The emerging high-resolution 3D printing technique called two-photon polymerization (2PP) enables to print devices bottom-up rapidly, contrary to the top-down lithography-based fabrication methods. In this work, various polymer microbeams are 3D printed and their resonant characteristics are analyzed to understand the origin of damping. The 2PP printed polymer resonators have shown less damping than other polymer devices reported earlier, with tensile-stressed clamped-clamped beams reaching a record quality factor of 1819. The resonant energy loss was dominant by bulk friction damping. These results pave the path towards using 3D printed microresonators as mass sensors with improved design and fabrication flexibility.

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Bacteria that are resistant to antibiotics present an increasing burden on healthcare. To address this emerging crisis, novel rapid antibiotic susceptibility testing (AST) methods are eagerly needed. Here, we present an optical AST technique that can determine the bacterial viability within 1 h down to a resolution of single bacteria. The method is based on measuring intensity fluctuations of a reflected laser focused on a bacterium in reflective microwells. Using numerical simulations, we show that both refraction and absorption of light by the bacterium contribute to the observed signal. By administering antibiotics that kill the bacteria, we show that the variance of the detected fluctuations vanishes within 1 h, indicating the potential of this technique for rapid sensing of bacterial antibiotic susceptibility. We envisage the use of this method for massively parallelizable AST tests and fast detection of drug-resistant pathogens.

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The capacity to precisely pipette femtoliter volumes of liquid enables many applications, for example, to functionalize a nanoscale surface and manipulate fluids inside a single-cell. A pressure-controlled pipetting method is the most preferred, since it enables the widest range of working liquids. However, precisely controlling femtoliter volumes by pressure is challenging. In this work, a new concept is proposed that makes use of axisymmetrical phaseguides inside a microfluidic channel to pipette liquid in discrete steps of known volume. An analytical model for the design of the femtopipettes is developed and verified experimentally. Femtopipettes are fabricated using a multi-scale 3D printing strategy integrating a digital light processing printed part and a two-photon-polymerization printed part. Three different variants are designed and fabricated with pipetting resolutions of 10 picoliters, 180 femtoliters and 50 femtoliters. As a demonstration, controlled amounts of a water-glycerol mixture were first aspirated and then dispensed into a mineral oil droplet.

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Reciprocating piezoelectric micropumps enable miniaturization in microfluidics for lab-on-a-chip applications such as organs-on-chips (OoC). However, achieving a steady flow when using these micropumps is a significant challenge because of flow ripples in the displaced liquid, especially at low frequencies or low flow rates (<50 µL/min). Although dampers are widely used for reducing ripples in a flow, their efficiency depends on the driving frequency of the pump. Here, we investigated multi-phase rectification as an approach to minimize ripples at low flow rates by connecting piezoelectric micropumps in parallel. The efficiency in ripple reduction was evaluated with an increasing number (n) of pumps connected in parallel, each actuated by an alternating voltage waveform with a phase difference of 2π/n (called multi-phase rectification) at a chosen frequency. We introduce a fluidic ripple factor ((Formula presented.)), which is the ratio of the root mean square ((Formula presented.)) value of the fluctuations present in the rectified output to the average fluctuation-free value of the discharge flow, as a metric to express the quality of the flow. The fluidic ripple factor was reduced by more than 90% by using three-phase rectification when compared to one-phase rectification in the 2–60 μL/min flow rate range. Analytical equations to estimate the fluidic ripple factor for a chosen number of pumps connected in parallel are presented, and we experimentally confirmed up to four pumps. The analysis shown can be used to design a frequency-independent multi-phase fluid rectifier to the desired ripple level in a flow for reciprocating pumps.

