Lab on a Chip最新文献

Single-cell analysis is essential for uncovering heterogeneous biological functions that arise from intricate cellular responses. Here, microfluidic droplet arrays enable high-throughput data collection through cell encapsulation in picoliter volumes, and the time-lapse imaging of these arrays further reveal functional dynamics and changes. However, accurate tracking of cell identities across time frames with large intervals in between remains challenging when droplets move significantly. Specifically, existing machine learning methods often depend on labeled data or require neighboring cells as reference; without them, these methods struggle to track droplets and cells across long distances within images with complex movement patterns. To address these limitations, we developed a pipeline that combines visual object detection, feature extraction via contrastive learning, and optimal transport-based object matching, minimizing the reliance on labeled training data. We validated our approach across various experimental and simulated conditions and were able to track thousands of water-in-oil microfluidic droplets over large distances and long intervals between frames (>30 min). We achieved high precision in previously untraceable scenarios, tracking 50 pl droplets in images with small, medium and large movements (corresponding to ∼126, ∼800 and ∼10 000 μm, respectively) with a success rate of correctly tracked droplets of >90% for average movements within 2–12 droplet diameters, and >60% for average movements of >100 droplet diameters. This workflow lays the foundation for the tracking of droplets over time in these arrays when large and complex movement patterns are present and where the uniqueness of the sample makes repeated experiments infeasible.

Effective nanoparticle (NP) manipulation is crucial for diverse nanotechnology applications, including biomolecule sorting, drug delivery optimization, and metallic material synthesis. However, continuous enrichment and focusing of NPs remain challenging. Here, we introduce a microfluidic method that leverages controlled electro-elasticity and Joule heating to achieve high-throughput, precise manipulation of NPs. This approach synergistically combines the advantages of microfluidics (high throughput, precision, and continuous operation) with the real-time control of electric fields, while mitigating adverse thermal effects. We demonstrate high-throughput focusing of 100 nm particles in a large, straight rectangular microchannel. The underlying mechanism, driven by slip velocity induced by the interplay of electric fields and viscoelastic flow, is investigated using nearly electrically neutral, surface-modified particles. We quantitatively determine optimal control parameters, including electric field strength, flow velocity, and polymer concentration. Furthermore, a simple dry-ice-based temperature control system enables focusing of NPs as small as 20 nm under high electric fields, effectively mitigating Joule heating. This method balances the need for high-energy input for NP control with the elimination of detrimental thermal energy. By controlling electro-elasticity and Joule heating, our approach overcomes limitations of existing NP manipulation techniques, providing a route towards rapid and gentle enrichment of diverse NP types.

Liquid microjets are widely used at X-ray free electron laser (XFEL) facilities to deliver a variety of samples to the pulsed X-ray focus for diffraction and spectroscopy experiments. Continuous jets waste sample between exposures, which is a major problem for many samples that are expensive or difficult to produce. Synchronizing microdroplets with the X-ray pulses can greatly improve the sample delivery efficiency by simultaneously reducing flow rate and producing a thicker sample. Here, we develop 3D-printed gas dynamic virtual nozzles (GDVN) designed to eject periodic droplets, and demonstrate synchronization with an external trigger of 1 kHz via piezoelectric transduction. A co-flowing helium sheath gas allows the droplets to eject into vacuum, which minimizes X-ray gas background scatter. Alternatively, the system can operate at atmospheric pressure without the need for humidity control. A control system enhances the synchronization such that 60% of droplet positions fall within 25% of the droplet diameter. Numerical simulations are presented that match well with experimental data and reveal recirculation patterns in the meniscus, along with a detailed view of the dynamics associated with onset of triggered synchronization. The system is designed such that it can be implemented at conventional end-stations at XFEL and synchrotron facilities with minimal modification.

