Prof. Andrew deMello

Principal Investigator

• +41 44 633 66 10

Andrew is currently Professor of Biochemical Engineering in the Department of Chemistry and Applied Biosciences at ETH Zurich and Head of the Institute for Chemical and Bioengineering. Prior to his arrival in Zurich he was Professor of Chemical Nanosciences and Head of the Nanostructured Materials and Devices Section in the Chemistry Department at Imperial College London.

He obtained a 1st Class Degree in Chemistry and PhD in Molecular Photophysics from Imperial College London in 1995 and subsequently held a Postdoctoral Fellowship in the Department of Chemistry at the University of California, Berkeley working with professor Richard Mathies.

His research interests cover a broad range of activities in the general area of microfluidics and nanoscale science. Primary specializations include the development of microfluidic devices for high-throughput biological and chemical analysis, ultra-sensitive optical detection techniques, nanofluidic reaction systems for chemical synthesis, novel methods for nanoparticle synthesis, the exploitation of semiconducting materials in diagnostic applications, the development of intelligent microfluidics and the processing of living organisms.

Andrew has given approximately 350 invited lectures at conferences and universities in North America, Europe, Africa and Asia (including 75 plenary or keynote lectures), has published 275 papers in refereed journals, and co-authored two books. He currently sits on the Editorial Boards of Analytical Chemistry, Advanced Materials Technologies Chemistry World, The Journal of Flow Chemistry and Chem (Cell Press) He is also co-founder of Molecular Vision Ltd, an Imperial College spin-out company developing low-cost diagnostic devices for use in the doctor's surgery and in the home.

Science originating from the deMello group has been recognized through the award of the 2002 SAC Silver Medal (Royal Society of Chemistry), the 2009 Clifford Paterson Medal from The Royal Society, the 2009 Corday Morgan Medal (Royal Society of Chemistry) and the 2007 Clark Memorial Lectureship (California State University). Most recently Andrew was awarded the 2012 Pioneers of Miniaturization Lectureship by Dow Corning and the RSC.

Journal

Holzner, G.; Du, Y.; Cao, X.; Choo, J.; deMello, A.; Stavrakis, S. An Optofluidic System with Integrated Microlens Arrays for Parallel Imaging Flow Cytometry. Lab on a Chip 2018, Accepted Manuscript

In recent years, high-speed imaging has become increasingly effective for the rapid analysis of single cells in flowing environments. Single cell imaging methods typically incorporate a minimum magnification of 10$\\\\times$ when extracting sizing and morphological information. Although information content may be significantly enhanced by increasing magnification, this is accompanied by a corresponding reduction in field of view, and thus a decrease in the number of cells assayed per unit time. Accordingly, the acquisition of high resolution data from wide field views remains an unsolved challenge. To address this issue, we present an optofluidic flow cytometer integrating a refractive, microlens array (MLA) for imaging cells at high linear velocities, whilst maximizing the number of cells per field of view. To achieve this, we adopt an elasto-inertial approach for cell focusing within an array of parallel microfluidic channels, each equipped with a microlens. We characterize the optical performance of the microlenses in terms of image formation, magnification and resolution using both ray-tracing simulations and experimental measurements. Results demonstrate that the optofluidic platform can efficiently count and magnify micron-sized objects up to 4 times. Finally, we demonstrate the capabilities of the platform as an imaging flow cyclometer, demonstrating the efficient discrimination of hB and Jurkat cells at throughputs up to 50,000 cells per second.

Dressler, O. J.; Howes, P. D.; Choo, J.; deMello, A. J. Reinforcement Learning for Dynamic Microfluidic Control. ACS Omega 2018, 3, 10084–10091

Recent years have witnessed an explosion in the application of microfluidic techniques to a wide variety of problems in the chemical and biological sciences. Despite the many considerable advantages that microfluidic systems bring to experimental science, microfluidic platforms often exhibit inconsistent system performance when operated over extended timescales. Such variations in performance are because of a multiplicity of factors, including microchannel fouling, substrate deformation, temperature and pressure fluctuations, and inherent manufacturing irregularities. The introduction and integration of advanced control algorithms in microfluidic platforms can help mitigate such inconsistencies, paving the way for robust and repeatable long-term experiments. Herein, two state-of-the-art reinforcement learning algorithms, based on Deep Q-Networks and model-free episodic controllers, are applied to two experimental “challenges,” involving both continuous-flow and segmented-flow microfluidic systems. The algorithms are able to attain superhuman performance in controlling and processing each experiment, highlighting the utility of novel control algorithms for automated high-throughput microfluidic experimentation.

Stavrakis, S.; Holzner, G.; Choo, J.; deMello, A. High-throughput microfluidic imaging flow cytometry. Current Opinion in Biotechnology 2019, 55, 36–43

Recently, microfluidic-based flow cytometry platforms have been shown to be powerful tools for the manipulation and analysis of single cells and micron-sized particles in flow. That said, current microfluidic flow cytometers are limited in both their analytical throughput and spatial resolution, due to their reliance on single point interrogation schemes. Conversely, high-speed imaging techniques can be applied to a wide variety of problems in which analyte molecules are manipulated at high linear velocities. Such an approach allows a detailed visualization of dynamic events through acquisition of a series of image frames captured with high temporal and spatial resolution. Herein, we describe some of the most significant recent advances in the development of multi-parametric, optofluidic imaging flow cytometry for the enumeration of complex cellular populations.

Yang, T.; Choo, J.; Stavrakis, S.; De Mello. Fluoropolymer Coated PDMS Microfluidic Devices for Application in Organic Synthesis. Chemistry - A European Journal 2018, 24, 11803–12100

In recent years there has been huge interest in the development of microfluidic reactors for the synthesis of small molecules and nanomaterials. Such reaction platforms represent a powerful and versatile alternative to traditional formats since they allow for the precise, controlled and flexible management of reactive processes. To date, the majority of microfluidic reactors used in small molecule synthesis have been manufactured using conventional lithographic techniques, from materials such as glasses, ceramics, stainless steel and silicon. Surprisingly, the fabrication of microfluidic devices from such rigid materials remains ill‐defined, complex, and expensive. Accordingly, the microfluidic toolkit for chemical synthesis would significantly benefit from the development of solvent‐resistant microfluidic devices that can be manufactured using soft‐lithographic prototyping methods. Whilst significant advances in the development of solvent‐resistant polymers have been made, only modest steps have been taken towards simplifying their use as microfluidic reactors. Herein, we emphasize the benefits of using a commercially available, amorphous perfluorinated polymer, CYTOP, as a coating with which to transform PDMS into a chemically inert material for use in organic synthesis applications. Its efficacy is demonstrated through the subsequent performance of photooxidation reactions and reactions under extremely acidic or basic conditions.

Lignos, I.; Morad, V.; Shynkarenko, Y.; Bernasconi, C.; Maceiczyk, R. M.; Protesescu, L.; Bertolotti, F.; Kumar, S.; Ochsenbein, S. T.; Masciocchi, N.; Guagliardi, A.; Shih, C.-J.; Bodnarchuk, M. I.; deMello, A. J.; Kovalenko, M. Exploration of Near-Infrared-Emissive Colloidal Multinary Lead Halide Perovskite Nanocrystals Using an Automated Microfluidic Platform. ACS Nano 2018, 12, 5504–5517

Hybrid organic–inorganic and fully inorganic lead halide perovskite nanocrystals (NCs) have recently emerged as versatile solution-processable light-emitting and light-harvesting optoelectronic materials. A particularly difficult challenge lies in warranting the practical utility of such semiconductor NCs in the red and infrared spectral regions. In this context, all three archetypal A-site monocationic perovskites—CH3NH3PbI3, CH(NH2)2PbI3, and CsPbI3—suffer from either chemical or thermodynamic instabilities in their bulk form. A promising approach toward the mitigation of these challenges lies in the formation of multinary compositions (mixed cation and mixed anion). In the case of multinary colloidal NCs, such as quinary CsxFA1–xPb(Br1–yIy)3 NCs, the outcome of the synthesis is defined by a complex interplay between the bulk thermodynamics of the solid solutions, crystal surface energies, energetics, dynamics of capping ligands, and the multiple effects of the reagents in solution. Accordingly, the rational synthesis of such NCs is a formidable challenge. Herein, we show that droplet-based microfluidics can successfully tackle this problem and synthesize CsxFA1–xPbI3 and CsxFA1–xPb(Br1–yIy)3 NCs in both a time- and cost-efficient manner. Rapid in situ photoluminescence and absorption measurements allow for thorough parametric screening, thereby permitting precise optical engineering of these NCs. In this showcase study, we fine-tune the photoluminescence maxima of such multinary NCs between 700 and 800 nm, minimize their emission line widths (to below 40 nm), and maximize their photoluminescence quantum efficiencies (up to 89%) and phase/chemical stabilities. Detailed structural analysis revealed that the CsxFA1–xPb(Br1–yIy)3 NCs adopt a cubic perovskite structure of FAPbI3, with iodide anions partially substituted by bromide ions. Most importantly, we demonstrate the excellent transference of reaction parameters from microfluidics to a conventional flask-based environment, thereby enabling up-scaling and further implementation in optoelectronic devices. As an example, CsxFA1–xPb(Br1–yIy)3 NCs with an emission maximum at 735 nm were integrated into light-emitting diodes, exhibiting a high external quantum efficiency of 5.9% and a very narrow electroluminescence spectral bandwidth of 27 nm.

Bezinge, L.; Maceiczyk, R. M.; Lignos, I.; Kovalenko, M. V.; deMello, A. J. Pick a Color MARIA: Adaptive Sampling Enables the Rapid Identification of Complex Perovskite Nanocrystal Compositions with Defined Emission Characteristics. ACS Applied Materials & Interfaces 2018, 10, 18869–18878

Recent advances in the development of hybrid organic–inorganic lead halide perovskite (LHP) nanocrystals (NCs) have demonstrated their versatility and potential application in photovoltaics and as light sources through compositional tuning of optical properties. That said, due to their compositional complexity, the targeted synthesis of mixed-cation and/or mixed-halide LHP NCs still represents an immense challenge for traditional batch-scale chemistry. To address this limitation, we herein report the integration of a high-throughput segmented-flow microfluidic reactor and a self-optimizing algorithm for the synthesis of NCs with defined emission properties. The algorithm, named Multiparametric Automated Regression Kriging Interpolation and Adaptive Sampling (MARIA), iteratively computes optimal sampling points at each stage of an experimental sequence to reach a target emission peak wavelength based on spectroscopic measurements. We demonstrate the efficacy of the method through the synthesis of multinary LHP NCs, (Cs/FA)Pb(I/Br)3 (FA = formamidinium) and (Rb/Cs/FA)Pb(I/Br)3 NCs, using MARIA to rapidly identify reagent concentrations that yield user-defined photoluminescence peak wavelengths in the green–red spectral region. The procedure returns a robust model around a target output in far fewer measurements than systematic screening of parametric space and additionally enables the prediction of other spectral properties, such as, full-width at half-maximum and intensity, for conditions yielding NCs with similar emission peak wavelength.

