Pouyan E. Boukany

Nanochannel electroporation delivers precise amounts of biomolecules into living cells

Many transfection techniques can deliver biomolecules into cells, but the dose cannot be controlled precisely. Delivering well-defined amounts of materials into cells is important for various biological studies and therapeutic applications. Here, we show that nanochannel electroporation can deliver precise amounts of a variety of transfection agents into living cells. The device consists of two microchannels connected by a nanochannel. The cell to be transfected is positioned in one microchannel using optical tweezers, and the transfection agent is located in the second microchannel. Delivering a voltage pulse between the microchannels produces an intense electric field over a very small area on the cell membrane, allowing a precise amount of transfection agent to be electrophoretically driven through the nanochannel, the cell membrane and into the cell cytoplasm, without affecting cell viability. Dose control is achieved by adjusting the duration and number of pulses. The nanochannel electroporation device is expected to have high-throughput delivery applications.

Shear banding or not in entangled DNA solutions depending on the level of entanglement

(Received 28 November 2007; final revision received 6 October 2008 Synopsis Entangled DNA solutions are ideal as a model system to examine nonlinear shear flow behavior. Even when the number of entanglements per chain, Z, is higher than 100, the solution is still soft enough with an elastic plateau modulus under 100 Pa and is thus amenable to experimental study by commercial rotational rheometry without ambiguity and uncertainty. We have investigated nonlinear flow behavior of three entangled DNA solutions with Z=24, 60, and 156, respectively, using a combination of particle-tracking velocimetric PTV and conventional rheometric measurements. We explore questions such as a whether shear banding also occurs in moderately entangled solutions, b whether creep results in development of nonlinear velocity profile, c whether shear banding produced in startup shear and creep persists at long times in steady state, and d whether these entangled solutions exhibit homogeneous shear at the upper end of the stress plateau region. We found that the first DNA solution Z=24 only shows transient weakly inhomogeneous shear and steady linear velocity profile. In the more entangled solutions Z=60 and 156, shear banding is observed in startup rate- and stress-controlled shear in the shear thinning regime. Shear homogeneity eventually returns at the upper end of the stress plateau shear

Universal Scaling Behavior in Startup Shear of Entangled Linear Polymer Melts

We have studied stress overshoot behavior in startup shear of four monodisperse polymer melts with a range of chain entanglement from Z=24 to 160 entanglement points per chain. In the elastic deformation regime defined by γτR>1, where τR is the Rouse relaxation time, (i) the peak shear stress σmax scales with the time tmax at the peak to −1/2 power, in contrast to an exponent of −1/4 in the viscoelastic regime (for γτR<1), (ii) σmax changes linearly with the elapsed strain at the stress peak γmax, which scales with the applied shear rate as γ1/3, (iii) a supermaster curve collapses time-dependent shear stress growth curves up to the stress maximum at all shear rates for all the four styrene-butadiene rubber samples.

A correlation between velocity profile and molecular weight distribution in sheared entangled polymer solutions

In this work we attempt to answer several questions concerning the flow characteristics of entangled polymer solutions in a sliding plate shearing cell. We explore (a) how the molecular weight distribution affects the velocity profile in simple shear, (b) whether the observed shear banding is consistent with a nonmonotonic constitutive model, (c) whether the flow response and velocity profiles are different in simple shear depending on the different modes of shear. Our results provide a comparison with recent reports on a polydisperse polymer sample [Tapadia and Wang, Phys. Rev. Lett. 96, 016001 (2006); Tapadia, et al., Phys. Rev. Lett. 96, 196001 (2006)] that revealed the first evidence for inhomogeneous shear during startup in cone-plate flow geometry of a rotational rheometer. Using a highly monodisperse sample, we observed the sample to partition into two fractions with different local shear rates instead of possessing a smooth spatial variation of the local shear rate as seen for the polydisperse sam...

Exploring the transition from wall slip to bulk shearing banding in well-entangled DNA solutions

In this study we have carried out a combination of rheometric and particle-tracking velocimetric (PTV) measurements to investigate nonlinear rheological behavior of three entangled DNA solutions (with ca. 150 entanglements per chain) and, in particular, to explore a transformation from slip-dominated steady-state flow to bulk shear inhomogeneity. In the stress plateau regime, an elastic recoil-like response occurs transiently at either interfaces or sample interior after stress overshoot during a startup shear. In both startup shear and creep mode, wall slip, bulk shear banding or a combination of both have been observed in both transient and steady states. The water-based solution shows massive wall slip allowing the bulk to remain in the Newtonian flow regime. Use of glycerol as a solvent can effectively reduce interfacial slip, permitting bulk shear banding to develop in both controlled-rate and controlled-stress modes. For the glycerol based solution, a sufficiently high Weissenberg number can attain in the rheometer where PTV observations reveal homogenous shear in steady state.

