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PubMed1994

Flow injection assay for the neurotoxin beta-ODAP using an immobilized glutamate oxidase reactor with prereactors to eliminate glutamate interferences.

The neurotoxic amino acid, beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP,ODAP) was oxidized by immobilized glutamate oxidase (GlOD) to produce hydrogen peroxide. The peroxide reacts with Trinder reagent in a reactor with immobilized horseradish peroxidase to form a red-colored quinone imine dye, which was detected spectrophotometrically at 512 nm. Determinations were made in a flow injection (FI) setup consisting of four packed-bed enzyme reactors containing GlOD (20 microL), catalase (20 microL), GlOD (250 microL), and peroxidase (50 microL) in series. Glutamate is oxidized quantitatively in the first reactor, but the hydrogen peroxide is destroyed in the second so that interferences from this substrate are removed. This step destroys only a few percent of the ODAP in the sample. Most of the remaining ODAP is oxidized in the third reactor. Injections of 20-microL ODAP standards gave a response curve which was linear within the range 10-650 microM. Phosphate buffer extracts of grass peas (lathyrus sativus) were purified by centrifugation and membrane filtration. Samples were injected into the FI setup to assay the toxin at a rate of 20 samples per hour. The beta-ODAP content of a batch of dry seed corresponded to 0.74% (w/w) with a relative standard deviation of 2.8%. Thermal treatment of ODAP standards at 80-90 degrees C reduced the response to 62% of that before heating. The decrease is due to beta<-->alpha isomerization, and the experiment thus confirms that the method is selective for the toxic beta-isomer.

Amino Acid Oxidoreductases↗
PubMed1995

Configurations of a flow injection system for perfusion studies of adherent cells.

This paper describes three configurations of a flow injection apparatus designed to be the fluidic drive for perfusion studies of cultured adherent cells. The apparatus was coupled to a flow-through perfusion chamber that was specifically designed for live cell perfusion using fluorescence microscopy as the detector. The instrument consists of two linear syringe pumps and a multiposition selector valve which, under computer control, allowed sequential injection, fluid switching, and flow injection to be performed with minimal system reconfiguration. When the apparatus is coupled to a dual inlet perfusion chamber, target cells can be exposed to very steep reagent pulses, while the traditional single inlet perfusion chamber allows more flexibility and provides a more gradual increase in reagent concentration. The most significant salient feature of the system is the ability to generate very steep pulses--a desirable feature for cell perfusion studies.

Cell Adhesion↗
PubMed2001

Continuous-flow, on-line monitoring of biospecific interactions using electrospray mass spectrometry.

A continuous-flow analytical screening system is presented using electrospray mass spectrometry to measure the interaction of biologically active compounds with soluble affinity proteins. The biochemical detection system is based on a solution-phase, homogeneous assay. In a first step, compounds to be screened (e.g., biotinylated compounds, concentration range 10-1,000 nmol/L) are injected into a continuous-flow reaction system and allowed to react with the affinity protein (e.g., streptavidin, concentration range 3-48 nmol/L). Subsequently, a reporter ligand (fluorescein-labeled biotin 96 nmol/L) is added to saturate the remaining free binding sites of the affinity protein and the concentration of unbound reporter ligand is measured using electrospray MS in the selectedion monitoring mode. The presence of active compounds in the sample results in an increase of the concentration of unbound reporter ligands. The feasibility of a homogeneous MS-based biochemical assay is demonstrated using streptavidin/biotin and anti-digoxigenin/digoxin as model systems. Compared to radioactive or fluorescence-based biochemical assays, the present assay format does not require the synthesis and purification of labels. Various analytical conditions were investigated to determine the ability of MS to measure the biochemical interactions. The availability of a single ligand that can be detected at 10-50 nmol/L concentrations by electrospray MS is sufficient to set up the biochemical assay. For the biospecific interactions studies, detection limits of 10-100 nmol/L were obtained.

Antigen-Antibody Reactions↗
PubMed2001

Development of a flow injection capillary chemiluminescent ELISA using an imprinted polymer instead of the antibody.

