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This paper describes the determination of limits of detection (LODs) of interactions between an antigen, human chorionic gonadotrophin (hCG), and antibodies, anti-[alpha]-hCG and anti-[beta]-hCG, using a sandwich assay by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). Randomly biotinylated antibodies were adsorbed onto a structured self-assembled monolayer (SAM)--streptavidin matrix, tethered to gold via a SAM consisting of biotinylated thiol molecules interspersed with hydroxyalkanethiol molecules. The influence of the concentration of biotinylated thiol on the binding of biotinylated antibody and its functionality, in terms of its ability to bind to the hCG antigen, was studied. This allowed determination of the optimum biotin--thiol mole fraction in the mixed thiol solution and consequently in the SAM, to maximize binding of hCG of the streptavidin-bound antibody. SPFS studies of the binding of a secondary fluorescently labeled antibody to hCG immobilized on the optimized SAM--streptavidin--antibody layer showed that a LOD of hCG of 2 mIU [mL.sup.-1] (4 x [10.sup.-12] mol [L.sup.-1]) could be realized. The system was further optimized by using a more oriented and organized surface by adsorbing monobiotinylated Fab--hCG in place of the whole antibody. A LOD of 0.3 mIU [mL.sup.-1] (6 x [10.sup.-13] mol [L.sup.-1]) was achieved for this system. This work illustrates the importance of antibody orientation, both on the planar surface and in terms of position of binding site, in maximizing sensor sensitivity.

A study demonstrates the use of the micro-patterning of thiol-terminated lipophilic self-assembled monolayers to support planar lipid bilayer membranes which satisfy the critical criteria required for use as potential biosensors. This new method involving the integration of microcontact printing and self-assembly of lipid vesicles enables the construction of a tethered biomimetic membrane. Experimental methods and results are discussed.

The article presents a new approach, which employs a modified nonwoven fabric as a thin film detection/response system for the pathogenic bacteria. The systems are shown to be widely used as responsive dressings with the release of antimicrobials.

The Surface Plasmon Resonance technique is employed for measuring the binding of cholera toxin to GM1 gangliosides on solid supported lipid bilayer vesicles. The analysis proves that europium(III) chloride can be easily used for the inhibition of such binding and subsequent membrane permeation.

In this paper the immobilization of small unilamellar DMPC/GM1 lipid vesicles containing a water-soluble bodipy dye is described. The binding of the complete αβ toxin expressed by Vibrio cholerae to the attached vesicles was measured using Surface Plasmon Resonance (SPR) and a value of the dissociation constant KD obtained. Further measurements showed that the interaction of both the αβ-toxin and the β-subunit alone resulted in the permeation of the lipid membrane, with release of a fluorophore contained within the vesicle being measured by combined SPR and Surface Plasmon enhanced Fluorescence Spectroscopy (SPFS). The leakage of dye through the membrane, measured by following the change in fluorescence, was fitted to a simple diffusion model. Finally, SPFS measurements of the effect of europium(III) chloride (EuCl3) showed that cholera toxin binding and subsequent membrane permeation could be blocked by 1 µmol dm-3 europium chloride. In view of the low oral toxicity of europium chloride, we speculate on the potential pharmaceutical applications of this molecule in the treatment of cholera infection. [ABSTRACT FROM AUTHOR]

Formation of crystalline biofilms following infection by Proteus mirabilis can lead to encrustation and blockage of long-term indwelling catheters, with serious clinical consequences. We describe a simple sensor, placed within the catheter drainage bag, to alert of impending blockage via a urinary color change. The pH-responsive sensor is a dual-layered polymeric "lozenge", able to release the self-quenching dye 5(6)-carboxyfluorescein in response to the alkaline urine generated by the expression of bacterial urease. Sensor performance was evaluated within a laboratory model of the catheterized urinary tract, infected with both urease positive and negative bacterial strains under conditions of established infection, achieving an average "early warning" of catheter blockage of 14.5 h. Signaling only occurred following infection with urease positive bacteria. Translation of these sensors into a clinical environment would allow appropriate intervention before the occurrence of catheter blockage, a problem for which there is currently no effective control method.

The early detection of wound infection in situ can dramatically improve patient care pathways and clinical outcomes. There is increasing evidence that within an infected wound the main bacterial mode of living is a biofilm: a confluent community of adherent bacteria encased in an extracellular polymeric matrix. Here we have reported the development of a prototype wound dressing, which switches on a fluorescent color when in contact with pathogenic wound biofilms. The dressing is made of a hydrated agarose film in which the fluorescent dye containing vesicles were mixed with agarose and dispersed within the hydrogel matrix. The static and dynamic models of wound biofilms, from clinical strains of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis, were established on nanoporous polycarbonate membrane for 24, 48, and 72 h, and the dressing response to the biofilms on the prototype dressing evaluated. The dressing indicated a clear fluorescent/color response within 4 h, only observed when in contact with biofilms produced by a pathogenic strain. The sensitivity of the dressing to biofilms was dependent on the species and strain types of the bacterial pathogens involved, but a relatively higher response was observed in strains considered good biofilm formers. There was a clear difference in the levels of dressing response, when dressings were tested on bacteria grown in biofilm or in planktonic cultures, suggesting that the level of expression of virulence factors is different depending of the growth mode. Colorimetric detection on wound biofilms of prevalent pathogens (S. aureus, P. aeruginosa, and E. faecalis) is also demonstrated using an ex vivo porcine skin model of burn wound infection.

