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PubMedDate not supplied

A reusable and specific protein A-coated piezoelectric biosensor for flow injection immunoassay.

A hydrophilic matrix of periodate-oxidized dextran was used as a double-sided linker to covalently immobilize Staphylococcus aureus protein A (SpA) molecules onto a poly-L-lysine-modified piezoelectric crystal surface to improve their stability, activity, and binding specificity with human immunoglobulin G (IgG) in flow injection assays. The prepared sensing crystals displayed best sensitivity and reusability at a flow rate of 140 microL/min. A human IgG concentration as low as 0.3 nM can be detected by this system. Up to 19 successive assay repetitions were achieved without significant loss of sensitivity using the same crystal. The analysis of adsorption kinetics indicates that such a preparation can greatly increase the amount of available active human IgG binding sites on immobilized SpA. Hardly any response arising from unspecific binding was detected. In addition, the sensing crystal prepared by this method was found to retain activity better than one prepared via direct deposition when stored in either wet or dry states. Finally, the prepared SpA-coated crystals were applied to the affinity immobilization of polyclonal goat anti-Schistosoma japonicum glutathione-S-transferase (GST) and were able to subsequently detect GST and its genetically engineered mutant either in a purified form or in the crude cell lysate.

Animals↗
PubMed2004

In vitro monitoring of nanogram levels of puerarin in human urine using flow injection chemiluminescence.

A rapid and sensitive chemiluminescence (CL) determination of puerarin with the flow injection technique was presented. It was found that puerarin could enhance the CL generated from luminol-KIO(4) system significantly. The increment of CL intensity was linear over the concentration of puerarin ranging from 0.3 to 100.0 ng mL(-1) (r(2)=0.9963), offering a detection limit as low as 0.1 ng mL(-1) (3 sigma). At a flow rate of 2.0 mL min(-1), one analysis cycle, including sampling and washing, could be accomplished in 20 s with a relative standard deviation of less than 5.0%. The experimental results demonstrated that the excretive amounts of puerarin reached its maximum in 3 h after taking 250 mL green drink containing 275 mg puerarin, and the puerarin excretive ratio during 24 h was 9.70% in the body of volunteers.

Flow Injection Analysis↗
PubMed2003

2D-IR correlation analysis of deteriorative process of traditional Chinese medicine 'Qing Kai Ling' injection.

The traditional Chinese medicine 'Qing Kai Ling' injection after deterioration is distinguished from the original formulation using Fourier-transform infrared spectroscopy (FTIR) and two-dimensional correlation infrared spectroscopy (2D-IR). The IR spectra of the injections before and after deterioration are so similar, that it is very difficult to distinguish them by using the conventional approach. However, the higher spectral resolution and more structural information provided by 2D-IR analysis makes the identification possible. According to the results of 2D correlation analysis, the band at 1611 cm(-1) in IR spectra, in fact, consists of the overlap of three bands at 1572, 1667 and 1729 cm(-1), which are assigned to the alkaloids (heterocyclic compounds), flavone derivatives, and carbonyl compounds in the injection, respectively. Interestingly, the autopeak at 1667 cm(-1) disappears in synchronous spectrum of the deteriorated 'Qing Kai Ling' injection. It is suggested that the deterioration of the injection in air at room temperature be due to the oxidation of the flavone compounds mainly. As all intensities of the correlation peaks involved with the band in 1059 cm(-1) decrease greatly, it is assigned to the thermal decomposition of the glucoside components. Hence, it can conclude that the oxidation of the flavone compounds and the decomposition of the glucoside components are some of the main causes concerning to the deterioration of 'Qing Kai Ling' injection at ambient temperature. Thus, the 2D-correlation analysis provides a powerful method for the quality control of the traditional Chinese medicine injection.

Drug Stability↗
PubMed2004

Microfluidic immunosensor systems.

