On-demand Droplet Generation Research Articles

Nucleic Acid Target Sensing Using a Vibrating Sharp-Tip Capillary and Digital Droplet Loop-Mediated Isothermal Amplification (ddLAMP).

Nucleic acid tests are key tools for the detection and diagnosis of many diseases. In many cases, the amplification of the nucleic acids is required to reach a detectable level. To make nucleic acid amplification tests more accessible to a point-of-care (POC) setting, isothermal amplification can be performed with a simple heating source. Although these tests are being performed in bulk reactions, the quantification is not as accurate as it would be with digital amplification. Here, we introduce the use of the vibrating sharp-tip capillary for a simple and portable system for tunable on-demand droplet generation. Because of the large range of droplet sizes possible and the tunability of the vibrating sharp-tip capillary, a high dynamic range (~2 to 6000 copies/µL) digital droplet loop-mediated isothermal amplification (ddLAMP) system has been developed. It was also noted that by changing the type of capillary on the vibrating sharp-tip capillary, the same mechanism can be used for simple and portable DNA fragmentation. With the incorporation of these elements, the present work paves the way for achieving digital nucleic acid tests in a POC setting with limited resources.

Drop-On-Demand Microdroplet Generation under Charge Injection by Corona Discharge.

In printing, microreactors, and bioassays, the precise control of micrometer-scale droplet generation is essential but challenging, often restricted by the equipment and nozzles used in traditional methods. We introduce a needle-plate electrode corona discharge technique that injects charges into an oil layer, enabling the precise manipulation of droplet polarization and splitting. This method allows for meticulous adjustment of microdroplet formation regarding location, size, and quantity by modulating the discharge voltage, discharge time, and electrode positioning. It enables the immediate initiation and cessation of droplet production, thereby facilitating on-demand droplet generation. Our study on the voltage-dependent droplet stretch coefficient shows that as the voltage increases, the droplets transition from controlled splitting to regular Taylor cone-like ejections, eventually reaching the Rayleigh limit and fully breaking apart. These advancements significantly improve microfluidic droplet manipulation, offering considerable benefits for applications in targeted drug delivery, rapid disease diagnostics, and precise environmental monitoring.

Label-free active single-cell encapsulation enabled by microvalve-based on-demand droplet generation and real-time image processing

Droplet microfluidics-based single-cell encapsulation is a critical technology that enables large-scale parallel single-cell analysis by capturing and processing thousands of individual cells. As the efficiency of passive single-cell encapsulation is limited by Poisson distribution, active single-cell encapsulation has been developed to theoretically ensure that each droplet contains one cell. However, existing active single-cell encapsulation technologies still face issues related to fluorescence labeling and low throughput. Here, we present an active single-cell encapsulation technique by using microvalve-based drop-on-demand technology and real-time image processing to encapsulate single cells with high throughput in a label-free manner. Our experiments demonstrated that the single-cell encapsulation system can encapsulate individual polystyrene beads with 96.3 % efficiency and HeLa cells with 94.9 % efficiency. The flow speed of cells in this system can reach 150 mm/s, resulting in a corresponding theoretical encapsulation throughput of 150 Hz. This technology has significant potential in various biomedical applications, including single-cell omics, secretion detection, and drug screening.

Coupling of droplet-on-demand microfluidcs with ESI/MS to study single-cell catalysis.

Droplet microfluidics provides an efficient method for analysing reactions within the range of nanoliters to picoliters. However, the sensitive, label-free and versatile detection with ESI/MS poses some difficulties. One challenge is the difficult association of droplets with the MS signal in high-throughput droplet analysis. Hence, a droplet-on-demand system for the generation of a few droplets can address this and other problems such as surfactant concentration or cross-contamination. Accordingly, the system has been further developed for online coupling with ESI/MS. To achieve this, we developed a setup enabling on-demand droplet generation by hydrodynamic gating, with downstream microscopic droplet detection and MS analysis. This facilitated the incorporation of 1-9 yeast cells into individual 1-5 nL droplets and the monitoring of yeast-catalysed transformation from ketoester to ethyl-3-hydroxybutyrate by MS. With our method a mean production rate of 0.035 ± 0.017 fmol per cell per h was observed with a detection limit of 0.30 μM. In conclusion, our droplet-on-demand method is a versatile and advantageous tool for cell encapsulation in droplets, droplet imaging and reaction detection using ESI/MS.

