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Droplet-based microfluidics

Droplet-based microfluidics is a subfield of microfluidics that involves the generation, manipulation, and analysis of discrete sub-microliter droplets suspended in an immiscible carrier fluid, such as oil, within microscale channels to enable precise control over chemical and biological reactions at high throughput.^[1] This technology leverages laminar flow and surface tension to produce uniform droplets ranging from picoliters to nanoliters in volume, often at rates exceeding thousands per second, allowing for the isolation and parallel processing of individual reactions in a compact, scalable format.^[2] The development of droplet-based microfluidics traces its origins to the late 1990s and early 2000s, building on foundational advances in microfluidic device fabrication and emulsion science.^[1] Key milestones include the 1997 demonstration of microchannel emulsification for monodisperse droplets by Kawakatsu et al., followed by the 2000 introduction of flow-focusing geometries for high-throughput generation by Umbanhowar et al., and early applications in 2003 for studying rapid kinetics in nanoliter volumes by Song et al.^[1] By the mid-2000s, innovations such as double-emulsion formation by Utada et al. in 2005 expanded capabilities for encapsulating aqueous cores in oil shells, paving the way for complex payload delivery.^[1] These advancements, often using soft lithography with materials like polydimethylsiloxane (PDMS), transformed the field from basic droplet production to integrated systems for real-time monitoring and sorting.^[2] At its core, the technology operates under low Reynolds number conditions, where viscous forces dominate over inertial ones, facilitating predictable droplet formation via methods like T-junctions, flow-focusing, or co-flow geometries.^[2] Droplet size and uniformity are governed by parameters such as the capillary number (Ca), interfacial tension, and flow rate ratios, with surfactants added to the carrier phase to stabilize emulsions against coalescence.^[1] Manipulation techniques include passive processes like fission and fusion driven by channel design, as well as active methods employing electric fields (electrowetting-on-dielectric, EWOD), acoustics, or optics for sorting and mixing.^[2] Detection often integrates fluorescence microscopy or mass spectrometry to analyze contents in these tiny volumes, enabling Poisson-distributed encapsulation for single-particle or single-cell studies.^[1] Droplet-based microfluidics has revolutionized applications across biology, chemistry, and materials science by minimizing reagent use and accelerating experimentation.^[1] In biology, it powers single-cell analysis, such as droplet-enabled single-cell RNA sequencing (Drop-seq) for transcriptomics and directed evolution of enzymes via compartmentalized screening.^[1] Chemically, it facilitates nanoparticle synthesis, polymer microparticle production, and high-throughput drug screening with femtoliter reactions.^[2] Biomedical uses extend to diagnostics, where droplets enable rapid pathogen detection (e.g., fungi in 1-2 hours) and point-of-care testing, as well as drug delivery systems like ultrasound-responsive microbubbles.^[2] Ongoing challenges include scaling production beyond laboratory settings and integrating with downstream analytics, but its versatility continues to drive innovations in personalized medicine and synthetic biology.^[1]

Fundamentals

Principles and definitions

Droplet-based microfluidics is a subfield of microfluidics that focuses on the generation, manipulation, and analysis of discrete droplets formed from immiscible fluids, typically serving as isolated reaction compartments for high-throughput processing of picoliter to nanoliter volumes in chemical and biological assays.^[3] This approach leverages microscale channels to create emulsions where droplets act as individual reactors, minimizing cross-contamination and enabling parallel operations at rates exceeding thousands per second. The system commonly employs two immiscible phases: an aqueous dispersed phase forming the droplets and a non-aqueous continuous carrier phase, such as fluorinated or mineral oil, or vice versa for oil-in-water emulsions.^[3] Surfactants are added to the carrier fluid to stabilize the interface and prevent coalescence, ensuring the droplets remain intact during downstream handling. A key feature is the production of highly monodisperse droplets, characterized by a coefficient of variation (CV) in size typically below 5%, which is essential for reproducible reaction kinetics and quantitative analysis.^[3] At the microscale, fluid dynamics are dominated by viscous and interfacial forces rather than inertia, as indicated by a low Reynolds number (Re ≪ 1), where Re = ρvd/μ (with ρ as fluid density, v as velocity, d as channel diameter, and μ as viscosity), resulting in laminar flow regimes that facilitate precise control over droplet formation and transport.^[4] The stability and shape of these droplets are governed by the Laplace pressure across the fluid interface, given by

\Delta P = \frac{2\gamma}{r}

where γ is the interfacial tension and r is the droplet radius; this pressure difference drives phenomena such as droplet breakup and merging. Droplet volume is controlled by adjusting the flow rate of the dispersed phase relative to the generation frequency, approximated as V ∼ Q_aqueous / f, where f is the droplet formation frequency, allowing tunability from femtoliters to microliters.^[4]

Historical development

The foundations of droplet-based microfluidics trace back to the 1980s, when early concepts in inkjet printing and emulsion science began to explore controlled droplet generation at microscales. Inkjet technologies, which produce droplets on the order of 10-100 micrometers through piezoelectric or thermal actuation, provided initial insights into precise liquid dispensing and served as precursors to microfluidic systems.^[5] Emulsion science, with its principles of stabilizing dispersed phases, further informed these developments, laying groundwork for later integration into microchannel devices.^[6] The field gained momentum in the late 1990s and early 2000s with seminal demonstrations of on-chip droplet generation. Key early milestones include the 1997 demonstration of microchannel emulsification for monodisperse droplets by Kawakatsu et al. and the 2000 introduction of flow-focusing geometries for high-throughput generation by Umbanhowar et al. In 2003, Anna et al. adapted flow-focusing geometries to microfluidic devices, enabling the production of highly monodisperse droplets by hydrodynamically squeezing the dispersed phase, which marked a key advance in scalable emulsion formation.^[3] Concurrently, Thorsen et al. in 2001 demonstrated T-junction configurations for generating uniform aqueous droplets in oil via shear forces, establishing a simple yet robust method for droplet production. These works, building on soft lithography techniques from Whitesides (1998), catalyzed the shift from single-phase to multiphase microfluidics. By 2006, Song et al.'s comprehensive review synthesized these advances, highlighting droplet microfluidics as a platform for high-throughput chemical reactions and establishing it as a distinct subfield.^[7] The 2010s saw rapid growth through integration with biological assays, particularly in nucleic acid quantification. Bio-Rad commercialized the first droplet digital PCR (ddPCR) system, the QX100, in 2011, leveraging thousands of partitioned droplets for absolute quantification without standards, which revolutionized gene expression analysis and pathogen detection.^[8] This era also expanded applications to single-cell encapsulation and screening, with over 64 reviews documenting the field's maturation by 2020.^[9] By the 2020s, innovations emphasized precision and automation, including AI-optimized device designs for enhanced droplet stability and throughput. In 2024, the NOVAsort platform introduced opto-volume-based sorting for error-free discrimination of droplets by size and fluorescence, enabling reliable single-bacterium analysis in high-density emulsions.^[10] These developments, up to 2025, reflect a transition toward intelligent, scalable systems for precision medicine and directed evolution.^[11]

