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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Liposomes

Classification: Reagent -> Liposomes

Citations 21

"Comparison Of Liposome Amplification And Fluorophor Detection In Flow Injection Immunoanalyses"
Anal. Chim. Acta 1997 Volume 354, Issue 1-3 Pages 23-28
Myoyong Lee*, Richard A. Durst and Rosie B. Wong

Abstract: A flow-injection liposome immunoanalysis (FILIA) system was developed for the measurement of imazethapyr herbicide. The liposome is a spherical vesicle encapsulating many marker molecules in an aqueous interior. By incorporating analyte-phospholipid conjugates into the bilayer, the liposome can competitively bind to anti-analyte antibody, immobilized in the immunoreactor column of the FILIA system. In this study, the analyte-tagged liposome containing a fluorescent dye, carboxyfluorescein, was compared to a single fluorophor-tagged analyte used for the generation of the analytical signal. The liposome enhanced sensitivity by 1000-fold compared to the single fluorescent molecule-tagged analyte. This is the first report to demonstrate directly the amplification of the response by liposomes in a flow-injection immunoassay.
Immunoassay Fluorescence

"Measuring Estrogens Using Flow Injection Immunoanalysis With Liposome Amplification"
Talanta 1993 Volume 40, Issue 12 Pages 1899-1904
Laurie Locascio-Brown* and Steven J. Choquette,

Abstract: A solid-phase competitive immunoassay is performed in flow injection analysis for the measurement of the hormone 17-β-estradiol. The flow injection analysis system incorporates a column-type reactor packed with solid silica particles onto which we have covalently immobilized the antigen 17-β-estradiol. Anti-estradiol is noncovalently conjugated to the liposome through a streptavidin-biotin linkage. When mixed with a sample containing the antigen, the antibody binding sites on the liposomes are complexed which reduces the binding of liposomes to the solid support in a concentration-dependent manner. Sequential immunoassays are performed on-column following a simple regeneration step.
Estrone Immunoassay

"Flow Injection Liposome Immunoanalysis (FILIA) For Alachlor"
Talanta 1994 Volume 41, Issue 10 Pages 1747-1753
Stuart G. Reeves*, Geoffrey S. Rule, Matthew A. Roberts, Alison J. Edwards and Richard A. Durst,

Abstract: A schematic diagram of the FILIA system is given. Samples of alachlor-tagged liposomes with a sulforhodamine B dye in TBS and alachlor in methanol were injected into a stream of TBS (200 µL/min) and onto a column (5 cm x 4.3 mm i.d.) packed with glass beads coated with anti-alachlor antibody (preparation details given). The column was washed for 5 min with 30 mM n-octyl-β-D-glucopyranosine to lyse the bound liposomes and release the fluorescent dye which was measured in a flow-through fluorimeter at 574 nm (excitation at 545 nm). The column was then washed with TBS for 5 min before the next sample injection. The effect of antibody concentration and flow rate on the sensitivity of the assay are discussed.
Alachlor Biological Immunoassay Fluorescence

"Development Of Flow Injection Liposome Immunoanalysis (FILIA) For Imazethapyr"
Talanta 1998 Volume 46, Issue 5 Pages 851-859
Myoyong Lee*, Richard A. Durst and Rosie B. Wong

Abstract: Imazethapyr is the herbicide developed for use in leguminous crops. In this study, flow-injection liposome immunoanalysis (FILIA) has been shown to be capable of measuring imazethapyr in a buffered solution with a detection limit of 0.1 ppb through the optimization process. Protein A coated glass beads covalently conjugated with antibody were contained in a glass column, and this column was used as an immunoreactor. Liposomes which encapsulated a fluorescent dye, sulforhodamine B (SRB) or carboxyfluorescein (CF), generated the analytical signal. By loading larger volumes of sample onto the column, it was shown that the detection limit could be lowered. Liposomes containing carboxyfluorescein gave more sensitive response and a lower detection limit than those with sulforhodamine B. Also, improved response was obtained by using a smaller flow cell in the fluorescence detector due to the reduced dilution effect.
Imazethapyr Immunoassay

"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

"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

"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

"Planar Waveguide Immunosensor With Fluorescent Liposome Amplification"
Anal. Chem. 1992 Volume 64, Issue 1 Pages 55-60
Steven J. Choquette, Laurie Locascio-Brown, and Richard A. Durst