A flow injection competitive assay analogous to enzyme immunoassays has been developed using a molecularly imprinted polymer instead of the antibody. A glass capillary was modified by covalently attaching an imprinted polymer to the inner capillary wall. The herbicide 2,4-dichlorophenoxyacetic acid was used as a model analyte. The analyte was labeled with tobacco peroxidase, and chemiluminescence was used for detection in combination with a photomultiplier tube or a CCD camera. In a competitive mode, the analyte-peroxidase conjugate was passed together with the free analyte through the polymer-coated capillary mounted in a flow system. After a washing step, the chemiluminescent substrate was injected and the bound fraction of the conjugate was quantified by measuring the intensity of the emitted light. Calibration curves corresponding to analyte concentrations ranging from 0.5 ng mL(-1) to 50 microg mL(-1) (2.25 nM-225 microM) were obtained. A lowered detection limit by 2 orders of magnitude was obtained when detection was done in discontinuous mode and the chemiluminescence light was conducted inside the photomultiplier tube by an optical fiber bundle, thus yielding a dynamic range of 5 pg mL(-1)-100 ng mL(-1) (22.5 pM-450 nM).

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Ultrasonic extraction followed by sonolysis-ozonolysis as a sample pretreatment method for determination of reactive arsenic toward sodium tetrahydroborate by flow injection-hydride generation AAS.

A new sample pretreatment method based on ultrasonic extraction in HCI medium and subsequent oxidation of the extracts by sonozone (i.e., sonolysis-ozonolysis) has been developed for determination of reactive arsenic toward sodium tetrahydroborate [mainly As(III) + As(V)] by flow injection-hydride generation atomic absorption spectrometry. This method avoids the use of intensive treatments with concentrated and corrosive acids, high pressures, and temperatures that are inherent with traditional wet or dry ashing procedures and entails reduced waste production and reagent consumption. A sonozone process at room temperature was optimized to break the bond of As to proteins and macromolecular constituents which was an essential requirement for effective reduction by L-cysteine prior to arsine generation. Spiking experiments showed that As(III), As(V), MMA, and DMA were fully recovered from several matrixes on applying the above treatment. On the other hand, a nonreducible As species such as arsenobetaine that is predominant in some biological samples remained unchanged. Application of the method to sediment, soil, fly ash, and plant CRMs demonstrated that, in general, a good agreement existed between certified and found As contents, thereby indicating the absence of nonreducible As forms. Low As recoveries were observed for fish CRMs, as a result of the nondegradability of arsenobetaine by sonozone. The detection limit of As in the samples investigated was in the range 0.19-2.8 microg g(-1).

Animals↗
PubMed2001

Determination of cabergoline by electrospray ionization tandem mass spectrometry: picogram detection via column focusing sample introduction.

An electrospray ionization tandem mass spectrometric method was developed for low-picogram detection of an ergot alkaloid, cabergoline, in coyote plasma extracts. Cabergoline is under investigation as an abortifacient in canid species. Central to the successful development of this method was the ability to introduce relatively large sample volumes into the mass spectrometer. This was achieved by focusing the analyte on a conventional high-performance liquid chromatography guard column prior to elution into the spectrometer. Volumes up to at least 900 microL could be injected onto the guard column using a 100% aqueous mobile phase. Cabergoline retained on the column was eluted as a discreet band into the mass spectrometer by the rapid addition of methanol (30%) to the mobile phase. As compared to flow injection sample introduction, the ability to inject larger sample volumes led to a greatly lowered detection limit. Using this technique and a modification of a previously reported extraction procedure, cabergoline could be determined in coyote plasma at concentrations as low as 9 pg of cabergoline/mL of plasma.

Animals↗
PubMed2002

A method for screening total mercury in water using a flow injection system with piezoelectric detection.

An automatic microgravimetric screening system based on piezoelectric detection and the use of acidic stannous chloride as reductant was developed for the fast detection and determination of total mercury in water. Reduced mercury is detected as an amalgam by using a gold-coated piezoelectric crystal, the sensor subsequently being regenerated by passing it through a peroxydisulfate solution. The gold-coated piezoelectric crystal is a highly efficient retention unit for the main soluble mercury species (inorganic, complexed, and organometallic) previously reduced to elemental mercury and is free of interferences from other metal ions. This detector exhibits good sensitivity: it allows the determination of mercury at sub-parts-per-billion concentration levels (0.30-1.00 microg/L). The precision, expressed as relative standard deviation, was +/- 2.7% (n = 11; P = 0.05) at 0.5 microg/L total mercury. The proposed method was successfully used as a rapid screening method for mercury monitoring in natural waters.

Electrochemistry↗
PubMed2002

Chemiluminometric sensor for simultaneous determination of L-glutamate and L-lysine with immobilized oxidases in a flow injection system.

