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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Analytical Biochemistry

  • Publisher: Elsevier
  • FAD Code: ANBI
  • CODEN: ANBCA2
  • ISSN: 0003-2697
  • Abbreviation: Anal. Biochem.
  • DOI Prefix: 10.1016/j.ab,10.1016/0003-2697
  • Language: English
  • Comments: Fulltext from 1960 V1

Citations 134

"Multicommutated Flow-through Optosensors Implemented With Photochemically Induced Fluorescence: Determination Of Flufenamic Acid"
Anal. Biochem. 2007 Volume 361, Issue 2 Pages 280-286
Javier López-Flores, María L. Fernández-De Córdova and Antonio Molina-Díaz

Abstract: This article describes a multicommutated flow injection-solid phase spectroscopy system implemented with photochemically induced fluorescence for the determination of flufenamic acid (FFA). A strongly fluorescent photoproduct is generated when FFA is irradiated online under UV light in a strong sulfuric medium. The photoproduct generated is retained on C18 silica gel (which fills the detection area of the flow cell) and directly monitored on the active solid support at 258/442 nm (?ex/?em). After maximum signal recording, the sensing zone is regenerated by eluting the retained photoproduct with an appropriate H2SO4/MeOH solution. The sensor, completely automated, is based on the use of three-way solenoid valves conveniently operated by a homemade multicommutation software written in Java language. The system is calibrated at 10 and 60 s for sampling time, showing detection limits of 1.28 x 10^-9 and 5.33 x 10^-10 mol L-1 and sampling rates of 38 and 28 h-1, respectively, with relative standard deviations of 0.9 and 1.2%. The applicability of the method is demonstrated for the determination of FFA in human serum, human urine, and a pharmaceutical preparation without any pre-treatment. Good recovery levels were achieved between 90.5 and 103.7%.

"Disposable Biosensor Based On Enzyme Immobilized On Au-chitosan-modified Indium Tin Oxide Electrode With Flow Injection Amperometric Analysis"
Anal. Biochem. 2007 Volume 360, Issue 2 Pages 288-293
Jiehua Lin, Wei Qu and Shusheng Zhang

Abstract: Indium tin oxide (ITO) electrode is used to fabricate a novel disposable biosensor combined with flow injection analysis for the rapid determination of H2O2. The biosensor is prepared by entrapping horseradish peroxidase (HRP) enzyme in colloidal gold nanoparticle-modified chitosan membrane (Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanning electron microscope, atomic force microscope, and electrochemical methods. Parameters affecting the performance of the biosensor, including concentrations of o-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under the optimal experimental conditions, H2O2 could be determined in the linear calibration range from 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n = 8). The amperometric response of the biosensor did not show an obvious decrease after the substrates were injected continuously 34 times into the flow cell. The prepared biosensor not only is economic and disposable, due to the low-cost ITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication in batch.

"Development Of Fast Fourier Transformation Continuous Cyclic Voltammetry As A Highly Sensitive Detection System For Ultra Trace Monitoring Of Penicillin V"
Anal. Biochem. 2007 Volume 360, Issue 2 Pages 175-181
Parviz Norouzi, Mohammad Reza Ganjali, Parandis Daneshgar, Taher Alizadeh and Ali Mohammadi

Abstract: Fast continuous cyclic voltammetry was used as a detection method for penicillin V in a flow injection system. Additionally, a special computer-based numerical calculation method (using fast Fourier transformation) providing enhancement of the analyte signal and noise reduction is introduced here. During the measurements, the potential waveform (consisting of the potential steps of cleaning, stripping, and the potential ramp) was continuously applied to an Au disk microelectrode (12.5 ?m in radius). In particular, the effects of accumulation potential, sweep rate, and delay time on the sensitivity and selectivity of the method were investigated. Eventually, the stripping time was found to be less than 300 ms, the detection limit of the method was 7.0 x 10^-12 M, and the associated relative standard deviation at 5.0 x 10^-6 M penicillin V was 2.3 for eight runs.

"Determination Of The DNA-binding Characteristics Of Ethidium Bromide, Proflavine, And Cisplatin By Flow Injection Analysis: Usefulness In Studies On Antitumor Drugs"
Anal. Biochem. 2006 Volume 355, Issue 2 Pages 157-164
A. Alonso, M.J. Almendral, Y. Curto, J.J. Criado, E. Rodríguez and J.L. Manzano

Abstract: Flow injection analysis was used to study the reactions occurring between DNA and certain compounds that bind to its double helix, deforming this and even breaking it, such that some of them (e.g., cisplatin) are endowed with antitumoral activity. Use of this technique in the merging zones and stopped-flow modes afforded data on the binding parameters and the kinetic characteristics of the process. The first compound studied was ethidium bromide (EtdBr), used as a fluorescent marker because its fluorescence is enhanced when it binds to DNA. The DNA-EtdBr binding parameters, the apparent intrinsic binding constant (0.31 ± 0.02 ?M-1), and the maximum number of binding sites per nucleotide (0.327 ± 0.009) were determined. The modification introduced in these parameters by the presence of proflavine (Prf), a classic competitive inhibitor of the binding of EtdBr to the DNA double helix, was also studied, determining the value of the intrinsic binding constant of Prf (KPrf = 0.119 ± 9 x 10^-3 ?M-1). Finally, we determined the binding parameters between DNA and EtdBr in the presence of the antitumor agent cisplatin, a noncompetitive inhibitor of such binding. This provided information about the binding mechanism as well as the duration and activity of the binding of the compound in its pharmacological use.

"Subattomole Oligonucleotide And P53 CDNA Determinations Via A High-resolution Surface Plasmon Resonance Combined With Oligonucleotide-capped Gold Nanoparticle Signal Amplification"
Anal. Biochem. 2006 Volume 354, Issue 2 Pages 220-228
Xin Yao, Xin Li, Freddy Toledo, Cecilia Zurita-Lopez, Margarita Gutova, Jamil Momand and Feimeng Zhou

Abstract: Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10^-20 mol, corresponding to 1.38 fM in a 15 µL solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyzes conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.

"Flow Injection Analysis-Rayleigh Light Scattering Detection For Online Determination Of Protein In Human Serum Sample"
Anal. Biochem. 2006 Volume 354, Issue 1 Pages 64-69
Ying Li, Lijun Dong, Weiping Wang, Zhide Hua, and Xingguo Chen

Abstract: A flow injection analysis (FIA) system combined with Rayleigh light scattering (RLS) detection is developed for the sensitive and rapid determination of protein concentration in human serum sample. This method is based on the weak intensity of RLS of Eriochrome Black T (EBT, 2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitronaphthalene-4-sulfonic acid sodium salt), which can be enhanced by the addition of protein in weakly acidic solution. The effects of pH and interfering species on the determination of protein were examined. Calibrations for protein, based on RLS intensity, were linear in the concentration ranges of 7-36 µg/ml for human serum album (HSA) and 8-44 µg/ml for bovine serum album (BSA). The detection limits of the method were found to be 0.882 and 2.507 µg/ml for HSA and BSA, respectively. A relative standard deviation of 0.76% (n = 5) was obtained with 20 µg/ml HSA standard solution. The FIA-RLS method was more stable than the general RLS method, and the average RSD value of FIA-RLS was less than that of the general RLS. The sample rate was determined to be 90 samples per hour.

"Flow Injection Analysis With Diode Array Absorbance Detection And Dynamic Surface Tension Detection For Studying Denaturation And Surface Activity Of Globular Proteins"
Anal. Biochem. 2006 Volume 351, Issue 1 Pages 100-113
Emilia Bramanti, Chiara Allegrini, Massimo Onor, Giorgio Raspi, Kristen J. Skogerboe and Robert E. Synovec

Abstract: In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-?l drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, ?-lactalbumin (?-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, ?-chymotrypsinogen A, ?-lactoglobulin (?-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, ?-Lac and ?-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.

"Comparative Modeling And Analysis Of Microfluidic And Conventional DNA Microarrays"
Anal. Biochem. 2006 Volume 348, Issue 2 Pages 284-293
James A. Benn, Jenny Hu, Bradley J. Hogan, Rebecca C. Fry, Leona D. Samson and Todd Thorsen

Abstract: A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min.

"Flow-injection Enzymatic Analysis For Glycerol And Triacylglycerol"
Anal. Biochem. 2005 Volume 346, Issue 2 Pages 234-240
Li-chen Wu and Chien-Ming Cheng

Abstract: A flow-injection enzymatic analytical system was developed for determination of glycerol and triacylglycerol based on enzymatic reactions in capillary followed by electrochemical detection. The hydrogen peroxide produced from the enzyme reaction was monitored by a platinum-based electrochemical probe. Different immobilization strategies on silica support were studied. The best and most effective configuration found for the measurement of glycerol and triacylglycerols in this system was the tandem connection of a lipase column and a silica-fused capillary column coimmobilized with glycerokinase (GK) and glycerol-3-phosphate oxidase (GPO). Lipase helps the breakdown of triacylglycerol to yield free fatty acids and glycerol, while glycerokinase catalyzes the adenosine-5-triphosphate-dependent phosphorylation of glycerol to yield α-glycerol phosphate, which can subsequently be oxidized by 3-glycerol phosphate oxidase to produce hydrogen peroxide. Response-surface methodology (RSM) was applied to optimize the proposed system for glycerol. Experiment settings were designed by central composite design to investigate the combined effects of pH, flow rate, reaction temperature, and ATP concentration on collected signals. The fitted model, per RSM, showed that the optimum conditions of the system are 2 mM ATP in 0.1 M carbonate buffer (pH 11.0), flow rate of 0.18 mL/min, temperature of 35°C, 20 µL of sample injection, and applied voltage of 0.650 V. The proposed biosensing system using lipase, GK, and GPO exhibited a flow-injection analysis peak response of 2.5 min and a detection limit of 5 x 10^-5 M glycerol (S/N = 3) with acceptable reproducibility (CV < 4.30%). It also had linear working ranges from 10 -4 to 10^-2 M for glycerol and from 10^-3 to 10^-2 M for triacylglycerol. The capillary enzyme reactor was stable up to 2 months in continuous operation, and it was possible to analyze up to 15 samples per hour. The present biosensing system holds promise for on-line detection of triacylglycerol in serum and glycerol content in fermented products.

"Evaluation Of The Total Antioxidant Capacity By Using A Multipumping Flow System With Chemiluminescent Detection"
Anal. Biochem. 2005 Volume 345, Issue 1 Pages 90-95
Silvia R.P. Meneses, Karine L. Marques, Cherrine K. Pires, Jo&atilde;o L.M. Santos, Eduarda Fernandes, Jos&eacute; L.F.C. Lima and Elias A.G. Zagatto

Abstract: An automated flow-based procedure for assessment of total antioxidant capacity was developed. It involved a multipumping flow system, a recent approach to flow analysis, and exploited the ability of selected compounds to inhibit the chemiluminescence reactions of luminol or lucigenin with hydrogen peroxide. The system included several discretely actuated solenoid micropumps as the only active components of the flow manifold. This enabled the reproducible insertion and efficient mixing of very low volumes of sample and reagents as well as the transportation of the sample zone toward a flow-through luminometer, where the chemiluminometric response was monitored. With luminol as the chemiluminogenic reagent, linearity of the analytical curves was noted up to 3.2 x 10^-4, 1.1 x 10^-3, and 8.8 x 10 -8 mol L-1 for Trolox, ascorbic acid, and resveratrol, respectively. With lucigenin, linear calibration plots up to 2 x 10 -5 mol L-1 of Trolox and 5.7 x 10^-5 mol L-1 of ascorbic acid were obtained. As favorable analytical figures of merit, the measurement precision (RSD typically between 0.2 and 2.0%, n = 10), low operational costs, low reagent consumption, sampling rate (160 and 70 h-1), and versatility should be highlighted. The proposed system can be used in distinct analytical circumstances without requiring physical reconfiguration.

"Continuous-flow/stopped-flow System Using An Immunobiosensor For Quantification Of Human Serum IgG Antibodies To Helicobacter Pylori"
Anal. Biochem. 2005 Volume 337, Issue 2 Pages 195-202
Germ&aacute;n A. Messina, Angel A.J. Torriero, Irma E. De Vito, Roberto A. Olsina and Julio Raba

Abstract: Conventional methods, such as gastric biopsy, enzyme-linked immunosorbent assay (ELISA), culture, require a long time for the determination of Helicobacter pylori infections. This study reports an amperometric immunoreactor for rapid and sensitive quantification of human serum immunoglobulin G (IgG) antibodies to H. pylori. Antibodies in the serum sample are allowed to react immunologically with the purified H. pylori antigens that are immobilized on a rotating disk. The bound antibodies are quantified by horseradish peroxidase (HRP) enzyme-labeled second antibodies specific to human IgG. HRP in the presence of hydrogen peroxide catalyzes the oxidation of hydroquinone to p-benzoquinone. The electrochemical reduction back to hydroquinone is detected on a glassy carbon electrode surface at -0.15 V. The electrochemical detection can be done within 1 min, and the analysis time does not exceed 30 min. The calculated detection limits for amperometric detection and the ELISA procedure are 0.6 and 1.9 U mL-1, respectively. The amperometric immunoreactors showed higher sensitivity and lower time consumed than did the standard spectrophotometric detection ELISA method. It can also be used for rapid analysis in conventional and field conditions in biological, physiological, and analytical practices.

"Determination Of The Refractive Index Increment (dn/dc) Of Molecule And Macromolecule Solutions By Surface Plasmon Resonance"
Anal. Biochem. 2004 Volume 333, Issue 2 Pages 273-279
Tathyana Tumolo, Lucio Angnes and Mauricio S. Baptista

Abstract: An automated method for dn/dc determination using a surface plasmon resonance instrument in tandem with a flow injection gradient system (FIG-SPR) is proposed. dn/dc determinations of small molecule and biomolecule, surfactant, polymer, and biopolymer solutions with precision around 1-2% and good accuracy were performed using the new method. dn/dc measurements were also carried out manually on a conventional SPR equipment with similar accuracy and precision. The FIG-SPR instrument is inexpensive and could be easily coupled to commercially available SPR and liquid chromatography instruments to obtain several properties of the solutions, which are based on measurements of refractive index.

"Automated Affinity Chromatography Measurements Of Compound Mixtures Using A Lab-on-valve Apparatus Coupled To Electrospray Ionization Mass Spectrometry"
Anal. Biochem. 2004 Volume 331, Issue 1 Pages 161-168
Yuko Ogata, Louis Scampavia, Tyan L. Carter, Erkang Fan and Franti&#353;ek Ture&#269;ek

Abstract: We report a fully automated affinity chromatography system using a lab-on-valve (LOV) apparatus coupled to an electrospray ionization ion-trap mass spectrometer (ESI-MS). The system allows simultaneous measurements of multiple ligand affinities to proteins immobilized on beads. Bead regeneration, column repacking, and repetitive measurements are achieved on the time scale of several minutes. In this study, the system was used to screen the binding of a peptide mixture to human and Trypanosoma brucei (T. brucei) truncated Pex5 (tPex5) proteins. Equilibrium dissociation constants (Kd) were measured for T. brucei tPex5 and compared to the values obtained by a fluorescence-based competition assay. The three peptides that showed affinity toward tPex5 had Kd values that were comparable in magnitude (within a factor of 5) and showed the same ranking order as those from manual fluorescence measurements. With 12 min of sample infusion, the entire sample-to-sample cycle takes about 15 min and can be repeated without any preparation between runs. For T. brucei tPex5 affinity measurements, 1 mg of protein was sufficient for 35 repetitive analyzes in the automated LOV-ESI-MS apparatus. The system allows rapid determination of Kd in the range of 10^-5-10 -7 M for sample mixtures and is suitable for screening a large number of compounds against multiple proteins.

"Liposome-based Microcapillary Immunosensor For Detection Of Escherichia Coli O157:H7"
Anal. Biochem. 2004 Volume 330, Issue 2 Pages 342-349
Ja-an Annie Ho, Hsiu-Wen Hsu and Ming-Ray Huang

Abstract: Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-β--glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 µL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 103-6 x 107 cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was [les]8.9%, which indicates an acceptable level of reproducibility for this newly developed method.

"Direct Determination Of Phosphate In Urine By Sequential-injection Analysis With Single On-line Dilution–calibration Method And Photometric Detection"
Anal. Biochem. 2004 Volume 330, Issue 2 Pages 193-198
D. G. Themelis, A. Economou, A. Tsiomlektsis and P. D. Tzanavaras

Abstract: In this work we report a sequential-injection (SI) analysis method for the direct determination of phosphate in urine with on-line dilution and photometric detection. The developed SI manifold incorporated an additional dilution coil (DC) connected to one of the ports of the selection valve. On-line dilution was performed by aspirating a zone of sample with volume VS from the sample line into the holding coil (HC), transferring volume VT of that zone into the DC, and, finally, aspirating a portion of VT with volume VA back into the HC. This dilution introduces an additional dispersion of the initial sample zone represented by the effective dispersion coefficient Deff which can be tailored to the desired value by appropriate selection of the volumes VS, VT, and VA. Actual detection of phosphates was performed by the molybdenum blue method with detection at 690 nm. By employing an on-line dilution step with a Deff=100, the calibration curve for phosphate was linear in the range 0.05 x 10^-2 to 3 x 10^-2 mol L-1 which covers the actual phosphate concentration in urine samples. The regression coefficient was r2=0.996, the relative standard deviation was sr=3.9% at the 1 x 10^-2 mol L-1 level (n=10), and the injection frequency was 30 injections h-1. The method was applied to urine samples with recoveries >97%.

"Study And Application Of Flow Injection Spectrofluorimetry With A Fluorescent Probe Of 2-(2-pyridil)-benzothiazoline For Superoxide Anion Radicals"
Anal. Biochem. 2004 Volume 326, Issue 2 Pages 176-182
Bo Tang, Li Zhang and Li-li Zhang

Abstract: This paper presents an automatic spectrofluorimetric method (flow injection spectrofluorimetry) using a novel fluorescent probe named H. Py. Bzt (2-(2-pyridil)-benzothiazoline) for determining superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by infrared and 1H nuclear magnetic resonance spectra. It could specially identify and trap O2-. and was oxidized by O2-. to form a strong fluorescence product. Based on this reaction, the flow injection spectrofluorimetric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species because the probe can be oxidized only by O2-. excluding H2O2. As a kind of simple, rapid, precise, sensitive and automatic technique, it was applied to measurement of SOD activity in scallion, garlic, and onion with satisfactory results.
Enzyme, superoxide dismutase Vegetable Vegetable Vegetable Fluorescence Optimization

"Mass Spectrometric Method For Detecting Carbon 13 Enrichment Introduced By Folate Coenzymes In Uric Acid"
Anal. Biochem. 2003 Volume 321, Issue 2 Pages 188-191
Gregory S. Gorman, Tsunenobu Tamura and Joseph E. Baggott

Abstract: Using [2-13C]uric acid as a test material, we developed a mass spectrometric procedure that detects and estimates the difference in 13C enrichment at the positions of carbons 2 and 8 of the purine ring. This method could replace radiochemical methods and could trace the incorporation of carbon fragments into the purine ring from 13C-labeled metabolites in humans.

"Atmospheric Pressure Chemical Ionization-mass Spectrometry Method To Improve The Determination Of Dansylated Polyamines"
Anal. Biochem. 2003 Volume 318, Issue 2 Pages 212-220
Fran&ccedil;ois Gaboriau, Ren&eacute; Havouis, Jacques-Philippe Moulinoux and Jean-Guy Delcros

Abstract: Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.

