Microfluidic platform for on-demand generation of spatially indexed combinatorial droplets

We propose a highly versatile and programmable nanolitre droplet-based platform that accepts an unlimited number of sample plugs from a multi-well plate, performs digitization of these sample plugs into smaller daughter droplets and subsequent synchronization-free, robust injection of multiple reagents into the sample daughter droplets on-demand. This platform combines excellent control of valve-based microfluidics with the high-throughput capability of droplet microfluidics. We demonstrate the functioning of a proof-of-concept device which generates combinatorial mixture droplets from a linear array of sample plugs and four different reagents, using food dyes to mimic samples and reagents. Generation of a one dimensional array of the combinatorial mixture droplets on the device leads to automatic spatial indexing of these droplets, precluding the need to include a barcode in each droplet to identify its contents. We expect this platform to further expand the range of applications of droplet microfluidics to include applications requiring a high degree of multiplexing as well as high throughput analysis of multiple samples.     

Microfluidic voltammetry: simulation of the chronoamperometric response of microband electrodes sited within microreactors

A computationally efficient fully implicit approach to the simulation of the chronoamperometric response of microband electrodes sited within microscale rectangular ducts is reported. The current response is reported for stagnant solution and where electrolyte is pumped through the cell under microfluidic control. The generality of the method is illustrated with reference to the simple case of a reversible one-electron-transfer reaction. The influence of flow rate and the effects of axial, lateral and normal diffusion upon the electrolysis current are examined and the results compared to approximate analytical behaviour where appropriate.Dedicated to Professor Dr. Alan Bond on the occasion of his 60th birthday.     

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 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 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 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 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.     

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.     

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 channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A

A selective aptameric sequence is adsorbed on a two-dimensional nanostructured metallic platform optimized for surface-enhanced Raman spectroscopy (SERS) measurements. Using nanofabrication methods, a metallic nanostructure was prepared by electron-beam lithography onto a glass coverslip surface and embedded within a microfluidic channel made of polydimethylsiloxane, allowing one to monitor in situ SERS fingerprint spectra from the adsorbed molecules on the metallic nanostructures. The gold structure was designed so that its localized surface plasmon resonance matches the excitation wavelength used for the Raman measurement. This optofluidic device is then used to detect the presence of a toxin, namely ochratoxin-A (OTA), in a confined environment, using very small amounts of chemicals, and short data acquisition times, by taking advantage of the optical properties of a SERS platform to magnify the Raman signals of the aptameric monolayer system and avoiding chemical labeling of the aptamer or the OTA target.

Perfluoroalkylation in flow microreactors: generation of perfluoroalkyllithiums in the presence and absence of electrophiles

Perfluoroalkyllithiums were effectively generated from perfluoroalkyl halides in the presence and absence of electrophiles using flow microreactor systems. The in situ trapping with electrophile is conducted at much higher temperatures than those required for batch macro reactors. The subsequent trapping method is quite effective for highly reactive electrophiles that are not compatible with the lithiation process.     

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.     

Microemulsions as microreactors for food applications

Major recent advances. Structured self-assembled liquids have been considered as efficient microreactors for organic and enzymatic reactions. Only recently scientists learned to use food-grade cosolvents and coemulsifiers together with hydrophilic non-ionic surfactants and to construct U-type phase diagrams with large isotropic regions ranging continuously from the oil-rich corner to the water-rich corner without any phase separation. The U-type microemulsions facilitate triggering and control of certain reactions by changing water activities. Maillard thermal degradation between sugars and amino acids is the main, and almost the only, chemical reaction that has been studied in food-grade microemulsions. Some examples of recent studies include: Maillard processes in binary structured fluids composed of monoglycerides of fatty acids and water forming microemulsions and lyotropic liquid crystalline structures; pseudoternary and pseudoquaternary W/O microemulsions; U-type microemulsions (W/O, O/W and bicontinuous microemulsions); enzymatic reactions aimed to prepare other surfactants such as sugar esters, monoglycerides and lysolecithins or triglycerides. Reactions in microreactors lead to unique new products. The reaction products and rates are controlled by the hydrophilicity/lipophilicity of the reagents (guest molecules), their molar ratios, type of oil phase, nature of surfactants and oil/surfactant ratios, nature of curvature and its elasticity (adjusted by cosolvent and coemulsifier) and by the water activity. The field is in its infancy and will need work of many more model reactions before it will be used in industrial food applications. Enzymatic reactions in non-food microemulsions are common practice but only few examples of food microemulsions as enzymatic microreactors have been extensively studied.     