Droplet Generation

T-junction formation

The T-junction method for droplet generation in microfluidics employs a geometry where the aqueous (dispersed) phase flows into a perpendicular channel intersecting the main channel carrying the oil (continuous) phase, forming a T-shaped junction. This configuration facilitates the initial contact and deformation of the aqueous fluid stream by the cross-flowing oil, leading to emulsification. The approach was first demonstrated by Thorsen et al. in 2001 using a microfluidic device to produce lipid vesicles, highlighting its potential for controlled two-phase flow interactions.^[12] Droplet breakup occurs primarily through shear forces exerted by the continuous phase on the growing dispersed phase interface, with the droplet detaching when it sufficiently obstructs the channel cross-section. In the low-capillary-number squeezing regime (Ca < 0.01), the obstruction induces a pressure gradient that aids pinch-off, while at higher Ca, dripping dominates via direct viscous shearing. In the squeezing regime, the droplet size scales as d / w \approx 1 + \alpha (Q_{\mathrm{aq}} / Q_{\mathrm{oil}}), where w is the channel width and α ≈ 1 is a geometric factor. In the dripping regime at higher Ca, the size decreases with increasing Ca, often following d / w \sim \mathrm{Ca}^{-\beta} with β ≈ 0.3–0.4. The capillary number is defined as \mathrm{Ca} = \mu U / \sigma, with \mu the continuous phase viscosity, U the average velocity, and \sigma the interfacial tension.^[13]^[14]^[15] Critical parameters include the flow rate ratio Q_{\mathrm{aq}}/Q_{\mathrm{oil}}, which must be kept below 1 (often 0.1–0.5) to promote stable monodisperse droplets by ensuring the dispersed phase does not overwhelm the continuous flow. Channel dimensions typically range from 10–100 μm in width and depth, enabling low Reynolds numbers (Re << 1) for laminar conditions and precise control over interfacial dynamics. Variations in viscosity ratio or interfacial tension further tune the breakup, but excessive dispersed phase flow can destabilize the process.^[13]^[16] This method excels in simplicity of fabrication via photolithography and PDMS molding, requiring no complex alignments or high pressures. However, at elevated production rates or Ca > 0.1, polydispersity increases, with size coefficients of variation (CV) typically 5–10%, limiting uniformity compared to more confined geometries.^[17]^[18]

Flow-focusing formation

Flow-focusing formation is a passive droplet generation technique in which an aqueous dispersed phase is injected through a central microchannel and symmetrically focused by two opposing streams of immiscible oil continuous phase, directing it toward a narrow constriction or orifice typically ranging from 5 to 50 μm in width. This geometry creates extensional hydrodynamic forces that thin the aqueous stream into a focused thread or tip, enabling the production of droplets smaller than the channel dimensions. The design contrasts with asymmetric shear-based methods by promoting uniform focusing, which enhances reproducibility. The droplet formation mechanism in the dripping regime proceeds in two stages: initial filling of the orifice by the aqueous phase, driven by viscous drag from the high-velocity oil streams, followed by rapid neck thinning and pinching-off dominated by interfacial tension forces. This process occurs at low capillary numbers (Ca ≈ 10^{-3} to 10^{-1}), where shear and pressure gradients balance surface tension to detach uniform droplets without forming long jets. Droplet size primarily scales with the flow rate ratio (Q_{aq}/Q_{oil}), viscosity ratio (μ_{aq}/μ_{oil}), and capillary number (Ca = μ_{oil} U / σ, where U is the oil velocity and σ is interfacial tension), often yielding diameters from 5 to 100 μm with polydispersity indices below 3% when oil flow rates are sufficiently high to ensure stable focusing. Key parameters influencing performance include the orifice geometry, which sets the minimum achievable droplet size, and flow rates, where increasing the oil-to-aqueous ratio (typically 5:1 to 20:1) reduces droplet diameter while maintaining monodispersity (CV < 3%). High oil velocities (up to several m/s) suppress instabilities, enabling production rates in the kilohertz range (up to 10 kHz), far exceeding other passive methods for certain applications. This high throughput and uniformity have made flow-focusing the basis for commercial systems, such as those developed by RainDance Technologies for digital PCR, which generate millions of picoliter-scale droplets per sample.

Co-flowing formation

Co-flowing formation utilizes a coaxial geometry consisting of concentric capillaries, where the inner capillary delivers the aqueous dispersed phase and the outer capillary supplies the surrounding oil continuous phase, enabling parallel flow without abrupt junctions.^[19] This setup, often fabricated from glass or polymers, allows the dispersed phase to form a steady jet enveloped by the continuous phase, promoting controlled emulsification suitable for larger or fragile droplets.^[20] The droplet formation mechanism is governed by the Rayleigh–Plateau instability, in which surface tension drives periodic perturbations along the jet, leading to its breakup into uniform droplets; the characteristic wavelength of the instability is typically 4.5 to 9 times the jet diameter, depending on viscosity ratio and flow conditions, as determined by linear stability analysis such as Tomotika's dispersion relation. In the dripping regime, droplets detach directly at the capillary tip, while longer jets in the jetting regime break farther downstream via this instability.^[19] Key parameters include the capillary number (Ca = μ v / σ), where values below 0.01 typically ensure the dripping regime for monodisperse droplets with coefficients of variation under 5%, and the jet length, which is modulated by the ratio of dispersed-to-continuous phase flow rates to control droplet size.^[20] Viscosity ratios and interfacial tension further influence stability, with balanced fluids yielding optimal uniformity. This approach offers advantages such as gentle handling of sensitive cargos like cells, owing to minimized shear stresses in the parallel flow configuration, making it ideal for biological applications including single-cell encapsulation.^[21] However, it suffers from lower throughput—often limited to hundreds of droplets per second—compared to focused geometries, and demands precise capillary alignment to avoid misalignment-induced polydispersity. Recent advances as of 2025 include active droplet generation methods, such as magnetic flow-focusing for size- and shape-controlled droplets, expanding beyond passive geometries for specialized applications.^[22]