"A Generic Assay For Phosphate-consuming Or -releasing Enzymes Coupled On-line To Liquid Chromatography For Lead Finding In Natural Products"
Anal. Biochem. 2003 Volume 316, Issue 1 Pages 118-126
T. Schenk, N. M. G. M. Appels, D. A. van Elswijk, H. Irth, U. R. Tjaden and J. van der Greef

Abstract: A generic continuous-flow assay for phosphate-consuming or -releasing enzymes coupled on-line to liquid chromatography (LC) has been developed. Operating the LC-biochemical assay in combination with mass spectrometry allows the fast detection and identification of inhibitors of these enzymes in complex mixtures. The assay is based on the detection of phosphate, released by the on-line continuous-flow enzymatic reaction, using a fluorescent probe. The probe consists of fluorophore-labeled phosphate-binding protein, which shows a strong fluorescence enhancement upon binding to inorganic phosphate. To detect very small changes of the phosphate concentration in a post-column enzymatic reaction medium, the enzymatic removal of phosphate impurities from solvents, reagents, and samples was optimized for application in continuous flow. The potential of the phosphate probe is demonstrated by monitoring the enzymatic activity, i.e., the phosphate release, from alkaline phosphatase. The selectivity of the phosphate readout, necessary to distinguish between phosphate containing substrate or product and free inorganic phosphate released after enzymatic conversion, is shown. The applicability of LC coupled to the enzymatic assay using the phosphate readout was demonstrated by detection of tetramisole in a plant extract as inhibitor of alkaline phosphatase. Parallel mass spectrometry allowed the simultaneous confirmation of the identity of the inhibitor.
Enzyme, alkaline phosphatase Plant Fluorescence Optimization Interferences

"Oligonucleotide Analysis By Sequential Injection Before Analysis (SIBA) Capillary Electrophoresis"
Anal. Biochem. 2003 Volume 313, Issue 1 Pages 183-185
William A. Williams, Amanda Hendrickson, Allison F. Gillaspy, David W. Dyer and Lisa A. Lewis

Abstract: Advancements since the mid 1980s [1, 2, 3, 4, 5, 6, 7, 8 and 9] now allow the use of CE1 to accurately assess the quality of synthetic oligonucleotides [10]. With the advent of multiplex oligonucleotide synthesizers, the requirement for increased-throughput CE analysis has become a necessity. However, CE applications for oligonucleotide quality control are typically low throughput (~25 samples/day). Therefore, we developed a sequential injection before analysis (SIBA) protocol to increase sample throughput approximately fourfold over that of traditional CE. Using the SIBA protocol, completely automated analysis of 96 25-mer oligonucleotides takes approximately 20 h. Using the SIBA protocol, an oligonucleotide is electrokinetically injected into the capillary inlet. The capillary is dipped in water to remove residual oligonucleotide and then immersed in the inlet and outlet electrophoresis buffer. Voltage is applied at 9 kV, electrophoresing the oligonucleotide toward the detector. After this first oligonucleotide travels a fraction of the distance to the detector, the cycle of electrokinetic injection, water dip, and electrophoresis is repeated until five oligonucleotides have been injected into the capillary. We attempted to implement additional injections prior to the final 30- to 60-min electrophoresis, but this resulted in a loss of resolution (data not shown). After 20 oligonucleotides (four runs) have been analyzed, the gel matrix is replaced.

"Detection Of Fumonisin B1: Comparison Of Flow-injection Liposome Immunoanalysis With High-performance Liquid Chromatography"
Anal. Biochem. 2003 Volume 312, Issue 1 Pages 7-13
Ja-an A. Ho and Richard A. Durst

Abstract: Fumonisins are secondary metabolites of the fungus Fusarium moniliforme, a common mycotoxin in corn, which are known to cause cancer in a number of experimental animals and have been linked to human esophageal cancer in China and South Africa. A high-performance liquid chromatographic (HPLC) method is currently the most widely used method for the quantitative determination of fumonisins. This method utilizes precolumn derivatization with o-phthaldialdehyde, isocratic elution, and fluorescence detection. In this study, the HPLC method was chosen as the reference method to evaluate the reproducibility and accuracy of FILIA (flow-injection liposome immunoanalysis) for the detection of the fumonisin B1 (FmB1). Studies indicate that a recovery of 86-90% could be obtained when commercial yellow cornmeal spiked with FmB1 was extracted in 75% methanol, which correlated favorably (correlation coefficient, r2=0.945) with the result of 80-92% obtained using the flow-injection liposome immunoanalysis (FILIA) system. The data suggest that the FILIA method is comparable to HPLC for the detection of fumonisins in corn, animal feeds, and human foods. Important features of FILIA as compared to HPLC are, most importantly, lower detection limit (ca. 25 x lower), and also less complex and faster sample preparation and therefore increased analytical throughput. In addition, 24 human corn-based foods and 6 animal feeds were examined for the presence of FmB1 using HPLC and FILIA.

"Modified Carbon Paste Electrodes For Flow Injection Amperometric Determination Of Isocitrate Dehydrogenase Activity In Serum"
Anal. Biochem. 2002 Volume 308, Issue 2 Pages 195-203
Pablo Rodr&iacute;guez-Granda, M. Jes&uacute;s Lobo-Casta&ntilde;&oacute;n, Arturo J. Miranda-Ordieres and Paulino Tu&ntilde;&oacute;n-Blanco

Abstract: A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1 x 10^-4M, with a detection limit of 5 x 10^-9M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150 h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.
Adsorption Enzyme

"Flow Injection Fluorescence Immunoassay For Gentamicin Using Sol-gel-derived Mesoporous Biomaterial"
Anal. Biochem. 2002 Volume 308, Issue 1 Pages 71-76
Huang-Hao Yang, Qing-Zhi Zhu, Hui-Ying Qu, Xiao-Lan Chen, Ma-Tai Ding and Jin-Gou Xu

Abstract: Sol-gel-derived mesoporous biomaterials were used for the first time in the flow injection fluorescence immunoassay system. Anti-gentamicin antibody was immobilized in a mesoporous sol-gel material using tetramethoxysilane as a precursor and poly(ethylene glycol) as a template. The sol-gel glass was used to develop an immunoaffinity column for the flow injection immunoassay of gentamicin. Little unspecific adsorption of gentamicin on the sol-gel and no antibody leaching under harsh elution conditions were found. The immunoassay is based on the competition between gentamicin and fluorescein isothiocyanate-labeled gentamicin for a limited number of encapsulated antibody binding sites. NaOH solution of 5 x 10^-3 mol/L is used for the regeneration of encapsulated antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 20 times without any loss of reactivity. Sample pre-concentration is not needed and a single assay can be performed within 10 min. The calibration for gentamicin has a working range of 250-5000 ng/mL with a detection limit of 200 ng/mL, which is close to that of the fluorescence immunoassay and fluorescence polarization immunoassay using the same reactants. Comparison of the results from this method with that obtained from HPLC showed an excellent correlation. [Journal Article; In English; United States]

"Flow Injection Spectrophotometric Determination Of The Antibiotic Fosfomycin In Pharmaceutical Products And Urine Samples After On-line Thermal-induced Digestion"
Anal. Biochem. 2002 Volume 304, Issue 2 Pages 244-248
Paraskevas D. Tzanavaras and Demetrius G. Themelis

Abstract: A new flow injection (FI) method for the precise and rapid spectrophotometric determination of the antibiotic fosfomycin (FMC) in urine and pharmaceutical samples is described. The method is based on the online quantitative thermal-induced digestion of the analyte prior to injection into the FI system. Ammonium persulfate was used as the oxidation reagent. The resulting orthophosphate ions were determined spectrophotometrically (lambda(max) = 690 nm) using the molybdenum blue approach. Chemical and FI variables that affected on-line oxidation were studied and optimized. The proposed method is very precise (s(r) = 1.2% at 1.0 x 10^-4 mol L-1 FMC, n = 12), offers a high sampling rate of 60 h-1, and allows for the determination of the analyte in the range 3.0 x 10^-6 to 3.0 x 10^-4 mol L-1 with a satisfactory 3s detection limit of 1.0 x 10^-6 mol L-1. Application of the proposed method to urine and pharmaceutical samples yielded accurate results with percentage recoveries in the range 96.4-102.5%.

"Comparison Of Different Protein Immobilization Methods On Quartz Crystal Microbalance Surface In Flow Injection Immunoassay"
Anal. Biochem. 2001 Volume 299, Issue 2 Pages 130-135
Yung-Chuan Liu, Chih-Ming Wang and Kuang-Pin Hsiung

Abstract: In this study, a quartz crystal microbalance (QCM) system operated repetitively in flow injection analysis (FIA) mode, is reported. Four immobilization approaches of seven different methods include: (i) physical adsorption; (ii) two thioamine thiolation methods, using cysteamine and cystamine for gold chemisorption and further coupling; (iii) two oxidized dextran spacer methods, coupling of cysteamine and cystamine thiolated QCM surface with periodate-oxidized dextran for further Schiff acid-base reaction; and (iv) two thiol-gold chemisorption-based self-assembled monolayer (SAM), applying short-chain, C-3, and longchain, C-11 mercapto fatty acids to insolubilize human serum albumin (HSA) on QCM surface. Effects of these protein immobilization methods on FIA immunoassay of anti-HSA were compared. At the 0.01 mg/ml anti-HSA level, the lowest analyte concentration tested, the SAM using 11-mercaptoundecanoic acid as QCM surface activating agent generated a larger frequency shift than the other immobilization methods. This implied that the use of thiolated long-chain fatty acid constructed as self-assembled monolayer may thereby potentially be a useful protein immobilization method in QCM-FIA application.

"Chiral Analysis Of Amino Acids Using Electrochemical Composite Bienzyme Biosensors"
Anal. Biochem. 2001 Volume 298, Issue 2 Pages 275-282
R. Dom&iacute;nguez, B. Serra, A. J. Reviejo and J. M. Pingarr&oacute;n

Abstract: The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes. Copyright 2001 Academic Press. [Journal Article; In English; United States]

"Determination Of L-cysteine In Amino Acid Mixture And Human Urine By Flow Injection Analysis With A Biamperometric Detector"
Anal. Biochem. 2001 Volume 297, Issue 2 Pages 170-176
Chuan Zhao, Juncai Zhang and Junfeng Song

Abstract: Based on the electrocatalytic oxidation of cysteine at pretreated platinum electrode and the flow injection biamperometry for irreversible couple, a novel electrochemical detector is proposed for the selective determination of cysteine in amino acid mixtures and human urine samples. A thin-layer flowthrough cell was used to achieve large electrode surface area to volume ratio. Two identical pretreated platinum electrodes were mounted in the cell with an applied potential difference of 10 mV. By coupling two independent and irreversible electrode processes, namely, the oxidation of cysteine and the reduction of platinum oxide, the biamperometric detection scheme has been established. The resulting current is linear to cysteine over the range 4 x 10^-7-4 x 10^-5 M with the detection limit 1 x 10^-7 M (15 pmol). The selectivity of the detector is tested by 55 foreign species including 26 ions, 11 amino acids, 6 vitamins, and 12 other compounds possibly found in urine. The detector performs well as a routine assay, showing high efficiency (180 samples/h) and good reproductivity shown by a RSD of 0.6% for eight repeated determinations of 2 x 10^-6 M cysteine. The urine samples are detected directly without the need of pretreatment or adding other reagents. (C) 2001 Academic Press.

"Calorimetric Analysis Of Cephalosporins Using An Immobilized TEM-1 β-lactamase On Ni2+ Chelating Sepharose Fast Flow"
Anal. Biochem. 2001 Volume 296, Issue 1 Pages 57-62
Ratana Lawung, Bengt Danielsson, Virapong Prachayasittikul and Leif B&uuml;low

Abstract: Two beta -lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta -lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow injection analysis of beta -lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta -lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone. (C) 2001 Academic Press.
Reactor

"Miniaturized Amperometric Biosensor Based On Xanthine Oxidase For Monitoring Hypoxanthine In Cell Culture Media"
Anal. Biochem. 2001 Volume 292, Issue 1 Pages 94-101
Lanqun Mao, Fang Xu, Qi Xu and Litong Jin

Abstract: Fabrication and characterization of miniaturized amperometric hypoxanthine biosensors are described and demonstrated for monitoring hypoxanthine in myocardial cell culture media. The sensors are based on xanthine oxidase (XO) immobilized on carbon fiber microelectrodes (CFMEs) using a composite film of Nafion and electropolymerized phenol (PPh), Nafion was used for XO immobilization because of its film hydrophobicity, enzyme-favored environment., and electrostatic interaction with XO, which was dispersed in Nafion film by immersing the Nafion-coated CFMEs in XO solution for 5 h, PPh film was formed as an overlay on Nafion and XO-modified CFMEs via electropolymerization. Hypoxanthine was measured with the sensor by the oxidation of enzymatic reaction products, hydrogen peroxide (H2O2), and uric acid (UA) at +0.60 V (vs Ag/AgCl). The use of Nafion and PPh as a matrix for XO immobilization yields enhanced specificity, sensitivity, and linearity toward hypoxanthine. A dynamic linear range of 5.0 muM to 1.8 mM was achieved with a calculated detection limit of 1.5 muM (S/N = 3) and a sensitivity of 3.144 nA/mM. In addition, the measurement was virtually interference-free from easily oxidizable species such as UA, ascorbic acid, physiological levels of neurotransmitters, and their principal metabolites. The biosensor was used to monitor hypoxanthine accumulation in myocardial cell culture media, in which the level of extracellular hypoxanthine was found to increase with ischemic tolerance, (C) 2001 Academic Press.
Selectivity Membrane

"An Amperometric Cellobiose Dehydrogenase-based Biosensor Can Be Used For Measurement Of Cellulase Activity"
Anal. Biochem. 2001 Volume 290, Issue 2 Pages 245-250
Lars Hild&eacute;n, Lars Eng, Gunnar Johansson, Sten-Eric Lindqvist and G&ouml;ran Pettersson

Abstract: The hemoflavoenzyme cellobiose dehydrogenase (CDH, EC 1.1.99.18) from Phanerochaete chrysosporium has been used in an amperometric redox polymer-based biosensor. Used in conjugation with a FIA. system this biosensor can replace colorimetric assays for measuring cellobiose liberated from cellulose in a series of cellulase-containing samples. The biosensor gave the same result as the Somogyi-Nelson method in a less time-consuming and laborious manner. The two methods showed about the same precision. (C) 2001 Academic Press.

"Determination Of L-carnitine By Flow Injection Analysis With NADH Fluorescence Detection"
Anal. Biochem. 2000 Volume 281, Issue 2 Pages 176-181
A. Manj&oacute;n, J. M. Ob&oacute;n and J. L. Iborra

Abstract: A flow injection analysis method for determining L-carnitine is reported. The system uses the enzyme L-carnitine dehydrogenase covalently immobilized to Eupergit C. The NADH produced by the action of the enzyme, which is proportional to the L-carnitine concentration, is quantified using fluorescence detection. The system response was rapid and had a wide range of linearity. At a flow rate of 0.2 ml/min, a detection limit of 1 µM (20 pmol) was obtained for L-carnitine, peak areas were linear up to 100 µM, and samples could be injected every 4 min. The method performed well as a routine assay, showing high sensitivity (54,000 AU µM), a precision of 0.96%, and the ability to carry out 144 consecutive assays with an RSD of 1,47% (good stability). Comparisons were made with other known methods for L-carnitine determination. Presence of D-carnitine had no effect on L-carnitine assay. The analysis was valid for determining L-carnitine concentrations in commercial pharmaceutical preparations

"Development Of An Offline Noncompetitive Flow Immunoassay For The Determination Of"
Anal. Biochem. 2000 Volume 279, Issue 1 Pages 46-54
E. Burestedta, S. Kjellstr&ouml;ma, J. Emn&eacute;usa and G. Marko-Varga

Abstract: A noncompetitive flow immunoassay system (FIA) for the analysis of interleukin-8 (IL-8) in cell samples was developed. Affinity interaction assays based on offline incubation of excess labeled antibodies and antigen (IL-8) were carried out. The residual unbound labeled antibody was trapped in an immunoaffinity column with immobilized IL-8 while the immunocomplex, labeled antibody/IL-8, was detected by a fluorescence detector. Two fluorophores, FLUOS and Cy5.5, were conjugated with IL-8 antibody. Optimization and comparison between the two fluorescent labeled antibodies were performed with regard to pH, antibody concentration, flow rate, injection volume, and association time. Additionally, a horseradish peroxidase enzyme label was used for the conjugation to the anti-IL-8. The enzyme substrate reaction was optimized with respect to temperature and length of the substrate reaction coil. The detection limits were found to be 200 amol using the FLUOS-labeled anti-IL-8 and 1 fmol using the Cy5.5 fluorescence label. The developed FIA technique was applied for the analysis of IL-8 in cell samples. Matrix-assisted laser desorption/ionization time-of- flight mass spectrometry was used to identify IL-8 in the cell samples. (C) 2000 Academic Press.

"Simultaneous Determination Of α-fetoprotein And Carcinoembryonic Antigen In Human Serum By Time-resolved Fluoroimmunoassay"
Anal. Biochem. 1999 Volume 276, Issue 1 Pages 81-87
Kazuko Matsumoto, Jingli Yuan, Guilan Wang and Hiroko Kimura

Abstract: A novel simultaneous measurement method for cw-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in human sera by time-resolved fluoroimmunoassay (TR-FIA) is described. The proposed approach combines the use of europium-labeled anti-AFP antibody for AFP TR-FIA and biotinylated anti-CEA antibody complexed to samarium-labeled streptavidin for CEA TR-FIA. A 96-well microtiter plate coated with a mixture of anti-AFP and anti-CEA monoclonal antibodies was used for the assay. After it was reacted with a solution containing AFP and CEA, a mixture of anti-AFP antibody labeled with BHHCT-Eu3+ and biotinylated anti-CEA antibody was added. The AFP concentration was determined by measuring the solid-phase fluorescence of the europium-labeled anti-AFP antibody at 615 nm. Then a BHHCT-Sm3+-labeled streptavidin-bovine serum albumin conjugate (SA-BSA) was added to react with the biotinylated anti-CEA antibody. After the reaction, the unreacted SA-BSA was washed out, and a 0.1 M NaOH solution containing 1.0 x 10^-5 M TOPO and 0.05% SDS was added to dissociate the samarium-labeled SA-BSA in the immune complex on the surface of the well into the solution. The CEA concentration was determined by measuring the solution fluorescence of 643 nm from the samarium-labeled SA-BSA. The present method gives detection limits of 0.07 ng/ml for AFP and 0.3 ng/ml for CEA. The coefficient variations of the method are less than 7%, and the recoveries are in the range of 90-110% for serum samples. The AFP and CEA concentrations in 27 human serum samples were determined by the present method as well as by single assay for comparison. A good correlation was obtained with the correlation coefficients of 0.990 for AFP and 0.973 for CEA.

"Flow Injection Analysis Of Binding Reaction Between Fluorescent Lectin And Cells"
Anal. Biochem. 1999 Volume 269, Issue 2 Pages 230-235
Yasuo Oda, Mitsuhiro Kinoshita, Katsuyoshi Nakayama, Shiho Ikeda and Kazuaki Kakehi

Abstract: A fluorimetric binding assay for lectin and yeast cells using the avidin-biotin system was previously reported. However, the true amt. of bound lectin could not be determined by this method due to difficulty in determination of the number of bound biotin molecules. In the present study, we have developed a method for assaying the binding reaction between fluorescent lectin and cells using a flow injection technique, which allows estimation of the amount of lectin bound to cells. An aliquot of the cell suspension was directly analyzed by injection into a flow injection system after the binding between the fluorescently labeled lectin and cells. The labeled lectins showed good linearity, at least over a range of 20-1000 ng as the injected amt. The intrinsic fluorescence of the labeled lectins did not change upon the binding. The binding reaction of the hydroxycoumarin-labeled lectins with yeast cells was rapid and reached an equilibrium. state within 10 min. Scatchard analysis showed that Saccharomyces cerevisiae cells contained approximately 1.3-1.6 x 10^8 binding sites per cell for Con A, Lycoris radiata agglutinin, and Tulipa gesneriana lectin with affinity constants of 3.2-4.7 x 10^6 M-1. The present method was applied to the study of binding between lectins and bacteria and mouse spleen cells. The assay method described here is highly sensitive and will be an alternative to assays using lectins labeled with radioisotopes. The procedure is quite simple and can be completed within 1 h.

