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PubMed2006

Microfluidic sensor for dynamic surface tension measurement.

A novel microfluidic sensor for measuring dynamic gas-liquid interfacial tension is reported. The device consists of a microfluidic chip with a microchannel network and an optical detection system. The sample is introduced into a main channel, while air is injected through a T-junction. Owing to the fixed flow rate ratio used for the sensor, surface tension is the only parameter determining bubble formation frequency, which can be measured by optical detection. Although the bubble is represented by a pulse in the output signal, the formation frequency is simply the frequency of the output signal. Measurements were carried out for aqueous solutions with different concentrations of the ionic surfactant cetyl trimethyl ammonium bromide. Surface tensions of these solutions were calibrated with a commercial tensiometer. The measurement results show a clear relationship between surface tension and formation frequency. The sensor can be used to identify the critical micelle concentration of the surfactant. The sensor potentially allows the use of a minute amount of sample compared with the relatively large amount required for existing commercial systems.

Equipment Design↗
PubMed2006

A sequential injection analysis/chemiluminescent plant tissue-based biosensor system for the determination of diamine.

In this paper, a new chemiluminescent plant tissue-based biosensor for diamine detection was presented by employing sequential injection analysis (SIA), which facilitates precise fluidic handling and lower consumption of sample and reagents. Pea-seedling tissue acted as the molecular recognition element and was packed in a mini-PTFE column and further incorporated in the SIA system. The analysis of diamines, such as putrescine and cadaverine, is based on an enzymatic conversion which takes place in the plant tissue column to produce hydrogen peroxide. The formed hydrogen peroxide was detected by a chemiluminescence reaction involving luminol and Co(2+). Under the optimal conditions, the linear calibration graphs were obtained within 0.2-80 microM (putrescine) and 0.5-100 microM (cadaverine). The detection limits of 0.03 and 0.06 microM were achieved for putrescine and cadaverine, respectively, along with the relative standard deviations of 2.14% and 3.08% (n=11) and a sampling frequency of 40 h(-1). The present biosensor has been used for the analysis of diamine in fish samples with an acceptable accuracy.

Biological Assay↗
PubMed2002

Atmospheric pressure ionization mass spectrometry of purine and pyrimidine markers of inherited metabolic disorders.

Purines and pyrimidines are of interest owing to their significance in processes in living organisms. Mass spectrometry is a promising analytical tool utilized in their analysis. Two atmospheric pressure ionization (API) methods (electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)) in both negative and positive modes applied to selected purine and pyrimidine metabolites (markers of inherited metabolic disorders) were studied. APCI is less sensitive to alkali metal cations present in a sample and offers higher response than ESI for studied compounds. Both of the techniques afford quasi-molecular ions, but fragmentation also occurs to a certain extent. However, the application of collision-induced dissociation of quasi-molecular ions is essential to confirm a certain metabolite in a sample. Fragmentation of both positive and negative ions was evaluated using multi-stage mass spectrometric experiments. Typical neutral losses correspond to molecules NH(3), H(2)O, HCN, CO, H(2)NCN, HNCO and CO(2). The ion [NCO](-) arises in the negative mode. The cleavage of the glycosidic C-N bond is characteristic for relevant metabolites. Other neutral losses (CH(2)O, C(2)H(4)O(2) and C(3)H(6)O(3)) originate from fragmentation of the glycosidic part of the molecules. In addition to fragmentation, the formation of adducts of some ions with applied solvents (H(2)O, CH(3)OH) was observed. The composition of the solution infused into the ion source affects the appearance of the mass spectra. Tandem mass spectra allow one to distinguish compounds with the same molecular mass (uridine-pseudouridine and adenosine-2'-deoxyguanosine). Flow injection analysis APCI-MS/MS was tested on model samples of human urines corresponding to adenosine deaminase deficiency and xanthine oxidase deficiency. In both cases, the results showed potential diagnostic usefulness.