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Resonant sensors hold great promise in measuring small masses, to enable future mass spectrometers, and small forces in applications like atomic and magnetic force microscopy. During the last decades, scaling down the size of resonators has led to huge enhancements in sensing resolution, but has also raised the question of what the ultimate limit is. Current knowledge suggests that this limit is reached when a resonator oscillates at the maximum amplitude for which its response is predominantly linear. We present experimental evidence that it is possible to obtain better resolutions by oscillation amplitudes beyond the onset of nonlinearities. An analytical model is developed that explains the observations and unravels the relation between ultimate sensing resolution and speed. In the high-speed limit, we find that the ultimate resolution of a resonator is improved when decreasing its damping. This conclusion contrasts with previous works, which proposed that lowering the damping does not affect or even harms the ultimate sensing resolution.

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Organ-on-a-chip (OoC) devices require the precise control of various media. This is mostly done using several fluid control components, which are much larger than the typical OoC device and connected through fluidic tubing, i.e., the fluidic system is not integrated, which inhibits the system’s portability. Here, we explore the limits of fluidic system integration using off-the-shelf fluidic control components. A flow control configuration is proposed that uses a vacuum to generate a fluctuation-free flow and minimizes the number of components used in the system. 3D printing is used to fabricate a custom-designed platform box for mounting the chosen smallest footprint components. It provides flexibility in arranging the various components to create experiment-specific systems. A demonstrator system is realized for lung-on-a-chip experiments. The 3D-printed platform box is 290 mm long, 240 mm wide and 37 mm tall. After integrating all the components, it weighs 4.8 kg. The system comprises of a switch valve, flow and pressure controllers, and a vacuum pump to control the diverse media flows. The system generates liquid flow rates ranging from 1.5 μ Lmin ^{- 1} to 68 μ Lmin ^{- 1} in the cell chambers, and a cyclic vacuum of 280 mbar below atmospheric pressure with 0.5 Hz frequency in the side channels to induce mechanical strain on the cells-substrate. The components are modular for easy exchange. The battery operated platform box can be mounted on either upright or inverted microscopes and fits in a standard incubator. Overall, it is shown that a compact integrated and portable fluidic system for OoC experiments can be constructed using off-the-shelf components. For further down-scaling, the fluidic control components, like the pump, switch valves, and flow controllers, require significant miniaturization while having a wide flow rate range with high resolution.

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The use of nanoparticles has been growing in various industrial fields, and concerns about their effects on health and the environment have been increasing. Hence, characterization techniques for nanoparticles are essential. Here, we present a silicon dioxide microfabricated suspended microchannel resonator (SMR) to measure the mass and concentration of nanoparticles in a liquid as they flow. We measured the mass detection limits of the device using laser Doppler vibrometry. This limit reached a minimum of 377 ag that correspond to a 34 nm diameter gold nanoparticle or a 243 nm diameter polystyrene particle, when sampled every 30 ms. We compared the fundamental limits of the measured data with an ideal noiseless measurement of the SMR. Finally, we measured the buoyant mass of gold nanoparticles in real-time as they flowed through the SMR. [Figure not available: see fulltext.].

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Piezoelectric micropumps enable miniaturization in microfluidics for lab-on-a-chip applications such as Organs-on-chips (OoC). However, achieving a steady flow with these micropumps is a significant challenge because of the reciprocating motion of the displacing component. Although dampers are widely preferred for reducing ripples, they are not efficient at low flow rates. Here, we propose a phased-actuation of piezoelectric micropumps connected in parallel and a damper to minimize ripples at low flow rates. We are able to reduce ripples by 80% with our proposed configuration compared to a micropump-only configuration between 10-50 μl/min flow rate range.@en