"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 Cell Microscopy

"Enzyme-based Flow Injection Analysis System For Glutamine And Glutamate In Mammalian Cell Culture Media"
Anal. Biochem. 1999 Volume 268, Issue 1 Pages 110-116
Christian Mayer, Alexandra Frauer, Thomas Schalkhammer and Fritz Pittner

Abstract: We present the setup of a flow injection analysis system designed for online monitoring of glutamate and glutamine. These amino acids represent a major energy source in mammalian cell culture. A cycling assay consisting of glutamate dehydrogenase and aspartate aminotransferase produces NADH proportional to the glutamate concentration. in the sample. NADH is then measured spectrophotometrically. Glutamine is determined by conversion to glutamate which is fed into the cycling assay. The conversion of glutamine to glutamate is catalyzed by asparaginase. Asparaginase was used in place of glutaminase due to its relatively high reactivity with glutamine and a pH optimum similar to that of glutamate dehydrogenase. The enzymes were immobilized covalently to activated controlled pore glass beads and integrated into the flow injection analysis system. The application of the immobilized enzymes and the tech. setup are presented in this paper. (c) 1999 Academic Press.

"Ferrocene-conjugated M-phenylenediamine Conducting Polymer-incorporated Peroxidase Biosensors"
Anal. Biochem. 1999 Volume 267, Issue 1 Pages 141-147
Ashok Mulchandani and Shengtian Pan

Abstract: The development and characteristics of a reagentless amperometric biosensor employing horseradish peroxidase incorporated in an electrochemically deposited ferrocene-modified phenylenediamine film on a glassy carbon electrode is reported. The horseradish peroxidase/poly(m-aminoanilinomethylferrocene)-modified glassy carbon electrode reagentless biosensor measured hydrogen peroxide and other organic peroxides in both aqueous and organic medium by reduction at a low applied potential of -0.05 V (vs Ag/AgCl) without interference from molecular oxygen. When modified with glucose oxidase, the new bienzyme electrode measured glucose sensitively and selectively, demonstrating the suitability of the above peroxide biosensor for other oxidoreductase enzyme-based biosensors.

"Oligosaccharide Dehydrogenase-catalyzed Assay For The Determination Of Polysaccharides"
Anal. Biochem. 1998 Volume 265, Issue 1 Pages 151-156
Gunilla S. Nilsson, Mats Andersson, Tautgirdas Ruzgas and Lo Gorton

Abstract: Oligosaccharide dehydrogenase (ODH), an enzyme known to have a broad selectivity for reducing sugars of low molecular weight, was investigated to determine its catalytic properties with larger polysaccharides. Six substrates were studied: pullulan standards with molecular weights of between 5,400 and 90,900, debranched starch, and dextran. In addition, maltotriose, isomaltotriose, maltose, and glucose were used as substrates for comparison. ODH catalyzed the oxidation of the large pullulans with a degree of polymerization of at least 560. Isomaltotriose and dextran were not oxidized. ODH activity for the pullulans, expressed as the rate constant K-ps, was only three times lower than that for maltose. When the oxidation of sugars with ODH was coupled to a color-forming reaction, quantitative spectrophotometric determination of sugars was possible using either Meldola's blue or N-methylphenazinium as electron accepters in combination with nitrotetrazolium blue. Linear calibration curves for maltose, maltotriose, and debranched starch were obtained using this ODH method and compared with curves from the conventional spectrophotometric copper sulfate method. This work demonstrates that ODH can be advantageously used for the determination of polysaccharides.
Polysaccharides Sugars, reducing Maltose Maltotriose Starch Amperometry LC Catalysis Method comparison

"A Combined Flow Injection Chemiluminescent Method For The Measurement Of Radical Scavenging Activity"
Anal. Biochem. 1998 Volume 264, Issue 2 Pages 291-293
Hong-Yeob Choi, Jin-Hyang Song and Dong-Ki Park

Abstract: The authors report a simple, rapid and reproducible flow injection-chemiluminescent method for radical scavenging activity. Chemiluminescence intensity is decreased by the presence of scavengers and the reduction is proportional to the concentration. and ability of scavengers. The flow injection-chemiluminescent method represents high reproducibility and anal. speed. The authors achieved relative standard deviation of 1.88%, and each run in less than 25 s. The present technique proves to be inexpensive, fast, and reliable. This flow injection-chemiluminescent technique could be applied as an alternative method for the measurement of the radical scavenging abilities of biological components. (c) 1998 Academic Press.
Gallic acid Chinese Chemiluminescence Indirect

"Study Of NADH Stability Using Ultraviolet-visible Spectrophotometric Analysis And Factorial Design"
Anal. Biochem. 1998 Volume 260, Issue 1 Pages 50-55
La&eacute;rcio Rover, Jr. a, Julio C. B. Fernandesa, Graciliano de Oliveira Netoa, Lauro T. Kubotaa, Edson Katekawab and S&iacute;lvia H. P. Serranob

Abstract: The chemical stability of nicotinamide adenine dinucleotide coenzyme (NADH/NAD(+)) and its derivatives (NADPH/NADP(+)) was investigated using changes in the UV-visible absorption spectra of these compounds. The spectra of cofactor (reduced form) were monitored at 340 nm wavelength in different buffers, showing a faster degradation in phosphate buffer. This was assigned to the adduct formation between phosphate and NADH (pyridine ring). The three-factor-two-level factorial design study evaluated the contributions: buffers (phosphate and Pipes 0.1 M), pH's (6.8 and 7.8), and temperature (25 and 30°C). The freshly prepared aqueous solutions of coenzyme were analyzed after 40 min under the different conditions (eight assays in triplicate) of the experiment. The main observed effects of the NADH oxidation were, in increasing order, pH, temperature, and buffer without appreciable interactive effects. Therefore, it was verified that the better conditions for cofactor use were alkaline media employing Pipes buffer or its derivatives and low temperatures.
Nicotinamide adenine dinucleotide oxidized Nicotinamide adenine dinucleotide reduced Spectrophotometry Factorial design Chemometrics Optimization

"An Electrochemical Enzyme Immunoassay For Chicken Luteinizing Hormone: Extension Of The Detection Limit By Adequate Control Of The Nonspecific Adsorption"
Anal. Biochem. 1998 Volume 259, Issue 2 Pages 167-175
Ying Qu, Luc R. Berghman and Frans Vandesande

Abstract: A noncompetitive heterogeneous enzyme immunoassay for the determination of chicken luteinizing hormone (LH) was equipped with an electrochemical endpoint in order to further enhance its sensitivity. The immunological principle of the original ELISA remained essentially unchanged, except for the fact that the peroxidase label was replaced by alkaline phosphatase, since in the upgraded version of the assay, p-aminophenyl phosphate was to be used as the substrate of alkaline phosphatase. Enzyme-generated p-aminophenol was injected into a flow injection system and detected amperometrically in a thin-layer flow cell with a glassy carbon electrode at 0.325 V vs Ag/AgCl. A classical problem associated with this type of solid-phase immunoassay is the adsorption of proteins other than the capture antibody to the solid phase. The detection sensitivity is therefore often limited by a large background signal observed in the absence of antigen. In the present study, an experiment was designed to examine in each step of the assay the contribution of each of the potential sources of background current. It was shown that the major contribution to the background current was caused by the nonspecific adsorption of biotinylated secondary antibody. Adsorption of the secondary antibody (biotinylated goat anti-rabbit IgG) to the capture antibody (mouse anti-chicken LHbeta) was clearly a case of specific aspecificity, whereas adsorption to the solid phase itself had to be treated as a nonspecific aspecificity. Addition of 0.25% mouse serum to the secondary antibody as a source of mouse immunoglobulin could overcome the cross-reaction and markedly reduced adsorption to capture antibody. The second part of nonspecific adsorption was eliminated by using combinations of Tween 20 and bovine serum albumin as blocking agents. Controlling the adsorption of the biotinylated secondary antibody in this way decreased the detection limit from 39 pg/ml in the original assay to 2.5 pg/ml in the electrochemical version. This way, the plasma volume of samples containing on the order of 1 ng/ml LH was reduced to less than 10 µL. The linear range was 2.5-625 pg/ml. The method allowed us to measure LH in buffer and in adult and juvenile chicken plasma. Copyright 1998 Academic Press.
Hormone, luteinizing 4-Aminophenol Blood Plasma Amperometry Electrode Immunoassay Interferences Immobilized antibody

"Determination Of Galactose And Galactocerebroside Using A Galactose Oxidase Column And Electrochemical Detector"
Anal. Biochem. 1998 Volume 258, Issue 1 Pages 103-108
Paul W. Stoecker, Paul Manowitz, Richard Harvey and Alexander M. Yacynych

Abstract: A method was developed to measure galactose and galactocerebroside using galactose oxidase immobilized on a solid resin. Galactose oxidase converts galactose and galactocerebroside to their corresponding aldehydes and H2O2, the latter being electroactive and measurable by electrochemical detection using DC amperometric detection. The minimal detection limits of galactose and galactocerebroside were 1 and 2 µM, respectively. The linear response to galactose and galactocerebroside was to at least 300 µM. About 100 samples can be measured per h using flow injection analysis The activity of sulfatidase (cerebroside-3-sulfate-3-sulfohydrolase), which converts sulfatide (sulfogalactocerebroside) to galactocerebroside, was measured, and its inhibition by O-phospho-L-tyrosine was determined.
Galactose Galactocerebroside Amperometry Sensor Resin Immobilized enzyme Interferences

"Determination Of Trehalose By Flow Injection Analysis Using Immobilized Trehalase"
Anal. Biochem. 1997 Volume 253, Issue 1 Pages 8-12
H. -D. Meyer zu D&uuml;ttingdorfa, B. Bachmannb, M. Buchholzc and W. Leuchtenbergerb

Abstract: A new method for the determination of trehalose by flow injection analysis (FIA) is described. The basic principle is the hydrolysis of the disaccharide trehalose into its monomer D-glucose by trehalase, a periplasmic enzyme of Escherichia coli. D-glucose is quantified spectrophotometrically after reaction with hexokinase and glucose-6-phosphate dehydrogenase. Trehalase is prepared by osmotic shock from a recombinant E, coli strain and precipitated with ammonium sulfate. The enzyme is immobilized on VA-Epoxy Biosynth from Riedel-de-Haen, The immobilization rate is about 60%. The FlA signals show a nonlinear dependence on the trehalose concentration. The resulting curve corresponds to a second-order polynomial that serves as a calibration function for test samples. Immobilized trehalase was used during a period of 4 months without any loss of suitability. Several samples of fermentation broth were tested. The results are verified by HPLC. Within an interval of 2 to 10 g/L trehalose the recovery is about 100-120% with a precision of 7% (coefficient of variation). (C) 1997 Academic Press. 10 References
Trehalose Fermentation broth Spectrophotometry Immobilized enzyme Kinetic Method comparison

"Detection And Characterization Of Phospholipase D By Flow Injection Analysis"
Anal. Biochem. 1997 Volume 244, Issue 1 Pages 55-61
M. Becker, U. Spohn and R. Ulbrich-Hofmann

Abstract: Phospholipase D was assayed by FIA (schematic diagram given) with, as substrate, liposomes prepared from phosphatidylcholine. The choline produced is converted in the presence of choline oxidase into betaine and H2O2 which was detected by Co(II)-catalyzed luminol chemiluminescence. A differential experimental set-up (diagram given) was used to study kinetic parameters of phospholipase D from Streptomycesc hromofuscus, Actinomadura spp. and Brassica oleratia. Calibration graphs were linear from 1-100 miu/ml. The detection limit was 75 pmol choline released/min which compared favourably with detection limits by amperometric (0.3 nmol), titrimetric (20 nmol) and spectrophotometric (50 nmol) detection methods. A precisely working automated system for the investigation of phospholipases D (PLDs, EC 3.1.4.4) from plant and microbial sources with flow injection analysis (FIA) has been developed. The two versions of the FIA setup described are based on the oxidation of choline liberated from phosphatidylcholine by PLD action and catalyzed by choline oxidase and the chemiluminescence detection of hydrogen peroxide produced by this reaction. The correlation between this chemiluminescence signal and the PLD activity was linear in the range between 1 and 100 mU/ml PLD. The sampling frequency was 12 samples per hour. This method was used to compare three different PLDs from cabbage and microbial sources with respect to their pH optima, temperature stability, effectors, and v/[S]-characteristics.
Enzyme, phospholipase D Vegetable Chemiluminescence Enzyme Automation Optimization Method comparison

"An Automatic Continuous-flow Aminopeptidase Detector And Its Applications"
Anal. Biochem. 1996 Volume 242, Issue 2 Pages 271-273
Koon-Sea Hui and Maria Hui

Abstract: Biological tissue samples were homogenized at 4°C with 50 mM Bicine hydrochloride buffer containing 0.2 mM DTT and centrifuged at 100 000 g for 1 h before being injected on to a Mono-Q column (5 cm x 5 mm i.d.) for elution with a gradient (1 ml/min) of NaCl in Bicine buffer. The eluate was mixed with buffer and added to 0.2 mM aminoacyl-2-naphthylamide (substrate) in buffer for fluorimetric detection at 250 nm (excitation) and 389 nm (emission). The calibration graph was linear for 10^-800 µg of puromycin-sensitive aminopeptidase, with a detection limit of 100 pg and an assay variation of the effect of phosphatase inhibitors on the enzyme preparation and the application of the assay is discussed.
Aminopeptidases Biological tissue LC Fluorescence Post-column derivatization Interferences

"Determination Of Amino-acid Isotope Ratios By Electrospray Ionization-mass Spectrometry"
Anal. Biochem. 1995 Volume 231, Issue 2 Pages 387-392
Michael B. Goshe and Vernon E. Anderson

Abstract: Isotope ratios of proline, leucine and arginine could be determined by direct flow injection analysis with an Extrel Benchmark MS instrument with an electrospray interface. Optimum tuning of the instrument for each amino-acid to obtain precise isotope ratios is described. Derivatization and GC were not required. Down to 50 nmol of amino-acids could be analyzed. The calibration graph was linear from 50-500 ng/µl of amino-acids in carrier solution. The RSD were 1.1-13.1% (n = 4 or 5).
Amino Acids Arginine Leucine l-Proline Mass spectrometry Isotope ratio

"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 Blood Serum Cell Spectrophotometry Method comparison Lachat Multichannel

"Studies On The Rate And Control Of Antibody Oxidation By Periodate"
Anal. Biochem. 1995 Volume 231, Issue 1 Pages 123-130
Wolfe C. A. C. and Hage D. S.

Abstract: The oxidation of antibody carbohydrate residues by periodate is a common approach for the site-specific immobilization or modification of antibodies for use in various bioanalytical methods. This study examined the time dependence of this oxidation process under a variety of pH, temperature, and concentration conditions. Polyclonal rabbit immunoglobulin G (IgG)was used as the model system for these studies. Flow injection analysis and a hydrazide label (Lucifer yellow CH) were used to monitor the progress of the oxidation reaction. It was found that the number of oxidized sites that were available for labeling could be varied between one and eight groups per antibody by adjusting the time, pH, periodate concentration, or reaction temperature. In each case, most of these groups were produced during the first 30-60 min of the reaction. A comparison was made between these results and those of previous studies that have examined the effects of periodate treatment on amino acid residues and antibody activity. From this work, general guidelines were developed for the control and optimization of antibody oxidation for use with assays that require either high or low levels of antibody modification.
Immunoglobulin G Spectrophotometry Optimization pH

"A Chemiluminescence Flow Injection Analysis Of Serum 3-hydroxybutyrate Using A Bioreactor Consisting Of 3-hydroxybutyrate Dehydrogenase And NADH Oxidase"
Anal. Biochem. 1995 Volume 229, Issue 1 Pages 133-138
Tabata M. and Totani M.

Abstract: Human serum (1 µL) was injected into a stream (0.6 ml/min) of 10 mM potassium phosphate buffer of pH 8.5 containing 0.7 µM FAD and 0.1 mM NAD. The stream passed through a column (2 cm x 2 mm i.d.) packed with ~102 mg (wet wt.) of 3-hydroxybutyrate dehydrogenase/NADH oxidase (5:1) co-immobilized on to porous alkylamine glass beads (80-120 mesh) using glutaraldehyde (preparation described). The H2O2 produced was reacted with luminol/hexacyanoferrate by mixing with streams (0.6 ml/min) of 0.7 mM luminol and 20 mM potassium hexacyanoferrate(III) and the chemiluminescence was measured. The calibration graph was linear for 1 pmol (detection limit) to 0.2 mM 3-hydroxybutyrate and the within- and day-to-day RSD were 1.2-2% and 1.7-2.7%, respectively (n = 10). The recoveries were 98-102%. The results agreed well with those obtained by the spectrophotometric method of Williamson et al. Ascorbic and uric acids interfered. The column activity decreased by 20% over five weeks (1500 runs). We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase. The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide. The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction. Among three immobilized enzyme columns, a co-immobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction. The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods. The co-immobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyzes.
3-hydroxybutyrate Blood Serum Chemiluminescence Immobilized enzyme Glass beads Interferences Method comparison

"Determination Of Intracellular Trehalose And Glycogen In Saccharomyces Cerevisiae"
Anal. Biochem. 1995 Volume 228, Issue 1 Pages 143-149
Schulze U., Larsen M. E. and Villadsen J.

Abstract: Saccharomyces cerevisiae cells were suspended in 1 mL 40 mM potassium acetate of pH 4.8. The cells were disintegrated in a bead mill at a vibration frequency of 150-1800/min for 30 min at 4°C. After centrifugation, the supernatant solution was hydrolyzed with either acid trehalase or amyloglycosidase for trehalose (I) and glycogen (II), respectively. The glucose produced was determined by FIA using a glucose oxidase method (Benthin et al., Anal. Chim. Acta, 1992, 261, 145). The results obtained compared well with those obtained by traditional methods and the RSD were 1.6% and 1.8% for I and II, respectively. A simple, sensitive and non-laborious enzyme-based method has been developed for determination of both trehalose and glycogen in yeast cells. The method is based on extraction of trehalose and glycogen into a 40 mM acetate buffer (pH 4.8) by mechanical disintegration of the cells in a bead mill. Subsequently, trehalose and glycogen can be hydrolyzed to glucose by the enzymes trehalase and amyloglycosidase, respectively. The formed glucose is quantified by a flow injection analyzer based on the enzyme glucose oxidase. The method gives results comparable to traditional methods but the simplicity of the analysis results in a much lower relative standard deviation. The excellent sensitivity of the glucose analyzer means that as little as 1 µg trehalose or glycogen can be determined which reduces the required sample volume. This makes the method ideal for physiological studies, e.g., of transients in continuous cultures of Saccharomyces cerevisiae. In addition, a consistent procedure has been derived for pretreatment and storage of samples.
Trehalose Glycogen Cell Electrode Biotechnology Immobilized enzyme

"Determination Of Sulfamethazine Residues In Milk By A Surface Plasmon Resonance-based Biosensor Assay"
Anal. Biochem. 1995 Volume 226, Issue 1 Pages 175-181
Sternesjo A., Mellgren C. and Bjorck L.

Abstract: The use of antibiotics and chemotherapeutics in animal husbandry has led to the occurrence of veterinary drug residues in all types of food of animal origin. Due to the specification of toxicologically based maximum residue levels for a large number of substances, existing control strategies need even faster and more sensitive methods to meet new and more rigorous regulations. The applicability of an immunosensor device for biospecific interaction analysis was investigated and the development of an assay for analysis of sulfamethazine (SMZ) in milk is described. SMZ was covalently immobilized to a carboxymethyldextran-modified gold film. Spiked samples with known concentrations of SMZ were prepared in HBS buffer and skim and raw milk for construction of standard curves. Polyclonal antibodies against SMZ were added to the sample and the immobilized surface was used to determine the amount of free antibodies by surface plasmon resonance detection. After each measurement the surface was regenerated by NaOH and HCl. In milk, the mean relative standard deviation of the assay was approximately 2% and the limit of detection less than 1 ppb. By introduction of a secondary sheep anti-rabbit antibody, the use of specific antibody could be reduced. Milk samples from the individual cow, herd, and tanker levels were analyzed and the relative standard deviations within each sample category were 4.4, 2.4, and 2.2%, respectively. The effect of some potential interferences, e.g., high somatic cells, bacterial contamination, and preservatives, was investigated. The results were not influenced in such a way that the risk for so-called false-positive findings was obvious.
Sulfamethazine Milk Sensor Sensor

"Peroxidase- And Tetracyanoquinodimethane-modified Graphite Paste Electrode For The Measurement Of Glucose/lactate/glutamate Using Enzyme-packed Bed Reactor"
Anal. Biochem. 1995 Volume 224, Issue 1 Pages 428-433
Pandey P. C. and Weetall H. H.

