Microfluidic platform for combinatorial synthesis in picolitre droplets

This paper presents a droplet-based microfluidic platform for miniaturized combinatorial synthesis. As a proof of concept, a library of small molecules for early stage drug screening was produced. We present an efficient strategy for producing a 7 × 3 library of potential thrombin inhibitors that can be utilized for other combinatorial synthesis applications. Picolitre droplets containing the first type of reagent (reagents A(1), A(2), …, A(m)) were formed individually in identical microfluidic chips and then stored off chip with the aid of stabilizing surfactants. These droplets were then mixed to form a library of droplets containing reagents A(1-m), each individually compartmentalized, which was reinjected into a second microfluidic chip and combinatorially fused with picolitre droplets containing the second reagent (reagents B(1), B(2), …, B(n)) that were formed on chip. The concept was demonstrated with a three-component Ugi-type reaction involving an amine (reagents A(1-3)), an aldehyde (reagents B(1-7)), and an isocyanide (held constant), to synthesize a library of small molecules with potential thrombin inhibitory activity. Our technique produced 10(6) droplets of each reaction at a rate of 2.3 kHz. Each droplet had a reaction volume of 3.1 pL, at least six orders of magnitude lower than conventional techniques. The droplets can then be divided into aliquots for different downstream screening applications. In addition to medicinal chemistry applications, this combinatorial droplet-based approach holds great potential for other applications that involve sampling large areas of chemical parameter space with minimal reagent consumption; such an approach could be beneficial when optimizing reaction conditions or performing combinatorial reactions aimed at producing novel materials.     

Microfluidic platform for the generation of organic-phase microreactors

Rapid prototyping photolithography of a thiolene-based resin was used to fabricate microfluidic devices stable to aliphatic and aromatic organic solvents. The swelling of the cross-linked polymer matrix in various organic solvents was quantified, and the solvent resistance properties of these microfluidic devices are described. Discrete droplets of hexanes and toluene of uniform size were generated in microfluidic devices inside a water matrix containing SDS surfactant (SDS = sodium dodecyl sulfate). Variation of water and organic flow rates in the fluidic channels was used to control droplet size and separation. Droplet composition could be controlled by varying flow rates of two joined organic streams. Organic-phase synthetic reactions within the droplets were demonstrated with the bromination of alkenes inside benzene droplets.     

Microfluidic chip: Next-generation platform for systems biology

Systems biology advocates the understanding of biology at the systems-level, which requires massive information of correlations among individual components in complex biological systems. Such comprehensive investigation entails the use of high-throughput analytical tools. Microfluidic technology holds high promise to facilitate the progress of biology by enabling miniaturization and upgrading of current biological research tools due to its advantages such as low sample consumption, reduced analysis time, high-throughput and compatible sizes with most biological samples. In this article, we documented the recent applications of microfluidic chips in biological researches at the molecular level, cellular level and organism level, serving the purpose for systems-level understanding of biology.     

Microfluidic fabrication of water-in-water droplets encapsulated in hydrogel microfibers

By combining microfiber spinning techniques with aqueous two phase system (ATPS), a rapid and simple strategy to fabricate water-in-water (w/w) droplets encapsulated in microfibers was proposed for the first time. Hydrophilic environment in hydrogel and the fiber format facilitates higher biocompatibility, convenient manipulation of the droplets and recycling of the contents inside droplets, which would have promising development in biological, pharmacological and environmental fields.     

Automated high-throughput generation of droplets

We report a microfluidic technique for high-throughput generation of droplets of nanolitre volume in parallel channels with online control of the volumes, volume fraction and distribution of droplet volumes with the use of two external valves.     