Abstract: A flow injection analysis (FIA) manifold (described) with an anti-theophylline derivatized planar waveguide optical sensor was developed. The planar waveguides were produced by immersing Corning 0211 cover slips into AgNO3 - NaNO3 (1:9) at 280°C for 15 min to 1 h, and monoclonal mouse anti-theophylline IgG was coupled to the waveguide surface. Samples of theophylline (50 µL) were injected into the FIA manifold, and the flow was stopped for 5 min to 1 h to allow equilibration of the sample to the sensor surface. After incubation, the flow cell was flushed, followed by detection of the specifically bound liposomes at 516 nm (excitation at 488 nm). Antibody activity was regenerated by adding 1-O-octyl β-D-glucopyranoside for 2 min, then rinsing for 20 min with phosphate-buffered saline solution (pH 7.4). Sensors capable of >15 sequential measurements demonstrated >10% precision.
Theophylline Sensor Sensor Fluorescence

"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

"Potentiometric Enzyme-amplified Flow Injection Analysis Detection System: Behaviour Of Free And Liposome-released Peroxidase"
Anal. Lett. 1989 Volume 22, Issue 5 Pages 1107-1124
Wu, T.G.;Bellama, J.M.;Durst, R.A.

Abstract: A three-channel peristaltic pump was used, with one channel pumping the carrier stream (0.5 M acetate buffer of pH 5.2, containing 1 M NaCl) and the other two, arranged to mix with each other and then with the carrier after sample injection, containing 4.8 mM H2O2 and the enzyme substrate, viz, 36 mM 4-fluoriphenol, both in acetate buffer solution Peroxidase in the sample catalyzed the oxidation of the substrate to F-, which was detected at an ion-selective electrode. Electrode fouling and slow recovery of electrode response were overcome with use of a separately pumped wash solution containing acetate buffer, 1 µM-NaF and 0.1% of Triton X-100. Sensitivity was increased by ~20%. To avoid loss of enzyme activity, sample solution were diluted with the acetate buffer containing 0.5% of gelatin. The reaction temperature was 25°C and the reaction coil was adjusted to give a reaction time of 3 min. The calibration graph covered the range from 0.2 to 3 miu of the enzyme per 100 µL injected; for 3 miu, the coefficient of variation was 1.5% (n = 16). A lower response, though still useful, was obtained when the enzyme was encapsulated in liposomes. In this instance, H2O2 also functioned as lysis reagent.
Enzyme, peroxidase Potentiometry Electrode

"Liposome Membrane Permeability Assay Using A Glucose Biosensor Flow Injection System"
Anal. Lett. 1995 Volume 28, Issue 9 Pages 1571-1578
Katsu, T.;Wei, H.;Hu, W.;Zhang, X.;Zhang, X.

Abstract: A suspension of liposomes containing glucose enclosed in the inner aqueous liposome phase (preparation described) was incubated with melittin or gramicidin S for 10 min at 30°C. The reaction mixture was injected into a carrier stream (3 mL/min) of 10 mM sodium phosphate buffer of pH 7 containing 0.15 M NaCl and released glucose was determined with use of a glucose analyzer incorporating a glucose electrode. The electrode was based on an O2 electrode with a BSA/glucose oxidase/glutaraldehyde-modified PTFE membrane (cf. Zhang et al., 'Collected papers in Biotechnology,' Chinese Chemical Industrial Press, Beijing, 1994, p. 22). The calibration graph for the biosensor system was linear for 0.1-7 mM glucose and the detection limit was 50 µM. The RSD was 2.8% at 4.75 mM glucose (obtained by disrupting liposomes with Triton X-100). The system was suitable for determining liposome membrane permeability.
Glucose Amperometry Electrode Sensor

"Flow Injection Liposome Immunoanalysis (FILIA) With Electrochemical Detection"
Electroanalysis 1995 Volume 7, Issue 9 Pages 838-845
Alison J. Edwards *, Richard A. Durst

Abstract: Alachlor (I) in environmental samples was covalently bonded to dipalmitoyl phosphatidylethnolamine using N-succinimidyl-S-acetylthioacetate as described by Siebert et al. (Anal. Chim. Acta 1993, 282, 297). The I-bonded lipid was then dissolved in an organic solvent (not specified) and reacted with aqueous 100 mM potassium ferrocyanide of pH 7.4. After sonication and removal of the organic solvent, the liposome suspension was reacted with further potassium ferrocyanide (no details given) at 45°C, filtered, gel-filtered and dialysed. The dialysed liposomes were then diluted in PBS of pH 7.4 and 20 µL injected onto a column (5 cm x 4.3 mm i.d.) containing I-antibody-immobilized glass beads (preparation details given), PBS as mobile phase (0.55 ml/min) and amperometric detection at +330 mV vs. Ag/AgCl. Calibration graphs were linear for 1-10% liposomes bound on-column, with a detection limit of 0.5 ng I on-column.
Alachlor Environmental Amperometry

