Generation of phospholipid vesicle-nanotube networks and transport of molecules therein

We describe micromanipulation and microinjection procedures for the fabrication of soft-matter networks consisting of lipid bilayer nanotubes and surface-immobilized vesicles. These biomimetic membrane systems feature unique structural flexibility and expandability and, unlike solid-state microfluidic and nanofluidic devices prepared by top-down fabrication, they allow network designs with dynamic control over individual containers and interconnecting conduits. The fabrication is founded on self-assembly of phospholipid molecules, followed by micromanipulation operations, such as membrane electroporation and microinjection, to effect shape transformations of the membrane and create a series of interconnected compartments. Size and geometry of the network can be chosen according to its desired function. Membrane composition is controlled mainly during the self-assembly step, whereas the interior contents of individual containers is defined through a sequence of microneedle injections. Networks cannot be fabricated with other currently available methods of giant unilamellar vesicle preparation (large unilamellar vesicle fusion or electroformation). Described in detail are also three transport modes, which are suitable for moving water-soluble or membrane-bound small molecules, polymers, DNA, proteins and nanoparticles within the networks. The fabrication protocol requires ∼90 min, provided all necessary preparations are made in advance. The transport studies require an additional 60-120 min, depending on the transport regime. © 2011 Nature America, Inc. All rights reserved

Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Programmable Assembly of DNA-Functionalized Liposomes by DNA

This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Nano, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/nn1030093Bionanotechnology involves the use of biomolecules to control both the structure and property of nanomaterials. One of the most studied examples is DNA-directed assembly of inorganic nanoparticles such as gold nanoparticles (AuNPs). However, systematic studies on DNA-linked soft nanoparticles, such as liposomes, are still lacking. We herein report the programmable assembly and systematic characterization of DNA-linked liposomes as a function of liposome size, charge, fluidity, composition, DNA spacer, linker DNA sequence, and salt concentration for direct comparison to DNA-directed assembly of AuNPs. Similar to the assemblies of AuNPs, sharp melting transitions were observed for liposomes where the first derivative of the melting curve full width at half-maximum (fwhm) is equal to or less than 1 °C for all of the tested liposomes, allowing sequence specific DNA detection. We found that parameters such as liposome size, charge, and fluidity have little effect on the DNA melting temperature. Cryo-TEM studies showed that programmable assemblies can be obtained and that the majority of the liposomes maintained a spherical shape in the assembled state. While liposome and AuNP systems are similar in many aspects, there are also important differences that can be explained by their respective physical properties.University of Waterloo || Natural Sciences and Engineering Research Council |

The multiple faces of self-assembled lipidic systems

Lipids, the building blocks of cells, common to every living organisms, have the propensity to self-assemble into well-defined structures over short and long-range spatial scales. The driving forces have their roots mainly in the hydrophobic effect and electrostatic interactions. Membranes in lamellar phase are ubiquitous in cellular compartments and can phase-separate upon mixing lipids in different liquid-crystalline states. Hexagonal phases and especially cubic phases can be synthesized and observed in vivo as well. Membrane often closes up into a vesicle whose shape is determined by the interplay of curvature, area difference elasticity and line tension energies, and can adopt the form of a sphere, a tube, a prolate, a starfish and many more. Complexes made of lipids and polyelectrolytes or inorganic materials exhibit a rich diversity of structural morphologies due to additional interactions which become increasingly hard to track without the aid of suitable computer models. From the plasma membrane of archaebacteria to gene delivery, self-assembled lipidic systems have left their mark in cell biology and nanobiotechnology; however, the underlying physics is yet to be fully unraveled

A Four Parameter Microfluidic Tandem SAW-IS Bio-Sensor

Surface Acoustic Resonance (SAR) biosensing has recently been proposed as a highly compact and robust alternative to the conventional SAW delay-line based biosensing. The device can also be presented as a one-port high frequency alternative to the QCM, employing SAW resonance. It enables simple one-port measurements at low powers, while offering robust integration with microfluidics and implementation in integrated sensor arrays. Here we discuss the SAR approach as a key enabling and demonstrate its integration with the impedance spectroscopy (IS) concept in a single microfluidic device. The IS is integrated within a SAW reflector formed as interdigitated electrode (IDE) capacitor. A test fixture with SAW and IS ports is designed and fabricated. Four sensitive parameters are deduced from the tandem sensor readout and employed in a proof of principle study of liposome layers and their interaction with Ca2+ ions