A chip-based platform for the in vitro generation of tissues in three-dimensional organization

We describe a multi-purpose platform for the three-dimensional cultivation of tissues. The device is composed of polymer chips featuring a microstructured area of 1-2 cm(2). The chip is constructed either as a grid of micro-containers measuring 120-300 x 300 x 300 microm (h x l x w), or as an array of round recesses (300 microm diameter, 300 microm deep). The micro-containers may be separately equipped with addressable 3D-micro-electrodes, which allow for electrical stimulation of excitable cells and on-site measurements of electrochemically accessible parameters. The system is applicable for the cultivation of high cell densities of up to 8 x 10(6) cells and, because of the rectangular grid layout, allows the automated microscopical analysis of cultivated cells. More than 1000 micro-containers enable the parallel analysis of different parameters under superfusion/perfusion conditions. Using different polymer chips in combination with various types of bioreactors we demonstrated the principal suitability of the chip-based bioreactor for tissue culture applications. Primary and established cell lines have been successfully cultivated and analysed for functional properties. When cells were cultured in non-perfused chips, over time a considerable degree of apoptosis could be observed indicating the need for an active perfusion. The system presented here has also been applied for the differentiation analysis of pluripotent embryonic stem cells and may be suitable for the analysis of the stem cell niche.     

Ceramic microreactors for heterogeneously catalysed gas-phase reactions

The high surface to volume ratio of microchannel components offers many advantages in micro chemical engineering. It is obvious, however, that the reactor material and corrosion phenomena play an important role when applying these components. For chemical reactions at very high temperatures or/and with corrosive reactants involved, microchannel components made of metals or polymers are not suited. Hence, a modular microreactor system made of alumina was developed and fabricated using a rapid prototyping process chain. With exchangeable inserts the system can be adapted to the requirements of various reactions. Two heterogeneously catalysed gas-phase reactions (oxidative coupling of methane, isoprene selective oxidation to citraconic anhydride) were investigated to check the suitability of the system at temperatures of up to 1000 degrees C. Apart from the high thermal and chemical resistance, the lack of any blind activity was found to be another advantage of ceramic components.     

Microfluidic generation of barcodes with in situ synthesized perovskite quantum dot encapsulation

Perovskite quantum dots(PQDs) are new class of optoelectronic materials, which have been widely studied for their extraordinary physical properties. Attempts to develop these materials are tending to make their fabrication much controllable and extend their values in different areas. Here, we present a novel strategy for one-step in situ synthesis of PQD-encapsulated barcode particles with the assistance of microfluidic technique. By changing the halide ratio in perovskite precursor solutions that emulsified in microfluidic devices, a series of PQDs with different colors have been successfully fabricated, which made them ideal materials as barcodes. Because of the stable encapsulation of ethyleneglycol dimethacrylate(EGDMA) resin, the PQD-encapsulated barcode particles were with no cytotoxicity and could be anti-quenched. It was demonstrated for the first time that the PQD-encapsutated barcode particles by microfluidics were valuable for multiplex biomolecular encoding and assays.These features indicate that the PQD-encapsutated barcode particles by microfluidics are ideal for many practical applications and have a broad prospect in biomedical field.     

Macroporous monolith supports for continuous flow capillary microreactors

A solid macroporous monolith is shown to be a suitable substrate for anchoring a palladium complex to obtain a continuous porous material suitable for conducting flow-through catalysis in capillary microreactors.     

Controlled enzyme-immobilisation on capillaries for microreactors for peptide mapping

In the present paper, the covalent immobilisation of the digesting enzyme trypsin has been achieved through photo-immobilisation on a portion of a silica capillary, thus leading to the construction of a capillary electrophoretic (CE)-microreactor for peptide mapping. The CE-microreactor is characterised by being a single piece, thus ensuring no fluidic or electrical leakage. The enzyme was immobilised with a surface density of 15.8 g/cm², the stability was high (80% after 38 days) and the rate of conversion was 0.2 ng/s. On-line protein mapping was tested with proteins of different dimensions, showing competitiveness in terms of time (completed map within 15 min) and exhaustive maps of small proteins. The results of the CE-microreactor and the potential to immobilise biocomponents easily on a desired portion of the capillary indicate further developments towards the construction of a variety of miniaturised enzymatic screening devices for high-throughput screening analysis.     

Timing controllable electrofusion device for aqueous droplet-based microreactors

This paper describes an electrofusion device for controlling the precise moment of fusion between droplets by applying an electric field. This device allows (i) accurate determination of the start of chemical/biological reactions, (ii) minimum contact of reactants with channel walls--eliminating surface absorption problems, (iii) easy fabrication and (iv) continuous observation of initiated reaction. We demonstrated the fusion of beta-galactosidase and fluorescein di-beta-D-galactopyranoside (FDG) droplets, and observed the enzymatic reaction using fluorescence microscopy. In addition, sequential fusion of pico-litre droplets was also accomplished.