Fabrication of Crescent Shaped Microparticles for Particle Templated Droplet Formation.

Crescent-shaped hydrogel microparticles are shown to template uniform volume aqueous droplets upon simple mixing with aqueous and oil media for various bioassays. This emerging "lab on a particle" technique requires hydrogel particles with tunable material properties and dimensions. The crescent shape of the particles is attained by aqueous two-phase separation of polymers followed by photopolymerization of the curable precursor. In this work, the phase separation of poly(ethylene glycol) diacrylate (PEGDA, Mw 700) and dextran (Mw 40 000) for tunable manufacturing of crescent-shaped particles is investigated. The particles' morphology is precisely tuned by following a phase diagram, varying the UV intensity, and adjusting the flow rates of various streams. The fabricated particles with variable dimensions encapsulate uniform aqueous droplets upon mixing with an oil phase. The particles are fluorescently labeled with red and blue emitting dyes at variable concentrations to produce six color-coded particles. The blue fluorescent dye shows a moderate response to the pH change. The fluorescently labeled particles are able to tolerate an extremely acidic solution (pH 1) but disintegrate within an extremely basic solution (pH 14). The particle-templated droplets are able to effectively retain the disintegrating particle and the fluorescent signal at pH 14.

Stopping Microfluidic Flow.

A cross-comparison of three stop-flow configurations-such as low-pressure (LSF), high-pressure open-circuit (OC-HSF), and high-pressure short-circuit (SC-HSF) stop-flow-is presented to rapidly bring a high velocity flow O(m s-1 ) within a microchannel to a standstill O(µm s-1 ). The performance of three stop-flow configurations is assessed by measuring residual flow velocities within microchannels having three orders of magnitude different flow resistances. The LSF configuration outperforms the OC-HSF and SC-HSF configurations within a high flow resistance microchannel and results in a residual velocity of <10 µm s-1 . The OC-HSF configuration results in a residual velocity of <150 µm s-1 within a low flow resistance microchannel. The SC-HSF configuration results in a residual velocity of <200 µm s-1 across the three orders-of-magnitude different flow resistance microchannels, and <100 µm s-1 for the low flow resistance channel. It is hypothesized that residual velocity results from compliance in fluidic circuits, which is further investigated by varying the elasticity of microchannel walls and connecting tubing. A numerical model is developed to estimate the expanded volumes of the compliant microchannel and connecting tubings under a pressure gradient and to calculate the distance traveled by the sample fluid. A comparison of the numerically and experimentally obtained traveling distances confirms the hypothesis that the residual velocities are an outcome of the compliance in the fluidic circuit.

Amphiphilic Particle-Stabilized Nanoliter Droplet Reactors with a Multimodal Portable Reader for Distributive Biomarker Quantification.

Compartmentalization, leveraging microfluidics, enables highly sensitive assays, but the requirement for significant infrastructure for their design, build, and operation limits access. Multimaterial particle-based technologies thermodynamically stabilize monodisperse droplets as individual reaction compartments with simple liquid handling steps, precluding the need for expensive microfluidic equipment. Here, we further improve the accessibility of this lab on a particle technology to resource-limited settings by combining this assay system with a portable multimodal reader, thus enabling nanoliter droplet assays in an accessible platform. We show the utility of this platform in measuring N-terminal propeptide B-type natriuretic peptide (NT-proBNP), a heart failure biomarker, in complex medium and patient samples. We report a limit of detection of ∼0.05 ng/mL and a linear response between 0.2 and 2 ng/mL in spiked plasma samples. We also show that, owing to the plurality of measurements per sample, "swarm" sensing acquires better statistical quantitation with a portable reader. Monte Carlo simulations show the increasing capability of this platform to differentiate between negative and positive samples, i.e., below or above the clinical cutoff for acute heart failure (∼0.1 ng/mL), as a function of the number of particles measured. Our platform measurements correlate with gold standard ELISA measurement in cardiac patient samples, and achieve lower variation in measurement across samples compared to the standard well plate-based ELISA. Thus, we show the capabilities of a cost-effective droplet-reader system in accurately measuring biomarkers in nanoliter droplets for diseases that disproportionately affect underserved communities in resource-limited settings.

A review on microfluidic-assisted nanoparticle synthesis, and their applications using multiscale simulation methods.

