High-throughput detection of ethanol-producing cyanobacteria in a microdroplet platform

Ethanol production by microorganisms is an important renewable energy source. Most processes involve fermentation of sugars from plant feedstock, but there is increasing interest in direct ethanol production by photosynthetic organisms. To facilitate this, a high-throughput screening technique for the detection of ethanol is required. Here, a method for the quantitative detection of ethanol in a microdroplet-based platform is described that can be used for screening cyanobacterial strains to identify those with the highest ethanol productivity levels. The detection of ethanol by enzymatic assay was optimized both in bulk and in microdroplets. In parallel, the encapsulation of engineered ethanol-producing cyanobacteria in microdroplets and their growth dynamics in microdroplet reservoirs were demonstrated. The combination of modular microdroplet operations including droplet generation for cyanobacteria encapsulation, droplet re-injection and pico-injection, and laser-induced fluorescence, were used to create this new platform to screen genetically engineered strains of cyanobacteria with different levels of ethanol production.     

Lipid-coated microdroplet array for in vitro protein synthesis

Monitoring complex biological assays such as in vitro protein synthesis over long periods in micrometer-sized cavities of poly(dimethyl siloxane) (PDMS) microfluidic devices requires a strategy that solves the adsorption and absorption problems on PDMS surfaces. In this study, we developed a technique that instantaneously arrays aqueous microdroplets coated with a phospholipid membrane within a single microfluidic device. The simple lipid bilayer coating effectively inhibits the adsorption of proteins and DNA, whereas the encapsulation of the droplet reduces the area in contact with the PDMS surface, resulting in decreased absorption in part. Although the size becomes smaller during the first few hours, a lipid-coated microdroplet array demonstrated a temporal stability of more than 20 h and a size uniformity of CV 3% in the device. Furthermore, we succeeded in expressing a green fluorescent protein by confining an in vitro translation system in the microdroplets, which was confirmed by scanning the fluorescence spectrum of the droplets, demonstrating that the lipid coat secured the synthetic reaction from the adsorption problem.     

Kinetic analysis of ion pair extraction of an alkyl sulfate anion across a liquid/liquid interface by fluorescence microspectroscopy and microelectrochemistry of single microdroplets

Mass transfer of an alkyl sulfate anion with a (ferrocenylmethyl)trimethylammonium cation from water into single oil microdroplets was kinetically studied by microcapillary injection, fluorescence microspectroscopy, and microelectrochemistry. The partitioning ratio and the extraction rate significantly depended on the alkyl chain length of the alkyl sulfate anion. The extraction rate for the n-dodecyl sulfate or n-tridecyl sulfate ion was proportional to both the alkyl sulfate ion and ferrocene derivative concentrations while that for the n-undecyl sulfate ion was proportional to the anion concentration alone. The results are discussed in terms of transfer of the individual ions across the microdroplet/water interface and adsorption/ion pair formation of the alkyl sulfate ion at the microdroplet/water interface.     

Magnetophoretic velocimetry of manganese(II) in a single emulsion droplet at the femtomole level.

We developed a new experimental technique named magnetophoretic velocimetry to determine a small amount of paramagnetic species in a single microdroplet. The magnetophoretic velocity of an aqueous droplet containing paramagnetic metal ion dispersed in an organic medium could response to a very small amount of the metal ion under an inhomogeneous magnetic field. The paramagnetic droplet (2 approximately 8 microm diam) used as a test sample in this study was the aqueous droplet of manganese(II) chloride dispersed in ethylbenzoate whose density was nearly equal to water. A pair of small Nd-Fe-B magnets placed with a gap of 400 microm generated an inhomogeneous magnetic field between the edges, at which the product of the magnetic flux density and the gradient, B(dB/dx), was as high as 410 T2 m(-1). When a silica capillary containing the emulsion was inserted into the gap between the magnets, the magnetophoretic migration of the droplets was observed with a video microscope. The magnetophoretic velocity divided by the squared radius of the droplet was proportional to the MnCl2 concentration in the droplet, as predicted by a theoretical calculation. The estimated detection limit in this simple method was lower than 10(-16) mol for manganese(II).     