Physical patterns represent potential surface cues for promoting osteogenic differentiation of stem cells and improving osseointegration of orthopedic implants. Understanding the early cell–surface interactions and their effects on late cellular functions is essential for a rational design of such topographies, yet still elusive. In this work, fluidic force microscopy (FluidFM) and atomic force microscopy (AFM) combined with optical and electron microscopy are used to quantitatively investigate the interaction of preosteoblasts with 3D-printed patterns after 4 and 24 h of culture. The patterns consist of pillars with the same diameter (200 nm) and interspace (700 nm) but distinct heights (500 and 1000 nm) and osteogenic properties. FluidFM reveals a higher cell adhesion strength after 24 h of culture on the taller pillars (32 ± 7 kPa versus 21.5 ± 12.5 kPa). This is associated with attachment of cells partly on the sidewalls of these pillars, thus requiring larger normal forces for detachment. Furthermore, the higher resistance to shear forces observed for these cells indicates an enhanced anchorage and can be related to the persistence and stability of lamellipodia. The study explains the differential cell adhesion behavior induced by different pillar heights, enabling advancements in the rational design of osteogenic patterns.

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Microfluidic organs-on-chips (OoCs) technology has emerged as the trend for in vitro functional modeling of organs in recent years. Simplifying the complexities of the human organs under controlled perfusion of required fluids paves the way for accurate prediction of human organ functionalities and their response to interventions like exposure to drugs. However, in the state-of-the-art OoC, the existing methods to control fluids use external bulky peripheral components and systems much larger than the chips used in experiments. A new generation of compact microfluidic flow control systems is needed to overcome this challenge. This study first presents a structured classification of OoC devices according to their types and microfluidic complexities. Next, we suggest three fundamental fluid flow control mechanisms and define component configurations for different levels of OoC complexity for each respective mechanism. Finally, we propose an architecture integrating modular microfluidic flow control components and OoC devices on a single platform. We emphasize the need for miniaturization of flow control components to achieve portability, minimize sample usage, minimize dead volume, improve the flowing time of fluids to the OoC cell chamber, and enable long-duration experiments.

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Surfactant losses by adsorption to rock surfaces make surfactant-based enhanced oil recovery economically less feasible. We investigated polyacrylate (PA) as a sacrificial agent in the reduction of anionic surfactant adsorption with focus on calcite surfaces by using quartz crystal microbalance with dissipation monitoring. It was found that the adsorption of the anionic surfactant alcohol alkoxy sulfate (AAS) followed a Langmuir adsorption isotherm, and the adsorbed amount reached saturation above its critical micellar concentration. Adsorption of PA was a much slower process compared to AAS adsorption. Increasing the calcium ion concentration also increased the amount of AAS adsorbed as well as the mass increase rate of PA adsorption. Experimental results combined with density functional theory calculations indicated that calcium cation bridging was important for anionic surfactant AAS and PA adsorption to calcite surfaces. To effectively reduce the amount of surfactant adsorption, it was needed to preflush with PA, rather than by a simultaneous injection. Preflushing with 30 ppm of PA gave a reduction of AAS adsorption of 30% under high salinity (HS, 31,800 ppm) conditions, compared to 8% reduction under low salinity (LS, 3180 ppm) conditions. In the absence of PA, the amount of adsorbed AAS was reduced by already 50% upon changing from HS to LS conditions. Lower calcium ion concentrations, as under LS conditions, contributed to this observation. On different mineral surfaces, PA reduced the AAS adsorption in the order of alumina > calcite > silica. These results offer important insights into mitigating surfactant adsorption using PA polyelectrolyte as sacrificial agent and contribute to improved flooding strategies with reduced surfactant loss.