High-performance flow-focusing geometry for spontaneous generation of monodispersed droplets

A high-performance flow-focusing geometry for spontaneous generation of monodispersed droplets is demonstrated. In this geometry, a two-phase flow is forced through a circular orifice integrated inside a silicon-based microchannel. The orifice with its cusp-like edge exerts a ring of maximized stress around the flow and ensures controlled breakup of droplets for a wide range of flow rates, forming highly periodic and reproducible dispersions. The droplet generation can be remarkably rapid, exceeding 10(4) s(-1) for water-in-oil droplets and reaching 10(3) s(-1) for oil-in-water droplets, being largely controlled by flow rate of the continuous phase. The droplet diameter and generation frequency are compared against a quasi-equilibrium model based on the critical Capillary number. The droplets are obtained despite the low Capillary number, below the critical value identified by the ratio of viscosities between the two phases and simple shear-flow.     

Continuous,on-demand generation and separation of diphenylphosphoryl azide

Diphenylphosphorylazide (DPPA) has been synthesized in microfluidics with near-100% yield in sub-3 minute residence time, affordably, and with a process design that minimizes hazards associated with hydrazoic acid (HN₃) production. A pilot-plant scale continuous process for the on-demand synthesis of diphenylphosphoryl azide (DPPA) that can readily be integrated with subsequent transformations was designed, built, and validated. Using Corning's Low Flow reactor system coupled to a membrane separator and in-line Fourier Transform Infrared (FTIR), DPPA was safely produced at a rate of 1?mol/hr as a 2.0?M anhydrous toluene stream. Continuous FTIR was able to reliably monitor product quality, purity and concentration, showcasing the ease and utility of this continuous flow process for manufacturing common, safe pharmaceutical precursors.     

Pneumatic handling of droplets on-demand on a microfluidic device for seamless processing of reaction and electrophoretic separation

Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.     

Microfluidic formation of pH responsive 5CB droplets decorated with PAA-b-LCP

We are reporting for the first time the pH responsiveness of liquid crystal (LC) microdroplets decorated with an amphiphilic block copolymer of PAA-b-LCP. We successfully demonstrated the adsorption of block copolymer on LC droplets by fluorescence microscopy and pH response to the radial-to-bipolar orientational change of the LC droplets by changing pH from 12 to 2 through the polarized optical microscope (POM). We believe that our results may pave the way for the generation of monodisperse droplets decorated by various amphiphilic block copolymers which respond to several kinds of the external stimuli. These developments may be important for potential applications of the LC droplets in sensing and encapsulation fields.     

Microfluidic device as a new platform for immunofluorescent detection of viruses

A bead-based microfluidic device was developed and demonstrated to achieve rapid and sensitive enzyme-linked immunosorbent assay (ELISA) with quantum dots as the labeling fluorophore for virus detection. In comparison to standard ELISA performed on the same virus, the minimal detectable concentration of the target virus was improved from 360 to 22 ng mL-1, the detection time was shortened from >3.25 h to <30 min, and the amount of antibody consumed was reduced by a factor of 14.3.     

Microfluidic platform with mass spectrometry detection for the analysis of phosphoproteins

The development of novel and reliable technologies for the analysis of proteins and their post-translational modifications, in particular, has recently received much attention and interest. The implementation of a fully integrated microfluidic device interfaced with MS detection for the analysis of phosphoproteins is presented in this paper. The microfluidic platform (3'x1.5') comprises two individual sample processing systems: one for performing direct sample infusion and one for performing microfluidic LC separations. Various MS detection strategies, specific for the study of post-translational modifications, were conducted using alpha-casein as a model protein. Neutral loss ion mapping, data-dependent triple-play and neutral loss analysis, and in situ dephosphorylation followed by LC separation and MS detection were performed. Consistent results in identifying phosphopeptides with conventional and microfluidic instrumentation have been obtained. Unlike with conventional instrumentation, however, the microfluidic device enabled the completion of each analysis from only a few microliters of sample, in approximately 10-15 min, and on a bioanalytical platform that facilitates multiplexing and disposability, and thus high-throughput, contamination-free analysis.     

