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PubMed1993

Size-exclusion chromatographic determination of beta-glucan with postcolumn reaction detection.

Using the specific interaction of beta-glucan with calcofluor dye as a detection method for size-exclusion chromatographic analysis, very simple direct extractions can be used for sample preparation. Either fluorescence or UV detection is employed. The calibration graphs are linear between 200 and 20 mg/l, although much more sensitive analyses are possible. The response remained independent of relative molecular mass in the range studied (1.7 x 10(6)-1.85 x 10(5)). Results for barley and malt show good correlation with those obtained by flow-injection analysis using calcofluor.

Chromatography, Gel↗
PubMed1996

Zeptomole-detecting biosensor for alkaline phosphatase in an electrochemical immunoassay for 2,4-dichlorophenoxyacetic acid.

A bienzyme substrate-recycling biosensor in a flow injection analysis system is described for the sensitive measurement of alkaline phosphatase (ALP) and applied to the fast readout of a competitive immunoassay for the widely used pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The phenol-indicating biosensor consists of a Clark-type electrode covered by a membrane with coentrapped tyrosinase and quinoprotein glucose dehydrogenase. ALP dephosphorylates phenyl phosphate to phenol (K(m) = 36 microM) outside the flow system. Phenol is oxidized in the sensor membrane by the oxygen-consuming tyrosinase via catechol to o-quinone. The quinone is reconverted to catechol by glucose dehydrogenase. This substrate cycling results in a 350-fold amplified sensor response to phenol. The oxygen consumption of the enzyme couple in the presence of phenol is monitored as a decrease in current. A total of 3.2 fM ALP (320 zmol/ 100 microL) has been detected after a 57.5 min incubation with phenyl phosphate. All involved reagents are stable over the time of measurement. The sensor does not produce any measurable blank signals. The immunoassay detects 0.1 microgram/L 2,4-D, the maximum concentration for pesticides allowed in drinking water by European Community regulations. The applicability of this biosensor for fast immunoassay readout is demonstrated by a 2 min incubation. By comparison, a standard photometric method (p-nitrophenyl phosphate) requires overnight incubation.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2002

Electrocatalytic amperometric determination of amitrole using a cobalt-phthalocyanine-modified carbon paste electrode.

Cobalt-phthalocyanine-modified carbon paste electrodes are shown to be excellent indicators for electrocatalytic amperometric measurements of triazolic herbicides such as amitrole, at low oxidation potentials (+0.40 V). The detection and determination of amitrole in flow injection analysis with a modified carbon paste electrode with Co-phthalocyanine is described. The concentrations of amitrole in 0.1 M NaOH solutions were determined using the electrocatalytic oxidation signal corresponding to the Co(II)/Co(III) redox process. A detection limit of 0.04 microg mL(-1) (4 ng amitrole) was obtained for a sample loop of 100 microL at a fixed potential of +0.55 V (vs. Ag/AgCl) in 0.1 M NaOH and a flow rate of 4.0 mL min(-1). Furthermore, the modified carbon paste electrodes offers reproducible responses in such a system, and the relative standard deviation was 3.3% using the same surface, 5.1% using different surface, and 6.9% using different pastes. The performance of the cobalt-phthalocyanine-modified carbon paste electrodes is illustrated here for the determination of amitrole in commercial formulations. The response of the electrodes is stable, with more than 80% of the initial retained activity after 50 min of continuous use.

Amitrole↗
PubMed1998

An optical biosensor for real-time chromatography monitoring: breakthrough determination.

The use of an optical biosensor for immunorecognition of protein products during affinity chromatography is discussed to provide rapid data describing the loading and subsequent breakthrough, followed by elution and fraction collection. The optical biosensor works by following in real-time the interaction of soluble ligate with an appropriate ligand attached to the optically active surface. The initial rate of interaction between soluble ligate and immobilized ligand has been shown to correlate well with ligate concentration. This method of analysis has also been shown to agree well with ELISA, the traditionally employed technique for immunoassay of protein products lacking, for example, catalytic activity. Forward prediction, using models of the breakthrough fitted to the real-time data, has enabled the column saturation point to be determined before it has been reached, thus enabling appropriate action to ensure minimal loss of protein product while improving column utilization efficiency. The biosensor, operated within a flow injection analysis regime, has been demonstrated to provide concentration data within 10 s, with a total assay turnaround of 30 s.