Berger, S.; Lattmann, E.; Aegerter-Wilmsen, T.; Hengartner, M.; Hajnal, A.; deMello, A.; Casadevall i Solvas, X. Long-term C. elegans immobilization enables high resolution developmental studies in vivo. Lab on a Chip 2018, 18, 1359–1368

Live-imaging of C. elegans is essential for the study of conserved cellular pathways (e.g. EGFR/Wnt signaling) and morphogenesis in vivo. However, the usefulness of live imaging as a research tool has been severely limited by the need to immobilize worms prior to and during imaging. Conventionally, immobilization is achieved by employing both physical and chemical interventions. These are known to significantly affect many physiological processes, and thus limit our understanding of dynamic developmental processes. Herein we present a novel, easy-to-use microfluidic platform for the long-term immobilization of viable, normally developing C. elegans, compatible with image acquisition at high resolution, thereby overcoming the limitations associated with conventional worm immobilization. The capabilities of the platform are demonstrated through the continuous assessment of anchor cell (AC) invasion and distal tip cell (DTC) migration in larval C. elegans and germ cell apoptosis in adult C. elegans in vivo for the first time.

Sevim, S.; Sorrenti, A.; Franco, C.; Furukawa, S.; Pané, S.; deMello, A. J. Self-assembled materials and supramolecular chemistry within microfluidic environments: From common thermodynamic states to non-equilibrium structures. Chemical Society Reviews 2018, 47, 3788–3803

Self-assembly processes are crucial in the bottom-up fabrication of hierarchical supramolecular structures and advanced functional materials. Their control has traditionally relied on encoded building blocks bearing suitable moieties for recognition and interaction, while targeting the thermodynamic equilibrium state. On the other hand, nature founds the creation of hierarchical organized materials with surprisingly complex biological functions on the ultimate control of reaction diffusion processes. Indeed, under non-equilibrium conditions (kinetic control), the spatio-temporal command of chemical gradients and reactant mixing during self-assembly (e.g. creation of non-uniform chemical environments) can strongly affect the outcome of self-assembly process (i.e. the materials prepared), thus enabling an ultimate control over materials properties and functions. In this tutorial review, we will show how the unique microscale physical conditions offered by microfluidic technologies, in the first place mixing only based on reagent diffusion, can advantageously be used to control the self-assembly of materials, and of supramolecular aggregates in solution, making possible the isolation of intermediate states, unprecedented non-equilibrium structures, as well as the emergence of novel functions. In particular, the selected examples will confirm that microfluidic devices are a valuable toolbox technology to unveil, understand and steer self-assembly pathways to a desired structure and/or property/function, as well as advanced processing tools for device fabrication and integration.

Pérez del Pino, A.; González-Campo, A.; Giraldo, S.; Peral, J.; György, E.; Logofatu, C.; deMello, A. J.; Puigmartí-Luis, J. Synthesis of graphene-based photocatalysts for water splitting by laser-induced doping with ionic liquids. Carbon 2018, 130, 48–58

The synthesis of metal-free graphene-based photocatalysts has received great attention recently due to their expected contributions to the development of solar-based hydrogen generation via water-splitting in a low cost and ecological manner. In this work, a new method for the generation of nitrogen-doped graphene-based powder employing an alternative solution to commonly used toxic and hazardous organic solvents is presented. The procedure involves ultraviolet pulsed laser irradiation of graphene oxide (GO) flakes dispersed in 1-butyl-3-methylimidazolium [bmim]-based ionic liquids using both chloride and acetate anions. The structural and compositional analysis using transmission electron mi- croscopy, X-ray photoelectron and infrared spectroscopy indicate that the irradiated GO becomes partially reduced and doped with graphitic, pyrrolic and pyridinic nitrogen species. Interestingly, the relative content of the nitrogen functionalities is controlled by the anion in the ionic liquid and its concentration, with the obtained graphene-based powders showing higher photocatalytic activity than GO. Furthermore, a remarkable synergistic effect is observed for GO-[bmim]-acetate powder (acting as co-catalyst) in combination with anatase TiO2 nanoparticles. The presented method opens new research avenues for the cost-effective mass production of graphene-based photocatalysts for water splitting applications.

Ugrinic, M.; Zambrano, A.; Berger, S.; Mann, S.; Tang, T.-Y. D.; deMello, A. Microfluidic Formation of Proteinosomes. Chemical Communications 2018, 54, 287–290

Herein we describe a novel microfluidic method for the generation of proteinosome micro-droplets, based on bovine serum albumin and glucose oxidase conjugated to PNIPAAm chains. The size of such water-in-oil droplets is regulated via control of the input reagent flow rate, with generated proteinosome populations exhibiting narrower size distributions than those observed when using standard bulk methodologies. Importantly, proteinosomes transferred from an oil- to an aqueous-environment remain intact, become fully hydrated and exhibit an increase in average size. Moreover, functional proteinosomes prepared via microfluidics exhibit lower Km values and higher enzymatic activities than proteinosomes produced by bulk methodologies.

Hoop, M.; Walde, C. F.; Riccò, R.; Mushtaq, F.; Terzopoulou, A.; Chen, X.-Z.; deMello, A. J.; Doonan, C. J.; Falcaro, P.; Nelson, B. J.; Puigmartí-Luis, J.; Pané, S. Biocompatibility characteristics of the metal organic framework ZIF-8 for therapeutical applications. Applied Materials Today 2018, 11, 13–21

Metal–organic frameworks (MOFs) are a class of crystalline materials constructed from organic linkers and inorganic nodes. MOFs typically possess ultra-high surface areas and pore volumes; thus, they are ideal candidates for biomedical applications. Zinc Imidazolate Framework 8 (ZIF-8) has been widely established in the literature as a potential candidate for on-demand drug delivery applications. Indeed, ZIF-8 has a remarkable loading capacity, stability in physiological environments, and tunable drug release properties. However, the use of ZIF-8 for in vivo applications requires a clear understanding of the interaction of ZIF-8 with biological tissue. In this work, we investigated the biocompatibility of ZIF-8 toward six different cell lines representing various body parts (kidney, skin, breast, blood, bones, and connective tissue). Our results suggest that ZIF-8 has no significant cytotoxicity up to a threshold value of 30 μg mL−1. Above 30 μg mL−1, the cytotoxicity is shown to result from the influence of released Zinc ions (Zn2+) on the mitochondrial ROS production. This adverse effect is responsible for cell cycle arrest in the G2/M phase due to irreversible DNA damage, ultimately initiating cellular apoptosis pathways. Due to this insight, we encapsulated a hormone, insulin, into ZIF-8 particles and then compared its drug delivery capabilities to the aforementioned cytotoxicity values. Our results suggest that ZIF-8 is suitable for therapeutic applications. Furthermore, this study establishes a clear understanding of the interaction of ZIF-8 and its constituents with various cell lines and highlights the important biocompatibility factors that must be considered for future in vivo testing.

Yang, T.; Stavrakis, S.; deMello, A. A High-Sensitivity, Integrated Absorbance and Fluorescence Detection Scheme for Probing Picoliter-Volume Droplets in Segmented Flows. Analytical Chemistry 2017, 89, 12880–12887

Droplet-based microfluidic systems that incorporate flowing streams of pL-volume droplets surrounded by a continuous and immiscible carrier phase have attracted significant recent attention due to their utility in complex chemical and biological experimentation. Analysis of pL-droplets, generated at kHz frequencies and moving at high linear velocities, is almost exclusively achieved using fluorescence-based detection schemes. To extend the applicability of such optical detection schemes we herein report the development of a simple and cost-effective optofluidic platform ,integrating liquid core PDMS waveguides, that allows the accurate measurement of absorbance within individual pL-volume droplets moving within segmented flows. Using such an approach, differential measurements of “sample “and “reference” droplets can be acquired at 1 kHz and yields detection limits of 400 nM for fluorescein in water. Significantly, the presented technique enables simultaneous fluorescence and absorbance interrogation of rapidly moving droplets in a fully automated manner. Proof of principle is demonstrated through the titration and monitoring of pH gradients in real time.

Holzner, G.; Stavrakis, S.; deMello, A. Elasto-Inertial Focusing of Mammalian Cells and Bacteria Using Low Molecular, Low Viscosity PEO Solutions. Analytical Chemistry 2017, 89, 11653–11663

The ability to manipulate biological cells is critical in a diversity of biomedical and industrial applications. Microfluidic-based cell manipulations provide unique opportunities for sophisticated and high-throughput biological assays such as cell sorting, rare cell detection, and imaging flow cytometry. In this respect, cell focusing is an extremely useful functional operation preceding downstream biological analysis, since it allows the accurate lateral and axial positioning of cells moving through microfluidic channels, and thus enables sophisticated cell manipulations in a passive manner. Herein, we explore the utility of viscoelastic carrier fluids for enhanced elasto-inertial focusing of biological species within straight, rectangular cross section microfluidic channels. Since the investigated polymer solutions possess viscosities close to that of water and exhibit negligible shear thinning, focusing occurs over a wide range of elasticity numbers and a large range of Reynolds numbers. With a view to applications in the robust focusing of cells and bacteria, we assess and characterize the influence of accessible focusing parameters, including blockage ratio, volumetric flow rate, cell concentration, and polymer chain length.

Rane, A. S.; Rutkauskaite, J.; deMello, A.; Stavrakis, S. High-Throughput Multi-parametric Imaging Flow Cytometry. Chem 2017, 3, 588–602

Flow cytometry, incorporating either point- or imaging-based detection schemes, is recognized to be the gold-standard tool for high-throughput manipulation and analysis of single cells in flow but is typically limited in either the number of cells that can be interrogated per unit of time or the resolution with which individual cells can be imaged. To address these limitations, we present a sheathless, microfluidic imaging flow cytometer incorporating stroboscopic illumination for blur-free cellular analysis at throughputs exceeding 50,000 cells/s. By combining inertial focusing of cells in parallel microchannels and stroboscopic illumination, the chip-based cytometer is able to extract multi-color fluorescence, bright-field, and dark-field images and perform accurate sizing of individual cells and analysis of heterogeneous cell suspensions while maintaining operational simplicity. To showcase the efficacy of the approach, we apply the method to the rapid enumeration of apoptotic cells and the high-throughput discrimination of cell-cycle phases.