Droplet Manipulation

Surfactant stabilization

In droplet-based microfluidics, surfactants are essential for stabilizing the oil-water interface of droplets, thereby preventing coalescence during generation, transport, and storage. By adsorbing to the interface, surfactants lower the interfacial tension and create a steric or electrostatic barrier that maintains droplet integrity over extended periods, often hours to days, under typical flow conditions. This stabilization is critical for applications requiring monodisperse populations of droplets, as even minor coalescence can compromise assay reproducibility.^[23] Common surfactant types include fluorinated compounds, such as commercial formulations like Pico-Surf, which are particularly effective in perfluorinated carrier oils due to their amphiphilic structure with fluorocarbon tails that strongly anchor at the interface. Silicone-based surfactants, exemplified by Span 80 (sorbitan monooleate), are favored for use with mineral or silicone oils, offering good stability in non-fluorinated systems while exhibiting lower cost and simpler handling. For biocompatibility in biological assays, polyethylene glycol (PEG)-based surfactants, often as diblock or triblock copolymers, are employed to reduce non-specific adsorption of biomolecules like proteins, enabling cleaner encapsulation of cells or reagents.^[23] The physical mechanism of stabilization involves rapid adsorption of surfactant molecules to the droplet surface, governed by kinetics where the surface coverage \Gamma is proportional to the bulk concentration C via \Gamma = k \cdot C, with k as the adsorption rate constant; this process typically occurs on millisecond timescales to match the fast formation rates in microfluidic channels. Adsorption reduces the interfacial tension \gamma by 50-90%, from typical values of 20-50 mN/m in pure oil-water systems to below 5 mN/m, facilitating smaller droplet sizes and uniform breakup during generation.^[23] Droplet stability is often assessed through metrics like coalescence time \tau, which scales exponentially with the energy barrier \Delta E as \tau \sim \exp(\Delta E / kT), where k is Boltzmann's constant and T is temperature; \Delta E arises from DLVO theory, balancing attractive van der Waals forces against repulsive electrostatic or steric interactions provided by the surfactant layer. Well-stabilized droplets can achieve \tau > 10^6 s, far exceeding experimental durations, though thin surfactant films (1-10 nm) must resist drainage under shear.^[23] A key challenge is surfactant partitioning into the aqueous droplet interior during prolonged assays, which depletes the interfacial layer, elevates local tension, and contaminates reactions—particularly problematic in small-volume (pL-nL) droplets where concentrations shift rapidly. Surfactant dynamics in microfluidic environments, including mass transfer and depletion, have been reviewed as of 2023. To address partitioning issues, custom block copolymers, such as PEG-fluoropolymer hybrids, have been developed as of 2025 to enhance partitioning at the interface and minimize aqueous solubility, improving long-term stability without compromising biocompatibility.^[24]^[25]

Reagent addition and fusion

In droplet-based microfluidics, one primary technique for reagent addition involves co-flow configurations prior to droplet formation, where parallel streams of the dispersed phase containing reagents and the continuous phase converge at the interface within coaxial or multi-inlet channels.^[21] This approach enables initial mixing through diffusion across the interface, with the characteristic diffusion time scaling as t \sim L^2 / D, where L is the droplet size (typically 10–100 μm) and D is the molecular diffusivity (on the order of 10^{-9}–10^{-10} m²/s for small molecules in aqueous media).^[26] The resulting droplets encapsulate pre-mixed reagents homogeneously, minimizing post-formation mixing needs and supporting applications requiring rapid reaction initiation, such as enzymatic assays.^[27] Droplet fusion provides a versatile method for combining reagents from separate pre-formed droplets, often achieved via dielectrophoresis (DEP) or hydrodynamic pairing. In DEP-based fusion, non-uniform electric fields generated by interdigitated electrodes induce attractive forces between adjacent droplets, destabilizing the surfactant-stabilized interface to promote coalescence.^[28] Typical operation involves applying voltages of 10–100 V in short pulses (1–10 ms) at frequencies around 1–10 kHz, achieving merging efficiencies exceeding 95% even with 20% polydispersity in droplet size.^[28] Hydrodynamic pairing, an alternative bias-free approach, relies on channel geometries like T-junctions or constrictions to synchronize droplet arrival and spacing, followed by optional electric pulses (600–1200 V peak-to-peak) for interface rupture.^[29] Recent advances in 2023–2024 have demonstrated near-100% pairing efficiency for frequency mismatches up to 10%, enabling scalable multi-step reactions without electrical pre-biasing.^[29] Surfactants play a critical role in modulating interface stability during these processes, as covered in stabilization techniques.^[21] For adding reagents to existing droplets without full fusion, pico-injection employs side channels intersecting the main flow path, where a pressure pulse drives reagent delivery through a narrow orifice destabilized by an electric field.^[30] This method injects controlled volumes (0.1–3 pL) using pressure differentials of 1–10 kPa, triggered at kilohertz rates synchronized to passing droplets via capacitance-based detection.^[30] The injected reagent merges rapidly with the droplet content due to interfacial rupture, supporting high-throughput combinatorial chemistry with sub-picoliter precision and minimal volume loss.^[30]