Atmospheric Pressure↗
PubMed2003

Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system.

A sequential injection analysis (SIA) is proposed for the simultaneous determination of L- and D-methotrexate (Mtx) using amperometric biosensors as detectors. A SIA system is proposed due to the highest precision and accuracy and lower consumption of sample and buffer. The amperometric biosensors used as detectors in SIA system were based on L-amino acid oxidase (L-AAOD) or/and L-glutamate oxidase (L-Glox) and horseradish peroxidase (HRP) for the assay of L-Mtx and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx were selected. The linear concentration ranges are of pmol/l to nmol/l magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D-Mtx with a frequency of 34 samples per hour.

Amino Acid Oxidoreductases↗
PubMed2004

Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3.

We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels. The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2. Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.

Bacillus anthracis↗
PubMed2003

Sequential injection analysis of nitrites and nitrates in human serum using nitrate reductase.

BACKGROUND: Nitrates and nitrites, as products of nitric oxide in human serum, are currently used as markers in many diseases. A sequential injection analysis (SIA) flow system, for their determination in serum, is presented. The exact timing of fluidic manipulations and the small volumes used in the automated SIA systems provide exquisite control of the reaction conditions and an economy in the biological fluid and enzymes used. METHODS: For nitrite determinations, 150 microl of sample and 50 microl of Griess reagent were sequentially aspirated to the system and sent to the detector. Nitrates were determined as nitrites after reduction through 0.09 U of nitrate reductase and 75 microl of NADPH. RESULTS: Nitrates and nitrites were determined at concentrations up to 4 and 3 mg/l, respectively, with relative standard deviation (rsd) <5%. Detection limits of 0.14 and 0.03 mg/l, respectively, were calculated. Statistical evaluation showed good agreement between the results obtained, for 15 deproteinized serum samples, with both the SIA system and the comparison batch procedure. CONCLUSIONS: The automatic developed method seems to be a good alternative for routine implementation since it is four times faster, and it requires one third of sample and one half of nitrate reductase than the comparison batch procedure.

Data Interpretation, Statistical↗
PubMed2000

AutoScan: an automated workstation for rapid determination of mass and tandem mass spectrometry conditions for quantitative bioanalytical mass spectrometry.

An automated flow injection analysis (FIA) mass spectrometry system (AutoScan) was developed to allow rapid unattended determination of optimal conditions during mass (ms) and tandem mass spectrometry (ms/ms) on new chemical entities (NCEs) arranged in 96-well plates. The 96-well plate is placed on the deck of a modified Gilson Multiprobe autosampler for injection into a PE Sciex API 2000 triple quadrupole mass spectrometer. A customized software interface is used to create the necessary scan experiments by associating each 96-well plate of NCEs to be scanned with an index file containing data on the identity of each analyte and its expected molecular weight. Analytes are injected four at a time into a custom injection manifold and conventional mass spectra are acquired in both polarities (+/-) using an alternating positive/negative Q1 scan function. The software determines the optimal polarity and definitive precursor ion for all analytes and uses the results to build the injection sequence for product ion scanning. The samples are automatically re-injected under MS/MS conditions, and product ion scans that loop among different collision energies are collected for each analyte. The resulting data are processed automatically and the optimal MS/MS transitions for each analyte are selected. A color-coded graphical interface facilitates data review. Any unusual ion transitions or transposition errors made during plate preparation are noted and corrected. Complete MS and MS/MS conditions are obtained for 96 compounds in about one hour and the resulting data are available for download as sample control injection sequence files.

Automation↗
PubMed2000

Data model for the elimination of matrix effects in enzyme-based flow-injection systems.

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.

Alanine Transaminase↗
PubMed1989

A chemiluminescence automatic analyser for the measurement of biological compounds.