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Micro- and nano-patterns are gaining increasing attraction in several fields ranging from nanoelectronics to bioengineering. The mechanical properties of the nanostructures (nanopillars, nanotubes, nanowires, etc.) are highly relevant for many applications but challenging to determine. Existing mechanical characterization methods require mounting the testing setup inside a scanning electron microscope (SEM) and additional sample modification. Here, we propose two atomic force microscopy (AFM) methods, based on contact mode imaging (CMI) and force spectroscopy imaging (FSI), to determine the mechanical characteristics of individual micro- and nanopillars as fabricated, without using SEM. We present the working principles of both methods and two case studies on nanopillars fabricated by additive manufacturing methods: two-photon polymerization (2PP) and electron beam induced deposition (EBID). Various mechanical parameters were determined using CMI and FSI, respectively. For the 2PP nanopillars, we measured the stiffness (13.5 ± 3.2 N/m and 15.9 ± 2.6 N/m), the maximum lateral force (883.0 ± 89.5 nN and 889.6 ± 113.6 nN), the maximum deflection (64.2 ± 13.6 nm and 58.3 ± 14.24 nm), the failure stress (0.3 ± 0.03 GPa and 0.3 ± 0.02 GPa), and the adhesion force (56.6 ± 4.5 µN and 58.6 ± 5.2 µN). For the EBID nanopillars, we measured the failure stress (2.9 ± 0.2 GPa and 2.7 ± 0.4 GPa). The similar results obtained using both techniques confirmed the efficacy and consistency of the methods. The proposed methodologies have the potential of enabling otherwise impossible measurements particularly when the specimens need to be tested under wet conditions, such as patterns for mechanobiological studies.

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The surface topography of implantable devices is of crucial importance for guiding the cascade of events that starts from the initial contact of the cells with the surface and continues until the complete integration of the device in its immediate environment. There is, however, limited quantitative information available regarding the relationships between the different stages of such cascade(s) and how the design of surface topography influences them. We, therefore, used direct laser writing to 3D-print submicron pillars with precisely controlled dimensions and spatial arrangements to perform a systematic study of such relationships. Using single-cell force spectroscopy, we measured the adhesion force and the work of adhesion of the preosteoblast cells residing on the different types of surfaces. Not only the adhesion parameters (after 2-60 s) but also the formation of focal adhesions was strongly dependent on the geometry and arrangement of the pillars: sufficiently tall and dense pillars enhanced both adhesion parameters and the formation of focal adhesions. Our morphological study of the cells (after 24 h) showed that those enhancements were associated with a specific way of cell settlement onto the surface (i.e., "top state"). The cells interacting with tall and dense pillars were also characterized by numerous thick actin stress fibers in the perinuclear region and possibly high internal stresses. Furthermore, living cells with highly organized cytoskeletal networks exhibited greater values of the elastic modulus. The early responses of the cells predicted their late response including matrix mineralization: tall and dense submicron pillars significantly upregulated the expression of osteopontin after 21 days of culture under both osteogenic and nonosteogenic conditions. Our findings paint a detailed picture of at least one possible cascade of events that starts from initial cell adhesion and continues to subsequent cellular functions and eventual matrix mineralization. These observations could inform the future developments of instructive surfaces for medical devices based on physical surface cues and early markers.

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Despite the potential of small-scale pillars of black titanium (bTi) for killing the bacteria and directing the fate of stem cells, not much is known about the effects of the pillars’ design parameters on their biological properties. Here, three distinct bTi surfaces are designed and fabricated through dry etching of the titanium, each featuring different pillar designs. The interactions of the surfaces with MC3T3-E1 preosteoblast cells and Staphylococcus aureus bacteria are then investigated. Pillars with different heights and spatial organizations differently influence the morphological characteristics of the cells, including their spreading area, aspect ratio, nucleus area, and cytoskeletal organization. The preferential formation of focal adhesions (FAs) and their size variations also depend on the type of topography. When the pillars are neither fully separated nor extremely tall, the colocalization of actin fibers and FAs as well as an enhanced matrix mineralization are observed. However, the killing efficiency of these pillars against the bacteria is not as high as that of fully separated and tall pillars. This study provides a new perspective on the dual-functionality of bTi surfaces and elucidates how the surface design and fabrication parameters can be used to achieve a surface topography with balanced bactericidal and osteogenic properties.

