Fluid and Air-Stable Lipopolymer Membranes for Biosensor Applications

Albertorio, Fernando; Diaz, Arnaldo J.; Yang, Tinglu; Chapa, Vanessa A.; Kataoka, Sho; Castellana, Edward T.; Cremer, Paul S.

doi:10.1021/la050871s

Cited by 134 publications

(191 citation statements)

References 66 publications

(107 reference statements)

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“…Bilayers cushioned on a soft polymer layer had been known to better preserve transmembrane protein mobility and function 32 , and had been used in fusion studies with viruses 33 . In addition, PEGylated bilayers seem to retain some ability to self-heal and are very robust 34,35 . First, a fraction of commercially available, lipid-linked PEG chains are included in the t-SUV membrane.…”

Section: Introductionmentioning

confidence: 99%

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Nikolaus

Karatekin

2016

JoVE

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In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. Video LinkThe video component of this article can be found at

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Section: Introductionmentioning

confidence: 99%

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Nikolaus

Karatekin

2016

JoVE

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“…41,42 The diffusion constant for the NBD-PE lipid was 3.4 ± 0.8 μm 2 /s and the mobile fraction was ~95 ± 2%. Moreover, individual SLBs were completely confined within their protein boundaries.…”

Section: Resultsmentioning

confidence: 99%

“…Fluorescence recovery after photobleaching (FRAP) 41,42 experiments were conducted to check the quality and fluidity of the SLBs. This was done using an inverted epifluorescence Nikon Eclipse TE2000-U microscope equipped with a 10× objective.…”

Section: Epifluorescence and Tirfmmentioning

confidence: 99%

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

2008

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A method has been designed for patterning supported phospholipid bilayers (SLBs) on planar substrates and inside microfluidic channels. To do this, bovine serum albumin (BSA) monolayers were formed via adsorption at the liquid/solid interface. Next, this interfacial protein film was selectively patterned by using deep UV lithography. Subsequently, SLBs could be deposited in the patterned locations by vesicle fusion. By cycling through this process several times, spatially addressed bilayer arrays could be formed with intervening protein molecules serving as twodimensional corrals. By employing this method, phospholipid bilayers containing various concentrations of ganglioside GM1 were addressed along the length of individual microfluidic channels. Therefore, the binding of GM1 with pentameric cholera toxin B (CTB) subunits could be probed. A seven-channel microfluidic device was fabricated for this purpose. Each channel was simultaneously patterned with four chemically distinct SLBs containing 0, 0.2, 0.5, and 2.0 mol % GM1, respectively. Varying concentrations of CTB were then introduced into each of the channels. With the use of total internal reflection fluorescence microscopy, it was possible to simultaneously abstract multiple equilibrium dissociation constants as a function of ligand density for the CTB-GM1 system in a single shot.Multivalent ligand-receptor interactions are ubiquitous on cell surfaces. They have a wide variety of consequences including the modulation of equilibrium dissociation constants, high receptor selectivity, and receptor clustering. 1 Multivalency can also play a direct role in signal transduction processes. 2 Examination of the underlying thermodynamics of ligandreceptor binding may lead to a greater understanding of its biological role and could provide insight into biomedical applications involving inhibitory drug design. 1,3,4 Unfortunately, high-throughput, low-protein consumption assays are not presently well enough developed in this field to afford rapid, systematic studies of binding data at two-dimensionally fluid bilayer interfaces. 5 In previous work it has been demonstrated that microfluidic devices can be designed for measuring binding affinities in multivalent systems. [5][6][7][8][9][10][11][12][13][14] In our previous setup, ligands were incorporated into supported lipid bilayers (SLBs) coated on the walls and floors of polydimethylsiloxane/glass microchannels. Linear arrays of channels were then monitored by total internal reflection fluorescence microscopy (TIRFM) 15 to obtain equilibrium dissociation constants in one-shot assays. This could be done by using the same surface chemistry in each microchannel while varying the solution concentration of the aqueous proteins. Such a setup made these assays relatively rapid, afforded high accuracy, and used only a few microliters of protein solution. Nevertheless, such platforms could be markedly improved by measuring multiple binding constants with various membrane chemistries simultaneously. This could be achie...

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“…The formation of planar lipid-based biomimetic platforms on gold surfaces has aroused the interest of many researchers [9][10][11][12]. One of the most limiting features of this type of assemblies has been the stability of the supported lipid bilayer (SLB) on gold which has led to complex approaches that require especially synthesized molecules, namely polyethylene glycol (PEG)-derivatives [13,14], thiolipids [6,15], biotinylated lipids [14][15][16][17] or other tether layers [18,19]. In this work we describe an alternative approach that takes full advantage of both the biophysical and the chemical properties of lipids in order to build a simple and efficient immunosensing platform.…”

Section: Introductionmentioning

confidence: 99%

Phospholipid/cholesterol/decanethiol mixtures for direct assembly of immunosensing interfaces

Almeida

Marquês

Liu

et al. 2015

Colloids and Surfaces B: Biointerfaces

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a b s t r a c tIn this work, a simple yet robust method to prepare lipid-based biosensing interfaces on gold using common lipids (a phospholipid and cholesterol) and an alkanethiol is reported. The lipids were carefully chosen to tailor the biophysical properties of the bilayer. The simplicity of the method relies on the incorporation of a small percentage of decanethiol in the lipid vesicles for a direct formation of a thiol-linked supported lipid bilayer, which is advantageous in several respects. It prevents the use of specially synthesized thiolipids and preserves the natural fluidity and dynamics of the lipids. As a consequence the whole arrangement is extremely stable regarding ionic strength changes and solution flow during surface plasmon resonance experiments. Moreover, we show that this interface is very effective on suppressing the nonspecific adsorption of proteins on the surface, and enables the covalent attachment of the recognition antibody. The subsequent detection of specific interaction toward antigen was monitored in real-time by SPR and confirmed by ellipsometric measurements. This lipid-based biosensing platform is versatile and can be adapted to the biorecognition reaction of interest.

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Fluid and Air-Stable Lipopolymer Membranes for Biosensor Applications

Cited by 134 publications

References 66 publications

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Multiplexing Ligand−Receptor Binding Measurements by Chemically Patterning Microfluidic Channels

Phospholipid/cholesterol/decanethiol mixtures for direct assembly of immunosensing interfaces

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