Recent years have witnessed an increased interest in the development of nanoparticles (NPs) owing to their potential use in a wide variety of biomedical applications, including drug delivery, imaging agents, gene therapy, and vaccines, where recently, lipid nanoparticle mRNA-based vaccines were developed to prevent SARS-CoV-2 causing COVID-19. NPs typically fall into two broad categories: organic and inorganic. Organic NPs mainly include lipid-based and polymer-based nanoparticles, such as liposomes, solid lipid nanoparticles, polymersomes, dendrimers, and polymer micelles. Gold and silver NPs, iron oxide NPs, quantum dots, and carbon and silica-based nanomaterials make up the bulk of the inorganic NPs. These NPs are prepared using a variety of top-down and bottom-up approaches. Microfluidics provide an attractive synthesis alternative and is advantageous compared to the conventional bulk methods. The microfluidic mixing-based production methods offer better control in achieving the desired size, morphology, shape, size distribution, and surface properties of the synthesized NPs. The technology also exhibits excellent process repeatability, fast handling, less sample usage, and yields greater encapsulation efficiencies. In this article, we provide a comprehensive review of the microfluidic-based passive and active mixing techniques for NP synthesis, and their latest developments. Additionally, a summary of microfluidic devices used for NP production is presented. Nonetheless, despite significant advancements in the experimental procedures, complete details of a nanoparticle-based system cannot be deduced from the experiments alone, and thus, multiscale computer simulations are utilized to perform systematic investigations. The work also details the most common multiscale simulation methods and their advancements in unveiling critical mechanisms involved in nanoparticle synthesis and the interaction of nanoparticles with other entities, especially in biomedical and therapeutic systems. Finally, an analysis is provided on the challenges in microfluidics related to nanoparticle synthesis and applications, and the future perspectives, such as large-scale NP synthesis, and hybrid formulations and devices.

Residue-free acoustofluidic manipulation of microparticles via removal of microchannel anechoic corner.

Surface acoustic wave (SAW)-based acoustofluidics has shown significant promise to manipulate micro/nanoscale objects for biomedical applications, e.g. cell separation, enrichment, and sorting. A majority of the acoustofluidic devices utilize microchannels with rectangular cross-section where the acoustic waves propagate in the direction perpendicular to the sample flow. A region with weak acoustic wave intensity, termed microchannel anechoic corner (MAC), is formed inside a rectangular microchannel of the acoustofluidic devices where the ultrasonic waves refract into the fluid at the Rayleigh angle with respect to the normal to the substrate. Due to the absence of a strong acoustic field within the MAC, the microparticles flowing adjacent to the microchannel wall remain unaffected by a direct SAW-induced acoustic radiation force (ARF). Moreover, an acoustic streaming flow (ASF) vortex produced within the MAC pulls the particles further into the corner and away from the direct ARF influence. Therefore, a residue of particles continues to flow past the SAWs without intended deflection, causing a decrease in microparticle manipulation efficiency. In this work, we introduce a cross-type acoustofluidic device composed of a half-circular microchannel, fabricated through a thermal reflow of a positive photoresist mold, to overcome the limitations associated with rectangular microchannels, prone to the MAC formation. We investigated the effects of different microchannel cross-sectional shapes with varying contact angles on the microparticle deflection in a continuous flow and found three distinct regimes of particle deflection. By systematically removing the MAC out of the microchannel cross-section, we achieved residue-free acoustofluidic microparticle manipulation via SAW-induced ARF inside a half-circular microchannel. The proposed method was applied to efficient fluorescent coating of the microparticles in a size-selective manner without any residue particles left undeflected in the MAC.

Flow lithography for structured microparticles: fundamentals, methods and applications.

