Ex Situ Integration of Multifunctional Porous Polymer Monoliths into Thermoplastic Microfluidic Chips.

A unique method for incorporating functional porous polymer monolith elements into thermoplastic microfluidic chips is described. Monolith elements are formed in a microfabricated mold, rather than within the microchannels, and chemically functionalized off chip before insertion into solvent-softened thermoplastic microchannels during chip assembly. Because monoliths may be trimmed prior to final placement, control of their size, shape, and uniformity is greatly improved over in-situ photopolymerization methods. A characteristic trapezoidal profile facilitates rapid insertion and enables complete mechanical anchoring of the monolith periphery, eliminating the need for chemical attachment to the microchannel walls. Off-chip processing allows the parallel preparation of monoliths of differing compositions and surface chemistries in large batches. Multifunctional flow-through arrays of multiple monolith elements are demonstrated using this approach through the creation of a fluorescent immunosensor with integrated controls, and a microfluidic bubble separator comprising a combination of integrated hydrophobic and hydrophilic monolith elements.

Lithographically defined macroscale modulation of lateral fluidity and phase separation realized via patterned nanoporous silica-supported phospholipid bilayers.

Using lithographically defined surfaces consisting of hydrophilic patterns of nanoporous and nonporous (bulk) amorphous silica, we show that fusion of small, unilamellar lipid vesicles produces a single, contiguous, fluid bilayer phase experiencing a predetermined pattern of interfacial interactions. Although long-range lateral fluidity of the bilayer, characterized by fluorescence recovery after photobleaching, indicates a nominally single average diffusion constant, fluorescence microscopy-based measurements of temperature-dependent onset of fluidity reveals a locally enhanced fluidity for bilayer regions supported on nanoporous silica in the vicinity of the fluid-gel transition temperature. Furthermore, thermally quenching lipid bilayers composed of a binary lipid mixture below its apparent miscibility transition temperature induces qualitatively different lateral phase separation in each region of the supported bilayer: The nanoporous substrate produces large, microscopic domains (and domain-aggregates), whereas surface texture characterized by much smaller domains and devoid of any domain-aggregates appears on bulk glass-supported regions of the single-lipid bilayer. Interestingly, lateral distribution of the constituent molecules also reveals an enrichment of gel-phase lipids over nanoporous regions, presumably as a consequence of differential mobilities of constituent lipids across the topographic bulk/nanoporous boundary. Together, these results reveal that subtle local variations in constraints imposed at the bilayer interface, such as by spatial variations in roughness and substrate adhesion, can give rise to significant differences in macroscale biophysical properties of phospholipid bilayers even within a single, contiguous phase.

Visualizing the growth and dynamics of liquid-ordered domains during lipid bilayer folding in a microfluidic chip.

A microfluidic platform enabling optical monitoring of bilayer lipid membrane formation by a new monolayer folding process is described. The thermoplastic chips integrate dried lipid films that are rehydrated by microfluidic perfusion, which enables delivery of lipid-laden air bubbles across a membrane-supporting aperture. As in traditional Montal-Mueller bilayer formation, lipid monolayers are delivered independently to each side of the aperture, thereby allowing asymmetric lipid composition in the resulting bilayer to be achieved. Confocal microscopy is used to image the monolayer folding process, and reveals the growth and dynamics of asymmetric liquid-ordered domains during bilayer stabilization.

Protecting, patterning, and scaffolding supported lipid membranes using carbohydrate glasses.

Disaccharides are known to protect sensitive biomolecules against stresses caused by dehydration, both in vivo and in vitro. Here we demonstrate how interfacial accumulation of trehalose can be used to (1) produce rugged supported lipid bilayers capable of near total dehydration; (2) enable spatial patterning of membrane micro-arrays; and (3) form stable bilayers on otherwise lipophobic substrates (e.g., metal transducers) thus affording protecting, patterning, and scaffolding of lipid bilayers.

A chitosan coated monolith for nucleic acid capture in a thermoplastic microfluidic chip.

A technique for microfluidic, pH modulated DNA capture and purification using chitosan functionalized glycidyl methacrylate monoliths is presented. Highly porous polymer monoliths are formed and subsequently functionalized off-chip in a batch process before insertion into thermoplastic microchannels prior to solvent bonding, simplifying the overall fabrication process by eliminating the need for on-chip surface modifications. The monolith anchoring method allows for the use of large cross-section monoliths enabling high flowrates and high DNA capture capacity with a minimum of added design complexity. Using monolith capture elements requiring less than 1 mm(2) of chip surface area, loading levels above 100 ng are demonstrated, with DNA capture and elution efficiency of 54.2% ± 14.2% achieved.

Microfluidic-enabled liposomes elucidate size-dependent transdermal transport.

Microfluidic synthesis of small and nearly-monodisperse liposomes is used to investigate the size-dependent passive transdermal transport of nanoscale lipid vesicles. While large liposomes with diameters above 105 nm are found to be excluded from deeper skin layers past the stratum corneum, the primary barrier to nanoparticle transport, liposomes with mean diameters between 31-41 nm exhibit significantly enhanced penetration. Furthermore, multicolor fluorescence imaging reveals that the smaller liposomes pass rapidly through the stratum corneum without vesicle rupture. These findings reveal that nanoscale liposomes with well-controlled size and minimal size variance are excellent vehicles for transdermal delivery of functional nanoparticle drugs.

Electro-optical BLM chips enabling dynamic imaging of ordered lipid domains.

Studies of lipid rafts, ordered microdomains of sphingolipids and cholesterol within cell membranes, are essential in probing the relationships between membrane organization and cellular function. While in vitro studies of lipid phase separation are commonly performed using spherical vesicles as model membranes, the utility of these models is limited by a number of factors. Here we present a microfluidic device that supports simultaneous electrical measurements and confocal imaging of on-chip bilayer lipid membranes (BLMs), enabling real-time multi-domain imaging of membrane organization. The chips further support closed microfluidic access to both sides of the membrane, allowing the membrane boundary conditions to be rapidly changed and providing a mechanism for dynamically adjusting membrane curvature through application of a transmembrane pressure gradient. Here we demonstrate the platform through the study of dynamic generation and dissolution of ordered lipid domains as membrane components are transported to and from the supporting annulus containing solvated lipids and cholesterol.

Optical detection of ion-channel-induced proton transport in supported phospholipid bilayers.

The integration of ion-channel transport functions with responses derived from nanostructured and nanoporous silica mesophase materials is demonstrated. Patterned thin-film mesophases consisting of alternating hydrophilic nanoporous regions and hydrophobic nanostructured regions allow for spatially localized proton transport via selective dimerization of gramicidin in lipid bilayers formed on the hydrophilic regions. The adjoining hydrophobic mesostructure doped with a pH sensitive dye reports the transport. The ease of integrating functional membranes and reporters through the use of patterned mesophases should enable high throughput studies of membrane transport.