High-throughput sorting of nanoliter droplets enabled by a sequentially addressable dielectrophoretic array

Droplet microfluidics has emerged as a powerful tool for a diverse range of biomedical and industrial applications such as single-cell analysis, directed evolution, and metabolic engineering. In these applications, droplet sorting has been effective for isolating small droplets encapsulating molecules, cells, or crystals of interest. Recently, there is an increased interest in extending the applicability of droplet sorting to larger droplets to utilize their size advantage. However, sorting throughputs of large droplets have been limited, hampering their wide adoption. Here, we report our demonstration of high-throughput fluorescence-activated droplet sorting of 1 nL droplets using an upgraded version of the sequentially addressable dielectrophoretic array (SADA), which we reported previously. The SADA is an array of electrodes that are individually and sequentially activated/deactivated according to the speed and position of a droplet passing nearby the array. We upgraded the SADA by increasing the number of driving electrodes constituting the SADA and incorporating a slanted microchannel. By using a ten-electrode SADA with the slanted microchannel, we achieved fluorescence-activated droplet sorting of 1 nL droplets at a record high throughput of 1752 droplets/s, twice as high as the previously reported maximum sorting throughput of 1 nL droplets.     

微流控芯片液滴生成与检测技术研究进展

微流控芯片液滴技术是一种操控微小体积液体的新技术,既可实现高通量微观样本的生成及控制,也可进行独立液滴的操作.分散的微液滴单元可作为理想的微反应器,在生物医药中的药物筛选、材料筛选和高附加值微颗粒材料合成领域展现出巨大的应用潜力.液滴微流控芯片是利用流体剪切力的改变,使互不相溶的两相流体在其界面处生成稳定、有序的液滴,...     

Partitioning invertase between a dilute water solution and generated droplets

Water droplets or mist occur naturally in the air at seashores. These water droplets carry inorganic and organic substances from the sea to the land via the air, creating fertile land in sandy coastal areas (1). The same phenomenon occurs in an air-fluidized bed bioreactor (2). In an air-fluidized bed reactor, proteins can be transferred from the bioreactor semisolid bulk phase to an enriched droplet phase. This protein transfer process (droplet fractionation) can be experimentally simulated by shaking a separatory funnel containing a dilute solution of a given protein, which can be an enzyme like invertase. The created droplets become richer in invertase (protein) than that of the original dilute solution. The droplets can then be coalesced by tranpping them and recovering the concentrated protein in the new liquid phase. Typically, in such a droplet fractionation process a collected enzyme can be degraded in its ability tocatalyze a chemical reaction. In this article, we explore whether the initial solution pH control variable can be adjusted to minimize the decrease of enzyme activity in this process. The protein droplet recovery problem is one in which the recovered amount of desired protein (enzyme) in the droplet is maximized, subject to the minimization of the enzyme activity loss. The partition coefficient, which is the ratio between the protein concentration in the droplets and the residual solution, is maximized at approx 4.8 and occurs at pH 3.0. Here, the partition coefficient for invertase decreases as the initial solution pH increases, between pH 3.0 and 8.0. Interestingly, the initial solution surface tension seems to be inversely proportional to the partition coefficient. The partition coefficien treachesa maximum value at a surface tension value of approx 63 mN/m at pH 3.0. The enzymatic activity of the initial, the residual, and the droplet solutions all decrease as the bulksolution pH increases. Adecrease of enzymatic activity was observed in the residual bulk solution when compared with that in the initial bulk solution at all pH levels. Also, up to 90% of the invertase activity was lost in the droplets when compared to the initial bulk solution.     

Droplet on a liquid substrate: Wetting,dewetting, dynamics,instabilities

Droplets on a liquid substrate (‘liquid lenses’) play an important role in various branches of engineering, including microfluidics, chemical engineering, environment protection, etc. In the present paper, we discuss basic phenomena characteristic for liquid lenses. We recall classical results on the shape of an equilibrium droplet and the kinds of droplet wetting. We overview briefly the main theoretical approaches used for the analysis of droplet dynamics, discuss the phenomena accompanying a droplet impact, physical effects used for droplet manipulations, and the factors that determine the interaction between droplets. We describe the main types of droplet instabilities leading to oscillations, self-propulsion, and disintegration of droplets. Some promising directions of further research are listed.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

An opinion on the multiscale nature of Covid-19 type disease spread

Recognizing the multiscale, interdisciplinary nature of the Covid-19 transmission dynamics, we discuss some recent developments concerning an attempt to construct a disease spread model from the flow physics of infectious droplets and aerosols and the frequency of contact between susceptible individuals with the infectious aerosol cloud. Such an approach begins with the exhalation event–specific, respiratory droplet size distribution (both airborne/aerosolized and ballistic droplets), followed by tracking its evolution in the exhaled air to estimate the probability of infection and the rate constants of the disease spread model. The basic formulations and structure of submodels, experiments involved to validate those submodels, are discussed. Finally, in the context of preventive measures, respiratory droplet–face mask interactions are described.     

