University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Cell

Classification: Cell

Citations 31

"Measurement Of Cellular Stimulation Through Monitoring PH Changes By Bead Injection Fluorescence Microscopy"
Talanta 2000 Volume 51, Issue 3 Pages 497-506
Ilkka Lähdesmäki, Craig Beeson, Gary D. Christian and Jaromir Ruzicka

Abstract: This work presents a method for extracellular and intracellular pH measurements in live cells based on a combination of the bead injection (BI) technique and fluorescence microscopy. For extracellular pH measurement, cells are grown on fluorescent beads, packed into a small column by a sequential injection instrument, and fluorescence intensity from the beads stained by the indicator is recorded by a fluorescence microscope. The method is applied to quantifying carbachol stimulation of Chinese hamster ovary (CHO) cells transfected with the mi muscarinic receptor and is verified by a glucose depletion experiment. The results yield an EC50 value of 1 µM for carbachol, which is in reasonable agreement with the literature value 3 µM determined by an existing potentiometric technique for measuring acid release. The intracellular measurement utilizes CHO M1 cells growing on non-fluorescent beads. For this method the cells rather than the beads are stained by incubating them in a Solution of the fluorescent pH indicator BCECF. The cells are also stimulated with carbachol and the intracellular pH dependent fluorescence from the cells is recorded. The results show dependence between intracellular pH changes and carbachol concentration and yield an EC50 value of 4 µM.
pH Microscopy Immobilized cell

"Flow Injection Microscopy For The Study Of Intracellular Calcium Mobilization By Muscarinic Agonists"
Anal. Biochem. 1999 Volume 268, Issue 2 Pages 377-382
Wendy Lee Connors and Jaromir Ruzicka

Abstract: The study of cellular response to chemical agonists is essential in understanding the complex functions mediated by cell surface receptors. Flow injection microscopy has been used with the CHO-M1-WT3 cell line and the fluorescent Ca2+ indicator Fura-2-AM to monitor mobilization of internal Ca2+. Repeated stimulation of cells mounted in an inverted radial flow chamber allows the direct comparison of relative intracellular Ca2+ mobilization with respect to agonist dose. The process of determining dose-response relationships is simplified since an entire dose-response curve can be constructed from a distinct set of cells. Use of flow injection lends precision to the application and removal of agonists while allowing cellular activity to be monitored throughout the stimulation and recovery processes. In this work, dose-response curves have been constructed for the muscarinic agonists carbachol, acetylcholine, and pilocarpine resulting in EC50 values of 1.7 µM, 56 nM, and 6.8 µM, respectively. (c) 1999 Academic Press.
Carbachol Acetylcholine Pilocarpine hydrochloride Microscopy

"Application Of Bioluminescence And Chemiluminescence In Biomedical Sciences"
Methods Enzymol. 2000 Volume 305, Issue 1 Pages 333-345
Larry J. Kricka

Abstract: A review discussing applications in lab. medicine, biomedical research, immunoassay and blotting, reporter-gene-based assays, cellular chemiluminescence and luminol- and lucigenin-enhanced chemiluminescence, and therapeutics. 135 references.
Cells Chemiluminescence Review

"A Flow Injection Renewable Surface Technique For Cell-based Drug Discovery Functional Assays"
Anal. Chem. 1999 Volume 71, Issue 6 Pages 1160-1166
Peter S. Hodder and Jaromir Ruzicka

Abstract: A novel now injection-renewable surface (FI-RS) technique is introduced for the execution of automated pharmacology-based assays on living cells. Cells are attached to microcarrier beads, which serve as the disposable and renewable surface with which the assay-is performed. The feasibility of this FI-RS technique is demonstrated by performing a functional assay using Chinese hamster ovary cells transfected with the rat muscarinic receptor (M1). The intracellular calcium elevation resulting from the agonist-receptor interaction is measured via a calcium-sensitive fluorescent probe (fura-2) and a fluorescence microscope photometry system. The FI apparatus allows reproducible and precise control of the concentration gradient of chosen muscarinic receptor agonists (carbachol, acetylcholine, pilocarpine) delivered to cells attached to microcarrier beads. The RS methodology eliminates problems associated with diminishing biological response vis-a-vis traditional functional assays that are performed repetitively on the same group of cells. Using this technique, reproducible responses are measured and pharmacologic parameters quantified that compare favorably to literature values. In addition, the use of the FI-RS functional assay as an analytical method for discrimination of agonists based on kinetic parameters is proposed.
Carbachol Pilocarpine hydrochloride Microscopy Bead suspension

