Digital microfluidics for cell-based assays

We introduce a new method for implementing cell-based assays. The method is based on digital microfluidics (DMF) which is used to actuate nanolitre droplets of reagents and cells on a planar array of electrodes. We demonstrate that this method is advantageous for cell-based assays because of automated manipulation of multiple reagents in addition to reduced reagent use and analysis time. No adverse effects of actuation by DMF were observed in assays for cell viability, proliferation, and biochemistry. A cytotoxicity assay using Jurkat T-cells was performed using the new method, which had approximately 20 times higher sensitivity than a conventional well plate assay. These results suggest that DMF has great potential as a simple yet versatile analytical tool for implementing cell-based assays on the microscale.     

A digital microfluidic approach to heterogeneous immunoassays

A digital microfluidic (DMF) device was applied to a heterogeneous sandwich immunoassay. The digital approach to microfluidics manipulates samples and reagents in the form of discrete droplets, as opposed to the streams of fluid used in microchannels. Since droplets are manipulated on relatively generic 2-D arrays of electrodes, DMF devices are straightforward to use, and are reconfigurable for any desired combination of droplet operations. This flexibility makes them suitable for a wide range of applications, especially those requiring long, multistep protocols such as immunoassays. Here, we developed an immunoassay on a DMF device using Human IgG as a model analyte. To capture the analyte, an anti-IgG antibody was physisorbed on the hydrophobic surface of a DMF device, and DMF actuation was used for all washing and incubation steps. The bound analyte was detected using FITC-labeled anti-IgG, and fluorescence after the final wash was measured in a fluorescence plate reader. A non-ionic polymer surfactant, Pluronic F-127, was added to sample and detection antibody solutions to control non-specific binding and aid in movement via DMF. Sample and reagent volumes were reduced by nearly three orders of magnitude relative to conventional multiwell plate methods. Since droplets are in constant motion, the antibody–antigen binding kinetics is not limited by diffusion, and total analysis times were reduced to less than 2.5 h per assay. A multiplexed device comprising several DMF platforms wired in series further increased the throughput of the technique. A dynamic range of approximately one order of magnitude was achieved, with reproducibility similar to the assay when performed in a 96-well plate. In bovine serum samples spiked with human IgG, the target molecule was successfully detected in the presence of a 100-fold excess of bovine IgG. It was concluded that the digital microfluidic format is capable of carrying out qualitative and quantitative sandwich immunoassays with a dramatic reduction in reagent usage and analysis time compared to macroscale methods.     

Recent advances in magnetic digital microfluidic platforms

Magnetic Digital microfluidics (DMF), which enables the manipulation of droplets containing different types of samples and reagents by permanent magnets or electromagnet arrays, has been used as a promising platform technology for bioanalytical and preparative assays. This is due to its unique advantages such as simple and “power free” operation, easy assembly, great compatibility with auto control systems, and dual functionality of magnetic particles (actuation and target attachment). Over the past decades, magnetic DMF technique has gained a widespread attention in many fields such as sample-to-answer molecular diagnostics, immunoassays, cell assays, on-demand chemical synthesis, and single-cell manipulation. In the first part of this review, we summarised features of magnetic DMF. Then, we introduced the actuation mechanisms and fabrication of magnetic DMF. Furthermore, we discussed five main applications of magnetic DMF, namely drug screening, protein assays, polymerase chain reaction (PCR), cell manipulation, and chemical analysis and synthesis. In the last part of the review, current challenges and limitations with magnetic DMF technique were discussed, such as biocompatibility, automation of microdroplet control systems, and microdroplet evaporation, with an eye on towards future development.     

Crosstalk-free colloidosomes for high throughput single-molecule protein analysis

Droplet microfluidics is a powerful platform for high-throughput single-molecule protein analysis. However, the issues of coalescence and crosstalk of droplets compromise the accuracy of detection and hinder its wide application. To address these limitations, a novel colloidosome-based method was presented by combining a Pickering emulsion with droplet microfluidics for single-molecule protein analysis. Utilizing the self-assembly of easily synthesized colloidal surfactant F-SiO_2 NPs at the water/oil interface, the colloidosomes are rigidly stabilized and can effectively avoid the leakage of fluorescent molecules. The crosstalk-free colloidosomes enable high-throughput single-molecule protein analysis, including heterogenous dynamic studies and digital detection. As a robust and accurate method, colloidosome-based microfluidics is promising as a powerful tool for a wide variety of applications, such as directed enzyme evolution, digital enzyme-linked immunosorbent assay(ELISA), and screening of antibiotics.     

