J. L. Gornall

Detecting DNA Folding with Nanocapillaries

We demonstrate for the first time the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. We show that glass capillaries can be pulled into nanocapillaries with diameters down to 45 nm. We study translocation of lambda -DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single lambda -DNA molecules. Our experiments prove that nanocapillaries are suitable for label-free analysis of DNA in aqueous solutions and viable alternatives to solid-state nanopores made by silicon nanotechnology.

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.

Probing DNA with micro-?and nanocapillaries and optical tweezers

We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

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.

High-speed video-based tracking of optically trapped colloids

We have developed an optical tweezer setup, with high-speed and real-time position tracking, based on a CMOS camera technology. Our software encoded algorithm is cross-correlation based and implemented on a standard computer. By measuring the fluctuations of a confined colloid at 6000? frames?s ? 1, continuously for an hour, we show our technique is a viable alternative to quadrant photodiodes. The optical trap is calibrated by using power spectrum analysis and the Stokes method. The trap stiffness is independent of the camera frame rate and scales linearly with the applied laser power. The analysis of our data by Allan variance demonstrates single nanometer accuracy in position detection.

Concentration-temperature superposition of helix folding rates in gelatin.

Using optical rotation as the primary technique, we have characterized the kinetics of helix renaturation in water solutions of gelatin. By covering a wide range of solution concentrations we identify a universal exponential dependence of folding rate on concentration and quench temperature. We demonstrate a new concentration-temperature superposition of data at all temperatures and concentrations, and build the corresponding master curve. The normalized rate constant is consistent with helix lengthening. Nucleation of the triple helix occurs rapidly and contributes less to the helical onset than previously thought.

Note: Direct force and ionic-current measurements on DNA in a nanocapillary

We have developed optical tweezers, with force measurements based on fast video tracking, for analysis and control of DNA translocation through nanocapillaries. Nanocapillaries are single-molecule biosensors with very similar characteristics to solid-state nanopores. Our novel experimental setup allows for ionic-current measurements in which the nanocapillary is oriented perpendicular to the trapping laser. Using video-based particle tracking, we are able to measure the position of DNA coated colloids at sub-millisecond resolution and in real-time. We present the first electrophoretic force and simultaneous ionic-current measurements of a single DNA molecule inside the orifice of a nanocapillary.

Combining Fluorescence Imaging and Ionic Current Detection in Nanocapillaries

We present here recent data showing simultaneous imaging and ionic current detection of DNA fluorescently labeled with SYTOX Orange. Using a fast Electron Multiplying CCD (EMCCD) camera, we have verified the link between ionic current translocation events and the movement of the DNA into the nanocapillary. Further work will focus on elucidating the nature of so-called ‘folding’ DNA events as well as studies on the balance of electrophoresis and electroosmosis in nanocapillaries.

Towards Simultaneous Force and Resistive Pulse Sensing in Protein Nanopores Using Optical Tweezers

Protein nanopores are highly suitable for single-molecule detection. They offer more reproducible, cost effective and well defined structures than solid-state alternatives with architectures often known from x-ray crystallography studies with sub-nanometer precision. Here we present a self-assembling hybrid nanopore system consisting of a protein nanopore embedded in a lipid membrane, supported across the tip of a nanopipette. Here, we show the insertion of Staphylococcus aureus toxin α-hemolysin into the supported membrane and the voltage-driven transport of single-stranded DNA homopolymers. Orientation of the nanopipette perpendicular to the optical trapping axis will allow for high resolution force measurements of macromolecular transport through protein nanopores.