Abstract: A flow injection analysis sensor for the measurement of glucose/lactate/glutamate is reported. The glucose oxidase/glutamate oxidase/lactate oxidase was immobilized on silanized controlled pore glass particles and packed into a Teflon column (i.d., 1.2 mm; length, 40 mm) to give a bed for glucose/lactate/glutamate. The hydrogen peroxide formed by the enzymatic reaction in the packed bed was monitored by a horseradish peroxidase- and tetracyanoquinodimethane (TCNQ)- modified graphite paste electrode at 50 mV vs Ag/AgCl. The glucose oxidase/lactate oxidase/glutamate oxidase were regenerated in the packed bed, whereas peroxidase was regenerated in the TCNQ-mediated graphite paste electrode by the oxidation of TCNQ. The oxidized TCNQ was electrochemically reduced at 50 mV vs Ag/AgCl. The cathodic current obtained by the reduction of TCNQ determined the concentration of the injected analytes in the packed bed. The system showed very rapid response. Response curves for the analysis of peroxide, glucose, lactate, and glutamate are reported.
Glucose Lactate Glutamate Sensor Electrode Immobilized enzyme Controlled pore glass Column

"Flow Injection Immunoassay For Albumin Using Thiophilic Gels"
Anal. Biochem. 1994 Volume 222, Issue 1 Pages 281-283
Palmer D. A., Evans M. and Miller J. N.

Abstract: Albumin standards (100 l) were incubated for 2 min with fluorescein isothiocyanate-labelled albumin (10 l) and sheep anti-albumin antiserum (100 l; 1:49 dilution in PBS of pH 7.4) and then injected in to a FIA system (Anal. Lett, 1993, 26, 2543) incorporating an immunoreactor packed with T-gel (Pierce and Warriner Chemical Co., Chester, UK) using phosphate buffer of pH 8 containing 0.5 M K2SO4 (binding buffer). When the unbound peak returned to the baseline, the flow was switched to the elution buffer (phosphate buffer of pH 8) to elute the bound complex. Flow was then switched back to binding buffer for re-equilibration. The binding and eluting flow rate were 2.4 ml/min and detection was at 525 nm (excitation at 495 nm). The calibration graph for albumin is shown. The RSD were 6.3-8.7% and the limit of detection was 10 g/ml.
Albumin Biological Immunoassay

"Tetracyanoquinodimethane-mediated Flow Injection Analysis Electrochemical Sensor For NADH Coupled With Dehydrogenase Enzymes"
Anal. Biochem. 1994 Volume 221, Issue 2 Pages 392-396
Pandey P. C.

Abstract: A flow injection analysis (FIA) sensor for the oxidation of NADH is reported. The system utilizes a graphite paste electrode modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for the oxidation of NADH to biologically active NAD+. Alcohol dehydrogenase/lactate dehydrogenase and NAD+ were coimmobilized in TCNQ-modified graphite paste using polyethylenimine to develop a FIA sensor for ethanol/lactate. The system responded rapidly with wide linearity. Response curves for ethanol/lactate and NADH are reported.
Nicotinamide adenine dinucleotide reduced Biological Electrode Biotechnology Sensor Redox Immobilized enzyme

"Online Enzymatic Amplification By Substrate Cycling In A Dual Bioreactor With Rotation And Amperometric Detection"
Anal. Biochem. 1994 Volume 220, Issue 2 Pages 297-302
Raba J. and Mottola H. A.

Abstract: An integrated dual enzyme reactor/detection system employing substrate cycling for the determination of L-lactate (I), is described (diagram given). Lactate oxidase (LOD) was immobilized onto a rotating disc in the lower part of the reactor and lactate dehydrogenase (LDH) was immobilized onto the top part (~1 mm from the rotating disc) by applying a film of oxidized Glycophase G/CPG-SSO (Corning Biological products, Medfield, MA, USA) to the reactor surfaces, and then treating it with 5% glutaraldehyde, followed by enzyme solution The substrate cycling is based on the LOD-catalyzed oxidation of I to pyruvate with production of H2O2, and the LDH-catalyzed regeneration of I from pyruvate with NADH. The H2O2 was detected at a stationary Pt-ring electrode held at +0.6 V vs. Ag/AgCl, positioned concentrically to the disc. The reactor was incorporated into a continuous-flow/stopped-flow/continuous-flow system. For a cell volume of 450 µL a sample size of 89 µL, 0.1 M phosphate buffer of pH 7.5 containing 0.55 mM NADH as carrier, a rotation rate of 900 rpm and 60 s of stopped-flow, detection limits of ~0.3 M I were achieved, with a RSD of 1.22%. Sample throughput was 30/h.
l-Lactate Biological Amperometry Electrode Electrode Stopped-flow

"High Performance Liquid Chromatographic Analysis Of Phospholipids From Different Sources With Combined Fluorescence And Ultraviolet Detection"
Anal. Biochem. 1994 Volume 220, Issue 1 Pages 172-180
Bernhard W., Linck M., Creutzburg H., Postle A. D., Arning A., Martincarrera I. and Sewing K. F.

Abstract: Phospholipids (PL) were dissolved in CHCl3/methanol (1:4), mixed with N-monomethylphosphatidylethanolamine (200-300 µM, internal standard, prep. described) and analyzed by HPLC on a column (5 cm x 4.6 mm i.d.) of Schambeck Nucleosil 5 NH2 (5 µm) with a similar guard column (1 cm x 4.6 mm i.d.) coupled to a second column (17.5 cm x 4.6 mm i.d.) of the same material via a switch valve enabling the second column to be bypassed for elution of the acidic PL. The columns were operated at 50°C with acetonitrile/methanol/H2O/50% aqueous methylphosphonic acid (146:50:3:0.06) adjusted to pH 6.3 with 25% aq NH4OH as mobile phase (1 ml/min) and detection at 205 nm. Post-column derivatization was performed with 250 µL of Brij 35, 50 mg of NaN3 and 150 µL of diphenylhexatriene (3 mM in THF) in 1 l of water (4.5 ml/min) in Teflon tubing (200 cm x 0.8 mm i.d.) at 50°C, for fluorescence detection at 460 nm (excitation at 340 nm). For fluorescence detection the calibration graphs were linear for up to 60 nmol of phosphatidylcholine (PC) species and up to 100 nmol of other PL; the detection limits were 0.5-2 nmol. The UV absorption differed among PL classes.
Phospholipids Biological tissue HPLC Fluorescence Spectrophotometry

"Determination Of Lipoxygenase Activity By Continuous-flow Amperometric Detection"
Anal. Biochem. 1994 Volume 219, Issue 1 Pages 154-155
Daniel R., Dorizon J. and Deprez D.

Abstract: The assay is based on continuous measurement of the fatty acid hydroperoxides based on the electrochemical reduction of the hydroperoxides at a Hg electrode. Soy-bean lipoxygenase (0.04 unit) was incubated at 25°C in 3 mL of aerated 0.05 M borate buffer (pH 9.2) containing 0.025% of Polysorbate 20. The reaction was initiated by adding 300 µM linoleic acid. The product was measured amperometrically by reduction at 0 V with a forced mercury-drop electrode vs. an SCE reference electrode and a Pt auxiliary electrode. The amperometric response was linear up to 200 µM linoleic acid 13-hydroperoxide formed. The method could be used to assay the enzyme in the presence of inhibitors.
Enzyme, lipoxygenase Linoleic hydroperoxide Amperometry Electrode

"Automated Determination Of Antibody Oxidation Using Flow Injection Analysis"
Anal. Biochem. 1994 Volume 219, Issue 1 Pages 26-31
Wolfe C. A. C. and Hage D. S.

Abstract: The oxidation of antibody carbohydrate residues is a common approach used for site-specific antibody immobilization or modification. In this study a flow injection analysis system (FIA) was developed for monitoring antibody oxidation. Antibodies were oxidized with periodate and the resulting aldehyde groups were labeled with Lucifer yellow CH (LyCH). The labeled antibodies were then injected onto an FIA system where the amount of LyCH label was determined by absorbance measurements at 428 nm and the amount of antibody was determined using an online bicinchoninic acid protein assay. The analysis time was 2 min per 20 µL sample injection. The limits of detection for rabbit immunoglobulin G (IgG) and LyCH were 1 x 10^-8 and 4 x 10^-7 M, respectively. The dynamic ranges for IgG and LyCH extended to 2 x 10^-5 and 7 x 10^-3 M. The within-run precision was±5% or less for both analytes. Studies with known LyCH/antibody mixtures indicated that the FIA system had greater accuracy than manual methods at high LyCH levels. One specific application studied for this system was its use in monitoring the time course of periodate-antibody oxidation.
Immunoglobulins Biological Spectrophotometry Method comparison Process monitoring

"Influence Of Flow Rate On Biosensors Based On Redox Enzymes Incorporated In A Redox Polymer Mounted In A Thin-layer Flow Cell"
Anal. Biochem. 1993 Volume 215, Issue 2 Pages 261-265
Elmgren M., Nordling M. and Lindquist S. E.

Abstract: Two enzyme electrodes, based on the redox enzymes glucose oxidase and cellobiose oxidase immobilized in redox polymers at electrode surfaces, were mounted in a thin-layer flow cell, with a Ag/AgCl reference electrode and a Pt counter electrode. Glucose or cellobiose sample solution were analyzed by either continuous-flow or substrate plug techniques. The catalytic steady-state current for continuous-flow measurements increased with increasing flow rate, due to convection. For injected plugs of substrate, the peak current was dependent both on convection and on time of contact between electrode and substrate solution The kinetic constants of the reaction vary with different experimental conditions, so that the useful concentration range of the sensor can be adjusted by varying both the type of film and the flow rate.
Electrode Sensor Optimization Kinetic Flowcell Redox

"A Combined Cellobiose Oxidase/glucose Oxidase Biosensor For HPLC Determination Online Of Glucose And Soluble Cellodextrines"
Anal. Biochem. 1993 Volume 214, Issue 2 Pages 389-396
Nordling M., Elmgren M., Stahlberg J., Pettersson G. and Lindquist S. E.

Abstract: Highly specific biosensors can be prepared by immobilizing flavin-containing oxidases in a redox polymer on an electrode surface. By combining one glucose oxidase and one cellobiose oxidase electrode in a flow cell we have made a sensor for flow injection analysis, or post-column quantification, of glucose, cellobiose, and higher cellodextrines in an HPLC system. Samples of different concentrations of glucose and cellobiose, separately or mixed, were injected into the mobile phase and the current response was recorded simultaneously from both electrodes. The recorded response peak heights could be used for calibration curves. The usable measuring ranges were roughly 50 µM - 50 mM for glucose and 5 µM - 80 mM for cellobiose. Soluble cellodextrines, Glc1-6, could be separated on a C18 column by isocratic elution and detected by the sensor.
Cellobiose Cellotriose Cellotetraose Cellopentaose Cellohexaose HPLC Sensor Electrode Apparatus Detector

"An Amperometric Flow Injection Analysis Biosensor For Glucose Based On Graphite Paste Modified With Tetracyanoquinodimethane"
Anal. Biochem. 1993 Volume 214, Issue 1 Pages 233-237
Pandey P. C., Glazier S. and Weetall H. H.

Abstract: A biosensor system using flow injection analysis (FIA) has been developed for the analysis of glucose in human serum. The system consists of the enzyme glucose oxidase incorporated into graphite paste modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for oxidation of the reduced enzyme at 200 mV vs Ag/AgCl. The flow injection assay described has detection limits of 2 mM glucose using a 100 µL sample injection through a 250 µL sample loop. Data are presented to show the effect of sample injection volume and flow rate on the response of the FIA sensor. The biosensor exhibited excellent reproducibility for 800 injections. The loss of response after 800 injections was due to leaching of TCNQ from the graphite paste. Each assay takes 3 min giving a sample throughput of 20 per hour at a flow rate of 30 ml/h. The sensor was applied to the determination of glucose in human serum. The glucose measurements are in good agreement with those of a commercially available spectrophotometric method. Data showing the effect of interfering substances, ascorbic acid and acetaminophen, on the response of the sensor are also reported. A biosensor system was developed comprising glucose oxidase incorporated into a graphite paste modified with tetracyanoquinodimethane which acted as an efficient mediator for the oxidation of the reduced enzyme at 200 mV vs. Ag/AgCl. Serum (100 µL) was injected into a stream of 0.1 M phosphate buffer of pH 7 (30 ml/h) in a flow cell and the enzyme electrode was maintained at 200 mV vs. Ag/AgCl. The calibration graph was linear up to 200 mM glucose with a detection limit of 2 mM glucose. Total time for the procedure was 3 h with a sample throughput of 20/h.
Glucose Serum Human Amperometry Sensor Electrode Electrode Method comparison Redox Interferences

"Enzymatic Amplification Of A Flow-injected Thermometric Enzyme-linked Immunoassay For Human Insulin"
Anal. Biochem. 1993 Volume 212, Issue 2 Pages 388-393
Mecklenburg M., Lindbladh C., Li H. S., Mosbach K. and Danielsson B.

Abstract: A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025µg/ml and a linear range from 0.05 to 2 µg/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon online analysis are discussed.
Insulin Sensor Immunoassay Enzyme

"A Flow Injection Biosensor System For The Amperometric Determination Of Creatinine: Simultaneous Compensation Of Endogenous Interferents"
Anal. Biochem. 1993 Volume 210, Issue 1 Pages 163-171
Rui C. S., Sonomoto K., Ogawa H. I. and Kato Y.

Abstract: Creatinine deaminase and L-glutamate oxidase were immobilized separately on propylamine - controlled pore glass with glutaraldehyde through a Schiff base, and glutamate dehydrogenase was coupled to succinate - controlled pore glass activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The two channel flow injection system (illustrated) comprised a peristaltic pump, injection valve, immobilized enzyme packed in a glass tube and a control tube of controlled pore glass with delay coil, a galvanic O electrode with flow cell, a potentiostat and a recorder. Urine samples were diluted (x 20) and portions (30 µL) were introduced into the system at a flow rate of 1.6 mL min-1. Double peak recording was obtained using the delay coil in one channel; one peak being creatinine, endogenous ammonia and glutamate, and the other being endogenous ammonia and glutamate. Calibration graphs were rectilinear from 0.1 (detection limit) to 2 mM creatinine with coefficient of variation of 2.3% (within-day; n = 12) and 3.9% (between-day; n = 8). Lower detection limits (~0.01 mM), suitable for creatinine determination in serum, were obtainable by increasing the injection volume, e.g., to 100 µL. A flow injection biosensor system was developed for the amperometric determination of creatinine based on coupled reactions of three sequentially aligned enzyme reactors, creatinine deiminase, glutamate dehydrogenase, and glutamate oxidase, using an oxygen electrode as the detector. To overcome the problem of endogenous ammonia and glutamate, the flow was split into two channels after the injector and rejoined before the glutamate dehydrogenase reactor. Double peak recording was obtained by setting a delay coil and a reference column in one of the two channels. The first peak gave the sum response of creatinine, endogenous ammonia, and glutamate, and the second that of endogenous ammonia and glutamate. By this method compensation for endogenous ammonia and glutamate, as well as for interfering ascorbic acid, was achieved simultaneously. The system gave linear calibrations up to 2 mM for the first peak and 3 mM for the second one. The lower detection limits were 0.1 and 0.02 mM for 35- and 100 µL injection of sample, respectively. One run was completed within 2 min. The system showed good reproducibility (<3%) and long operational stability (> 1300 runs). The assay results of creatinine in urine showed good correlation with those obtained from the chemical method of Jaffe.
Creatine Urine Amperometry Sensor Kinetic Immobilized enzyme Interferences Controlled pore glass

"Peptide Mapping Using EOF-driven Capillary Isoelectric Focusing"
Anal. Biochem. 1993 Volume 208, Issue 2 Pages 323-329
Mazzeo J. R., Martineau J. A. and Krull I. S.

Abstract: Capillary isoelectric focusing (CIEF) in uncoated and commercially available coated capillaries was applied to the separation of tryptic peptides from bovine and chicken cytochrome c. This is the first report of peptide mapping by CIEF. Before separation, the pIs of individual peptides expected to be formed during the digest were calculated, and then the separation pattern obtained was correlated with the calculated pIs. This correlation was reasonably good for some peptides, but not as good for others. However, differentiation of the two species digests was easily achieved based on the CIEF map. The major limitation of the method is the fact that uv detection at 280 nm must be employed, meaning that only tryptophan and tyrosine containing peptides will be detected, since the ampholytes used to generate the pH gradient absorb below 280 nm. Post-column derivatization of the peptides is one solution to this problem. Migration time reproducibility was on the order of 2%, and run times were less than 20 min for the entire pH gradient range in the uncoated capillary. In the coated capillary, superior resolution was obtained at the expense of longer run times.
Peptides Spectrophotometry Isoelectric focusing Post-column derivatization pH gradient

"An Enzyme-reactor For Electrochemical Monitoring Of Choline And Acetylcholine: Applications In High Performance Liquid Chromatography, Brain Tissue, Microdialysis And Cerebrospinal Fluid"
Anal. Biochem. 1992 Volume 204, Issue 2 Pages 305-310
Frans Flentge, Kor Venema, Tineke Koch and Jakob Korf

Abstract: 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 online 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.
Choline Acetylcholine Brain Cerebrospinal Fluid HPLC Electrochemical analysis Post-column derivatization Immobilized enzyme Dialysis

"Solubilized Substrates For The Online Measurement Of Lipases By Flow Injection Analysis During Chromatographic Enzyme Purification"
Anal. Biochem. 1992 Volume 202, Issue 1 Pages 16-24
Gabriele C. Chemnitius, Helmut Erdmann* and Rolf D. Schmid

Abstract: A modular flow injection system was applied in the online assay of lipases. Substrate emulsion and lipase solution were simultaneously injected into carrier stream 1 and carrier stream 2, respectively, the carrier streams were combined and passed through a thermostated reaction coil before being passed to the photometer. 1,2-O-didodecyl-rac-glycero-3-glutaric resorufin ester was the preferred substrate with a reaction coil temperature of 37°C. With a combination of stopped-flow technique and zone sampling and detection at 571 nm, calibration graphs were rectilinear up to 800 IU mL-1 of lipase from Staphylococcus carnosus. Results for lipases from other species are presented. A flow injection analysis (FIA) system for the online measurement of lipases in chromatography processes was developed. The photometrically detectable substrates, p-nitrophenyl palmitate, S,O,O'-tripropyryl-1-thioglycerol, and 1,2-O-dilauryl-rac-glycero-3- glutaric resorufin ester were investigated. Different detergents and qualities of assay emulsions were tested for optimal results in FIA applications. Emphasis was placed on increasing the stability of the assay emulsion. Lipases of different origin and specificity were detected. The linear detection range was adapted to the requirements of the chromatography purifn. procedures. The connection of the FIA with a fast-protein liquid chromatography system permitted the automatization of lipase purifn. by monitoring protein content, salinity, and enzyme activity of the effluent from column chromatography.
Enzyme, lipase LC Spectrophotometry Heated reaction Stopped-flow Zone sampling Emulsion Surfactant

"High Performance Liquid Chromatographic Determination Of Catecholamines And Their Precursors And Metabolites In Human Urine And Plasma By Post-column Derivatization Involving Chemical Oxidation Followed By Fluorescence Reaction"
Anal. Biochem. 1992 Volume 200, Issue 2 Pages 332-338
Hee-Kyoung Jeon, Hitoshi Nohta and Yosuke Ohkura

