pH-driven dual sensing: A single fluorescent probe for real-time food spoilage tracking and microfluidic sorting of acid-producing bacteria.

Natural killer cell encapsulation in core-shell proteinic adhesive microcapsules for persistent and localized release of cytotoxic factors.

Understanding microbial growth and metabolism under controlled environments is critical for both fundamental research and bioprocess development. In this study, we present a cost-effective droplet-based microfluidic device enabling high-throughput screening of GFP expression in Escherichia coli (E. coli) under varying glucose concentrations. Fluorinated Ethylene Propylene (FEP) tubing was selected for its low cost and compatibility with stable water-in-oil emulsification, facilitating robust droplet generation. The system achieved a 33333-fold reduction in media consumption compared to traditional Erlenmeyer flask cultures. Growth kinetics and GFP expression were assessed in both Erlenmeyer flasks and microdroplets, showing high qualitative correlation between platforms. Low glucose levels (5-10 g/L) supported rapid initial growth and early GFP production, followed by a fluorescence decline due to nutrient depletion. In contrast, higher glucose concentrations (25-50 g/L) prolonged the exponential phase and enhanced GFP production per unit biomass, though growth was slowed by overflow metabolism. In microdroplets, delayed GFP expression at 25 and 50 g/L were observed, and parallel bioreactor experiments confirmed that this delay is caused by oxygen limitation at high glucose concentrations. Importantly, the microfluidic device enables controlled variation of oxygen availability simply by adjusting droplet size or generation frequency, providing a powerful means to probe oxygen-sensitive metabolic behaviors. These results validate the microfluidic platform's ability to mimic Erlenmeyer flask-scale dynamics, while uniquely allowing precise modulation of oxygen transfer conditions at the microscale. The system offers a reliable, miniaturized alternative for optimizing microbial bioprocesses with drastically reduced reagent use and increased experimental throughput.

Droplet microfluidics enables high-throughput single-cell analysis but encounters inherent challenges in antibiotic susceptibility testing (AST), primarily stemming from inadequate control over single-bacterium encapsulation and droplet manipulation. Herein, we report an integrated droplet microfluidic platform that achieves precise bacterial encapsulation via electrocontrolled injection. Flow parameters were systematically optimized to generate highly monodisperse droplets with excellent morphological stability, while an integrated pressure-volume calibration method coupled with a minimum electroinjection threshold ensured accurate regulation of injection volume and reliable intradroplet reagent delivery. By coencapsulating Escherichia coli harboring the pGLO plasmid and resazurin, dual-fluorescence monitoring was realized via GFP fluorescence, indicating bacterial viability and proliferation, as well as resorufin fluorescence, which is produced from the reduction of resazurin, reflecting metabolic activity. This platform enables controllable encapsulation down to the single-bacterium level and completes AST within 3 h via fluorescence readout. Integrating miniaturization, high throughput, and low reagent consumption, this multichannel droplet microfluidic system provides a robust and efficient tool for rapid antimicrobial resistance detection, with considerable potential for clinical translation.

Directed evolution of enzymes at the crossroads of tradition and innovation.

Precise and noncontact temperature control within microfluidic droplets is critical for microreactor applications. However, conventional acoustothermal platforms heavily rely on expensive, patterned piezoelectric substrates. To address this limitation, our study introduces a novel and cost-effective methodology for droplet thermal regulation utilizing laterally excited Lamb waves (driven at 1 MHz) on universal, nonpiezoelectric glass substrates. We demonstrate that droplet temperature elevation fundamentally arises from the conversion of input acoustic power into localized heat, primarily via the viscous dissipation of radiated longitudinal waves dynamically coupled with acoustic streaming-induced convection. Systematic quantitative analysis of glycerol-water droplets (20-50 μL) revealed that a rapid, stable thermal plateau of 31.4 °C is achieved within 100 s at 1.10 W input power. Furthermore, increasing fluid viscosity (from 10% to 100% glycerol) significantly enhances viscous dissipation, leading to a continuous increase in temperature (at 1.0 W) and improving spatial thermal homogeneity. Coupled numerical simulations validated the experimental thermal fields, revealing that an initial peak acoustic streaming velocity of 0.12 m/s effectively redistributes thermal energy, which subsequently suppresses the streaming intensity to 0.08 m/s due to acoustic-to-thermal energy conversion. By decoupling acoustic generation from the microfluidic substrate, this lateral-excitation methodology provides a highly tunable, low-cost platform for precise spatiotemporal thermal management, significantly advancing the scalable deployment of droplets as isolated bioreactors and bioparticle carriers.

