John D. Brennan

Self‐quenching of nitrobenzoxadiazole labeled phospholipids in lipid membranes

The emission intensity, wavelength, and lifetime of the fluorophore nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD‐PE) are sensitive to the local environmental structure when this species is present as a component of an amphiphilic membrane. Alterations of the physical and electrostatic structure of a membrane can result in changes in the fluorescence signal owing to changes in the extent of self‐quenching of the probe. To investigate self‐quenching, NBD‐PE was incorporated into monolayers and vesicles composed of Egg phosphatidylcholine at concentrations of 0.1 to 50 molu2009%. Monolayer samples were dipcast onto glass slides at a pressure of 35 mNu2009m−1. Both the integrated intensity per fluorophore (quantum yield) from vesicles and dipcast monolayers, and the mean fluorescence lifetime from vesicles decreased as the concentration of fluorophore in the membranes was increased. At all concentrations studied the decay of NBD‐PE fluorescence was fitted to two discrete exponentials, and both lifetim...

Control of ion transport across bilayer lipid membranes by adjustment of surface charge associated with phase domain structures

Abstract Interest continues in the use of immobilized lipid membranes of mixed lipid composition to prepare chemically modified electrodes. Fundamental investigations of the structure and mechanism of the analytical function of such systems are scarce. In this work ion conductivities of bilayer lipid membranes formed from mixtures of egg phosphatidylcholine and dipalmitoylphosphatidic acid were evaluated to determine the effect of surface charge and phase domain formation on the process of ion translocation. Ion conductivity was controlled by the surface distribution of ions at the membrane solution interface as predicted from electrical double-layer theory. It was found that the conductivity of the membranes could be approximated as a linear function of the weight percentage composition of the charged lipid. The conductivity was observed to alter drastically at a lipid composition containing a minimum of 25% phosphatidic acid as this component within the membrane was increased. This was attributed to the presence of a phase transition induced by the phosphatidic acid. At concentrations of the acid less than 25%, ion conduction occurred through zones that were enriched in the charged lipid. At higher concentrations of the acid, the average surface charge was the predominant factor which determined the magnitude of ion conductivity. The adjustment of pH to control the degree of ionization of the phosphatidic acid had a similar effect to the variation of the amount of the acidic phospholipid within the membrane of experiments done at fixed pH. An electrochemical method based on ion permeability is proposed for the determination of the p K a value for a charged lipid within a planar bilayer lipid membrane.

Fluorescence transduction of an enzyme-substrate reaction by modulation of lipid membrane structure

Abstract The emission intensity of the fluorophore nitrobenzoxadiazoledipalmitoylphosphatidylethanolamine (NBD-PE) is sensitive to local environmental structure when this species is used as a component of a phospholipid membrane. The physical and electrostatic structure of a membrane may be modulated by selective chemical reactions, and the resulting alteration in fluorescence intensity provides transduction of such selective chemical processes. One example is the reaction between the extrinsic membrane-associated enzyme acetylcholinesterase (AChE) and the substrate acetylcholine (ACh), which produces an increase in hydronium ion activity at the surface of a lipid membrane. A mechanism of transduction of the enzymatic reaction by lipid monolayer membranes was investigated by spectrofluorimetric methods and fluorescence microscopy. Mixed monolayers composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA) which contained 30 mol-% or more of DPPA and 1 mol-% of NBD-PE provided transduction of the AChEue5f8ACh reaction. Reaction of micromolar concentrations of ACh with AChE-monolayer systems induced increases in fluorescence intensity of up to 50%. Direct observation of the microscopic structure of lipid monolayers on a time scale of minutes showed that the reaction did not drastically affect the distribution of coexisting microscopic phase domains that were present in the monolayers The fluorescence imaging and spectroscopic results did indicate that massive structural reorganization at a molecular level probably occurred in a period of seconds. The results are consistent with an electrostatic mechanism of perturbation of the structure of the monolayer in which local pH gradients associated with the reaction of AChE with substrate altered the extent of ionization of DPPA in the headgroup zone of the membrane.