Weidenbacher, L.; Abrishamkar, A.; Rottmar, M.; Guex, A. G.; Maniura-Weber, K.; deMello, A. J.; Ferguson, S. J.; Rossi, R. M.; Fortunato, G. Electrospraying of microfluidic encapsulated cells for the fabrication of cell-laden electrospun hybrid tissue constructs. Acta Biomaterialia 2017, 64, 137–147

The fabrication of functional 3D tissues is a major goal in tissue engineering. While electrospinning is a promising technique to manufacture a structure mimicking the extracellular matrix, cell infiltration into elec- trospun scaffolds remains challenging. The robust and in situ delivery of cells into such biomimetic scaffolds would potentially enable the design of tissue engineered constructs with spatial control over cellular distri- bution but often solvents employed in the spinning process are problematic due to their high cytotoxicity. Herein, microfluidic cell encapsulation is used to establish a temporary protection vehicle for the in situ delivery of cells for the development of a fibrous, cell-laden hybrid biograft. Therefore a layer-by-layer process is used by alternating fiber electrospinning and cell spraying procedures.Both encapsulation and subsequent electrospraying of capsules has no negative effect on the viability and myogenic differentiation of murine myoblast cells. Propidium iodide positive stained cells were analyzed to quantify the amount of dead cells and the presence of myosin heavy chain positive cells after the processes was shown. Furthermore, encapsulation successfully protects cells from cytotoxic solvents (such as dimethyl- formamide) during in situ delivery of the cells into electrospun poly(vinylidene fluoride-co-hexafluoropropylene) scaffolds. The resulting cell-populated biografts demonstrate the clear potential of this approach in the creation of viable tissue engineering constructs.

Maceiczyk, R. M.; Hess, D.; Chiu, F. W. Y.; Stavrakis, S.; deMello, A. J. Differential detection photothermal spectroscopy: towards ultra-fast and sensitive label-free detection in picoliter & femtoliter droplets. Lab on a Chip 2017, 17, 3654–3663

Despite the growing importance of droplet-based microfluidics in high-throughput experimentation, few current methods allow the sensitive measurement of absorbance within rapidly moving droplets. To address this significant limitation, we herein present the application of differential detection photothermal interferometry (DDPI) for single-point absorbance quantification in pL- and fL-volume droplets. To assess the efficacy of our approach, we initially measure absorbance in 100 pL droplets at frequencies in excess of 1 kHz and determine a detection limit of 1.4 μmol L−1 for Erythrosin B (A = 3.8 × 10−4). Subsequently, we apply the method to the analysis of fL-volume droplets and droplets generated at frequencies in excess of 10 kHz. Finally, we demonstrate the utility of DDPI as a detection scheme for colorimetric assays. Specifically, we extract the Michaelis–Menten constant for the reaction of β-galactosidase and chlorophenol-red-β-D-galactopyranoside and monitor the metabolomic activity of a population of HL-60 cells at the single cell level. Results establish single-point absorbance detection as a powerful, sensitive and rapid alternative to fluorescence for a wide range of assays within segmented flows.

Abrishamkar, A.; Rodríguez-San-Miguel, D.; Rodríguez Navarro, J. A.; Rodriguez-Trujillo, R.; Amabilino, D. B.; Mas-Ballesté, R.; Zamora, F.; deMello, A. J.; Puigmarti-Luis, J. Microfluidic-based Synthesis of Covalent Organic Frameworks (COFs): A Tool for Continuous Production of COF Fibers and Direct Printing on a Surface. Journal of Visualized Experiments 2017

Covalent Organic Frameworks (COFs) are a class of porous covalent materials which are frequently synthesized as unprocessable crystalline powders. The first COF was reported in 2005 with much effort centered on the establishment of new synthetic routes for its preparation.To date, most available synthetic methods for COF synthesis are based on bulk mixing under solvothermal conditions. Therefore, there is increasing interest in developing systematic protocols for COF synthesis that provide for fine control over reaction conditions and improve COF processability on surfaces, which is essential for their use in practical applications. Herein, we present a novel microfluidic-based method for COF synthesis where the reaction between two constituent building blocks, 1,3,5-benzenetricarbaldehyde (BTCA) and 1,3,5-tris(4- aminophenyl)benzene (TAPB), takes place under controlled diffusion conditions and at room temperature. Using such an approach yields sponge-like, crystalline fibers of a COF material, hereafter called MF-COF. The mechanical properties of MF-COF and the dynamic nature of the approach allow the continuous production of MF-COF fibers and their direct printing onto surfaces. The general method opens new potential applications requiring advanced printing of 2D or 3D COF structures on flexible or rigid surfaces.

Ding, Y.; Choo, J.; deMello, A. J. From single-molecule detection to next-generation sequencing: microfluidic droplets for high-throughput nucleic acid analysis. Microfluidics and Nanofluidics 2017, 21

Droplet-based microfluidic technologies have proved themselves to be of significant utility in the performance of high-throughput chemical and biological experiments. By encapsulating and isolating reagents within femtoliter–nanoliter droplet, millions of (bio) chemical reactions can be processed in a parallel fashion and on ultra-short timescales. Recent applications of such technologies to genetic analysis have suggested significant utility in low-cost, efficient and rapid workflows for DNA amplification, rare mutation detection, antibody screening and next-generation sequencing. To this end, we describe and highlight some of the most interesting recent developments and applications of droplet-based microfluidics in the broad area of nucleic acid analysis. In addition, we also present a cursory description of some of the most essential functional components, which allow the creation of integrated and complex workflows based on flowing streams of droplets.

Chiu, D. T.; deMello, A. J.; Di Carlo, D.; Doyle, P. S.; Hansen, C.; Maceiczyk, R. M.; Wootton, R. C. R. Small but Perfectly Formed? Successes, Challenges, and Opportunities for Microfluidics in the Chemical and Biological Sciences. Chem 2017, 2, 201–223

Microfluidic systems are pervasive in many areas of experimental science, but what are the real advantages of this technology? We describe some of the fea- tures and properties that make microfluidic devices unique experimental tools. In addition to pointing out some of the less effective uses of this technology, we assess the most successful applications of microfluidics over the last two de- cades and highlight the areas where they had the greatest impact. We also pro- pose applications where microfluidic systems could be applied to the greatest effect in the future.

Maceiczyk, R.; Shimizu, H.; Müller, D.; Kitamori, T.; deMello, A. A Photothermal Spectrometer for Fast and Background-Free Detection of Individual Nanoparticles in Flow. Analytical Chemistry 2017, 89, 1994–1999

Sensitive detection and quantification of individual plasmonic nanoparticles is critical in a range of applications in the biological, nanomaterials, and analytical sciences. Although a wide range of techniques can be applied to the analysis of immobilized particles, high-throughput analysis of nanoscale species in flow is surprisingly underdeveloped. To address this shortcoming, we present an ultrasensitive, background-free technique based on the photothermal effect and termed differential detection photothermal interferometry (DDPI). We show, both theoretically and experimentally, that DDPI can specifically extract either the phase or amplitude of a photothermal signal. We then quantitatively detect 10 and 20 nm diameter gold nanoparticles at femtomolar concentrations and at linear flow speeds of 10 mm/s. In the case of 50 nm gold particles, we operate at an even higher linear flow speed of 100 mm/s, corresponding to an analyzed volume of more than 1 nL/s. This allows quantification of particle content at attomolar to femtomolar concentrations and counting rates between 0.1 and 400 particles per second. Finally, we confirm that the signal follows the size-dependent variations predicted by Mie theory.

Casadevall i Solvas, X.; deMello, A. Particle concentration influences inertial focusing in Multiorifice Flow Fractionation microfluidic devices. Matters 2016

Multiorifice Flow Fractionation (MOFF) devices have been used for the separation of microparticles and cells according to their size and the degree of inertia (i.e. Re number). Herein an additional parameter, particle concentration, is reported to affect the performance of MOFF when the remaining conditions are unchanged. Particularly, at low concentrations focusing occurs efficiently at the center of these devices, while at higher concentrations particles tend to accumulate near the channel walls. This indicates that particle-particle interactions are a key component in the performances of these devices.

Rubio-Martinez, M.; Imaz, I.; Domingo, N.; Abrishamkar, A.; Mayor, T. S.; Rossi, R. M.; Carbonell, C.; deMello, A. J.; Amabilino, D. B.; Maspoch, D.; Puigmartí-Luis, J. Freezing the Nonclassical Crystal Growth of a Coordination Polymer Using Controlled Dynamic Gradients. Advanced Materials 2016, 28, 8150–8155

Manually engineered self-assembled structures have for many years been investigated under equilibrium conditions so that their most stable forms are reached, until recently. There has been a growing interest in obtaining and studying non-equilibrium self-assembled structures. The primary reason for this is that non-equilibrium structures (which are typically formed transiently under a constant influx of energy) can offer a broad number of intriguing opportunities in the development of novel materials and systems with advanced functionalities. For example, transient and/or steady-state self-assembled structures generated far from equilibrium are the basis of many sophisticated functions observed in living systems, e.g. DNA replication and/or cell division. Nonetheless, the controlled synthesis and study of intermediate, self-assembled structures is still a major challenge, which currently limits advancements in materials development and technology. Herein we show for first time a methodology that can be efficiently used to synthesize, isolate and study out-of-equilibrium crystal structures employing controlled and diffusion limited microfluidic environments. Unlike studies conducted with conventional mixing procedures in a flask, we prove experimentally and with numerical simulations that microfluidic technologies can undoubtedly fine-tune reaction times and reagents concentration profiles; factors that enable obtaining out of-equilibrium self-assembled crystal forms.

