Monodisperse particles of varying size, shape, and deformability were produced using two microfluidic strategies. For both strategies, monodisperse emulsion droplets of a crosslinkable solution were generated via flow-focusing. Subsequently, droplets were crosslinked either on chip or in an external bath. On-chip gelation resulted in spherical particles; varying the degree of crosslinking varied the deformability systematically. The optimized flow-focusing device design separated the production of monodisperse aqueous alginate droplets and the on-chip introduction of crosslinking ions. Two features were then adapted to target softer particles: the dispersed phase design and the polymer choice. The alternative design used a sheathed dispersed phase, with the polymer solution surrounding an unreactive viscous core, which generated alginate particles with a softer core. Poly(N-isopropylacrylamide) (pNIPAM) allowed access to a broad range of moduli. The resulting spherical particles were characterized using capillary micromechanics to determine the shear (G) and compressive (K) moduli. Particles with G = 0.013 kPa to 26 kPa and K = 0.221 kPa to 34.9 kPa were obtained; the softest particles are an order of magnitude softer than those previously reported. The second approach, based on earlier work by Hu et al., produced axisymmetric, non-spherical particles with fore-aft asymmetry. Alginate drops were again formed in a flow-focusing device but were crosslinked off-chip in an external gelation bath. By changing the bath viscosity, crosslinker concentration, and outlet height, the falling droplets deformed differently during gelation, resulting in a variety of shapes, such as teardrop, mushroom, and bowl shapes.

Vesicles provide an attractive model system to understand the deformation of living cells in response to mechanical forces. These simple, enclosed lipid bilayer membranes are suitable for complementary theoretical, numerical, and experimental analysis. A recent study [Narsimhan, Spann, Shaqfeh, J. Fluid Mech., 2014, 750, 144] predicted that intermediate-aspect-ratio vesicles extend asymmetrically in extensional flow. Upon infinitesimal perturbation to the vesicle shape, the vesicle stretches into an asymmetric dumbbell with a cylindrical thread separating the two ends. While the symmetric stretching of high-aspect-ratio vesicles in extensional flow has been observed and characterized [Kantsler, Segre, Steinberg, Phys. Rev. Lett., 2008, 101, 048101] as well as recapitulated in numerical simulations by Narsimhan et al., experimental observation of the asymmetric stretching has not been reported. In this work, we present results from microfluidic cross-slot experiments observing this instability, along with careful characterization of the flow field, vesicle shape, and vesicle bending modulus. The onset of this shape transition depends on two non-dimensional parameters: reduced volume (a measure of vesicle asphericity) and capillary number (ratio of viscous to bending forces). We observed that every intermediate-reduced-volume vesicle that extends forms a dumbbell shape that is indeed asymmetric. For the subset of the intermediate-reduced-volume regime we could capture experimentally, we present an experimental phase diagram for asymmetric vesicle stretching that is consistent with the predictions of Narsimhan et al.

In this paper, we use rheometry and flow visualization to study the dynamics of the interface between shear bands in a wormlike micellar solution sheared between concentric cylinders, i.e., in a Taylor–Couette (TC) cell, and to evaluate the stress diffusion coefficient and the stress correlation length in the Johnson–Segalman model. Two wormlike micellar solutions are studied: an aqueous solution of CTAB–NaNO3 and a solution of CPCl–NaSal in brine. These systems are highly elastic, exhibit Maxwellian behavior in linear viscoelasticity experiments, and shear banding in nonlinear experiments [S. Lerouge, et al., Soft Matter, 2008, 4, 1808–1819, M. A. Fardin, et al., Soft Matter, 2012, 8(39), 10072–10089, P. Ballesta, et al., J. Rheol., 2007, 51, 1047]. A large, custom-built, computer controlled TC cell allows us to rotate both cylinders independently and to visualize the flow in the r–z plane using a CCD camera. At low shear rates, the flow is stable and the fluid appears homogeneous throughout the gap between the cylinders. Above a critical shear rate, a shear banding transition occurs. This manifests itself in the formation of two distinct bands in the r–z plane, with an interface between the two bands. For sufficiently high ramp speeds, multiple steps of interface evolution are identified, as noted by Radulescu, Lerouge, and others [O. Redulescu, et al., Europhys. Lett., 2003, 62, 230, S. Lerouge, et al., Soft Matter, 2008, 4, 1808–1819]. We quantify the interface travel using direct visualization and use this measure, as well as superposition rheometry [P. Ballesta, et al., J. Rheol., 2007, 51, 1047], to determine the stress diffusion coefficient D and the stress correlation length ζ in the Johnson–Segalman model. These parameters are evaluated at different temperatures, shear rates, and gap sizes. We find that the stress diffusion coefficient and the stress correlation length exhibit a strong dependence on the gap of the Taylor–Couette cell for both shear-banding systems. For the CTAB–NaNO3 system, we report a linear dependence of the stress diffusion coefficient on temperature for the parameter range considered. In addition, we find that for this system, the stress diffusion coefficient is independent of shear rate. For the CPCl–NaSal system, we observe the same color changes in the sample reported by others on extended light exposure; however, we find that different histories of light exposure do not affect the measured stress diffusion coefficient.

