Structure-Specific Liquid Crystal Anchoring Induced by the Molecular Combing of Short Oligonucleotides.

Surface-immobilized oligonucleotides were "combed" by meniscus motion and exposed to a nematic liquid crystal (LC). Although the oligonucleotides were as short as 16 bases, they were apparently oriented by this process and, in turn, successfully biased the orientation of the adjacent LC material. Single-stranded DNA (ssDNA) induced LC orientation in the combing direction, while hybridized double-stranded DNA (dsDNA) rotated the azimuthal LC orientation by ∼30° from the combing direction. The sensitivity of the chiral response to mixed ssDNA/dsDNA surfaces was characterized by employing complementary DNA that was longer than the immobilized DNA, resulting in single-stranded overhangs of various lengths. A rotated LC orientation was observed even when more than 70% of the DNA was single-stranded, and the transition from the rotated to nonrotated response was apparently discontinuous as a function of ssDNA surface coverage. These phenomena represent a sensitive DNA hybridization detection strategy that can potentially comprise a multiplexed assay.

Receptor-Mediated Liposome Fusion Kinetics at Aqueous/Liquid Crystal Interfaces.

Membrane fusion events are essential to cell biology, and a number of reductionist systems have been developed to mimic the behavior of these biological motifs. One such system monitors the DNA hybridization-mediated fusion of liposomes with the liquid crystal (LC) interface by observing changes in LC orientation using a simple optical detection scheme. We have systematically explored key parameters of this system to determine their effects on individual elementary steps of the complex fusion mechanism. The liposome composition, specifically the degree of lipid unsaturation and PE content, decreased the bilayer rigidity, thereby increasing the rate of vesicle rupture under the stress applied by DNA hybridization. In contrast, the presence of cholesterol had the opposite effect on the mechanical properties of the bilayer, and hence of the membrane fusion rates. The accessibility of receptor moieties (i.e., complementary DNA oligonucleotides) affected the fusion kinetics by modulating the rate of hybridization events. DNA accessibility was controlled by systematic variation of the length of the DNA receptor molecules and the thickness of the steric barrier comprised of adsorbed PEGylated lipids. These results provide design rules for understanding the trade-offs between response kinetics and other important system properties, such as nonspecific adsorption. Moreover, these findings improve our understanding of the biophysical properties of membrane fusion, an important process in both natural and model systems used for bioassay and bioimaging applications.

DNA Hybridization-Mediated Liposome Fusion at the Aqueous Liquid Crystal Interface.

The prominence of receptor-mediated bilayer fusion in cellular biology motivates development of biomimetic strategies for studying fusogenic mechanisms. An approach is reported here for monitoring receptor-mediated fusion that exploits the unique physical and optical properties of liquid crystals (LC). PEG-functionalized lipids are used to create an interfacial environment capable of inhibiting spontaneous liposome fusion with an aqueous/LC interface. Then, DNA hybridization between oligonucleotides within bulk phase liposomes and a PEG-lipid monolayer at an aqueous/LC interface is exploited to induce receptor-mediated liposome fusion. These hybridization events induce strain within the liposome bilayer, promote lipid mixing with the LC interface, and consequently create an interfacial environment favoring re-orientation of the LC to a homeotropic (perpendicular) state. Furthermore, the bi-functionality of aptamers is exploited to modulate DNA hybridization-mediated liposome fusion by regulating the availability of the appropriate ligand (i.e., thrombin). Here, a LC-based approach for monitoring receptor (i.e., DNA hybridization)-mediated liposome fusion is demonstrated, liposome properties that dictate fusion dynamics are explored, and an example of how this approach may be used in a biosensing scheme is provided.

On-demand droplet fusion: a strategy for stimulus-responsive biosensing in solution.

A novel strategy is reported for biochemically controlled fusion of oil-in-water (O/W) droplets as an in-solution sensor for biological targets. Inspired by the SNARE complex in cells, the emulsions were stabilized by a combination of phospholipids, phospholipid-poly(ethylene glycol) conjugates, and cholesterol-anchored oligonucleotides. Prior to oligonucleotide binding, the droplets were stable in aqueous media, but hybridization of the oligonucleotides in a zipperlike fashion was shown to initiate droplet fusion. Using image analysis of content mixing of dye-loaded droplets, fusion specificity was studied and optimized as a function of interfacial chemistry. Changing the orientation of the anchored oligonucleotides, using long-chain phospholipids (C18 and C22), and binding a complementary oligonucleotide slowed or even halted fusion completely. Based on these studies, a sensor for the biomarker thrombin was designed using competitive binding of aptamer strands, with droplet fusion increasing as a function of thrombin addition in accordance with a simple binding model, with sensitivity down to 100 nM and with results in as little as 15 min. Future efforts will focus on utilizing this mechanism of content mixing to facilitate highly sensitive detection via modalities such as magnetoresistance or chemiluminescence.

Liquid crystal reorientation induced by aptamer conformational changes.

Aptamer-ligand binding events, involving small molecule targets, at a surfactant-laden aqueous/liquid crystal (LC) interface were found to trigger a LC reorientation that can be observed in real-time using polarized light. The response was both sensitive and selective: reorientation was observed at target concentrations on the order of the aptamer dissociation constant, but no response was observed in control experiments with target analogues. Circular dichroism and resonance energy transfer experiments suggested that the LC reorientation was due to a conformational change of the aptamer upon target binding. Specifically, under conditions where aptamer-ligand binding induced a conformational change from a relaxed random coil to more intricate secondary structures (e.g., double helix, G-quadruplex), a transition from planar to homeotropic LC orientation was observed. These observations suggest the potential for a label-free LC-based detection system that can simultaneously respond to the presence of both small molecules and nucleic acids.

Mixed alkylsilane functionalized surfaces for simultaneous wetting and homeotropic anchoring of liquid crystals.

Surfaces functionalized with a self-assembled monolayer (SAM) formed from a mixture of two alkylsilanes with different chain lengths have been designed to simultaneously improve the liquid crystal (LC) wettability and promote homeotropic anchoring of the LC. Most chemically functionalized surfaces (e.g., long alkyl chain SAMs) that promote homeotropic alignment of LC possess low surface energy and result in poor LC wettability, inhibiting LC infiltration into microstructured surfaces and sometimes resulting in LC dewetting from the surface. However, a surface modified with a mixed SAM of octadecyltriethoxysilane (C18) and ethyltriethoxysilane (C2) exhibited very low LC contact angle while providing homeotropic anchoring. Ellipsometry was used to correlate the bulk concentration of C18 in the deposition solution to the surface coverage of C18 in the mixed monolayer; these bulk and surface concentrations were found to be equal within experimental uncertainty. The LC contact angle was found to depend nonmonotically with the surface coverage density, with a minimum (14.4 ± 0.1°) at a C18 surface coverage of 0.26 ± 0.08. Homeotropic LC anchoring was achieved at a C18 surface coverage of ≥0.11 ± 0.04, in the regime where a minimum in the LC contact angle was observed. The practical application of this approach to surface modification was demonstrated using a micropillar array sensor substrate. When the array was functionalized with a conventional C18 SAM, the LC did not infiltrate the array and exhibited a contact angle of 47.4 ± 0.5°. However, the LC material successfully infiltrated and wetted the same microstructured substrate when functionalized with a C18/C2 mixed SAM, while still exhibiting the desired homeotropic anchoring.