Abstract: Urine or plasma was mixed with isoproterenol and 3,4-dihydroxyphenylpropionic acid (internal standards) and HClO4. After centrifugation, the supernatant solution was adjusted to pH 1.5 to 2.0 with 2 M K carbonate and centrifuged. The supernatant solution was applied to a Toyopak IC-SP S cartridge, eluted with 1.5 M KCl in 100 mM HCl - methanol (93:7; for urine) or 2 M NaClO4 - methanol (93:7; for plasma). Eluates were analyzed by HPLC (cf. Anal. Sci., 1991, 7, 257). The calibration graphs were rectilinear for 2 to 1000 pmol of L-dopa and 0.2 to 200 pmol for other catecholamines. The coefficient of variation were 1.9 to 5.4 and 3.2 to 7.3% for urine and plasma, respectively. Detection limits were from 0.5 to 95 pmol mL-1.
Catecholamines Plasma Human Urine HPLC Fluorescence Post-column derivatization

"Determination Of Organophosphorus And Carbamate Insecticides By Flow Injection Analysis"
Anal. Biochem. 1992 Volume 200, Issue 1 Pages 187-194
Satish Kumaran* and C. Tran-Minh

Abstract: For determination of the cited groups of insecticides, substrate solution (0.5 mM acetylcholine in 2.5 mM HEPES buffer solution of pH 8.0 containing 20 mM MgCl2, 100 mM NaCl and 0.01% of gelatin; 250 µL) was injected into a carrier stream (0.45 mL min-1) of HEPES buffer solution and the mixture was passed through a 10 cm single bead string reactor containing acetylcholinesterase (I) immobilized on glass beads (0.5 to 0.75 mm). The H+ produced was detected by a pH electrode with a wall-jet entry to assay I activity. Insecticide sample solution was passed through the reactor for 15 min instead of the working buffer. The working buffer was then reintroduced into the carrier line and the substrate solution was injected again to determine I activity. The concentration. of insecticide was determined by the inhibition of enzyme activity. I was reactivated by passing 20 µM 2-pyridine aldoxime methiodide solution through the reactor for 15 to 20 min. The method was applied in the analysis of simulated seawater. Calibration graphs are presented for paraoxon and malathion. Detection limits ranged from 0.5 ppb for malathion to 275 ppb for bromophos-methyl. A flow injection system, incorporating an acetylcholinesterase (AChE) single bead string reactor (SBSR), for the determination of some organophosphorus (azinphos-Et, azinphos-Me, bromophos-Me, dichlorovos, fenitrothion, malathion, paraoxon, parathion-Et, and parathion-Me) and carbamate insecticides (carbofuran and carbaryl) is presented. The detector is a simple pH electrode with a wall-jet entry. Variations in enzyme activity due to inhibition are measured from pH changes when the substrate (acetylcholine) is injected before and after the passage of the solution containing the insecticide. The percentage inhibition of enzyme activity is correlated to the insecticide concentration. Several parameters influencing the performance of the system are studied and discussed. The detection limits of the insecticides ranged from 0.5 to 275 ppb. The determination of these compounds was conducted in Hepes buffer and a synthetic seawater preparation The enzyme reactor can be regenerated after inhibition with a dilute solution of 2-PAM and be reused for analysis. The immobilized enzyme did not lose any activity up to 12 weeks when stored at 4°C.
Insecticides Paraoxon Malathion azinphos-methyl Azinphos ethyl Bromophos Dichlorophos Fenitrothion Ethylparathion Methylparathion Carbofuran Carbaryl Sea Electrode Immobilized enzyme Glass beads Single bead string reactor Buffer

"Semi-automated Flow Injection Procedure For Acetylcholinesterase And Cholinesterase Activities"
Anal. Biochem. 1992 Volume 200, Issue 1 Pages 176-179
Ignacio L&oacute;pez Garc&iacute;a, Fernando Ruiz Abell&aacute;n and Manuel Hern&aacute;ndez C&oacute;rdoba

Abstract: A stopped-flow flow injection method is described (details and diagram of apparatus given) for the assay of cholinesterase (I) and acetylcholinesterase (II) based on the Ellman colorimetric method. For determination of I, serum (100 µL) was diluted to 10 mL with phosphate buffer solution For determination of II, 25 µL of blood was mixed with 1 mL of isotonic saline. The erythrocytes, pelleted by centrifugation, were haemolysed in 1 mL of water and the haemolysate was analyzed. Sample solution (50 µL) was injected into a carrier stream (0.1 mL min-1) comprising 2 mM acetylthiocholine and 0.2 mM 5,5'-dithiobis-(2-nitrobenzoic acid) in 0.1 M phosphate buffer solution (pH 7.2) and the resulting stream was passed through a 46 cm reaction coil at 37°C to a flow cell where the flow was stopped to allow detection at 410 nm. The calibration graphs for I and II were rectilinear and the FIA was significantly more sensitive than the manual procedure. A stopped-flow FIA adaptation of the classical Ellman's colorimetric procedure for the measurement of acetylcholinesterase or cholinesterase activity is described. The samples are injected into a flow analytical system which is provided with an electronic timer and an electrovalve in order to stop the flow when the sample is inside the measurement cell. In this way, the absorbance-time relationship is obtained with a savings of sample, time, and reagents.
Acetylcholinesterase Enzyme, cholinesterase Blood Serum Blood Spectrophotometry Heated reaction Stopped-flow Buffer Valve Method comparison

"High Performance Liquid Chromatographic Determination Of Pyrophosphate In The Presence Of A 20,000-fold Excess Of Orthophosphate"
Anal. Biochem. 1991 Volume 199, Issue 2 Pages 279-285
Norimasa Yoza, Izumi Akazaki, Tetsuya Nakazato, Nobuyuki Ueda, Hiroki Kodama and Akira Tateda

Abstract: Mixed samples of orthophosphate (I) and pyrophosphate (II) (100 µL) were injected into the carrier stream of 0.23 M or 0.18 M KCl and 0.1% Na4EDTA (1 mL min-1) and was passed through a column (25 cm x 4 mm) of TSK-gel SAX (10 µm). The eluate was mixed with a reagent stream (0.5 mL min-1) of Mo(V) - Mo(VI) for reaction in a reaction coil (15 m x 0.5 mm) of PTFE at 140°C. The cooled mixture was analyzed at 330 to 800 nm. The presence of a 20,000-fold excess of I did not interfere seriously. The working range was from 0.3 to 500 µM I and II, with coefficient of variation of 2 to 10%.
Pyrophosphate HPLC Column EDTA Heated reaction Interferences

"Quantitation Of DNA And RNA In Crude Tissue Extracts By Flow Injection Analysis"
Anal. Biochem. 1991 Volume 199, Issue 1 Pages 137-141
Elaine M. Caldarone and Lawrence J. Buckley

Abstract: An automated two-dye flow injection analysis system to quantitate DNA and RNA in crude extracts of tissues is described. The method uses the fluorochrome dyes ethidium bromide and Hoechst 33258. DNA concentration is determined directly from its fluorescence in Hoechst dye. RNA is estimated from fluorescence in ethidium bromide after subtraction of the fluorescence due to DNA. This method has several advantages: a simple extraction procedure, a low detection limit (0.01 µg DNA and 0.10 µg RNA), automation, and a high sample throughput. Tissue was extracted with N-dodecanoylsarcosine (I) solution in Tris - EDTA buffer solution to give a final I concentration. in the supernatant solution of 0.1%. A diagram of the flow injection system is presented. DNA was determined from its fluorescence at 458 nm with 4-~5-(4-methylpiperazin-1-yl)[2,5'-bi-1H-benzimidazol]-2'-yl~phenol (excitation at 356 nm), and RNA from its fluorescence at 600 nm with homidium bromide (excitation at 525 nm). A DNA - homidium bromide calibration graph was also constructed and used to correct the RNA fluorescence for the presence of DNA. The detection limits were 0.01 µg for DNA and 0.1 µg for RNA. The method was applied to eggs, larvae and tissues of fish.
DNA RNA Fish Tissue Fish Fish Fluorescence Spectrophotometry

"Flow Injection Determination Of Proteins Using Enhanced Peroxyoxalate Chemiluminescence Applied To The Determination Of Immunoglobin G And Albumin In Serum"
Anal. Biochem. 1991 Volume 197, Issue 2 Pages 340-346
R. deLavalle and M. L. Grayeski

Abstract: The intensity of the chemiluminescence (CL) signal from an aqueous peroxyoxalate CL reaction can be significantly enhanced in the presence of various proteins with hydrophobic sites. A flow injection measurement for various hydrophobic proteins based on this CL enhancement was developed. The enhancement is due to the inclusion of the CL species in the favorable environment provided by the protein's hydrophobicity, which results in efficient light production. Various protein structures were evaluated; the degree of enhancement depends on the protein structure and CL reaction conditions. The CL enhancement measurement in the flow injection system is made after the introduction of the protein solution to the main phosphate buffer stream followed by the addition of the CL reagent streams: (1) hydrogen peroxide in water and (2) 8-anilino-1-naphthalene sulfonic acid and 4,4'-oxalylbis-(trifluoromethylsulfonylimino)ethylene bis(4-methyl morpholinium trifluoromethane sulfonate) in acetonitrile. Although prior separation of proteins is required before the measurement, the advantage of this approach is increased sensitivity without derivatization of the protein. The enhancement was demonstrated for several proteins, including antibodies, which suggests that this approach may be generally applicable to a variety of measurements, including immunoassay determinations. This CL enhancement was used to develop a simple and accurate flow injection measurement for the determination of albumin and IgG in human serum. A flow injection system (diagram presented) is presented for the determination of hydrophobic proteins, based on enhanced peroxyoxalate chemiluminescence. The protein sample was carried in the main phosphate buffer stream (pH 7.5) to mix with 4,4'-oxalyldisulfonylbis(trifluoromethylimino)diethylenebis-(4-methylmorpholinium trifluoromethanesulfonate) - 8-anilinonaphthalene-1-sulfonic acid reagent followed by H2O2. The resulting light emission was detected by a photomultiplier tube. Separation of proteins before measurement was achieved on open protein G - Sepharose 4 fast-flow columns. The method was applied to the development of an automated method to determine albumin and IgG in human serum.
Proteins Albumin Immunoglobulin G Serum Human Chemiluminescence Buffer Hydrophobic membrane

"Luminescent Immobilized Enzyme Test Systems For Inorganic Pyrophosphate: Assays Using Firefly Luciferase And Nicotinamide-mononucleotide Adenylyl Transferase Or Adenosine-5'-triphosphate Sulfurylase"
Anal. Biochem. 1991 Volume 197, Issue 1 Pages 266-272
Bruce A. Barshop*, David T. Adamson

Abstract: Cell culture growth medium was analyzed automatically with use of a luminescent immobilized enzyme test system (described) in which the sample was mixed with NAD - luciferin (I) or adenylophosphosulfate - I and passed through a column of immobilized NMN adenylytransferase or ATP sulfurylase, respectively, with continuous-flow luminescence detection. The limit of detection was 50 nM-pyrophosphate and the coefficient of variation was 15.3%. The method was applied to analysis of fibroblasts and lymphocytes.
Pyrophosphate Luminescence Automation Column Immobilized enzyme

"Determination Of Serum Cholinesterase Activity By Liquid Chromatography With Electrochemical Detection"
Anal. Biochem. 1991 Volume 196, Issue 2 Pages 377-384
R. Brent Miller and C. LeRoy Blank

Abstract: Serum was incubated at room temperature for 10 min in phosphate buffer solution (pH 7.2) containing ethylhomocholine, choline and acetylcholine. After addition of HClO4 and centrifugation, the supernatant solution was subjected to HPLC on a C18 (3 µm ODS) column (10 cm x 3.2 mm). The mobile phase (0.9 mL min-1) was Tris buffer solution (pH 7.5) containing acetic acid, tetramethylammonium chloride, Na octyl sulfate, NaN3, EDTA and acetonitrile. Post-column derivatization was effected with choline oxidase and acetylcholinesterase with electrochemical detection at 0.5 V vs. Ag - AgCl. The limit of detection was 3 pmol of choline or 0.1 µmol mL-1 h-1 of cholinesterase. The calibration graph was rectilinear for up to 269 µmol mL-1 h-1, with a coefficient of variation of 1.7%.
Enzyme, cholinesterase Blood Serum HPLC Electrochemical analysis Buffer C18 Column Heated reaction Post-column derivatization

"Reversed-phase Ion-pair High Performance Liquid Chromatography Method For Bovine Testicular Hyaluronidase Digests Using Post-column Derivatization With 2-cyanoacetamide And Ultra-violet Detection"
Anal. Biochem. 1991 Volume 196, Issue 1 Pages 183-191
Jeffrey A. Cramer and Leonard C. Bailey

Abstract: Bovine testicular hyaluronidase was incubated with hyaluronic acid hexasaccharide, at 37°C. After addition of the mobile phase [0.03 M tetrabutylammonium hydroxide (pH 9.0) - acetonitrile (4:1)], the mixture was subjected to HPLC on a column (15 cm x 4.1 mm) of Hamilton PRP-1 polymeric C18. The mobile phase flow rate was 0.8 mL min-1 and the eluant was mixed with 1% 2-cyanoacetamide in 0.2 M Na borate buffer (pH 9.0) for 2 min at 100°C before cooling and detection at 276 nm. The calibration graphs were rectilinear for 10 to 400 nmol of C-4 and C-6 oligomers and the coefficient of variation were 1.5 to 5.7%.
HPLC Spectrophotometry Buffer C18 Column Heated reaction Post-column derivatization

"Flow Injection Analysis Of Lactose Using Covalently Immobilized β-galactosidase, Mutarotase, And Glucose Oxidase/peroxidase On A 2-fluoro-1-methylpyridinium Salt-activated Fractogel Support"
Anal. Biochem. 1991 Volume 194, Issue 1 Pages 16-24
Dyer Narinesingh, Valerie A. Stoute, Gershwin Davis and That T. Ngo

Abstract: Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized β-galactosidase or β-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of α-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 µg/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed. Milk was incubated at 25°C for 10 min with concentrated HCl and then gravity-filtered. The filtrate was adjusted to pH 6.5 and mixed with 3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone in phosphate buffer solution (pH 6.5). The mixture was subjected to flow injection analysis at 33°C on a column of β-galactosidase in series with a bioreactor of glucose oxidase - peroxidase, with detection at 590 nm. The calibration graph was rectilinear for 160 µg mL-1 of lactose and the coefficient of variation was 2.7 to 3.8% (n = 3). Between 30 and 40 samples per h can be analyzed.
Lactose Milk Spectrophotometry Buffer Column Filtration Immobilized enzyme Method comparison pH Reactor Heated reaction

"Determinations Of Lactate And Lactate Dehydrogenase Activity In Serum With The Flow Injection Analysis System Involving Immobilized Enzyme Column And Chemiluminescence"
Anal. Biochem. 1991 Volume 193, Issue 1 Pages 112-117
Masayoshi Tabata, Masayuki Totani* and Takashi Murachi

Abstract: The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods. Serum was applied to a column of lactate oxidase - catalase immobilized on alkylamine glass beads. The eluate was incubated at 37°C for 2 min with phosphate buffer solution (pH 7.5) containing pyruvate and NADH. The mixture was applied to a lactate oxidase column and the eluate was subjected to chemiluminometric determination (J. Appl. Biochem., 1984, 6, 251). Lactate in serum was determined as above but the sample was applied directly on to the lactate oxidase column. The calibration graph was rectilinear for 5.6 mM lactate and the detection limit was 10 µM. For lactate dehydrogenase, the calibration graph was rectilinear for 1840 IU L-1 and the within- and day-to-day coefficient of variation were 3.0 and 3.9%, respectively. The results correlated well (r = 0.974) with those obtained on a Hitachi Model 726 discrete automatic analyzer. with use of pyruvate as substrate.
Lactate Enzyme, lactate dehydrogenase Blood Serum Chemiluminescence Buffer Column Glass beads Heated reaction Immobilized enzyme

"Flow Injection Analysis With Electrochemical Detection Of Reduced Nicotinamide Adenine Dinucleotide Using 2,6-dichloroindophenol As A Redox Coupling Agent"
Anal. Biochem. 1991 Volume 192, Issue 1 Pages 243-250
Hua T. Tang, Kiamars Hajizadeh, H. Brian Halsall* and William R. Heineman*

Abstract: The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 µM and with a detection limit of 0.38 µM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.
Nicotinamide adenine dinucleotide oxidized Blood Serum Electrochemical analysis Electrode Voltammetry Electrode Voltammetry Interferences Redox

"Comparison Of Methods For Following Alkaline Phosphatase Catalysis: Spectrophotometric Versus Amperometric Detection"
Anal. Biochem. 1991 Volume 192, Issue 1 Pages 90-95
Robert Q. Thompson*, George C. Barone, III, H. Brian Halsall, and William R. Heineman

Abstract: Alkaline phosphatase (I) catalysis was performed in a flow injection system with 4-aminophenyl phosphate and 4-nitrophenyl phosphate as substrates for amperometric and spectrophotometric detection, respectively; the products of the two reactions were 4-aminophenol (II) and 4-nitrophenol (III). Calibration graphs for II (0.025 to 1 µM) and III (0.2 to 4 µM) showed that the detection limit for II in the amperometric method (7 nM) was ~20-fold better than that for III in the spectrophotometric method. Also, 0.05 µg L-1 of I could be detected by the amperometric method, which was almost an order of magnitude better than by the spectrophotometric method. The amperometric detection method was specially suited to small sample volume since electrochemical cells can be reduced in volume much more readily than can spectrophotometric cells and without loss in sensitivity.
Enzyme, alkaline phosphatase Amperometry Spectrophotometry Catalysis Method comparison Small sample

"Determination Of Proteolytic Enzymes By Flow Injection Analysis"
Anal. Biochem. 1991 Volume 192, Issue 1 Pages 70-73
Pierre Nicolas, Annick Lamy and Sylviane Reymond

Abstract: Quantitation of proteolytic enzymes using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide has been adapted to flow injection analysis. This procedure has been developed using two different proteases: subtilisin and chymotrypsin. For both enzymes the influence of substrate concentration on spectrophotometric response has been studied. The assay is based on the merging zones technique combined with a washing step. Results are obtained in less than 15 s and samples may be run at a rate of 90/h with good reproducibility. A linear relation between peak heights and enzyme concentrations was observed for 0-0.15 Anson unit/liter of subtilisin and for 0-30 mg/liter of a commercial preparation of chymotrypsin. The method requires only small sample volumes, and the consumption of the chromogenic substrate is reduced to a minimum by using intermittent pumping. The flow injection system described for the determination of subtilisin (I) and chymotrypsin (II) is based on the merging-zones principle with intermittent pumping; N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide was used as substrate. High sampling rates were achieved by incorporating a second pump to flush the system with a wash solution Injection of samples into the buffer stream activated a pump that immediately delivered the substrate at the merging point and propelled the sample into the system. When the top of the peak had been recorded, the first pump was stopped and a second one was started, thereby delivering the wash buffer at a higher rate. The absorbance was monitored at 405 nm. The calibration graphs were rectilinear in the ranges 0.5 to 2 mM I and 0.75 to 2 mM II. Results were obtained in 15 s and samples could be analyzed at 90 h-1 with good reproducibility.
Enzymes, proteolytic Buffer Intermittent pumping Merging zones Small sample

"Fluorometric Determination Of Urinary Kynurenic Acid By Flow Injection Analysis Equipped With A"
Anal. Biochem. 1990 Volume 190, Issue 1 Pages 88-91
Ken-ichi Mawatari, Fumio Iinuma and Mitsuo Watanabe