A chemiluminometric flow-through sensor for simultaneous determination of L-glutamate (Glu) and L-lysine (Lys) in a single sample has been developed. Immobilized uricase, immobilized peroxidase, support material, coimmobilized glutamate oxidase/peroxidase, support material, and coimmobilized lysine oxidase/peroxidase were packed sequentially in a transparent PTFE tube, and the tube was placed in front of a photomultiplier tube as a flow cell. A three-peak recording was obtained by one injection of the sample solution. The peak height of the first peak was due to the concentrations of urate and other reductants in the sample; the immobilized uricase was used to decompose urate, and the hydrogen peroxide produced was decomposed with a luminol-hydrogen peroxide reaction by immobilized peroxidase. The peak heights of the second and third peaks were free from the interferences from the reductants and were dependent only on the concentrations of Glu and Lys, respectively. Calibration graphs for Glu and Lys were linear at 40-1,000 and 50-1,200 nM, respectively. The sampling rate was 11/h without carryover. The sensor was stable for two weeks. The sensor system was applied to the simultaneous determination of Glu and Lys in serum.

Artifacts↗
PubMed2002

A flow injection on-line multiplexed sorption preconcentration procedure coupled with flame atomic absorption spectrometry for determination of trace lead in water, tea, and herb medicines.

One of the limitations in previous flow injection (FI) sorption preconcentration procedures in a knotted reactor (KR), which have been carried out exclusively with a single continuous sample injection over a certain period, is the relatively low retention efficiency (typically 40-50%). Although the sensitivity of such systems could be improved by properly increasing sample preconcentration time, sample loading flow rate, or both, further improvement of the sensitivity has been limited by the narrow linearity of the relationship between signal intensity and preconcentration time or sample loading time. In this work, a novel on-line FI multiplexed sorption preconcentration procedure with repetitive sample injections was developed to overcome the above problems in the previous systems. In contrast to previous FI preconcentration systems, the proposed multiplexed preconcentration procedure evenly divides a single longer sample injection step into several shorter substeps while the total preconcentration time is still kept constant. To demonstrate its merits, the proposed FI on-line KR multiplexed sorption preconcentration system was combined with flame atomic absorption spectrometry (FAAS) for determination of trace lead in water, tea, and herb medicines. The lead in the sample solution on-line reacted with ammonium pyrrolidine dithiocarbamate, and the resultant analyte complex was sorbed on the inner walls of the KR. The residual sample solution was then removed from the KR with an air flow. The above two steps were repeated eight times with a total preconcentration time of 120 s. The sorbed analyte was eluted from the KR with 4.5 mol L(-1) HCl for on-line FAAS detection. The present multiplexed preconcentration procedure with eight repetitive sample injections for a total preconcentration time of 120 s gave a retention efficiency of 92%, twice that obtained by one single sample injection preconcentration (47%). In addition, the linear ranges of the diagrams of absorbance against sample loading flow rate and sample loading time were extended, offering more potential for achieving high sensitivity by increasing sample loading rates or sample loading time compared to the previous one single continuous sample injection preconcentration procedure. At a sample loading flow rate of 3.6 mL min(-1) for a total preconcentration period of 120 s, an enhancement factor of 57 and a detection limit (3sigma) of 8 microg L(-1) were obtained. The precision was 1.4% (RSD, n = 11) at the 200 microg L(-1) level. The developed method was successfully applied to the determination of trace lead in various water samples, herb medicines, and a certified tea reference material.

Flow Injection Analysis↗
PubMed2002

Sampling BIAS at channel junctions in gated flow injection on chips.

The commonly used gated injection scheme was examined and found to suffer from multiple levels of electrokinetic sampling bias, including a new type based on transradial electrokinetic selection (TREKS). TREKS occurs as analytes of differing electrophoretic mobilities migrate around the corner at a channel junction in a microchip. The overall sample bias in gated injection was shown to be time-dependent and resulted in a larger sample bias against components of negative electrophoretic mobility. A new injection procedure for microchip devices based on interstream diffusion at zero potential is proposed. Diffusion of molecules into the separation channel is the main driving force for this type of injection. The new scheme is shown to be useful for injection of complex samples with multiply charged components, such as peptide mixtures. This procedure allows sampling of volumes from 12 to 45 pL, reproducible retention times (RSD < 1.5%), and reproducible peak areas (RSD < 2.3%).

Electrophoresis↗
PubMed2003

An autonomous nutrient analyzer for oceanic long-term in situ biogeochemical monitoring.