"Manipulating And Monitoring Biomolecular Interactions With Conducting Electroactive Polymers"
Adv. Mater. 2002 Volume 14, Issue 13-14 Pages 953-961
G.G. Wallace, L.A.P. Kane-Maguire

Abstract: Biointeractions with for instance amino acids and DNA can be monitored and regulated using conductive electroactive polymers. This article describes several of these biointeractions and the effect of doping on them. The progress in practical applications and simple device fabrication is also mentioned. The Figure shows an optical micrograph of living (healthy) red blood cells incorporated into a conducting polymer during polymerization.
Amino Acids DNA Electrode

"Automated System For Multichannel Flow Injection Analysis"
Anal. Chim. Acta 1994 Volume 292, Issue 3 Pages 281-295
U. Spohn*, J. van der Pol, R. Eberhardt, B. Joksch and Ch. Wandrey

Abstract: The design of a flexible, multichannel flow injection analyzer. for the automated sequential determination of up to six analytes is described. A computer program, FIACCO, was developed for the control of the hardware, signal evaluation and recording. The hardware included four piston pumps, a three-channel peristaltic pump, multiway valves, two injection valves and a fluorescence detector. The program was designed to operate six enzyme reactors in parallel, with independent control of pH, flow rates and residence time in the reactors, with use of single or double step stopped-flow. The system can be used for the implementation of a wide variety of FIA procedures. All switching times and time courses can be freely programmed and independently controlled, and sample pre-conditioning, separation steps and residence times are controlled precisely. Residence times and reaction conditions can be controlled in wide ranges and changed during the assay procedures. The system was applied to the analysis of an animal cell culture for ammonia (after gas dialysis separation), glucose, glutamine, glutamate and lactate.
Ammonia Glucose Glutamine Glutamate Lactate Dialysis Multichannel Stopped-flow

"Lactate, Glutamate And Glutamine Biosensors Based On Rhodinized Carbon Electrodes"
Anal. Chim. Acta 1994 Volume 295, Issue 3 Pages 243-251
S. F. Whitea, A. P. F. Turnera,*, U. Bilitewskib, R. D. Schmidb and J. Bradleyb

Abstract: Amperometric enzyme sensors for lactate, glutamate and glutamine were prepared using rhodinized carbon electrodes (6 mm diameter). The lactate sensors were fabricated using lactate oxidase immobilized in hydroxyethylcellulose, overlaid with a cellulose acetate membrane. The glutamate and glutamine sensors were fabricated by immobilizing glutamate oxidase and glutamate oxidase/glutaminase, respectively, with glutaraldehyde. The sensors were tested at a potential of +400 mV vs. Ag/AgCl using a FIA system with 0.1 M sodium phosphate/0.1 M KCl buffer (0.2 ml/min, pH 7 or 5.5 for the glutamine sensor). The sensors exhibited a linear response from 0.1-1.5 mM and a RSD (n = 3) of 1.5% within the linear range. The effects of long-term storage at 4°C on the sensors were measured. The responses of the lactate, glutamate and glutamine sensors decreased to 44, 29 and 29% of their initial values after 25, 20 and 12 days of storage, respectively. The feasibility of using these sensors for mammalian cell culture monitoring is discussed.
Glutamate Glutamine Lactate Sensor Electrode Electrode

"Development Of Enzyme-cartridge Flow Injection Analysis For Industrial Process Monitoring. 3. Application For Monitoring Of Recombinant Animal Cell Cultivations"
Anal. Chim. Acta 1995 Volume 315, Issue 1-2 Pages 153-157
H. Jürgensb, R. Akhnoukha, G. Kretzmera and K. Schügerla,*

Abstract: The application of L-amino acid oxidase (L-ASOD) for monitoring of amino acids during the cultivation of the recombinant insect cell line, Spodoptera frugiperda, and the production of β-galactosidase as well as the application of L-lactate monooxidase (L-LMO) for the monitoring of the lactate concentration during the cultivation of the adherent recombinant cell line, Baby Hamster Kidney (BHK) and production of Antithrombin III (AT III) are presented.
Amino acids, L l-Lactate Immobilized enzyme Process monitoring

"Characterization Of Planar Concentration Gradients In A Sequential Injection System For Cell-perfusion Studies"
Analyst 1993 Volume 118, Issue 10 Pages 1235-1240
Cy H. Pollema and Jaromir Ruzicka