Incubation methods

In droplet-based microfluidics, incubation methods are essential for allowing biochemical reactions, cell growth, or enzymatic processes to proceed within isolated aqueous droplets suspended in an immiscible carrier oil, maintaining their integrity over controlled periods. These methods balance the need for high-throughput processing with precise environmental regulation, such as temperature and humidity, to prevent droplet coalescence or evaporation. Common approaches include off-chip and on-chip strategies, each suited to different experimental durations and complexities. Off-chip incubation involves collecting generated droplets into external containers, such as syringes, vials, or Petri dishes, for subsequent environmental control. This method enables extended incubation times, such as up to 4 days at 37°C in a CO₂ incubator for cell viability assays, with over 80% cell survival maintained due to the stability provided by fluorinated surfactants. For polymerase chain reaction (PCR) applications, droplets can be subjected to thermal cycling between room temperature and 95°C using conventional thermal cyclers, facilitating digital PCR workflows. However, re-injection of incubated droplets into microfluidic devices for downstream analysis carries risks of contamination from external handling or emulsion instability. Humidity control is often implemented during off-chip storage to minimize evaporation, particularly for longer incubations exceeding 24 hours. On-chip incubation, in contrast, integrates storage directly within the microfluidic device to preserve high throughput and reduce handling steps. Droplets are directed into meandering channels, known as delay lines, or dedicated storage traps, where residence time t is determined by t = L / v, with L as the channel length and v as the flow velocity, allowing for tunable incubation periods such as 15 minutes in a 1.5 m long channel. These designs often feature expanded cross-sections or multi-layer fabrication to mitigate back-pressure and accommodate droplet expansion during reactions. Temperature regulation on-chip is achieved using integrated Peltier elements for rapid heating and cooling rates up to 7–8°C/s, or external water baths for stable isothermal conditions around 37°C, ensuring uniform thermal profiles across the device. Humidity is managed through sealed channels or oil overlays to limit water loss, supporting incubations up to 24 hours with less than 2% droplet size variation. Recent advancements include trapped droplet arrays, which enable long-term single-cell incubation on the order of hours to days by immobilizing droplets in addressable nanowells or microcages using dielectrophoretic or hydrodynamic trapping. For instance, a 2025 droplet-digital platform achieves over 80% recovery and approximately 90% viability for incubated cells over 7 days, facilitating high-throughput phenotypic analysis without off-chip transfer.^[31] These arrays support dynamic environmental control, including precise temperature maintenance at 37°C, and integrate seamlessly with prior reagent addition steps for in-droplet reactions.

Sorting techniques

Sorting techniques in droplet-based microfluidics allow for the selective isolation of droplets containing desired contents, such as cells or biomolecules, from large populations generated at high throughput. These methods rely on physical forces to deflect or redirect droplets at sorting junctions, often triggered by upstream detection signals like fluorescence. Common approaches include hydrodynamic, dielectrophoretic, magnetic, and optical techniques, each suited to different droplet properties and carrier fluids.^[32] Hydrodynamic sorting employs fluid flow manipulation through valves or bifurcations to separate droplets primarily by size. Pneumatic valves, formed by deforming polydimethylsiloxane (PDMS) channels with air pressure, block or redirect flow paths to sort droplets into separate outlets, achieving throughputs of 1–250 Hz with accuracies exceeding 98%. Bifurcation-based designs use channel geometry to exploit differences in droplet velocity or size, enabling passive size-based separation without external actuation. For instance, pinched microchannels have been used to profile droplet sizes continuously.^[32]^[33] Dielectrophoretic (DEP) sorting applies alternating current (AC) electric fields, typically at frequencies of 1–10 MHz, to induce label-free deflection of droplets based on their dielectric properties relative to the carrier fluid. The DEP force acting on a droplet is approximated as F \sim \epsilon \nabla (E^2), where \epsilon is the permittivity of the medium and E is the electric field strength, causing droplets to move toward or away from field maxima depending on the Clausius-Mossotti factor. Electrodes embedded near sorting junctions generate non-uniform fields to pull selected droplets across streamlines, with systems achieving up to 30 kHz throughput and over 99% accuracy. Seminal implementations include fluorescence-activated DEP sorters for enzyme evolution screens.^[34]^[35]^[32] Magnetic sorting leverages magnetic field gradients to manipulate droplets containing paramagnetic particles or ferrofluids, enabling selective extraction without electrical contact. External permanent magnets or electromagnets produce gradients of 0.1–1 T, generating forces on the order of piconewtons to pull droplets toward collection channels, with throughputs ranging from 0.5–100 Hz and accuracies of 58–95%. This method is particularly useful for sorting cell-laden droplets, as demonstrated in systems separating microalgal cells based on concentration. The magnetophoretic force scales with the cube of particle radius and the square of the field strength.^[36]^[32] Optical sorting uses laser-induced forces for precise, on-demand droplet deflection, often integrated with fluorescence detection for content-based selection. Laser-based thermocapillary effects or optical tweezers generate localized heating or radiation pressure to alter droplet trajectories, though throughputs are typically below 1 Hz with 100% accuracy in controlled setups. A notable advancement is the 2024 NOVAsort system, which combines opto-volume fluorescence detection with interdigitated electrodes for error-free sorting based on size and intensity thresholds, achieving over 99% accuracy at 235 Hz and processing more than 800,000 droplets per hour. This addresses polydispersity issues in traditional optical methods.^[32]^[37] Non-traditional magnetic approaches extend sorting to aqueous carriers, avoiding oil phases for biocompatible applications. In all-aqueous droplet systems using aqueous two-phase emulsions, magnetic nanoparticles enable manipulation via gradients, as shown in 2023 designs with fluorinated magnetic particles for interfacial assembly and actuation in water-based media.^[38] These methods support gentle sorting of sensitive cargos like cells, with forces tuned by particle coating and field strength.