"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

"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

"Kinetic And Equilibrium Studies Of Porphyrin Interactions With Unilamellar Lipidic Vesicles"
Biochemistry 1994 Volume 33, Issue 32 Pages 9447-9459
Katerina Kuzelova and Daniel Brault

Abstract: The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped-flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) constants relative to albumin. In both systems studied, a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32°C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.
Porphyrin Serum Human Fluorescence

"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

"Liposome Immunoassay Of Anti-asialo-GM1 Antibody Detected By Kinetic Method Of Analysis In Flow Injection System"
Bunseki Kagaku 1991 Volume 40, Issue 11 Pages 697-703
Masamitsu KATAOKA, Hirohisa ABE, Yoshio UMEZAWA, Tatsuji YASUDA

Abstract: An immunoassay technique using an immunoreaction at a liposome membrane surface is described. Multilamellar liposomes comprised dipalmitoylphosphatidylcholine(DPPC), cholesterol(Chol) and anti-asialo-GM1(GA1) antigen in the molar ratio 1:1:0.1. Molybdate ions entrapped in the liposomes as marker ions were released from the liposomes by a complement-mediated immunoreaction, and acted as a catalyst for promoting the hydrogen peroxide-iodide ion redox reaction. The most suitable pH of the kinetic reaction and concentrations of hydrogen peroxide and sodium iodide for the determination of molybdate ion were found to be 1, 4.7 x 10^-3 and 1.0 x 10^-3M, respectively. To minimize the sample volume, a flow-injection method was adopted. The immunoreaction was carried out as follows. A mixed solution of sample anti-GA1 antibody, liposomes and complement were incubated at 37.5°C for 1 h. The resulting solution was injected into the kinetic-FIA system. The decrease in the number of iodide ions by the molybdate ion-catalyzed reaction was monitored using an iodide ion-selective electrode. The marker ion release was specific for the anti-GA1 antibody, and depended on the presence of a complement. The present method can be used to detect as low as 103 to 104 dilution of anti-GA1.
Antibody, anti-asialo-GM1 Immunoassay

"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

"Determination Of Imazethapyr Using Capillary Column Flow Injection Liposome Immunoanalysis"
J. Agric. Food Chem. 1996 Volume 44, Issue 12 Pages 4032-4036
Myoyong Lee and and Richard A. Durst

Abstract: A sensitive immunoanalysis system was developed for the quantitation of imazethapyr, the active ingredient in PURSUIT herbicide. Imazethapyr [5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid] is one of the imidazolinone class of herbicides. The assay was based on sequential competitive binding of imazethapyr and liposomes for a limited number of antibody binding sites. A capillary tube (20 cm x 0.53 mm i.d.) with immobilized antibody was used as the immunoreactor column. Liposomes that entrap fluorescent molecules as the detectable label provide instantaneous, rather than time-dependent, enhancement, common with enzyme immunoassays. In this study, liposomes encapsulated carboxyfluorescein dye and were made antigenic by incorporating in the bilayer a phospholipid that had the analyte conjugated to its polar head group. The calibration curve for imazethapyr in Tris-buffered saline solution had a working range of 0.1-100 ng/mL. In the range between 1 and 100 ng/mL, recoveries from fortified tap and pond water samples ranged from 93 to 114%. Filtration was the only step needed for sample cleanup, and an assay could be performed in <10 min.
Imazethapyr Pond Immunoassay

"Liposomes And Immunoassays"
J. Immunol. Methods 1997 Volume 204, Issue 2 Pages 105-133
H. A. H. Rongen*, A. Bult and W. P. van Bennekom

Abstract: Various aspects of the application of liposomes as a label in immunoassays are reviewed. Methods for the preparation of liposomes, from the basic film method to the more advanced dehydration-rehydration method, are discussed. Furthermore, the markers used in liposome labels, as well as the methods to conjugate liposomes to antigens or antibodies, are summarized. Liposome immunoassays are applied as homogeneous or heterogeneous assays. Homogeneous assays often rely on the lytic activity of complement on antibody-associated liposomes. Another group of homogeneous assays utilizes the inhibitory action of antibodies on the activity of conjugates of mellitin (a bee venom protein) with a hapten. Free mellitin conjugates are able to lyse liposomes effectively. Heterogeneous liposome immunoassays, performed either competitively or non-competitively, resemble more closely standard enzyme linked immunosorbent assays, with the enzyme being replaced by a liposome label. Washing steps are used to separate antigen-specifically bound liposomes from unbound liposomes. All bound liposomes are lysed with a detergent, giving an instantaneous amplification. Flow injection liposome immunoassays and liposome immunosensors are also described as examples of other possible immunoassay formats.
Sensor Immunoassay

"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