Lab on a Chip最新文献

We present a microfluidic solution for improved tumor cell analysis based on selection-free isolation of nucleated cells from whole blood. It consists of a high-density silicon microcavity array combined with the novel fluidic strategy of microfluidic decanting. This enables multistep on-chip staining protocols comprising sample loading–blocking–extracellular staining–fixation–permeabilization and intracellular staining to quantify tumor cells. The performance of the workflow was investigated and proven by spiking colon cancer cell lines into whole blood for the detection of the epithelial tumor markers EpCAM and cytokeratin. Total cell recovery rates of ≥95% were achieved for different sample species. The method allows for rapid reagent exchange within 10 s each almost without cell loss compared to approximately 50% cell loss in reference centrifugal processing. The isolation of nucleated cells resulted in a high intra-assay precision with a CV of 2% and a single cell per well distribution of 90%, which is consistent with the theoretical estimate using Poisson statistics. The linearity of the method was demonstrated over three orders of magnitude with r² = 0.9998. These results demonstrate a highly efficient approach for the quantification of tumor cells from whole blood that could be integrated into automated point-of-care devices in the future.

Organ-on-chip (OOC) devices are an emerging New Approach Method in both pharmacology and toxicology. Such devices use heterotypic combinations of human cells in a micro-fabricated device to mimic in vivo conditions and better predict organ-specific toxicological responses in humans. One drawback of these devices is that they are often made from polydimethylsiloxane (PDMS), a polymer known to interact with hydrophobic chemicals. Due to this interaction, the actual dose experienced by cells inside OOC devices can differ strongly from the nominal dose. To account for these effects, we have developed a comprehensive model to characterize chemical–PDMS interactions, including partitioning into and diffusion through PDMS. We use these methods to characterize PDMS interactions for 24 chemicals, ranging from fluorescent dyes to persistent organic pollutants to organophosphate pesticides. We further show that these methods return physical interaction parameters that can be used to accurately predict time-dependent doses under continuous-flow conditions, as would be present in an OOC device. These results demonstrate the validity of the methods and model across geometries and flow rates.

The rapid growth in data generation presents a significant challenge for conventional storage technologies. DNA storage has emerged as a promising solution, offering substantially greater storage density and durability. However, the current DNA data writing process is costly and labor-intensive, hindering the commercialization of DNA data storage. In this study, we present a digital microfluidics (DMF) platform integrated with E47 DNAzyme ligation chemistry to develop a programmable, cost-effective, and automated DNA data writing process. Our method utilizes pre-synthesized single-stranded DNA as building blocks, which can be assembled into diverse DNA sequences that encode desired data. By employing DNAzymes as biocatalysts, we enable an enzyme-free ligation process at room temperature, significantly reducing costs compared to traditional enzyme-based methods. Our proof-of-concept demonstrates an automated DNA writing process with the reduced reagent input, providing an alternative solution to the high costs associated with current DNA data storage methods. The high specificity of ligation using DNAzymes obviates the need for storing each unique DNA block in its own reservoir, which greatly reduces the total number of reservoirs required to store the starting material. This simplifies the overall layout, and the associated plumbing of the DMF platform. To adapt the conventional column-purification required ligation on the DMF platform, we introduce a DNAzyme-cleavage-assisted bead purification assay. This method employs 17E DNAzymes to cleave and release biotinylated DNA from streptavidin beads, followed by a one-pot ligation with E47 DNAzymes to assemble the desired DNA strands. Our study represents a significant advancement in DNA data storage technology, offering a cost-effective and automated solution that enhances scalability and practicality for commercial DNA data storage applications.