Abstract: This paper describes the characterization of a perfusion chamber that is coupled with a sequential injection system and is being designed for live-cell perfusion. The apparatus consists of a multi-port valve, a peristaltic pump, a perfusion chamber and an epifluorescence microscope. The entire system is computer controlled and temperature regulated. The parameters discussed are the concentration-time profiles with regard to the volume of reagent used and the position of the cell in the perfusion chamber. Other parameters discussed include the stopped-flow compliance, reproducibility and symmetry of the concentration gradients formed. The system is shown to be suitable for two modes of perfusion; the first in which all cells are exposed to the same concentration of reagent, and the second in which cells are exposed to a gradient of concentrations. All characterization is performed with use of bulk fluorescein as a tracer, and a correlation is made between the bulk flow and the response within the cellular environment by using 5-[N-(octadecanoyl)amino]fluorescein. The cited system consisted of a bidirectional peristaltic pump, a holding coil, a multiposition valve and a transfer line connecting the valve to the fountain cell perfusion chamber (diagram given). Detection was performed using an epifluorescence microscope and the entire system was computer controlled and temperature regulated. The system provided cells with a perfusing buffer at controlled temperature and flow rate and permitted the exposure of cells to impulses of reagents of defined concentration for well-defined periods. The perfusion system was characterized by exploring the bulk flow behavior and its relationship to the stimulation of cells adhered within the perfusion chamber, using bulk fluorescein as tracer. A correlation was made between the bulk flow and the response within the cellular environment using 5-[N-(octadecanoyl)amino]fluoroscein. The concentration-time profiles were studied with respect to the volume of reagent used and the position of the cell in the perfusion chamber. The stopped-flow compliance, the reproducibility and the symmetry of the concentration gradients formed were also investigated. Results are discussed.
Microscopy Gradient technique Sequential injection Perfusion Stopped-flow Fountain cell Computer

"Pulse Voltammetry In Single Cells Using Platinum Microelectrodes"
Anal. Chem. 1992 Volume 64, Issue 11 Pages 1264-1268
Ta Kung Chen, Yau Yi Lau, Danny K. Y. Wong, and Andrew G. Ewing

Abstract: A multiple-pulse voltammetric technique is described wherein a large anodic cleaning potential and a large cathodic activation potential are applied before each detection potential pulse. The detection pulse is stepped linearly through a selected potential range in order to obtain a voltammogram. Application of the technique is exemplified by voltammetric studies on K4Fe(CN)6, glucose, dopamine, dihydroxyphenylacetic acid, catechol and ascorbic acid with use of either a 10- or a 75-m diameter Pt-disc working electrode. The technique has also been used for in vivo measurements of dopamine concentration. in single-cell cytoplasm of snail by implantation of a 2-m diameter Pt-ring electrode. By use of this technique electrode response was degraded by only ~30% after continuous voltammetry for 40 min inside a single nerve cell.
Sulfur compounds Voltammetry Electrode Amperometry Electrode

"Characterization Of Glucose Microsensors For Intracellular Measurements"
Anal. Chem. 1992 Volume 64, Issue 18 Pages 2160-2163
Takayuki Abe, Yau Yi Lau, and Andrew G. Ewing

Abstract: Ultra-small glucose sensors were constructed by the immobilization of glucose oxidase on 2 µm diameter platinized carbon ring electrodes. The electrodes were oxidized in phosphate buffer at 1.1 V vs. NaCl - SCE for 15 min, followed by sequential immersion in glucose oxidase solution in phosphate buffer for 3 min, buffer solution for 3 h, bovine serum albumin solution and glutaraldehyde solution In tests using a flow injection system, the sensors had response times as low as 270 ms and there was a rectilinear relationship between electrode tip diameter and response time. Glucose concentration. in the sub-mM range was measured at atmospheric O levels, but at low O concentration. (0.2 mM) there was interference by O. Easily oxidized neurotransmitters such as dopamine interfered, and the sensor response was not affected by pH in the range 6.0 to 8.0. The sensor was used for the detection of glucose in single-cell cytoplasm of the large dopamine cell of the pond snail. The current response increased when 2 pl of 3 M glucose was injected.
Glucose Electrode Electrode Sensor Interferences

"Specific Flow Injection Sandwich Binding Assay For IgG Using Protein A And A Fusion Protein"
Anal. Chem. 1993 Volume 65, Issue 23 Pages 3368-3371
Wiebke Brandes, Hans Eckhard Maschke, and Thomas Scheper