A Heck-type coupling for the synthesis of novel bridged metallochlorin-fullerene C-60 dyads

A short and convenient synthesis of metallochlorin-C-60 dyads based on a Heck-type hetero coupling at the 3(2) position of a chlorin is described. p-Bromobenzaldehyde was treated with Zn-metalated 13(2)-demethoxycarbonylmethylpheophorbide a, using a palladium acetate/LiCl catalyst mixture under phase-transfer conditions in DMF at 70 degrees C. The resulting asymmetric olefin was obtained in a high trans/cis ratio. The desired trans isomer was separated and subsequently transformed into a donor-acceptor dyad by a 1,3-dipolar cycloaddition to C-60 in the presence of sarcosine in refluxing toluene. The resulting dyads are expected to undergo efficient photoinduced electron transfer and can potentially be utilized in solar energy conversion devices. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A Heck-type coupling for the synthesis of novel bridged metallochlorin-fullerene C-60 dyads

A short and convenient synthesis of metallochlorin-C-60 dyads based on a Heck-type hetero coupling at the 3(2) position of a chlorin is described. p-Bromobenzaldehyde was treated with Zn-metalated 13(2)-demethoxycarbonylmethylpheophorbide a, using a palladium acetate/LiCl catalyst mixture under phase-transfer conditions in DMF at 70 degrees C. The resulting asymmetric olefin was obtained in a high trans/cis ratio. The desired trans isomer was separated and subsequently transformed into a donor-acceptor dyad by a 1,3-dipolar cycloaddition to C-60 in the presence of sarcosine in refluxing toluene. The resulting dyads are expected to undergo efficient photoinduced electron transfer and can potentially be utilized in solar energy conversion devices. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Dynamic microcompartmentalization of giant unilamellar vesicles by sol gel transition and temperature induced shrinking/swelling of poly(N-isopropyl acrylamide)

Giant unilamellar vesicles (GUVs) were microinjected with aqueous solutions of poly(N-isopropyl acrylamide) (PNIPAAm). Temperature-dependent sol-gel phase transitions of the solutions, followed by shrinking and swelling of the resulting hydrogel, were studied in the presence of a variety of co-solutes within the GUV. Reversible formation of a dense, spherical hydrogel structure (compartment) was observed in all cases with defined shrinking/swelling behaviour at temperatures above the lower critical solution temperatures (LCSTs). Nanotube-mediated merging of two vesicles with thus formed compartments resulted in a single GUV with two internalized hydrogel structures. As an application example, we demonstrate how fluorescent nanoparticles can be immobilized in such gel structures

Light-activated desorption of photoactive polyelectrolytes from supported lipid bilayers

Phospholipid vesicles and supported bilayers have emerged as a promising platform for the development of biorecognition devices. To expand the capabilities of such biochips, it becomes desirable to direct and control the assembly of lipid structures into more sophisticated architectures. As one step toward this goal, we demonstrate the photoregulated desorption of a new class of polymer from lipid bilayers. The neutral, hydrophobic polymer resides within the bilayer under mild pH and ambient conditions. However, it contains side groups that can undergo excited state proton transfer (ESPT). The polymer therefore behaves as a polyelectrolyte when exposed to IN light. With the ensuing increase in hydrophilicity, the molecule is spontaneously ejected from the bilayer. Quartz crystal microbalance measurements with dissipation monitoring (QCM-D) have recorded this process and have shown that a rapid buffer exchange during light exposure results in efficient removal of the polymer from the system. Three polymers were tested in all: a polyanion, a polyeation, and a polyzwitterion. A one-step approach to the synthesis of the monomer, performed under relatively mild reaction conditions, made it possible to synthesize each polymer in one step