Droplet microfluidics is indispensable for precision fabrication of microscale functional materials, with broad applications in biomedical engineering, flexible electronics, and materials science. However, conventional microchannel-based systems suffer from high cost, complex fabrication, and nozzle-orifice-dependent size limitation, as well as the lack of robustness and flexibility to process complex fluids. To address these critical challenges, this research developed a modular acoustic streaming vortex (MASV) platform assembled from commercial off-the-shelf components. By synergistically modulating piezoelectric driving (PD) and amplitude modulation (AM) frequencies, the platform achieves ultra-broad size tunability (13-750 μm) of monodisperse microdroplets with a fixed nozzle (inner diameter of outlet ∼0.16 mm). Furthermore, studies have demonstrated that the MASV platform can efficiently process complex fluids (liquid metals and glycerol solutions) while exhibiting millisecond-scale real-time tunability and stable continuous operation for up to 8 hours. Notably, the study found that ejecting regime transition from dripping to jetting can be realized by reducing two-phase interfacial tension, thus supporting programmable fabrication of functional microfibers. This low-cost, versatile platform integrates modularity, nozzle-independent size control, complex fluid compatibility, and long-term stability, holding significant potential in flexible production of functional micromaterials.

Increasing the complexity of self-assembled supramolecular nanostructures necessitates considered design and synthesis of the precursor molecular components as well as precise control over the reaction microenvironment. Conventional batch techniques are susceptible to kinetic trapping of intermediate species, resulting in reduced target yields for progressively complex assemblies. In contrast, the microenvironmental control offered by microfluidics enables local thermodynamic minima to be circumvented, enabling self-assembly processes to proceed to completion. Here, the dynamic covalent assembly of imine-based molecular ladders and branched nanostructures is achieved through droplet microfluidics. Droplets of an organic solution composed of a solvent (chloroform), a multi-role Lewis acidic reagent (scandium triflate), and complementary oligo(peptoid) precursor strands bearing amine and aldehyde pendant groups are generated in an aqueous solution. Downstream hydrodynamic microtraps immobilized the droplets in the flowing aqueous phase, hence the media around the droplet is constantly replaced, thereby avoiding coevolution of the two phases and enabling continuous extraction of reagent from the entrapped droplet into the surrounding microflow via interfacial diffusion. As the reagent concentration is altered, so are the equilibrium conditions. At high reagent concentration, the amine and aldehyde condensation reaction product between the reactive pendant groups affixed to the oligomeric precursor species is supported. As the concentration drops, Sc3+-catalyzed imine bond rearrangement occurs, providing an error correction mechanism, enabling the generated ladder constructs to come into registry which were confirmed by off-chip analysis of the collected droplets using MALDI-MS. Furthermore, the assembly of three complementary oligo(peptoid) precursor strands into three-way, imine-based nanostructures was achieved.

Environmental microbial bioprospecting enabled by a Raman fingerprinting and functional sorting on a microfluidic static droplet array.

Since their discovery, bacteriophages-viruses that infect bacteria-have been invaluable to molecular biology and biotechnology. Renewed interest in phage-based antimicrobials, driven by the global antibiotic resistance crisis, highlights the need for improved quantitative tools. While conventional double-layer plaque assays (DLA) have provided foundational insights, they are limited by their inability to monitor infection dynamics over time and the inflexibility in experimental setups. Here, we present a high-throughput droplet microfluidics platform to quantify individual phage infection events. By co-encapsulating individual phages and bacteria in microfluidic droplets, we precisely control key experimental parameters such as exposure time and the ratio of phages to bacteria. This approach enables direct quantification of lysis events and measurement of lysis kinetics without interference from further progeny-driven infection processes inherent to bulk cultures. Applicable to diverse phage-host systems, this method offers a dynamic and accurate framework for studying phage biology and supports the development of phage-based antimicrobial strategies.