Animals↗
PubMed2007

Piezoelectric quartz crystal microbalance sensor for trace aqueous cyanide ion determination.

Using selective reaction chemistry, our present research has developed an online, real-time sensor capable of monitoring toxic cyanide at both drinking water standard and environmental regulatory concentrations. Through the use of a flow cell, aqueous samples containing cyanide are reacted with a gold electrode of a piezoelectric crystal to indirectly sense cyanide concentration by the dissolution of metallic gold. The quartz crystal is an AT-cut wafer sandwiched between two neoprene O-rings within the liquid flow cell. The presence of cyanide in solution results in the selective formation of a soluble dicyano-gold complex according to the Elsner reaction: 4Au + 8CN- + 2H2O + O2 <=> 4Au(CN)2- + 4OH-. The resulting loss of gold from the electrode is detected by the piezoelectric crystal as a resonant frequency change. Since free cyanide is a weak acid (pKa = 9.3), available protons compete for cyanide ligands. Therefore, increased sample pH provides higher sensitivity. The detection limits at pH 12 are 16.1 and 2.7 ppb for analysis times of 10 min and 1 h, respectively. The incorporation of the flow cell improves both analyte sensitivity and instrument precision, with an average signal intensity drift of only 5% over a 2-h analysis. The calibrations show excellent linearity over a variety of cyanide concentrations ranging from low ppb to hundreds of ppm. This detection method offers the advantage of selectively detecting cyanides posing a biohazard while avoiding detection of stable metal cyanides. This aspect of the system is based on competitive exchange of available metals and gold with cyanide ligands. Stable metal cyanide complexes possess a higher formation constant than cyanoaurate. This detection system has been configured into a flow injection analysis array for simple adaptation to automation. Anions commonly found in natural waters have been examined for interference effects. Additionally, the sensor is free from interference by aqueous cyanide analogues including thiocyanate. The developed detection system provides rapid cyanide determinations with little sample preparation or instrument supervision.

Journal Article↗
PubMed2000

Chemiluminescent reaction of fluorescent organic compounds with KHSO5 using cobalt(II) as catalyst and its first application to molecular imprinting

The decomposition of peroxomonosulfate (HSO5-) has been investigated by chemiluminescence (CL). A weak CL was observed during mixing the HSO5- solution with the Co2+ solution in unbuffered conditions. An appropriate amount of fluorescent organic compounds (FOCs), such as dansyl amino acids and pyrene, was added to the KHSO5/Co2+ solution, a strong CL was recorded. A possible CL mechanism, based on studies of the fluorescence, CL, and UV-visible spectra and comparison of Co3+ oxidation ability with the SO4.- radical ion, was discussed. The CL from HSO5-/Co2+ is the emission of singlet oxygen produced from the catalytic decomposition of HSO5-. It was suggested that the decomposition of HSO5- in aqueous solution with Co2+ proceeds via one-electron transfer to yield SO4.- radical ion. The FOC was attacked by SO4.- radical ion and oxidized to decompose into small molecules. During this proceeding, CL emission was given out. The present CL system has been developed as a flow injection analysis for FOCs. The detection limits (S/N = 3) were in the concentration range 10(-9)-10(-7) M for FOCs. Oxidation decomposition and CL emission of the analytes have been used in the molecular imprinting recognition. As an initial attempt, dansyl-L-phenylalanine was used as a template molecule and methacrylic acid and 2-vinylpyridine were used as functional monomers. The network copolymer imprinted with dansyl-L-phenylalanine exhibits an affinity for the template molecule. When the flowing streams of HSO5- and Co2+ solutions mixing through the molecularly imprinted polymer particles filled the flow cell, the template molecule, dansyl-L-phenylalanine reacted with the HSO5-/Co2+ solution and CL was emitted. The dansyl-L-phenylalanine was decomposed during the CL process, and the cavities of a defined shape and an arrangement of functional groups complementary to the template in the polymer were left for the next sample analysis.

