Pillar-structured 3D inlets fabricated by dose-modulated e-beam lithography and nanoimprinting for DNA analysis in passive, clogging-free, nanofluidic devices.

We present the fabrication of three-dimensional inlets with gradually decreasing widths and depths and with nanopillars on the slope, all defined in just one lithography step. In addition, as an application, we show how these micro- and nanostructures can be used for micro- and nanofluidics and lab-on-a-chip devices to facilitate the flow and analyze single molecules of DNA. For the fabrication of 3D inlets in a single layer process, dose-modulated electron beam lithography was used, producing depths between 750 nm and 50 nm along a 30 µm long inlet, which is additionally structured with nanometer-scale pillars randomly distributed on top, as a result of incomplete exposure and underdevelopment of the resist. The fabrication conditions affect the slope of the inlet, the nanopillar density and coverage. The key parameters are the dose used for the electron beam exposure and the development conditions, like the developer's dilution, stirring and development time. The 3D inlets with nanostructured pillars were integrated into fluidic devices, acting as a transition between micro and nanofluidic structures for pre-stretching and unfolding DNA molecules, avoiding the intrusion of folded molecules and clogging the analysis channel. After patterning these structures in silicon, they can be replicated in polymer by UV nanoimprinting. We show here how the inlets with pillars slow down the molecules before they enter the nanochannels, resulting in a 3-fold decrease in speed, which would translate to an improvement in the resolution for DNA optical mapping.

Quantitative Assessment of Endosomal Escape of Various Endocytosed Polymer-Encapsulated Molecular Cargos upon Photothermal Heating.

Encapsulated molecular cargos are efficiently endocytosed by cells. For cytosolic delivery, understanding the dynamic process of cargos release from the carrier vehicles used for encapsulation and the lysosomes where the carrier vehicles are trapped (which in general is the bottleneck), followed by diffusion in the cytosol is important for improving drug/gene delivery strategies. A methodology is reported to image this process on a millisecond scale and to quantitatively analyze the data. Polyelectrolyte capsules with embedded gold nanostars to encapsulate 43 fluorescent molecular cargos with diverse properties, ranging from small fluorophores to fluorescently labeled proteins, siRNA, etc., are used. By short laser irradiation intracellular release of the molecular cargos from endocytosed capsules into the cytosol is triggered, and their intracellular spreading is imaged. Most of the released molecular cargos evenly distribute inside the entire cell, while others are enriched in certain cell compartments. The time the different molecular cargos take to distribute within cells, i.e., the spreading time, is used as a quantifier. Quantitative analysis reveals that intracellular spread cannot be described by free diffusion, but is determined by interaction of the molecular cargo with intracellular components.