Yashina, A.; Lignos, I.; Stavrakis, S.; Choo, J.; deMello, A. J. Scalable production of CuInS2/ZnS quantum dots in a two-step droplet-based microfluidic platform. Journal of Materials Chemistry C 2016, 4, 6401–6408

We report the scalable formation of CuInS2/ZnS nanocrystals using a two-stage microfluidic reactor integrated with a real-time optical detection system, which is able to monitor reaction parameters prior and subsequent to the addition of the shell material. By injecting a ZnS single source precursor in droplets containing CuInS2 cores and without the need of purification steps, we are able to obtain core-shell nanocrystal populations emitting between 580 and 760 nm with significant narrower size distributions (90–95 nm) than for the same material systems synthesized on the macroscale. In-line monitoring allowed for rapid assessment of optimum reaction parameters (Cu/In, S/(Cu+In), Zn/(Cu+In) molar ratios, temperatures and reaction time) and enabled the formation of CuInS2/ZnS nanocrystals with high photoluminescence quantum yields (∼ 55%) within a few seconds. We believe that this synthetic methodology will be of significant utility in controllable production of ternary and quaternary metal chalcogenides, complex core-shell and doped nanostructures.

Maceiczyk, R. M.; Bezinge, L.; deMello, A. J. Kinetics of nanocrystal synthesis in a microfluidic reactor: theory and experiment. Reaction Chemistry & Engineering 2016, 1, 261–271

The processes occurring during nanocrystal nucleation and growth are currently not well understood. Herein, we theoretically and experimentally investigate the growth kinetics in colloidal nanocrystal synthesis. Using a novel microfluidic reactor integrating independent modules for nucleation and growth, we demonstrate the controlled, direct synthesis of high quality nanocrystals in high yield. For CdSe nanocrystals, we find that size tuning solely by variation of the reaction time and temperature does not yield product populations of optimal size dispersion or yield. Instead, we present an improved method for the synthesis of bespoke nanocrystals that relies on the controlled addition of precise amounts of additional precursor subsequent to nucleation and fine tuning of the reaction time and temperature in the second stage. Real-time spectroscopic monitoring of the produced crystals in conjunction with kinetic simulations confirms the close correspondence between the model and the experiment and elegantly quantifies the effects of temperature, concentration, additives and surfactants on conversion, growth and diffusion rates within the model framework. We show that the conversion of the precursor to a monomer follows a first order rate law and that the growth rate has a stronger temperature dependence than the conversion rate. Moreover, the surfactant concentration retards the reaction by inhibiting diffusion to the growing crystals whilst maintaining a uniform conversion rate. Finally, we demonstrate that diphenylphosphine, a common additive in CdSe synthesis, enhances the reaction rate by accelerating precursor conversion.
» REACTION CHEMISTRY AND ENGINEERING COVER 2016

Martino, C.; Vigolo, D.; Solvas, X. C. i; Stavrakis, S.; deMello, A. J. Real-Time PEGDA-Based Microgel Generation and Encapsulation in Microdroplets. Advanced Materials Technologies 2016, 1, 1600028

Herein, a method is reported that combines droplet-based microfluidics and microscope projection photolithography for the generation of poly ethylene glycol diacrylate microgels and their encapsulation within pL-volume droplets. By implementing continuous-flow photolithography in the vicinity of a cross junction, the real-time generation and in situ encapsulation of fiber-like structures within pL-volume aqueous microdroplets is demonstrated. The effect of UV excitation is assessed at varying distances from the cross junction for both constant and pulsed UV excitation modes, as a route to controlling microfiber length. Finally, UV excitation within trapped droplets is explored and how the combination of the two techniques can lead to the generation of 3D patterned microstructures is demonstrated, opening new avenues for the rapid generation of inner scaffolding of artificial cell facsimiles.

Müller, D.; Cattaneo, S.; Meier, F.; Welz, R.; de Vries, T.; Portugal-Cohen, M.; Antonio, D. C.; Cascio, C.; Calzolai, L.; Gilliland, D.; de Mello, A. Inverse supercritical fluid extraction as a sample preparation method for the analysis of the nanoparticle content in sunscreen agents. Journal of Chromatography A 2016, 1440, 31–36

We demonstrate the use of inverse supercritical carbon dioxide (scCO2) extraction as a novel method of sample preparation for the analysis of complex nanoparticle-containing samples, in our case a model sunscreen agent with titanium dioxide nanoparticles. The sample was prepared for analysis in a simplified process using a lab scale supercritical fluid extraction system. The residual material was easily dispersed in an aqueous solution and analyzed by Asymmetrical Flow Field-Flow Fractionation (AF4) hyphenated with UV- and Multi-Angle Light Scattering detection. The obtained results allowed an unambiguous determination of the presence of nanoparticles within the sample, with almost no background from the matrix itself, and showed that the size distribution of the nanoparticles is essentially maintained. These results are especially relevant in view of recently introduced regulatory requirements concerning the labeling of nanoparticle-containing products. The novel sample preparation method is potentially applicable to commercial sunscreens or other emulsion-based cosmetic products and has important ecological advantages over currently used sample preparation techniques involving organic solvents.

Ding, Y.; Qiu, F.; Casadevall i Solvas, X.; Chiu, F.; Nelson, B.; deMello, A. Microfluidic-Based Droplet and Cell Manipulations Using Artificial Bacterial Flagella. Micromachines 2016, 7, 25

Herein, we assess the functionality of magnetic helical microswimmers as basic tools for the manipulation of soft materials, including microdroplets and single cells. Their ability to perform a range of unit operations is evaluated and the operational challenges associated with their use are established. In addition, we also report on interactions observed between the head of such helical swimmers and the boundaries of droplets and cells and discuss the possibilities of assembling an artificial swimming microorganism or a motorized cell.

Lignos, I.; Stavrakis, S.; Nedelcu, G.; Protesescu, L.; deMello, A. J.; Kovalenko, M. V. Synthesis of Cesium Lead Halide Perovskite Nanocrystals in a Droplet-Based Microfluidic Platform: Fast Parametric Space Mapping. Nano Letters 2016, 16, 1869–1877

Prior to this work, fully inorganic nanocrystals of cesium lead halide perovskite (CsPbX3, X = Br, I and Cl and Cl/Br and Br/I mixed halide systems), exhibiting bright and tunable photoluminescence, have been synthesized using conventional batch (flask-based) reactions. Unfortunately, our understanding of the parameters governing the formation of these nanocrystals is still very limited due to extremely fast reaction kinetics and multiple variables involved in ion-metathesis-based synthesis of such multinary halide systems. Herein, we report the use of a droplet-based microfluidic platform for the synthesis of CsPbX3 nanocrystals. The combination of online photoluminescence and absorption measurements and the fast mixing of reagents within such a platform allows the rigorous and rapid mapping of the reaction parameters, including molar ratios of Cs, Pb and halide precursors, reaction temperatures and reaction times. This translates into enormous savings in reagent usage and screening times when compared to analogous batch synthetic approaches. The early-stage insight into the mechanism of nucleation of metal halide nanocrystals suggests similarities with multinary metal chalcogenide systems, albeit with much faster reaction kinetics in the case of halides. Furthermore, we show that microfluidics-optimized synthesis parameters are also directly transferrable to the conventional flask-based reaction.

Gao, R.; Cheng, Z.; deMello, A. J.; Choo, J. Wash-free magnetic immunoassay of the PSA cancer marker using SERS and droplet microfluidics. Lab on a Chip 2016, 16, 1022–1029

We report a novel wash-free magnetic immunoassay technique for prostate-specific antigen (PSA) that uses a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. The magnetic bar embedded in a droplet-based microfluidic system segregates the free and bound SERS tags by splitting the droplets into two smaller parts. The presence of PSA targets leads more SERS tags to immunocomplex in one droplet so that fewer SERS tags remain in another supernatant solution droplet. Thus, SERS signal measurement enables the quantitative evaluation of PSA markers. This approach can provide a rapid and sensitive assay that is applicable for PSA cancer markers in serum without any washing. Specifically, SERS signals were measured at 174 droplets per minute and averaged for quantitative evaluation of PSA. The limit of detection (LOD) determined by our SERS-based microdroplet sensor was estimated to be below 0.1 ng mL−1, which is significantly below the clinical cut-off value for the diagnosis of prostate cancer. In addition, because the entire assay can be carried out automatically, only a minimal amount of sample is needed. Accordingly, the approach is expected to be useful as a potential clinical tool for the early diagnosis of prostate cancer.

Abrishamkar, A.; Paradinas, M.; Bailo, E.; Rodriguez-Trujillo, R.; Pfattner, R.; Rossi, R. M.; Ocal, C.; deMello, A. J.; Amabilino, D. B.; Puigmartí-Luis, J. Microfluidic Pneumatic Cages: A novel approach for in-chip crystal trapping, manipulation and controlled chemical treatment. Journal of Visualized Experiments 2016

The precise localization and controlled chemical treatment of structures on a surface are significant challenges for common laboratory technologies. Herein, we introduce a microfluidic-based technology, employing a double-layer microfluidic device, which can trap and localize in situ and ex situ synthesized structures on microfluidic channel surfaces. Crucially, we show how such a device can be used to conduct controlled chemical reactions onto on-chip trapped structures and we demonstrate how the synthetic pathway of a crystalline molecular material and its positioning inside a microfluidic channel can be precisely modified with this technology. This approach provides new opportunities for the controlled assembly of structures on surface and for their subsequent treatment.

Martino, C.; Statzer, C.; Vigolo, D.; deMello, A. J. Controllable Generation and Encapsulation of Alginate Fibers Using Droplet-Based Microfluidics. Lab on a Chip 2016, 16, 59–64

Herein we demonstrate the segmentation of alginate solution streams to generate alginate fibers of precisely controllable lengths between 200 and 1000 μm. Moreover, we demonstrate the subsequent encapsulation of the formed fibers within pL-volume microdroplets, produced within the same microfluidic device, in a direct manner. Finally, we show immediate and complete on- chip gelation of alginate fibers in a rapid and reproducible fashion.

Stanley, C. E.; Grossmann, G.; Casadevall i Solvas, X.; deMello, A. J. Soil-on-a-Chip: Microfluidic platforms for environmental organismal studies. Lab on a Chip 2016, 16, 228–241

Soil is the habitat of countless organisms and encompasses an enormous variety of dynamic environmental conditions. While it is evident that a thorough understanding of how organisms interact with the soil environment may have substantial ecological and economical impact, current laboratory-based methods depend on reductionist approaches that are incapable of simulating natural diversity. The application of Lab-on-a-Chip or microfluidic technologies to organismal studies is an emerging field, where the unique benefits afforded by system miniaturisation offer new opportunities for the experimentalist. Indeed, precise spatiotemporal control over the microenvironments of soil organisms in combination with high-resolution imaging has the potential to provide an unprecedented view of biological events at the single-organism or single-cell level, which in turn opens up new avenues for environmental and organismal studies. Herein we review some of the most recent and interesting developments in microfluidic technologies for the study of soil organisms and their interactions with the environment. We discuss how so-called “Soil-on-a-Chip” technology has already contributed significantly to the study of bacteria, nematodes, fungi and plants, as well as inter-organismal interactions, by advancing experimental access and environmental control. Most crucially, we highlight where distinct advantages over traditional approaches exist and where novel biological insights will ensue.