Microfluidic platforms have emerged as powerful tools for efficient intracellular delivery of exogenous cargo. While droplet microfluidics coupled with cell mechanoporation shows significant potential, its broader adoption is often hindered by channel clogging and limited scalability. To address these challenges, we developed a parallelized droplet-based cell mechanoporation platform with integrated bypass channels. This architecture stabilizes internal pressure and mitigates clogging-induced failure, ensuring robust and continuous operation. The platform achieves delivery efficiencies exceeding 98% and cell viabilities above 80% at a throughput of 2 × 107 cells/h, enables highly efficient mRNA transfection (~98%), and supports CRISPR/Cas9-mediated CD3 knock-out. Collectively, these results establish parallelized droplet cell mechanoporation as a scalable and reliable strategy for intracellular delivery with applicability in cell engineering and therapeutic development.

The translation of 3D multicellular systems into clinical applications has been constrained by the need to balance physiological relevance and scalability. Current biofabrication methods primarily depend on passive cell aggregation or capillary- and viscosity-limited segmentation, resulting in stochastic heterogeneity that limits high-throughput screening (HTS). Here, we present OsciSphere, a chip-free droplet microfluidic platform that utilizes Weber-number-driven inertial forces to enable deterministic bioassembly of uniform 3D multicellular systems. Through programmable oscillatory acceleration, OsciSphere achieves precise, high-frequency droplet generation in standard well plates, eliminating the requirement for complex microfabrication. We demonstrate the versatility of this platform by generating miniaturized multicellular tumor spheroids (µMCTs) for drug screening, tissue-derived organoids (µTDOs) for pharmacological studies, and patient-derived organoids (µPDOs) that support tumor-immune co-cultures. In comparison to conventional Matrigel domes, OsciSphere-assembled 3D multicellular systems display improved uniformity, viability, and chemosensitivity. The platform's scalability enabled the screening of 49 commensal gut bacterial secretomes, leading to the identification of Eubacterium species that modulate cancer apoptotic pathways. Furthermore, µPDOs generated with OsciSphere support efficient infiltration of autologous PBMCs, enabling quantitative assessment of PD-1 blockade. This platform provides a robust, accessible approach to bridging the gap between complex tissue modeling and large-scale functional screening in precision oncology.

Single-cell analysis technologies are pivotal in unraveling complex bihological mechanisms, yet existing platforms are often limited to sequencing-based end-point measurements, which fail to capture live cell dynamics. Here, we present a microfluidic-microelectronic device, the Microfluidic Dielectrophoretic Arresting System (MiDAS) that employs dielectrophoresis (DEP) for high-throughput single-cell and droplet trapping in a compact array. We tested multiple trap geometries, including a 20µm-diameter DEP trap for polymer microbeads, fungal and mammalian cells, and a 40µm-diameter trap for water-in-oil droplets. The platform demonstrates broad sample compatibility, reliably immobilizing cells and beads of varying sizes. By integrating optical imaging and Raman spectroscopy, we enable interrogation of individual cells with temporal resolution. We describe different modes of MiDAS operation to trap and manipulate single-cells or reverse emulsion droplets on demand, with applications in droplet microfluidics. Our MiDAS platform's simple fabrication, robust performance, and broad compatibility with diverse sample types position it as a versatile tool with transformative potential for single-cell analysis, offering researchers an innovative approach to interrogate cellular dynamics with precision and throughput.

Background: Osteosarcoma (OS) is an aggressive bone tumor. The lack of physiologically relevant three-dimensional models that recapitulate the native tumor microenvironment hampers drug development and mechanistic studies. The study aimed to develop bone-mimetic microspheres for the construction of an OS model. Materials and Methods: We employed droplet microfluidics to fabricate bone-mimetic microspheres (named MSHA) from a composite of gelatin methacryloyl, polyethylene glycol diacrylate, and nano-hydroxyapatite (nHA). MNNG/HOS cells were cultured on MSHA microspheres and subsequently evaluated for their bioactivity and capabilities of stemness, migration, and invasion. Results: The microfluidic platform enabled efficient and scalable production of highly uniform MSHA microspheres with controlled sizes. MNNG/HOS cells cultured on MSHA maintained high viability and spontaneously formed compact tumor spheroids after 7 days. Compared with two-dimensional cultures, cells cultured on these microsphere-based platforms exhibited enhanced migration and invasion capacities, along with increased expression of relevant biomarkers. RNA sequencing further revealed the activation of cancer-related pathways. Notably, the incorporation of nHA into microspheres amplified these malignant phenotypes, potentially through the activation of ECM-receptor interaction and calcium signaling pathways. Conclusions: The microfluidics-fabricated MSHA microspheres, as biomimetic three-dimensional culture scaffolds, offer a promising platform for applications in mechanistic studies of osteosarcoma progression and drug screening.