Coflowing aqueous and oil-based ferrofluid streams exposed to a magnetic field.

We report the transition between stream and droplet regimes in a coflow of an aqueous stream and oil-based ferrofluid. The transition between stream and droplet regimes is typically attained by controlling the capillary numbers (Ca) of the phases. Remarkably, we experimentally evidence a transition between the regimes by adjusting the exposure of the system to a magnetic field, with Ca fixed. We represent the various regimes: stable coflow, interface deformation, and droplet generation, in terms of the magnetic bond number (Bom) and the ratio of capillary numbers of the phases (Car). The different regimes are a consequence of the interplay of the magnetic, viscous, and interfacial tension forces, represented by the two dimensionless numbers. We explain the regimes in terms of the magnetic pinch-off (τmp) and advection (τa) time scales: for τmp ≫ τa a stable coflow is observed, for τmp ∼ τa interface deformation is observed, and for τmp ≪ τa droplet breakup is observed. We study the interface deformation and droplet size from experiments and predict the same from theoretical scaling. We find the interface deformation increases and the droplet size decreases with increases in Bom and Car. The present study may find applications in magnetic field-assisted on-demand droplet generation in microfluidics.

Modulation of coaxial cone-jet instability in active co-flow focusing

The breakup of coaxial cone-jet interfaces to compound droplets in axisymmetric co-flow focusing (CFF) upon actuation is studied through numerical simulations. Due to the coupling effect of double interfaces, the response behaviours of coaxial cone-jet flow to actuation are more complex than those of a single-layered interface structure. Particularly, the coaxial jet presents totally different response modes between weak and strong interface coupling situations. In this work, the phase diagrams of response modes for coaxial jet breakup are depicted, considering the effect of perturbation frequency, amplitude and liquid flow rates. In particular, the breakup of a coaxial jet can be synchronized with actuation within a frequency range containing the natural breakup frequency, resulting in uniform compound droplets with a single core inside the shell, and the size of droplets can be adjusted by frequency. As the perturbation frequency exceeds the upper critical value, the external perturbation is unable to dominate the jet breakup, while below the lower critical frequency, the jet breaks up with multiple droplets generated in one period. The perturbation amplitude mainly affects the jet breakup length and also leads to the transition between different response modes. The coaxial cone upstream of the orifice can act as a buffer layer, regulating the perturbation amplitude of the coaxial jet downstream. The degree of buffering effect is affected by the perturbation frequency and amplitude. As the perturbation amplitude approaches unity, the decrease of perturbation frequency leads to the intermittent jet behaviour from the cone tip with a vibrating manner of the coaxial cone. Based on the linear instability analysis on the simplified single jet models for weak-coupled and strong-coupled jets, scaling analyses are carried out, which predict the jet breakup length and the natural frequency and critical frequency for the synchronized breakup. Finally, a strong pulse is added on the perturbation to produce compound droplets with a controllable number of cores. The present work provides valuable guidance for the practical application of on-demand compound droplet generation through active CFF.

Development of Robust On-Demand Droplet Generation System Using 3-D Image Reconstruction as Feedback

Droplet-based microfluidics is an emerging interdisciplinary field with broad applications. On-demand droplet generation with precise size allows performing sensitive detection and quantitative analysis with high throughput and separate units, while the essential precondition is characterizing the volume of droplet accurately. Generally, fluorescence or bright-field imaging can be adopted to provide the length or width of the droplet from 2-D images, and then derive an idealized model to approximately estimate the droplet volume, which would always lead to steady-state errors with ignoring the irregular curvature of droplet surface in the depth direction. In this work, we propose the usage of real-time quantitative phase imaging (QPI) technique for the 3-D detection of flowing droplets to provide a precise measurement of droplet volume which can serve as feedback signal. Comparative experiments with a passive droplet generation system confirm that our proposed method can offer higher accuracy and monodispersity of on-demand droplet generation, together with enhanced stability and anti-interference to resist internal or external perturbations, which could promise a predictable outcome in many microfluidics-based systems.

Highly Uniform Droplet Generation Enabled by Microfluidic Impact Printing and Its Application in Lyophilized Microbeads Preparation.