Kang, D.-K.; Gong, X.; Cho, S.; Kim, J.; Edel, J. B.; Chang, S.-I.; Choo, J.; deMello, A. J. 3D Droplet Microfluidic Systems for High-Throughput Biological Experimentation. Analytical Chemistry 2015, 87, 10770–10778

Herein, we describe the development of a multilayer droplet microfluidic system for creating concentration gradients and generating microdroplets of varying composition for high-throughput biochemical and cell-based screening applications. The 3D droplet-based microfluidic device consists of multiple PDMS layers, which are used to generate logarithmic concentration gradient reagent profiles. Parallel flow focusing structures are used to form picoliter-sized droplets of defined volumes but of varying composition. As proof of concept, we demonstrate rapid enzymatic activity assays and drug cytotoxicity assays on bacteria. The 3D droplet-based microfluidic platform has the potential to allow for high-efficiency and high-throughput analysis, overcoming the structural limitations of single layer microfluidic systems.

Chiu, F. W. Y.; Bagci, H.; Fisher, A. G.; deMello, A. J.; Elvira, K. S. A microfluidic toolbox for cell fusion. Journal of Chemical Technology & Biotechnology 2016, 91, 16–24

Cellular fusion is a key process in many fields ranging from historical gene mapping studies and monoclonal antibody production, through to cell reprogramming. Traditional methodologies for cell fusion rely on the random pairing of different cell types and generally result in low and variable fusion efficiencies. These approaches become particularly limiting where substantial numbers of bespoke one-to-one fusions are required, for example for in-depth studies of nuclear reprogramming mechanisms. In recent years, microfluidic technologies have proven valuable in creating platforms where the manipulation of single cells is highly efficient, rapid and controllable. These technologies also allow the integration of different experimental steps and characterisation processes into a single platform. Although the application of microfluidic methodologies to cell fusion studies is promising, current technologies that rely on static trapping are limited both in terms of the overall number of fused cells produced and their experimental accessibility. Here we review some of the most exciting breakthroughs in core microfluidic technologies that will allow the creation of integrated platforms for controlled cell fusion at high throughput.

van Swaay, D.; Tang, T.-Y. D.; Mann, S.; de Mello, A. Microfluidic Formation of Membrane-Free Aqueous Coacervate Droplets in Water. Angewandte Chemie International Edition 2015, 54, 8398–8401

We report on the formation of coacervate droplets from poly(diallyldimethylammonium chloride) with either adenosine triphosphate or carboxymethyl-dextran using a microfluidic flow-focusing system. The formed droplets exhibit improved stability and narrower size distributions for both coacervate compositions when compared to the conventional vortex dispersion techniques. We also demonstrate the use of two parallel flow-focusing channels for the simultaneous formation and co-location of two distinct populations of coacervate droplets containing different DNA oligonucleotides, and that the populations can coexist in close proximity up to 48 h without detectable exchange of genetic information. Our results show that the observed improvements in droplet stability and size distribution may be scaled with ease. In addition, the ability to encapsulate different materials into coacervate droplets using a microfluidic channel structure allows for their use as cell-mimicking compartments.

Lignos, I.; Stavrakis, S.; Kilaj, A.; deMello, A. J. Millisecond-Timescale Monitoring of PbS Nanoparticle Nucleation and Growth Using Droplet-Based Microfluidics. Small 2015, 11, 4009–4017

The early-time kinetics (<1 s) of lead sulfide (PbS) quantum dot formation are probed using a novel droplet-based microfluidic platform, which allows for high-throughput and real-time optical analysis of the reactive process with millisecond time resolution. The reaction platform enables the concurrent investigation of the emission characteristics of PbS quantum dots and a real-time estimation of their size and concentration during nucleation and growth. These investigations reveal a two-stage mechanism for PbS nanoparticle formation. The first stage corresponds to the fast conversion of precursor species to PbS crystals, followed by the growth of the formed particles. The growth kinetics of the PbS nanoparticles follow the Lifshitz–Slyozov–Wagner model for Ostwald ripening, allowing direct estimation of the rate constants for the process. In addition, the extraction of absorption spectra of ultrasmall quantum dots is demonstrated for first time in an online manner. The droplet-based microfluidic platform integrated with online spectroscopic analysis provides a new tool for the quantitative extraction of high temperature kinetics for systems with rapid nucleation and growth stages.

Dressler, O. J.; Yang, T.; Chang, S.-I.; Choo, J.; Wootton, R. C. R.; deMello, A. J. Continuous and low error-rate passive synchronization of pre-formed droplets. RSC Advances 2015, 5, 48399–48405

A microfluidic droplet-handling architecture for the synchronization of asynchronous, mis-matched, pre-formed droplet streams is demonstrated. This architecture is shown to be robust to variations in droplet input frequencies, whilst still producing highly reliable synchronisation. The operational phase space with regards to droplet size disparity is explored and the long-term operational stability of the system confirmed. Specifically, the microfluidic platform to synchronise droplet streams at a rate of 33 Hz over extended periods of time and with an error rate less than 0.2%.
» Continuous synchronisation of droplets

Poulsen, C. E.; Wootton, R. C. R.; Wolff, A.; deMello, A. J.; Elvira, K. S. A microfluidic platform for the rapid determination of distribution coefficients by gravity assisted droplet-based liquid-liquid extraction. Analytical Chemistry 2015, 87, 6265–6270

The determination of pharmacokinetic properties of drugs, such as the distribution coefficient (D) is a crucial measurement in pharmaceutical research. Surprisingly, the conventional (gold standard) technique used for D measurements, the shake-flask method, is antiquated and unsuitable for the testing of valuable and scarce drug candidates. Herein, we present a simple microfluidic platform for the determination of distribution coefficients using droplet-based liquid–liquid extraction. For simplicity, this platform makes use of gravity to enable phase separation for analysis and is 48 times faster and uses 99% less reagents than performing an equivalent measurement using the shake-flask method. Furthermore, the D measurements achieved in our platform are in good agreement with literature values measured using traditional shake-flask techniques. Since D is affected by volume ratios, we use the apparent acid dissociation constant, pK′, as a proxy for intersystem comparison. Our platform determines a pK′ value of 7.24 ± 0.15, compared to 7.25 ± 0.58 for the shake-flask method in our hands and 7.21 for the shake-flask method in the literature. Devices are fabricated using injection molding, the batchwise fabrication time is <2 min per device (at a cost of \$1 U.S. per device), and the interdevice reproducibility is high.

Gao, R.; Ko, J.; Cha, K.; Ho Jeon, J.; Rhie, G.; Choi, J.; deMello, A. J.; Choo, J. Fast and sensitive detection of an anthrax biomarker using SERS-based solenoid microfluidic sensor. Biosensors and Bioelectronics 2015, 72, 230–236

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-d-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the acccuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples.

Hess, D.; Rane, A.; deMello, A. J.; Stavrakis, S. High-Throughput, Quantitative Enzyme Kinetic Analysis in Microdroplets using Stroboscopic Epifluorescence Imaging. Analytical Chemistry 2015, 87, 4965–4972

Droplet-based microfluidic systems offer a range of advantageous features for the investigation of enzyme kinetics, including high time resolution and the ability to probe extremely large numbers of discrete reactions while consuming low sample volumes. Kinetic measurements within droplet-based microfluidic systems are conventionally performed using single point detection schemes. Unfortunately, such an approach prohibits the measurement of an individual droplet over an extended period of time. Accordingly, we present a novel approach for the extensive characterization of enzyme–inhibitor reaction kinetics within a single experiment by tracking individual and rapidly moving droplets as they pass through an extended microfluidic channel. A series of heterogeneous and pL-volume droplets, containing varying concentrations of the fluorogenic substrate resorufin β-d-galactopyranoside and a constant amount of the enzyme β-galactosidase, is produced at frequencies in excess of 150 Hz. By stroboscopic manipulation of the excitation laser light and adoption of a dual view detection system, “blur-free” images containing up to 150 clearly distinguishable droplets per frame are extracted, which allow extraction of kinetic data from all formed droplets. The efficiency of this approach is demonstrated via a Michaelis–Menten analysis which yields a Michaelis constant, Km, of 353 μM. Additionally, the dissociation constant for the competitive inhibitor isopropyl β-d-1-thiogalactopyranoside is extracted using the same method.

Pirbodaghi, T.; Vigolo, D.; Akbari, S.; deMello, A. Investigating the fluid dynamics of rapid processes within microfluidic devices using bright-field microscopy. Lab on a Chip 2015, 15, 2140–2144

The widespread application of microfluidic devices in the biological and chemical sciences requires the implementation of complex designs and geometries, which in turn leads to atypical fluid dynamic phenomena. Accordingly, a complete understanding of fluid dynamics in such systems is key in the facile engineering of novel and efficient analytical tools. Herein, we present an inexpensive and accurate approach for studying the fluid dynamics of rapid processes within microfluidic devices using bright-field microscopy with white light illumination. Specifically, we combine Ghost Particle Velocimetry and the detection of moving objects in automated video surveillance to track submicron size tracing particles via cross correlation between the speckle patterns of successive images. The efficacy of the presented technique is demonstrated by measuring the flow field over a square pillar (80 μm × 80 μm) in a 200 μm wide microchannel at high volumetric flow rates. Experimental results are in excellent agreement with those obtained via computational fluid dynamics simulations. The method is subsequently used to study the dynamics of droplet generation at a flow focusing microfluidic geometry. A unique feature of the presented technique is the ability to perform velocimetry analysis of ultra high-speed phenomena, which is not possible using micron-resolution particle image velocimetry (μPIV) approaches based on confocal or fluorescence microscopy.

Martino, C.; Lee, T. Y.; Kim, S.-H.; deMello, A. J. Microfluidic Generation of PEG-b-PLA Polymersomes Containing Alginate-based Core Hydrogel. Biomicrofluidics 2015, 9, 024101

Herein, we demonstrate a novel method for the generation of monodisperse cell-like structures containing a biocompatible hydrogel matrix surrounded by a membrane responsive to chemical cues. Specifically, we employ droplet-based microfluidics to generate PEG-PLA polymersomes encapsulating alginate in liquid form. We investigate alginate core gelation by creating an osmotic pressure gradient across the polymeric membrane that, through expansion, allows the passage of calcium ions. The effects of calcium concentration on the core gelation are explored.