Abstract: The assay was designed for monitoring the production of IgG by hybridoma cells that produce only one IgG. The solution of IgG in 0.1 M potassium phosphate buffer of pH 7 was injected into a stream (0.6 or 1.2 ml/min) of 0.1 M potassium phosphate buffer of pH 7/1 mM MgCl2/1 mM dithioerythritol which flowed through a column (7 mm i.d.) containing protein A immobilized on CNBr-activated Sepharose. An Escherichia coli cell lysate containing protein A/β- galactosidase fusion protein (prep. described) was injected via the same valve and the system (with the protein A column bypassed) was washed with 0.1% SDS/10 mM NaOH before the eluate from the protein A column passed to a column containing glucose oxidase and catalase immobilized on Sepharose and injection of lactose solution in the assay buffer. Glucose formed by the action of β-galactosidase was oxidized on the bi-enzyme column and the total heat evolved during the enzymatic reactions was proportional to the amount of IgG bound to the protein A column. The method was applied to rabbit IgG and to mouse IgG2a and IgG2b. The sensitivity decreased in this order and could be improved by decreasing the flow rate from 1.2 to 0.6 ml/min; the range for mouse IgG2a was 0.2-2 mg/ml. A sandwich-type flow injection binding assay for quantitation of various IgG's was developed. The assay is based on the pseudoimmunological reaction between protein A from Staphylococcus aureus and immunoglobulin G from different species. Protein A immobilized on a solid support and a fusion protein of protein A and β-galactosidase from Escherichia coli are used for detection. The fusion protein is produced with a temperature-inducible recombinant E. coli strain. A sandwich is formed by subsequent injection of IgG and fusion protein into the buffer stream flowing through the immobilized protein A column. The amount of enzyme activity bound is proportional to the amount of IgG bound and is measured by pumping a lactose solution as substrate for β-galactosidase through the protein A column. Lactose is converted to glucose and galactose. The detector is an enzyme thermistor that measures the heat evolved in the enzymatic conversion of glucose by coimmobilized glucose oxidase and catalase. The assay takes 16 min at a flow rate of 0.6 mL min-1 with a lower detection limit of 33 pmol per injection of rabbit IgG. The precision of replicate measurements has a standard deviation of 4-5%, and the column can be used for more than 50 cycles.
Immunoglobulin G Thermistor Immobilized protein Sepharose beads

"Automated Analysis Of 2,3-diamino-2,3-dideoxy-D-glucuronic Acid By Cation-exchange Chromatography With Fluorimetric Post-column Derivatization"
Anal. Biochem. 1989 Volume 176, Issue 1 Pages 63-65
Mary F. Verostek*, Lee E. Bartholomew

Abstract: 2,3-Diamino-2,3-dideoxy-D-glucuronic acid (I) was separated from glucosamine (II), galactosamine (III) and amino-acids on a column (18 cm x 9 mm) of Dionex DC-6A at 50°C, with 0.35 M Na citrate buffer (pH 5.28) containing 0.2 M H3BO3 as mobile phase (7.0 mL h-1). The amino-sugars in the eluate were derivatized by addition of phthalaldehyde and fluorescence was measured at >400 nm (excitation at 370 nm). Down to 50 to 100 pmol each of I, II and III could be detected. The calibration graph was rectilinear from 5 to 40 nmol for I. The method was used to determine I in acid hydrolysates of bacterial cell walls.
Acids HPLC Fluorescence Post-column derivatization

"Simultaneous Determination Of Nitrate And Nitrite In Biological Samples By Multichannel Flow Injection Analysis"
Anal. Biochem. 1995 Volume 231, Issue 2 Pages 383-386
Phillip F. Pratt, Kasem Nithipatikom and William B. Campbell

Abstract: In a multichannel flow injection analyzer. (a modified Automated QuikChem Ion Analyzer, Lachat Instruments Inc., Milwaukee, WI, USA; modifications described), the sample was divided into two channels, one representing total nitrite (obtained by Cd reduction of nitrate to nitrite) and the second representing only nitrite. The absorbance of the product of the reaction of nitrite with Greiss reagent was measured at 540 nm. The detection limit was 25 nM-nitrite or -nitrate. The calibration graph was linear from 25 nM to 20 µM nitrite. Recovery from bovine coronary endothelial cells was 95% for both ions. The method was applied to the determination of the ions in serum and bovine coronary artery endothelial cells and rat cerebellar granule cells. An automated method for the simultaneous determination of nitrite and nitrate in biological samples by using a multichannel flow injection analyzer has been developed. The method was based on the reaction of nitrite with Greiss reagent. The sample solution was injected and equally divided into two channels; channel one (1) represented total nitrite obtained by cadmium reduction of nitrate to nitrite while channel two (2) represented only nitrite. The absorbance of the color product was measured by photometric detectors with 540-nm filters. This method combines high reproducibility of sample introduction via flow injection and sensitivity of spectrophotometric detection. The detection limit is 25 nM for both nitrite and nitrate. The chemistry manifolds are constructed of Teflon tubing which, along with a low- pressure Flowfit connector system, provides for low maintenance, ease of use, and high sample throughput. We demonstrated that the system can be used for the determination of both nitrate and nitrite in a variety of biological samples as well as a comparison of the results from this system and the HPLC system.
Nitrate Nitrite Spectrophotometry Method comparison Lachat Multichannel

"Determination Of Glutamine In Mammalian Cell Cultures With A Flow Injection Analysis - Wall-jet Electrode System"
Anal. Lett. 1995 Volume 28, Issue 4 Pages 593-603
Huang, Y.L.;Khoo, S.B.;Yap, M.G.S.