Journal Article↗
PubMed2003

Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature.

OBJECTIVE: Although it has been shown recently that acetylcholine (ACh)-induced vasodilation of forearm resistance vessels is predominantly mediated by nitric oxide, direct biochemical evidence for eNOS stimulation by bradykinin (BK) in the human arterial circulation is still lacking. Therefore, the present study was designed to test the hypothesis that in the human forearm vasculature eNOS stimulation significantly contributes to BK-induced vasodilation. METHODS: BK was infused in the presence and absence of the NOS inhibitor L-NMMA (8 micromol/min) into the brachial artery of 16 healthy volunteers and the effects compared to muscarinergic eNOS stimulation following acetylcholine infusion. Forearm blood flow (FBF) was measured by venous occlusion plethysmography, and plasma nitrite (NO(2)(-)), which represents a sensitive and specific marker of regional eNOS activity, was determined in the antecubital vein and brachial artery by flow injection analysis. Nitric oxide production was calculated as product of the veno-arterial difference of NO(2)(-) concentration times FBF. RESULTS: Kininergic (BK: 20, 60, 200 ng/min) as well as muscarinergic (ACh: 1, 3, 10 microg/min) stimulation resulted in a dose-dependent increase in FBF and NO(2)(-) in each individual. The relationship between FBF and NO production upon BK infusion was comparable to that obtained with ACh (r = 0.98; n = 64, p < 0.01). Moreover, NOS inhibition reduced both flow responses and NO production (BK: 54 and 75 %; ACh: 57 and 72 %) to a similar extent. CONCLUSIONS: These data provide direct biochemical evidence for the involvement of eNOS in bradykinin-induced vasodilation of forearm resistance vessels in humans.

Acetylcholine↗
PubMed2004

Multidimensional analysis of denatured milk proteins by hydrophobic interaction chromatography coupled to a dynamic surface tension detector.

Multidimensional analysis of denatured milk proteins is reported using high-performance liquid chromatography (HPLC) combined with dynamic surface tension detection (DSTD). A hydrophobic interaction chromatography (HIC) column (a TSK-Gel Phenyl-5PW column, TosoBiosep), in the presence of 3.0 M guanidine hydrochloride (GdmHCl) as denaturing agent is employed as the mobile phase. Dynamic surface tension is measured through the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 4 s) is achieved, for each eluting drop of 4 s length, providing insight into both the kinetic and thermodynamic behavior of molecular orientation processes at the liquid-air interface. An automated calibration procedure and data analysis method is applied with the DSTD system, which allows two unique solvents to be used, the HIC mobile phase for the sample and a second solvent (water for example) for the standard, permitting real-time dynamic surface tension data to be obtained. Three-dimensional data is obtained, with surface tension as a function of drop time first converted to surface pressure, which is plotted as a function of the chromatographic elution time axis. Experiments were initially performed using flow injection analysis (FIA) with the DSTD system for investigating commercial single standard milk proteins (alpha-lactalbumin, beta-lactoglobulin, alpha-, beta-, kappa-casein and a casein mixture) denatured by GdmHCl. These FIA-DSTD experiments allowed the separation and detection conditions to be optimized for the HIC-DSTD experiments. Thus, the HIC-DSTD system has been optimized and successfully applied to the selective analysis of surface-active casein fractions (alpha s1- and beta-casein) in a commercial casein mixture, raw milk samples (cow's, ewe's and goat's milk) and other diary products (yogurt, stracchino, mozzarella, parmesan cheese and chocolate cream). The different samples were readily distinguished based upon the selectivity provided by the HIC-DSTD method. The selectivity advantage of using DSTD relative to absorbance detection is also demonstrated.

