed. These sensors, utilised as detectors in a flow system, were applied for the analysis of chloride in pharmaceutical solutions. The quality of the results obtained was evaluated by comparison with those provided by the reference method and no significant statistical differences at the 95% confidence level were observed. The simultaneous attainment of two measurements permitted the standardisation of results in real time and the detection of failures in the procedure.

Calibration↗
PubMed2004

Direct injection of solid-phase extraction eluents onto silica columns for the analysis of polar compounds isoniazid and cetirizine in plasma using hydrophilic interaction chromatography with tandem mass spectrometry.

Isoniazid and cetirizine do not retain well on reversed-phase columns due to their high polarity. Silica columns, when operated under hydrophilic interaction conditions, do provide excellent retention of these compounds. We have developed simple and proof of concept analytical methods for the analysis of isoniazid and cetirizine in animal and human plasma, respectively. Both methods employed the approach of direct injection of solid-phase extraction (SPE) organic eluents onto silica columns for analysis, thus eliminating evaporation and reconstitution steps that are typically needed for reversed-phase liquid chromatographic analysis. Isoniazid was extracted from animal plasma samples using a Waters Oasis HLB 96-well plate and then eluted with acetonitrile, while cetirizine was extracted from human plasma with a Waters MCX mu-Elute plate and then eluted with acetonitrile containing 5% concentrated ammonium hydroxide. The direct injection of the SPE eluent onto the analytical column was necessary since significant loss of isoniazid was found during the evaporation and reconstitution steps. The method for isoniazid also enabled ultra-fast analysis due to the relatively low back-pressure exhibited by silica columns even under high flow conditions. Both methods show good linearity, accuracy and precision covering the range of 10-2000 ng/mL of isoniazid, and 1-1000 ng/mL of cetirizine in plasma. Substantial time savings were realized as a result of both the elimination of the evaporation and reconstitution steps and the fast chromatographic analysis.

Animals↗
PubMed1993

Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models.

Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction. The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes. Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci. 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior. Unlike the wild-type protein (Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure. The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained. The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.

Apoproteins↗
PubMed2000

Determination of perchlorate at trace levels in drinking water by ion-pair extraction with electrospray ionization mass spectrometry.

Perchlorate has been added to the U.S. Environmental Protection Agency's Drinking Water Contaminant Candidate List (CCL). The present work describes the analysis of perchlorate in water by liquid-liquid extraction followed by flow injection electrospray mass spectrometry (ESI/MS). Cationic surfactants, mostly alkyltrimethyl-ammonium salts, are used to ion-pair aqueous perchlorate, forming extractable ion pairs. The cationic surfactant associates with the perchlorate ion to form a complex detectable by ESI/MS. The selectivity of the extraction and the mass spectrometric detection increases confidence in the identification of perchlorate. The method detection limit for perchlorate based on 3.14 sigma n-1 of seven replicate injections was 100 ng L-1 (parts per trillion). Standard addition was used to quantitate perchlorate in a drinking water sample from a contaminated source, and the concentration determined agreed within experimental error with the concentration determined by ion chromatography.

Flow Injection Analysis↗
PubMed2003

Performance of capillary gel electrophoretic analysis of oligonucleotides coupled on-line with electrospray mass spectrometry.

Synthetic oligonucleotides (ODNs) are routinely analyzed using capillary gel electrophoresis (CGE) for size-sieving based separations as well as electrospray mass spectrometry (ESI-MS) for identification. On-line coupling of these methods is therefore desired in order to combine the analytical capabilities provided by both methods. Performance of on-line CGE-ESI-MS systems is influenced by various parameters, and choice of optimal conditions is crucial for successful coupling experiments. In this study, we explore characteristics of the on-line coupled CGE-ESI-MS system for ODN analysis. Effects of CGE buffer concentration, capillary length, separation and orifice voltage on CGE separation and MS detection of a phosphodiester ODN mixture were examined. Attention was paid to the influence of the interface, such as geometry of capillary alignment, sheath liquid flow-rate and sheath liquid composition on performance of the system.

Acetonitriles↗
PubMed2006

Serum iohexol analysis by micellar electrokinetic capillary chromatography.

A simple and rapid ( approximately 4 min) method for the measurement of iohexol in serum for assessing the glomerular filtration rate is described. It is based on direct serum injection on the capillary by MEKC. The method is linear between 8 and 260 mg/L, with an RSD of peak height of 2.9%. Several simple steps have contributed to an improved daily precision, such as choosing a high pH buffer, increasing the SDS concentration, frequent standardization, and eliminating any sample pretreatment.

Blood Chemical Analysis↗