Research on droplet generation technology with a large volume range and high uniformity has great significance in biomedical engineering. We explored the influence of both the liquid level height in the reservoir and the flow resistance distribution on the droplet volumes besides the nozzle size for the microfluidic impact printing (MIP) method. Finally, this droplet generation technology is applied to the preparation of lyophilized microbeads with high uniformity in volume. The coefficient of variation (CV) of the droplet volumes can be down to 0.55%. By adjusting the flow resistance distribution of the microchannels and the nozzle size, the droplet volume generated by the MIP system ranges from 0.4 nL to 610 nL. On-demand droplet generation with high uniformity in the range of subnanoliter to submicroliter is achieved. Furthermore, this droplet generation technology is successfully applied to the preparation of highly uniform lyophilized microbeads. This large volumetric range and highly uniform droplet generation technology has tremendous application potential in fields such as digital PCR detection, pharmaceutic preparation, and the construction of concentration arrays. Furthermore, it can also be used as a high-precision liquid distribution method for processing liquid in a submicroliter.

Experimental investigation on the performances of a valve-based and on-demand droplet generator producing droplets in a wide size range

Many aspects of our daily lives are affected by the generation of water droplets, and it is important to controllably produce droplets with a wide size range in various applications. In this paper, we describe an on-demand droplet generating system based on a high-speed two-way solenoid valve. A nozzle made of stainless steel tubing is fit into one port of the valve, and the other port is connected to the fluid reservoir by which the pressure can be controlled via a pressure regulator. When the liquid is pressurized and the valve is opened with a short pulse voltage, trace amounts of liquid will be ejected from the nozzle to form a droplet. Droplet generation is captured using a high-speed camera to measure the dimension and velocity and to evaluate the performances of the generator, such as repeatability and stability. We demonstrate the influences of applied pressure and pulse width of driving voltage on droplet generation. It is shown that the droplet generator is capable of producing droplets in a wide size range for a given nozzle (e.g., about 0.7–2.2 mm for a 1.0 mm diameter nozzle). A single droplet is stably generated at Z = 268.1, obviously higher than the published data and the Weber number of a fluid jet (Wej) ranging from 2.1 to 5.6. The droplet generator presented here will be useful for research related to large droplets, such as freezing rain in atmospheric science and supercooled large drops in aircraft icing.

Analytical model of the dynamic behavior of a vibrating mesh nebulizer for optimal atomization efficiency

This work deals with the assessment of optimal working condition of a mesh nebulizer on-demand droplet generation. The purpose of this paper is to introduce a model of vibrating mesh nebulizer to identify optimal working regime from a mechanical point of view. The influence of geometrical parameters and mechanical properties of both the piezoelectric component, vibrating plate and geometry of perforations is investigated and discussed based on an analytical model of stepped plate. This model is validated by 2D-axisymmetric finite element computations. The main trends identified by the model are recovered by the simulation and enable to formulate design rules to maximize the atomization efficiency with respect to a given set of mechanical and geometrical parameters.

Model-based feedback control for on-demand droplet dispensing system with precise real-time phase imaging

Recent years have witnessed the advances of droplet-based microfluidics as a powerful tool for high-throughput assays in biology, chemistry, material and environmental sciences. Precise and effective droplet generation is critical for the practical applications of droplet-based microfluidics. Although many passive and active droplet generation methods have been proposed and demonstrated, there are still challenges towards the implementation of droplet-on-demand in terms of system complexity, precision and robustness. In this work, we propose and demonstrate a dual-pressure-pulse (DPP) driven droplet generation method incorporated with model-based feedback control to achieve on-demand droplet generation. Based on the study of droplet formation dynamics in a flow-focusing configuration using real-time quantitative phase imaging (QPI) technique, we validate the proposed dual-pressure-pulse (DPP) driven droplet generation concept, for which the physical model of droplet formation can be greatly simplified. With this approach, model-based feedback control can be implemented for DPP actuation to achieve on-demand droplet generation with relatively high system response and low steady-state error.

A low-cost, programmable, and multi-functional droplet printing system for low copy number SARS-CoV-2 digital PCR determination