Electrophoresis of a charged droplet in a dielectric liquid for droplet actuation

Electrophoretic motion of a charged droplet in a dielectric fluid under an electric field has been investigated experimentally for use as a microdroplet actuation method. The effects of the droplet size, electric field strength, and electrolyte concentration and ion species on the charging of an aqueous droplet have been examined. The amount of electrical charging has been measured by two different methods: indirect measurement using the image analysis of droplet motion and direct measurement using the electrometer. Quantitative comparison of the droplet charge measured experimentally and the theoretical value of a perfectly conductive sphere shows that an aqueous droplet is less charged than the corresponding perfectly conductive sphere. The limiting effect on electrical charging is more significant for an electrolyte droplet, and the effect is positively correlated to the electrolyte concentration rather than the ion species. This implies that the low electrical conductivity of water is not a major cause of the limiting effect. The scaling law of the charging amount for a deionized water droplet nearly follows that of the perfect conductor, whereas for an electrolyte droplet, the scaling law exponent is slightly higher. Some advantages and potentials of the current droplet actuation method are also discussed in comparison with the conventional ones.     

Sizing subcellular organelles and nanoparticles confined within aqueous droplets

This article describes two complementary techniques, single-particle tracking and correlation spectroscopy, for accurately sizing nanoparticles confined within picoliter volume aqueous droplets. Single-particle tracking works well with bright particles that can be continuously illuminated and imaged, and we demonstrated this approach for sizing single fluorescent beads. Fluorescence correlation spectroscopy detects small intensity bursts from particles or molecules diffusing through the confocal probe volume, which works well with dim and rapidly diffusing particles or molecules; we demonstrated FCS for sizing synaptic vesicles confined in aqueous droplets. In combination with recent advances in droplet manipulations and analysis, we anticipate this capability to size single nanoparticles and molecules in free solution will complement existing tools for probing cellular systems, subcellular organelles, and nanoparticles.     

Determination of aqueous solubility and surface adsorption of polycyclic aromatic hydrocarbons by laser multiphoton ionization

Polycyclic aromatic hydrocarbons (PAHs) are of considerable analytical interest due to their environmental effects. On-line monitoring of these compounds based on microdroplet sampling requires accurate characterization of their solubilities and surface adsorption. A new method to determine aqueous solubility and surface adsorption of PAHs is suggested, using a combination of microdroplet sampling and multiphoton ionization-based fast-conductivity (MPI-FC) techniques. Controlled droplet contamination by PAHs was performed by external deposition upon direct contact with renewable water droplets (?10 μL). Our approach relies on the finding that, at the onset of aqueous equilibrium solubility, X(E), a sharp increase in the detected photocharges is recorded. We show that this is directly related to enhanced surface adsorption of a particular PAH material. This behavior manifests itself in slope variation of the respective calibration curves. The PAH materials used, which have provided about four decades variation in X(E) figures, were anthracene, perylene, pyrene, and phenanthrene. Applicability of the method for studying the surface excess and adsorption is specified. The MPI-FC technique provides an easy analytical tool for measuring low aqueous solubility of organic compounds and characterization of their adsorption, with no need for laborious sample preparation routines.     

基于电液动力学方法的非连续药物传输系统微液滴形成和中和效应分析(英文)

应用电液动力学方法形成微液滴是目前非连续药物传输微系统发展中的关键问题.本文介绍了一种基于电液动力学原理,可有效形成微液滴的药物传输系统.系统参数包括:外加电场1 500 V,两电极距离100μm,针管内径20μm,整体体积为2 mm×2 mm×3 mm.基于微电液动力学原理、微流体效应、带电颗粒中和和药品物理性质,详细分析了微液滴的形成过程,理论计算出微液滴的尺寸和形成时间.根据设计,需要8.5 ms可形成体积为2.83 nL的微小液滴.此项研究提供了一种基于电液动力学方法的可重复、稳定、可控地形成微流体的药物传输微系统.     