Calibration↗
PubMedDate not supplied

Fast screening for drugs of abuse by solid-phase extraction combined with flow-injection ionspray-tandem mass spectrometry.

A fast analytical approach for the simultaneous quantitative screening for illicit drugs in serum and urine without tedious chromatographic separation steps was developed by combining solid-phase extraction (SPE) followed by flow-injection analysis (FIA) with ionspray-ionization and tandem mass spectrometry (MS-MS) detection using a PE Sciex API 300 triple-quadrupole MS. MS-MS analysis was performed by sequentially isolating the precursor ions of the analytes and their deuterated standards with subsequent fragmentation and monitoring of one fragment ion for each substance. A multiple-reaction monitoring experiment was set up for morphine (MO), codeine (COD), amphetamine (AMP), benzoylecgonine (BZE), and their deuterated analogues. For method evaluation, serum samples spiked with 2-1000 ng of each drug and deuterated standards were extracted by mixed-mode SPE, redissolved in CH3CN-NH4OAc-buffer, and directly injected by flow injection into the ionspray source. The specificity of this new method was demonstrated by testing compounds with similar chemical structure for interferences from the analytes of interest (e.g., dihydromorphone, morphine glucuronide, and 6-monoacetylmorphine with MO; dihydrocodeine and hydrocodone with COD; cocaine [COC] and ecgonine methylester with BZE; methamphetamine with AMP). The possibility of interferences of such compounds with the FIA-ionspray-MS-MS screening method is discussed. Spiked serum samples and serum and urine samples from drug addicts and victims of drug abuse were analyzed with FIA-MS-MS and, after derivatization, with gas chromatography-mass spectrometry (GC-MS). Comparable quantitative results were obtained with both methods; no interferences with metabolites or other compounds were found. The FIA-ionspray-MS-MS method is a fast, quantitative, sensitive, and highly specific alternative method to drug-screening by immunoassays, high-performance liquid chromatography, and GC-MS. It can be used for the simultaneous detection of different drugs and metabolites such as opiates, COC, AMP derivatives, and many other drugs.

Chromatography, Gas↗
PubMed2004

Towards single molecule analysis in PDMS microdevices: from the detection of ultra low dye concentrations to single DNA molecule studies.

In this paper, we report on the performance of electrophoretical separation and laser-induced fluorescence (LIF) detection of dyes and fluorescently labeled biomolecules in poly(dimethylsiloxane) (PDMS) microdevices. The dyes fluorescein and fluorescein isothiocyanate (FITC) have been separated effectively in nM concentrations. Fluorescein injections gave linear concentration response in the range from 4 to 100 pM. As ultimate detection sensitivity, 100 fM injected fluorescein was obtained. Further, 100 fM injected fluorescein could be detected. This is to our knowledge the smallest electrokinetically injected dye concentration detected on a microchip. Injection studies of fluorescently labeled avidin revealed a theoretical detection limit of 25 nM for laser-induced fluorescence detection in good agreement with separations in glass chips. Furthermore, the injection of several and even one single DNA molecule using a PDMS cross injector has been demonstrated as well as free solution separation of lambda- and T2-DNA (60 pM each) in periodically structured channels.

Biopolymers↗
PubMed1984

Direct injection of plasma and urine in automated analysis of catecholamines by coupled-column liquid chromatography with post-column derivatization.

Adrenaline, noradrenaline and dopamine have been quantified by direct injection of plasma and urine in a liquid chromatographic system comprising three columns, one packed with a boronic acid gel and two with reversed-phase material. The catecholamines were selectively adsorbed on the boronic acid gel and separated by ion-pair chromatography on the reversed-phase columns. Dopamine was detected by coulometry, while noradrenaline and adrenaline were detected by fluorimetry as trishydroxyindoles after post-column coulometric oxidation and alkaline rearrangement. The reaction rate constants of the post-column reactions were determined by a flow injection analysis approach. Limits of detection were 0.04, 0.05 and 1.6 pmol for noradrenaline, adrenaline and dopamine, respectively. Endogenous plasma levels of noradrenaline and adrenaline could be quantified with a precision (RSD) of 2-4%.