A feedback control system for high-fidelity digital microfluidics

Digital microfluidics (DMF) is a technique in which discrete droplets are manipulated by applying electrical fields to an array of electrodes. In an ideal DMF system, each application of driving potential would cause a targeted droplet to move onto an energized electrode (i.e., perfect fidelity between driving voltage and actuation); however, in real systems, droplets are sometimes observed to resist movement onto particular electrodes. Here, we implement a sensing and feedback control system in which all droplet movements are monitored, such that when a movement failure is observed, additional driving voltages can be applied until the droplet completes the desired operation. The new system was evaluated for a series of liquids including water, methanol, and cell culture medium containing fetal bovine serum, and feedback control was observed to result in dramatic improvements in droplet actuation fidelity and velocity. The utility of the new system was validated by implementing an enzyme kinetics assay with continuous mixing. The new platform for digital microfluidics is simple and inexpensive and thus should be useful for scientists and engineers who are developing automated analysis platforms.     

A digital microfluidic method for multiplexed cell-based apoptosis assays

Digital microfluidics (DMF), a fluid-handling technique in which picolitre-microlitre droplets are manipulated electrostatically on an array of electrodes, has recently become popular for applications in chemistry and biology. DMF devices are reconfigurable, have no moving parts, and are compatible with conventional high-throughput screening infrastructure (e.g., multiwell plate readers). For these and other reasons, digital microfluidics has been touted as being a potentially useful new tool for applications in multiplexed screening. Here, we introduce the first digital microfluidic platform used to implement parallel-scale cell-based assays. A fluorogenic apoptosis assay for caspase-3 activity was chosen as a model system because of the popularity of apoptosis as a target for anti-cancer drug discovery research. Dose-response profiles of caspase-3 activity as a function of staurosporine concentration were generated using both the digital microfluidic method and conventional techniques (i.e., pipetting, aspiration, and 96-well plates.) As expected, the digital microfluidic method had a 33-fold reduction in reagent consumption relative to the conventional technique. Although both types of methods used the same detector (a benchtop multiwell plate reader), the data generated by the digital microfluidic method had lower detection limits and greater dynamic range because apoptotic cells were much less likely to de-laminate when exposed to droplet manipulation by DMF relative to pipetting/aspiration in multiwell plates. We propose that the techniques described here represent an important milestone in the development of digital microfluidics as a useful tool for parallel cell-based screening and other applications.     

A digital microfluidic platform for primary cell culture and analysis

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.     

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.     

Some recent advances in the design and the use of miniaturized droplet-based continuous process: applications in chemistry and high-pressure microflows

This mini-review focuses on two different miniaturizing approaches: the first one describes the generation and use of droplets flowing within a millifluidic tool as individual batch microreactors. The second one reports the use of high pressure microflows in chemistry. Millifluidics is an inexpensive, versatile and easy to use approach which is upscaled from microfluidics. It enables one to produce hierarchically organized multiple emulsions or particles with a good control over sizes and shapes, as well as to provide a convenient data acquisition platform dedicated to slow or rather fast chemical reactions, i.e., from hours to a few minutes. High-pressure resistant devices were recently fabricated and used to generate stable droplets from pressurized fluids such as supercritical fluid-liquid systems. We believe that supercritical microfluidics is a promising tool to develop sustainable processes in chemistry.     

A robust approach to studying the adsorption of Pluronic F108 on nonporous membranes

A method for poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) desorption from synthetic nonporous polymeric membranes, using hexane:isopropanol treatment and subsequent colorimetric quantification, is described. The polymers polysulfone, poly(vinyldiene fluoride), and poly(ether imide) were used to fabricate solid adsorption matrices. The desorbed Pluronic F108 forms a color complex with ammonium ferrothiocyanate (NH4FeSCN) and is based on partitioning of a chromophore present in NH4FeSCN from an aqueous phase to a chloroform phase in the presence of Pluronic. The protocols for Pluronic desorption and detection are simple, sensitive, inexpensive, rapid, and reproducible over a wide range of Pluronic coating concentrations and membrane surface chemistries. A linear response over the concentration range from 3 to 130 microg ml(-1) is obtained. The adsorption isotherms for flat sheet membranes are also described and the Langmuir equation provides the best fit for the adsorption data obtained within the concentration range studied. The absence of any significant interference from certain proteins, vitamins, carbohydrates, plasma, and halogenated derivatives makes the assay equally suitable for the estimation of Pluronic F108 in the attendant Pluronic conjugates or in biomedical applications. Using nonporous hollow fine fibers and capillary membranes as model curved substrates we were also able to correlate an increase in the radius of curvature with a corresponding increase in the surface interfacial adsorption of Pluronic F108.     