Droplet digital polymerase chain reaction (ddPCR) is a rapidly developing technology used for accurate, quantitative analysis of rare samples. However, ddPCR has only been implemented in research field but rarely in clinical trials due to its relatively high cost and negative user experiences compared with qPCR. We developed a novel programmable on-demand droplet generator based on a microfluidic adaptive printing system (MAP-ddPCR) to create a cost-effective ddPCR process. This process easily produces low-volume, spot-on-demand droplet dispensing and data analysis using simple equipment and workflow. Compared with the existing droplet generation systems that rely on surface-assistant, the proposed MAP system generates a variety of droplet arrays on regular non-surface-treated glass slides. This system directly processes PCR and performs data analysis without requiring a secondary droplets transfer. The static and independent properties of each droplet dramatically avoid cross-contamination during PCR, provide the opportunity to trace droplets in real-time and simplify the analysis. We demonstrated that the MAP-ddPCR produces reliable data using gradient concentrations of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human genomic cDNA. These concentrations were further verified by quantitative PCR (qPCR). In addition, a very low viral load of SARS-CoV-2 was precisely detected and quantified by the MAP-ddPCR system. Finally, this system is affordable and simpler to integrate compared to other more expensive commercial digital PCR methods. Therefore, the proposed MAP-ddPCR system is expected to have a significant impact on market applications.

Microwave Heating Induced On-Demand Droplet Generation in Microfluidic Systems.

In this note, we report a simple, new method for droplet generation in microfluidic systems using integrated microwave heating. This method enables droplet generation on-demand by using microwave heating to induce Laplace pressure change at the interface of the two fluids. The distance between the interface and junction and microwave excitation power have been found to influence droplet generation. Although this method is limited in generating droplets with a high rate, the fact that it can be integrated with microwave sensing that can be used as the feedback to tune the supply flow of materials presents unique advantages for applications that require dynamic tuning of material properties in droplets.

A versatile droplet on demand generator based on active pressure control.

The development of a novel on-demand droplet generator is reported in this paper based on off-the-shelf electromechanical components. With the ability to produce a broad range of droplet sizes, this device was designed to have minimum components without compromising reliability. The generator performance was assessed by producing water droplets for fire dynamics and suppression studies. Droplet formation at the tip of the nozzle was visualized using high-speed imaging. Stable and unstable regions can be identified easily through the images. Interchangeable nozzles can be used, allowing different droplet sizes. The setting parameters of the device allow accurate control of droplet size and initial speed. The device controls the liquid pressure actively and precisely. The active control of pressure and the ability to have specific droplet ejection parameters ensured the repeatability of droplet conditions. Characterization of the device is presented along with the calibration data, which can be collected in an efficient methodology. These data enable the selection of droplet size and speed when required. This system could easily be implemented in other research areas. Hence, the intention is to share and highlight the generator features for its use in a range of other scientific applications.

Experimental investigation of on-demand ferrofluid droplet generation in microfluidics using a Pulse-Width Modulation magnetic field with proposed correlation

Micro-magnetofluidics offers a promising tool to regulate the drop formation process with versatile applications in engineering and biomedicine. In the present study, on-demand ferrofluid drop generation at a T-junction is investigated utilizing a magnetic pulse. Also, a novel method for ferrofluid droplet formation is introduced using a non-uniform Pulse-Width Modulation (PWM) magnetic field. A novel mechanism of drop generation named “beating regime” was seen for the first time in which the ferrofluid moves back and forth before the breakup. The effect of the magnetic induction, continuous phase flow rate, duty cycle, and applied frequency on the generation frequency and drop diameter was investigated under the PWM magnetic field and compared with those under the DC magnetic field. The results showed that greater values of drop diameter and generation frequency are obtained either when the magnetic induction and/or duty cycle increases or when the applied frequency decreases. The regime maps of ferrofluid droplet formation were presented for different magnetic Bond and Capillary numbers under both PWM and DC magnetic fields and compared with each other. Finally, a correlation was presented to estimate the dimensionless drop diameter on the basis of four nondimensionalized parameters, showing a 7.2 % average relative error.

Experimental study on a solenoid valve-based generator for droplet generation

An on-demand droplet generator is developed based on a high-speed and 2-way solenoid valve and studied experimentally in this paper. It is found that the valve-based droplet generator has good controllability, repeatability and stability. Under a suitable applied pressure, a single droplet can be generated by opening the valve with a short voltage pulse. Besides, the droplet size can be conveniently controlled by the applied liquid pressure and pulse width of the control signal. The results show that the present device successes to avoid the limitation of an appropriate outlet pressure required at nozzle tip for most droplet generators in the literature and is capable of producing droplets in a wide size range for a fixed nozzle diameter.

Electric Triggering for Enhanced Control of Droplet Generation.

