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Efficient mixing in microfluidic systems is bottlenecked by laminar flow and diffusion-dominated transport, limiting the performance of biochemical assays such as conversion rates of enzymatic processes. Here, we present an acoustofluidic micromixer based on two-photon polymerization (2PP)-printed three-dimensional micro-needles that act as ultrasonic microresonators to generate localized acoustic streaming. The freeform arrangement of the microneedles enables integration into diverse microfluidic channel layouts and produces strong, tunable streaming vortices that enhance transport. We characterize the streaming flow fields as a function of actuation frequency, and demonstrate rapid and voltage-dependent mixing with a 2.5-fold enhancement. When applied to the alkaline phosphatase-fluorescein diphosphate (ALP-FDP) reaction, acoustic mixing doubles the fluorescein signal under optimal conditions and significantly reduces the assay time and channel footprint required in continuous flow. These resultsdemonstrate that 3D microneedle-based acoustofluidics is a compact, versatile, and integrable solution for enhancing transport-limited biochemical processes in lab-on-a-chip systems.

The droplet-microfluidic system under acoustic-field actuation represents a classical multiphase flow problem with broad relevance to engineering and biomedical applications. When an acoustic wave interacts with a viscous droplet suspended in another liquid, it induces acoustic streaming both inside and outside the droplet and exerts acoustic radiation stresses that deform its shape. In this work, we present a comprehensive analytical and numerical framework to investigate the coupled dynamics of droplet shape evolution, acoustic fields and acoustic streaming. The study considers droplets stably positioned within a standing wave at either a pressure node or a velocity node, undergoing deformations analogous to the classical Taylor regime. Our analytical model, supported by numerical simulations, reveals that the acoustic field is predominantly governed by low-order modes, up to the quadrupole. Furthermore, the acoustic-streaming pattern is dictated by the density contrast between the droplet and the surrounding fluid, whereas both density and compressibility contrasts control the droplet deformation. We also demonstrate that impedance mismatch is not the fundamental criterion for acoustic scattering. Instead, mismatches in density, or compressibility, or both, serve as the primary mechanisms. In addition, we develop a phase diagram illustrating droplet shape-deformation and streaming-pattern regimes as functions of the material properties (density ratio and compressibility ratio). These results provide deep mechanistic insights for the design and control of efficient droplet-acoustofluidic platforms and lay the groundwork for next-generation ultrasound-driven strategies for precise droplet manipulation in soft-matter and microfluidic applications.

Heteroresistance (HR) is an antibiotic resistance phenotype characterized by the presence of rare resistant subpopulations (frequency ≈ 10⁻⁷ to 10⁻⁴) within a main susceptible bacterial population. During antibiotic exposure, these subpopulations can be enriched and cause treatment failure. Standard antibiotic susceptibility testing (AST) often fails to detect HR, and the current gold-standard population analysis profile (PAP) test is labor-intensive and time-consuming. We present a digital phenotyping approach combining droplet microfluidics with image texture to detect HR from clinical isolates, including Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii) and Gram-positive (Staphylococcus aureus) bacteria isolated from bloodstream infections. Our method achieves detection at subpopulation frequencies as low as 10⁻⁶ in 12 to 30 h, depending on bacterial species, which is faster than the PAP test, together with single-cell resolution and high-throughput. This computationally assisted microfluidic platform enables rapid and accurate identification of HR, representing a step toward targeted antibiotic therapy in critical infections.

Introduction: The spatial variation of biochemical signals, known as chemical gradients, is a critical regulator of cellular activity across a spectrum of biological phenomena. These gradients act as directional signals, influencing processes ranging from embryonic morphogenesis and immune response to tissue regeneration and disease pathogenesis. Consequently, the capacity to manipulate and precisely define the distribution of signaling molecules, particularly labile growth factors (GF) and cytokines, is paramount for deciphering cellular behavior and advancing therapeutic interventions. Conventional 2D cell culture methods fail to generate stable, spatially defined gradients, limiting physiological relevance. To overcome this, we developed a polydimethylsiloxane (PDMS) microfluidic device incorporating microstructures created via two-photon polymerization [1]. This system enables the sustained generation of chemical gradients, providing a robust tool for studying their impact on the development and sprouting of 3D cellular constructs.

Materials and methods: The gradient-on-chip platform, fabricated from PDMS, was designed to establish controlled GF gradients. Fluorophore-tagged dextran was used to characterize the establishment of the gradient. Furthermore, to validate the functionality of the gradient, 3D cellular constructs such as spheroid and organoid were cultured within the device for 7–10 days under a regulated flow of nutrients. Confocal laser scanning microscopy and real-time imaging, conducted within a stage-top incubator, were used to monitor cellular responses such as sprouting and morphological changes. Quantitative image analysis was performed to assess gradient effects.

Results: The selected GFs promote growth, sprouting, and invasion. We specifically investigated: 1) how 3D cellular constructs sprouting and migration respond to these gradients and 2) whether temporal changes in chemical gradients correlate with cellular behavior [2,3]. In contrast to static cultures, cells subjected to growth factor gradients demonstrated enhanced proliferation, vascular-like networks, and a higher degree of organized structure. Morphological analysis revealed spatial differences in sprouting and differentiation. Quantitative analysis confirmed significant effects of biochemical gradients on the development of 3D cellular constructs.

Discussion: The microfluidic device is crucial in generating stable and tunable biochemical gradients, providing a more physiologically relevant environment than traditional static culture models. By precisely controlling gradient formation, this platform enables the study of dynamic cellular behaviors, such as sprouting, migration, and tissue organization. 