Detection and Analysis

Optical and fluorescence methods

Optical and fluorescence methods are essential for real-time monitoring and analysis in droplet-based microfluidics, enabling the detection of droplet contents, sizes, and dynamics through label-based and label-free approaches. These techniques leverage light-matter interactions to provide high sensitivity and throughput, crucial for applications like single-cell assays and high-content screening. Fluorescence-based methods, in particular, dominate due to their ability to encode multiple analytes via distinct dyes, while optical imaging offers insights into physical properties without labels. Fluorescence detection often employs encoded dyes such as FAM (excitation ~495 nm, emission ~520 nm) and ROX (excitation ~587 nm, emission ~607 nm) for multiplexing, allowing simultaneous monitoring of multiple reactions or biomarkers within droplets. For instance, in droplet digital PCR, FAM/ROX ratios enable absolute quantification of nucleic acids across thousands of partitions. Photomultiplier tubes (PMTs) serve as detectors, achieving sensitivities down to ~10^{-12} M for fluorophores, which supports single-molecule detection in small droplet volumes (~1 nL). This high sensitivity arises from PMTs' low noise and high quantum efficiency, facilitating real-time readout at rates up to several kHz. Microscopy techniques complement fluorescence for detailed visualization. Brightfield microscopy measures droplet size and morphology by analyzing transmitted light intensity, providing rapid assessment of uniformity in flows exceeding 1000 droplets/s. Confocal microscopy enhances this with optical sectioning for 3D imaging of internal structures, such as cell distributions within droplets, though at lower throughputs (~100-500 droplets/s) due to scanning requirements. These methods integrate seamlessly with microfluidic channels, often using LED illumination for compactness and cost-effectiveness. Image analysis in these systems relies on algorithms like droplet tracking velocimetry (DTV), which computes velocity as v = \frac{\Delta x}{\Delta t} from sequential frames to monitor droplet trajectories and flow dynamics. DTV processes digital videos to extract size, shape, and speed, enabling feedback for downstream manipulations like sorting. Key challenges include photobleaching, where prolonged excitation degrades fluorophores, reducing signal fidelity in long incubations; mitigation strategies involve pulsed illumination or photostable dyes. Recent advances, such as 2025 AI-enhanced morphology detection, use deep learning to analyze brightfield images of droplet communities, identifying bacterial interactions with >95% accuracy without labels, thus addressing photobleaching limitations in microbial studies.

Spectroscopic methods

Spectroscopic methods in droplet-based microfluidics enable label-free analysis of chemical composition by probing molecular vibrations and scattering, providing fingerprint-like signatures for biomolecules and reaction products without disrupting droplet integrity. These techniques, primarily Raman and infrared (IR) absorption spectroscopies, offer non-destructive interrogation of sub-nanoliter volumes, complementing fluorescence-based detection by revealing intrinsic molecular structures rather than relying on exogenous labels. Vibrational spectroscopies are particularly suited for droplet systems due to their ability to capture real-time changes in functional groups during synthesis or biological processes. Raman spectroscopy stands out as a label-free method for identifying biomolecules, with characteristic peaks in the 1000–3000 cm⁻¹ region corresponding to C–H stretches, amide bands, and other vibrational modes unique to proteins, lipids, and nucleic acids. This fingerprint region allows discrimination of cellular components or metabolites encapsulated in droplets, enabling applications like single-cell phenotyping. Signal enhancement is achieved through surface-enhanced Raman scattering (SERS) by integrating metallic nanostructures, such as gold nanoparticles, into the droplets, yielding enhancement factors up to 10⁶, which lowers detection limits to attomolar concentrations for analytes like proteins or small molecules. Infrared absorption spectroscopy, particularly in the mid-IR range (2.5–20 μm), targets functional groups such as C=O, O–H, and N–H bonds, providing complementary information to Raman on molecular identity and concentration. However, strong water absorption in the mid-IR (e.g., at ~1650 cm⁻¹ for O–H bending) limits direct transmission measurements in aqueous droplets, often requiring attenuated total reflectance or evanescent wave configurations to minimize interference. Recent advancements employ fiber-optic coupling, such as chalcogenide or silica-clad fibers for evanescent wave sensing, allowing in-droplet probing with path lengths of millimeters while bypassing bulk water absorption, as demonstrated in setups for monitoring organic solvents or biomolecules. Current throughput for spectroscopic analysis in flowing droplet systems typically ranges from 1–10 droplets per second, constrained by acquisition times (seconds per spectrum) and droplet spacing to avoid crosstalk. This enables screening of hundreds to thousands of droplets per hour, suitable for high-content assays like enzyme kinetics or microbial metabolism. In 2024, innovations in droplet trapping—using hydrodynamic or dielectrophoretic traps within microfluidic channels—facilitated in-situ Raman spectroscopy, extending interrogation times to minutes per droplet for deeper spectral resolution without halting overall flow, as shown in under-oil platforms for multi-phase reactions. Data processing in these methods relies on multivariate techniques like principal component analysis (PCA) to classify spectra amid noise from carrier fluids or surfactants. PCA reduces dimensionality by identifying principal components that capture variance in peak intensities, enabling automated differentiation of droplet contents—such as healthy versus stressed cells—with accuracies exceeding 90% when combined with linear discriminant analysis. This approach handles the high-dimensional datasets from ensemble measurements, supporting scalable classification in droplet workflows.

Mass spectrometry interfaces

Mass spectrometry (MS) interfaces enable the coupling of droplet-based microfluidics to high-sensitivity analyte identification, allowing label-free analysis of complex mixtures from picoliter-scale volumes generated in droplets. This integration facilitates high-throughput screening and detailed molecular characterization, particularly for biomolecules like proteins, peptides, and metabolites, by ionizing and detecting analytes directly from microfluidic outputs. Key advantages include minimal sample loss and compatibility with downstream applications such as single-cell proteomics and metabolomics.^[39] Electrospray ionization (ESI) is a primary interface for droplet microfluidics, where droplets are evaporated to form a charged aerosol that is directly infused into the MS. In this process, aqueous droplets encapsulated in an immiscible carrier oil are directed to a nano-ESI tip, often integrated into polydimethylsiloxane (PDMS) chips, where the solvent evaporates, concentrating the analytes before applying a high voltage (typically 2-5 kV) to generate charged droplets that desolvate into gas-phase ions. Flow rates for such systems range from 1-10 μL/min, enabling analysis rates up to 30 Hz for ultrahigh-throughput applications. This method supports segmented flow configurations, where droplets act as discrete reaction vessels, preserving sample integrity during transfer.^[39]^[40] Matrix-assisted laser desorption/ionization (MALDI) provides an alternative interface, particularly suited for offline analysis, by depositing and drying droplets on a target plate for pulsed laser ionization. Droplets, typically 4 nL in volume, are generated via T-junction or flow-focusing in microfluidic chips and accumulated on a stainless-steel MALDI plate using an xy-stage for precise spotting; the carrier oil evaporates, concentrating analytes, and a matrix solution (e.g., α-cyano-4-hydroxycinnamic acid with additives like trichloroacetic acid) is applied to form co-crystallized spots. Ionization occurs via a laser pulse at 355 nm, desorbing and ionizing analytes for time-of-flight (TOF) MS detection, achieving enhanced homogeneity and signal intensity compared to conventional spotting methods. This approach improves detection limits through the focusing effect of microfluidics, enabling reproducible analysis of low-abundance species.^[41] Common interfaces include nano-ESI tips fabricated from stainless-steel capillaries (e.g., 76 μm inner diameter) pulled to fine emitters and nano-ESI with segmented flow, where oil-sheathed aqueous plugs minimize cross-contamination and maintain droplet stability during transfer to the MS inlet. These setups achieve sensitivities in the femtomole range for proteins and peptides, with detection limits as low as 150 pg per droplet for complex samples like cell lysates. Merits encompass high throughput (e.g., up to 2.85 million samples per day) and compatibility with various MS platforms, though challenges include droplet coalescence under high voltage and the need for fast-scanning analyzers to preserve resolution.^[39]^[41]^[40] Recent advancements, such as direct infusion from droplets into nano-ESI MS, have expanded applications to metabolomics, enabling real-time profiling of low-volume samples without chromatographic separation. For instance, optimized oil phases and ESI parameters have facilitated high-sensitivity detection of metabolites in single cells, addressing challenges in ion suppression and throughput for complex biological matrices. These developments, highlighted in 2023 reviews, underscore the potential for droplet-MS in drug discovery and synthetic biology by providing selective, label-free analysis at rates exceeding 10 Hz.^[39]