Organ-on-a-chip and microfluidic systems offer new ways to overcome limitations from traditional in vitro models in preclinical studies. However, the lack of standardization and important non-specific binding of tested drugs to devices commonly made of polydimethylsiloxane (PDMS) still slow down their full integration into industrial research pipelines. The goal of this study is to develop a standardized 3D-printed biochip with low-binding properties using perfluoropolyether (PFPE), allowing long-time dynamic cultures of in situ formed cellular spheroids. We first documented the non-specific binding of molecules relevant for pharmaceutical companies and mechanical and surface properties of PFPE as compared with PDMS. A new microstructured biochip was then designed and 3D-printed in PFPE to offer a 400 μL chamber containing 384 microwells. The 3D-printing fabrication protocol has been detailed considering key parameters such as UV exposure time or postcuring. Finally, 384 HepG2/C3a spheroids were formed per chip under dynamic conditions and maintained for 11 days. The high viability, functionality and polarization of the spheroids cultured in these printed PFPE biochips showed the relevance of this new microphysiological system as an alternative to PDMS devices.

Optical microheating technologies have revealed how biological systems sense heating and cooling at the microscopic scale. Sensing is based on thermosensitive biochemical reactions that frequently engage membrane proteins, Ca²⁺ channels, and pumps to convert sensing information as the Ca²⁺ signalling in cells. These findings highlight the feasibility of thermally manipulating intracellular Ca²⁺ signalling. However, how the thermosensitive Ca²⁺ signalling would behave, particularly in multicellular systems, remains elusive. In this study, to extend the ability of the spatiotemporal temperature control by optical microheating technologies, we propose holographic heating microscopy. Water-absorbable infrared (IR) laser light is modulated by a reflective liquid crystal on a silicon spatial light modulator (LCOS-SLM). A computer-generated hologram displayed on the LCOS-SLM modulates the spatial phase pattern of the IR laser light to generate predesigned temperature gradients at the microscope focal plane. The holographic heating microscopy visualises how thermosensitive Ca²⁺ signalling is generated and propagated in MDCK cells, rat hippocampal neurons, and rat neonatal cardiomyocytes. Moreover, the optical control of the temporal temperature gradient reveals the cooling-rate dependency of Ca²⁺ signalling in HeLa cells. These findings demonstrate the extended ability of holographic heating microscopy in investigating cellular thermosensitivities and thermally manipulating cellular functions.

The establishment of physiologically relevant in vitro skin models remains a fundamental challenge in tissue engineering, particularly concerning reliable drug screening platforms. Despite advances in conventional skin equivalents, matrix contraction has substantially impeded long-term experimental studies. Here, we report a novel non-contracting full-thickness skin equivalent incorporating a microvascular-like endothelial network that addresses these constraints. We employed an engineered porous scaffold that limits matrix contraction and supports development of a microvascular-like network. The porous support eliminated macroscopic contraction (100% area retention vs. 11.9% previous), enabling extended dermal maturation and stable long-term ALI culture. Sequential seeding of human umbilical vein endothelial cells (HUVEC), dermal fibroblasts, and keratinocytes produced a stable, interconnected vascular architecture. Network identity and perfusability were confirmed by CD31/CD144 immunofluorescence and fluorescent microsphere perfusion. This configuration permits prolonged culture stability and reproducible pharmacological assessments. The model's efficacy was evaluated through an atopic dermatitis (AD) pathological model. Upon pro-inflammatory cytokine stimulation (IL-4, IL-13, IL-22), comprehensive analyses revealed significant alterations in stratum corneum morphology, epidermal protein expression, and atopic-specific biomarkers (IL6, TSLP, CA2). Cytokine-dependent recruitment and dermal localization of HL-60 cells, demonstrated superior physiological relevance compared to avascular models. This platform represents a significant advancement in skin tissue engineering, providing a sophisticated tool for investigating dermatological pathologies and pharmacological responses, while offering a viable alternative to traditional animal testing.