Simultaneous generation of multiple aqueous droplets in a microfluidic device

This paper describes a microfluidic platform for the on-demand generation of multiple aqueous droplets, with varying chemical contents or chemical concentrations, for use in droplet based experiments. This generation technique was developed as a complement to existing techniques of continuous-flow (streaming) and discrete-droplet generation by enabling the formation of multiple discrete droplets simultaneously. Here sets of droplets with varying chemical contents can be generated without running the risk of cross-contamination due to the isolated nature of each supply inlet. The use of pressure pulses to generate droplets in parallel is described, and the effect of droplet size is examined in the context of flow rates and surfactant concentrations. To illustrate this technique, an array of different dye-containing droplets was generated, as well as a set of droplets that displayed a concentration gradient of a fluorescent dye.     

Investigating the effects of precursor concentration and gelling parameters on droplet-based generation of Ca-Alginate microgels: identifying new stable modes of droplet formation

Droplet-based microfluidics is an attractive approach for producing microgels due to its high potential to control the size and shape of the particles and precisely entrap the substances within the hydrogel matrix. However, the microfluidic generation of monodisperse microgels with desired structures is still challenging. Indeed, the rheological and interfacial properties of the immiscible fluids, as well as the adopted gelling strategy, play important roles in microfluidic methods. Herein, sodium alginate droplets with different concentrations are generated via a microfluidic device with a flow-focusing unit. Besides, a combined in situ and ex situ strategy is optimized to crosslink sodium alginate droplets in the presence of calcium ions. The effects of alginate concentration and junction width in the flow focusing unit are investigated on droplet size and droplet formation regimes. It is observed that by increasing the alginate concentration, the dripping regime of droplet formation may be transformed to one of the binary dripping or quasijetting regimes. In the binary dripping regime, two successive different-sized droplets are generated in each period of droplet formation, which leads to low monodispersity in the collected droplets. However, the droplets produced in the quasijetting regime are interestingly monodisperse and also smaller than those of the dripping and binary dripping regimes. The breakup dynamics of the alginate thread is also analyzed with a computational fluid dynamics (CFD) code. This analysis discloses that the viscous stresses, as well as the viscous dissipation, have important roles in controlling the stable modes of droplet formation.     

Spontaneous generation of emulsion droplets by autonomous fluid-pumping using the gas permeability of poly(dimethylsiloxane) (PDMS)

Conventional droplet-based microfluidic systems require expensive, bulky external apparatuses, such as electric power supplies and pressure-driven pumps for fluid transportation. This study demonstrates an alternative way to produce emulsion droplets by autonomous fluid-handling based on the gas permeability of poly(dimethylsiloxane) (PDMS). Furthermore, basic concepts of fluid-handling are expanded to control the direction of the microfluid in the microfluidic device. The alternative pumping energy resulting from the high gas permeability of PDMS is used to generate water-in-oil (W/O) emulsions, which require no additional structures apart from microchannels. We can produce emulsion droplets by simple loading of the oil and aqueous solutions into the inlet reservoirs. During the operation of the microfluidic device, changes in droplet size, volumetric flow rate, and droplet generation frequency were quantitatively analyzed. As a result, we found that changes in the wetting properties of the microchannel greatly influence the volumetric flow rate and droplet generation frequency. This alternative microfluidic approach for preparing emulsion droplets in a simple and efficient manner is designed to improve the availability of emulsion droplets for point of care bioanalytical applications, in situ synthesis of materials, and on-site sample preparation tools.     

Electrical actuation of dielectric droplets by negative liquid dielectrophoresis

This paper presents an electrical actuation scheme of dielectric droplet by negative liquid dielectrophoresis. A general model of lumped parameter electromechanics for evaluating the electromechanical force acting on the droplets is established. The model reveals the influence of actuation voltage, device geometry, and dielectric parameter on the actuation force for both conductive and dielectric medium. Using this model, we compare the actuation forces for four liquid combinations in the parallel-plate geometry and predict the low voltage actuation of dielectric droplets by negative dielectrophoresis. Parallel experimental results demonstrate such electric actuation of dielectric droplets, including droplet transport, splitting, merging, and dispending. All these dielectric droplet manipulations are achieved at voltages < 100 V_rms. The frequency dependence of droplet actuation velocity in aqueous solution is discussed and the existence of surfactant molecules is believed to play an important role by realigning with the AC electric field. Finally, we present coplanar manipulation of oil and water droplets and formation of oil-in-water emulsion droplet by applying the same low voltage.     