Journal Article↗
PubMed2005

An electro-osmotic micro-pump based on monolithic silica for micro-flow analyses and electro-sprays.

A high-pressure electro-osmotic micro-pump fabricated by a sol-gel process is reported as a fluid-driving unit in a flow-injection analysis (FIA) system. The micro FIA system consists of a monolithic micro-pump on a glass slide (2.5 x 7.5 cm), a micro-injector, and a micro-sensor (2.5 x 1.5 cm). The monolithic silica matrix has a continuous skeleton morphology with micrometer-sized through-pores. The micrometer-size pores with a large negative surface charge density build up a large pressure under a DC electric field to drive fluid through the downstream units. A novel Nafion joint for the downstream cathode eliminates flow into the electrode reservoir and further enhances pressure build-up. The measured pump-pressure curve indicated a maximum pressure of 0.4 MPa at flow rate of 0.4 microL min(-1) at 6 kV. Despite the large voltage, the small current transmission area through the monolith produced a negligible current (less than 100 microA) that did not generate bubbles or ion contaminants. The flow rate can be precisely controlled in the range 200 nL to 2.5 microL min(-1) by varying the voltage from 1 to 6 kV. The high pump pressure and the large current-free DC field also enabled the pump to act as an electro-spray interface with a downstream analytical instrument.

Journal Article↗
PubMed1992

An enzyme-reactor for electrochemical monitoring of choline and acetylcholine: applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebrospinal fluid.

A sandwich-type enzyme reactor in which the enzymes are physically immobilized in a minimal dead space between two cellulose membranes, resulting in improved sensitivity, was developed for the electro-chemical detection of choline (Ch) and acetylcholine (ACh). The reactor contains the enzymes choline oxidase with or without acetylcholine esterase, for the detection of ACh and Ch, respectively. For the HPLC analysis of Ch and ACh the detection system was coupled post column. Levels of Ch and ACh of rat striatum tissue and human cerebrospinal fluid were found to be similar to those determined with published methods. Because of low back pressure--a further advantage of the reactor--the detection system could also be directly coupled to the outlet of a microdialysis device, allowing the on-line real-time measurement of extracellular brain Ch. The versatility of the enzyme reactor for the monitoring of analytes in HPLC eluates, flow injection analysis, with or without prepurification, is emphasized. The usefulness of the reactor-detector system in biomedical applications is illustrated by the measurement of increases of rat striatal extracellular Ch following cardiac arrest.

Acetylcholine↗
PubMed2005

Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans.

A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n=10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n=351, p<0.001): 351+/-13 (0 RF), 261+/-10 (1 RF), 253+/-11 (2 RF), 222+/-18 (3 RF), and 171+/-29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n=12) compared to healthy volunteers (n=12). Nitrite correlated significantly with FMD (r=0.56, p<0.001) and inversely with IMT (r= -0.49, p<0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease.

Adult↗
PubMed2004

Amino acid analysis using disposable copper nanoparticle plated electrodes.

A disposable copper nanoparticle-plated screen-printed carbon electrode (designated as Cun-SPE(100-nm)) provides a new material for the determination of native amino acids. All 20 underivatized amino acids can be sensitively determined at 0.0 V vs. Ag/AgCl in pH 8 phosphate buffer solution. The precisely controlled copper nanoparticles can boost up the CuIIO/CuI2O redox signal on the working surface without any prior pretreatment procedure. The formation of a reversible 1:1 CuIIO-amino acid complex on the Cun-SPE(100-nm) was proposed to play a key role in the reaction mechanism. Stable detection responses were obtained for all amino acids by flow injection analysis with detection limits (S/N = 3) that lie in the range of 24 nM-2.7 microM. Selected amino acids from six representative chemical natures were separated by HPLC and detected at the Cun-SPE(100-nm) with promising results.