微液滴微流控芯片:微液滴的形成、操纵和应用

微流控芯片中形成的微液滴粒径均一、可控,与传统的连续流体系相比,具有能实现试剂的快速混合、通量更高等优点.本文介绍了微流控芯片中由微通道控制的微液滴的形成、分裂、合并、混合、分选和捕获等微液滴操纵技术,以及微液滴技术在纳米粒子、聚合物微粒的合成、纳米粒子自组装、蛋白质结晶研究和DNA、细胞分析等领域的研究进展.     

Intelligent droplet tracking with correlation filters for digital microfluidics

Tracking the movement of droplets in digital microfluidics is essential to improve its control stability and obtain dynamic information for its applications such as point-of-care testing, environment monitoring and chemical synthesis. Herein, an intelligent, accurate and fast droplet tracking method based on machine vision is developed for applications of digital microfluidics. To continuously recognize the transparent droplets in real-time and avoid the interferes from background patterns or inhomogeneous illumination, we introduced the correlation filter tracker, enabling online learning of the multi-features of the droplets in Fourier domain. Results show the proposed droplet tracking method could accurately locate the droplets. We also demonstrated the capacity of the proposed method for estimation of the droplet velocity as faster as 20 mm/s, and its application in online monitoring the Griess reaction for both colorimetric assay of nitrite and study of reaction kinetics.     

Rapid Chemical Reaction Monitoring by Digital Microfluidics‐NMR: Proof of Principle Towards an Automated Synthetic Discovery Platform

Microcoil nuclear magnetic resonance (NMR) has been interfaced with digital microfluidics (DMF) and is applied to monitor organic reactions in organic solvents as a proof of concept. DMF permits droplets to be moved and mixed inside the NMR spectrometer to initiate reactions while using sub‐microliter volumes of reagent, opening up the potential to follow the reactions of scarce or expensive reagents. By setting up the spectrometer shims on a reagent droplet, data acquisition can be started immediately upon droplet mixing and is only limited by the rate at which NMR data can be collected, allowing the monitoring of fast reactions. Here we report a cyclohexene carbonate hydrolysis in dimethylformamide and a Knoevenagel condensation in methanol/water. This is to our knowledge the first time rapid organic reactions in organic solvents have been monitored by high field DMF‐NMR. The study represents a key first step towards larger DMF‐NMR arrays that could in future serve as discovery platforms, where computer controlled DMF automates mixing/titration of chemical libraries and NMR is used to study the structures formed and kinetics in real time.     

Digital microfluidics: a versatile tool for applications in chemistry, biology and medicine

Digital microfluidics (DMF) has recently emerged as a popular technology for a wide range of applications. In DMF, nanoliter to microliter droplets containing samples and reagents can be manipulated to carry out a range of discrete fluidic operations simply by applying a series of electrical potentials to an array of patterned electrodes coated with a hydrophobic insulator. DMF is distinct from microchannel-based fluidics as it allows for precise control over multiple reagent phases (liquids and solids) in heterogeneous systems with no need for complex networks of connections, microvalves, or pumps. In this review, we discuss the most recent developments in this technology with particular attention to the potential benefits and outstanding challenges for applications in chemistry, biology, and medicine.     

Quantification of surface-bound proteins by fluorometric assay: Comparison with quartz crystal microbalance and amido black assay

Protein adsorption is of major and widespread interest, being useful in the fundamental understanding of biological processes at interfaces through to the development of new materials. A number of techniques are commonly used to study protein adhesion, but few are directly quantitative. Here we describe the use of Nano Orange, a fluorometric assay, to quantitatively assess the adsorption of bovine fibrinogen and albumin onto model hydrophilic (OH terminated) and hydrophobic (CH3 terminated) surfaces. Results obtained using this method allowed the calibration of previously unquantifiable data obtained on the same surfaces using quartz crystal microbalance measurements and an amido black protein assay. Both proteins were found to adsorb with higher affinity but with lower saturation levels onto hydrophobic surfaces. All three analytical techniques showed similar trends in binding strength and relative amounts adsorbed over a range of protein concentrations, although the fluorometric analysis was the only method to give absolute quantities of surface-bound protein. The versatility of the fluorometric assay was also probed by analyzing protein adsorption onto porous superhydrophobic and superhydrophilic surfaces. Results obtained using the assay in conjunction with these surfaces were surface chemistry dependent. Imbibition of water into the superhydrophilic coatings provided greater surface area for protein adsorption, although the protein surface density was less than that found on a comparable flat hydrophilic surface. Superhydrophobic surfaces prevented protein solution penetration. This paper demonstrates the potential of a fluorometric assay to be used as an external calibration for other techniques following protein adsorption processes or as a supplemental method to study protein adsorption. Differences in protein adsorption onto hydrophilic vs superhydrophilic and hydrophobic vs superhydrophobic surfaces are highlighted.     