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Functional gradients are material transitions that are found in nature and are known to result in materials with superior properties and multiple functionalities. The emerging multi-material 3D printing (=additive manufacturing, AM) techniques provide a powerful tool for the design and fabrication of bioinspired functionally graded materials (FGMs). In particular, the spatial distribution of materials can be controlled at the level of individual volumetric pixels (voxels i.e., cubes with side lengths of 20–40 μm), thereby ensuring accuracy, reliability, and reproducibility of the obtained properties and allowing for systematic studies of how various design variables affect the deformation and fracture behaviors of FGMs. Here, we designed, 3D printed, and mechanically tested tensile and notched FGMs specimens with step-wise (i.e., 5-, 10-, and 15-steps) and continuous (sigmoid and linear) gradients. The deformation and fracture mechanisms of these FGM composites were studied using digital image correlation, digital microscopy, and scanning electron microscopy. We further characterized the chemical composition and local mechanical properties of FGM composites using XPS and nanoindentation measurements, respectively. Tensile test specimens with a continuous gradient (i.e., linear) exhibited much higher Young's moduli (≈3-folds) and ultimate strengths (≈2-folds) but lower elongations (≈2-folds drop) as compared to those with stepwise gradients (i.e., 5-steps). Similarly, notched specimens with linear gradients exhibited 2-folds higher values of the stiffness and fracture stress, but 1.5-folds lower fracture strains as compared to those with 5-steps gradients. Although we found non-uniform highly concentrated strain distributions in all specimens, FGMs with linear gradients showed a smoother strain distribution and smaller crack blunting zones as compared to those with stepwise gradients. Our results imply that for stiffness and strength linear-gradient perform better than abrupt hard-soft-hard specimens.

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One of the methods to create sub-10 nm resolution metal-composed 3D nanopillars is electron beam-induced deposition (EBID). Surface nanotopographies (e.g., nanopillars) could play an important role in the design and fabrication of implantable medical devices by preventing the infections that are caused by the bacterial colonization of the implant surface. The mechanical properties of such nanoscale structures can influence their bactericidal efficiency. In addition, these properties are key factors in determining the fate of stem cells. In this study, we quantified the relevant mechanical properties of EBID nanopillars interacting with Staphylococcus aureus (S. aureus) using atomic force microscopy (AFM). We first determined the elastic modulus (17.7 GPa) and the fracture stress (3.0 ± 0.3 GPa) of the nanopillars using the quantitative imaging (QI) mode and contact mode (CM) of AFM. The displacement of the nanopillars interacting with the bacteria cells was measured by scanning electron microscopy (50.3 ± 9.0 nm). Finite element method based simulations were then applied to obtain the force-displacement curve of the nanopillars (considering the specified dimensions and the measured value of the elastic modulus) based on which an interaction force of 88.7 ± 36.1 nN was determined. The maximum von Mises stress of the nanopillars subjected to these forces was also determined (3.2 ± 0.3 GPa). These values were close to the maximum (i.e., fracture) stress of the pillars as measured by AFM, indicating that the nanopillars were close to their breaking point while interacting with S. aureus. These findings reveal unique quantitative data regarding the mechanical properties of nanopillars interacting with bacterial cells and highlight the possibilities of enhancing the bactericidal activity of the investigated EBID nanopillars by adjusting both their geometry and mechanical properties.@en

Fabricating large areas of geometrically complex and precisely controlled topographies is required for the studies of cell behavior on patterned surfaces. Direct laser writing (DLW) is an advanced 3D-fabrication technique, which facilitates the manufacturing of structures within various scales (from a few hundred nanometers to millimeters). However, this method requires improvements in the accuracy and reproducibility of the submicron and nanoscale features that are printed over a large area. Here, we present a scheme to both improve the uniformity of the printed submicron patterns and decrease the printing time. The effects of various processing parameters (e.g., laser power and writing field) on the dimensions and uniformity of submicron pillars as well as on their Young’s modulus and surface wettability were assessed. Decreasing the writing field to 33 × 33 μm2 significantly improved the uniformity of submicron pillars that were printed over an area of 4 mm2 in a single-step process. Preosteoblast cells (MC3T3-E1) were used to assess the cytocompatibility of the used material (IP-L780 resin) with a focus on cell morphology, cell proliferation, cytoskeletal organization, and the elastic modulus of the cells. The cells cultured for 2 days on the submicron pillars showed a polarized shape and a higher Young’s modulus of the area corresponding to the nucleus relative to those cultured on flat surfaces. Taken together, the results of the current study clearly show that the submicron patterns created using DLW are both cytocompatible and could modulate the morphology and mechanical properties of cells. This work paves the way for direct printing of submicron features with controlled Young’s moduli over large areas in a single-step process, which is necessary for systematically studying how such patterns modulate cellular functions.@en