Electroporation of dye-labeled bio-molecules into bacteria has proven to be a valuable route for single-molecule tracking in living cells. However, control over cell viability, electroporation efficiency, and environment conditions before, during, and after electroporation is difficult to achieve in bulk experiments. Here, a microfluidic platform is presented capable of single-cell electroporation with in situ microscopy and demonstrate delivery of DNA into bacteria. Via real time observation of the electroporation process, it is found that the effect of electrophoresis plays an important role when performing electroporation in a miniaturized platform and show that its undesired action can be balanced by using bipolar electrical pulses. It is suggested that a low temperature of the sample during electroporation is important for cell viability due to temperature-dependant viscoelastic properties of the cell membrane. It is further found that the presence of low conductive liquid between cells and the electrodes leads to a voltage divider effect that strongly influences the success of on-chip electroporation. Finally, it is concluded that electroporation is a highly stochastic process and envision that the microfluidic system presented here, capable of single-cell read-out, can be used for further fundamental studies to increase the understanding of the electroporation process in bacterial cells.

Culturing living cells in three-dimensional environments increases the biological relevance of laboratory experiments, but requires solutes to overcome a diffusion barrier to reach the centre of cellular constructs. We present a theoretical and numerical investigation that brings a mechanistic understanding of how microfluidic culture conditions, including chamber size, inlet fluid velocity and spatial confinement, affect solute distribution within three-dimensional cellular constructs. Contact with the chamber substrate reduces the maximally achievable construct radius by 15%. In practice, finite diffusion and convection kinetics in the microfluidic chamber further lower that limit. The benefits of external convection are greater if transport rates across diffusion-dominated areas are high. Those are omnipresent and include the diffusive boundary layer growing from the fluid-construct interface and regions near corners where fluid is recirculating. Such regions multiply the required convection to achieve a given solute penetration by up to 100, so chip designs ought to minimize them. Our results define conditions where complete solute transport into an avascular three-dimensional cell construct is achievable and applies to real chambers without needing to simulate their exact geometries.

Chemical gradients play important roles in biological systems, influencing responses to injury, disease, and therapeutic delivery. Precisely accessing substances within a local gradient in the tissue microenvironment is crucial for manipulating key cellular processes and understanding the resulting biological responses. However, establishing localized and sustained gradients of often unstable growth factors (GF) and cytokines using standard cell culture methods can be challenging. Microfluidic platforms offer a promising approach for studying a wide range of medically relevant pathways. We have previously described the successful development of a stable gradient within a microfluidic device, comprising a PDMS-based microfluidic device with integrated physical microstructures fabricated using two-photon polymerization [1].Here, we have used this device to study the effect of chemical gradients of fibroblast growth factor (FGF2) and epidermal growth factor (EGF) on human lung fibroblast spheroids encapsulated and maintained inside the microfluidic device for 7-14 days. Those GF were chosen as FGF2 have been shown to promote spheroid growth and increase the production of collagen, an extracellular matrix component [2]. While EGF has been shown to promote spheroid growth and enhance cell spreading and invasion [3].Therefore, we are monitoring the spatiotemporal response of cells to GF gradients within spheroids. Specifically, we are investigating: 1) how cell movement and migration respond to these gradients at the spheroid periphery versus the core; 2) whether collagen accumulates preferentially in certain regions; and 3) whether changes in chemical gradients over time correlate with changes in cell behavior within the spheroids. We analyzed and characterized these parameters within a microfluidic device under controlled environmental conditions in a stage-top incubator, using confocal laser scanning microscopy with precisely controlled nutrient and GF flow. This device provides a more physiological and controlled in vitro model compared to traditional 2D cell cultures for understanding the key cellular processes under defined chemical gradients.

Vessel-on-chip models are an attractive alternative as a more physiologically relevant platform to study vascular disease and drug diffusion through the blood vessel walls than the standard in vitro and in vivo models currently in use. Current vessel-on-chip models often rely on the creation of vascular networks in a hydrogel, which allows for flow through of the internal lumen but not outside the vessel wall.

In this work, we developed a single hydrogel, high-resolution, perfusable vessel-on-chip model made from fully biocompatible hydrogel. The structure is designed for dual-flow, i.e., one flow through the inner lumen and another on the outside of the channel. This makes it possible to use this as a model for different biological barriers, such as the blood-brain-barrier, as well as diffusion through the channel wall itself. The hydrogel structure is fabricated directly on-chip by using 2-photon 3D printing (NanoOne 250, UpNano, Austria) with a commercial matrigel-based hydrogel resin (HydrobioINX U200, BIO INX, Belgium). The combination of 2-photon 3D printing and this hydrogel resin allows for resolutions down to 10 µm [1], making it possible to fabricate biomimetic vascular structures on a biologically relevant size scale. 

The microfluidic chip used consists of a 500 µm thick PDMS layer with the desired design cut-out, sandwiched between two microscopy cover glasses. The non-polymerised hydrogel resin is loaded into the chip, and the inlets are plugged to avoid evaporation during the fabrication process. Printing times for the desired structures is relatively fast, ranging between minutes to just over 1 hour for more complex designs. Development of the finished structures is a critical step and is achieved by gently flushing out the non-polymerised hydrogel resin overnight via syringe pumps. The chip is placed inside a cell culture incubator at 37 °C for the duration of this process. 

In conclusion, we have developed a method to fabricate perfusable single-hydrogel structures directly inside microfluidic chips using 2-photon 3D printing. The vessel model is capable of dual-flow, making it possible to study biological vascular barriers on-chip.