Commercial ultrafiltration and dialysis membranes have broad pore size distributions and are over 1,000 times thicker than the molecules they are designed to separate, leading to poor size cut-off properties, filtrate loss within the membranes, and low transport rates. Nanofabricated membranes have great potential in molecular separation applications by offering more precise structural control, yet transport is also limited by micrometre-scale thicknesses. This limitation can be addressed by a new class of ultrathin nanostructured membranes where the membrane is roughly as thick (approximately 10 nm) as the molecules being separated, but membrane fragility and complex fabrication have prevented the use of ultrathin membranes for molecular separations. Here we report the development of an ultrathin porous nanocrystalline silicon (pnc-Si) membrane using straightforward silicon fabrication techniques that provide control over average pore sizes from approximately 5 nm to 25 nm. Our pnc-Si membranes can retain proteins while permitting the transport of small molecules at rates an order of magnitude faster than existing materials, separate differently sized proteins under physiological conditions, and separate similarly sized molecules carrying different charges. Despite being only 15 nm thick, pnc-Si membranes that are free-standing over 40,000 microm2 can support a full atmosphere of differential pressure without plastic deformation or fracture. By providing efficient, low-loss macromolecule separations, pnc-Si membranes are expected to enable a variety of new devices, including membrane-based chromatography systems and both analytical and preparative microfluidic systems that require highly efficient separations.

Mechanical cues and substrate interaction affect the manner in which cells adhere, spread, migrate and form tissues. With increased interest in tissue-on-a-chip and co-culture systems utilizing porous membranes, it is important to understand the role of disrupted surfaces on cellular behavior. Using a transparent glass membrane with defined pore geometries, we investigated endothelial fibronectin fibrillogenesis and formation of focal adhesions as well as development of intercellular junctions. Cells formed fewer focal adhesions and had shorter fibronectin fibrils on porous membranes compared to non-porous controls, which was similar to cell behavior on continuous soft substrates with Young’s moduli seven orders of magnitude lower than glass. Additionally, porous membranes promoted enhanced cell-cell interactions as evidenced by earlier formation of tight junctions. These findings suggest that porous membranes with discontinuous surfaces promote reduced cell-matrix interactions similarly to soft substrates and may enhance tissue and barrier formation.

Porous nanocrystalline silicon (pnc-Si) is a 15 nm thin freestanding membrane material with applications in small-scale separations, biosensors, cell culture and lab-on-a-chip devices. Pnc-Si has already been shown to exhibit high permeability to diffusing species and selectivity based on molecular size or charge. In this report we characterize properties of pnc-Si in pressurized flows. We compare results to long-standing theories for transport through short pores using actual pore distributions obtained directly from electron micrographs. Measurements are in agreement with theory over a wide range of pore sizes and porosities and at orders-of-magnitude higher than those exhibited by commercial ultrafiltration and experimental carbon nanotube membranes. We also show that pnc-Si membranes can be used in dead-end filtration to fractionate gold nanoparticles and protein size ladders with better than 5 nm resolution, insignificant sample loss, and little dilution of the filtrate. These performance characteristics, combined with scalable manufacturing, make pnc-Si filtration a straightforward solution to many nanoparticle and biological separation problems.

Porous membranes enable the partitioning of cellular microenvironments in vitro, while still allowing physical and biochemical crosstalk between cells, a feature that is often necessary for recapitulating physiological functions. This article provides an overview of the different membranes used in tissue barrier and cellular co-culture models with a focus on experimental design and control of these systems. Specifically, we discuss how the structural, mechanical, chemical, and even the optical and transport properties of different membranes bestow specific advantages and disadvantages through the context of physiological relevance. This review also explores how membrane pore properties affect perfusion and solute permeability by developing an analytical framework to guide the design and use of tissue barrier or co-culture models. Ultimately, this review offers insight into the important aspects one must consider when using porous membranes in tissue barrier and lab-on-a-chip applications.