An autonomous nutrient analyzer in situ (ANAIS) has been developed to monitor nitrate, silicate, and phosphate concentrations while deployed at sea at pressure (down to 1000 m). Detection is made by spectrophotometry. The instrument uses solenoid-driven diaphragm pumps to propel the sample, the standards, and the reagents through a microconduit, flow injection-style thermostated manifold. The analyzers are placed in an equipressure container filled with oil. The analyzers operate until a pressure of 100 bar and show a linear response up to 40 microM nitrate, 150 microM silicate, and 5 microM phosphate with a detection limit less than 0.1, 0.5, and 0.1 microM and an accuracy of 1, 1, and 3% for nitrate, silicate, and phosphate, respectively. The measurement protocol includes three steps over 13 min: rinsing with the sample stream, reagents introduction, and absorbance detection. Field tests comprise ANAIS nitrate, silicate, and phosphate testing alone in the surface ocean. Phosphate results are not yet fully satisfactory. The instrument implemented on top of a YOYO vertical eulerian profiler was then deployed successfully in the northwestern Mediterranean Sea acquiring 30 nitrate profiles between 200 and 1100 m over a 15-day period. This chemical analyzer can be a valuable observing asset adapted on any type of oceanographic platform.

Automation↗
PubMed2003

Selective measurement of ultratrace methylmercury in fish by flow injection on-line microcolumn displacement sorption preconcentration and separation coupled with electrothermal atomic absorption spectrometry.

A novel nonchromatographic speciation technique for ultratrace methylmercury in biological materials was developed by flow injection microcolumn displacement sorption preconcentration and separation coupled on-line with electrothermal atomic absorption spectrometry (ETAAS). In the developed technique, Cu(II) was first on-line complexed with diethyldithiocarbamate (DDTC), and the resultant Cu-DDTC was presorbed onto a microcolumn packed with the sorbent from a cigarette filter. Selective preconcentration of methylmercury (MeHg) in the presence of Hg(II), ethylmercury (EtHg), and phenylmercury (PhHg) was achieved at pH 6.8 through loading the sample solution onto the microcolumn due to a displacement reaction between MeHg and the presorbed Cu-DDTC. The retained MeHg was subsequently eluted with 50 microL of ethanol and on-line determined by ETAAS. Interferences from coexisting heavy metal ions with lower stability of their DDTC complexes relative to Cu-DDTC were minimized without the need of any masking reagents. No interferences from 5.5 mg L(-1) Cu(II), 4.5 mg L(-1) Cd(II), 2.5 mg L(-1) Cr(III), 3 mg L(-1) Fe(III), 10 mg L(-1) Ni(II), 10 mg L(-1) Pb(II), and at least 25 mg L(-1) Zn(II) were observed for the determination of MeHg at the 50 ng L(-1) level (as Hg). With the consumption of only 3.4 mL of sample solution, an enhancement factor of 75, a detection limit of 6.8 ng L(-1) (as Hg) in the digest (corresponding to 3.4 ng g(-1) in original solid sample for a final 50 mL of digest of 0.1 g of solid material), and a precision (RSD, n = 13) of 2.3% for the determination of methylmercury at the 50 ng L(-1) (as Hg) level were achieved at a sample throughput of 30 samples h(-1). The recoveries of methylmercury spike in real fish samples ranged from 97 to 108%. The developed technique was validated by determination of methylmercury in a certified reference material (DORM-2, dogfish muscle), and was shown to be useful for the determination of methylmercury in real fish samples.

Adsorption↗
PubMed2003

Silver-induced enhancement of thiochrome-based peroxide measurements.

Thiamine is presently one of the most attractive substrates used for sensitive fluorometric measurements of peroxides. Thiochrome (TC), a highly fluorescent product, is formed in enzyme-mediated oxidations. It is assumed that H(2)O(2) is nearly quantitatively converted to TC. The reaction cannot differentiate H(2)O(2) from many other peroxides such as methylhydroperoxide (MHP); to perform differential measurements, H(2)O(2) can first be selectively destroyed by a suitable catalyst such as MnO(2). In substituting Ag(2)O for MnO(2) to accomplish the selective destruction of H(2)O(2), we achieved the stated objective but were puzzled by a 3-fold increase in the MHP response in the presence of Ag(2)O. It was soon discovered that traces of dissolved Ag(+) and Hg(2+) can dramatically increase the yield of TC in this reaction from either H(2)O(2) or MHP; the normal yield in fact is only 20%. We present here a reaction scheme and kinetic model that adequately describes this behavior and should provide a path to substantially increase the sensitivity of this important assay method.

Flow Injection Analysis↗
PubMed2005

Application of a liposomal bioluminescent label in the development of a flow injection immunoanalytical system.