Abstract: The cited system (diagram given) incorporated a reactor (2 cm x 5 mm i.d.) containing glutamate dehydrogenase (GDH) immobilized on aminopropyl-derivatized controlled-pore glass beads (AMP-CPG; 120-200 mesh) which was situated upstream of a second reactor (3 cm x 1 mm i.d.) packed with glutamate oxidase and glutaminase co-immobilized on AMP-CPG (immobilization methods given). Cell culture supernatants were pumped at 0.42 ml/min into a carrier stream (1 ml/min) of 0.1 M potassium phosphate buffer of pH 5.5. The glutamine was converted to H2O2 in the second reactor; the GDH reactor eliminated interference from up to 0.08 g/l glutamate. H2O2 was determined at the wall-jet electrode (Gunasingham and Tan, Anal. Chim. Acta, 1990, 234, 321) at +0.65 V vs. Ag/AgCl (using 3 mm Pt disc working and 5 mm vitreous C disc counter electrodes). The calibration graph was linear from 0.01-0.2 g/l glutamine and the RSD was 5.1%. No detection limit is given. Recoveries were 98%. After 70 determinations 70% of the initial reactor activity remained. The results generally agreed well with those obtained by HPLC.
Glutamine Amperometry Electrode Electrode Immobilized enzyme Controlled pore glass Glass beads Interferences Method comparison

"Amperometry And Cyclic Voltammetry Of Tyrosine And Tryptophan-containing Oligopeptides At Carbon-fibre Microelectrodes Applied To Single-cell Analysis"
Electroanalysis 1997 Volume 9, Issue 3 Pages 203-208
Charina D. Paras, Robert T. Kennedy*

Abstract: The electrode was prepared by inserting a 9 µm carbon fiber inside a glass capillary which was then pulled to a fine tip. The carbon fiber was kept in place by immersing the capillary tip in epoxy. After polishing the electrode surface, FIA measurements were made at a potential of 0.8 V vs. SCE using 60 mM phosphate buffer of pH 7.4 as the carrier stream (1.5 ml/min). Melanocytes were isolated from the pituitary neurointermediate lobe and the peptide hormones were extracted, incubated (details given) and determined. The stability of the electrode was improved by electrochemical pretreatment involving potential scanning from -1.0 to +1.0 V at 300 V/s for 2 min. Selectivity of the peptides was achieved by cyclic voltammetry over the range 0 to 1.2 V using a scan rate of 800 V/s. The experimental parameters were optimized for the detection of the melanocyte-stimulating hormone secreted from single melanocytes during exocytosis.
Peptides, oligo Amperometry Voltammetry Electrode Buffer Selectivity Optimization

"Ruthenium Catalyst For Amperometric Determination Of Insulin At Physiological PH"
J. Electroanal. Chem. 1997 Volume 425, Issue 1-2 Pages 191-199
Waldemar Gorskia, Craig A. Aspinwalla, Jonathan R. T. Lakeyb and Robert T. Kennedya,*

Abstract: The potential of a carbon-fiber microelectrode in 0.20 mM RuCl3/10 mM HClO4 was cycled between -0.85 and +0.65 V at 100 V/s for 15 min. When the resulting microelectrode was maintained at +0.65 V vs. Na-saturated SCE in a flow injection system in a carrier stream of 0.15 M NaCl/10 mM phosphate buffer of pH 7.40, the calibration graph was linear for 0.1-1 µM insulin (sample volume ~1 mL) and the detection limit was 23 nM. The 90% response time was M-insulin was stable for 3 days. The microelectrode was used to detect the exocytosis of insulin from single pancreatic β-cells. A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and -0.85 V vs. SSCE at 100 V s-1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH 7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 µM (five points) had slopes of 72±2 pA µM-1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t(90%)) was 40 ms or less. The electrode response to 0.2 µM insulin was stable for three days, The modified electrode was used for amperometric detection of exocytosis from individual pancreatic β-cells. 37 References
Insulin Amperometry Electrode Electrode