Abstract: A flow injection analysis involving a photochemical reaction and fluorometric detection has been developed for the determination of urinary kynurenic acid. Kynurenic acid was found to fluoresce on irradiation with ultraviolet light at pH 7.2 in the presence of hydrogen peroxide. This method was applied to flow injection analysis using a new procedure involving a 'bypass line' for the simultaneous determination of urinary kynurenic acid and background fluorescence. The calibration graph showed linearity over the range of 0.20 to 120 pmol. For pretreatment of urinary kynurenic acid, a PRE-SEP C18 cartridge was used. The mean recovery of kynurenic acid from urine was 94.5%. The content of urinary kynurenic acid was 13.0±2.68 µmol/day. There was good correlation (r = 0.9729) between values determined by flow injection analysis and high performance liquid chromatography. Urine, diluted 25-fold with 0.2 M Na2HPO4 - 0.1 M citric acid buffer (pH 3.2), was applied to a PRE-SEP C18 cartridge and kynurenic acid (I) was eluted with phosphate buffer solution (pH 7.2) containing 20% methanol. The eluate was subjected to flow injection analysis with 0.07 M phosphate buffer (pH 7.2) containing 25 mM H2O2 - methanol (8:2) as carrier solution (1.2 mL min-1). After injection into the carrier solution the stream was split; one part was passed through a photochemical reaction coil and the second part, for background correction, bypassed the reaction coil. Fluorescence detection was at 465 nm (excitation at 370 nm). The calibration graph was rectilinear for 0.20 to 120 pmol of I. Recovery was 94.5% and results correlated well (r = 0.973) with those by HPLC.
Kynurenic acid Urine HPLC Fluorescence Photochemistry Calibration Dilution pH Method comparison

"Determination Of Tin In Biological Samples Using Gaseous Hydride Generation - Inductively Coupled Plasma Atomic Emission Spectrometry"
Anal. Biochem. 1990 Volume 190, Issue 1 Pages 71-77
Katsuhiko Yokoi, Mieko Kimura and Yoshinori Itokawa

Abstract: Liver, brain, testis or kidney was homogenized and heated with HNO3 and HClO4 in Kjeldahl flasks until white fumes appeared. Water was added with heating until the solution was clear and colorless. An aliquot of the solution was treated with trichloroacetic acid (I) and diluted with water to 0.2% in I. This solution was mixed, in a continuous-flow hydride-generation system (diagram given), with 1% I solution and then with 0.5% NaBH4 in 0.1% NaOH solution and passed through a gas - liquid separator (design presented) and the hydride, in an Ar-stream, was determined by ICP-AES at 189.989 nm. The limit of detection was 30 pg mL-1 of Sn, mean recovery was 87 to 99% and the coefficient of variation was 1.2%.
Tin Liver Brain Kidney Testis Spectrophotometry Dilution Phase separator Detection limit Volatile generation Volatile generation Kjeldahl

"Flow Injection Analysis Of Serum Urea Using Urease Covalently Immobilized On 2-fluoro-1-methylpyridinium Salt-activated Fractogel And Fluorescence Detection"
Anal. Biochem. 1990 Volume 188, Issue 2 Pages 325-329
D. Narinesingh, R. Mungal and T. T. Ngo

Abstract: Serum samples were analyzed for their urea content using fluorescence flow injection analysis incorporating an immobilized urease bioreactor and a gas permeable separator. The urease was immobilized under mild and facile conditions to a hydrophilic 2-fluoro-1-methylpyridinium-activated support. The ammonia released as a result of urease-catalyzed urea hydrolysis diffused through a gas permeable membrane into a constant stream of o-phthaldehyde solution to form a highly fluorescent product with lambda ex at 340 nm and lambda em at 455 nm. Up to 25 serum samples can be analyzed per hour. The within-day coefficient of variation (CV) was 1.12% and the day-to-day CV was 1.25% for serum containing 10.50 mg urea nitrogen dl-1. The bioreactor shows excellent storage (at 4°C) and operational stabilities (at 37°C). Urea was determined in serum by flow injection analysis (illustrated) with urease immobilized on 2-fluori-1-methylpyridinium toluene-4-sulfonate-activated Fractogel. The ammonia released diffuses through a gas-permeable membrane into a stream of phthalaldehyde. The fluorescence of the resulting product is measured at 455 nm (excitation at 340 nm). Calibration graphs were rectilinear up to 1400 mg dl-1 and the detection limit was 0.1 mg dl-1. Recovery was 98 to 103% and coefficient of variation were 1.5%.
Urea Blood Serum Fluorescence Enzyme Immobilized enzyme Gas diffusion Membrane Calibration Detection limit

"Determination Of Phosphatidylcholine In A Flow Injection System Using Immobilized Enzyme Reactors"
Anal. Biochem. 1990 Volume 187, Issue 2 Pages 240-245
Mohammed Masoom, Rita Roberti and Luciano Binaglia

Abstract: Two alternative procedures are described for the quantitative determination of phosphatidylcholine in a flow injection system utilizing immobilized enzymes. Phospholipase C from Bacillus cereus and phospholipase D from cabbage were covalently bound to the surface of controlled-pore glass beads and the enzyme-derivatized beads were packed in small columns. In the first procedure, the phospholipase C column was connected with a second column containing coimmobilized alkaline phosphatase and choline oxidase. In the alternative procedure, the column packed with immobilized phospholipase D was connected with a column packed with immobilized choline oxidase. The hydrogen peroxide produced through the action of choline oxidase in both flow injection systems was detected amperometrically. Both procedures are suitable for an accurate and rapid quantitation of phosphatidylcholine. The sensitivity of the method based on phospholipase C and alkaline phosphatase is higher than that using phospholipase D. Quantitation of phosphatidylcholine at the nanomole level can be easily obtained using the first methodology. Membrane lipids were extracted from brain by the method of Folch et al. (J. Biol. Chem., 1957, 226, 497) and dissolved in CHCl3 - methanol (2:1). The solution was evaporated in vacuo and the lipid residue was dissolved in 0.1 M Tris - HCl buffer of pH 7.5 containing 0.3% of Triton X-100 and 0.4 mM ZnCl2. The suspension was injected into a stream (pH 6.5) of 30 mM CaCl2 containing 0.3% of Triton X-100 and 20 mM diethylbarbitone and passed through columns of phospholipase C immobilized on glass beads and of alkaline phosphatase and choline oxidase immobilized on glass beads before electrochemical detection with a vitreous-carbon electrode at +0.6 V. The calibration graph was rectilinear for 0.05 to 2 mM phosphatidylcholine. The sensitivity was higher than that of a flow injection method using phospholipase D.
Phosphatidylcholine Amperometry Electrochemical analysis Electrode Immobilized enzyme Enzyme Column Controlled pore glass Sensitivity Membrane Buffer pH Triton X Glass beads Surfactant

"Kinetics Of Protease Hydrolysis Of Extended Peptide Substrates: Measurement By Flow Injection Analysis"
Anal. Biochem. 1990 Volume 186, Issue 2 Pages 269-272
Georges L. Chong, Lawrence C. Davis* and Gerald R. Reeck

Abstract: A flow injection analysis (FIA) system was developed to study the enzyme-catalyzed hydrolysis of synthetic peptides, each of which contained one scissile bond. The concentrations of α-amino groups in reactions mixtures were determined by FIA with o-phthalaldehyde as a fluorescence reagent. The method allows a rapid, precise, and sensitive determination of kinetic constants for proteases acting on extended peptide substrates.
Enzyme, protease Fluorescence Kinetic Enzyme

"Metalloprotein Separation And Analysis By Directly Coupled Size Exclusion High Performance Liquid Chromatography Inductively Coupled Plasma Mass Spectroscopy"
Anal. Biochem. 1990 Volume 186, Issue 2 Pages 187-201
A. Z. Mason*, S. D. Storms and K. D. Jenkins

Abstract: The feasibility of using directly coupled size exclusion high performance liquid chromatography inductively coupled plasma mass spectroscopy (HPLC/ICP-MS) for the separation and subsequent elemental analysis of metalloproteins in biological samples has been studied. Data, on up to eight elements, was acquired simultaneously and the reconstructed elemental profiles from the chromatographed samples were quantified by flow injection analysis. Absolute and relative detection limits, reproducibility, operational dynamic range, and linearity of response were initially evaluated by analyzing standards of metallothionein protein of known elemental composition for Cd, Zn, and Cu. There was evidence of displacement of Zn from the protein during chromatography and the substitution of Cu sequestered from the mobile phase. Cd associated with the protein was fully recovered during chromatography. Memory effects, due to protein adsorption to the glassware in the torch box, were minimal and there was no degradation of the resolution of the chromatographed peak during extended transport through the HPLC/ICP-MS interface. The versatility of the technique has been demonstrated by the quantitative multi-element analysis of cytosolic metal-binding proteins separated from the polychaete worm Neanthes arenaceodentata. Fidelity of analysis has been demonstrated by two independent procedures: first, by comparing the elemental profiles obtained by directly aspirating the HPLC eluant into the ICP-MS to those obtained by collecting fractions and quantifying the metal content of the proteins in the conventional analytical mode; second, by comparing the stable isotopic profiles for 114Cd obtained by simultaneous ICP-MS analysis with radiometric profiles of 109Cd obtained by counting radioactivity associated with collected fractions.(ABSTRACT TRUNCATED AT 250 WORDS)
Proteins, metallo Mass spectrometry HPLC Detection limit Linear dynamic range Interface Multielement

"Determination Of Methylguanidine In Plasma And Urine By High Performance Liquid Chromatography With Fluorescence Detection Following Post-column Derivatization"
Anal. Biochem. 1990 Volume 184, Issue 2 Pages 213-218
Venkata K. Boppana*, Gerald R. Rhodes* and David P. Brooks

Abstract: Plasma (1 ml) was mixed with 0.2 µg of ethylguanidine as internal standard, adjusted to pH 11 with 26 µL of 1 M NaOH and applied to a 1 mL weak-cation-exchange extraction column, which was washed with water before elution with methanolic 1% trifluoroacetic acid. After evaporation under N, the residue was dissolved in 0.2 mL of the mobile phase [0.06 M chloroacetate buffer (pH 4.0) - methanol (17:3)], and a 0.1 mL portion was subjected to HPLC on an Ultrasil cation-exchange silica (10 µm) column (25 cm x 4.6 mm) at 60°C with a mobile phase flow rate of 0.75 mL min-1, post-column mixing at 70°C with NaOH and ninhydrin solution and fluorescence detection with a 470 nm cut-off filter (excitation at 390 nm). Urine was diluted tenfold with water before injection of a 20 µL portion. Calibration graphs were rectilinear for 1 to 1000 ng mL-1 of methylguanidine in plasma and 0.1 to 600 µg mL-1 in urine, and the detection limit was 100 pg. The intra- (n = 5) and inter-day (n = 15) coefficient of variation for 10, 100 and 1000 ng mL-1 of methylguanidine in plasma were 2.49, 1.30 and 1.38% and 7.51, 5.20 and 7.98%, respectively.
Methylguanidine Blood Plasma Urine HPLC Fluorescence Post-column derivatization Silica Detection limit

"Flow Injection Fluorometry Of Protein Using Hypochlorite-thiamine Reagent"
Anal. Biochem. 1990 Volume 184, Issue 1 Pages 184-188
Toshio Yokoyamaa, Noriko Nakamurab and Toshio Kinoshitac

Abstract: This paper describes a flow injection protein assay based on the formation of N-chlorides. Thiamine, which gives fluorescent thiochrome on reaction with N-chlorides, is used as a reagent. The protein sample is first mixed with the carrier solution containing sodium hypochlorite to chlorinate peptide bonds. The fluorescence reagent, containing thiamine and sodium nitrite, is then delivered to the mixture; the sodium nitrite decomposes active chlorine. The assay is sensitive, reproducible, and linear over a range from 20 ng to 2 µg of bovine serum albumin. The fluorescence intensity reflects the correct amount of protein because the thiochrome formed is proportional to the number of peptide bonds. The method is based on that of Kinoshita et al. (Chem. Pharm. Bull., 1976, 24, 2901; 1980, 28, 641). The flow injection system is described and illustrated. Injected protein samples are heated in a spiral coil at 70°C with NaClO solution in a 50 mM phosphate buffer carrier stream of pH 7.5 containing 0.1% of Brij 35 (total available Cl content 0.05%). After this, the N-chloroproteins formed are mixed with phosphate-buffered 0.5% NaNO2 - 0.005% thiamine hydrochloride solution in a second reactor coil at 70°C. The unconsumed ClO- is thereby destroyed and the thiamine to oxidized by the N-chloroproteins to thiochrome. The flow stream is then cooled to 25°C and the fluorescence is measured at 440 nm (excitation at 370 nm). The response is rectilinear for 20 ng to 2 µg of protein (e.g., bovine serum albumin, for which the detection limit is 10 ng). Some compounds interfere, e.g., those with NH-groups (e.g., tryptophan, proline and some purine bases); it is suggested that interfering compounds can be removed by prior HPLC.
Protein HPLC Fluorescence Sensitivity Linear dynamic range Buffer pH Detection limit Interferences

"Determination Of Inorganic Phosphate By Flow Injection Method With Immobilized Enzymes And Chemiluminescence Detection"
Anal. Biochem. 1989 Volume 182, Issue 2 Pages 366-370
Hideki Kawasaki, Katsumi Sato, Jyunko Ogawa, Yukio Hasegawa and Hidetaka Yuki

Abstract: Inorganic phosphate (I) was determined by flow injection analysis with use of two glass columns (0.9 cm x 3 mm), one packed with purine-nucleoside phosphorylase-coated controlled-pore glass beads (AMP-500; 120 to 200 mesh), and the second packed with xanthine oxidase and urate oxidase-coated beads (preparation described). The carrier solution (1 mL min-1) was 25 µM inosine in 10 mM HEPES - NaOH buffer (pH 7.5) and the H2O2 produced was determined with use of 10 µM luminol and 0.8 µM peroxidase in 50 mM carbonate buffer (pH 10.5) and chemiluminescence detection. The calibration graph was rectilinear for 5 to 250 pmol of K2HPO4 and the detection limit was 500 fmol. The within-assay coefficient of variation was 1.03 to 9.36%. The method was applied in determination of I in DNA.
Phosphate DNA Chemiluminescence Immobilized enzyme Glass Column Controlled pore glass Buffer

"Determination Of Thiobarbituric Acid-reactive Substances In Oxidized Lipids By High Performance Liquid Chromatography With A Post-column Reaction System"
Anal. Biochem. 1989 Volume 182, Issue 1 Pages 116-120
Kazuaki Yoden* and Toshihiro Iio

Abstract: Free malonaldehyde (I) and other thiobarbituric acid-reactive substances were determined in oxidized lipids (prep. described) by HPLC on a column (15 cm x 6 mm) of Inertsil ODS-2 (5 µm) with linear gradient elution (0.7 mL min-1) from aqueous 50 to 100% methanol over 30 min, and held at 100% methanol for 15 min. The eluate was mixed (0.7 mL min-1) with 0.5% 2-thiobarbituric acid in 0.05 M HCl in a reaction coil (20 m x 0.4 mm) at 55°C and the resulting red pigment was determined fluorimetrically at 553 nm (excitation at 515 nm). The calibration graph was rectilinear up to 4 nM-I and the detection limit was 0.5 nM. The procedure was applied in the analysis of thermally oxidized methyl linoleate, the degradation products of methyl linoleate hydroperoxides and the oxidation products of rat liver microsomes.
Thiobarbituric acid Liver HPLC Fluorescence Post-column derivatization Column Gradient technique Calibration Detection limit

"Flow Injection Binding Assays: A Way To Increase The Speed In Binding Analyses"
Anal. Biochem. 1989 Volume 181, Issue 2 Pages 379-382
Bo Mattiasson, Per Berd&eacute;n and Torbj&ouml;rn G. I. Ling

Abstract: A flow injection binding assay for glucosides and mannosides is described. The sample was mixed with enzyme label (horseradish peroxidase) and passed through a column of immobilized concanavalin A. Bound activity was measured by passing substrate (to peroxidase) and chromogenic reagent through the column and monitoring the effluent spectrophotometrically. To speed up the assay, the sample was applied in pulses and the substrate was included in the column regeneration buffer. Calibration graphs for glucosides and mannosides were rectilinear for 0.02 to 2 mM.
Carbohydrates Glycoproteins Glycosides Mannosides Spectrophotometry Enzyme Column Immobilized reagent Buffer Injection technique Chromogenic reagent

"Continuous-flow Automated Assay For Iodimetric Estimation Of Hydroperoxides"
Anal. Biochem. 1989 Volume 176, Issue 2 Pages 353-359
Sian M. Thomas*, Wendy Jessup, Janusz M. Gebicki

Abstract: Lipid hydroperoxides in aqueous samples or heptane extracts of biological materials were determined by an automated continuous-flow iodimetric method. The reagent was 1% KI in methanol - acetic acid (2:1) (made 1 mM with respect to desferal, used to inactivate contaminating Fe) and the wash solution was 2% acetic acid. The formation of tri-iodide ions was measured at 360 nm. All operations were protected from light. The detection limit was 60 pmol of hydroperoxide; coefficient of variation were 3, 3.6 and 2.5% (n = 10) for serum, aqueous lipid and heptane extracts of biological material, respectively. Results correlated well with those by the manual iodimetric assay (r = 0.999).
Hydroperoxides, lipid Blood Serum Spectrophotometry Detection limit Method comparison

"Determination Of Acetylcholine And Choline By Flow Injection With Immobilized Enzymes And Fluorimetric Or Luminometric Detection"
Anal. Biochem. 1989 Volume 176, Issue 2 Pages 221-227
Jan R&iacute;cn&yacute;, Jil&iacute; Coupek and Stanislav Tucek

Abstract: Acetylcholine and choline were determined in solution and tissue extracts by a continuous-flow system with a sequence of enzyme reactors containing immobilized acetylcholinesterase, choline oxidase and peroxidase. Additional reactors with immobilized choline oxidase and catalase were used to remove choline and choline-generated H2O2 from samples when acetylcholine was being determined. Acetylcholinesterase, choline oxidase and catalase were immobilized on Separon HEMA E and peroxidase was immobilized on Separon HEMA (for fluorimetric detection) or on a coiled glass capillary or on controlled-pore glass beads (for luminometric detection). Detection was by measuring fluorescence generated from the reaction of H2O2 with 3-(4-hydroxyphenyl)propionic acid in presence of peroxidase, or measuring luminescence generated from the reaction of H2O2 with luminol in presence of peroxidase. Calibration graphs were rectilinear up to 100 pmol of acetylcholine with fluorescence detection. Detection limits were 10 or 25 pmol of acetylcholine with the coiled-capillary peroxidase or controlled-pore-glass peroxidase columns, respectively, with luminometric detection, and 0.2 pmol with fluorimetric detection.
Acetylcholine Choline Biological tissue Luminescence Fluorescence Immobilized enzyme Catalysis Controlled pore glass Glass beads Detection limit Column

"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 Cell HPLC Fluorescence Post-column derivatization

"Determination Of Glutathione In Biological Material By Flow Injection Analysis Using An Enzymic Recycling Reaction"
Anal. Biochem. 1988 Volume 174, Issue 2 Pages 489-495
F. A. M. Redegeld*, M. A. J. van Opstal, E. Houdkamp and W. P. van Bennekom

Abstract: Rat hepatocyte preparation or rat liver homogenate was deproteinized with HClO4. For determination of oxidized glutathione(I), N-ethylmaleimide was added; the excess was destroyed by alkaline hydrolysis (pH 11.2; 3 M potassium phosphate buffer) and HClO4 was added to neutralize. After centrifugation, the supernatant solution was injected into a carrier buffer (0.4 M potassium phosphate of pH 6.75) for flow injection analysis. The reagent solution (each at 0.2 mL min-1) were 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) in carrier buffer and 0.5 mM NADPH containing 3 iu mL-1 of glutathione reductase (in carrier buffer). The mixture flowed at 0.6 mL min-1 through a single-bead string reactor for enzymatic recycling before measurement of absorbance at 412 nm. The calibration graphs were rectilinear up to 200 and 400 pmol of oxidized and reduced I, respectively. The detection limit was 1 pmol. Glutathione was determined in isolated rat hepatocytes and results correlated well with those by spectrophotometry (r = 0.977). A sensitive and specific assay for glutathione using a recycling reaction followed by spectrophotometric detection in a flow injection analysis system is presented. The proposed method provides specific amplification of the response to glutathione by combined use of the enzyme GSSG reductase and the chromogenic reagent 5,5'-dithiobis(2-nitrobenzoic acid). Both oxidized (GSSG) and reduced (GSH) glutathione are detected, so that GSSG must be determined separately after alkylation of the GSH with N-ethylmaleimide. The sensitivity is controlled by the number of times the cycle occurs and therefore by the residence time of the sample in the reactor. This time depends on the reactor length and the flow rate. The influence of residence time, temperature, and enzyme concentration on the response has been studied and the optimum reaction conditions have been selected. The sample throughput is as high as 30 h-1 and the detection limit is 1 pmol GSH at a signal-to-noise ratio of 3. The method has been evaluated by the quantification of GSH and GSSG in isolated hepatocytes. A high correlation between the new flow injection analysis method and the original spectrophotometric batch assay has been found (slope = 1.039, intercept = 0.6, n = 216, r = 0.977). The main advantages of the proposed method are high sample throughout, high sensitivity, and good reproducibility.
Glutathione Liver Rat Spectrophotometry Calibration Method comparison Optimization Chromogenic reagent

