A review on emulsification via microfluidic processes

Emulsion is a disperse system with two immiscible liquids, which demonstrates wide applications in diverse industries. Emulsification technology has advanced well with the development of microfluidic process. Compared to conventional methods, the microfluidics-based process can produce controllable droplet size and distribution. The droplet formation or breakup is the result of combined effects resulting from interfacial tension, viscous, and inertial forces as well as the forces generated due to hydrodynamic pressure and external stimuli. In the current study, typical microfluidic systems, including microchannel array, T-shape, flow-focusing, co-flowing, and membrane systems, are reviewed and the corresponding mechanisms, flow regimes, and main parameters are compared and summarized.     

Breakup dynamics for high‐viscosity droplet formation in a flow‐focusing device: Symmetrical and asymmetrical ruptures

The breakup mechanism of high‐viscosity thread for droplet formation in a flow‐focusing device is investigated using a high‐speed digital camera. Aqueous solution of 89.5%‐glycerol is used as the dispersed phase, while silicone oil as the continuous phase. The breakup process of the dispersed thread presents two categories: symmetrical rupture and asymmetrical rupture. Furthermore, the rupture behavior could be divided into two stages: the squeezing stage controlled by the squeezing pressure and the pinch‐off stage controlled by viscous stresses of both phases and surface tension. Specifically, it suggests that the differences in the shape of the liquid–liquid interface and the dynamics in the two breakup processes are caused by the disparity of the strain field at the point of detachment. Moreover, the thinning rate and the dynamics of the dispersed thread change with the viscosity of the continuous phase, but are less dependent of the flow rate of the continuous phase. © 2015 American Institute of Chemical Engineers AIChE J, 62: 325–337, 2016     

A high-throughput screen for porphyrin metal chelatases: application to the directed evolution of ferrochelatases for metalloporphyrin biosynthesis

Porphyrins are of particular interest in a variety of applications ranging from biocatalysis and chemical synthesis to biosensor and electronic technologies as well as cancer treatment. Recently, we have developed a versatile system for the high-level production of porphyrins in engineered E. coli cells with the aim of diversifying substitution patterns and accessing porphyrin systems not readily available through chemical synthesis. However, this approach failed to produce significant amounts of the metalloporphyrin in vivo from overproduced protoporphyrin due to insufficient metal insertion. Therefore, we systematically assessed the activity of the B. subtilis ferrochelatase in vivo and in vitro. A true high-throughput-screening approach based on catalytic in vivo ferrochelatase activity was developed by using fluorescence-activated cell sorting (FACS). This assay was used to screen a library of 2.4 x 10(6) ferrochelatase mutants expressed in protoporphyrin-overproducing recombinant E. coli cells. Several selected protein variants were purified, and their improved catalytic activity was confirmed in vitro. In addition to ferrochelatase activity, metal transport into E. coli was identified as another limitation for in vivo heme overproduction. Overexpression of the metal transporter zupT as part of the assembled pathway increased the overall metalloporphyrin production twofold. This report represents the most exhaustive in vitro evolution study of a ferrochelatase and demonstrates the effectiveness of our novel high-throughput-screening system for directed evolution of ferrochelatases based on their catalytic activity.     

Development of microfluidic devices for biomedical and clinical application

This review focuses on the development and use of microfluidic devices within a clinical setting. The underlying theoretical background of microfluidics is briefly elucidated. The materials and techniques used to fabricate the devices and their applicability to the clinical environment are described. The current research in this area is appraised and projections for future applications are discussed. Copyright © 2010 Society of Chemical Industry     

A simple and inexpensive microfluidic electrolysis cell

A single channel microfluidic electrolysis cell based on inexpensive materials and fabrication techniques is described. The cell is characterised using the electrochemistry of the Fe(CN)₆³⁻/Fe(CN)₆⁴⁻ couple and its application in electrosynthesis is illustrated using the methoxylation reactions of N-formylpyrrolidine and 4-t-butyltoluene. It is shown that the reactions can be carried out with a good conversion in a single pass. The device, as described, allows the production of several mmol/hour of the methoxylated products.     