A compact automated analyser which could analyse constituents in biological fluids with a small sample volume and in a short time has been developed. The instrument was composed of a flow injection analysis system equipped with chemiluminometric detection and an immobilized enzyme column reactor used in combination. Chemiluminescence has high sensitivity, and its reaction proceeds very quickly. Furthermore, an immobilized enzyme column reactor can produce a sufficient amount of hydrogen peroxide from compounds in serum in a short time. When enzymes are used as reagents for the analysis of substances in blood or blood serum, the final signals emitted by different enzyme reactions are usually not only hydrogen peroxide but also ammonia, NAD(P)H and so on. However, the practical chemiluminescence method for ammonia and NAD(P)H has not been established. We have discovered a new practical method for ammonia and NAD(P)H using an enzyme column reactor consisting of both immobilized L-glutamate dehydrogenase and L-glutamate oxidase. The determinations of glucose and uric acid in serum by chemiluminometry after production of hydrogen peroxide by the respective oxidases are presented. A newly chemiluminometric determination of ammonia, NAD(P)H and its applications to other enzymatic analyses that give ammonia and NAD(P)H as a final signal are also described.

Ammonia↗
PubMedDate not supplied

A new on-line micellar-enhanced photochemically-induced fluorescence method for determination of phenylurea herbicide residues in water.

A new flow injection analysis micellar-enhanced photochemically induced fluorescence (FIA-MEPIF) method was developed for the determination of four phenylurea herbicides, including isoproturon, neburon, linuron and diuron. On-line photoconversion under UV irradiation of these herbicides into strongly fluorescent photoproducts was performed in buffered aqueous solutions and in the presence of surfactants [sodium dodecyl sulphate (SDS) or cetyl trimethyl ammonium chloride (CTAC)] at micellar concentrations. The MEPIF and FIA parameters were optimized. The analytical figures of merit for the determination of the four herbicides were satisfactory, with concentration linear dynamic ranges over about one to two orders of magnitude, detection limits of 0.33-0.92 mg[sol ]L and relative standard deviations of 1.3-11%, according to the compound. Application to the analysis of fortified tap water samples yielded good recovery values (91-103%, according to the herbicide). Our results show that the on-line MEPIF-FIA method is simple, versatile, sensitive and can be easily applied to quantify pollutant residues in multiresidue systems.

Calibration↗
PubMed2004

Development of an automated water toxicity biosensor using Thiobacillus ferrooxidans for monitoring cyanides in natural water for a water filtering plant.

An on-line biosensor consisting of immobilized Thiobacillus ferrooxidans and an oxygen electrode was developed for automated monitoring of acute toxicity in water samples. T. ferrooxidans is an obligatory acidophilic, autotrophic bacterium and derives its energy by the oxidation of ferrous ion, elemental sulfur, and reduced sulfur compounds including metal sulfides. The assay is based on the monitoring of a current increase by addition of toxicoids, which is caused by the inhibition of bacterial respiration and decrease in oxygen consumption. Optimum cell number on the membrane was 5.0 x 10(8) cells. The steady-state current was obtained when concentration of FeSO4 was above 3.6 mM at pH 3. The sensor response of T. ferrooxidans immobilized membrane for 5.0 microM KCN was within an error of 10% for 30 membranes. A linear relationship was obtained at KCN concentration in the range of 0.5-3.0 microM in a flow-type monitoring system. Minimum detectable concentrations of KCN, Na2S, and NaN3 were 0.5, 1.2, and 0.07 microM, respectively. The monitoring system contained two biosensors and these sensors were cleaned with sulfuric acid (pH 1.5) twice a day. This treatment could remove fouling on microbial immobilized membrane by natural water and ferrous precipitation in the flow cell. This flow-type monitoring sensor was operated continuously for 5 months. Also, T. ferrooxidans immobilized membrane can be stored for one month at 4 degrees C when preserved with wet absorbent cotton under argon gas.

Biosensing Techniques↗
PubMed2007

Flow modeling in a novel non-perfusion conical bioreactor.

