Simple reconstitution of protein pores in nano lipid bilayers.

We developed a new, simple and robust approach for rapid screening of single molecule interactions with protein channels. Our glass nanopipets can be fabricated simply by drawing glass capillaries in a standard pipet puller, in a matter of minutes, and do not require further modification before use. Giant unilamellar vesicles break when in contact with the tip of the glass pipet and form a supported bilayer with typical seal resistances of ∼140 GΩ, which is stable for hours and at applied potentials up to 900 mV. Bilayers can be formed, broken, and re-formed more than 50 times using the same pipet enabling rapid screening of bilayers for single protein channels. The stability of the lipid bilayer is significantly superior to that of traditionally built bilayers supported by Teflon membranes, particularly against perturbation by electrical and mechanical forces. We demonstrate the functional reconstitution of the E. coli porin OmpF and α-hemolysin in a glass nanopipet supported bilayer. Interactions of the antibiotic enrofloxacin with the OmpF channel have been studied at the single-molecule level, demonstrating the ability of this method to detect single molecule interactions with protein channels. High-resolution conductance measurements of protein channels can be performed with low sample and buffer consumption. Glass nanopipet supported bilayers are uniquely suited for single-molecule studies as they are more rigid and the lifetime of a stable membrane is on the scale of hours, closer to that of natural cell membranes.

Real-time particle tracking at 10,000 fps using optical fiber illumination.

We introduce optical fiber illumination for real-time tracking of optically trapped micrometer-sized particles with microsecond time resolution. Our light source is a high-radiance mercury arc lamp and a 600 µm optical fiber for short-distance illumination of the sample cell. Particle tracking is carried out with a software implemented cross-correlation algorithm following image acquisition from a CMOS camera. Our image data reveals that fiber illumination results in a signal-to-noise ratio usually one order of magnitude higher compared to standard Köhler illumination. We demonstrate position determination of a single optically trapped colloid with up to 10,000 frames per second over hours. We calibrate our optical tweezers and compare the results with quadrant photo diode measurements. Finally, we determine the positional accuracy of our setup to 2 nm by calculating the Allan variance. Our results show that neither illumination nor software algorithms limit the speed of real-time particle tracking with CMOS technology.

Helix-coil transition of gelatin: helical morphology and stability.

By combining optical rotation with thermal characterization and rheological measurements, we have studied triple helix formation in water and ethylene glycol solutions of gelatin. We find the enthalpy change per unit helix required for the transition from triple helix to random coil is independent of the concentration of helices in solution and the temperature at which the helices form. Helices formed in ethylene glycol are less stable than those formed in water solutions as, unlike water, ethylene glycol is too large a molecule to mediate interchain hydrogen bonds. The storage modulus has a universal dependence on helix concentration in both solvents but, due to a reduction in helix length, the critical concentration at which an elastic gel forms is smaller in ethylene glycol.

Mechanically induced helix-coil transition in biopolymer networks.

The quasi-equilibrium evolution of the helical fraction occurring in a biopolymer network (gelatin gel) under an applied stress has been investigated by observing modulation in its optical activity. Its variation with the imposed chain extension is distinctly nonmonotonic and corresponds to the transition of initially coiled strands to induced left-handed helices. The experimental results are in qualitative agreement with theoretical predictions of helices induced on chain extension. This new effect of mechanically stimulated helix-coil transition has been studied further as a function of the elastic properties of the polymer network: crosslink density and network aging.