Cancer is a serious disease in human beings, and its high lethality is mainly due to the invasion and metastasis of cancer cells. Clinically, the accumulation and high orientation of collagen fibrils were observed in cancerous tissue, which occurred not only at the location of invasion but also at 10–20 cm from the tumor. Studies indicated that the invasion of cancer cells could be guided by the oriented collagen fibrils, even in a dense matrix characterized by difficulty degradation. So, the orientation of collagen fibrils is closely related to invasion by cancer cells. However, the formation of the orientation of collagen fibrils remains insufficiently studied. A microfluidic chip-based collagen fibril tissue model was established to demonstrate its underlying mechanism. In this article, the dynamic mechanism of collagen fibril reconstruction from free orientation to high orientation was investigated at the mesoscopic dynamic level. In the experiment, the mechanical forces from interstitial flow and cell deformation were confirmed as significant factors for collagen fibril remodeling. Additionally, enzymes were confirmed as an another inducer to reconstruct the morphology of collagen fibrils, the mechanism of which was chemical degradation and recombination. Interstitial flow combined with an enzyme is an excellent combination for remodeling the distal collagen fibrils of a tumor, and this phenomenon was caught in a microfluidic platform with a micro-dose. This study to some extent answers the question of the kinetic mechanism of collagen fibril remodeling, and is expected to provide support for further proposed strategies to inhibit the orientation reconstruction of collagen fibrils and cancer treatment and prognosis.

The cell membrane is a crucial biological interface to consider in biomedical research, as a significant proportion of drugs interacts with this barrier. While understanding membrane–drug interactions is important, existing in vitro platforms for drug screening predominantly focus on interactions with whole cells or tissues. This preference is partly due to the instability of membrane-based systems and the technical challenges associated with buffer replacement around lipid membranes formed on microfluidic chips. Here, we introduce a novel microfluidic design capable of forming stable freestanding lipid bilayers with efficient replacement of the media in their local environment for molecular delivery to the membrane. With the use of bubble traps and resistance channels, we achieved sufficient hydrodynamic control to maintain membrane stability during the membrane formation and the molecular delivery phases. As a proof of concept, we successfully formed 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayers on the chip and delivered the antibiotic azithromycin at low (5 μM) and high (250 μM) doses. Using optical tweezers, we characterized how azithromycin influenced the membrane elastic properties, including tension and bending rigidity. This microfluidic device is a versatile tool that can deliver various buffers, molecules or nano-/microparticles to freestanding membranes, and study the resulting impact on the membranes' properties.

The gut-skin axis has emerged as a crucial mediator of skin diseases, with mounting evidence highlighting the influence of gut microbiota on skin health. However, investigating these mechanisms has been hindered by the lack of experimental systems that enable direct study of gut microbiota-skin interactions. Here, we present the gut microbe-skin chip (GMS chip), a novel microfluidic platform designed to model microbiome-gut-skin axis interactions. The GMS chip allows the coculture of intestinal epithelial cells (Caco-2), human epidermal keratinocytes (HEKa), and gut microbes with fluidic connection mimicking the blood flow. We validated that the gut compartment, with a self-sustaining oxygen gradient, enabled coculturing gut bacteria such as Escherichia coli (E. coli) and Lactobacillus rhamnosus GG (LGG), and the skin cells properly differentiated in the chip in the presence of fluid flow. Disruption of intestinal epithelial integrity by dextran sodium sulfate (DSS) combined with lipopolysaccharides (LPS) selectively decreased skin cell viability while sparing gut cells. Notably, pretreatment with LGG showed a protective effect against the skin cell damage by enhancing the intestinal barrier function. The GMS chip effectively recapitulates the influence of gut microbiota on skin health, representing a pivotal step forward in studying gut-skin axis mechanisms and the role of the gut microbiome in skin diseases.

We introduce a robust multilayer dielectric stack for digital microfluidic chips to withstand the humid conditions of cell culture incubators for at least 60 days. Consisting of a combination of 1 μm polyvinylidene difluoride and 5 μm SU-8 layers, the stack demonstrated high breakdown voltages up to 1600 V and minimal surface currents <30 nA at 100 V. Long-term stability and precision in liquid handling enabled us to study macrophage phenotype modulation, pro-inflammatory response induction in macrophage population with single cell cytokine quantification and testing of a potentially anti-inflammatory drug candidate TCB-2 and its influence on macrophage phenotype, morphology, and cytokine release. The multilayer dielectric stack offers a durable solution for long-term biological assays on digital microfluidic platforms.