The integration of DNAzyme and droplet-based microfluidics is advancing pathogen detection toward higher sensitivity and throughput. However, this interdisciplinary convergence still faces multiple technical challenges. Based on practical applications, this article systematically outlines current key bottlenecks. It also proposes corresponding strategies and envisions future pathways for intelligent and precision-oriented integration.

Droplet microfluidics has been widely used in biological, chemical, and medical research owing to its advantages of miniaturization, high throughput, and low reagent consumption. However, limited sensitivity and optical path length in on-chip absorbance detection remain major challenges for droplet-based microfluidic analysis. Traditional absorbance detection suffers from low sensitivity due to the extremely short optical path in microfluidic channels, while existing optical path extension methods have drawbacks such as complex fabrication, easy droplet rupture, or strict incident angle requirements. To address these issues, this study developed a droplet microfluidic absorbance detection platform integrating optical fibers, on-chip micromirrors, external fluidic actuation, and an absorbance detection module. Microchannel sidewalls filled with low-melting-point metal act as mirrors; the multi-reflection optical path, combined with optical fibers and micromirrors, compensates for insufficient light manipulation and effectively extends the absorption path length, improving sensitivity and accuracy. Using this method, the detection limit for methylene blue solution was 20 μM, and the sensitivity for Escherichia coli (E. coli) suspension was doubled compared with traditional Nanodrop OD600 measurement. This device features low fabrication difficulty and cost and stable detection, providing a proof-of-concept strategy for enhanced absorbance detection in droplet microfluidic systems.

Simultaneous high-frequency generation and low-voltage electrostatic manipulation in droplet microfluidics

High-accuracy object classification in droplet microfluidics using novel Ghost-EMA networks

Droplet-based synthesis of microencapsulated phase change hydrogels via electro-coalescence.

Droplet manipulation constitutes a fundamental operation in numerous bio-microfluidic applications, including but not limited to medical diagnostics and targeted drug delivery. Among the various technologies developed for this purpose, magnetic digital microfluidics (MDMF) has emerged as a compelling approach due to its inherent advantages of contamination-free actuation, low cost, and configurational flexibility. Nevertheless, conventional MDMF remains constrained by its reliance on bulky instrumentation and substantial power consumption for generating controllable magnetic fields, which limit its in-field applications. To address these limitations, this work presents a programmable and portable electromagnetic microfluidic droplet manipulation platform that synergistically integrates static and dynamic magnetic fields to enable non-contact, high-precision droplet control under ultra-low power conditions. The proposed system comprises an electromagnetic actuation module, a permanent magnet, and a glass substrate coated with Teflon film. The entire system is secured by a PMMA support structure, within which a glass substrate is mounted and spatially separated from the permanent magnet. The PMMA support is fabricated using a milling process, offering a simple manufacturing procedure and high structural reusability and reproducibility. The control logic is implemented on a field-programmable gate array (FPGA) development board, facilitating fully autonomous operation powered by a standard battery. The platform operates at a low voltage of 3.5 V and a driving current of 180 mA, corresponding to a total power consumption of merely 0.63 W, while achieving robust manipulation of droplets in the volume range of 0.5 to 5 μL. A maximum average droplet velocity of up to 0.6 cm/s was attained under optimal conditions. The proposed platform offers a scalable and energy-efficient solution for portable droplet-based assays and holds significant promise for integration into point-of-care diagnostic tools and field-ready biochemical analysis systems. The platform demonstrates excellent operational stability and reproducibility, as validated by repeated actuation experiments with a positioning deviation of approximately 0.1 mm under optimized conditions. The fabrication process also exhibits high reliability with consistent performance across multiple experimental runs.