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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Richard A. Durst

Abbrev:
Durst, R.A.
Other Names:
Address:
BioAnalytical Chemistry Laboratories, Department of Food Science and Technology, Cornell University, Geneva, NY 14456-0462, USA
Phone:
+1-315-787-2297
Fax:
+1-315-787-2284

Citations 10

"Liposome-based Flow Injection Enzyme-immunoassay For Theophylline"
Microchim. Acta 1990 Volume 100, Issue 3-4 Pages 187-195
Tai -Guang Wu and Richard A. Durst

Abstract: A peristaltic pump was used to supply, in 0.1 M Tris buffer (pH 7.2) as carrier, a standard solution of theophylline (I) or plasma sample, in the same buffer, to a column (17.8 cm x 2.5 mm) of glass beads coupled to monoclonal anti-theophylline antibodies. The injector (Rheodyne type 7010) then delivered a solution of liposomes that encapsulated horse-radish peroxidase and had been sensitized with 4-(1,3-dimethylxanthin-8-yl)butyric acid (II). Competition between I and II for the antibodies occured, and unbound liposomes were eluted for post-column reaction with H2O2 and 4-fluoriphenol. This reaction caused release of F-, which was determined with an Orion model 69-09 ion-selective electrode. The column was then washed with glycine - HCl solution to dissociate the antigen - antibody complex and reactivate the column. Calibration graphs are presented for two liposome compositions (10 and 20 miu mL-1 of enzyme activity). I can be detected over the concentration. range 0.2 to 4000 ng mL-1, i.e., a detection limit of 100 fmol in a 0.1 mL sample. For an activity of 10 miu mL-1, the coefficient of variation (n = 6) was 4.6% at the level of 4.3 ng mL-1. The assay takes ~10 min.
Theophylline Blood Plasma Immunoassay Electrode Buffer pH Column Enzyme Liposomes Calibration Glass beads Detection limit

"Liposome-based Flow Injection Immunoassay System"
J. Res. Natl. Bur. Stand. 1988 Volume 93, Issue 6 Pages 663-665
Laurie Locascio-Brown, Anne L. Plant, and Richard A. Durst

Abstract: A flow injection system is described (with diagram) in which fluorophore-encapsulating liposomes covalently bound to an antigen compete with analyte antigen for Fab' fragments of the antibody covalently bound to non-porous glass beads (60 to 80 mesh) in a glass column (9.95 cm x 2 mm). The flow properties and stability of the liposomes are discussed.
Immunoassay Glass beads Liposomes

"Liposome-based Flow Injection Immunoassay For Determining Theophylline In Serum"
Clin. Chem. 1993 Volume 39, Issue 3 Pages 386-391
Laurie Locascio-Brown, Anne L. Plant, Ruth Chesler, Martin Kroll, Mark Ruddel, and Richard A. Durst

Abstract: The flow injection system described (with diagram) comprises a microprocessor, an autosampler, an immunoreactor column, a fluorimetric detector and associated valves and pumps. Theophylline was used as a model to demonstrate the feasibility of the method. Phosphate-buffered saline (pH 7.4) was passed through the column containing immobilized monoclonal anti-theophylline antibodies (~2 x 10^-12 binding sites column-1), liposomes (preparation described) containing 80 to 100 mM carboxyfluorescein and sample solution were injected on to the column to compete for the binding sites, the column was then washed with detergent solution for 6.5 min to release the dye which was detected fluorimetrically at 515 nm (excitation at 490 nm). The calibration graph was rectilinear from 0.025 to 0.4 mg L-1 of theophylline. The determination limit was 39 nM theophylline. The results correlated well with those obtained with use of a commercially available fluorescence polarization method. The cited method may also be applied to the determination of small haptens and large proteins. We developed a method for quantitatively determining theophylline in serum, using a heterogeneous immunoassay called flow injection immunoanalysis. The reaction involves competition between serum theophylline and theophylline-labeled liposomes. Separation occurs on a solid-phase reactor column containing immobilized antibody to theophylline incorporated in a flow injection system. Subsequent lysis of the bound liposomes provides sensitive detection of the analyte. Effective regeneration of the immobilized antibody activity allows the reactor to be reused for hundreds of sequential samples. Comparison of the results of the flow injection immunoassay method with results obtained with a commercially available fluorescence polarization method showed an excellent correlation.
Theophylline Blood Serum Immunoassay Clinical analysis Liposomes