"Online Monitoring Of Mammalian-cell And Yeast Fermentations With A Commercial Biochemical Analyzer"
Biotechnol. Techniq. 1997 Volume 11, Issue 6 Pages 427-430
N. Vriezen and J.P. van Dijken

Abstract: A commercial analyzer was tested for online monitoring of fermentations. A new sample block was constructed to effectively degas the fermentation broth. The robust analyzer accurately measured glucose up to 110 mmol/l and lactate up to 21 mmol/l at a frequency of 1 measurement per 2 minutes directly in suspensions of mammalian and yeast cells. 8 References
Glucose Lactate Process monitoring Suspension

"Flow Injection Analysis Quantifies Multidrug Resistance Phenotype"
Cancer Lett. 1995 Volume 97, Issue 1 Pages 93-98
P. V. Venkatesan and B. Nagarjan*

Abstract: Flow injection analysis (FIA of DNA damage, repair and drug accumulation was employed to examine the relation between DNA damage, drug resistance and cell survival. Resistant sublines to adriamycin (P388/ADR), mitoxantrone (P388/MTN) and drug sensitive (P388/S) leukemic cells were exposed to different concentrations of adriamycin. The subtle difference in tumor response between sensitive and resistant cells was well differentiated and the results were comparable with other methods of measuring DNA strand-break and repair. Drug concentrations as low as 5 x 10^-11 M could be measured by FIA. In addition the method also enables assessment of cross- resistance/sensitivity. The speed, sensitivity and reproducibility make FIA a good technique for routine monitoring of tumor cell response to DNA damaging agents.
Adriamycin Process monitoring

"Carrier-based Ion-selective Electrodes And Bulk Optodes. 2. Ionophores For Potentiometric And Optical Sensors"
Chem. Rev. (ACS) 1998 Volume 98, Issue 4 Pages 1593-1687
Philippe Bühlmann, Ernö Pretsch, and Eric Bakker

Abstract: A review, with 1051 refs., is given on individual carrier-based ion-selective electrodes and bulk optodes, and is ordered according to the analyte for which they were developed.
Magnesium Electrode Field effect transistor Potentiometry Optrode Apparatus Detector

"Flow-cytometric Assay For Intracellular Non-protein Thiols Using Mercury Orange"
Cytometry 1988 Volume 9, Issue 6 Pages 529-532
Jose Enrique O'Connor, Dr. Bruce F. Kimler*, Michael C. Morgan, Kathryn J. Tempas

Abstract: The level of nonprotein thiols was assayed in individual mammalian cells using flow cytometry. Previous determinations of glutathione (GSH, the most abundant nonprotein thiol in most cells) by flow cytometry were based on UV laser excitation of fluorochromes. Because of several shortcoming of UV excitation, an assay for GSH using visible light is of interest. Selective staining of nonprotein thiols with mercury orange (a mercurial compound that binds stoichiometrically to sulfhydryl groups) was obtained by restricting the staining time. By using various drugs that affect GSH levels and overall thiol levels in cells, it was shown that GSH is the primary thiol group being stained. Thus a quick, specific technique using mercury orange has been developed for the flow cytometric determination of nonprotein thiols and preferentially for GSH in individual mammalian cells. Cells were suspended in ice-cold phosphate-buffered saline (PBS), and the suspension was centrifuged at 1500 rpm for 5 min. The cell pellet was suspended in 0.1 mM mercury orange in acetone, and the suspension was incubated at 0°C for 5 min and then centrifuged at 1500 rpm for 5 min. The pellet was suspended in ice-cold PBS, and the suspension was analyzed for glutathione on a Coulter EPICS V flow cytometer. The laser was tuned at 488 nm and operated at 50 mW, and glutathione was determined from the fluorescence at >570 nm, which was well correlated with the cell size as measured by forward-angle light scattering.
Glutathione Fluorescence

"Flow Injection Fluorescence Microscopy Applied To A Rapid Cell Surface Immunoassay"
Cytometry 1995 Volume 19, Issue 1 Pages 70-76
Cy H. Pollema, Åke Lernmark, Jaromir Ruzicka

Abstract: A perfusion system for fluorescence microscopy that utilized a flow injection system was developed and used to study cell surface antibody binding on viable cells grown in monolayer cultures on coverslips. A polyclonal cell-specific antiserum used to probe the cell surface was monitored by indirect immunofluorescence. The flow injection system was completely automatic and allowed controlled perfusion of cell surfaces with the desired sequence of antibodies. The individual steps of the indirect assay were studied to determine the binding behavior of the primary cell surface antibody, the labeled second antibody, and control of nonspecific binding. Under stopped-flow conditions, the second antibody was maximally bound within 10 min, while constant mixing of the second antibody solution over the cells resulted in maximal binding within as little as 6 min. A primary antibody contact time of 2 min followed by a wash and then exposure to the second antibody for 2 min showed that this fast automated procedure could distinguish specific from nonspecific binding of cell surface antibodies to the same set of cells for several repeated exposures. The flow injection fluorescence microscopy system can be automated to allow screening for cell surface antibodies and to study their interaction with specific cell surface antigens.
Antibodies Microscopy Immunoassay Perfusion Stopped-flow