Electrochemical methods

Electrochemical methods in droplet-based microfluidics enable the detection of redox-active species through electron transfer at integrated electrodes, offering label-free, high-throughput analysis with minimal sample volumes. These techniques leverage the confined environment of droplets to monitor electrochemical signals from analytes such as enzymes, metabolites, and small molecules, providing insights into reaction kinetics and concentrations without optical interference from carrier fluids.^[42] Amperometry is the predominant electrochemical technique in droplet systems, measuring the steady-state current generated by the oxidation or reduction of analytes at a constant applied potential. The current i is governed by the diffusion-limited equation i = n F A D C / \delta, where n is the number of electrons transferred, F is Faraday's constant, A is the electrode area, D is the diffusion coefficient, C is the analyte concentration, and \delta is the diffusion layer thickness. This method has been applied to detect thiocholine from acetylcholinesterase activity in droplets, achieving limits of detection around 0.5 μM for glucose oxidase substrates.^[42]^[43]^[44] Impedimetric detection complements amperometry by using alternating current (AC) impedance spectroscopy to probe capacitance and resistance changes at droplet interfaces, particularly for non-redox events like biomolecular binding. In digital microfluidic platforms, impedance shifts arise from alterations in the electrode-droplet interface, enabling sensitive immunoassays with detection limits as low as 0.07 IU/mL for rubella IgG. This approach is especially useful for monitoring droplet composition without direct Faradaic processes.^[42]^[45]^[46] Integration of microelectrodes, typically 10-50 μm in width, into microfluidic channels facilitates seamless detection during droplet flow, with materials like platinum or gold ensuring compatibility with aqueous-oil phases. These compact designs support portable point-of-care devices, as demonstrated in systems quantifying droplet velocity, size, and redox content simultaneously via chronoamperometric responses.^[47]^[42] Recent advances, highlighted in 2025 reviews, include chronoamperometry for real-time enzyme kinetics in droplets, achieving sub-second resolution (e.g., 0.05 s) for Michaelis-Menten parameters in reactions like catalase-mediated hydrogen peroxide decomposition. This enables inhibition assays with IC50 values in the μM range, using volumes under 50 μL, and can be combined briefly with fluorescence for multi-modal validation of kinetic profiles.^[42]^[48]^[49]

Applications

Single-cell and microbial analysis

Droplet-based microfluidics enables the isolation of individual cells or microbes by encapsulating them within aqueous droplets suspended in an immiscible oil phase, allowing high-throughput analysis while minimizing cross-contamination. This approach is particularly valuable for studying heterogeneous populations, such as microbial communities, where traditional bulk methods obscure individual behaviors.^[50] Encapsulation efficiency follows Poisson statistics, where the probability of single-cell occupancy is maximized when the average number of cells per droplet (λ) is approximately 1, given by λ = cell density × droplet volume; to favor single occupancy, λ is typically kept below 1, yielding up to 37% single-cell droplets at the theoretical optimum.^[51] Passive encapsulation relies on random distribution at the droplet formation junction, while active methods, such as dielectrophoresis or hydrodynamic focusing, can enhance precision beyond Poisson limits.^[52] For microbial applications, droplet sizes are tuned to 10–100 μm to accommodate bacterial dimensions, ensuring gentle shear forces that preserve cell integrity during formation.^[53] Once encapsulated, single cells or microbes can be cultured within nutrient-laden media droplets, supporting growth and phenotypic studies in a confined microenvironment that mimics natural niches. Biocompatible surfactants, such as fluorinated polyethers (e.g., PFPE-PEG), stabilize droplets during extended incubation, enabling viability rates exceeding 90% for bacteria over 24–48 hours by preventing coalescence and osmotic stress.^[54] This setup facilitates observation of growth dynamics, such as division rates or metabolic responses, at scales unattainable in bulk cultures, with on-chip incubation methods allowing real-time monitoring without off-device transfer.^[50] Key challenges in droplet-based single-cell and microbial analysis include channel clogging from cell aggregates or debris, which can be mitigated by incorporating passive filters upstream of droplet generators, and inherent heterogeneity in cell size or shape that leads to variable occupancy and reduced sorting efficiency.^[55] Device materials like polydimethylsiloxane (PDMS) offer flexibility for rapid prototyping but suffer from protein adsorption and gas permeability issues, while glass provides superior optical clarity and chemical inertness at the cost of higher fabrication complexity.^[56] These factors necessitate optimized flow rates (typically 1–10 μL/min) and surfactant concentrations (1–2% w/v) to balance throughput with reliability.^[57] Recent advancements include 2024 platforms for single-bacterium analysis, such as integrated electrohydrodynamic dispensers that generate on-demand droplets for precise encapsulation and downstream functional assays like biosurfactant production screening.^[58] In 2025, deep learning models integrated with droplet imaging have enabled label-free analysis of microbial morphology in co-culture communities, using morphology-based classification to quantify interactions like phage-bacteria dynamics from brightfield Z-stack images without fluorescent labeling.^[59] These innovations expand applications to ecological microbiology, revealing hidden diversity in unculturable species.^[60]