Serial femtosecond crystallography (SFX) is a powerful technique that uses X-ray free-electron lasers (XFEL) to determine structures of biomolecular complexes. Specifically, it benefits the study of atomic resolution structures of large membrane protein complexes and time-resolved reactions with crystallography. One major drawback of SFX studies with XFELs is the consumption of large amounts of a protein crystal sample to collect a complete X-ray diffraction data set for high-resolution crystal structures. This increases the time and resources required for sample preparation and experimentation. The intrinsic pulsed nature of all current X-ray sources is a major reason why such large amounts of sample are required. Any crystal sample that is delivered in the path of the X-ray beam during its "off-time" is wasted. To address this large sample consumption issue, we developed a 3D printed microfluidic system with integrated metal electrodes for water-in-oil droplet generation to dynamically create and manipulate aqueous droplets. We demonstrate on-demand droplet generation using DC potentials and the ability to tune the frequency of droplet generation through the application of AC potentials. More importantly, to assist with the synchronization of droplets and XFEL pulses, we show that the device can induce a phase shift in the base droplet generation frequency. This novel approach to droplet generation has the potential to reduce sample waste by more than 95% for SFX experiments with XFELs performed with liquid jets and can operate under low- and high-pressure liquid injection systems.

Microfluidic on-demand droplet generation, storage, retrieval, and merging for single-cell pairing.

A multifunctional microfluidic platform combining on-demand aqueous-phase droplet generation, multi-droplet storage, and controlled merging of droplets selected from a storage library in a single integrated microfluidic device is described. A unique aspect of the technology is a microfluidic trap design comprising a droplet trap chamber and lateral bypass channels integrated with a microvalve that supports the capture and merger of multiple droplets over a wide range of individual droplet sizes. A storage unit comprising an array of microfluidic traps operates in a first-in first-out manner, allowing droplets stored within the library to be analyzed before sequentially delivering selected droplets to a downstream merging zone, while shunting other droplets to waste. Performance of the microfluidic trap is investigated for variations in bypass/chamber hydrodynamic resistance ratio, micro-chamber geometry, trapped droplet volume, and overall flow rate. The integrated microfluidic platform is then utilized to demonstrate the operational steps necessary for cell-based assays requiring the isolation of defined cell populations with single cell resolution, including encapsulation of individual cells within an aqueous-phase droplet carrier, screening or incubation of the immobilized cell-encapsulated droplets, and generation of controlled combinations of individual cells through the sequential droplet merging process. Beyond its utility for cell analysis, the presented platform represents a versatile approach to robust droplet generation, storage, and merging for use in a wide range of droplet-based microfluidics applications.

A digital acoustofluidic device for on-demand and oil-free droplet generation

We report a digital acoustofluidic device for on-demand and oil-free droplet generation. By applying a programmed radio frequency signal to a circular interdigital transducer, the dynamic focused acoustic pressure profiles generated rise up and dispense sample liquids from a reservoir to dynamically eject the droplets into the air. Our device allows droplets to be dispensed on demand with precisely controlled generation time and sequence, and accurate droplet volume. Moreover, we also demonstrate the generation of a droplet with a volume of 24 pL within 10 ms, as well as the encapsulation of a single cell into droplets. This acoustofluidic droplet generation technique is simple, biocompatible, and enables the on-demand droplet generation and encapsulation of many different biological materials with precise control, which is promising for single cell sampling and analysis applications.

Numerical study on droplet generation in axisymmetric flow focusing upon actuation

In the pure axisymmetric flow focusing (AFF), it is difficult to generate uniform droplets due to the random breakup of bulk flows. Therefore, applying external perturbations is a desirable approach to control the monodispersity of the droplets and makes it possible to produce uniform droplets at relatively high flow rates. In the present study, the effects of different external perturbations (waveform shape, frequency f and amplitude A) on the droplet generation are numerically investigated. When the focused phase is singly actuated, the size of the uniform droplets can be obtained and manipulated by adjusting f and A. In particular, the jet breakup has the same frequency as the external perturbation in the generation of uniform droplets. There exists a cutoff frequency beyond which the perturbation cannot control the jet breakup, even with large A. This is found to be associated with the critical condition for the onset of the Rayleigh-Plateau instability. In addition, the reservoir effect of the cone in the AFF effectively reduces the influence of the perturbation on the liquid supply to the liquid jet, accounting for the presence of jetting at low f and large A. Moreover, we apply the perturbations either singly to the focusing phase or simultaneously to the focused and focusing phases and assess their effects on the jet breakup. Finally, the square waveform perturbations acting on the droplet generation are discussed. The present work provides a guidance of the actuation-aided AFF for practical applications of on-demand droplet generation.