Acoustofluidic technologies enable precise manipulation of microscale objects using travelling and standing sound waves in physiological fluids, offering exciting capabilities for biomedical and chemical applications. In particular, surface acoustic wave-based devices have shown great promise for on-chip micromanipulation, but their planar transducer configuration limits the usable workspace near the microchannel surface. Here, we present a novel acoustofluidic platform based on a digitally addressable array of piezoelectric micromachined ultrasound transducers (PMUTs) that generate bulk acoustic waves and acoustic traps within three-dimensional (3D) fluidic chambers. Through a combination of finite element modelling and experimental measurements, we quantify the acoustic field distribution and study acoustic trap formation dynamics. We demonstrate deterministic 3D levitation of particles in water at rest and under continuous flow by generating standing acoustic waves across the height of the chamber. Our results show that 30 μm polystyrene particles can be levitated to a pressure node generated 640 μm above the surface with less than 6% positional error. The system applies in-plane acoustic radiation forces as high as 90 pN to keep the particles in the trap under flow rates up to 40 μL min⁻¹. We leverage spatiotemporal modulation of the acoustic field for continuous planar transport of microparticle aggregates. PMUT arrays are microfabricated using conventional cleanroom techniques, thus can be readily integrated with compact fluidic systems. Our work lays the foundation for the development of reconfigurable and scalable acoustofluidic micromanipulation systems, with broad potential for lab-on-chip technologies.

Spirulina (Arthrospira platensis) is a valuable cyanobacterium used for various applications, including health supplements, cosmetics, biofertilizers, carbon capture, and biofuels. Efficient monitoring of microalgae growth in photobioreactors is crucial for optimizing yields in large-scale culturing. Existing monitoring systems take samples from the bioreactor at different intervals and perform the visualization and quantification of algae growth parameters. In this work, a microfluidic platform is mounted on a tubular photobioreactor, and the system continuously monitors the growth behavior of Spirulina over several days, with algal development captured on demand. Furthermore, the microfluidic sensor is fabricated using a novel xurography-based approach on photopolymer sheets. It captures real-time micrographs of algae continuously for 5 days (over 120 hours) under two different conditions: open-loop and closed-loop. In the open-loop configuration, the sensor hydrostatically taps the algal medium from the bioreactor at regular intervals. In contrast, the closed-loop sensor continuously (24/7) circulates the culture medium through the microchip for visualization without the use of any driving mechanism. From the micrographs, algal cell density, cell count, and trichome length are estimated continuously, and all parameters exhibited an increasing trend over time. Importantly, the cell density obtained from the microfluidic sensor closely matches with the conventional benchmark glass slide method, with an error of less than 3.3%. The microfluidic monitoring platform is found to be low-cost, accurate, fast, and efficient compared to existing systems, and moreover, it is easily amenable to automation.

Sickle cell disease (SCD) is characterized by the polymerization of hemoglobin S (HbS) upon deoxygenation, leading to the formation of sickled red blood cells (RBCs) with reduced deformability. Under hypoxic conditions, the impaired RBC behavior significantly contributes to vaso-occlusive events, hemolysis, and end-organ damage. Consequently, RBC deformability serves as a pivotal hemorheological biomarker for evaluating disease severity and therapeutic response. The OcclusionChip, a microfluidic assay, measures RBCs deformability through microcapillary occlusion. However, its current hypoxic assay relies on a complex nitrogen gas setup, rendering it bulky, expensive, and unsuitable for point-of-care diagnostic use. Here, we optimized a chemically induced hypoxia assay using sodium metabisulfite (SMB) within the OcclusionChip platform and validated the hypoxia occlusion index (HOI) as a robust measure of RBC deformability in SCD. Optimal hypoxia conditions were established, replicating nitrogen-induced hypoxia without affecting RBC membrane integrity, reactive oxygen species (ROS) levels, or phosphatidylserine (PS) exposure. Under these conditions, RBCs from individuals with heterozygous (HbAS), HbSC, and HbSS genotypes showed significantly higher HOI compared to healthy controls (HbAA), correlating strongly with clinical biomarkers in SCD. Additionally, the HOI assay effectively assessed the efficacy of therapeutic agents, including hemoglobin-oxygen affinity modifiers (GBT021601, GBT440) and protein kinase R (PKR) activators (PKR-3, FT4202), which significantly reduced OI in SCD RBCs. Notably, combination therapies showed enhanced effectiveness, highlighting the assay's potential for optimizing treatment regimens. This study establishes the chemically induced hypoxia OcclusionChip assay as a reliable and clinically useful tool for evaluating RBC deformability in SCD, with significant potential to improve personalized treatment strategies and thus patient outcomes.