"Continuous Spectrophotometric Assay For The Enzymatic Marker Glucose 6-phosphatase"
Anal. Biochem. 1988 Volume 174, Issue 1 Pages 32-37
Maria Stio, Paolo Vanni* and Giancarlo Pinzauti

Abstract: A continuous spectrophotometric assay for glucose 6-phosphatase is described. The method uses glucose dehydrogenase and mutarotase as ancillary enzymes. Glucose 6-phosphatase activity is measured by following NADH formation at 340 nm. The method is linear, at least up to 38 mU in the test which corresponds to a ΔE of 0.24 min-1, when the enzyme is assayed in a microsomal fraction. We also discuss the method's suitability for subcellular fractionation. No other continuous assay for this important enzymatic marker of the endoplasmic reticulum is currently available.
Enzyme, glucose 6-phosphatase Spectrophotometry

"A Stopped-flow Assay For Glycogen Phosphorylase Appropriate To Measure Catalytic Activity At High Enzyme Concentrations"
Anal. Biochem. 1988 Volume 173, Issue 2 Pages 235-240
M. Company, J. Zuluaga, P. Mart&iacute;nez and J. S. Jim&eacute;nez*

Abstract: Glycogen phosphorylase (EC 2.4.1.1) may be assayed in the glycogen degradation direction by a continuous spectrophotometric method. The formation of glucose 1-phosphate from glycogen and phosphate produces a controlled change of pH which can be measured by the changes in absorbance of phenol red added to the system. The procedure may be conveniently applied to a stopped-flow spectrophotometer to measure the rate of the reaction. Therefore the activity of the enzyme may be determined at low conventional concentrations and, by the same technique, at high enzyme concentrations approaching those supposed to exist in vivo.
Enzyme, glycogen phosphorylase Stopped-flow

"Immobilization Of Linamarase And Its Use In The Determination Of Bound Cyanide In Cassava Using Flow Injection Analysis"
Anal. Biochem. 1988 Volume 172, Issue 1 Pages 89-95
D. Narinesingh*, D. Jaipersad and I. Chang-Yen

Abstract: β-Glucosidase was immobilized (details given) on 2-fluoro-N-methylpyridinium-activated Fractogel support; the gel was washed and packed in nylon columns (5 cm x 2 mm) for use in flow injection analysis. Cassava leaves or tubers were extracted with H3PO4, the extracts were filtered under vacuum and the filtrates were adjusted to pH 7.2 with 1 M NaOH and centrifuged at 3000 rpm for 5 min. Portions (90 µl) of the supernatant solution were injected into the flow injection system, at 30°, and passed (0.33 mL min-1) into the reactor column, or a blank column to correct for free CN-. The solution were then mixed with picrate reagent of pH 10.8 and passed through a coil at 85°C and the absorbance of the complex formed was measured at 525 nm. The calibration graph was rectilinear, for both free CN- and CN- liberated from linamarin, from 1 to 10 mM; the detection limit was 67 ppm. Interference from glucose and acetone was negligible. Extracts from the tubers (cortex and parenchyma) and leaves of Manihot esculenta Crantz (cassava) were analyzed for their releasable cyanide content using flow injection analysis incorporating an immobilized linamarase bioreactor. Linamarase was immobilized under very mild conditions to an activated 2-fluoro-N-methylpyridinium Fractogel support. The released cyanide, which was monitored spectrophotometrically at 525 nm using an alkaline picrate reagent, was found to be highest in the cortex and lowest in the parenchyma.
Cyanide Leaves Tubers Sample preparation Spectrophotometry Interferences Heated reaction Immobilized enzyme Interface

"Liquid Chromatographic Determination Of Amino-acids Using A Hollow-fiber Membrane Reactor"
Anal. Biochem. 1988 Volume 171, Issue 2 Pages 398-403
Jun Haginaka* and Junko Wakai

Abstract: Eighteen common amino-acids were determined by HPLC on a column (30 cm x 4.6 mm) of Develosil ODS-5 (5 µm) with a guard column (3 cm x 4.6 mm) of the same packing material. The mobile phase (0.8 mL min-1) was a five-step gradient (described) from 0 to 80% of 15 mM Na octanesulfonate - 21 mM H3PO4 - 9 mM NaH2PO4 - methanol (1:1:1:6; pH 4.2 in 20:20:20:1; pH 2.8). Post-column derivatization was carried out at 60°C by reaction with phthalaldehyde and 2-mercaptoethanol (combined with hypochlorite oxidation for proline), introduced into the main stream with use of sulfonated and aminated hollow-fiber membrane reactors, respectively. Derivatives were detected fluorimetrically at 450 nm (excitation at 340 nm). Detection limits were 0.4 to 20 pmol for the 18 amino-acids. The method was applied in the determination of amino-acids in protein hydrolysates.
Amino Acids HPLC Fluorescence Heated reaction Hollow fiber membrane Post-column derivatization

"Sequestration Electrochemistry: The Interaction Of Chlorpromazine And Human Orosomucoid"
Anal. Biochem. 1988 Volume 171, Issue 2 Pages 290-293
D. Scott Wright, Mark L. Friedman, Sarah H. Jenkins, William R. Heineman* and H. Brian Halsall*

Abstract: A simple and rapid method is presented for determination of the association constants and stoichiometries describing ligand macromolecule interactions. Based on flow injection analysis and electrochemical detection by amperometry, the only requirements for direct measurements are that the ligand have redox properties and that these properties change upon binding to the macromolecule. Bound ligand may then be measured in the presence of free ligand. Detection limits are of the order of 2 pmol of ligand or less, a level that should provide access to previously unmeasurable systems. For the exemplary system, chlorpromazine and human orosomucoid, K0ass was determined as 0.39 x 10^6 M-1 with 0.76 chlorpromazine binding sites of this affinity per orosomucoid molecule.
Chlorpromazine Orosomucoid Human Blood Amperometry Stability constants

"Direct Nanogram Quantitation Of Nucleic Acid And Protein With A Continuous-flow Microcell"
Anal. Biochem. 1988 Volume 169, Issue 1 Pages 138-141
Jingdong Zhu, Abbas Rashidbaigi and Sidney Pestka

Abstract: The samples were injected into the post-column flow stream of an HPLC system as the stream was pumped past a diode-array detector set at 260 nm for nucleic acids and 280 nm for proteins. Response was rectilinear between 3 ng and 7 µg of DNA, 10 ng and 10 µg of RNA and 10 ng and 80 µg of bovine serum albumin. The method was simple, required only 5 to 10 µL of sample solution, and was rapid (2 samples min-1).
Nucleic acids, ribo Nucleic acids, deoxyribo Albumin Cow Serum Spectrophotometry Post-column derivatization

"Detection Of Polyols And Sugars By Cuprammonium Ion In The Presence Of Strong Base"
Anal. Biochem. 1987 Volume 165, Issue 2 Pages 392-398
D. B. McKay, G. P. Tanner, D. J. Maclean* and K. J. Scott

Abstract: The cuprammonium reagent of Grimble et al. (Ibid., 1983, 128, 422) was modified so that final concentration. of active constituents in the reaction mixture were: 1.0 mM Cu(II), 0.4 M NH3 and 0.2 M NaOH (in excess); the absorbance (at 280 nm) of the modified reagent blank was <50% of that of previous methods. The new reagent was most sensitive for the colorimetric determination of polyols, especially D-glucitol, but it also produced a better color yield for some aldoses and disaccharides than the original reagent. It was used for post-column HPLC detection of various sugars and polyols; calibration graphs for D-glucitol, D-mannitol and D-glucose were rectilinear from 40 to 180 nmol, 0 to 150 nmol and 0 to 300 nmol, respectively. The method was applied in analysis of fungal mycelia.
Polyols Sugars d-Glucose d-Mannitol d-Glucitol Fungus HPLC Spectrophotometry Post-column derivatization

"A Flow Microcalorimetric Method For Enzyme Activity Measurements: Application To Dihydrofolate Reductase"
Anal. Biochem. 1987 Volume 165, Issue 2 Pages 341-348
Lucas Sica, Robert Gilli, Claudette Briand and Jean Claude Sari*

Abstract: A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 x 10^-4 units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.
Enzyme, dihydrofolate reductase Calorimetry

"Microsequence Analysis Of Peptides And Proteins. 6. A Continuous-flow Reactor For Sample Concentration And Sequence Analysis"
Anal. Biochem. 1987 Volume 163, Issue 2 Pages 517-529
John E. Shively, Phillip Miller and Michael Ronk

Abstract: We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2-3 cm) containing 5-10 mg of Polybrene-coated, spherical, porous silica (100-200 µm particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0 mL samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95-100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.
Peptides Proteins Clinical analysis Reactor

"Measurement Of Protein Using Flow Injection Analysis With Bicinchoninic Acid"
Anal. Biochem. 1987 Volume 161, Issue 1 Pages 152-156
Lawrence C. Davis* and Gary A. Radke

Abstract: The bicinchoninic acid reagent developed by Pierce Chemical Co. was used in the determination of proteins in a flow injection analyzer comprising ~8 ft of PTFE tubing (0.5 mm i.d.) with a peristaltic pump, a controlled-temperature water bath (reaction temperature was 72°C), and a spectrophotometer with flow cell. The sensitivity was comparable with that of the Lowry method. Results were obtained in <1 min at a rate of up to 60 h-1. Sample size was 5 to 20 µL and the response was rectilinear up to 10 µg of protein. We have used the bicinchoninic acid reagent developed by Pierce Chemical Co. to measure proteins in a simple flow injection analyzer. The sensitivity is comparable to that of the Lowry method and no pipetting of reagents is needed. Results are obtained in less than 1 min and samples may be run at a rate of 60/h. The response is linear over a range of protein concentration (0-10 µg) and sample size (5-20 µL) convenient for most analytical requirements. A peristaltic pump, a controlled-temperature water bath, and a spectrophotometer with flow cuvette are the only special apparatus required.
Proteins Industrial Pharmaceutical Spectrophotometry Heated reaction Method comparison

"Fluorimetric Determination Of Guanidino-compounds By New Post-column Derivatization System Using Reversed-phase Ion-pair High Performance Liquid Chromatography"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 392-398
Yoshie Kobayashi, Hiroaki Kubo and Toshio Kinoshita

Abstract: Nine guanidino-compounds and creatinine in serum were separated in 25 min by reversed-phase ion-pair HPLC with post-column derivatization on a column (15 cm x 4.6 mm) of Nucleosil C8 (5 µm). The mobile phase (1 mL min-1) was 1 mM Na 1,2-naphthoquinone-4-sulfonate (fluorogenic reagent) and 15 mM Na octanesulfonate in water - acetonitrile - methanol (92:3:5) adjusted to pH 4.0 or 3.5 with acetic acid; 1 M NaOH was delivered at 0.5 mL min-1 to a reaction coil (5 m x 0.5 mm) and the fluorescent products formed were detected at 505 nm (excitation at 355 nm). The detection limit was <20 ng and recovery from serum was 93 to 108%. The method was accurate and reproducible and could be used for routine determinations in haemodialysis patients. Guanidino-drugs could also be determined by using this system.
Drugs Guanidino compounds Blood Serum HPLC Fluorescence Heated reaction Post-column derivatization Dialysis

"Amino-acid Analysis By High Performance Liquid Chromatography Of A Single Stained Protein Band From A Polyacrylamide Gel"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 362-367
Yoko Hashimoto, Sadako Yamagata* and Taro Hayakawa*

Abstract: A single stained band containing ~5 µg of protein was cut from a polyacrylamide gel and hydrolyzed in the presence of mercaptoacetic acid. The hydrolysate was analyzed by HPLC at 55°C on a column (15 cm x 4 mm) of sulfonated polystyrene cation-exchange resin (7 µm) with a pre-column (25 cm x 4 mm) of Dowex 50W-16 resin and step gradient elution with 0.2 M Na citrate (pH 3.15) - ethanol (93:7), 0.6 M Na citrate (pH 10.0) and 0.2 M NaOH at 0.3 mL min-1. Fluorimetric detection was at 450 nm (excitation at 348 nm) after post-column derivatization with phthalaldehyde and NaClO. Ammonia did not interfere. The recovery of methionine was inconsistent and recoveries of tryptophan, isoleucine, threonine and serine were low. Bovine serum albumin, ribonuclease B, ovalbumin, pepsin and chymotrypsinogen A were analyzed by this method with good agreement with theoretical values.
Amino Acids Methionine Tryptophan Isoleucine Threonine Serine Albumin Enzyme, ribonuclease b Ovalbumin Pepsin Chymotrypsinogen, A Cow Serum HPLC Fluorescence Interferences Post-column derivatization

"Selective Determination Of Lithium In Biological Fluids Using Flow Injection Analysis"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 243-250
G. E. Pacey, Y. P. Wu and K. Sasaki

Abstract: The batch extraction of the cryptand 211 - Li - resazurin complex into 1,2-dichloroethane was carried out at 24°C for 10 min and the absorbance of the organic phase was measured at 630 nm. The chromogenic aza-12-crown-4 batch extraction of Li into CHCl3 was carried out at 24°C for 10 min and the absorbance of the organic phase was measured at 400 nm. Both systems were successfully automated by using a PTFE flow injection system (diagram presented). For the cryptand 211 - resazurin system with 1000 ppm of Na present the calibration graph was rectilinear for 0.7 to 2.1 ppm of Li for batch extraction (0.28 to 1.12 ppm for flow injection) and for the chromogenic aza-12-crown-4 system it was rectilinear for 0.3 to 2 ppm of Li (both methods). The methods were used in the determination of Li in serum and urine, with flow injection giving the better precision and accuracy. Good agreement was observed with known values and AAS results.
Lithium Blood Serum Urine Spectrophotometry Method comparison Theory

"Nucleic Acid Quantitation By Continuous-flow Fluorimetry"
Anal. Biochem. 1986 Volume 154, Issue 2 Pages 638-642
Michael G. Murray and Herbert E. Paaren

Abstract: A solution of the fluorochrome (ethidium bromide or Hoechst 33258 dye) in 10 mM Tris - HCl (pH 7.5) - 10 mM NaCl - 1 mM EDTA is pumped through a pulse-damping coil to an injection valve, equipped with a constant-volume fill loop. The sample solution is injected into the fluorochrome stream and the mixture is pumped to a fluorescence detector. Samples (5 µL) ranging in concentration from 0.05 to 40 µg mL-1 of nucleic acid can be analyzed accurately at a rate of 3 to 5 min-1. The samples can be recovered.
Nucleic acids Fluorescence Pulse dampener

"Picomole Analysis Of Glutathione, Glutathione Disulfide, Glutathione S-sulfonate And Cysteine S-sulfonate By High Performance Liquid Chromatography"
Anal. Biochem. 1985 Volume 151, Issue 2 Pages 418-423
Douglas A. Keller and Daniel B. Menzel

Abstract: Glutathione(I), glutathione disulfide(II), glutathione S-sulfonate(III) and cysteine S-sulfonate(IV) were separated by HPLC on a column of Spherisorb 5 SAX strong anion exchanger with non-linear gradient elution with 0 to 30% of 144 mM Li citrate - 1 mM EDTA (pH 5.3) in 5 mM citric acid - 1 mM EDTA; determination was by post-column derivatization with phthalaldehyde and 2-mercaptoethanol at 70°C, and fluorescence detection at 430 to 470 nm (excitation at 305 to 395 nm). The detection limits were 10, 10, 50 and 200 pmol for I, II, III and IV, respectively; the respective recoveries were 102.6, 103.3, 95.5 and 99.7%. The method was applied to the analysis of supernatant solution obtained after homogenization of lung tissue with HClO4.
Glutathione Glutathione disulfide Glutathione S sulfonate S-sulfonatecysteine Lung HPLC Fluorescence Heated reaction Post-column derivatization

"Lowry Protein Determination By Automated Flow Injection Analysis For Bovine Serum Albumin And Hepatitis B Surface Antigen"
Anal. Biochem. 1985 Volume 151, Issue 2 Pages 309-314
Ronald A. Salerno, Cathy Odell, Nancy Cyanovich, Bernard P. Bubnis, Wayne Morges and Alan Gray

Abstract: The Lowry method for quantitation of protein was adapted to automated flow injection analysis. The procedure was developed using two different pure proteins: bovine serum albumin and hepatitis B surface antigen. The system was optimized for reagent concentration, pH, gain, temperature, sample volume, and output. The response of each protein was affected differently by temperature. The reaction slopes and absorbance values of the proteins were similar at 90°C to allow quantitation of hepatitis surface antigen against bovine serum albumin. Advantages of the automated flow injection analysis Lowry procedure include: rapid analyzes (90 samples/h), small sample volume (30 µL, 100 µL), fast response (20 s), reproducibility (less than or equal to 2% CV within an assay and 3 to 6% CV among assays), sensitivity (5 µg), and high correlation (99.8%) with manual assay. After a 30-min set-up period, the analyzer was available to assay protein on demand throughout the day, making it suitable for process and quality control testing. Bovine serum albumin(I) or hepatitis B surface antigen(II) was determined by the Lowry method adapted for use with flow injection analysis. The sample was mixed with the alkaline tartrate copper reagent and heated at 90°C in a reaction coil (100 cm x 0.7 mm). The solution was mixed with Folin - Ciocalteu reagent in a second reaction coil (60 cm x 0.5 mm) and the absorbance of the mixture was measured at 600 nm. The pH was 10.5. The calibration graphs were rectilinear for 10 to 80 and 10 to 40 µg mL-1 of I and II, respectively, and the limit of detection was 5 µg mL-1; the within- and between-assay coefficient of variation were 2 and 3 to 6%, respectively. The results correlated well (r = 0.998) with those obtained by the manual method.
Proteins Albumin Cow Serum Spectrophotometry Heated reaction Optimization Tecator

"Amperometric Assays Of Total And Free Cholesterols In Serum By The Combined Use Of Immobilized Cholesterol Esterase And Cholesterol Oxidase Reactors And Peroxidase Electrode"
Anal. Biochem. 1985 Volume 149, Issue 2 Pages 387-391
Toshio Yao, Minoru Sato, Yoshiaki Kobayashi and Tamotsu Wasa

Abstract: A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2 µL sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2-160 mg/dl and to total cholesterol in the range 3-300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively. Cholesterol esterase and cholesterol oxidase were separately immobilized on silica packed into columns (length 10 and 30 cm, respectively). The injection of a sample (2 µL) containing cholesterol (free and/or esterified) resulted in generation of H2O2 by the immobilized enzymes, and the H2O2 was used to generate Fe(CN)63- from Fe(CN)64- in an immobilized peroxidase electrode. The Fe(CN)63- could be measured amperometrically at low potential (-50 mV vs. silver - Ag+), so that there was little interference from other normal constituents of serum. Free cholesterol was assayed by omission of the cholesterol esterase reactor. The peak current was rectilinearly related to cholesterol concentration. in the range 2 to 160 mg dl-1 or cholesteryl palmitate concentration. in the range 3 to 300 mg dl-1. About 80 samples h-1 could be assayed for free cholesterol or 40 h-1 for total cholesterol. Within-assay coefficient of variation were <1.5%. Results agreed well with those from a chemical method. The reactors and the electrode retained >90% of their original activities after repetitive use for 4 and 2 months, respectively.
Cholesterol, free Serum Human Amperometry Electrode Interferences Immobilized enzyme Silica

"Flow Injection Analysis Of Catecholamine Secretion From Bovine Adrenal Medulla Cells On Microbeads"
Anal. Biochem. 1985 Volume 144, Issue 1 Pages 218-227
Michelle Herrera, Lung-Sen Kao, David J. Curran and Edward W. Westhead