Nucleic acid processing

Droplet-based microfluidics has revolutionized nucleic acid processing by enabling high-throughput partitioning, amplification, and analysis of DNA and RNA at the single-molecule level. This approach leverages emulsion droplets to isolate nucleic acids, facilitating precise control over reactions and minimizing cross-contamination. Key applications include polymerase chain reaction (PCR) amplification, library preparation for sequencing, and directed evolution of genetic variants, all of which benefit from the scalability and compartmentalization offered by droplet systems.^[61] In droplet digital PCR (ddPCR), nucleic acid samples are partitioned into thousands of monodisperse water-in-oil droplets, typically around 20,000 per 20 μL reaction, following Poisson statistics to achieve absolute quantification without standard curves. This method provides high precision, with dynamic ranges spanning 1 to 100,000 copies and the ability to detect rare alleles at frequencies as low as 0.001%. Partitioning efficiency exceeds 95%, ensuring reliable encapsulation and enabling applications such as copy number variation analysis and rare mutation detection. The workflow uses conventional TaqMan assays, processing up to 2 million reactions in a 96-well format for enhanced throughput compared to traditional qPCR.^[61]^[62] For sequencing library preparation, emulsion PCR (ePCR) within droplets amplifies single DNA molecules attached to beads, producing clonal clusters for downstream analysis. Introduced in early next-generation sequencing platforms, ePCR encapsulates fragments in emulsions containing approximately 2 × 10^6 droplets per milliliter, with about 30% occupancy yielding up to 450,000 amplified beads per reaction and 10^7 copies per bead. This technique mitigates biases from bulk amplification and has been integral to high-coverage genome sequencing, such as achieving 40-fold coverage of a 580 kb bacterial genome. More recent adaptations integrate ePCR with microfluidic droplet generation for single-cell applications, enhancing uniformity and yield.^[63]^[64] Advanced sequencing methods like UDA-seq further exploit droplet microfluidics through combinatorial indexing, combining droplet-specific barcoding with a post-indexing step to scale multimodal single-cell analysis. Published in 2025, UDA-seq adapts existing droplet platforms (e.g., 10x Genomics) to generate up to 38 million barcode combinations, reducing doublet rates to below 1.23% and enabling profiling of over 150,000 cells from a single sample or 200,000 across multiple tissues. This yields throughputs exceeding 10^6 reads per experiment while maintaining data quality comparable to commercial standards, facilitating large-scale RNA and multi-ome sequencing.^[65] Directed evolution of nucleic acids in droplets involves in-droplet mutagenesis, expression, and selection, allowing iterative screening of vast variant libraries. Droplet systems enable compartmentalization of individual variants, with throughputs reaching 10^6 to 10^9 variants per round via fluorescence-activated sorting at rates up to 30 kHz. For instance, evolution of aptamers like iSpinach has involved 4–9 cycles, identifying high-affinity binders from libraries exceeding 10^9 members, while antibody engineering against PD-1 achieved similar scales in two cycles using yeast display within droplets. This approach accelerates discovery of optimized sequences for therapeutics and biosensors.^[66] Recent advances in 2021 have introduced error-corrected droplet sequencing protocols, incorporating unique molecular identifiers (UMIs) and dual-nucleotide substitutions to mitigate sequencing errors in barcode and UMI detection. These methods, such as scBUC-seq adaptations, achieve high-throughput correction, reducing error rates in single-cell droplet data and enabling accurate variant calling in complex libraries. Such innovations enhance reliability for low-input nucleic acid analysis, bridging gaps in sensitivity for rare event detection.^[67]

Protein and biomolecule characterization

Droplet-based microfluidics has emerged as a powerful platform for characterizing proteins and biomolecules, particularly in elucidating their structures and interactions at high throughput. By encapsulating proteins in picoliter- to nanoliter-scale aqueous droplets suspended in an immiscible carrier fluid, these systems enable precise control over reaction conditions, minimize sample consumption, and facilitate rapid screening of vast parameter spaces. This approach is especially valuable for structural biology, where traditional methods often require microliter volumes and extended timelines, limiting scalability. A key application is protein crystallization, which is essential for X-ray crystallography to determine atomic structures. Droplet microfluidics supports vapor diffusion setups by generating alternating droplets of protein solution and reservoir buffer within a water-permeable oil carrier, allowing controlled dehydration and equilibration. For instance, in a composite PDMS/glass capillary system, lysozyme droplets lost approximately 50% of their volume over 24 hours, leading to crystal formation in over half of the trials within two days. This method has enabled on-chip X-ray diffraction analysis, with crystals diffracting to resolutions around 1.8 Å. High-throughput screening is a hallmark, with platforms generating over 1,300 crystallization trials using just 10 μL of protein solution in under 20 minutes—equating to more than 10^3 screens per day—vastly accelerating the exploration of chemical space for challenging targets.^[68] Binding assays in droplets further advance biomolecule interaction studies, quantifying affinities such as dissociation constants (K_d). Fluorescence anisotropy measurements exploit the change in rotational diffusion of a fluorescent ligand upon binding to a larger protein, detectable within individual nanoliter droplets. In one system, droplets encoding varying protein concentrations were analyzed in-line, yielding high-resolution binding curves for human Rad51 (HumRadA) variants interacting with a fluorescently labeled BRC4 peptide, with K_d values ranging from 4 to 670 nM and precision within 10% error. This droplet format supports multiplexed titrations at rates of four per minute, using as little as 100 data points per curve, and extends to crude cell lysates for direct affinity screening without purification.^[69] Despite these advances, challenges persist in droplet-based approaches. Distinguishing true crystals from amorphous or microcrystalline precipitates is critical, as precipitation often appears as grainy or birefringent aggregates that mimic crystals under initial imaging; techniques like dye uptake (e.g., with Chromotrope 2B) or birefringence under polarized light help confirm crystalline order, but require integration with downstream validation such as X-ray diffraction. In cell-free expression systems within droplets, maintaining protein viability and activity during encapsulation and incubation poses additional hurdles, including surfactant-induced denaturation and limited oxygen diffusion, which can reduce yields for folding-sensitive biomolecules. Recent innovations address some limitations, such as a 2024 droplet microfluidics platform for time-resolved serial crystallography, which generates uniform microcrystals for X-ray screening at synchrotron sources, enabling dynamic structural studies of protein reactions with sub-millisecond resolution.^[70]