Compartmentalization of electrophoretically separated analytes in a multiphase microfluidic platform

Herein, we describe the monolithic integration of a multiphase microfluidic system to a microcapillary gel electrophoresis (μCGE) architecture for the complete isolation and storage of separated analyte bands. Within this platform, analyte molecules are separated using microchannel gel electrophoresis, and the eluted bands are stored in a sequence of approximately 40-600 encapsulating microdroplets. Importantly, employing such a system allows for total control of droplet size, shape, and composition. This approach is utilized to separate, optically detect, and encapsulate two fluorescent analytes from a composite sample mixture. Further to this, we subsequently investigate the potential of the system to be used as a concentration gradient generator through analysis of the segmented analyte bands and droplet composition.     

Fabrication of Micropatterned Dipeptide Hydrogels by Acoustic Trapping of Stimulus‐Responsive Coacervate Droplets

Acoustic standing waves offer an excellent opportunity to trap and spatially manipulate colloidal objects. This noncontact technique is used for the in situ formation and patterning in aqueous solution of 1D or 2D arrays of pH‐responsive coacervate microdroplets comprising poly(diallyldimethylammonium) chloride and the dipeptide N‐fluorenyl‐9‐methoxy‐carbonyl‐D‐alanine‐D‐alanine. Decreasing the pH of the preformed droplet arrays results in dipeptide nanofilament self‐assembly and subsequent formation of a micropatterned supramolecular hydrogel that can be removed as a self‐supporting monolith. Guest molecules such as molecular dyes, proteins, and oligonucleotides are sequestered specifically within the coacervate droplets during acoustic processing to produce micropatterned hydrogels containing spatially organized functional components. Using this strategy, the site‐specific isolation of multiple enzymes to drive a catalytic cascade within the micropatterned hydrogel films is exploited.     

Electro‐Assisted Bioprinting of Low‐Concentration GelMA Microdroplets

Low‐concentration gelatin methacryloyl (GelMA) has excellent biocompatibility to cell‐laden structures. However, it is still a big challenge to stably fabricate organoids (even microdroplets) using this material due to its extremely low viscosity. Here, a promising electro‐assisted bioprinting method is developed, which can print low‐concentration pure GelMA microdroplets with low cost, low cell damage, and high efficiency. With the help of electrostatic attraction, uniform GelMA microdroplets measuring about 100 μm are rapidly printed. Due to the application of lower external forces to separate the droplets, cell damage during printing is negligible, which often happens in piezoelectric or thermal inkjet bioprinting. Different printing states and effects of printing parameters (voltages, gas pressure, nozzle size, etc.) on microdroplet diameter are also investigated. The fundamental properties of low‐concentration GelMA microspheres are subsequently studied. The results show that the printed microspheres with 5% w/v GelMA can provide a suitable microenvironment for laden bone marrow stem cells. Finally, it is demonstrated that the printed microdroplets can be used in building microspheroidal organoids, in drug controlled release, and in 3D bioprinting as biobricks. This method shows great potential use in cell therapy, drug delivery, and organoid building.     

A unified analytical model for the flight distance of flying microdroplet before solidification

Heat dissipation and solidification of a flying microdroplet are omnipresent in nature and industrial applications. The current study lacks a model to determine the critical flight distance before the microdroplet solidification, which is crucial for powder production and microdroplet jet printing in additive manufacturing. This study investigated the transient velocity and temperature attenuation of a flying microdroplet. Both the velocity and temperature of the microdroplet were found to be decayed exponentially with the flying time. Based on energy conversion and flying velocity integration, an analytical model was proposed to determine the critical flight distances that the microdroplet begins and ends to solidify. Moreover, the parameters affecting the critical flight distance were unified into a nondimensional formula. Finally, we validated the proposed models experimentally through the deposited morphologies of copper microdroplets induced by laser pulses.     