Self‐similar breakup of viscoelastic thread for droplet formation in flow‐focusing devices

The self‐similarity of the breakup of viscoelastic dispersed thread for droplet formation in flow‐focusing devices is investigated experimentally. A high‐speed camera is used to capture the evolution and angles of the cone‐shaped liquid‐liquid interface. The self‐similar profiles for the liquid‐liquid interface are obtained by normalizing the interface with the minimum width of the dispersed thread. The breakup of the dispersed thread transfers from a self‐similar power law scaling stage with an exponent of 0.36 to a self‐similar exponential scaling stage. The asymptotic cone angles prior to final breakup are consistent with the value of 125.5° and 151°, respectively. The viscoelasticity inhibits the development of finite‐time singularity for the breakup of the liquid‐liquid interface at microscale, similar to the capillary breakup at macroscale. The results demonstrate that the breakup of the viscoelastic dispersed thread for droplet formation exhibits self‐similarity at microscale. © 2017 American Institute of Chemical Engineers AIChE J, 2017     

Personalized Single‐Cell Encapsulation Using E‐Jet 3D Printing with AC‐Pulsed Modulation

With the growing therapeutic importance of cell microcarriers, there has been a rise in the need to develop technologies that facilitate efficient microencapsulation of cells, currently limited by a lack of straightforward and low‐cost strategies for single‐cell isolation and printing. Thus, the aim of this study is to develop a gentle and cell‐compatible electro‐hydrodynamic jet 3D printing technique to facilitate the efficient microencapsulation of cells in hydrogel microspheres, and investigate the effects of parameters (flow rate, voltage frequency, nozzle diameter, working distance, and substrate velocity) on the printing process. Stable microspheres are obtained by regulating these parameters to balance various forces, with control of their diameters in the range of 100–600 µm. The study demonstrates that under optimized conditions, the technique is able to successfully encapsulate cells within hydrogel microspheres with high viability over a wide range of diameters. This 3D printing technique expands the potential utility of microspheres into additional biological applications, such as cancer biology and drug screening. It can also be used as an effective platform for the production of tumor spheroids, generating multicellular spheroid models in vitro or for injectable cell delivery.     

A descriptive force‐balance model for droplet formation at microfluidic Y‐junctions

In a previous article, we studied the basics of emulsification in microfluidic Y‐junctions, however, without considering the effect of viscosity of the disperse phase. As it is known from investigations on many different microstructures that viscosity and viscosity ratio are governing parameters for droplet size, we here investigate whether this is also the case for microfluidic Y‐junctions and do so for a wide range of process conditions. The investigated Y‐junctions have a width of 19.9 or 12.8 μm and a depth of 5.0 μm, and the formed monodisperse droplets (CV < 1%) are between 3 and 20 μm. We varied the disperse‐phase viscosity using different oils (1–105 mPa s), and continuous‐phase viscosity using glycerol–water and ethanol–water mixtures (1.0–6.2 mPa s), which corresponds to disperse‐to‐continuous‐phase viscosity ratios from 0.4 to 105.0. Through the variation of the liquids, also a range in interfacial tensions (12–55 mN m^?1) is assessed. The disperse‐phase flow rate is varied from 0.039 to 18.0 μL h^?1, the continuous‐phase flow rate from 1.39 μL h^?1 to 0.41 mL h^?1, and this corresponds to flow rate ratios from 1.1 × 10^?3 to 0.14, which is once again based on wide range of conditions. For all these conditions, in which droplets are formed in the dripping and jetting regime, the droplet size could be described with a model based on the existing force‐balance model, but now extended to incorporate the cross‐sectional area of the droplet and the resistance with the wall. Surprisingly enough, it was found that the droplet size is not influenced by the disperse‐phase viscosity, or the viscosity ratio, but it is dominated by the resistance with the wall and the continuous‐phase properties. Because of this, emulsification with Y‐junctions is intrinsically simpler than any other shear‐based method as droplet size is only determined by the continuous phase. © 2010 American Institute of Chemical Engineers AIChE J, 2010     

Evaluation of a centrifuged double y-shape microfluidic platform for simple continuous cell environment exchange

We have demonstrated the efficacy of a microfluidic medium exchange method for single cells using passive centrifugal force of a rotating microfluidic-chip based platform. At the boundary of two laminar flows at the gathering area of two microfluidic pathways in a Y-shape, the cells were successfully transported from one laminar flow to the other, without mixing the two microfluidic mediums of the two laminar flows during cell transportation, within 5 s with 1 g (150 rpm) to 36.3 g (900 rpm) acceleration, with 93.5% efficiency. The results indicate that this is one of the most simple and precise tools for exchanging medium in the shortest amount of time.     

Activity‐Fed Translation (AFT) Assay: A New High‐Throughput Screening Strategy for Enzymes in Droplets

There is an increasing demand for the development of sensitive enzymatic assays compatible with droplet‐based microfluidics. Here we describe an original strategy, activity‐fed translation (AFT), based on the coupling of enzymatic activity to in vitro translation of a fluorescent protein. We show that methionine release upon the hydrolysis of phenylacetylmethionine by penicillin acylase enabled in vitro expression of green fluorescent protein. An autocatalytic setup where both proteins are expressed makes the assay highly sensitive, as fluorescence was detected in droplets containing single PAC genes. Adding a PCR step in the droplets prior to the assay increased the sensitivity further. The strategy is potentially applicable for any activity that can be coupled to the production of an amino acid, and as the microdroplet volume is small the use of costly reagents such as in vitro expression mixtures is not limiting for high‐throughput screening projects.     