"Coaxial Flow Mixer For Real-time Monitoring Of Cellular Responses In Flow Injection Cytometry"
Cytometry 1996 Volume 25, Issue 2 Pages 200-204
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Abstract: Improved time resolution of kinetic cellular events in now cytometry is demonstrated by using a coaxial flow-mixing device integrated within a flow injection (FI) system. The instrument is used in combination with a Becton Dickinson FACS Analyzer for on-fine reagent addition, rapid sample mixing, and temperature control of cell suspensions. The coaxial flow device can instantaneously (<60 ms) mix reagent and sample streams, allowing cytometric analysis of subsecond events to be performed. Kinetic measurements can be performed on the FACS analyzer in a variable time range of from 100 ms to 3 min. The system also allows the collection of unlimited cellular events at a specific incubation time point. Because the system operates continuously and no boost in core flow is required, disturbances of flow conditions are avoided. The capabilities of the flow injection cytometer have been demonstrated by the determination of internal (Ca2+)-immobilization in Jurkat T lymphocytes perfused internally with INDO-1 and stimulated by ionomycin.
Calcium(2+) Flow cytometry Coaxial mixer Kinetic

"Flow Injection Fluorescence Microscopy: A Novel Tool For The Study Of Cells Through Controlled Perfusion"
Experimen. Cell Res. 1993 Volume 205, Issue 2 Pages 197-204
Kurt M. Scudder, Gary D. Christian and Jaromir Ruzicka

Abstract: Current methods of microscope stage perfusion do not take full advantage of existing technology for precise fluid control. The concept of flow injection, used extensively by analytical chemists, is described and its application to the fluorescence microscopic study of cultured cells is proposed. Using this technique, cells may be exposed to single or multiple reagent zones of almost any profile, sequence, and duration, with computer-controlled precision. A flow injection system is employed in conjunction with a novel perfusion chamber-the fountain cell. The ability of the flow injection system to perfuse cells with a reagent with a reproducibility of 1% RSD is demonstrated. The system was used to monitor changes in calcium levels in baby hamster kidney cells loaded with FURA-2 as a result of stimulation with a precisely timed concentration of ionomycin. The unique feature of the technique is that it allows a series of responses of a given cell to be directly compared to each other.
Microscopy Perfusion

"Fast Online Flow Injection Analysis System For IgG Monitoring In Bioprocesses"
J. Biotechnol. 1997 Volume 59, Issue 1-2 Pages 145-153
Martin Reinecke and Thomas Scheper*

Abstract: An automated immunoassay, with one affinity component immobilized on a solid surface, has been developed to monitor the production of different immunoglobulins during mammalian cell cultivation processes. The whole analysis device is based on the principle of flow injection analysis (FIA) and a cartridge with the immobilized affinity component is implemented into the FIA system. This cartridge is filled with a carrier material to which protein G is covalently bound. After sample injection, binding of the IgG on the protein G within the cartridge takes place while after a washing step, the IgGs are eluted by a pH shift, and the IgG concentration is monitored via fluorescence. In the automated immunoassay, undiluted cell free samples from the reactor or from down-stream processing can be analyzed directly. Due to the separation the IgG can be detected without interference from other sample components by protein fluorescence. The results are obtained with analysis times below 6 min. Sample volumes of less than 100 µL may be used. The assay is sensitive to concentrations from 5 up to 500 µg mL-1. Using this FIA-System, immunoglobulins G, produced in different media, were successfully monitored. The results of the assay were validated by ELISA.
Immunoglobulin G Fluorescence Immunoassay Method comparison Immobilized protein Interferences Process monitoring

"Assessment Of Cross-resistance In Adriamycin- And Mitoxantrone-resistant Cells By Flow Injection Analysis"
J. Clin. Biochem. Nutr. 1995 Volume 19, Issue 3 Pages 123-130
Venkatesan, P.V.;Nagarajan, B.

