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%.

Membrane protrusion coarsening and nanotubulation within giant unilamellar vesicles.

Hydrophobic side groups on a stimuli-responsive polymer, encapsulated within a single giant unilamellar vesicle, enable membrane attachment during compartment formation at elevated temperatures. We thermally modulated the vesicle through implementation of an IR laser via an optical fiber, enabling localized directed heating. Polymer-membrane interactions were monitored using confocal imaging techniques as subsequent membrane protrusions occurred and lipid nanotubes formed in response to the polymer hydrogel contraction. These nanotubes, bridging the vesicle membrane to the contracting hydrogel, were retained on the surface of the polymer compartment, where they were transformed into smaller vesicles in a process reminiscent of cellular endocytosis. This development of a synthetic vesicle system containing a stimuli-responsive polymer could lead to a new platform for studying inter/intramembrane transport through lipid nanotubes.

Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation.

This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (s/n) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.

Droplets for ultrasmall-volume analysis.

By using methods that permit the generation and manipulation of ultrasmall-volume droplets, researchers are pushing the boundaries of ultrasensitive chemical analyses. (To listen to a podcast about this feature, please go to the Analytical Chemistry Web site at pubs.acs.org/ancham.).

Fabrication improvements for thermoset polyester (TPE) microfluidic devices.

Thermoset polyester (TPE) microfluidic devices were previously developed as an alternative to poly(dimethylsiloxane) (PDMS) devices, fabricated similarly by replica molding, yet offering stable surface properties and good chemical compatibility with some organics that are incompatible with PDMS. This paper describes a number of improvements in the fabrication of TPE chips. Specifically, we describe methods to form TPE devices with a thin bottom layer for use with high numerical aperture (NA) objectives for sensitive fluorescence detection and optical manipulation. We also describe plasma-bonding of TPE to glass to create hybrid TPE-glass devices. We further present a simple master-pretreatment method to replace our original technique that required the use of specialized equipment.

Fabrication of thermoset polyester microfluidic devices and embossing masters using rapid prototyped polydimethylsiloxane molds.

Plastics are increasingly being used for the fabrication of Lab-on-a-Chip devices due to the variety of beneficial material properties, affordable cost, and straightforward fabrication methods available from a range of different types of plastics. Rapid prototyping of polydimethylsiloxane (PDMS) devices has become a well-known process for the quick and easy fabrication of microfluidic devices in the research laboratory; however, PDMS is not always an appropriate material for every application. This paper describes the fabrication of thermoset polyester microfluidic devices and masters for hot embossing using replica molding techniques. Rapid prototyped PDMS molds are convienently used for the production of non-PDMS polymeric devices. The recessed features in the cast polyester can be bonded to a second polyester piece to form an enclosed microchannel. Thermoset polyester can withstand moderate amounts of pressure and elevated temperature; therefore, the cast polyester piece also can be used as a master for embossing polymethylmethacrylate (PMMA) microfluidic systems. Examples of enclosed polyester and PMMA microchannels are presented, and we discuss the electroosmotic properties of both types of channels, which are important for analytical applications such as capillary electrophoresis.

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.