"Liposome-enhanced Flow Injection Immunoanalysis"
Biotechnology 1988 Volume 6, Issue 3 Pages 266-269
Anne L. Plant, Laurie Locascio-Brown, Marius V. Brizgys and Richard A. Durst

Abstract: A review with 4 references. Flow injection immunoanalysis, antibody immobilization, and liposomes for signal enhancement are discussed.
Clinical analysis Fluorescence Immunoassay Biotechnology Computer Immobilized enzyme Immobilized reagent Liposomes

"Use Of Protein A In A Liposome-enhanced Flow Injection Immunoassay"
Anal. Proc. 1994 Volume 31, Issue 11 Pages 339-340
Geoffrey S. Rule, Derek A. Palmer, Stuart G. Reeves and Richard A. Durst

Abstract: A glass column (10 cm x 6 mm i.d.) containing controlled-pore glass coated with protein A was used in the cited immunoassay for alachlor (I). The column was activated by injecting anti-I antibody (raised in rabbits) onto the protein A matrix. Alachlor (1 mg/ml in methanol) was diluted with TBS of pH 7.4 and injected onto the column. Liposomes (100 mM sulforhodamine B in TBS encapsulated in lumen) were then injected onto the column. A detergent solution (octyl β-L-glucopyranoside) was then passed through the column. The fluorescence generated by the released sulforhodamine B was measured at 572 nm (excitation at 556 nm). The antibody was then removed with 20% acetic acid (pH 2.2) and the column reconditioned for the next analysis by returning the mobile phase to pH 7.4 TBS. Assays were conducted at a flow rate of 0.8 ml/min. Each analysis took 11 min. The detection limit was 10 ng of I injected.
Alachlor Biological Immunoassay Fluorescence Controlled pore glass Liposomes

"Liposome Immunoanalysis For The Environment"
Anal. Eur. 1996 Volume 45, Issue 1 Pages 48-50
Durst, R.A.;Reeves, S.G.

Abstract: The use of liposomes in immunoassays to provide encapsulated markers as signal enhancers is presented. The liposomes are spherical bi-layer vesicles that can entrap a water-soluble marker during their formation. The markers can be visible dyes, fluorescent dyes, chemi/bioluminescent substrates, electroactive species or enzymes. The liposome surface contains an analyte tag which binds to a cell surface antibody, the marker is then measured directly (visible dyes) or the liposome is lysed before measurement can be performed. The liposomes can be used in an enzyme-linked flow injection liposome immunoanalysis (FILIA) and a system for the detection of alachlor is outlined. The system used an immunoaffinity column containing immobilized antibodies and had a detection limit of 5 ppb alachlor. The liposome markers can also be mixed with sample and applied to plastic-backed nitrocellulose containing an antibody competition zone and a liposome capture zone to produce test strips, the color of the liposome capture zone being measured. The use of test strips for multi-analyte assays is discussed.
Liposome Environmental Immunoassay Liposomes

"Liposome Flow Injection Immunoassay: Model Calculations Of Competitive Immunoreactions Involving Univalent And Multivalent Ligands"
Anal. Chem. 1991 Volume 63, Issue 18 Pages 2007-2011
William T. Yap, Laurie Locascio-Brown, Anne L. Plant, Steven J. Choquette, Viola Horvath, and Richard A. Durst