Controlled generation of droplets using an electric field in a flow-focusing paper-based device

Droplet-based microfluidics is a modular platform in high-throughput single-cell and small sample analyses. However, this droplet microfluidic system was widely fabricated using soft lithography or glass capillaries, which is expensive and technically demanding for various applications, limiting use in resource-poor settings. Besides, the variation in droplet size is also restricted due to the limitations on the operating forces that the paper-based platform is able to withstand. Herein, we develop a fully integrated paper-based droplet microfluidic platform for conducting droplet generation and cell encapsulation in independent aqueous droplets dispersed in a carrier oil by incorporating electric fields. Through imposing an electric field, the droplet size would decrease with increasing the electric field and smaller droplets can be produced at high applied voltage. The droplet diameter can be adjusted by the ratio of inner and outer flow velocities as well as the applied electric field. We also demonstrated the proof of concept encapsulation application of our paper device by encapsulating yeast cells under an electric field. Using a simple wax printing method, carbon electrodes can be integrated on the paper. The integrated paper-based microfluidic platform can be fabricated easily and conducted outside of centralized laboratories. This microfluidic system shows great potential in drug and cell investigations by encapsulating cells in resource-limited environments.     

A new droplet breakup phenomenon in electrokinetic flow through a microchannel constriction

A completely new droplet breakup phenomenon is reported for droplets passing through a constriction in an electrokinetic flow. The breakup occurs during the droplet shape recovery process past the constriction throat by the interplay of the dielectrophoretic stress release and the interface energy for droplets with smaller permittivity than that of the ambient fluid. There are conditions for constriction ratios and droplet size that the droplet breakup occurs. The numerical predictions provided here require experimental verification, and then can give rise to a novel microfluidic device design with novel droplet manipulations.     

Electrochemical Detection of Droplets in Microfluidic Devices: Simultaneous Determination of Velocity,Size and Content

Microfluidic devices were designed to electrochemically detect in a two‐phase flow the velocity, size and content of aqueous droplets containing redox species. The principle of these determinations is based on the analysis of a unique chronoamperometric response recorded during the passage of a droplet over channel microelectrodes. Two configurations of electrochemical cell with different geometries were investigated both theoretically and experimentally. Velocity and size of droplets, as well as internal recirculating convection within droplets, were evaluated from chronoamperometric curves by specific transition times depending on the cell configuration. In addition, the droplet content was probed from the Faradaic current controlled by mass transport and by internal hydrodynamic regime. For droplet velocity and size, experimental data were systematically compared to optical measurements. All the results demonstrated the high performance of the electrochemical detection reached under these conditions. They successfully validate the concept of self‐consistent electrochemical detections of aqueous droplets within microchannels for the simultaneous determination of their velocity, size and content.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应(PCR)技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21μm的液滴.利用"玻璃天花板"的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹～10⁵copies/μL范围内呈现良好的线性关系(R²=0.998).该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

The head-on colliding process of binary liquid droplets at low velocity: high-speed photography experiments and modeling

The experimental and theoretical studies are reported in this paper for the head-on collisions of a liquid droplet with another of the same fluid resting on a solid substrate. The droplet on the hydrophobic polydimethylsiloxane (PDMS) substrate remains in a shape of an approximately spherical segment and is isometric to an incoming droplet. The colliding process of the binary droplets was recorded with high-speed photography. Head-on collisions saw four different types of response in our experiments: complete rebound, coalescence, partial rebound with conglutination, and coalescence accompanied by conglutination. For a complete rebound, both droplets exhibited remarkable elasticity and the contact time of the two colliding droplets was found to be in the range of 10–20 ms. With both droplets approximately considered as elastic bodies, Hertz contact theory was introduced to estimate the contact time for the complete rebound case. The estimated result was found to be on the same order of magnitude as the experimental data, which indicates that the present model is reasonable.     