Covalent immobilization of amphiphilic monolayers containing urease onto optical fibers for fluorimetric detection of urea

Abstract Bifunctional 11 or 12 carbon chain length amphiphiles with a triethoxy chlorosilane group at one terminus and an amine functionality at the other terminus are covalently immobilized onto planar quartz wafers and optical fibers. A small amount of the fluorescent probe nitrobenzaoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) is partitioned into the membranes from an aqueous suspension. For coated wafers or fibers placed into aqueous solutions, alterations of pH change the physical and electrostatic structure of the membranes, which in turn alters the emission intensity of the NBD-PE owing to changes in self-quenching. The fluorescence intensity decreases as the degree of ionization of headgroups within the membrane decreases, consistent with an increase in self-quenching. Samples which are immobilized onto optical fibers have better sensitivity to changes of pH than those immobilized onto planar wafers. The enzyme urease is covalently linked onto the functional groups at the surface of some membranes to investigate immobilized membranes that are chemically selective. A small amount of NBD-PE is partitioned from water into the membrane either before or after immobilization of urease. Addition of urea to this system produces ammonia and carbonic acid, and results in changes in fluorescence intensity from the immobilized layer owing to alteration of surface charge at the membrane. The detection system is sensitive to changes in the bulk concentration of urea as small as 20 μM, with a limit of detection of 40 μM of urea.

Fluorescence transduction of an enzyme-substrate reaction by covalently immobilized monolayers of amphiphiles

Abstract The emission intensity of the fluorophore nitrobenzoxadiazoledipalmitoylphosphatidylethanolamine (NBD-PE) is sensitive to the local environmental structure when this species is present as a component of an amphiphilic membrane. The physical and electrostatic structure of a membrane may be modulated by selective reactions which induce electrostatic changes at the surface of the membrane. The resulting change in the fluorescence signal may then be used to monitor the concentration of an analyte which is present in solution. The membrane-associated enzyme acetylcholinesterase (AChE) hydrolyses acetylcholine (ACh) to produce acetic acid and choline. The reaction of ACh with AChE was detected using a monolayer consisting of fatty acids of C 16 chain length which were covalently attached to quartz wafers and which contained a small amount of NBD-PE (partitioned from water into the membrane). The enzyme-substrate reaction produced decreases in fluorescence intensity from the monolayer, detection of 2 μM ACh. The mechanism of transduction of the enzymatic reaction was investigated using spectrofluorimetric mehtods and fluorescence microscopy.

Optimization of the Self-Quenching Response of Nitrobenzoxadiazole Dipalmitoylphosphatidylethanolamine in Phospholipid Membranes for Biosensor Development

Incorporation of the lipid-conjugated fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) into bilayer lipid membranes (BLMs) provides a matrix wherein changes in the structure of the membrane can be transduced into changes in fluorescence intensity or lifetime. In the work reported here, a comparison was made between an empirical model recently developed by our group to account for alterations in the fluorescence lifetime and average fluorescence intensity of NBD-PE as a result of self-quenching and an earlier alternative model which describes self-quenching of membrane-bound chlorophyll a. Our model showed the more satisfactory correlation with self-quenching data obtained from lipid membranes containing 1 to 50 mol % of NBD-PE. This model was used to determine the optimum initial surface concentration of NBD-PE to be incorporated into phospholipid membranes for biosensor development. Optimization was based on the magnitude of the change in fluorescence intensity as a function of changes in the local concentration of the probe. The presence of acidic headgroups in the membrane results in negligible improvement in sensitivity, while a heterogeneous membrane structure greatly enhances the signal magnitude. Experimental results did not provide accurate optimum concentrations, although two NBD-PE surface concentrations were found to yield close agreement with theoretically predicted optimum surface concentrations of 0.027 ± 0.001 and 0.073 ± 0.001 molecules NBD-PE nm−2.

Bilayer lipid membranes as electrochemical switches in reactions involving alteration of surface charge

Abstract Recent reports have indicated that some hydrolytic enzyme reactions at bilayer lipid membranes (BLMs) formed from a mixture of dipalmitoylphosphatidic acid (DPPA) and egg phosphatidyl choline (PC) can provide an electrochemical response in the form of a single transient of current. The time required for the appearance of a transient of constant magnitude has been observed to depend on substrate concentration, and the results suggest that BLMs may produce signals suitable for the development of chemically-selective switches. The mechanism of transient signal generation has not been established. Transient signals appear only when using BLMs prepared from mixtures of DPPA/PC, and are associated with pH changes at the surfaces of membranes. This work reports results of electrochemical studies of BLMs and fluorescence studies of monolayer lipid membranes, which are based on a continuous variation of pH in bulk solution to alter the surface charge density due to DPPA at the membrane surface. The origin of the transient current is due to a combination of double layer reorganization and lipid head group reorganization, and the magnitude and reproducibility of response can be adjusted by selection of DPPA/PC composition.