Immunosensing microfluidic devices are reviewed. Devices are commonly fabricated in glass, silicon, and polymers, with polymers seeing greater attention in recent years. Methods have been developed to immobilize antibodies and other molecules and resist non-specific adsorption through surface modification. The most common detection method is fluorescence, followed by electrochemistry. Various microfluidic designs have been reported for immunoassay applications. The observed trends in microfluidic immunoassay applications closely resemble the trends of general immunoassays, where large molecules are detected principally through a sandwich procedure, while competitive assays are used to detect smaller molecules. The following future trends are suggested: more sensitive detection, increased integration and miniaturization, multianalyte analysis, more robust reagents and devices, and increased functionality of surface treatments.

Biosensing Techniques↗
PubMed2005

Microfluidics for flow cytometric analysis of cells and particles.

This review describes recent developments in microfabricated flow cytometers and related microfluidic devices that can detect, analyze, and sort cells or particles. The high-speed analytical capabilities of flow cytometry depend on the cooperative use of microfluidics, optics and electronics. Along with the improvement of other components, replacement of conventional glass capillary-based fluidics with microfluidic sample handling systems operating in microfabricated structures enables volume- and power-efficient, inexpensive and flexible analysis of particulate samples. In this review, we present various efforts that take advantage of novel microscale flow phenomena and microfabrication techniques to build microfluidic cell analysis systems.

Animals↗
PubMed2006

Flow injection potentiometric determination of chlorpromazine.

New chlorpromazine selective electrodes with a tubular arrangement and no internal reference solution are proposed. Selective membranes are of poly(vinyl chloride) (PVC) with the tetraphenylborate.chlorpromazine (TPB.CPZ) ion-exchanger dissolved in o-nitrophenyl octyl ether (oNPOE). Analytical features of the electrodes were evaluated on a single-channel flow assembly having 500 microl injection volumes and flow-rates of 4.5 ml min(-1). For a carrier solution of 3.3 x 10(-3)M in sodium sulphate, Nernstian response was observed over the concentration range 1.0 x 10(-5) to 1.0 x 10(-2)M. Average slopes were about 59 mV decade(-1) and squared correlation coefficients were >0.9984. Slight hiper-Nernstian behaviour was observed in buffer solutions of 4.4 pH; average slopes were of 62.06 mV decade(-1). The electrode displayed a good selectivity for CPZ, with respect to, several foreign inorganic and organic species. The selective electrodes were successfully applied to the analysis of pure solutions and pharmaceutical preparations. Proposed method allows the analysis of 84 samples h(-1), producing wastewaters of low toxicity. The proposed method offers the advantage of simplicity, accuracy, applicability to coloured and turbid samples, and automation feasibility.

Antipsychotic Agents↗
PubMed1998

Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase.

The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow-injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to microM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 ml min-1 and 37 degrees C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10(-10) M (amplification factor; more than 10,000). This chemically amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to microM) of uric acid in highly diluted body fluids in which the concentration of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4 degrees C in 10 mM TRIS-HCl buffer, pH 9.0.

Dithiothreitol↗
PubMed2003

Analysis of glucose and lactate in hippocampal dialysates of rats during the operant conditioned reflex using microdialysis.

Changes of extracellular glucose and lactate in hippocampus for freely moving rats during the operant conditioned reflex were examined simultaneously. Samples of the dialysate were assayed for both glucose and lactate using in vivo microdialysis and a microbore flow injection analysis-immobilized enzyme reactor-electrochemical detection (FIA-IMER-ECD) system. Microdialysis samplings were conducted in a Skinner box where lights were delivered as conditioned stimuli (CS) paired with foot shocks as unconditioned stimuli (US). In the treatment group the concentration of glucose and lactate showed no fluctuations during the whole process. However, in the control group in which the rats were exposed to many foot shocks, lactate levels decreased by 19% below baseline during the behavioral session and glucose showed a delayed decrease (by 18%). Compared with glucose, lactate can immediately indicate the dynamic changes in brain.

Animals↗
PubMed2003

Micro sequential injection: automated insulin derivatization and separation using a lab-on-valve capillary electrophoresis system.