Structured microparticles, with unique shapes, customizable sizes, multiple materials, and spatially-defined chemistries, are leading the way for emerging 'lab on a particle' technologies. These microparticles with engineered designs find applications in multiplexed diagnostics, drug delivery, single-cell secretion assays, single-molecule detection assays, high throughput cytometry, micro-robotics, self-assembly, and tissue engineering. In this article we review state-of-the-art particle manufacturing technologies based on flow-assisted photolithography performed inside microfluidic channels. Important physicochemical concepts are discussed to provide a basis for understanding the fabrication technologies. These photolithography technologies are compared based on the structural as well as compositional complexity of the fabricated particles. Particles are categorized, from 1D to 3D particles, based on the number of dimensions that can be independently controlled during the fabrication process. After discussing the advantages of the individual techniques, important applications of the fabricated particles are reviewed. Lastly, a future perspective is provided with potential directions to improve the throughput of particle fabrication, realize new particle shapes, measure particles in an automated manner, and adopt the 'lab on a particle' technologies to other areas of research.

Engineering Design of Concentric Amphiphilic Microparticles for Spontaneous Formation of Picoliter to Nanoliter Droplet Volumes.

Simple mixing of aqueous and oil solutions with amphiphilic particles leads to the spontaneous formation of uniform reaction volumes (dropicles) that can enable numerous applications in the analysis of biological entities (e.g., cells and molecules). Approaches to manufacture such amphiphilic particles are just starting to be investigated. Here, we investigate the tunable manufacturing of concentric amphiphilic particles, with outer hydrophobic and inner hydrophilic layers, fabricated by flowing reactive precursor streams through a 3D printed device with coaxial microfluidic channels, and curing the structured flow by UV exposure through a photomask. The dimensions of the engineered amphiphilic particles, including height, inner and outer diameters, and thicknesses of the hydrophobic and hydrophilic layers, are precisely controlled by modulating the UV exposure time, the precursor flow rate ratios, and the size of the channel in the exposure region. The particle design is systematically engineered to hold a wide range of droplet volumes, that is, from a few hundred picoliters to several nanoliters. We show that the particle size can be significantly reduced from previous reports to not only hold subnanoliter drops but the shape can also be tuned to increase the seeding density and orientation of dropicles within a well plate for imaging and analysis.

Acoustofluidic generation of droplets with tunable chemical concentrations.

The dynamic control of the chemical concentration within droplets is required in numerous droplet microfluidic applications. Here, we propose an acoustofluidic method for the generation of a library of aqueous droplets with the desired chemical concentrations in a continuous oil phase. Surface acoustic waves produced by a focused interdigital transducer interact with two parallel laminar streams with different chemical compositions. Coupling the acoustic waves with the flow streams results in the controlled acoustofluidic mixing of the aqueous solutions through the formation of acoustic streaming flow-induced microvortices. The mixed streams are split at a bifurcation, and one of the streams with a precisely controlled chemical concentration is fed into a T-junction to produce droplets with tunable chemical concentrations. The periodic acoustofluidic mixing of the aqueous streams enables the generation of a droplet library with a well-defined inter-droplet concentration gradient. The proposed method is a promising tool for the on-chip dynamic control of in-droplet chemical concentrations and for next-generation droplet microfluidic applications.

Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays.

Reactions performed in uniform microscale volumes have enabled numerous applications in the analysis of rare entities (e.g. cells and molecules). Here, highly monodisperse aqueous droplets are formed by simply mixing microscale multi-material particles, consisting of concentric hydrophobic outer and hydrophilic inner layers, with oil and water. The particles are manufactured in batch using a 3D printed device to co-flow four concentric streams of polymer precursors which are polymerized with UV light. The cross-sectional shapes of the particles are altered by microfluidic nozzle design in the 3D printed device. Once a particle encapsulates an aqueous volume, each "dropicle" provides uniform compartmentalization and customizable shape-coding for each sample volume to enable multiplexing of uniform reactions in a scalable manner. We implement an enzymatically-amplified immunoassay using the dropicle system, yielding a detection limit of <1 pM with a dynamic range of at least 3 orders of magnitude. Multiplexing using two types of shape-coded particles was demonstrated without cross talk, laying a foundation for democratized single-entity assays.

Microparticle self-assembly induced by travelling surface acoustic waves.