丙烯酰胺在聚乙二醇水溶液中聚合产品的微观形态

采用偶氮类水溶性引发剂2,2′-偶氮二异丙基咪唑啉二盐酸盐(VA044)引发丙烯酰胺(AM)在聚乙二醇(PEG)水溶液中的双水相聚合;研究了引发剂、单体、聚乙二醇浓度及温度对最终产品中聚丙烯酰胺(PAM)液滴形态、尺寸的影响.随着引发剂浓度的增加,液滴由球状变为细长条状;随着温度的上升,球状液滴逐渐趋于条状,然后又重新趋于球状;在初始单体浓度较低时,PAM液滴滴径分布较窄,当其浓度增加后,滴径呈多峰分布;随着PEG浓度的增加,聚合物液滴趋于球状。     

Crystallization of condensation droplets on a liquid surface

Highly ordered microporous two-dimensional membranes have been obtained from polymer solutions (Widawski et al. (1994) Nature 369: 397–399). Recently, a mechanism for the formation of such membranes was proposed, involving water vapour condensation (induced by the rapid evaporation of the volatile solvent) onto the surface of solutions and the formation of floating water droplets. Unfortunately, the droplets growth process was not observed, and consequently only qualitative information was reported. In the present paper, results of light-scattering experiments with this system are reported. The formation of water droplets growing at the surface of the solution has been observed and the evolution with time of the mean droplet radius has been found to be described by a power law with an exponent of 1/3, proving that no coalescence processes occur. This particular behaviour is attributed to the precipitation of the polymer at the water/solution interface and to the formation of a mechanically resistant polymer layer encapsulating each droplet. In this way, water droplets behave like solid particles, allowing compact sheets to be formed. The presence of important surface currents is believed to promote the formation of “polycrystal” and “monocrystal” patterns. Received: 4 January 1999 Accepted in revised form: 15 February 1999     

A simplified PDMS microfluidic device with a built-in suction actuator for rapid production of monodisperse water-in-oil droplets

We previously established an automatic droplet-creation technique that only required air evacuation of a PDMS microfluidic device prior to use. Although the rate of droplet production with this technique was originally slow (∼10 droplets per second), this was greatly improved (∼470 droplets per second) in our recent study by remodeling the original device configuration. This improvement was realized by the addition of a degassed PDMS layer with a large surface area-to-volume ratio that served as a powerful vacuum generator. However, the incorporation of the additional PDMS layer (which was separate from the microfluidic PDMS layer itself) into the device required reversible bonding of five different layers. In the current study, we aimed to simplify the device architecture by reducing the number of constituent layers for enhancing usability of this microfluidic droplet generator while retaining its rapid production rate. The new device consisted of three layers. This comprised a degassed PDMS slab with microfluidic channels on one surface and tens of thousands of vacuum-generating micropillars on the other surface, which was simply sandwiched by PMMA layers. Despite its simplified configuration, this new device created monodisperse droplets at an even faster rate (>1000 droplets per second).     

流动聚焦型微流控体系中的液滴的图案化排列和转变模式

流体在微流通道中形成剪切流场（低雷诺数）．不同于宏观体系,由于剪切力和表面张力的竞争作用,产生的液滴在微尺度下的微流通道中形成特殊的排列现象---周期性类似“晶格”排列现象．设计了新型流动聚焦型微流控芯片,分析研究在微流体系中液滴周期性图案化排列和转变机理性,液滴排列模式受两方面因素影响：水油两相的流速比值和微通道尺寸．当微通道宽度为250或300 μm时,液滴形成单层分散,双层和单层挤压排列．当微通道宽度为350 μm 时,液滴会形成单层分散到三层排列到双层挤压最后到单层挤压排列．当出口通道宽度增加到400 μm时,甚至出现了液滴四层排列的现象．同时研究了各个液滴排列模式的“转变点”.     

水滴在超疏水植物叶片上的沉积方法和机理研究进展

自然界中有很多超疏水植物叶片, 水滴撞击在这些表面时极易产生溅射和反弹, 造成农用化学品喷雾施药时药物的大量损失, 利用率低下, 从而重复喷洒施药. 农用化学品过度使用将造成食品安全、 农药残留、 水资源浪费及环境生态污染等问题. 因此, 增加水滴在超疏水植物叶片表面的沉积效率对提高农药利用率尤为重要. 本文从分析水滴在超疏水表面的撞击动力学特征开始, 结合添加助剂后液滴的物理化学性质, 系统阐述了水滴在超疏水植物叶片上的沉积方法和机理, 并提出筛选助剂和研究机理不仅要考虑助剂性质还要结合基底结构、 撞击动力学特征等因素, 而且还要考虑单水滴尺寸大小、 基底运动和弹性及环境因素等对沉积的影响. 本文对农药喷洒及生物医学、 机械工程、 涂料喷涂和油墨打印等领域均有指导意义和应用价值.