A flow injection liposome immunoanalytical system was developed using biotin as the model analyte and liposomal aequorin as the label. Aequorin is a photoprotein isolated from luminescent jellyfish (notably Aequorea victoria) and other marine organisms that emits visible light in the presence of a trace of Ca2+. Because of this characteristic, the aequorin complex has been used as an intracellular Ca2+ indicator. In this study, a bioluminescent label was designed by encapsulating aequorin inside the cavity of the liposome, whose outer surface was sensitized with the analyte of interest. The analyte-tagged liposomal aequorin was employed in the development of a heterogeneous bioluminescence immunoassay for the model analyte biotin. The proposed immunoassay was based on the competition between the model biotin and aequorin-encapsulating, biotin-tagged liposomes for a limited number of anti-biotin antibody-binding sites. The anti-biotin antibodies were immobilized via protein A in a capillary immunoreactor column, and 30% MeOH was used for the regeneration of antibody-binding sites after each measurement, which allowed the immunoreactor to be used for up to 50 sequential sample injections without any loss of reactivity. The calibration curve for biotin in Tris-buffered saline solution had a linear range of 1 x 10(-11)-1 x 10(-3) M. The detection limit of the assay was 50 pg (equivalent to 200-microL injection of 1 x 10(-9) M). This study demonstrates the procedures for the encapsulation of the photoprotein aequorin into the liposome, which can be used as a sensitive label in bioluminescence immunoassays for biotin or in other applications.

Aequorin↗
PubMed2005

Thick membrane, solid contact ion selective electrode for the detection of lead at picomolar levels.

A new approach for decreasing the lower detection limit of a lead ion selective electrode (ISE) is presented. The ISE is designed using nonfunctionalized porous glassy carbon loaded with ionophore/plasticizer/additive cocktail. This material acts both as the support for the liquid polymeric membrane and as the signal transducer of the ISE. The high purity of the glassy carbon, together with its high conductivity, allows for the development of a thick, low-resistance composite membrane. This sensor element enables the continuous measurement of lead down to picomolar levels, with very small detection limit deterioration due to the lead ion transport within the bulk of the thick membrane.

Flow Injection Analysis↗
PubMed2006

Continuous ultrasound-assisted extraction coupled to flow injection-pervaporation, derivatization, and spectrophotometric detection for the determination of ammonia in cigarettes.

A dynamic system for the continuous removal of ammonia from cigarettes with ultrasound assistance and iterative change of the flow direction of the extractant through the sample cell has been developed. A 0.1-g sample of cigarette was subjected to 7 min of ultrasound-assisted extraction (application and duration of pulse 0.7 s, output amplitude 85% of the converter nominal amplitude), and 1 M NaOH solution was used both as extractant and as carrier in the dynamic system. The ultrasound-assisted extractor was coupled to a pervaporation unit through a flow injection interface in order to develop a fully automated method. In arriving at the pervaporator, the ammonia is transferred from the donor-carrier stream to an acceptor stream, where the classical Berthelot reaction takes place--thus favoring pervaporation. The blue complex formed is spectrophotometrically monitored at 655 nm. The method was applied to the determination of ammonia in a selection of 10 European cigarette brands and Kentucky Reference 2R4F cigarettes.

Ammonia↗
PubMed2006

Biosensor based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/amperometric detection of organophosphate pesticides and nerve agents.

A highly sensitive flow injection amperometric biosensor for organophosphate pesticides and nerve agents based on self-assembled acetylcholinesterase (AChE) on a carbon nanotube (CNT)-modified glassy carbon (GC) electrode is described. AChE is immobilized on the negatively charged CNT surface by alternatively assembling a cationic poly(diallyldimethylammonium chloride) (PDDA) layer and an AChE layer. Transmission electron microscopy images confirm the formation of layer-by-layer nanostructures on carboxyl-functionalized CNTs. Fourier transform infrared reflectance spectrum indicates the AChE was immobilized successfully on the CNT/PDDA surface. The unique sandwich-like structure (PDDA/AChE/PDDA) on the CNT surface formed by self-assembling provides a favorable microenvironment to keep the bioactivity of AChE. The electrocatalytic activity of CNT leads to a greatly improved electrochemical detection of the enzymatically generated thiocholine product, including a low oxidation overvoltage (+150 mV), higher sensitivity, and stability. The developed PDDA/AChE/PDDA/CNT/GC biosensor integrated into a flow injection system was used to monitor organophosphate pesticides and nerve agents, such as paraoxon. The sensor performance, including inhibition time and regeneration conditions, was optimized with respect to operating conditions. Under the optimal conditions, the biosensor was used to measure as low as 0.4 pM paraoxon with a 6-min inhibition time. The biosensor had excellent operational lifetime stability with no decrease in the activity of enzymes for more than 20 repeated measurements over a 1-week period. The developed biosensor system is an ideal tool for online monitoring of organophosphate pesticides and nerve agents.

Acetylcholinesterase↗