A model for micro/ultrafiltration cell deactivation in cell‐recycle reactors

Mechanical stress due to micro/ultrafiltration has been suggested by a number of authors as one of the sources for cell deactivation in continuous cell‐recycle reactors. This work introduces cell deactivation in the modelling of cell‐recycle fermenters. A two‐population based model is used. It is shown that the kinetic parameters obtained from chemostat fermentations describe accurately the experimental results and the apparent deviations to the Luedeking–Piret relationship at low growth rates are explained. The model is applied to the data of two authors with dilution rates (D) ranging 0.2 and 1.05 h⁻¹ and using either Lactobacillus rhamnosus or Lactococcus cremoris cells. Although only data for lactic fermentation were used, the proposed model is of general applicability and not restricted to a type of fermentation, a given apparatus configuration or Newtonian fluids. The development of improved procedures for biological reactor modelling, such as presented in this work, wall allow more adequate and efficient design and operation of these reactors. © 2000 Society of Chemical Industry     

Ultrahigh‐Throughput FACS‐Based Screening for Directed Enzyme Evolution

Directed enzyme evolution has proven to be a powerful tool for improving a range of properties of enzymes through consecutive rounds of diversification and selection. However, its success depends heavily on the efficiency of the screening strategy employed. Fluorescence‐activated cell sorting (FACS) has recently emerged as a powerful tool for screening enzyme libraries due to its high sensitivity and its ability to analyze as many as 10⁸ mutants per day. Applications of FACS screening have allowed the isolation of enzyme variants with significantly improved activities, altered substrate specificities, or even novel functions. This review discusses FACS‐based screening for enzymatic activity and its potential application for the directed evolution of enzymes, ribozymes, and catalytic antibodies.     

人M-CSF与SCF融合蛋白的构建及其在昆虫细胞中的表达

应用重组DNA技术构建M CSF与SCF的融合基因并将其克隆于昆虫杆状病毒转移载体 pVL13 92中 ,通过与野生型苜蓿夜蛾核型多角体病毒 (AcNPV)DNA共转染草地夜蛾细胞Sf9,融合基因插入AcNPV基因组。重组病毒感染单层Sf9细胞后 ,表达产物分泌到胞外培养液中 ,用MTT比色法和TF 1细胞株可检测到表达产物与IL 3的协同效应。上述研究为开发具有应用价值的新型细胞融合因子奠定了基础     

Digital Microfluidics for Manipulation and Analysis of a Single Cell

The basic structural and functional unit of a living organism is a single cell. To understand the variability and to improve the biomedical requirement of a single cell, its analysis has become a key technique in biological and biomedical research. With a physical boundary of microchannels and microstructures, single cells are efficiently captured and analyzed, whereas electric forces sort and position single cells. Various microfluidic techniques have been exploited to manipulate single cells through hydrodynamic and electric forces. Digital microfluidics (DMF), the manipulation of individual droplets holding minute reagents and cells of interest by electric forces, has received more attention recently. Because of ease of fabrication, compactness and prospective automation, DMF has become a powerful approach for biological application. We review recent developments of various microfluidic chips for analysis of a single cell and for efficient genetic screening. In addition, perspectives to develop analysis of single cells based on DMF and emerging functionality with high throughput are discussed.     

A microelectrochemical scanning flow cell with downstream analytics

The combination of a capillary based microelectrochemical flow cell system and downstream UV–vis analytics allows obtaining synchronized electrochemical and spectroscopic data in a fully automated mode. This method combination can be generally applied to microelectrochemical studies in which an electrochemical species is released or consumed during the electrochemical reaction. For the example of pure zinc surfaces, the characterization of the integrated spectroscopic system is presented with a Zn²⁺ detection limit below 0.1 μmol l⁻¹ using Zincon as complexing agent. A parameter screening of the effect of pH in the range of 6.6–9.0 in borate buffer reveals a linear increase in zinc dissolution with proton concentration but a distinct step in the open circuit potential from the active state (around −700 mV SHE, pH 6.6–7.1) to the passive state (around −300 mV SHE, pH 7.4–9.0) indicating the formation of a closed passive layer. This mechanism is strongly influenced by sulfate anions which increase the dissolution rate of the passive film and promote the active state as monitored by the dissolution profile and OCP (open circuit potential) values. Within the scope of this parameter variation, the congruency between OCP transients, potentiodynamic sweeps and time resolved dissolution profiles is discussed.