Ring-shaped nanoparticle assembly and cross-linking on lipid vesicle scaffolds.

We show the assembly of carboxylate-modified polystyrene nanoparticles into flexible circular, ring-shaped structures with micrometer sized diameters around the base of surface-adhered lipid vesicles. The rings remain around the vesicles but disintegrate when the lipid membranes are dissolved in detergent. The aqueous medium allows carbodiimide-based cross-linking chemistry to be applied to the particle assemblies resulting in the preservation of the rings even after the lipid compartments are dissolved.

Protocells: Milestones and Recent Advances.

The origin of life is still one of humankind's great mysteries. At the transition between nonliving and living matter, protocells, initially featureless aggregates of abiotic matter, gain the structure and functions necessary to fulfill the criteria of life. Research addressing protocells as a central element in this transition is diverse and increasingly interdisciplinary.

Manipulation of Lipid Membranes with Thermal Stimuli.

We describe a protocol for the assembly and application of infrared (IR-B) laser-based set-ups to be used for localized heating of solid-supported planar and vesicular lipid membrane assemblies.

Dynamic Range Expansion of the C-Reactive Protein Quantification with a Tandem Giant Magnetoresistance Biosensor.

In this study, we report a convenient analytical method for a full-range quantification of the C-reactive protein (CRP), a blood biomarker of infection and cardiovascular events. We determine CRP over the entire diagnostically relevant concentration range in undiluted human blood serum in a single test, using a tandem giant magnetoresistance (GMR) sensor. The tandem principle combines a sandwich assay and a competitive assay, which allows for the discrimination of the concentration values resulting from the multivalued dose-response curve ("Hook" effect), which characterizes the one-step sandwich assay at high CRP concentrations.

A tandem giant magnetoresistance assay for one-shot quantification of clinically relevant concentrations of N-terminal pro-B-type natriuretic peptide in human blood.

We report a microfluidic sandwich immunoassay constructed around a dual-giant magnetoresistance (GMR) sensor array to quantify the heart failure biomarker NT-proBNP in human plasma at the clinically relevant concentration levels between 15 pg/mL and 40 ng/mL. The broad dynamic range was achieved by differential coating of two identical GMR sensors operated in tandem, and combining two standard curves. The detection limit was determined as 5 pg/mL.

Generation of interconnected vesicles in a liposomal cell model.

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca solution from the micropipette tip.

Microfluidic technology for investigation of protein function in single adherent cells.

Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency.

Styrene maleic acid copolymer induces pores in biomembranes.

We investigated the interactions between styrene-maleic acid (SMA) copolymers and phospholipid bilayers, using confocal microscopy and surface acoustic wave resonance (SAR) sensing. For the first time we experimentally observed and followed pore formation by SMA copolymers in intact supported bilayers and unilamellar vesicles, showing that fluorescein, a water-soluble organic compound with a mean diameter of 6.9 Å, can traverse the membrane.

Characterization of Binding of Magnetic Nanoparticles to Rolling Circle Amplification Products by Turn-On Magnetic Assay.

The specific binding of oligonucleotide-tagged 100 nm magnetic nanoparticles (MNPs) to rolling circle products (RCPs) is investigated using our newly developed differential homogenous magnetic assay (DHMA). The DHMA measures ac magnetic susceptibility from a test and a control samples simultaneously and eliminates magnetic background signal. Therefore, the DHMA can reveal details of binding kinetics of magnetic nanoparticles at very low concentrations of RCPs.

Homogeneous Differential Magnetic Assay.

Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal.

Molecular Lipid Films on Microengineering Materials.

In this study, we have systematically investigated the formation of molecular phospholipid films on a variety of solid substrates fabricated from typical surface engineering materials and the fluidic properties of the lipid membranes formed on these substrates. The surface materials comprise of borosilicate glass, mica, SiO, Al (native oxide), AlO, TiO, ITO, SiC, Au, Teflon AF, SU-8, and graphene. We deposited the lipid films from small unilamellar vesicles (SUVs) by means of an open-space microfluidic device, observed the formation and development of the films by laser scanning confocal microscopy, and evaluated the mode and degree of coverage, fluidity, and integrity.

Volume-amplified magnetic bioassay integrated with microfluidic sample handling and high- SQUID magnetic readout.

A bioassay based on a high- superconducting quantum interference device (SQUID) reading out functionalized magnetic nanoparticles (fMNPs) in a prototype microfluidic platform is presented. The target molecule recognition is based on volume amplification using padlock-probe-ligation followed by rolling circle amplification (RCA). The MNPs are functionalized with single-stranded oligonucleotides, which give a specific binding of the MNPs to the large RCA coil product, resulting in a large change in the amplitude of the imaginary part of the ac magnetic susceptibility.