Microfluidic large-scale integration on a chip for mass production of monodisperse droplets and particles

In this study, we report the mass production of monodisperse emulsion droplets and particles using microfluidic large-scale integration on a chip. The production module comprises a glass microfluidic chip with planar microfabricated 16-256 droplet-formation units (DFUs) and a palm-sized stainless steel holder having several layers for supplying liquids into the inlets of the mounted chip. By using a module having 128 cross-junctions (i.e., 256 DFUs) arranged circularly on a 4 cm x 4 cm chip, we could produce droplets of photopolymerizable acrylate monomer at a throughput of 320.0 mL h(-1). The product was monodisperse, having a mean diameter of 96.4 microm, with a coefficient of variation (CV) of 1.3%. Subsequent UV polymerization off the module yielded monodisperse acrylic microspheres at a throughput of approximately 0.3 kg h(-1). Another module having 128 co-flow geometries could produce biphasic Janus droplets of black and white segments at 128.0 mL h(-1). The product had a mean diameter of 142.3 microm, with a CV of 3.3%. This co-flow module could also be applied in the mass production of homogeneous monomer droplets.     

Reversible Diels-Alder reactions for the generation of dynamic combinatorial libraries

Condensation reactions between various dienes and dienophiles have been screened for reversibility. Functionalized fulvenes, bearing in particular biological groups, and cyanolefins have been found to react rapidly and reversibly, in the temperature range from -10 to +50 degrees C. These results pave the way for the development of dynamic combinatorial libraries based on reversible Diels-Alder chemistry.     

Microfluidic generation of multifunctional quantum dot barcode particles

We develop a new strategy to prepare quantum dot (QD) barcode particles by polymerizing double-emulsion droplets prepared in capillary microfluidic devices. The resultant barcode particles are composed of stable QD-tagged core particles surrounded by hydrogel shells. These particles exhibit uniform spectral characteristics and excellent coding capability, as confirmed by photoluminescence analyses. By using double-emulsion droplets with two inner droplets of distinct phases as templates, we have also fabricated anisotropic magnetic barcode particles with two separate cores or with a Janus core. These particles enable optical encoding and magnetic separation, thus making them excellent functional barcode particles in biomedical applications.     

2D and 3D spatially addressed arrays for high-throughput automated synthesis of combinatorial libraries

One of the key elements in the drug discovery process is the use of automation to synthesize libraries of compounds for biological screening. The "split-and-mix" approaches in combinatorial chemistry have been recognized as extremely powerful techniques to access large numbers of compounds, while requiring only few reaction steps. However, the need for effective encoding/deconvolution strategies and demands for larger amounts of compounds have somewhat limited the use of these techniques in the pharmaceutical industry. In this paper, we describe a concept of directed sort and combine synthesis with spatially arranged arrays of macroscopic supports. Such a concept attempts to balance the number of reaction steps, the confidence in compound identity, and the quantity of synthesized compounds. Using three-dimensional arrays of frames each containing a two-dimensional array of macroscopic solid supports, we have conceptualized and developed a modular semiautomated system with a capacity of up to 100 000 compounds per batch. Modularity of this system enables flexibility either to produce large diverse combinatorial libraries or to synthesize more focused smaller libraries, both as single compounds in 12-15 micromol quantities. This method using sortable and spatially addressed arrays is exemplified by the synthesis of a 15 360 compound library.     

Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel

Deciphering the signaling pathways that govern stimulation of na?ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na?ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.     

Microfluidic platform for studying the anti-cancer effect of ursolic acid on tumor spheroid

At present, the probability that a new anti-tumor drug will eventually succeed in clinical trials is extremely low. In order to make up for this shortcoming, the use of a three-dimensional (3D) cell culture model for secondary screening is often necessary. Cell spheroid is the easiest 3D model tool for drug screening. In this study, the microfluidic chip with a microwell array was manufactured, which could allow the formation of tumor spheroids with uniform size and easily retrieve cell spheroids from the chip. Cell spheroids were successfully cultured for over 15 days and the survival rate was as high as 80%. Subsequently, cellular response to the ursolic acid (UA) was observed on the chip. Compared to the monolayer culture cells in vitro, the tumor spheroids showed minor levels of epithelial-mesenchymal transition fluctuation after drug treatment. The mechanism of cell spheroid resistance to UA was further verified by detecting the expression level of upstream pathway proteins. But the invasive ability of tumor spheroids was attenuated when the duration of action of UA extended. The anti-cancer effect of UA was innovatively evaluated on breast cancer by using the microfluidic device, which could provide a basis and direction for future preclinical research on UA.     