Abstract: The use of liposomes as detectable reagents in solid-phase immunoassays has been explored in a flow injection immunoanalysis (FIIA) system. Model calculations are presented for FIIA based on the competitive binding of univalent analyte and multivalent liposomes to immobilized antibodies. Parameters such as binding constants, concentrations of liposomes and antibody, and steric hindrance are considered for their relative effects on detectable liposome signal response to analyte concentrations. Qualitative comparisons of the model with the experimental data are made. A mathematical model is derived for competitive binding of theophylline (I) and liposomes to anti-I antibodies immobilized on a glass bead in flow injection systems. The model was applied in the optimization of a concentration.-dependent flow injection immunoassay. Preliminary experimental results agreed with predicted results.
Liposome Theophylline Immunoassay Glass beads Optimization Liposomes

"Liposome Flow Injection Immunoassay: Implications For Sensitivity, Dynamic Range, And Antibody Regeneration"
Anal. Chem. 1990 Volume 62, Issue 23 Pages 2587-2593
Laurie Locascio-Brown, Anne L. Plant, Viola Horvath, and Richard A. Durst

Abstract: We have developed a liposome-based flow injection immunoassay (FIIA) system for quantitation of a clinical analyte, theophylline. With very minor changes in assay format, this procedure can also be used for the quantitation of anti-theophylline. Automated sequential analyzes were performed at room temperature with picomole sensitivity and a day-to-day coefficient of variation of less than 5% for aqueous solutions. The system components include liposomes that contain fluorophores in their aqueous centers and an immobilized-antibody reactor column. The immunoreactor was regenerated hundreds of times over 3 months of continuous use with no measurable loss of antibody activity. The two assay formats studied produced distinct dynamic ranges for their respective analytes. The special advantages of using flow injection analysis for immunoassays and of using liposomes in FIIA are discussed.
Theophylline Immunoassay Linear dynamic range Sensitivity Automation Column Immobilized reagent Activity Liposomes

"Behavior Of Liposomes In Flow Injection Systems"
Anal. Chem. 1988 Volume 60, Issue 8 Pages 792-797
Laurie Locascio-Brown, Anne L. Plant, and Richard A. Durst

Abstract: Liposomes, containing entrapped water-soluble molecules, were tested in continuous-flow systems as a means of signal enhancement of the entrapped analyte. Liposomes containing carboxyfluorescein were detected fluorimetrically at 515 nm (excitation at 450 nm). Peak profiles were obtained for the system with use of a 187 µL sample loop, a packed bead column, a knitted delay tube and a straight open-bore tube; the flow rate was 0.5 mL min-1. The effect of these parameters on the peak profiles was discussed.
6-Carboxyfluorescein Fluorescence Immobilized reagent Mixing coil Liposomes Reactor Sensitivity Knotted reactor Signal enhancement

"Radiometric And Fluorimetric Determination Of Aminosilanes And Protein Covalently Bound To Thermally Pre-treated Glass Substrates"
Anal. Chim. Acta 1990 Volume 228, Issue 1 Pages 107-116
Laurie Locascio-Brown, Anne L. Plant and Richard A. Durst, Marius V. Brizgys

Abstract: Derivatization of soda-lime glass spheres with aminosilanes and the stability of these groups at near-physiological pH in flow streams of aqueous buffered solution were studied. The extent of silanization was determined by a radioactive tracer method and a method based on a fluorescent marker, which confirmed the presence of immobilized and adsorbed amines in the nmol range. A method for covalently attaching bovine serum albumin to the beads via a cross-linking reagent which reacts selectively with amines is described. Thermal pre-treatment of the glass before derivatization enhanced suface derivatization with aminosilanes. Less than monolayer films prepared with monofunctional silanes were stable, after initial conditioning, with a 3% loss over 24 h in constantly flowing solution at pH 8, allowing the design of reusable immunoassay systems which were readily calibrated.
Protein Aminosilanes Immunoassay Radiochemical Fluorescence pH Buffer Immobilized reagent Glass beads Calibration