We have developed a bioreactor vessel design which has the advantages of simplicity and ease of assembly and disassembly, and with the appropriately determined flow rate, even allows for a scaffold to be suspended freely regardless of its weight. This article reports our experimental and numerical investigations to evaluate the performance of a newly developed non-perfusion conical bioreactor by visualizing the flow through scaffolds with 45 degrees and 90 degrees fiber lay down patterns. The experiments were conducted at the Reynolds numbers (Re) 121, 170, and 218 based on the local velocity and width of scaffolds. The flow fields were captured using short-time exposures of 60 microm particles suspended in the bioreactor and illuminated using a thin laser sheet. The effects of scaffold fiber lay down pattern and Reynolds number were obtained and correspondingly compared to results obtained from a computational fluid dynamics (CFD) software package. The objectives of this article are twofold: to investigate the hypothesis that there may be an insufficient exchange of medium within the interior of the scaffold when using our non-perfusion bioreactor, and second, to compare the flows within and around scaffolds of 45 degrees and 90 degrees fiber lay down patterns. Scaffold porosity was also found to influence flow patterns. It was therefore shown that fluidic transport could be achieved within scaffolds with our bioreactor design, being a non-perfusion vessel. Fluid velocities were generally same of the same or one order lower in magnitude as compared to the inlet flow velocity. Additionally, the 90 degrees fiber lay down pattern scaffold was found to allow for slightly higher fluid velocities within, as compared to the 45 degrees fiber lay down pattern scaffold. This was due to the architecture and pore arrangement of the 90 degrees fiber lay down pattern scaffold, which allows for fluid to flow directly through (channel-like flow).

Bioreactors↗
PubMed1990

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum.

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.

Amino Acid Oxidoreductases↗
PubMed2007

Carbon nanotube/poly(methyl methacrylate) (CNT/PMMA) composite electrode fabricated by in situ polymerization for microchip capillary electrophoresis.

We describe the development and application of a novel carbon nanotube/poly(methyl methacrylate) (CNT/PMMA) composite electrode as a sensitive amperometric detector of microchip capillary electrophoresis (CE). The composite electrode was fabricated by the in situ polymerization of a mixture of CNTs and prepolymerized methyl methacrylate in the microchannel of a piece of fused silica capillary under heat. The performance of this unique system was demonstrated by the separation and detection of phenolic pollutants and purines. The new CNT-based CE detector offered significantly lower operating potentials, yielded substantially enhanced signal-to-noise characteristics, and exhibited resistance to surface fouling and, hence, enhanced stability. Long-term stability and reproducibility with relative standard deviations of less than 5 % for the peak current (n=20) were also demonstrated. The simplicity and significant performance exhibited by the CNT/PMMA composite electrode indicate great promise for conventional CE, flowing-injection analysis, and other microfluidic analysis systems.

Electrochemistry↗
PubMed2005

Head-column field-amplified sample stacking in a capillary electrophoresis-flow injection system.

A simple, effective, and continuous online concentration method for the sensitive detection of alkaloids applying CE-flow injection analysis with head-column field-amplified sample stacking was developed. A series of samples was continuously introduced into the capillary by electrokinetic means without interrupting the high voltage. A short water plug was introduced by the EOF at the capillary inlet end prior to sample introduction. Under optimum conditions, 15-fold improvement in concentration sensitivity was achieved, giving an LOD of about 0.67 and 0.73 microg/mL for ephedrine (E) and pseudoephedrine (PE), respectively. The separation could be achieved within 4 min and sample throughput rate could reach up to 7/h. The repeatability (defined as RSD) was 3.62, 1.51% with peak area evaluation and 1.30, 2.58% with peak height evaluation for E and PE, respectively. This method has been successfully applied to the analysis of commercial pharmaceutical preparations containing E and PE, and the recoveries were 92.3-102.4%.