Peridynamic Modeling of Ruptures in Biomembranes.

We simulate the formation of spontaneous ruptures in supported phospholipid double bilayer membranes, using peridynamic modeling. Experiments performed on spreading double bilayers typically show two distinct kinds of ruptures, floral and fractal, which form spontaneously in the distal (upper) bilayer at late stages of double bilayer formation on high energy substrates. It is, however, currently unresolved which factors govern the occurrence of either rupture type.

Spatial characterization of a multifunctional pipette for drug delivery in hippocampal brain slices.

Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies.

New Method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues.

A rapid microfluidic technique for integrated viability determination of adherent single cells.

Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity.

Nanopatterning of mobile lipid monolayers on electron-beam-sculpted Teflon AF surfaces.

Direct electron-beam lithography is used to fabricate nanostructured Teflon AF surfaces, which are utilized to pattern surface-supported monolayer phospholipid films with 50 nm lateral feature size. In comparison with unexposed Teflon AF coatings, e-beam-irradiated areas show reduced surface tension and surface potential. For phospholipid monolayer spreading experiments, these areas can be designed to function as barriers that enclose unexposed areas of nanometer dimensions and confine the lipid film within.

A heating-superfusion platform technology for the investigation of protein function in single cells.

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system.

Lab on a Biomembrane: rapid prototyping and manipulation of 2D fluidic lipid bilayers circuits.

Lipid bilayer membranes are among the most ubiquitous structures in the living world, with intricate structural features and a multitude of biological functions. It is attractive to recreate these structures in the laboratory, as this allows mimicking and studying the properties of biomembranes and their constituents, and to specifically exploit the intrinsic two-dimensional fluidity. Even though diverse strategies for membrane fabrication have been reported, the development of related applications and technologies has been hindered by the unavailability of both versatile and simple methods.

Probing enzymatic activity inside single cells.

We report a novel approach for determining the enzymatic activity within a single suspended cell. Using a steady-state microfluidic delivery device and timed exposure to the pore-forming agent digitonin, we controlled the plasma membrane permeation of individual NG108-15 cells. Mildly permeabilized cells (~100 pores) were exposed to a series of concentrations of fluorescein diphosphate (FDP), a fluorogenic alkaline phosphatase substrate, with and without levamisole, an alkaline phosphatase inhibitor.

A multifunctional pipette for localized drug administration to brain slices.

We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells.

Thermal migration of molecular lipid films as a contactless fabrication strategy for lipid nanotube networks.

We demonstrate the contactless generation of lipid nanotube networks by means of thermally induced migration of flat giant unilamellar vesicles (FGUVs), covering micro-scale areas on oxidized aluminum surfaces. A temperature gradient with a reach of 20 μm was generated using a focused IR laser, leading to a surface adhesion gradient, along which FGUVs could be relocated. We report on suitable lipid-substrate combinations, highlighting the critical importance of the electrostatic interactions between the engineered substrate and the membrane for reversible migration of intact vesicles.

An optofluidic temperature probe.

We report the application of a microfluidic device for semi-contact temperature measurement in picoliter volumes of aqueous media. Our device, a freely positionable multifunctional pipette, operates by a hydrodynamic confinement principle, i.e.

Fluctuations in Tat copy number when it counts the most: a possible mechanism to battle the HIV latency.

The HIV-1 virus can enter a dormant state and become inactive, which reduces accessibility by antiviral drugs. We approach this latency problem from an unconventional point of view, with the focus on understanding how intrinsic chemical noise (copy number fluctuations of the Tat protein) can be used to assist the activation process of the latent virus. Several phase diagrams have been constructed in order to visualize in which regions of the parameter space noise can drive the activation process.

Kinetics of diffusion-mediated DNA hybridization in lipid monolayer films determined by single-molecule fluorescence spectroscopy.

We use single-molecule fluorescence microscopy to monitor individual hybridization reactions between membrane-anchored DNA strands, occurring in nanofluidic lipid monolayer films deposited on Teflon AF substrates. The DNA molecules are labeled with different fluorescent dyes, which make it possible to simultaneously monitor the movements of two different molecular species, thus enabling tracking of both reactants and products. We employ lattice diffusion simulations to determine reaction probabilities upon interaction.

Single-cell electroporation using a multifunctional pipette.

We present here a novel platform combination, using a multifunctional pipette to individually electroporate single-cells and to locally deliver an analyte, while in their culture environment. We demonstrate a method to fabricate low-resistance metallic electrodes into a PDMS pipette, followed by characterization of its effectiveness, benefits and limits in comparison with an external carbon microelectrode.