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.

Toward Brain-on-a-Chip: Human Induced Pluripotent Stem Cell-Derived Guided Neuronal Networks in Tailor-Made 3D Nanoprinted Microscaffolds.

Brain-on-a-chip (BoC) concepts should consider three-dimensional (3D) scaffolds to mimic the 3D nature of the human brain not accessible by conventional planar cell culturing. Furthermore, the essential key to adequately address drug development for human pathophysiological diseases of the nervous system, such as Parkinson's or Alzheimer's, is to employ human induced pluripotent stem cell (iPSC)-derived neurons instead of neurons from animal models. To address both issues, we present electrophysiologically mature human iPSC-derived neurons cultured in BoC applicable microscaffolds prepared by direct laser writing. 3D nanoprinted tailor-made elevated cavities interconnected by freestanding microchannels were used to create defined neuronal networks-as a proof of concept-with two-dimensional topology. The neuronal outgrowth in these nonplanar structures was investigated, among others, in terms of neurite length, size of continuous networks, and branching behavior using z-stacks prepared by confocal microscopy and cross-sectional scanning electron microscopy images prepared by focused ion beam milling. Functionality of the human iPSC-derived neurons was demonstrated with patch clamp measurements in both current- and voltage-clamp mode. Action potentials and spontaneous excitatory postsynaptic currents-fundamental prerequisites for proper network signaling-prove full integrity of these artificial neuronal networks. Considering the network formation occurring within only a few days and the versatile nature of direct laser writing to create even more complex scaffolds for 3D network topologies, we believe that our study offers additional approaches in human disease research to mimic the complex interconnectivity of the human brain in BoC studies.