Abstract: The cross-resistance pattern of daunorubicin (DNR) in adriamycin (ADR)- and mitoxantrone (MTN)-resistant sublines was examined by measuring DNA strand-break and drug accumulation by flow injection analysis (FIA). Drug accumulation and retention were lower in both the resistant sublines compared with their values for sensitive ones. The cytotoxicity studies showed that ADR- and MTN-resistant cells were 15 and 12 fold cross-resistant to DNR. A significant reduction in DNR-induced DNA damage was observed in both the resistant sublines even at the same intracellular concentration, suggesting the role of altered topoisomerase II activity in resistance. A good positive correlation between cytotoxicity and strand-break results emphasizes the significance of DNA strand-break measurement to monitor development of drug resistance. (14 references)
DNA Drugs Clinical analysis

"Continuous-flow NMR Bioreactor For Invivo Studies Of Microbial Cell-suspensions With Low Biomass Concentrations"
J. Magn. Reson. 1992 Volume 98, Issue 3 Pages 654-659
A. A. de Graaf, R. M. Wittig, U. Probsta, J. Strohhaecker, S. M. Schoberth and H. Sahm

Abstract: The authors have initiated in vivo NMR studies using microbial cultures with a low biomass concentration (up to 10 mg dry weight (dw)/ml), because these suspensions can be handled quite well in a reproducible manner. To keep the measuring time within acceptable limits, the authors had to develop a new experimental setup that has an NMR sensitivity (defined as signal-to-noise ratio per unit time) as high as possible.
Nuclear magnetic resonance In vivo monitoring

"Study Of The UV Spectrophotometric Determination Of Fumaric Acid And The Interfering Effect Of Some Dicarboxylic Acids"
Sb. Vys. Sk. Chem. Technol. Praze 1988 Volume 22H, Issue 1 Pages 115-129
Kult, K.;Kuzilek, V.;Vrbsky, J.

Abstract: Optimum conditions for the direct UV determination of fumaric acid(I) in aqueous solution comprise pH 8 (acetate buffer) and absorbance measurement at 250 nm. Beer's law is obeyed for 0.04 to 72 mM I, and the limit of determination is 11.6 µM. The method has been used to determine I in cell mixtures for the enzymatic production of L-malic acid from I. Up to 10-fold and 20-fold amounts of malic and succinic acids, respectively, do not interfere; malic acid interferes by absorbing at 250 nm. The technique can be used for detection in flow injection analysis.
Fumaric acid Spectrophotometry Interferences

"Cell Stimulus And Lysis In A Microfluidic Device With Segmented Gas-Liquid Flow"
Anal. Chem. 2005 Volume 77, Issue 11 Pages 3629-3636
Jamil El-Ali, Suzanne Gaudet, Axel G&uuml;nther, Peter K. Sorger and Klavs F. Jensen

Abstract: We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using α-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.
Microfluidic Air segmentation

"Capacitance Measurements Of Antibody-antigen Interactions In A Flow System"
Anal. Chem. 1997 Volume 69, Issue 18 Pages 3651-3657
Christine Berggren and Gillis Johansson

Abstract: Capacitive immunosensors were made by coupling monoclonal antibodies to thioctic acid, which had self-assembled on a gold electrode. Surface areas that were not covered were plugged with 1-dodecanethiol to make the layer dense and insulating. Cyclic voltammetry showed that the hexacyanoferrate redox reactions were blocked by this procedure. The capacitance of the electrode was evaluated from the current transients obtained when a potentiostatic step was applied. The immunosensor was placed in a flow system, and a capacitance decrease could be observed after injection of an unlabeled antigen. It was linear over almost three decades when plotted vs the logarithm of the antigen concentration. Human chorionic gonadotropin hormone could be determined in the range 1 pg/mL-1 ng/mL, with a detection limit of 0.5 pg/mL (15 10^-15 M). A similar response was obtained with immobilized F(ab')2 fragments. No cross-reactivity was observed with the thyrotropic hormone, which has one chain in common with gonadotropin. Monoclonal antibodies toward interleukin-2 immobilized on the immunosensor gave also a response over 1 pg/mL-1 ng/mL, with a detection limit of 1 pg/mL. An immunosensor with monoclonal antibodies toward human albumin gave a calibration curve with lower slope than the other proteins but still with a detection limit of 1 pg/mL.
Antigens Capacitance Sensor Detector

"Automated Measurement Of Phosphatases"
Anal. Biochem. 1969 Volume 32, Issue 3 Pages 355-361
A.L. Tappel

Abstract: Automated phosphate determination was used to quantitate the amount of the specific marker enzymes acid phosphatase, glucose-6-phosphatase, and Mg2+-activated ATPase found in subcellular fractions. The versatility of the automated phosphate determination and various parameters affecting the analyzes are discussed.
Phosphate