Borates↗
PubMed2005

Fabrication and performance of a three-dimensionally adjustable device for the amperometric detection of microchip capillary electrophoresis.

A microchip CE-amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an AD cell containing a one-dimensionally adjustable disk detection electrode in a Plexiglas holder. It facilitates the precise 3-D alignment between the channel outlet and the detection electrode without a complicated 3-D manipulator. The performance of this unique system was demonstrated by separating five aromatic amines (1,4-phenyldiamine, aniline, 2-methylaniline, 4-chloroaniline, and 1-naphthylamine) of environmental concern. Factors influencing their separation and detection processes were examined and optimized. The five analytes have been well separated within 140 s in a 74 cm long separation channel at a separation voltage of +2500 V using a 10 mM phosphate buffer (pH 3.5). Highly linear response is obtained for the five analytes over the range 20-200 microM with the detection limits ranging from 0.46 to 1.44 microM, respectively. The present system demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9). The new approach for the microchannel-electrode alignment should find a wide range of applications in CE, flowing injection analysis, and other microfluidic analysis systems.

1-Naphthylamine↗
PubMed2006

Carbon nanotube/poly(methyl methacrylate) composite electrode for capillary electrophoretic measurement of honokiol and magnolol in Cortex Magnoliae Officinalis.

This paper describes the development and the application of a novel carbon nanotube/poly(methyl methacrylate) (CNT/PMMA) composite electrode as a sensitive amperometric detector of CE. The composite electrode was fabricated on the basis of the in situ polymerization of a mixture of CNT and prepolymerized methylmethacrylate in the microchannel of a piece of fused-silica capillary under heat. The performance of this unique system has been demonstrated by separating and detecting honokiol and magnolol in traditional Chinese medicine, Cortex Magnoliae Officinalis. Factors influencing their separation and detection processes were examined and optimized. Honokiol and magnolol were well separated within 7 min in a 40 cm long capillary at a separation voltage of 15 kV using a 50 mM borate buffer (pH 9.2). The new CNT-based CE detector offered significantly lower operating potentials, yielded substantially enhanced S/N characteristics, and exhibited resistance to surface fouling and hence enhanced stability. It demonstrated long-term stability and reproducibility with RSDs of less than 5% for the peak current (n = 9) and should also find a wide range of applications in microchip CE, flowing injection analysis, and other microfluidic analysis systems.

Biphenyl Compounds↗
PubMed2003

Exact mass measurement on an electrospray ionization time-of-flight mass spectrometer: error distribution and selective averaging.

An automated, accurate and reliable way of acquiring and processing flow injection data for exact mass measurement using a bench-top electrospray ionization time-of-flight (ESI-TOF) mass spectrometer is described. Using Visual Basic programs, individual scans were selected objectively with restrictions on ion counts per second for both the compound of interest and the mass reference peaks. The selected "good scans" were then subjected to two different data-processing schemes ("combine-then-center" and "center-then-average"), and the results were compared at various ion count limit settings. It was found that, in general, the average of mass values from individual scans is more accurate than the centroid mass value of the combined (same) scans. In order to acquire a large number of good scans in one injection (to increase the sampling size for statistically valid averaging), an on-line dilution chamber was added to slow down the typically rapid mass chromatographic peak decay in flow-injection analysis. This simple addition worked well in automation without the need for manual sample dilution. In addition, by dissolving the reference compound directly into the mobile phase, manual syringe filling can be eliminated. Twenty-seven samples were analyzed with the new acquisition and process routines in positive electrospray ionization mode. For the best method found, the percentage of samples with RMS error less than 5 ppm was 100% with repetitive injection data (6 injections per sample), and 95% with single injection data. Afterwards, 31 other test samples were run (with MW ranging from 310 to 3493 Da, 21 samples in ESI+ and 10 in ESI- mode) and processed with similar parameters and 100% of them were mass-calculated to RMS error less than 5 ppm also.

Automation↗