Chemical synthesis and materials

Droplet-based microfluidics serves as a powerful platform for chemical synthesis and materials fabrication by using emulsion droplets as confined reaction templates, enabling precise control over particle size, composition, and morphology at the micro- and nanoscale. This approach leverages the generation of monodisperse droplets through flow-focusing or T-junction geometries, where reagents are encapsulated and reactions such as polymerization, precipitation, or crosslinking occur within the droplets to yield uniform materials. Unlike bulk methods, droplet templating minimizes reagent consumption, enhances reaction uniformity, and allows high-throughput production, making it ideal for synthesizing advanced materials like particles, gels, and crystals.^[71]^[72] Microparticles are commonly synthesized via polymerization within oil-in-water or water-in-oil emulsion droplets, where monomers are emulsified and subsequently polymerized using thermal, UV, or chemical initiators to form solid structures with diameters typically ranging from 1 to 100 μm. For instance, poly(methyl methacrylate) (PMMA) microparticles are produced by free-radical polymerization of methyl methacrylate monomers in droplet templates, yielding highly monodisperse spheres suitable for optical and chromatographic applications. This method ensures low polydispersity indices (often <5%) and enables incorporation of functional additives, such as dyes or nanoparticles, during the emulsification step.^[71]^[73]^[74] Nanoparticles are fabricated using emulsion precipitation techniques in droplet microfluidics, where rapid mixing within confined volumes drives nucleation and growth of inorganic or hybrid structures. Silica nanoparticles, for example, are synthesized by hydrolyzing tetraethyl orthosilicate precursors in aqueous droplets dispersed in oil, resulting in particles of 10-200 nm with narrow size distributions for use in catalysis and drug delivery. Similarly, quantum dots such as CdSe or PbS are formed through controlled precipitation of semiconductor precursors in double-emulsion droplets, achieving size-tunable emission wavelengths (e.g., 500-700 nm) and high quantum yields (>50%) due to the uniform reaction conditions.^[71]^[75] Gels, particularly microgels, are created through droplet crosslinking, where polymer solutions in droplets are solidified via ionic or covalent bonds to form porous, swellable networks. Alginate microgels, a prominent example, are generated by extruding sodium alginate solutions into calcium chloride-containing collection baths after droplet formation, producing spherical gels of 20-100 μm with tunable mechanical properties for encapsulation and separation applications. This on-chip or off-chip crosslinking allows for high encapsulation efficiency (>90%) and biocompatibility, with gel stiffness adjustable by varying alginate concentration (1-3 wt%) or crosslinking time.^[76]^[77] Liquid crystal microcapsules are templated in droplets to encapsulate nematic or cholesteric phases within polymeric shells, enabling applications in flexible displays where birefringence effects produce color or contrast under electric fields. Microfluidic emulsification of liquid crystal oils in aqueous surfactants followed by interfacial polymerization forms capsules of 10-50 μm, offering improved stability and response times compared to bulk methods. Recent optimizations in 2023 have focused on phase transfer dynamics in lyotropic systems, using droplet coalescence to study and control transitions between isotropic and crystalline phases, enhancing capsule uniformity for display technologies.^[78]^[79] Extraction processes in droplet microfluidics often employ aqueous two-phase systems (ATPS), where incompatible polymers like polyethylene glycol and dextran partition solutes between phases within droplets, facilitating selective separation without organic solvents. Droplet-based ATPS enables rapid mass transfer (equilibration in seconds) and high throughput (>1000 droplets/s), with partition coefficients tunable by phase composition for purifying chemicals or biomolecules. This method supports continuous extraction in flow, achieving >95% recovery for targeted species in applications like purification of dyes or metal ions.^[80]^[81] Droplet templating also extends to crystal synthesis, where supersaturated solutions in droplets promote nucleation and growth under controlled conditions, yielding monodisperse crystals for pharmaceuticals and materials science. For example, inorganic crystals like calcium carbonate are formed by mixing precursors in double emulsions, with droplet size dictating crystal dimensions (1-10 μm) and polymorphism. This confined environment reduces metastable phases and enables screening of crystallization conditions at high throughput, as demonstrated in seminal works on microfluidic crystallizers.^[82]^[83]

Diagnostic and therapeutic uses

Droplet-based microfluidics has emerged as a powerful platform for diagnostic applications, particularly in detecting antibiotic resistance in clinical samples. In 2025, researchers developed a droplet microfluidic system capable of identifying rare antibiotic-resistant subpopulations in Escherichia coli from bloodstream infections at frequencies as low as 10⁻⁶, enabling rapid phenotypic screening without prior cultivation.^[84] This approach encapsulates bacteria in picoliter droplets with growth media and antibiotics, allowing for high-sensitivity detection of heteroresistant cells that traditional methods often miss, thus accelerating personalized treatment decisions for infections.^[85] In drug discovery, droplet microfluidics facilitates high-throughput screening of cell-free expression systems, integrating machine learning for optimization. The 2025 DropAI platform uses droplet-based reactors to screen vast libraries of transcription-translation biocircuits, achieving economic efficiency through AI-guided selection of optimal gene expression conditions.^[86] By encoding variants with fluorescent colors and decoding via imaging, DropAI processes up to 100,000 reactions per run, identifying high-yield protein producers that enhance therapeutic development pipelines.^[87] For therapeutic uses, droplet microfluidics enables precise microencapsulation of drugs and cells, improving targeted delivery. This technique generates uniform microparticles loaded with active pharmaceutical ingredients, such as nanoparticles or biologics, for controlled release in applications like cancer therapy and regenerative medicine.^[88] Additionally, integrated droplet devices serve as biosensors for detecting mycotoxins in food and environmental samples, with 2025 advancements incorporating microfluidic channels for on-site, ultrasensitive assays that combine extraction and signal amplification.^[89] Droplet platforms support ultrahigh-throughput screening, routinely exceeding 10⁶ assays per day through automated droplet generation and analysis at rates of thousands per second.^[90] However, translating this to clinical settings faces challenges, including standardization of droplet stability, integration with regulatory-approved workflows, and cost-effective scaling for routine diagnostics beyond research labs.^[91]

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