Liquid crystal droplets as a hosting and sensing platform for developing immunoassays

In this paper, we report an immunoassay in which probe proteins are immobilized on the surface of liquid crystal (LC) droplets rather than on solid surfaces. The advantage of this immunoassay is that the binding of antibodies to the probe proteins can be transduced by the LC droplets directly without the need for additional steps. For example, when we incubate the LC droplets decorated with immunoglobulin G (IgG) in a solution containing anti-IgG (AIgG), these droplets change their orientations from radial to bipolar configuration. In contrast, when we incubate the IgG-LC droplets in a solution containing anti-human serum albumin (AHSA), no changes are observed. The change of orientational configuration indicates the formation of the antigen-antibody immunocomplex on the surface of the LC droplets. Using LC droplet immunoassays, we successfully detect antibody concentrations as low as 0.01 μg/mL for AIgG and 0.02 μg/mL for AHSA. Because the immunoassay using LC droplets is label-free and gives a unique optical response, it has the potential to be further developed as a portable and low-cost immunoassay.     

High-speed, clinical-scale microfluidic generation of stable phase-change droplets for gas embolotherapy

In this study we report on a microfluidic device and droplet formation regime capable of generating clinical-scale quantities of droplet emulsions suitable in size and functionality for in vivo therapeutics. By increasing the capillary number-based on the flow rate of the continuous outer phase-in our flow-focusing device, we examine three modes of droplet breakup: geometry-controlled, dripping, and jetting. Operation of our device in the dripping regime results in the generation of highly monodisperse liquid perfluoropentane droplets in the appropriate 3-6 μm range at rates exceeding 10(5) droplets per second. Based on experimental results relating droplet diameter and the ratio of the continuous and dispersed phase flow rates, we derive a power series equation, valid in the dripping regime, to predict droplet size, D(d) approximately equal 27(Q(C)/Q(D))(-5/12). The volatile droplets in this study are stable for weeks at room temperature yet undergo rapid liquid-to-gas phase transition, and volume expansion, above a uniform thermal activation threshold. The opportunity exists to potentiate locoregional cancer therapies such as thermal ablation and percutaneous ethanol injection using thermal or acoustic vaporization of these monodisperse phase-change droplets to intentionally occlude the vessels of a cancer.     

Microfluidic platform for electrophysiological studies on Xenopus laevis oocytes under varying gravity levels

Voltage clamp measurements reveal important insights into the activity of membrane ion channels. While conventional voltage clamp systems are available for laboratory studies, these instruments are generally unsuitable for more rugged operating environments. In this study, we present a non-invasive microfluidic voltage clamp system developed for the use under varying gravity levels. The core component is a multilayer microfluidic device that provides an immobilisation site for Xenopus laevis oocytes on an intermediate layer, and fluid and electrical connections from either side of the cell. The configuration that we term the asymmetrical transoocyte voltage clamp (ATOVC) also permits electrical access to the cytosol of the oocyte without physical introduction of electrodes by permeabilisation of a large region of the oocyte membrane so that a defined membrane patch can be voltage clamped. The constant low level air pressure applied to the oocyte ensures stable immobilisation, which is essential for keeping the leak resistance constant even under varying gravitational forces. The ease of oocyte mounting and immobilisation combined with the robustness and complete enclosure of the fluidics system allow the use of the ATOVC under extreme environmental conditions, without the need for intervention by a human operator. Results for oocytes over-expressing the epithelial sodium channel (ENaC) obtained under laboratory conditions as well as under conditions of micro- and hypergravity demonstrate the high reproducibility and stability of the ATOVC system under distinct mechanical scenarios.