Maceiczyk, R. M.; Lignos, I. G.; deMello, A. J. Online detection and automation methods in microfluidic nanomaterial synthesis. Current Opinion in Chemical Engineering 2015, 8, 29–35

Microfluidic reactors are increasingly being recognized as a promising tool for the synthesis of bespoke and high quality nanomaterials. Herein, we discuss currently available methods for interfacing microfluidic reactors with online analysis systems such as photothermal, fluorescence, absorbance, X-ray, backscattering and correlation spectroscopies. Integration of appropriate on-line detection methods enables the facile extraction of information relating to size, shape and chemical composition of the formed nanoparticles, thus greatly enhancing control over the synthetic process. Furthermore, we discuss recent approaches aimed at implementing ‘intelligent’ algorithms that use such extracted information for optimization and parameter space evaluation. Lastly, we provide brief opinion about future directions of this emerging field.

Ding, Y.; Casadevall i Solvas, X.; deMello, A. The “V-junction”: a novel structure for high-speed generation of bespoke droplet flows. The Analyst 2015, 140, 414–421

We present the use of microfluidic “V-junctions” as a droplet generation strategy that incorporates enhanced performance characteristics when compared to more traditional “T-junction” formats. This includes the ability to generate target-sized droplets from the very first one, efficient switching between multiple input samples, the production of a wide range of droplet sizes (and size gradients) and the facile generation of droplets with residence time gradients. Additionally, the use of V-junction droplet generators enables the suspension and subsequent resumption of droplet flows at times defined by the user. The high degree of operational flexibility allows a wide range of droplet sizes, payloads, spacings and generation frequencies to be obtained, which in turn provides for an enhanced design space for droplet-based experimentation. We show that the V-junction retains the simplicity of operation associated with T-junction formats, whilst offering functionalities normally associated with droplet-on-demand technologies.
» Analyst 2015 Cover

Robinson, T.; Valluri, P.; Kennedy, G.; Sardini, A.; Dunsby, C.; Neil, M. A. A.; Baldwin, G. S.; French, P. M. W.; de Mello, A. J. Analysis of DNA binding and nucleotide flipping kinetics using two-color two-photon fluorescence lifetime imaging microscopy. Analytical Chemistry 2014, 86, 10732–10740

Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.

Martino, C.; Berger, S.; Wootton, R. C. R.; deMello, A. J. A 3D-Printed Microcapillary Assembly for Facile Double Emulsion Generation. Lab on a Chip 2014, 14, 4178–4182

The design, fabrication and testing of facile microcapillary device assembly, suitable for monodisperse double emulsion production is reported. The interface is fabricated in a direct and rapid manner via 3D printing and shown to be robust in the controllable generation of both single and double emulsions at high generation frequencies.

Stanley, C. E.; Stöckli, M.; van Swaay, D.; Sabotič, J.; Kallio, P. T.; Künzler, M.; deMello, A. J.; Aebi, M. Probing bacterial-fungal interactions at the single cell level. Integrative Biology, 2014, 6, 935–945

Interactions between fungi and prokaryotes are abundant in many ecological systems. A wide variety of biomolecules regulate such interactions and many of them have found medicinal or biotechnological applications. However, studying a fungal-bacterial system at a cellular level is technically challenging. New microfluidic devices provided a platform for microscopic studies and for long-term, time-lapse experiments. Application of these novel tools revealed insights into in the dynamic interactions between the basidiomycete Coprinopsis cinerea and Bacillus subtilis. Direct contact was mediated by polar attachment of bacteria to only a subset of fungal hyphae suggesting a differential competence of fungal hyphae and thus differentiation of hyphae within a mycelium. The fungicidal activity of Bacillus subtilis was monitored at a cellular level and showed a novel mode of action on fungal hyphae.

Maceiczyk, R. M.; deMello, A. J. Fast and Reliable Metamodeling of Complex Reaction Spaces Using Universal Kriging. The Journal of Physical Chemistry C 2014, 118, 20026–20033

We report the application of metamodeling algorithms based on Universal Kriging for the controlled synthesis of compound semiconductor nanoparticles. Application of such a metamodel allows the prediction of reaction outcomes at arbitrary points within sparsely sampled parameter spaces as a function of reaction conditions. To demonstrate the applicability of Universal Kriging to chemical reaction screening within microfluidic reaction systems CdSe and CdSeTe quantum dots were synthesized by using a segmented flow capillary reactor. Variation of input reagent flows (to control reagent concentrations and reaction residence times) and online spectroscopic monitoring of product characteristics was achieved in a fully automated manner. The resulting fluorescence spectra are analyzed to extract the fwhm, wavelength maximum, and intensity of the band-edge emission. These values are subsequently used as inputs for the Universal Kriging metamodeling algorithm to predict the reactor output at arbitrary points within accessible parameter space. Results demonstrate that the algorithm can predict reaction outcomes with high accuracy and reliability.

Phillips, T. W.; Lignos, I. G.; Maceiczyk, R. M.; deMello, A. J.; deMello, J. C. Nanocrystal synthesis in microfluidic reactors: where next?. Lab on a Chip 2014, 14, 3172-3180

The past decade has seen a steady rise in the use of microfluidic reactors for nanocrystal synthesis, with numerous studies reporting improved reaction control relative to conventional batch chemistry. However, flow synthesis procedures continue to lag behind batch methods in terms of chemical sophistication and the range of accessible materials, with most reports having involved simple one- or two-step chemical procedures directly adapted from proven batch protocols. Here we examine the current status of microscale methods for nanocrystal synthesis, and consider what role microreactors might ultimately play in laboratory-scale research and industrial production.

Choi, J.-W.; Lee, S.; Lee, D.-H.; Kim, J.; deMello, A. J.; Chang, S.-I. Integrated pneumatic micro-pumps for high-throughput droplet-based microfluidics. RSC Advance, 2014, 4, 20341–20345

Droplet-based microfluidic systems have recently emerged as powerful experimental tools in the chemical and biological sciences. In conventional droplet-based microfluidics, controlled droplet generation is normally achieved using precision syringe pumps, where the sample is delivered to the microdevice using external tubing that possesses an appreciable dead volume. Accordingly, there is an unmet need for a droplet generation system that does not require the use of syringe pumps. Herein, we report the integration of pneumatic micro-pumps with droplet-based microfluidic systems and their subsequent use in high-throughput biological experimentation. The efficacy of the system is demonstrated by investigating the interaction and the binding inhibition between angiogenin and the anti-angiogenin antibody with a dissociation constant (KD) value of 9.1 ± 3.5 nM and a half maximal inhibitory concentration (IC50) value of 12.2 ± 2.5 nM, respectively.

Lignos, I.; Protesescu, L.; Stavrakis, S.; Piveteau, L.; Speirs, M. J.; Loi, M. A.; Kovalenko, M. V.; deMello, A. J. Facile Droplet-based Microfluidic Synthesis of Monodisperse IV–VI Semiconductor Nanocrystals with Coupled In-Line NIR Fluorescence Detection. Chemistry of Materials 2014, 26, 2975–2982

We describe the realization of a droplet-based microfluidic platform for the controlled and reproducible synthesis of lead chalcogenide (PbS, PbSe) nanocrystal quantum dots (QDs). Monodisperse nanocrystals were synthesized over a wide range of experimental conditions, with real-time assessment and fine-tuning of material properties being achieved using NIR fluorescence spectroscopy. Importantly, we show for the first time that real-time monitoring of the synthetic process allows for rapid optimization of reaction conditions and the synthesis of high quality PbS nanocrystals, emitting in the range of 765–1600 nm, without any post-synthetic processing. The segmented-flow capillary reactor exhibits stable droplet generation and reproducible synthesis of PbS nanocrystals with high photoluminescence quantum yields (28%) over extended periods of time (3–6 h). Furthermore, the produced NIR-emitting nanoparticles were successfully used in the fabrication of Schottky solar cells, exhibiting a power conversion efficiency of 3.4% under simulated AM 1.5 illumination. Finally, the droplet-based microfluidic platform was used to synthesize PbSe nanocrystals having photoluminescence peaks in the range of 860–1600 nm, showing the exceptional control and stability of the reactor.

Kim, J.; Chang, S.-I.; deMello, A. J.; O’Hare, D. Integration of monolithic porous polymer with droplet-based microfluidics on a chip for nano picoliter volume sample analysis. Nano Convergence 2014, 1

In this paper, a porous polymer nanostructure has been integrated with droplet-based microfluidics in a single planar format. Monolithic porous polymer (MPP) was formed selectively within a microfluidic channel. The resulting analyte bands were sequentially comartmentalised into droplets. This device reduces band broadening and the effects of post-column dead volume by the combination of the two techniques. Moreover it offers the precise control of nano/picoliter volume samples.

Berger, S.; Stawikowska, J.; van Swaay, D.; deMello, A. Continuous Suspension of Lipids in Oil by the Selective Removal of Chloroform via Microfluidic Membrane Separation. Industrial & Engineering Chemistry Research 2014, 53, 9256–9261

A continuous flow method for the suspension of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids in oil using a microfluidic platform is presented. The system consists of a microfluidic device housing a semipermeable membrane, a vacuum pump, and a syringe pump. Separation is achieved using a counter current flow of chloroform and a lipid containing oil stream, driven by the syringe pump and vacuum. Using such a system, a high efficiency extraction method was realized through the use of a semipermeable polydimethylsiloxane (PDMS) membrane on an anodized aluminum oxide (AAO) support. For a liquid flow rate of 5 μL/min, an air flow rate of 100 mL/min, and initial chloroform concentrations between 0.245 and 1.619 M, extraction rates of 93.5% to 97.9% and a retentate stream purity of between 99.79% and 99.29% were achieved.