We present an acoustofluidic method based on travelling surface acoustic waves (TSAWs) to induce self-assembly of microparticles inside a microfluidic channel. The particles are trapped above an interdigitated transducer, placed directly beneath the microchannel, by the TSAW-based direct acoustic radiation force (ARF). This approach was applied to trap 10 µm polystyrene particles, which were pushed towards the ceiling of the microchannel by 72 MHz TSAWs to form single- and multiple-layer colloidal structures. The repair of cracks and defects within the crystal lattice occurs as part of the self-assembly process. The sample flow through the first inlet can be switched with a buffer flow through the second inlet to control the number of particles assembled in the crystalline structure. The constant flow-induced Stokes drag force on the particles is balanced by the opposing TSAW-based ARF. This force balance is essential for the acoustics-based self-assembly of microparticles inside the microchannel. Moreover, we studied the effects of varying input voltage and fluid flow rate on the position and shape of the colloidal structure. The active self-assembly of microparticles into crystals with multiple layers can be used in the bottom-up fabrication of colloidal structures with dimensions greater than 500 µm × 500 µm, which is expected to have important applications in various fields.

In-droplet microparticle washing and enrichment using surface acoustic wave-driven acoustic radiation force.

Washing and enrichment of particles and cells are crucial sample preparation procedures in biomedical and biochemical assays. On-chip in-droplet microparticle washing and enrichment have been pursued but remained problematic due to technical difficulties, especially simultaneous and precise control over the droplet interface and in-droplet samples. Here, we have achieved a breakthrough in label-free, continuous, on-demand, in-droplet microparticle washing and enrichment using surface acoustic waves. When exposed to the acoustic field, the droplet and suspended particles experience acoustic radiation force arising from inhomogeneous wave scattering at the liquid/liquid and liquid/solid interfaces. Based on these acoustophoretic phenomena, we have demonstrated in-droplet microparticle washing and enrichment in an acoustofluidic device. We expect that the proposed acoustic method will offer new perspectives to sample washing and enrichment by performing the operation in microscale droplets.

Sheathless Focusing and Separation of Microparticles Using Tilted-Angle Traveling Surface Acoustic Waves.

Sheathless focusing and separation of microparticles is an important preprocessing step in various biochemical assays in which enriched sample isolation is critical. Most of the previous microfluidic particle separation techniques have used sheath flows to achieve efficient sample focusing. The sheath flow dilutes the analyte and requires additional microchannels and accurate flow control. We demonstrated a tilted-angle traveling surface acoustic wave (taTSAW)-based sheathless focusing and separation of particles in a continuous flow. The proposed device consists of a piezoelectric substrate with a pair of interdigitated transducers (IDTs) deposited at two different angles relative to the flow direction. A Y-shaped polydimethylsiloxane (PDMS) microchannel having one inlet and two outlet ports was positioned on top of the IDTs such that the acoustic energy coupling into the fluid was maximized and wave attenuation by the PDMS walls was minimized. The two IDTs independently produced high-frequency taTSAWs, which propagated at ±30° with respect to the flow direction and imparted a direct acoustic radiation force onto the target particles. A sample mixture of 4.8 and 3.2 µm particles was focused and then separated by the actuation of the IDTs at 194 and 136 MHz frequencies, respectively, without using an additional sheath flow. The proposed taTSAW-based particle separation device offered a high purity >99% at the both outlets over a wide range of flow speeds (up to 83.3 mm/s).

Vertical Hydrodynamic Focusing and Continuous Acoustofluidic Separation of Particles via Upward Migration.

A particle suspended in a fluid within a microfluidic channel experiences a direct acoustic radiation force (ARF) when traveling surface acoustic waves (TSAWs) couple with the fluid at the Rayleigh angle, thus producing two components of the ARF. Most SAW-based microfluidic devices rely on the horizontal component of the ARF to migrate prefocused particles laterally across a microchannel width. Although the magnitude of the vertical component of the ARF is more than twice the magnitude of the horizontal component, it is long ignored due to polydimethylsiloxane (PDMS) microchannel fabrication limitations and difficulties in particle focusing along the vertical direction. In the present work, a single-layered PDMS microfluidic chip is devised for hydrodynamically focusing particles in the vertical plane while explicitly taking advantage of the horizontal ARF component to slow down the selected particles and the stronger vertical ARF component to push the particles in the upward direction to realize continuous particle separation. The proposed particle separation device offers high-throughput operation with purity >97% and recovery rate >99%. It is simple in its fabrication and versatile due to the single-layered microchannel design, combined with vertical hydrodynamic focusing and the use of both the horizontal and vertical components of the ARF.

On-demand acoustic droplet splitting and steering in a disposable microfluidic chip.

