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We demonstrate a novel array-based diagnostic platform comprising lipid/polydiacetylene (PDA) vesicles embedded within a transparent silica-gel matrix. The diagnostic scheme is based upon the unique chromatic properties of PDA, which undergoes blue-red transformations induced by interactions with amphiphilic or membrane-active analytes. We show that constructing a gel matrix array hosting PDA vesicles with different lipid compositions and applying to blood plasma obtained from healthy individuals and from patients suffering from disease, respectively, allow distinguishing among the disease conditions through application of a simple machine-learning algorithm, using the colorimetric response of the lipid/PDA/gel matrix as the input. Importantly, the new colorimetric diagnostic approach does not require a priori knowledge on the exact metabolite compositions of the blood plasma, since the concept relies only on identifying statistically significant changes in overall disease-induced chromatic response. The chromatic lipid/PDA/gel array-based "fingerprinting" concept is generic, easy to apply, and could be implemented for varied diagnostic and screening applications.

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AIM: Development of a new chromatic (colorimetric/fluorescence) bacterial sensor, for rapid, sensitive and versatile detection of bacterial proliferation. METHODS AND RESULTS: We constructed agarose-embedded chromatic films which produce dramatic colour changes and fluorescence transformations in response to bacterial growth. The sensing constructs comprise glass-supported Langmuir-Schaeffer phospholipid/polydiacetylene films that undergo both blue-red transformations and induction of intense fluorescence following interactions with bacterially secreted amphiphilic compounds that diffuse through the agarose. The agarose matrix coating the sensor film further contains growth nutrients, facilitating signal amplification through promotion of bacterial culture proliferation. The agarose layer also constitutes an effective barrier for reducing background signals not associated with the bacteria. We demonstrate the applications of the new sensor for the detection of Gram-negative and Gram-positive bacteria, and for screening specimens of physiological fluids (blood and urine) and foods (meat) for bacterial contaminations. CONCLUSIONS: The experiments demonstrate that the new agarose-embedded film constructs are capable of bacterial detection through visible colour transitions and fluorescence emission recorded in conventional apparatuses. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrated a new simple chromatic platform for bacterial detection, based on the generation of easily recorded colour and fluorescence changes. The new bacterial detection scheme is highly generic and could be employed for varied practical uses, in which, rapid reporting on bacterial presence is required.

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The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.

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PURPOSE: This work aims to demonstrate a novel chemical assay for rapid screening and analysis of the mode of action of membrane interaction by penetration enhancers. METHODS: The new bio-mimetic membrane assembly, consisting of supramolecular aggregates of lipids and conjugated polydiacetylene, undergoes visible and quantifiable blue-red color transitions upon interaction with penetration enhancers. RESULTS: The new colorimetric model has been employed to examine various classes of penetration enhancers, including 1-dodecylhexahydro-2H-azepin-2-one (Azone), oleic acid, propylene-glycol, menthol, ethoxyglycol-diethyleneglycol-monoethyl-ether (Transcutol), polysorbate-polyethylenesorbitan-monolaurate (Tween-20), and the drug 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one (Diazepam). The assay enables to evaluate the validity of various observations and hypotheses proposed in previous studies regarding permeation enhancement activities. Our results suggest, for example. that propylene glycol (PG) by itself does not interfere with membranes, but rather exhibits synergistic effect in combination with other penetration enhancers. Similarly, our data demonstrate that Transcutol does not independently interact with membranes. The colorimetric system also indicates that interaction of penetration enhancers with membranes depend upon the lipid phase, as well as the self-assembly properties of the enhancer molecules. CONCLUSIONS: The new biomimetic model membrane system can be applied for rapid screening of the activities of penetration enhancers, and provides insight into the mechanisms of permeability of membrane-active compounds.

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Biomolecular recognition of antigens and epitopes by antibodies is a fundamental event in the initiation of immune response and plays a central role in a variety of biochemical processes. Peptide binding requires, in many cases, presentation of the peptides at interfaces, such as protein surfaces, cellular membranes, and synthetic polymer surfaces. We describe a novel molecular system in which interactions between antibodies and peptide epitopes displayed at a biomimetic membrane interface can be detected through induction of visible, rapid color transitions. The colorimetric assembly consists of a phospholipid/polydiacetylene matrix anchoring a hydrophobic peptide displaying the epitope at its N-terminus. The colorimetric transitions observed in the assembly, corresponding to perturbation of the polydiacetylene framework, are induced only upon recognition of the displayed epitope by its specific antibody present in the aqueous solution. Significantly, the color changes occur after a single mixing step, without further chemical reactions or enzymatic processing. The new molecular system could be utilized for studying antigen-antibody interactions and peptide-protein recognition, epitope mapping, and rapid screening of biological and chemical libraries.

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Supramolecular chemical assemblies composed of polydiacetylene (PDA) exhibit rapid colorimetric transitions upon specific interactions with a variety of biological analytes in aqueous solutions. Among the analytes that give rise to the unique blue-red color changes are lipophilic enzymes, antibacterial peptides, ions, antibodies, and membrane penetration enhancers. The chemical assemblies include conjugated PDA, responsible for the chromatic transitions, and the molecular recognition elements, which are either chemically or physically associated with the PDA. Thus, by incorporation of specific recognition elements, the system can be designed in ways allowing for highly selective identification of analytes. In particular, receptors and epitopes can be incorporated within the sensor assembly, which then determine the specificity of the colorimetric transitions. The PDA-based molecular assemblies are robust and can be readily applied to diagnosis of physiological molecules and for rapid screening of chemical and biological libraries, for example, in 96 well-plate platforms.

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Interactions between peptides and lipid membranes play major roles in numerous physiological processes, such as signaling, cytolysis, formation of ion channels, and cellular recognition. We describe a new colorimetric technique for studying peptide-membrane interactions. The new assay is based on supramolecular assemblies composed of phospholipids embedded in a matrix of polydiacetylene (PDA) molecules. The phospholipid/PDA vesicle solutions undergo visible color changes upon binding of membrane peptides. Experiments utilizing various analytical techniques confirm that the blue-to-red color transitions of the phospholipid/PDA vesicles are directly related to adoption of helical conformations by the peptides and their association with the lipids. Spectroscopic data indicate that the colorimetric transitions are correlated with important molecular parameters, such as the degree of penetration of the peptides into lipid bilayers, and the mechanisms of peptide-lipid binding. The results suggest that the new colorimetric assay could be utilized for studying interactions and organization of membrane peptides.

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The increased resistance of various bacteria toward available antibiotic drugs has initiated intensive research efforts into identifying new sources of antimicrobial substances. Short antibiotic peptides (10-30 residues) are prevalent in nature as part of the intrinsic defense mechanisms of most organisms and have been proposed as a blueprint for the design of novel antimicrobial agents. Antimicrobial peptides are generally believed to kill bacteria through membrane permeabilization and extensive pore-formation. Assays providing rapid and easy evaluation of interactions between antimicrobial membrane peptides and lipid bilayers could significantly improve screening for substances with effective antibacterial properties, as well as contribute to the elucidation of structural and functional properties of antimicrobial peptides. Here we describe a colorimetric sensor in which particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids were shown to exhibit striking color changes upon interactions with antimicrobial membrane peptides. The color changes in the system occur because of the structural perturbation of the lipids following their interactions with antimicrobial peptides. The assay was also sensitive to the antibacterial properties of structurally and functionally related peptide analogs.

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