Amino Acids↗
PubMed2005

Microfluidic pH-sensing chips integrated with pneumatic fluid-control devices.

This paper presents a microfluidic chip capable of performing precise continuous pH measurements in an automatic mode. The chip is fabricated using micro-electro-mechanical-systems (MEMS)-based techniques and incorporates polydimethylsiloxane (PDMS) microstructures, pH-sensing electrodes and pneumatic fluid-control devices. Through its enhanced microchannel design and use of pneumatic fluid-control devices, the microfluidic chip reduces the dead volume of the sample and increases the pumping rate. The maximum pumping rate of the developed micro-pump is 28 microL/min at an air pressure of 10 psi and a driving frequency of 10 Hz. The total sample volume consumed in each sensing operation is just 0.515 microL. As a result, the developed chip reduces the sample volume compared to conventional large-scale pH-sensing systems. The microfluidic chip employs the electrochemical sensing method to conduct precise pH level measurements. The sensing electrodes are fabricated by sputtering a layer of SiO(2)-LiO(2)-BaO-TiO(2)-La(2)O(3) (SLBTLO) onto platinum (Pt) electrodes and the pH value of the sample is evaluated by measuring the potential difference between the sensing electrodes and a reference electrode. Additionally, the integration of the microfluidic chip with a pneumatic fluid-control device facilitates automatic sample injection and a continuous sensing operation. The developed system provides a valuable tool with which to examine pH values in a wide range of biomedical and industrial applications.

Electrochemistry↗
PubMed2006

Determination of Pb(II) with a dithizone-modified carbon paste electrode.

A dithizone (DTZ) modified carbon paste electrode was developed for the sensitive and selective determination of Pb(II) using differential pulse anodic stripping voltammetry as well in batch as in FIA system. The analysis procedure is based on an open circuit accumulation step in a stirred sample solution. This was followed by a medium exchange to a clean solution and subsequently by a proper anodic stripping. The analytical performance was evaluated with respect to the quantity of modifier in the paste, accumulation time, background electrolyte, Pb(II) concentration and other variables. When the accumulation time applied was 5 minutes, linear calibration graphs were obtained in the concentration range 1 x 10(-7)-1 x 10(-5) M and 8 x 10(-8)-5 x 10(-6) M for batch and Flow Injection Analysis (FIA), respectively. The detection limits found were 8.65 x 10(-8) M in batch and 4.45 x 10(-8) M in FIA. A convenient and rapid renewal of electrode surface allows the use of a single modified electrode surface in multiple analytical determinations. Several coexisting metals ions such as Cd(II), Hg(II), Cu(II) and Zn(II) had no interference on the determination of Pb(II). The proposed method was applied in the determination of lead in soils located in the vicinity of metallurgic transformation industry. The results obtained were in accordance to the ones supplied by Atomic Absorption Spectroscopy (AAS).

Calibration↗
PubMed2004

Analysis of herbicide Krovar I by liquid chromatography with atmospheric pressure chemical ionization mass spectrometry.

A simple, very efficient method is presented for routine analysis of herbicide Krovar I (active components bromacil and diuron) in water and soil samples. Water samples were extracted by liquid-liquid extraction with dichloromethane (DCM) as extraction solvent. For soil samples two different extraction techniques were compared: microwave-assisted solvent extraction and a shaking technique using a platform shaker. Extracts were analyzed by high performance liquid chromatography using a water:methanol gradient. Liquid chromatography was coupled with atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) for quantification of bromacil and diuron. Optimization of the APCI-MS was done by using standards in the flow injection analysis mode (FIA). Method detection limit for liquid samples for bromacil is 0.04 microg L(-1) and for diuron 0.03 microg L(-1). Method detection limit for soil samples is 0.01 microg g(-1) dry weight for both compounds. Results of analysis of field samples of water and soil are also presented.

Atmospheric Pressure↗