Automated sampling and fluorogenic derivatization of islet proteins (insulin, proinsulin, c-peptide) are separated and analyzed by a novel lab-on-valve capillary electrophoresis (LOV-CE) system. This fully integrated device is based on a micro sequential injection instrument that uses a lab-on-valve manifold to integrate capillary electrophoresis. The lab-on-valve manifold is used to perform all microfluidic tasks such as sampling, fluorogenic labeling, and CE capillary rejuvenation providing a very reliable system for reproducible CE separations. Fluorescence detection was coupled to an epiluminescence fluorescence microscope using a customized capillary positioning plate. This customized plate incorporated two fused-silica fiber optic probes that allow for simultaneous absorbance and fluorescence detection, extending the utility of this device. Derivatization conditions with respect to the sequence of addition, timing, injection position, and volumes were optimized through iterative series of experiments that are executed automatically by software control. Reproducibility in fluorogenic labeling was tested with repetitive injections of 3.45 mM insulin, yielding 1.3% RSD for peak area, 0.5% RSD for electromigration time, and 2.8% RSD for peak height. Fluorescence detection demonstrated a linear dynamic range of 3.43 to 6.87 microM for insulin (r2 = 0.99999), 0.39 to 1.96 pM for proinsulin (r2 = 0.99195) and 260 to 781 nM for c-peptide (r2 = 0.99983). By including hydrodynamic flushing immediately after the detection of the last analyte, the sampling frequency for islet protein analysis was increased. Finally, an in vitro insulin assay using rat pancreatic islet excretions was tested using this lab-on-valve capillary electrophoresis system.

Autoanalysis↗
PubMed2002

Flow-injection electrospray ionization mass spectrometry of crude cell extracts for high-throughput bacterial identification.

Flow-injection electrospray ionization mass spectrometry (FI-ESI-MS) of unfractionated cell-free extracts obtained from bacterial cells suspended in a solvent mixture was investigated as a rapid analytical method for reproducible, high-throughput bacterial identification. Five bacterial strains (two Escherichia coli, two Bacillus spp. and one Brevibacillus laterosporus) were studied in this investigation. Axenically grown bacterial cells were suspended in an acidic organic solvent and the cell-free extract was sequentially injected into a solvent flow stream that was sprayed into the ionization chamber of the ESI-MS. The spectra produced contained reproducible information, which was useful for discriminating between the bacteria. Tandem mass spectrometry was used to characterize further the peaks, and at least three classes of macromolecules, namely phospholipids, glycolipids, and proteins, were found to contribute most to the spectral information. Bacterial extracts stored under different conditions gave very similar mass spectra for each of the five bacterial strains, indicating that the extracts were stable even at room temperature for up to 24 h, with no loss of information content, which has obvious implications for automated high-throughput analysis. An analysis of the components of the extracting solvent mixture and their effects on the spectral information showed that acetonitrile contributes most significantly to the extraction process and hence to the information content of the spectra.

Bacteria↗
PubMed2006

Flow-injection chemiluminescence sensor for determination of isoniazid in urine sample based on molecularly imprinted polymer.

In this paper, molecularly imprinted polymer (MIP) of isoniazid is synthesized through thermal radical copolymerization of metharylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) in the presence of isoniazid template molecules. A novel flow injection chemiluminescence sensor for isoniazid determination is developed by packing the isoniazid-MIP into the flow cell as recognition elements. Isoniazid could be selectively adsorbed by the MIPs and the adsorbed isoniazid was sensed by its great enhancing effect on the weak CL reaction between luminol and periodate which were mixed in the flow cell. The enhanced CL intensity is linear in the range 2x10(-9) to 2x10(-7) g/mL and the detection limit is 7x10(-10) g/mL (3sigma) isoniazid with a relative standard deviation 2.8% (n=9) for 8x10(-8) g/mL. The sensor is reversible and reusable. It has a great improvement in sensitivity and selectivity for CL analysis. As a result, the sensor has been successfully applied to determination of isoniazid in human urine. At the same time, the binding characteristic of the polymer to isoniazid was evaluated by batch method and the dynamic method, respectively.