Abstract: Bovine adrenal medullary cells have been cultured on microbeads which are placed in a low-volume flow system for measurements of stimulation-response parameters. Electronically controlled stream switching allows stimulation of cells with pulse lengths from 1 s to many minutes; pulses may be repeated indefinitely. Catecholamines secreted are detected by an electrochemical detector downstream from the cells. This flow injection analysis technique provides a new level of sensitivity and precision for measurement of kinetic parameters of secretion. A manual injection valve allows stimulation by higher levels of stimulant in the presence of constant low levels of stimulant. Such experiments show interesting differences between the effects of K+ and acetylcholine on cells partially desensitized to acetylcholine.
Catecholamines Cell Biochemical analysis Electrochemical analysis Kinetic Microbeads

"Selective Post-column Phthalaldehyde-derivatization System For The Determination Of Histamine In Biological Material By High Performance Liquid Chromatography"
Anal. Biochem. 1985 Volume 144, Issue 1 Pages 98-103
Stig Allenmark, Sture Bergstr&ouml;m and Lennart Enerb&auml;ck

Abstract: Rat tissue and blood samples were treated with 0.4 M HClO4 and then centrifuged. The supernatant solution was adjusted to pH 6.5 with K2CO3 and subjected to HPLC on a column (15 cm x 4.6 mm) of Nucleosil C18 (5 µm) with a mobile phase (0.4 mL min-1) of 5 mM pentanesulfonic acid in 15 mM citrate buffer containing 5% of methanol, and electrochemical detection (for electroactive biogenic amines) or fluorimetric detection by post-column derivatization with phthalaldehyde and emission measurements at 418 nm (excitation at 350 nm). The calibration graph was rectilinear for 0.5 to 135 pmol of histamine injected, and the detection limit was 0.25 pmol.
Histamine Blood Rat Tissue HPLC Electrochemical analysis Fluorescence Post-column derivatization

"Triple-column Ion-exchange Physiological Amino-acid Analysis With Fluorescent Detection: Baseline Characterization Of Human Cerebrospinal Fluid"
Anal. Biochem. 1984 Volume 143, Issue 1 Pages 82-94
Thomas N. Ferraro and Theodore A. Hare*

Abstract: Samples of cerebrospinal fluid were deproteinized with aqueous 20% sulfosalicylic acid and centrifuged. The supernatant solution was then subjected to HPLC on three columns (100 cm x 2 mm) of cation-exchange resin, at 60°C, 55°C and 52°C, with Li citrate buffer solution as the mobile phases (0.25 mL min-1), post-column derivatization with phthalaldehyde and fluorimetric detection. The three columns were used to elute acidic, basic and neutral amino-acid fractions, with an overlap in the profiles. Recoveries of 98.7±3.6% were obtained, with coefficient of variation (n = 9) of <5%. The calibration graphs were rectilinear between 0.2 and 5 nmol.
Amino Acids Cerebrospinal Fluid HPIC Fluorescence Post-column derivatization

"Chemically Coupled Spectrophotometric Assays Based On Flow Injection Analysis. Determination Of Nitrogenase By Assays For Creatine, Ammonia, Hydrazine, Phosphate, And Dithionite"
Anal. Biochem. 1984 Volume 140, Issue 2 Pages 434-442
Lawrence C. Davis* and Gary A. Radke

Abstract: Micromethods of direct chemical coupling have been developed for several different enzyme reactions, using the principles of flow injection analysis. Samples of 1-25 µL are injected into a flowing stream of color-forming reagents and the peak of color change is measured after about 1 min. Alternatively, continuous slow infusion of a reacting system (5-100 µL/min) gives a continuous change of color which can be monitored to derive enzyme reaction rates. These techniques are highly sensitive, requiring a few nanomoles of the substance being detected. Phosphate, ammonia, dithionite, creatine, and hydrazine have been measured. Consumption of reagents is less than 75 mL per hour; typical sample throughout is 30-40 samples per hour by the injection method, and 5 samples per hour by continuous infusion. The procedure has been applied to nitrogenase, continuously monitoring creatine produced from creatine phosphate by creatine kinase which is used to supply a constant level of ATP for nitrogenase. In this way nitrogenase activity can be determined over a wide range of enzyme concentrations. Production of inorganic phosphate directly from ATP, by injection of formaldehyde-quenched samples, was used when coupling to creatine kinase was not possible. Both injection of aliquots and continuous infusion were used for detection of hydrazine during nitrogenase reduction of azide, and the injection method has been used for ammonia assay during dinitrogen reduction. Dithionite oxidation was measured directly from decolorization of iodine, after trapping both dithionite and bisulfite with formaldehyde. The construction of the flow injection apparatus is briefly described; reagents were pumped from disposable plastic 50 mL syringes. The following reactions of nitrogenase were used: hydrolysis of ATP to form phosphate (determined with molybdate), reduction of N3- to give hydrazine (determined with 4-dimethylaminobenzaldehyde), oxidation of S2O42- to SO32- (reaction quenched with formaldehyde, determination by reaction with iodine), reduction of N2 to NH3 (determined by the indophenol blue reaction), and formation of creatine from reaction mixtures containing creatine phosphate and creatine kinase (reaction quenched with alkali, determination with biacetyl and 1-naphthol). The methods permit the analysis of 30 to 40 samples h-1, with sample volume of 1 to 25 µl.
Ammonia Creatine Dithionite Hydrazine Enzyme, nitrogenase Phosphate Spectrophotometry Method comparison

"High Performance Liquid Chromatographic Separation And Quantification Of Alanopine And Strombine In Crude Tissue Extracts"
Anal. Biochem. 1984 Volume 139, Issue 2 Pages 413-417
G. B. Fiore, C. V. Nicchitta and W. R. Ellington*

Abstract: Perchloric acid extracts of tissues of marine invertebrates were analyzed by HPLC on a Waters amino-acid column (25 cm x 4.6 mm) packed with strong cation exchanger. The mobile phase was 0.1 M citrate buffer (pH 3.0) at 0.4 mL min-1 and detection was by post-column derivatization with NaClO (at 60°C) and phthalaldehyde in the presence of 2-mercaptoethanol, with subsequent fluorimetry at 418 nm (excitation at 340 nm). Retention times of alanopine (2,2'-iminodipropionic acid) and strombine (2-methyliminodiacetic acid) were 4.7 and 5.4 min, respectively. Response was rectilinear from 0.05 to 0.25 nmol.
Alanopine Strombine Tissue HPLC Fluorescence Post-column derivatization Heated reaction

"Optimal Conditions For 4-hydroxybenzoyl- And 2-furoyl-hydrazine As Reagents For The Determination Of Carbohydrates, Including Ketosamines"
Anal. Biochem. 1984 Volume 139, Issue 1 Pages 205-211
Michael Lever, Trevor A. Walmsley, Robert S. Visser and Sandra J. Ryde

Abstract: Optimum conditions were determined for reaction of aromatic hydrazides with specific carbohydrates and ketosamines as a basis for their selective determination. Reagent concentration, reaction pH and catalyst concentration. (e.g., Bi(III) as its tartrate complex) were optimized for the cited reagents; absorbance changes with time were monitored at 410 nm. A continuous-flow apparatus was developed for monitoring reactions between ketosamines and the furohydrazide reagent. Relative absorbances (with reference to glucose) for a range of carbohydrates are tabulated.
Carbohydrates Ketosamines Spectrophotometry Catalysis

"Assay Of Biological Thiols By A Combination Of High Performance Liquid Chromatography And Post-column Reaction With 6,6'-dithiodi(nicotinic Acid)"
Anal. Biochem. 1984 Volume 138, Issue 1 Pages 95-98
Junko Nishiyama and Toyo Kuninori

Abstract: Thiols were separated by HPLC on a reversed-phase column (25 cm x 4.6 mm) packed with Fine Sil C18-10, with, as mobile phase, 33 mM KH2PO4 adjusted to pH 2.2 with H3PO4 or 33 mM sodium phosphate of pH 6.8; detection was by post-column derivatization with 6,6'-dithiodi(nicotinic acid) and measurement of the absorbance of the released 6-mercaptonicotinic acid at 344 nm. A comparison was made with post-column derivatization with 5,5'-dithiobis-(2-nitrobenzoic acid). Cysteine, cysteamine, homocysteine, glutathione and penicillamine were determined (detection limit 0.1 nmol); ergothioneine, 2-thiouracil and thiolhistidine could be separated but not determined. The method was applied to the assay of glutathione in human erythrocytes and in E. coli.
Cysteine Cysteamine Homocysteine Glutathione Penicillamine Human Bacteria HPLC Spectrophotometry Post-column derivatization

"Ethylated Fluoresceins: Assay Of Cytochrome P-450 Activity And Application To Measurements In Single Cells By Flow Cytometry"
Anal. Biochem. 1983 Volume 133, Issue 1 Pages 46-57
Arthur G. Miller

Abstract: Four ethylated derivatives of fluorescein were synthesized and characterized by spectroscopic means. Two of the compounds were specifically metabolized by mouse-liver PAH-induced cytochrome P-450 to yield fluorescein, which has 15 times the fluorescence intensity of the original substrate. One of the derivatives, ethoxyfluorescein ethyl ester, could be applied in flow cytometric analysis and in sorting of intact viable cells on the basis of their cytochrome P-450 activity.
Cytochrome P-450 Biochemical analysis Flow cytometry Fluorescence

"High-sampling-rate Automated Continuous-flow Fluorimetric Technique For Analysis Of Nanogram Levels Of Histamine In Biological Samples"
Anal. Biochem. 1983 Volume 133, Issue 1 Pages 16-29
B. Lebel

Abstract: The method is based on that of Shore et al. (J. Pharmacol. Exp. Ther., 1959, 127, 182), automated to allow 60 analyzes in 1 h. Flow schemes, one with and the other without dialysis, are presented. Histamine is caused to react with phthalaldehyde, and the fluorescence of the product is measured at 450 nm (excitation at 360 nm); peak heights and baseline corrections are calculated by a computerized system. The limit of detection is 25 pg, and the rectilinear calibration range is 0 to 5 µg mL-1 of histamine. The method has been applied to whole blood, tissues and cell cultures.
Histamine Whole Biological tissue Cell Fluorescence Dialysis

"Continuous-flow Detector For Cytochrome P-450 And Cytochrome P-420"
Anal. Biochem. 1983 Volume 132, Issue 1 Pages 26-33
R. F. Bonner, P. M. Bungay, L. R. Pohl and R. V. Branchflower

Abstract: The detector (illustrated and fully described) samples eluate from an h.p.l.c column, reduces haem proteins by reaction with S2O42-, then splits the reduced stream into two parts, one of which is caused to react with CO; the difference in absorbance between the resulting streams is measured every 5 s at 420, 450 and 490 nm. Results are calculated and recorded automatically. The detection limit for cytochrome P-450 and its degradation product (cytochrome P-420) is 30 pmol mL-1. Advantages of the method over manual methods are listed.
Cytochrome P-450 Cytochrome P-420 Blood HPLC Spectrophotometry Apparatus Post-column derivatization

"Automated Assay Of α-amylase Inhibitor Proteins By Continuous-flow Analysis"
Anal. Biochem. 1983 Volume 130, Issue 2 Pages 506-513
David H. Strumeyer* and Bradford R. Fisher

Abstract: A procedure for the automated assay of purified amylase inhibitors was developed. Samples were analyzed at a rate of 60/h using ferricyanide reagent to monitor the suppression of the release of reducing groups from a solution of starch by a calibrated α-amylase reagent. In addition to the sequential analysis of individual samples, the use of gradients permitted the continuous analysis of the effect of substrate and of inhibitor concentration. Also described are some of the effects of starch and inhibitor concentration and time of preincubation on the amylase-inhibitor reaction. The procedure was also suitable for the assay of samples of amylase and should be applicable to the determination of the effects of inhibitors on other enzymes which release reducing sugars.
Proteins

"Determination Of Protein-bound Phosphate By Continuous-flow Analysis: Automated Procedure For Determination Of Alkali-labile Phosphate"
Anal. Biochem. 1983 Volume 130, Issue 2 Pages 311-320
Kathrin McCollum and George Taborsky*

Abstract: A method is described for the specific, quantitative determination of protein-bound phosphorus by a continuous flow procedure using a Technicon AutoAnalyzer. It is based on the exceptional alkali lability of serine phosphate linkages to -elimination when the serine residues are present in a polypeptide chain. The results are reproducible within about 3, 5, or 10%, respectively, when the analytical sample contains about 100, 10, or 3 nmol of protein-bound P. The presence of less than 1 nmol protein-bound P can be detected. The method tolerates wide variations of the pH and ionic composition of the sample, making it suitable for the automatic, serial analysis of chromatographic effluent fractions. Low-molecular-weight phosphomonoesters, ribonucleic acid (phosphodiester), and nucleotide phosphates (pyrophosphate) do not react measurably. Carboxyphenyl phosphate is partially hydrolyzed (10-15%). In contrast, the release of P from various phosphoproteins is quantitative.
Phosphate

"Automated Analysis Of Alditols By Anion-exchange Chromatography With Photometric And Fluorimetric Post-column Derivatization"
Anal. Biochem. 1983 Volume 128, Issue 2 Pages 429-437
Susumu Honda*, Masaye Takahashi, Seiko Shimada, Kazuaki Kakehi and Sigetake Ganno

Abstract: Eight alditols were separated in ~80 min as their borate complexes by stepwise elution with three borate buffers on a column packed with Hitachi 2633 resin. The alditols in the eluate were derivatized automatically to colored, fluorescent products by applying sequential reactions of periodate oxidation and Hantzsch condensation, and the products were detected either photometrically or fluorimetrically. This automated method allowed simultaneous determination of 20-500 and 20-200 nmol amounts of alditols by photometric and fluorimetric monitorings, respectively. The lower limits of detection were ~2 and 0.5 nmol, respectively. The interference by aldoses was slight. Aldoses may be also determined as alditols by direct injection of aqueous solutions to which excess amounts of sodium borohydride have been added. This method was applied with success to urinary alditol assay and to molecular weight determination by end group analysis.
Alditols HPIC Fluorescence Spectrophotometry Post-column derivatization

"Flow Cytometry: Rapid Biochemical Analysis Of Single Cells"
Anal. Biochem. 1982 Volume 125, Issue 2 Pages 225-242
Howard S. Kruth*

Abstract: Flow cytometry is a dynamic technology with rapidly increasing applications in medicine and biology. The process of staining analytes with specific dyes and of measuring them in individual cells differs from conventional biochemical analysis in which heterogeneous cells are analyzed as a group and results are expressed as average values per cell. An average value, however, may not be representative of each cell within a population. Consider the example of a cell population comprising two subpopulations: one subpopulation consists of cells deficient in analyte while the other contains cells enriched in the same analyte. Analysis of these heterogeneous cells as a group fails to reveal two different subpopulations.
Biochemical analysis Flow cytometry Review

"Continuous-flow System For Measurement Of Calcium Ion Activities In The Fast Kinetic Mode"
Anal. Biochem. 1981 Volume 115, Issue 1 Pages 1-6
Hubert Affolter and Ernesto Carafoli

Abstract: A continuous-flow system, suitable for the fast kinetic measurement of changes in Ca2+ ion activity with specific electrodes, is described. Provided that the ionic strength of the measuring medium is at least 20 m, the system has a time resolution of 2 ms and a detection limit of 0.2 Ca2+. As an example of a possible biochemical application, some measurements of the initial rate of Ca2+ uptake by lobster muscle sarcoplasmic reticulum are presented.
Calcium Biological tissue Electrode Kinetic Activity

"Calcium Measurement: Picomole Quantitation By Continuous-flow Colorimetry"
Anal. Biochem. 1981 Volume 114, Issue 2 Pages 288-293
Gerald G. Vurek

Abstract: A new microcolorimeter with a 220 nL volume, 1.1-cm light path cuvet and solid-state photometric components is described. It has been used in a continuous-flow analysis system for the colorimetric measurement of total calcium in biologic fluids. The sensitivity is 0.5 pmol and it is linear beyond 50 pmol.
Calcium Blood Spectrophotometry

"Continuous-flow Analysis Of 3α-hydroxysteroids Using Immobilized 3α-hydroxysteroid Dehydrogenase"
Anal. Biochem. 1981 Volume 112, Issue 2 Pages 239-243
R. Bovara, G. Carrea, P. Cremonesi and G. Mazzola

Abstract: The 3-hydroxysteroid dehydrogenase from Pseudomonas testosteroni was coimmobilized with diaphorase on cellulose beads and used to prepare a continuous-flow µreactor. Thus it was possible to determine urinary and serum 3-OH steroids with spectrophotometric monitoring in the visible region. The increased stability of the enzymes following immobilization and the mechanical characteristics of the matrix made the reactor usable for at least 20 days. The assay speed was 10-15 samples per hour. The results obtained with immobilized enzymes followed closely those obtained with free 3-hydroxysteroid dehydrogenase. The method is sensitive, reliable, and economical.
3-α-Hydroxysteroids Urine Immobilized enzyme

"Determination Of Inorganic Phosphate. Method For Determination Of Phosphatase Activities By Continuous-flow System"
Anal. Biochem. 1980 Volume 102, Issue 2 Pages 353-357
Hannu K. Penttinen

Abstract: A sensitive and accurate method for the determination of inorganic phosphate is described. The method enables the estimation of 10 nanomoles of inorganic phosphate with a coefficient of variation of 3.6% for ten replicates. The method is suitable for the estimation of the activities of thiamine triphosphatase, adenosine triphosphatase, and alkaline and acid phosphatase by a continuous flow system.
Acid phosphatase Enzyme, alkaline phosphatase Activity

"The Peroxidase-catalyzed Oxidation Of NADH As A Indicator Reaction For Repetitive Determinations By Sample Injection In Closed Flow-through Systems: The Determination Of LDH In Blood Serum"
Anal. Biochem. 1979 Volume 97, Issue 2 Pages 255-263
D. P. Nikolelis, Chih-duen C. Painton and Horacio A. Mottola*

Abstract: The air oxidation of reduced nicotinamide adenine dinucleotide (NADH), catalyzed by peroxidase, provides a useful 'indicator reaction' for the determination of both NADH and serum lactate dehydrogenase (LDH). Application of this indicator reaction for repetitive determinations by sample injection into a continuously circulated reagent mixture, and by monitoring of oxygen depletion with an amperometric sensor, is described. At determination rates of 260 determinations/h, NADH and LDH have been determined in the range of 3.8 x 10^-4 to 7.6 x 10^-3 M and 70 to 700 U/mL, respectively, and with relative errors and standard deviations (population) of about 2%. LDH has been determined in human blood serum and the injection procedure compared with a modified Wroblewski-Ladue procedure; the Pearson correlation coefficient was found to be 0.999 (40 samples). Recovery studies are also reported for LDH in serum calibration references and controls.
Enzyme, lactate dehydrogenase Blood Serum Amperometry Clinical analysis Closed loop Enzyme

"Rapid Analysis Of Discrete Samples: The Use Of Nonsegmented Continuous-flow"
Anal. Biochem. 1976 Volume 70, Issue 1 Pages 167-173
Kent K. Stewart, Gary R. Beecher and P.E. Hare

Abstract: The principles and design of a high speed discrete sample analyzer are presented. Instead of air segmentation, small-bore tubing, low sample volumes, and high flow rates are used. The analyzer was operated at rates up to 120 samples/hr with return to baseline between samples. When trypsin standards were analyzed, relative standard deviations of <1% were attained.
Trypsin Biochemical analysis Spectrophotometry Theory Enzyme

"A Method For Automated Analyses Of The Activities Of Trypsin, Chymotrypsin And Their Inhibitors"
Anal. Biochem. 1973 Volume 51, Issue 1 Pages 11-18
Kent K. Stewart

Abstract: Methods for the automated analyzes of the activity of trypsin and chymotrypsin and their inhibitors using -BAPA and -GPNA, respectively, as substrates are described.
Trypsin Enzyme, chymotrypsin Spectrophotometry

"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 Cell