Experiments with gradients of soluble bioactive species have significantly advanced with microfluidic developments that enable cell observation and stringent control of environmental conditions. While some methodologies rely on flow to establish gradients, others opt for flow-free conditions, which is particularly beneficial for studying non-adherent and/or shear-sensitive cells. In flow-free devices, bioactive species diffuse either through resistive microchannels in microchannel-based devices, through a porous membrane in membrane-based devices, or through a hydrogel in gel-based devices. However, despite significant advancements over traditional methods such as Boyden chambers, these technologies have not been widely disseminated in biological laboratories, arguably due to entrenched practices and the intricate skills required for conducting microfluidic assays. Here, we developed microfluidic platforms integrating barriers with Quake-type pneumatic microvalves in place of microgrooves, membranes, or gels. One set of microvalves is used to maintain flow-free conditions and another set to regulate diffusion between a central channel housing the specimen of interest and sink/source reservoirs. This configuration enables stringent control over residual flows, precise spatial–temporal regulation of gradient formation, and exceptional gradient stability, maintained over extended periods via automated refilling of source and sink reservoirs. The gradient establishment was validated using fluorescent tracers with molar masses of 0.3–40 kDa, while cellular assays demonstrated the chemotactic response of primary human neutrophils swimming toward FMLP. The fabrication of microfluidic devices remains standardly demanding, but experimentation can be fully automated thanks to microvalves, making it accessible to non-expert users. This work presents a robust microfluidic approach for generating tunable gradients with stringent control over flow-free, time-zero, and long-term conditions and its automation and accessibility may promote adoption in academic and biomedical settings especially for non-adherent specimens.

Diffusion-based microfluidic gradient generators (DMGGs) are essential for various in vitro studies due to their ability to provide a convection-free concentration gradient. However, these systems, often referred to as membrane-based DMGGs, exhibit delayed gradient formation due to the incorporated flow-resistant membrane. This limitation substantially hinders their application in dynamic and time-sensitive studies. Here, we accelerate the gradient response in DMGGs by removing the membrane and implementing new geometrical configurations to compensate for the membrane's role in suppressing parasitic flows. We introduce these novel configurations into two microfluidic designs: the H-junction and the Y-junction. In the H-junction design, parasitic flow is redirected through a bypass channel following the gradient region. The Y-junction design features a shared discharge channel that allows converging discharge flow streams, preventing the buildup of parasitic pressure downstream of the gradient region. Using hydraulic circuit analysis and fluid dynamics simulations, we demonstrate the effectiveness of the H-junction and Y-junction designs in suppressing parasitic pressure flows. These computational results, supported by experimental data from particle image velocimetry, confirm the capability of our designs to generate a highly stable, accurate, and convection-free gradient with rapid formation. These advantages make the H-junction and Y-junction designs ideal experimental platforms for a wide range of in vitro studies, including drug testing, cell chemotaxis, and stem cell differentiation.

There is a growing demand for reliable, efficient, and easily integrated micropumps for microfluidics. Despite the demonstrated potential of acoustic wave-driven devices for on-chip pumping, current prototypes lack the practicality and integratability for deployment in microfluidic systems. This study presents an acoustic micropump based on the resonance of arrays of on-substrate sharp-edge micropillars prepared in a fluid-filled channel and driven by a piston ultrasonic transducer. At an operating frequency of 80.5 kHz and a driving voltage of 54 V_p-p, a flow rate of 16.2 μL min^-1 is achieved in a downstream straight channel with dimensions 12(L) × 0.6(W) × 0.2(H) mm³. The corresponding pumping pressure exceeds 1.3 kPa, more than an order of magnitude higher than its predecessors. In experimental demonstrations, two micropumps are employed as feeding units for an acoustofluidic particle separation device based on tilted-angle standing surface acoustic waves (TaSSAWs). The current micropump exhibits advantages of high pumping pressure, fast response time, and high reliability, making it a promising pumping unit for lab-on-a-chip systems.