基于声表面波传感技术的微液滴检测方法

为了提高声表面波(SAW)技术用于微液滴体积测量的精度,提出了一种差分面积拟合法.根据声表面波信号的特点分析了差分面积拟合法的合理性,在自行研制的声表面波液滴体积检测系统上对该方法的应用进行了验证.采用两种黏性不同的微液滴,分别为5～9μL的微水滴和微油滴进行了测量.对测得的信号分别采用曲线峰值法和差分面积拟合法与微液滴大小进行回归.实验结果表明,采用差分面积拟合法进行回归得到的模型的相关系数相比采用峰值法提高了4%以上,同时对两种微液滴的预测均方根误差(RMSEP)减小了37%以上,从而证明了该方法的有效性.     

Simultaneous spatial and spectral imaging of lasing droplets

We present an experimental technique that allows the simultaneous spatial imaging and spectral analysis of falling droplets that exhibit lasing. Single droplet investigations serve as, among other purposes, a preliminary study for spray and combustion researchers. The described setup provides a valuable tool for the evaluation of microdroplet investigations with laser-spectroscopic techniques that rely on laser-induced fluorescence (LIF) or similar spectroscopical phenomena. The emphasis is that both spatial and spectral information are obtained from single-shot images of a falling droplet. Furthermore, combining spatial imaging and a spatially resolving optical multichannel analyzer makes a pointwise rastering of the droplets spectrum possible. This allows for the (almost) unambiguous determination of sources of influence on the spectrum of these droplets-such as geometrical distortion and lasing, nondissolved tracer lumps, and similar phenomena. Although the focus is on the experimental technique itself, we supplement detailed studies of lasing in falling microdroplets. These results were obtained with the aim of developing a system for measuring temperature distributions in droplets and sprays. In the light of these results the practice of calibrating a droplets spectrum by use of a bulk liquid sample needs to be critically reviewed.     

Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets

This paper describes a method, which combines optical trapping and microfluidic-based droplet generation, for selectively and controllably encapsulating a single target cell or subcellular structure, such as a mitochondrion, into a picoliter- or femtoliter-volume aqueous droplet that is surrounded by an immiscible phase. Once the selected cell or organelle is encased within the droplet, it is stably confined in the droplet and cannot be removed. We demonstrate in droplet the rapid laser photolysis of the single cell, which essentially "freezes" the state that the cell was in at the moment of photolysis and confines the lysate within the small volume of the droplet. Using fluorescein di-beta-d-galactopyranoside, which is a fluorogenic substrate for the intracellular enzyme beta-galactosidase, we also assayed the activity of this enzyme from a single cell following the laser-induced lysis of the cell. This ability to entrap individual selected cells or subcellular organelles should open new possibilities for carrying out single-cell studies and single-organelle measurements.     

Dynamics of Formation and Parameters of a Fog upon Abrupt Expansion of a Compressed Vapor–Gas Volume

This paper deals with an experimental investigation of structural changes in the compressed vapor–gas volume of a water vapor–hydrogen–air system, which are caused by an abrupt expansion of the volume due to the loss of integrity of the confinement shell. A homogeneous system transforms to a heterogeneous water vapor–microdroplets–hydrogen–air mixture behind the rarefaction wave. The microdroplet sizes and their bulk concentration are measured during the formation of a fog. A method is proposed for estimating the volume fraction of microdroplets from the measurements of light absorption on one wavelength of the IR region.     