Zhao, Y.; Pereira, F.; deMello, A. J.; Morgan, H.; Niu, X. Droplet-based in situ compartmentalization of chemically separated components after isoelectric focusing in a Slipchip. Lab on a Chip 2014, 14, 555–561

Isoelectric focusing (IEF) is a powerful and widely used technique for protein separation and purification. There are many embodiments of microscale IEF that use capillary or microfluidic chips for the analysis of small sample volumes. Nevertheless, collecting the separated sample volumes without causing remixing remains a challenge. Herein, we describe a microfluidic Slipchip device that is able to efficiently compartmentalize focused analyte bands in situ into microdroplets. The device contains a microfluidic “zig-zag” separation channel that is composed of a sequence of wells formed in the two halves of the Slipchip. The analytes are focused in the channel and then compartmentalised into droplets by slipping the chip. Importantly, sample droplets can be analysed on chip or collected for subsequent analysis using electrophoresis or mass spectrometry for example. To demonstrate this approach, we perform IEF separation using standard markers and protein samples, with on-chip post-processing. Compared to alternative approaches for sample collection, the method avoids remixing, is scalable and is easily hyphenated with the other analytical methods.

Gong, X.; Patil, A. V.; Ivanov, A. P.; Kong, Q.; Gibb, T.; Dogan, F.; deMello, A. J.; Edel, J. B. Label-Free In-Flow Detection of Single DNA Molecules using Glass Nanopipettes. Analytical Chemistry 2014, 86, 835–841

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

Dressler, O. J.; Maceiczyk, R. M.; Chang, S.-I.; deMello, A. J. Droplet-Based Microfluidics: Enabling Impact on Drug Discovery. Journal of Biomolecular Screening 2014, 19, 483–496

Over the past two decades, the application of microengineered systems in the chemical and biological sciences has transformed the way in which high-throughput experimentation is performed. The ability to fabricate complex microfluidic architectures has allowed scientists to create new experimental formats for processing ultra-small analytical volumes in short periods and with high efficiency. The development of such microfluidic systems has been driven by a range of fundamental features that accompany miniaturization. These include the ability to handle small sample volumes, ultra-low fabrication costs, reduced analysis times, enhanced operational flexibility, facile automation, and the ability to integrate functional components within complex analytical schemes. Herein we discuss the impact of microfluidics in the area of high-throughput screening and drug discovery and highlight some of the most pertinent studies in the recent literature.

Elvira, K. S.; i Solvas, X. C.; Wootton, R. C. R.; deMello, A. J. The past, present and potential for microfluidic reactor technology in chemical synthesis. Nature Chemistry 2013, 5, 905–915

The past two decades have seen far-reaching progress in the development of microfluidic systems for use in the chemical and biological sciences. Here we assess the utility of microfluidic reactor technology as a tool in chemical synthesis in both academic research and industrial applications. We highlight the successes and failures of past research in the field and provide a catalogue of chemistries performed in a microfluidic reactor. We then assess the current roadblocks hindering the widespread use of microfluidic reactors from the perspectives of both synthetic chemistry and industrial application. Finally, we set out seven challenges that we hope will inspire future research in this field.

Niu, X.; Pereira, F.; Edel, J. B.; de Mello, A. J. Droplet-Interfaced Microchip and Capillary Electrophoretic Separations. Analytical Chemistry 2013, 85, 8654–8660

Both capillary and chip-based electrophoresis are powerful separation methods widely used for the separation of complex analytical mixtures in the fields of genomics, proteomics, metabolomics, and cellular analysis. However their utility as basic tools in high-throughput analysis and multidimensional separations has been hampered by inefficient or biased sample injection methods. Herein, we address this problem through the development of a simple separation platform that incorporates droplet-based microfluidic module for the encapsulation of analytes prior to the analytical separation. This method allows for the precise and reproducible injection of pL to nL volume isolated plugs into an electrophoretic separation channel. The developed platform is free from inter sample contamination, allows for small sample size, high-throughput analysis, and can provide quantitative analytical information.

Cho, S.; Kang, D.-K.; Sim, S.; Geier, F.; Kim, J.-Y.; Niu, X.; Edel, J. B.; Chang, S.-I.; Wootton, R. C. R.; Elvira, K. S.; deMello, A. J. Droplet-based microfluidic platform for high-throughput, multi-parameter screening of photosensitizer activity. Analytical Chemistry 2013, 85, 8866–8872

We present a fully integrated droplet-based microfluidic platform for the high-throughput assessment of photodynamic therapy photosensitizer (PDT) efficacy on Escherichia coli. The described platform is able to controllably encapsulate cells and photosensitizer within pL-volume droplets, incubate the droplets over the course of several days, add predetermined concentrations of viability assay agents, expose droplets to varying doses of electromagnetic radiation, and detect both live and dead cells online to score cell viability. The viability of cells after encapsulation and incubation is assessed in a direct fashion, and the viability scoring method is compared to model live/dead systems for calibration. Final results are validated against conventional colony forming unit assays. In addition, we show that the platform can be used to perform concurrent measurements of light and dark toxicity of the PDT agents and that the platform allows simultaneous measurement of experimental parameters that include dark toxicity, photosensitizer concentration, light dose, and oxygenation levels for the development and testing of PDT agents.

Pereira, F.; Niu, X.; deMello, A. J. A Nano LC-MALDI Mass Spectrometry Droplet Interface for the Analysis of Complex Protein Samples. PLoS ONE 2013, 8, e63087

The integration of matrix-assisted laser desorption ionization (MALDI) mass spectrometry with an upstream analytical separations (such as liquid chromatography and electrophoresis) has opened up new opportunities for the automated investigation of complex protein and peptide mixtures. The ability to efficiently analyze complex proteomic mixtures in this manner is primarily determined by the ability to preserve spatial discrimination of sample components as they leave the separation column. Current interfacing methods are problematic in this respect since minimum fraction volumes are limited to several microliters. Herein we show for the first time an LC-MALDI interface based on the formation, processing and destruction of a segmented flow. The interface consists of a droplet-generator to fractionate LC effluent into nL-volume droplets and a deposition probe that transfers the sample (and MALDI matrix) onto a conventional MALDI-MS target. The efficacy of the method is demonstrated through the analysis of Trypsin digests of both BSA and Cytochrome C, with a 50% enhancement in analytical performance when compared to conventional interface technology.

Gielen, F.; van Vliet, L.; Koprowski, B. T.; Devenish, S. R. A.; Fischlechner, M.; Edel, J. B.; Niu, X.; deMello, A. J.; Hollfelder, F. A Fully Unsupervised Compartment-on-demand Platform for Precise Nanolitre Assays of Time-Dependent Steady-State Enzyme Kinetics and Inhibition. Analytical Chemistry 2013, 85, 4761–4769

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection system results in a fully automated analytical platform. Michaelis-Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond β-glucosidase can be generated based on 24 time courses taken at different substrate concentrations with a total volume consumption of only 1.4 µL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 minutes; droplet production (5 minutes), initial reading of the droplet sequence (5 minutes), droplet fusion to initiate the reaction and read-out over time (10 minutes). Similarly the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured and Ki values were determined. In both cases the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite by >104-fold volume reduction, from micro- to nanoliters.

van Swaay, D.; deMello, A. Microfluidic methods for forming liposomes. Lab on a Chip 2013, 13, 752

Liposome structures have a wide range of applications in biology, biochemistry, and biophysics. As a result, several methods for forming liposomes have been developed. This review provides a critical comparison of existing microfluidic technologies for forming liposomes and, when applicable, a comparison with their analogous macroscale counterparts. The properties of the generated liposomes, including size, size distribution, lamellarity, membrane composition, and encapsulation efficiency, form the basis for comparison. We hope that this critique will allow the reader to make an informed decision as to which method should be used for a given biological application.

Elvira, K. S.; Wootton, R. C. R.; Reis, N. M.; Mackley, M. R.; deMello, A. J. Through-wall mass transport as a modality for safe generation of singlet oxygen in continuous flows. ACS Sustainable Chemistry & Engineering 2013, 1, 209–213

Singlet oxygen, a reactive oxygen species, has been a basic synthetic tool in the laboratory for many years. It can be generated either through a chemical process or most commonly via a photochemical process mediated by a sensitizing dye. The relative paucity of singlet oxygen employment in fine chemical industrial settings can be attributed to many factors, not least the requirement for excessive quantities of oxygenated organic solvents and the dangers that these represent. Microcapillary films (MCFs) are comprised of multiple parallel channels embedded in a plastic film. In this study, MCFs are employed as flow reactor systems for the singlet oxygen mediated synthesis of ascaridole. No gaseous oxygen is supplied directly to the reaction, rather mass transport occurs exclusively through the reactor walls. The rate of production of ascaridole was found to be strongly dependent on the partial pressure of oxygen present within the reaction system. This methodology significantly simplifies reactor design, allows for increased safety of operation, and provides for space–time yields over 20 times larger than the corresponding bulk synthesis.

Yamazaki, M.; Krishnadasan, S.; deMello, A. J.; deMello, J. C. Non-emissive plastic colour filters for fluorescence detection. Lab on a Chip 2012, 12, 4313–4320

We report the fabrication of non-emissive short- and long-pass filters on plastic for high sensitivity fluorescence detection. The filters were prepared by overnight immersion of titania-coated polyethylene terephthalate (PET) in an appropriate dye solution – xylene cyanol for short-pass filtering and fluorescein disodium salt for long-pass filtering – followed by repeated washing to remove excess dye. The interface between the titania and the dye molecule induces efficient quenching of photo-generated excitons in the dye molecule, reducing auto-fluorescence to negligible values and so overcoming the principal weakness of conventional colour filters. Using the filters in conjunction with a 505 nm cyan light-emitting diode and a Si photodiode, dose-response measurements were made for T8661 Transfluosphere beads in the concentration range 1 × 10^9 to 1 × 10^5 beads μL−1, yielding a limit of detection of 3 × 10^4 beads μL−1. The LED/short-pass filter/T8661/long-pass filter/Si-photodiode combination reported here offers an attractive solution for sensitive, low cost fluorescence detection that is readily applicable to a wide range of bead-based immunodiagnostic assays.

Goyder, M. S.; Willison, K. R.; Klug, D. R.; DeMello, A. J.; Ces, O. Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins. BMB Reports 2012, 45, 233–238

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electro- phoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes fol- lowed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reprodu- cibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chro- matography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This meth- od is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout plat- forms for studies of the ribosomal proteome.