On-chip droplet splitting is one of the fundamental droplet-based microfluidic unit operations to control droplet volume after production and increase operational capability, flexibility, and throughput. Various droplet splitting methods have been proposed, and among them the acoustic droplet splitting method is promising because of its label-free operation without any physical or thermal damage to droplets. Previous acoustic droplet splitting methods faced several limitations: first, they employed a cross-type acoustofluidic device that precluded multichannel droplet splitting; second, they required irreversible bonding between a piezoelectric substrate and a microfluidic chip, such that the fluidic chip was not replaceable. Here, we present a parallel-type acoustofluidic device with a disposable microfluidic chip to address the limitations of previous acoustic droplet splitting devices. In the proposed device, an acoustic field is applied in the direction opposite to the flow direction to achieve multichannel droplet splitting and steering. A disposable polydimethylsiloxane microfluidic chip is employed in the developed device, thereby removing the need for permanent bonding and improving the flexibility of the droplet microfluidic device. We experimentally demonstrated on-demand acoustic droplet bi-splitting and steering with precise control over the droplet splitting ratio, and we investigated the underlying physical mechanisms of droplet splitting and steering based on Laplace pressure and ray acoustics analyses, respectively. We also demonstrated droplet tri-splitting to prove the feasibility of multichannel droplet splitting. The proposed on-demand acoustic droplet splitting device enables on-chip droplet volume control in various droplet-based microfluidic applications.

Microfluidic flow switching via localized acoustic streaming controlled by surface acoustic waves.

We propose an acoustic flow switching device that utilizes high-frequency surface acoustic waves (SAWs) produced by a slanted-finger interdigitated transducer. As the acoustic field induced by the SAWs was attenuated in the fluid, it produced an acoustic streaming flow in the form of a pair of symmetrical microvortices, which induced flow switching between two fluid streams in a controlled manner. The microfluidic device was composed of a piezoelectric substrate attached to a polydimethylsiloxane (PDMS) microchannel having an H-shaped junction that connected two fluid streams in the middle. The two immiscible fluids, separated by the PDMS wall, flowed in parallel, briefly came in contact at the junction, and separated again into the downstream microchannels. The acoustic streaming flow induced by the SAWs rotated the fluid streams within the microchannel cross-section, thereby altering the respective positions of the two fluids and directing them into the opposite flow paths. The characteristics of the flow switching mechanism were investigated by tuning the input voltage and the flowrates. On-demand acoustic flow switching was successfully achieved without additional moving parts inside the microchannel. This technique may be useful for fundamental studies that integrate complex experimental platforms into a single chip.

A Pumpless Acoustofluidic Platform for Size-Selective Concentration and Separation of Microparticles.

We have designed a pumpless acoustofluidic device for the concentration and separation of different sized particles inside a single-layered straight polydimethylsiloxane (PDMS) microfluidic channel. The proposed device comprises two parallel interdigitated transducers (IDTs) positioned underneath the PDMS microchannel. The IDTs produce high-frequency surface acoustic waves that generate semipermeable virtual acoustic radiation force field walls that selectively trap and concentrate larger particles at different locations inside the microchannel and allow the smaller particles to pass through the acoustic filter. The performance of the acoustofluidic device was first characterized by injecting into the microchannel a uniform flow of suspended 9.9 µm diameter particles with various initial concentrations (as low as 10 particles/mL) using a syringe pump. The particles were trapped with ∼100% efficiency by a single IDT actuated at 73 MHz. The acoustofluidic platform was used to demonstrate the pumpless separation of 12.0, 4.8, and 2.1 µm microparticles by trapping the 12 and 4.8 µm particles using the two IDTs actuated at 73 and 140 MHz, respectively. However, most of the 2.1 µm particles flowed over the IDTs unaffected. The acoustofluidic device was capable of rapidly processing a large volume of sample fluid pumped through the microchannel using an external syringe pump. A small volume of the sample fluid was processed through the device using a capillary flow and a hydrodynamic pressure difference that did not require an external pumping device.

Acoustic Wave-Driven Functionalized Particles for Aptamer-Based Target Biomolecule Separation.