Investigation of a phase transition in a single optically levitated microdroplet by Raman-Mie scattering

Light-scattering measurements of optically levitated microdroplets containing three components, glycerin, water, and ammonium sulfate, are presented. Evaporation of the microdroplet is studied by means of morphology-dependent resonances observed in both Raman spectra as well as elastically scattered light and by the simultaneous measurement of the laser power. The phase transition from the liquid to the solid state of ammonium sulfate inside the microdroplet is observed by means of morphology-dependent resonances and Raman scattering.     

High-Performance Unidirectional Manipulation of Microdroplets by Horizontal Vibration on Femtosecond Laser-Induced Slant Microwall Arrays

The high-performance unidirectional manipulation of microdroplets is crucial for many vital applications including water collection and bioanalysis. Among several actuation methods (e.g., electric, magnetic, light, and thermal actuation), mechanical vibration is pollution-free and biocompatible. However, it suffers from limited droplet movement mode, small volume range (V_Max/V_Min < 3), and low transport velocity (≤11.5 mm s⁻¹) because the droplet motion is a sliding state caused by the vertical vibration on the asymmetric hydrophobic microstructures. Here, an alternative strategy is proposed—horizontal vibration for multimode (rolling, bouncing/reverse bouncing, converging/diffusing, climbing, 90^o turning, and sequential transport), large-volume-range (V_Max/V_Min ≈ 100), and high-speed (≈22.86 mm s⁻¹) unidirectional microdroplet manipulation, which is ascribed to the rolling state on superhydrophobic slant microwall arrays (SMWAs) fabricated by the one-step femtosecond laser oblique ablation. The unidirectional transport mechanism relies on the variance of viscous resistance induced by the difference of contact area between the microdroplet and the slant microwalls. Furthermore, a circular, curved, and “L”-shaped SMWA is designed and fabricated for droplet motion with particular paths. Finally, sequential transport of large-volume-range droplets and chemical mixing microreaction of water-based droplets are demonstrated on the SMWA, which demonstrates the great potential in the field of microdroplet manipulation.     

Biocatalytic Self‐Assembly of Nanostructured Peptide Microparticles using Droplet Microfluidics

Uniformly‐sized, nanostructured peptide microparticles are generated by exploiting the ability of enzymes to serve (i) as catalysts, to control self‐assembly within monodisperse, surfactant‐stabilized water‐in‐oil microdroplets, and (ii) as destabilizers of emulsion interfaces, to enable facile transfer of the produced microparticles to water. This approach combines the advantages of biocatalytic self‐assembly with the compartmentalization properties enabled by droplet microfluidics. Firstly, using microfluidic techniques, precursors of self‐assembling peptide derivatives and enzymes are mixed in the microdroplets which upon catalytic conversion undergo molecular self‐assembly into peptide particles, depending on the chemical nature of the precursors. Due to their amphiphilic nature, enzymes adsorb at the water‐surfactant‐oil interface of the droplets, inducing the transfer of peptide microparticles from the oil to the aqueous phase. Ultimately, through washing steps, enzymes can be removed from the microparticles which results in uniformely‐sized particles composed of nanostructured aromatic peptide amphiphiles.     

液液两相流法制备海藻酸钠包覆的液态金属微滴

本文以室温液态金属(GaInSn)为分散相、海藻酸钠(NaAlg)溶液(1wt%)为连续相,采用液液两相流方法,在竖直共轴微通道中,制备得到NaAlg凝胶包覆的多个GaInSn微滴,具有单分散、尺寸一致的特点。GaInSn/NaAlg两相流存在四种流型:分散相滴流、分散相柱塞流、连续相滴流和连续相射流。GaInSn微滴的包覆模式三种:Squeezing、Dripping和Compound Jetting,其中Dripping和Compound Jetting是主要的包覆模式。在较低的GaInSn流量下,NaAlg流量增加到一定程度后,包覆模式由Dripping转变Compound Jetting。固定两相流量比,随着两相流量的同比例增加,GaInSn微滴的特征频率呈线性增加、包覆个数增加、特征长度变化不显著。