Hydronium ion sensitivity of surface stabilized stearic acid membranes prepared by Langmuir-Blodgett monolayer transfer

Abstract Alteration of the physical and electrostatic structure of fatty acid membranes may be monitored using the emission intensity of the fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE). Stearic acid/NBD-PE monolayers were deposited onto quartz wafers by Langmuir-Blodgett dipcasting from the air-water interface. The membranes were investigated spectrofluorimetrically and by fluorescence microscopy. Modulation of the pH at the surface of these membranes resulted in alterations of the fluorescence intensity. The results are consistent with an electrostatic mechanism of perturbation of the structure of the monolayer. Alteration of local pH gradients resulted in changes of the extent of ionization and affected the structure of the hydration layer and/or the hydrogen bond network at the membrane-solution interface. Fluorescence microscopy showed that the structures present on dipcast wafers were altered drastically during titrations as a result of mechanical agitation from stirring.

Transduction of the reaction between urea and covalently immobilized urease by fluorescent amphiphilic membranes

Abstract Amphiphiles with chain lengths of 12 and 16 carbons having a triethoxy chlorosilane group at one terminus were covalently immobilized by reaction with hydroxyl groups on the surfaces of planar quartz wafers and optical fibres. The enzyme urease was covalently immobilized onto either carboxylic acid or amine functionalities at the other terminus of the immobilized amphiphiles, resulting in a surface coverage of about 60% of a close-packed monolayer of protein. A small amount (2–3 mol.%) of the fluorescent probe nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) was partitioned into the membranes from an aqueous suspension following immobilization of the urease. Addition of urea to coated wafers or optical fibers placed into aqueous solutions resulted in substantial changes of fluorescence intensity from both the carboxylic acid and amine functionalized membranes. A 20μM change in the concentration of urea could be detected, with a limit of detection of 40μM of urea. The sensitivity degraded ten-fold over a period of 7 days when the samples were stored in buffer at a temperature of 4°C. An investigation into the mechanism of the fluorescence response revealed that local alterations of pH at the surface of membranes due to enzymatic hydrolysis of urea resulted in changes in the extent of ionization of both the membrane and the urease. The resulting changes in the electrostatic interactions between the membrane and the enzyme produced alterations in the rotational mobility of the amphiphiles and fluorophores which affected the self-quenching of NBD-PE.

Towards a homogeneous fluorescence assay for a herbicide: characterization of the interactions of fusilade, bromomethylmethoxy coumarin derivatized fusilade, and anti-fusilade IgG antibody

Abstract The hapten 2-(4-(5-trifluoromethyl-2-pyridyloxy)phenoxy)-propionic acid (fusilade), was derivatized with the fluorescent probe 4-bromomethyl-7-methoxycoumarin (BrMmC). The labelled hapten (fusilade-coumarin, F-C) was incubated with antifusilade IgG and the fluorescence spectra, intensity and time resolved fluorescence decay of the coumarin derivative were measured to study the complexation process of the hapten-antibody system. The fluorescence decay was fit to a two component decay. The magnitude and proportion of these components remained constant but a three-fold increase in intensity was observed during the complexation process. No shifts in the wavelength of the maximum emission occurred during the complexation. The mechanism of the fluorescence intensity response was studied, and it was determined that the increase in intensity observed during the direct fluorescence assay was not due to changes of mobility, polarity or viscosity in the vicinity of the probe. A full examination of fluorescence quenching processes indicated formation of non-emissive ground state complexes (dimers or higher order aggregates) at probe concentrations above 10 −6 M. A homogeneous competitive binding assay indicated that the system did not demonstrate competitive binding as the addition of non-fluorescent fusilade did not result in a displacement of labelled fusilade and thus a decrease in intensity. An assay involving mixing of varying amounts of unlabelled fusilade with a known concentration of F-C in the presence of anti-fusilade IgG was determined to be feasible.