We developed a hybrid microfluidic device that utilized acoustic waves to drive functionalized microparticles inside a continuous flow microchannel and to separate particle-conjugated target proteins from a complex fluid. The acoustofluidic device is composed of an interdigitated transducer that produces high-frequency surface acoustic waves (SAW) and a polydimethylsiloxane (PDMS) microfluidic channel. The SAW interacted with the sample fluid inside the microchannel and deflected particles from their original streamlines to achieve separation. Streptavidin-functionalized polystyrene (PS) microparticles were used to capture aptamer (single-stranded DNA) labeled at one end with a biotin molecule. The free end of the customized aptamer15 (apt15), which was attached to the microparticles via streptavidin-biotin linkage to form the PS-apt15 conjugate, was used to capture the model target protein, thrombin (th), by binding at exosite I to form the PS-apt15-th complex. We demonstrated that the PS-apt15 conjugate selectively captured thrombin molecules in a complex fluid. After the PS-apt15-th complex was formed, the sample fluid was pumped through a PDMS microchannel along with two buffer sheath flows that hydrodynamically focused the sample flow prior to SAW exposure for PS-apt15-th separation from the non-target proteins. We successfully separated thrombin from mCardinal2 and human serum using the proposed acoustofluidic device.

Acoustothermal tweezer for droplet sorting in a disposable microfluidic chip.

Precise control over droplet position within a microchannel is fundamental to droplet microfluidic applications. This article proposes acoustothermal tweezer for the control of droplet position, which is based on thermocapillary droplet migration actuated by acoustothermal heating. The proposed system comprises an acoustothermal heater, which is composed of a slanted finger interdigital transducer patterned on a piezoelectric substrate and a thin PDMS membrane, and a PDMS microchannel. In the proposed system, droplets moving in a droplet microfluidic chip experience spatiotemporally varying thermal stimuli produced by acoustothermal heating and thus migrate laterally. In comparison to previous methods for droplet sorting, the acoustothermal tweezer offers significant advantages: first, the droplet position can be manipulated in two opposite directions, which enables bidirectional droplet sorting to one of three outlets downstream; second, precise control over the droplet position as well as improved droplet lateral displacement on the order of hundreds of micrometers can be achieved in a deterministic manner, thereby enabling multichannel droplet sorting; third, the PDMS microfluidic chip is disposable and thus can be easily replaced since it is attached to the substrate by reversible bonding, which allows the acoustothermal heater to be reused. Given these advantages, the proposed droplet sorting system is a promising droplet microfluidic lab-on-a-chip platform for tunable, on-demand droplet position control.

On-Demand Droplet Capture and Release Using Microwell-Assisted Surface Acoustic Waves.

We demonstrate an acoustofluidic platform that uses surface acoustic waves (SAWs) for the facile capture of droplets inside microwells and their on-demand release. When the ac signal applied to the device is tuned to modulate the location of the SAW, the SAW-based acoustic radiation force retracts or pushes the droplets into or out of one of three microwells fabricated inside a microchannel to selectively capture or release the droplet.

Particle Separation inside a Sessile Droplet with Variable Contact Angle Using Surface Acoustic Waves.

A sessile droplet of water carrying polystyrene microparticles of different diameters was uniformly exposed to high frequency surface acoustic waves (SAWs) produced by an interdigitated transducer (IDT). We investigated the concentration behavior of the microparticles as the SAWs generated a strong acoustic streaming flow (ASF) inside the water droplet and exerted a direct acoustic radiation force (ARF) on the suspended particles, the magnitude of which depended upon the particle diameter. As a result of the ARF, the microparticles were concentrated according to their diameters at different positions inside the sessile droplet placed in the path of the SAW, right in front of the IDT. The microparticle concentration behavior changed as the sessile droplet contact angle with the substrate was varied by adding surfactant to the water or by gradually evaporating the water. The positions at which the smaller and larger microparticles were concentrated remained distinguishable, even at very different experimental conditions. The long-term exposure of the droplets to the SAWs was accompanied by the gradual evaporation of the carrier fluid, which dynamically changed the droplet contact angle as well as the concentration of particles. Complete evaporation of the fluid left behind several concentrated yet separated clusters of particles on the substrate surface. The effect of the droplet contact angle on particles' concentration behavior and consequent separation of particles has been uniquely studied in this SAW-based report.