Large Laterally Ordered Nanochannel Arrays from DNA Combing and Imprinting

One-dimensional nanostructures such as nanochannels (and nanotubes) are characterized by extremely small transverse size and resultant high degree of spatial confinement that endow them a unique set of properties. When patterned laterally, these nanostructures are widely used as critical transport devices for a variety of applications such as sensing, nanomanipulation, and information processing.[1–8] While numerous fabrication techniques have been developed, few can generate large and highly ordered arrays of both nanochannels and nanowires with no defects and low-cost. The most notable high-resolution lithographic techniques include electron beam lithography (EBL) and focused ion beam milling (FIB),[9–13] but they are associated with either low throughput or high-cost. Another lithographic technique, nanoimprint lithography (NIL), is of high throughput and relatively low-cost, but it requires the use of highly specialized equipment and molds prepared typically by EBL.[14– 17] Many inexpensive techniques have been developed, but they are inadequate in terms of high precision, low defect rate, or large area fabrication of both nanochannels/tubes and nanowires/strands.[7,18–25] Moreover, these nanostructures need to be connected to the micro/macroscale structures, such as reservoirs and channels, to form functional devices. This is not a trivial task and the lack of a low-cost solution to this problem significantly limits the applicability of many nanoconstructs.

Interfacial Stick-Slip Transition in Simple Shear of Entangled Melts

This article describes a systematic investigation of a discontinuous interfacial stick-slip transition (SST) in simple shear of monodisperse entangled 1,4-polybutadiene (PBD) and polyisoprene (PIP) melts with different molecular weights and architecture, using a specially designed controlled-force shear rheometer. The magnitude of the transition is found to be determined by the level of chain entanglement. Specifically, the dependence of extrapolation length b on molecular weight as b∼Mw3.4 and of the melt viscosity as b∼η is consistent with the observations based on capillary rheometric studies [X. Yang et al., Rheol. Acta 37, 415–423 (1998)]. The interfacial nature of the flow behavior is explicitly demonstrated by a surface treatment of the shearing plates and dependence of the abrupt increase of the apparent shear rate on the gap distance as well as by particle tracking velocimetry. The critical stress for different molecular weights of PBD and PIP is about 0.2 and 0.1MPa, respectively, independent of...

Nonendocytic delivery of lipoplex nanoparticles into living cells using nanochannel electroporation.

The delivery of biomolecules, including siRNAs (≈21 bp) and large plasmids (≈10 kbp), into living cells holds a great promise for therapeutic and research applications. Lipoplex nanoparticles are popular nanocarriers for gene delivery. In conventional transfection methods, the cellular uptake of lipoplex nanoparticels occurs through the endocytosis process. The entrapment of lipoplex nanoparticles into endocytic vesicle is a major barrier in achieving efficient gene silencing and expression. Here, a novel nanochannel electroporation (NEP) method is employed to facilitate the cellular uptake and release of siRNAs/DNAs from lipoplexes. First, it is demonstrated that in a NEP device, lipoplex nanoparticles can be injected directly into the cell cytoplasm within several seconds. Specifically, it is found that lipoplexes containing MCL-1 siRNA delivered by NEP can more efficiently down-regulate the expression of MCL-1 mRNA in A549 cancer cells than conventional transfection. Quantum dot-mediated Förster resonance energy transfer (QD-FRET) reveals that lipoplexes delivered via NEP can directly release siRNA in the cytoplasm without going through the endocytosis route, which unravels the responsible mechanism for efficient gene delivery. Furthermore, the advantage of combining NEP with lipoplex nanoparticles by the successful delivery of large plasmids (pCAG2LMKOSimO, 13 kbp) into CHO cells is demonstrated.

Letter to the Editor: Sufficiently entangled polymers do show shear strain localization at high enough Weissenberg numbers

This Letter concludes that the recent data of Li et al. [J. Rheol. 57, 1411–1428 (2013)] are entirely consistent with the previous observations of the occurrence and absence of shear banding during startup shear and nonquiescent relaxation after large stepwise shear. In other words, based on the linear viscoelastic characteristics of these solutions depicted in Fig. 5(a) of Li et al., we find their results to follow from the previous analysis: One insufficiently entangled solution naturally exhibited homogeneous shear under the explored conditions. The two more entangled solutions did not exhibit shear banding and nonquiescent relaxation, because the samples appear to have significant polydispersity in the molecular weight distribution and because the applied shear rates were much lower than those needed to produce shear banding. Thus, the observations of Li et al. support rather than refute the existing knowledge concerning nonlinear rheological responses of entangled polymer solutions to startup and stepwise shear.

Nature of steady flow in entangled fluids revealed by superimposed small amplitude oscillatory shear

We carry out a systematic investigation into steady-state shear behavior of six entangled solutions based on a superposition of continuous shear and small amplitude oscillatory shear (SAOS). During steady shear in the shear thinning regime, the superimposed SAOS frequency sweep measurements reveal characteristics of viscous liquids, e.g., terminal dynamics, on the experimental time scale of the reciprocal shear rate. The residual entanglement network retains the same level of elastic stiffness as the equilibrium system does. Consistent with the convective constraint release idea, chains in the network are forced to pass around each other as they must do so to undergo steady flow. When such a sample is examined at significantly short time scales, chains are unable to pass around and the signature of this residual entanglement is that the storage modulus is greater than the loss modulus at higher frequencies than the applied shear rate. The particle-tracking velocimetric observations confirm that whether sh...