Simultaneous Multipass Resistive-Pulse Sensing and Fluorescence Imaging of Liposomes.

Simultaneous multipass resistive-pulse sensing and fluorescence imaging have been used to correlate the size and fluorescence intensity of individual E. coli lipid liposomes composed of E. coli polar lipid extracts labeled with membrane-bound 3,3-dioctadecyloxacarbocyanine (DiO) fluorescent molecules. Here, a nanopipet serves as a waveguide to direct excitation light to the resistive-pulse sensing zone at the end of the nanopipet tip. Individual DiO-labeled liposomes (>50 nm radius) were multipassed back and forth through the orifices of glass nanopipets' 110-150 nm radius via potential switching to obtain subnanometer sizing precision, while recording the fluorescence intensity of the membrane-bound DiO molecules. Fluorescence was measured as a function of liposome radius and found to be approximately proportional to the total membrane surface area. The observed relationship between liposome size and fluorescence intensity suggests that multivesicle liposomes emit greater fluorescence compared to unilamellar liposomes, consistent with all lipid membranes of the multivesicle liposomes containing DiO. Fluorescent and nonfluorescent liposomes are readily distinguished from each other in the same solution using simultaneous multipass resistive-pulse sensing and fluorescence imaging. A fluorescence "dead zone" of ∼1 µm thickness just outside of the nanopipet orifice was observed during resistive-pulse sensing, resulting in "on/off" fluorescent behavior during liposome multipassing.

Monitoring the escape of DNA from a nanopore using an alternating current signal.

We present the use of an alternating current (AC) signal as a means to monitor the conductance of an alpha-hemolysin (alphaHL) pore as a DNA hairpin with a polydeoxyadenosine tail is driven into and released from the pore. Specifically, a 12 base pair DNA hairpin attached to a 50-nucleotide poly-A tail (HP-A(50)) is threaded into an alphaHL channel using a DC driving voltage. Once the HP-A(50) molecule is trapped within the alphaHL channel, the DC driving voltage is turned off and the conductance of the channel is monitored using an AC voltage. The escape time, defined as the time it takes the HP-A(50) molecule to transport out of the alphaHL channel, is then measured. This escape time has been monitored as a function of AC amplitude (20 to 250 mV(ac)), AC frequency (60-200 kHz), DC drive voltage (0 to 100 mV(dc)), and temperature (-10 to 20 degrees C), in order to determine their effect on the predominantly diffusive motion of the DNA through the nanopore. The applied AC voltage used to monitor the conductance of the nanopore has been found to play a significant role in the DNA/nanopore interaction. The experimental results are described by a one-dimensional asymmetric periodic potential model that includes the influence of the AC voltage. An activation enthalpy barrier of 1.74 x 10(-19) J and a periodic potential asymmetry parameter of 0.575 are obtained for the diffusion at zero electrical bias of a single nucleotide through alphaHL.