Fabrication of high-aspect-ratio PDMS microfluidic devices with conventional SU-8 based soft photolithography is challenging, and often, the thickness of the master from which PDMS replicas are molded is non-uniform. Here, we present an optimized, low cost, fast prototyping microfabrication technique to make deep (up to 500 μm) and high-aspect-ratio (up to 10) microfluidic channels by producing masters by laminating a single or multiple layers of a thin dry film photoresist onto metal wafers. In particular, we explore the required exposure energy for different film thicknesses as well as the highest achievable channel depths and aspect ratios. The homogeneity of the depth of PDMS channels formed using these masters is quantified and found to be remarkably uniform over distances of 20 mm or more. The importance of the processing parameters, such as the exposure energy and development time on final feature size, wall angle, and channel aspect ratio, is investigated. In addition, we report some failure cases, the potential reasons, and strategies for making optimized devices. Potentially, deep microfluidic channels with a wide range of aspect ratios can be used to make long, homogenous separation devices that can be used in cell sorting, filtration, and flow cytometry. We believe the protocols we outline here will be of great utility to the microfluidics community.

This review is a practical guide for experimentalists interested in specifically labeling internal sequences on double-stranded (ds) DNA molecules for single-molecule experiments. We describe six labeling approaches demonstrated in a single-molecule context and discuss the merits and drawbacks of each approach with particular attention to the amount of specialized training and reagents required. By evaluating each approach according to criteria relevant to single-molecule experiments, including labeling yield and compatibility with cofactors such as Mg2+, we provide a simple reference for selecting a labeling method for given experimental constraints. Intended for non-specialists seeking accessible solutions to DNA labeling challenges, the approaches outlined emphasize simplicity, robustness, suitability for use by non-biologists, and utility in diverse single-molecule experiments.

We demonstrate the feasibility of a single-molecule microfluidic approach to both sequence detection and obtaining kinetic information for restriction endonucleases on dsDNA. In this method, a microfluidic stagnation point flow is designed to trap, hold, and linearize double-stranded (ds) genomic DNA to which a restriction endonuclease has been pre-bound sequence-specifically. By introducing the cofactor magnesium, we determine the binding location of the enzyme by the cleavage process of dsDNA as in optical restriction mapping, however here the DNA need not be immobilized on a surface. We note that no special labeling of the enzyme is required, which makes it simpler than our previous scheme using stagnation point flows for sequence detection. Our accuracy in determining the location of the recognition site is comparable to or better than other single molecule techniques due to the fidelity with which we can control the linearization of the DNA molecules. In addition, since the cleavage process can be followed in real time, information about the cleavage kinetics, and subtle differences in binding and cleavage frequencies among the recognition sites, may also be obtained. Data for the five recognition sites for the type II restriction endonuclease EcoRI on λ-DNA are presented as a model system. While the roles of the varying fluid velocity and tension along the chain backbone on the measured kinetics remain to be determined, we believe this new method holds promise for a broad range of studies of DNA–protein interactions, including the kinetics of other DNA cleavage processes, the dissociation of a restriction enzyme from the cleaved substrate, and other macromolecular cleavage processes.

The ability to strongly and sequence-specifically attach modifications such as fluorophores and haptens to individual double-stranded (ds) DNA molecules is critical to a variety of single-molecule experiments. We propose using modified peptide nucleic acids (PNAs) for this purpose and implement them in two model single-molecule experiments where individual DNA molecules are manipulated via microfluidic flow and optical tweezers, respectively. We demonstrate that PNAs are versatile and robust sequence-specific tethers.