Flow Injection Analysis↗
PubMed2003

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection.

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.

Adrenergic beta-Agonists↗
PubMed2005

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry.

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.

Acetylcholine↗
PubMed2001

On-line mercury and methylmercury pre-concentration by adsorption of their dithiophosphoric acid diacyl ester chelates on a C18 column and cold-vapor atomic-absorption detection.

The pre-concentration of mercury(II) and methylmercury by adsorption of their dithiophosphoric acid diacyl ester (DDTP) chelates on a C18 column, then detection with cold-vapor atomic-absorption spectrometry was investigated. Conditions such as sample pH, reductant and chelating agent flow and concentration, and eluent and carrier gas flow were optimized. Optimization was performed by use of evolutionary operation with a proper factorial design. At a sample flow of 5.3 mL min(-1) and a loading time of 4.5 min, column adsorption efficiency ranged from 88 to 93% for both species. Detection limits down to 10 ng L(-1) were obtained at a sample throughput of 12 h(-1). There was good agreement between found and certified values in the analysis of certified reference materials after their microwave-assisted mineralization with HNO3 and H2O2.

Chelating Agents↗
PubMed1999

Simultaneous determination of paracetamol, caffeine and acetylsalicylic acid by means of a FI ultraviolet pls multioptosensing device.

A simple and rapid analytical procedure is proposed for the simultaneous determination of caffeine (CF), acetylsalicylic acid (ASA) and paracetamol (PCT) in pharmaceutical preparations by partial least-squares (PLS) treatment of a flow-through multisensor based on the integration of the retention and UV detection of the analytes on a solid support. Diode-array spectrophotometry has been used to obtain spectra (240-350 nm) of the analytes retained on C18 bonded phase beads packed in a flow cell. By using a 0.5% pH 1 HClO4 solution as the carrier, the multisensor responds linearly in the measuring range without requiring additional reagents or derivatization processes and the active microzone is regenerated by using methanol as eluting agent. Spectra of the corresponding analytes were used to provide multivariate data for the multivariate procedure. The statistical parameters obtained by the application of PLS methods at different reaction times were analysed, from which the optimum reaction time for the simultaneous determination of the analytes was selected. In the analysis of real and synthetic samples, precise and accurate values were obtained.

Acetaminophen↗
PubMed1991

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA.

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product analysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was successfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.

Animals↗
PubMed2000

Trace analysis of estrogenic chemicals in sewage effluent using liquid chromatography combined with tandem mass spectrometry.

A rapid, selective, sensitive, and reproducible method for the analysis of environmental estrogens, either natural or synthetic, present in samples from sewage treatment works (STW) has been developed. Isolation of these compounds from STWs was performed by applying a simple extraction procedure using an ENVI-CARB cartridge (a graphitized non porous carbon black) as the solid-phase extraction (SPE) system. Liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) and tandem mass spectrometry was used for detection. For the multiple reaction monitoring (MRM) mode, the [M + H](+) ion of estrone and the [M + H-H(2)O](+) ions of 17beta-estradiol, estriol and 17alpha-ethynylestradiol were selected as the precursors for collisionally induced dissociation (CID). The average recoveries from sewage final effluent samples ranged from 84 to 93% for low levels, and from 89 to 95% for high levels. The precision of the method ranged from 11 to 8% for low level and from 9 to 7% for high level samples. The lower level of quantitation for these estrogens in STW samples was determined at 0.5 ng/L for 17beta-estradiol and 17alpha-ethynylestradiol, and 1 ng/L for estrone and estriol, based on 1-L aliquots of sewage treatment works water, using the optimum tuning parameters for each individual selected precursor ion/product ion transition. Compared to a previous gas chromatography/mass spectrometry (GC/MS) method for the analysis of ten STW samples, this method was shown to provide higher sensitivity and lower time consumption.

Calibration↗