Suspended microfluidic resonators enable detection of fluid density and viscosity with high sensitivity. Here, a two-legged suspended microchannel resonator that probes pico-litres of liquid is presented. The higher resonant modes (flexural and torsional) were explored for increased sensitivity and resolution. Unlike other reported microchannel resonators, this device showed an increase in the quality factor with resonant frequency value. The performance of the resonator was tested by filling the channel with three liquids, one at a time, over a density range of 779 - 1110 Kg/m^3 and a viscosity range of 0.89 - 16.2 mPa.s. The highest resolution obtained was 0.011% change in density. Measurements with torsional mode showed an improvement of about six times in sensitivity and about fifteen times in resolution compared to the first flexural mode. When the empty channel was filled with liquids of different viscosity, the quality factor of the first flexural mode remained overall constant with a variation below 3.3% between the fluids, and confirming the inherent property of suspended microchannel resonators. However, it significantly decreased for second flexural and torsional modes. No noticeable difference was observed in the quality factor between different liquid viscosities for all modes.@en

Microvalves are important flow-control devices in many standalone and integrated microfluidic applications. Polydimethylsiloxane (PDMS)-based pneumatic microvalves are commonly used but they generally require large peripheral connections that decrease portability. There are many alternatives found in the literature that use Si-based microvalves, but variants that can throttle even moderate pressures (1) tend to be bulky (cm-range) or consume high power. This paper details the development of a low-power, normally-open piezoelectric microvalve to control flows with a maximum driving pressure of 1, but also retain a small effective form-factor of 5x5x1.8. A novel combination of rapid prototyping methods like stereolithography and laser-cutting have been used to realize this device. The maximum displacement of the fabricated piezoelectric microactuator was measured to be 8.5 at 150. The fabricated microvalve has a flow range of 0–90 at 1 inlet pressure. When fully closed, a leakage of 0.8 open-flow was observed with a power-consumption of 37.5. A flow resolution of 0.2— De-ionized (DI) water was measured at 0.5 pressure.@en

We introduce a two-channel microfluidic atomic force microscopy (AFM) cantilever that combines the nanomechanical sensing functionality of an AFM cantilever with the ability to manipulate fluids of picolitres or smaller volumes through nanoscale apertures near the cantilever tip. Each channel is connected to a separate fluid reservoir, which can be independently controlled by pressure. Various systematic experiments with fluorescent liquids were done by either injecting the liquids from the on-chip reservoir or aspirating directly through the nanoscale apertures at the tip. A flow rate analysis of volume dosing, aspiration and concentration dosing inside the liquid medium was performed. To understand the fluid behaviour, an analytical model based on the hydrodynamic resistance, as well as numerical flow simulations of single and multi-phase conditions were performed and compared. By applying pressures between -500 mbar and 500 mbar to the reservoirs of the probe with respect to the ambient pressure, flow rates ranging from 10 fl s-1 to 83 pl s-1 were obtained inside the channels of the cantilever as predicted by the analytical model. The smallest dosing flow rate through the apertures was 720 fl s-1, which was obtained with a 10 mbar pressure on one reservoir and ambient pressure on the other. The solute concentration in the outflow could be tuned to values between 0% and 100% by pure convection and to values between 17.5% and 90% in combination with diffusion. The results prove that this new probe enables handling multiple fluids with the scope to inject different concentrations of analytes inside a single living cell and also perform regular AFM functionalities.

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