Recent advances in microfluidic production of Janus droplets and particles

Microfluidic production of multicompartmental emulsion droplets and particles has received considerable attention of late. In particular, droplets having two physically and chemically distinct segments (so-called Janus droplets) and the anisotropic particles synthesized from these droplets, are becoming increasingly popular because of their novel and promising properties, which make them suitable for use in numerous applications, including for controlled drug release, display devices, and self-assembly. So far, a range of interesting anisotropies have been accorded to Janus droplets and particles via microfluidics; these span from chromatic, magnetic, and hydrophobic–hydrophilic characteristics to selective degradation properties. Here, we summarize and discuss the recent trends related to Janus droplets and particles produced through microfluidic processing. We also review the parallelization technologies being developed for scaling up microfluidic emulsification in the industry.     

Droplet microfluidics based microseparation systems

Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.     

A new angle on pluronic additives: advancing droplets and understanding in digital microfluidics

Biofouling in microfluidic devices limits the type of samples which can be handled and the duration for which samples can be manipulated. Despite the cost of disposing fouled devices, relatively few strategies have been developed to tackle this problem. Here, we have analyzed a series of eight amphiphilic droplet additives, Pluronic coblock polymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO), as a solution to biofouling in digital microfluidics using serum-containing cell culture media as a model fluid. Our analysis shows that species with longer PPO chains are superior for enabling droplet motion and reducing biofouling. Two of the tested species, L92 and P105, were found to lengthen device lifetimes by 2-3 times relative to additives used previously when used at optimal concentrations. Pluronics with low PEO content such as L92 were found to be cytotoxic to an immortalized mammalian cell line, and therefore we recommend that Pluronic additives with greater or equal to 50% PEO composition, such as P105, be used for digital microfluidic applications involving cells. Finally, contact angle measurements were used to probe the interaction between Pluronic-containing droplets and device surfaces. Strong correlations were found between various types of contact angle measurements and the capacity of additives to reduce biofouling, which suggests that contact angle measurements may be useful as a tool for rapidly screening new candidates for the potential to reduce biofouling. We propose that this study will be useful for scientists and engineers who are developing digital microfluidic platforms for a wide range of applications involving protein-containing solutions, and in particular, for applications involving cells.     

Versatile Preparation of Nonspherical Multiple Hydrogel Core PAM/PEG Emulsions and Hierarchical Hydrogel Microarchitectures

The preparation of nonspherical materials composed of separated multicomponents by droplet‐based microfluidics remains a challenge. Based on polymerization‐induced phase separation and droplet coalescence in microfluidics, we prepared emulsions of variously shaped PAM/PEG core/shell droplets and hydrogels composed of two separated components, which show flexible and transformable hierarchical structures and microarchitectures. We find that AM/PEG aqueous droplets form a core/shell structure after polymerization resulting from phase separation. Thus multicore/shell droplets are easily produced by coalescence of core/shell structures. By changing the polymerization temperature and the flow rate, the morphology of the multicore droplets and the hydrogel can be easily adjusted. The hydrogels exhibit apparent anisotropy and different protein release rates depending on their structures. The preparation technique is simple and versatile and the resulting hydrogels have potential applications in many fields.     

Monitoring entering and spreading of emulsion droplets at an expanding air/water interface: a novel technique

The entering and spreading of emulsion droplets at quiescent and expanding air/water interfaces was studied using a new apparatus consisting of a modified Langmuir trough in which the air/water interface can be continuously expanded by means of rollers in the place of traditional barriers. When sodium caseinate and whey protein isolate-stabilized emulsion droplets were injected under the surface of sodium caseinate and whey protein isolate solutions, respectively, it appeared that the droplets entered the air/water interface only if the air/water surface pressure did not exceed a threshold value of approximately 15 mN/m. This condition was satisfied either under quiescent conditions for low protein concentrations or by continuous expansion of the interface at higher protein concentrations. According to equilibrium thermodynamics, entering of the droplets and the formation of lenses should occur for all the systems investigated, but this was not observed. At surface pressures higher than approximately 15 mN/m, immersed emulsion droplets were metastable. This is probably due to a kinetic barrier caused by the formation of a thin water film bounded by protein adsorption layers between the emulsion droplet and the air/water interface.