Liposomes containing lipids and polydiacetylene (PDA) are hybrid systems encompassing both a fluid phospholipid membrane and a polymer scaffold (PDA). However, the biophysical role of PDA in such liposomes is not well understood. In this report, we studied the effects of photopolymerization of PDA on the stability of lipid-PDA liposomes, and their sensitivity to selected purified toxins and bacterial supernatants, using a fluorescence assay. Of the three different types of liposomes with variable lipid chain lengths that were chosen, the degree of polymerization had a significant impact on the long-term stability, and response, to external microbial exotoxins secreted by pathogenic bacteria, namely, Staphylococcus aureus and Pseudomonas aeruginosa. The degree of polymerization of TCDA played an important role in lipid-chain-length-dependent stabilization of lipid-PDA liposomes, as well as in their response to bacterial toxins of S. aureus and P. aeruginosa.

Phospholipid vesicles have been the focus of attention as potential vehicles for drug delivery, as they are biomimetic, easy to produce, and contain an aqueous compartment which can be used to carry hydrophilic material, such as drugs or dyes. Lipid vesicles used for this purpose present a particular challenge, as they are not especially stable and can rapidly break down and release their contents away from the target area, especially at physiological temperatures/environments. This study aims to investigate optimum methods for vesicle stabilization where the vesicles are employed as part of a system or technology that signals the presence of pathogenic bacteria via the effect of secreted cytolytic virulence factors on a sensor interface. A number of approaches have been investigated and are presented here as a systematic study of the long-term (14 day) stability at 37 °C, and at various pHs. The response of vesicles, both in suspension and within hydrogels, to Staphylococcus aureus (RN 4282) and Pseudomonas aeruginosa (PAO1) whole bacteria, and supernatants from overnight cultures of both (containing secreted proteins but free of cells), was measured via a sensitive encapsulated carboxyfluorescein release assay. The results showed that lipid chain length, cholesterol concentration, and stabilization via photopolymer stable components were critical in achieving stability. Finally, dispersion of the optimum vesicle formulation in hydrogel matrixes was investigated, culminating in the in vivo demonstration of a simple prototype wound dressing.

This paper describes the modification of nonwoven fabric such that it responds by releasing an encapsulated antimicrobial from within an attached vesicle in response to two species of pathogenic bacteria (Staphylococcus aureus MSSA 476 and Pseudomonas aeruginosa PAO1), but does not respond to nonpathogenic Escherichia coli DH5alpha. This concept is based on the generalization that a majority of pathogenic bacteria secrete virulence factors such as toxins and lipases that actively damage cell membranes, typically observed as tissue damage around infected wounds, while nonpathogenic bacteria do not (or not at high concentration). The eventual aim of this work is to produce responsive dressings which release antimicrobials and change color only on infected wounds. This paper details preliminary approaches to achieving this goal, including vesicle-bacteria studies in aqueous suspension, and fluorescence imaging of fluorescein containing vesicles lysed by S. aureus and P. aeruginosa, but not by E. coli.

Tethered bilayer lipid membranes are stable solid supported model membrane systems. They can be used to investigate the incorporation and function of membrane proteins. In order to study ion translocation mediated via incorporated proteins, insulating membranes are necessary. The architecture of the membrane can have an important effect on both the electrical properties of the lipid bilayer as well as on the possibility to functionally host proteins. Alpha-hemolysin pores have been functionally incorporated into a tethered bilayer lipid membrane coupled to a gold electrode. The protein incorporation has been monitored optically and electrically and the influence of the molecular structure of the anchor lipids on the insertion properties has been investigated.

This article describes a method by which intact vesicles can be chemically attached to hydrolyzed maleic anhydride films covalently bound to plasma-polymerized SiO2 on Au substrates. Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) combined with surface plasmon resonance spectroscopy (SPR) was used to monitor the activation of plasma-deposited maleic anhydride (pp-MA) film with EDC/NHS and the subsequent coupling of lipid vesicles. The vesicles were formed from a mixture of phosphatidylcholine and phosphatidylethanolamine lipids, with a water-soluble fluorophore encapsulated within. Vesicle attachment was measured in real time on plasma films formed under different pulse conditions (plasma duty cycle). Optimum vesicle attachment was observed on the pp-MA films containing the highest density of maleic anhydride groups. Phospholipase A2 was used to lyse the surface-bound vesicles and to release the encapsulated fluorophore.

This letter describes a new method for studying the interaction of the membrane-lysing enzyme phospholipase A(2) (PLA(2)) with phospholipid bilayers by simultaneous measurements of enzyme binding and vesicle lysis using surface plasmon resonance (SPR) and permeabilization using surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The PLA(2) inhibitor dimethyl-eicosadienoic acid was incorporated into the surface-bound vesicles and support bilayer in order to study its role in preventing PLA(2)-mediated vesicle lysis. This methodology has a generic applicability for the study of a range of membrane-disrupting agents.

This letter describes how the irreversible attachment of bovine serum albumin (BSA) to films of plasma-polymerized maleic anhydride can be measured by an indirect antibody-binding assay and how this attachment appears to be strongly affected by the polymerization conditions. Surface plasmon resonance (SPR) was used to follow the binding of the antibody, anti-bovine serum albumin (aBSA), to protein-modified plasma-polymerized maleic anhydride films. It was found that BSA could be irreversibly bound to polymers made under pulse plasma conditions, but BSA did not bind to polymers made under continuous wave conditions. Moreover, the degree of antibody binding, which is directly related to the quantity of BSA on the polymer, correlated with the plasma duty cycle (t(on)/t(on) + t(off)): lower duty cycle pulse plasma conditions gave greater BSA attachment. We speculate that BSA is being covalently bound to the polymer via the reaction of amine groups on lysine residues in BSA with the retained anhydride group functionality in the polymer.