The extraordinary permeability and manufacturability of ultrathin silicon-based membranes are enabling devices with improved performance and smaller sizes in such important areas as molecular filtration and sensing, cell culture, electroosmotic pumping, and hemodialysis. Because of the robust chemical and mechanical properties of silicon nitride (SiN), several laboratories have developed techniques for patterning nanopores in SiN using reactive ion etching (RIE) through a template structure. These methods however, have failed to produce pores small enough for ultrafiltration (<100 nm) in SiN and involve templates that are prone to microporous defects. Here we present a facile, wafer-scale method to produce nanoporous silicon nitride (NPN) membranes using porous nanocrystalline silicon (pnc-Si) as a self-assembling, defect free, RIE masking layer. By modifying the mask layer morphology and the RIE etch conditions, the pore sizes of NPN can be adjusted between 40 nm and 80 nm with porosities reaching 40%. The resulting NPN membranes exhibit higher burst pressures than pnc-Si membranes while having 5× greater permeability. NPN membranes also demonstrate the capacity for high resolution separations (<10 nm) seen previously with pnc-Si membranes. We further demonstrate that human endothelial cells can be grown on NPN membranes, verifying the biocompatibility of NPN and demonstrating the potential of this material for cell culture applications.

Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100–300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives.

flax 3,4 & Thomas R. Gaborski 2,4 ✉ extracellular vesicles (eVs) are membrane vesicles secreted by cells and can modulate biological activities by transferring their content following uptake into recipient cells. Labelling of EVs is a commonly used technique for understanding their cellular targeting and biodistribution. A reliable fluorescent technique needs to preserve the size of EVs since changes in size may alter their uptake and biodistribution. Lipophilic fluorescent dye molecules such as the PKH family have been widely used for EV labelling. Here, the effect of PKH labelling on the size of EVs was systematically evaluated using nanoparticle tracking analysis (NTA), which is a widely used technique for determining the size and concentration of nanoparticles. NTA analysis showed a size increase in all the PKH labelling conditions tested. As opposed to lipophilic dye molecules, no significant shift in the size of labelled EVs was detected with luminal binding dye molecules such as 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE, hereinafter CFSE). This finding suggests that PKH labelling may not be a reliable technique for the tracking of EVs.

We have developed electroosmotic pumps (EOPs) fabricated from 15-nm-thick porous nanocrystalline silicon (pnc-Si) membranes. Ultrathin pnc-Si membranes enable high electroosmotic flow per unit voltage. We demonstrate that electroosmosis theory compares well with the observed pnc-Si flow rates. We attribute the high flow rates to high electrical fields present across the 15-nm span of the membrane. Surface modifications, such as plasma oxidation or silanization, can influence the electroosmotic flow rates through pnc-Si membranes by alteration of the zeta potential of the material. A prototype EOP that uses pnc-Si membranes and Ag/ AgCl electrodes was shown to pump microliter per minute-range flow through a 0.5-mm-diameter capillary tubing with as low as 250 mV of applied voltage. This silicon-based platform enables straightforward integration of low-voltage, on-chip EOPs into portable microfluidic devices with low back pressures.lectroosmotic flow results from the interaction between an electric field and the diffuse layer of ions at a charged surface. In capillaries or pores, the migration of the diffuse layer toward the oppositely charged electrode causes the bulk fluid within the channel to flow through viscous drag. Electroosmotic pumps (EOPs) are designed to generate high flow rates in microchannels using these principles (1, 2). EOPs present a number of advantages over mechanical pumps, including the lack of mechanical parts, pulse-free flows, and ease of control through electrode actuation. EOPs have been suggested as pumps for cooling circuits (3) and microfluidic devices that aid in drug delivery (4, 5) or diagnostics (2, 6). Microfluidic devices enable the miniaturization of multistep laboratory processes into small, low-cost, disposable units (6, 7). The inclusion of multiple steps into a single device increases the need for the precision pumping of fluids on-chip.High voltages (>1 kV) are often required for direct current (dc) EOPs to achieve sufficient flow rates in microchannels (8, 9). However, devices with high-voltage EOPs require bulky external power supplies and a skilled technician to operate, which defeats the ease of use and portability aims of a microfluidic diagnostic tool. For these reasons, the development of a low-voltage EOP is a current focus in the literature. Several recent low-voltage EOPs have been fabricated from porous silicon (10), alumina (11-13), track-etched polymer (14), and carbon nanotube membranes (15). These low-voltage EOPs are much thinner than their highvoltage predecessors (60-350 μm compared with >10 mm). Yao et al. suggest that further thinning of EOPs will enable better voltage-specific characteristics (16). Here, we examine the electroosmotic pumping by nanoporous membranes that are more than two orders of magnitude thinner than any membrane material previously used in an EOP.We have recently developed an ultrathin (15-30 nm), nanoporous membrane material called porous nanocrystalline silicon (pnc-Si) (17). pnc-Si membranes are fabricated on silicon wafers usin...