Yashina, A.; Meldrum, F.; deMello, A. Calcium carbonate polymorph control using droplet-based microfluidics. Biomicrofluidics 2012, 6, 022001

Calcium carbonate (CaCO3) is one of the most abundant minerals and of high importance in many areas of science including global CO2 exchange, industrial water treatment energy storage, and the formation of shells and skeletons. Industrially, calcium carbonate is also used in the production of cement, glasses, paints, plastics, rubbers, ceramics, and steel, as well as being a key material in oil refining and iron ore purification. CaCO3 displays a complex polymorphic behaviour which, despite numerous experiments, remains poorly characterised. In this paper, we report the use of a segmented-flow microfluidic reactor for the controlled precipitation of calcium carbonate and compare the resulting crystal properties with those obtained using both continuous flow microfluidic reactors and conventional bulk methods. Through combination of equal volumes of equimolar aqueous solutions of calcium chloride and sodium carbonate on the picoliter scale, it was possible to achieve excellent definition of both crystal size and size distribution. Furthermore, highly reproducible control over crystal polymorph could be realised, such that pure calcite, pure vaterite, or a mixture of calcite and vaterite could be precipitated depending on the reaction conditions and droplet-volumes employed. In contrast, the crystals precipitated in the continuous flow and bulk systems comprised of a mixture of calcite and vaterite and exhibited a broad distribution of sizes for all reaction conditions investigated.

Elvira, K. S.; Leatherbarrow, R.; Edel, J.; deMello, A. Droplet dispensing in digital microfluidic devices: Assessment of long-term reproducibility. Biomicrofluidics 2012, 6, 022003

We report an in-depth study of the long-term reproducibility and reliability of droplet dispensing in digital microfluidic devices (DMF). This involved dispensing droplets from a reservoir, measuring the volume of both the droplet and the reservoir droplet and then returning the daughter droplet to the original reservoir. The repetition of this process over the course of several hundred iterations offers, for the first time, a long-term view of droplet dispensing in DMF devices. Results indicate that the ratio between the spacer thickness and the electrode size influences the reliability of droplet dispensing. In addition, when the separation between the plates is large, the volume of the reservoir greatly affects the reproducibility in the volume of the dispensed droplets, creating “reliability regimes.” We conclude that droplet dispensing exhibits superior reliability as inter-plate device spacing is decreased, and the daughter droplet volume is most consistent when the reservoir volume matches that of the reservoir electrode.

Choi, J.-W.; Kang, D.-K.; Park, H.; deMello, A. J.; Chang, S.-I. High-Throughput Analysis of Protein–Protein Interactions in Picoliter-Volume Droplets Using Fluorescence Polarization. Analytical Chemistry 2012, 84, 3849–3854

Droplet-based microfluidic systems have emerged as a powerful platform for performing high-throughput biological experimentation. In addition, fluorescence polarization has been shown to be effective in reporting a diversity of bimolecular events such as protein–protein, DNA–protein, DNA–DNA, receptor–ligand, enzyme–substrate, and protein–drug interactions. Herein, we report the use of fluorescence polarization for high-throughput protein–protein interaction analysis in a droplet-based microfluidic system. To demonstrate the efficacy of the approach, we investigate the interaction between angiogenin (ANG) and antiangiogenin antibody (anti-ANG Ab) and demonstrate the efficient extraction of dissociation constants (KD = 10.4 ± 3.3 nM) within short time periods.

Stanley, C. E.; Wootton, R. C. R.; deMello, A. J. Continuous and segmented flow microfluidics: Applications in high-throughput chemistry and biology. CHIMIA International Journal for Chemistry 2012, 66, 88–98

This account highlights some of our recent activities focused on developing microfluidic technologies for application in high-throughput and high-information content chemical and biological analysis. Specifically, we discuss the use of continuous and segmented flow microfluidics for artificial membrane formation, the analysis of single cells and organisms, nanomaterial synthesis and DNA amplification via the polymerase chain reaction. In addition, we report on recent developments in small-volume detection technology that allow access to the vast amounts of chemical and biological information afforded by microfluidic systems.

Wootton, R. C. R.; deMello, A. J. Microfluidics: Analog-to-digital drug screening. Nature 2012, 483, 43–44

Current methods for screening libraries of compounds for biological activity are rather cumbersome, slow and imprecise. A method that breaks up a continuous flow of a compound\\\'s solution into droplets offers radical improvements.

Gong, X.; Miller, P. W.; Gee, A. D.; Long, N. J.; de Mello, A. J.; Vilar, R. Gas–Liquid Segmented Flow Microfluidics for Screening Pd-Catalyzed Carbonylation Reactions. Chemistry - A European Journal 2012, 18, 2768–2772

Go with the (segmented) flow: A gas–liquid microfluidic reactor system has been developed to study Pd-catalyzed carbonylation reactions over a range of flow regimes and reaction conditions (see picture). The segmented gas–liquid flow regime, in comparison to annular flow, enables reactions to be studied over longer reaction times and without the buildup of unwanted Pd particles.

Gielen, F.; deMello, A. J.; Edel, J. B. Dielectric Cell Response in Highly Conductive Buffers. Analytical Chemistry 2012, 84, 1849–1853

We present a novel method for the identification of live and dead T-cells, dynamically flowing within highly conductive buffers. This technique discriminates between live and dead (heat treated) cells on the basis of dielectric properties variations. The key advantage of this technique lies in its operational simplicity, since cells do not have to be resuspended in isotonic low conductivity media. Herein, we demonstrate that at 40 MHz, we are able to statistically distinguish between live and dead cell populations.

Cho, S.-W.; Kang, D.-K.; Choo, J.-B.; Demllo, A. J.; Chang, S.-I. Recent advances in microfluidic technologies for biochemistry and molecular biology. BMB Reports 2011, 44, 705–712

Advances in the fields of proteomics and genomics have necessitated the development of high-throughput screening methods (HTS) for the systematic transformation of large amounts of biological chemical data into an organized database of knowledge. Microfluidic systems are ideally suited for high-throughput biochemical experimentation since they offer high analytical throughput, consume minute quantities of expensive biological reagents, exhibit superior sensitivity and functionality compared to traditional micro-array techniques and can be integrated within complex experimental work flows. A range of basic biochemical and molecular biological operations have been transferred to chip-based microfluidic formats over the last decade, including gene sequencing, emulsion PCR, immunoassays, electrophoresis, cell-based assays, expression cloning and macromolecule blotting. In this review, we highlight some of the recent advances in the application of microfluidics to biochemistry and molecular biology.

Xize Niu, Fabrice Gielen, Joshua B. Edel and Andrew J. deMello. A microdroplet dilutor for high-throughput screening. Nature Chemistry, 2011, 3, 437-442.

Pipetting and dilution are universal processes used in chemical and biological laboratories to assay and experiment. In microfluidics such operations are equally in demand, but difficult to implement. Recently, droplet-based microfluidics has emerged as an exciting new platform for high-throughput experimentation. However, it is challenging to vary the concentration of droplets rapidly and controllably. To this end, we developed a dilution module for high-throughput screening using droplet-based microfluidics. Briefly, a nanolitre-sized sample droplet of defined concentration is trapped within a microfluidic chamber. Through a process of droplet merging, mixing and re-splitting, this droplet is combined with a series of smaller buffer droplets to generate a sequence of output droplets that define a digital concentration gradient. Importantly, the formed droplets can be merged with other reagent droplets to enable rapid chemical and biological screens. As a proof of concept, we used the dilutor to perform a high-throughput homogeneous DNA-binding assay using only nanolitres of sample.
» A microfluidic droplet dilutor
» Nature Chemistry 2011 Cover

deMello, A.; Morgan, H. 10th Anniversary Issue: UK. Lab on a Chip 2011, 11, 1191–1192

The United Kingdom has a long history of innovation and development, much of which has its roots in the Industrial Revolution of the 18th and 19th centuries. Starting with the mechanisation of the textile manufacturing industry, major advances, particularly in the mechanical and chemical engineering fields, soon followed. One of the early pioneers of mechanical systems was James Watt who made significant improvements to steam engines, turning them into reliable, powerful, energy efficient machines that would drive the manufacturing centres of the country. His partner John Roebuck was one of the early pioneers (along with figures such as William Henry Perkin) to develop new methods for the mass production of chemicals, laying the foundations for the chemical industry as we know it today. Indeed, by the end of the 19th century Britain had large industrial plants manufacturing a large array of chemicals, and was exporting products and technology all over the world.

Wootton, R. C. R.; deMello, A. J. Microfluidics: Exploiting elephants in the room. Nature 2010, 464, 839–840

Microfluidic devices have many applications in chemistry and biology, but practical hitches associated with their use are often overlooked. One such device that optimizes catalysts tackles these issues head-on.

Schaerli, Y.; Wootton, R. C.; Robinson, T.; Stein, V.; Dunsby, C.; Neil, M. A. A.; French, P. M. W.; deMello, A. J.; Abell, C.; Hollfelder, F. Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets. Analytical Chemistry 2009, 81, 302–306

We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 × 106-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.
» Continuous flow PCR in droplets

Lanigan, P. M. P.; Chan, K.; Ninkovic, T.; Templer, R. H.; French, P. M. W.; de Mello, A. J.; Willison, K. R.; Parker, P. J.; Neil, M. a. A.; Ces, O.; Klug, D. R. Spatially selective sampling of single cells using optically trapped fusogenic emulsion droplets: a new single-cell proteomic tool. Journal of The Royal Society Interface 2008, 5, S161–S168

We present a platform for the spatially selective sampling of the plasma membrane of single cells. Optically trapped lipid-coated oil droplets (smart droplet microtools, SDMs), typically 0.5–5 mm in size, composed of a hexadecane hydrocarbon core and fusogenic lipid outer coating (mixture of 1,2-dioleoyl-phosphatidylethanolamine and 1,2-dioleoyl-sn-glycero-3- phosphatidylcholine) were brought into controlled contact with target colon cancer cells leading to the formation of connecting membrane tethers. Material transfer from the cell to the SDM across the membrane tether was monitored by tracking membrane-localized enhanced green fluorescent protein.

Alexander Iles, Matthew Habgood, Andrew J. de Mello and Robert C. R. Wootton. A Simple technique for microfluidic heterogeneous catalytic\r\nhydrogenation reactor fabrication. Catalysis Letters, 2007, 114, 71-74.

A simple process is described for the facile production of microfluidic reactors with built-in metallic catalysts. This approach\r\nprovides considerable reductions in cost and complexity when compared to existing catalytic chip designs. The process involves the\r\nsputtering of catalytic metals into the channels of microfluidic reactors prior to device bonding. The utility of such microreactors as\r\nenvironments for heterogenous catalytic hydrogenations was tested and demonstrated by applying them to the on-chip reduction of\r\nbutyraldehyde to butanol and benzyl alcohol to benzaldehyde. The use of such built-in systems as microreactors for specific\r\nprocesses was shown to have considerable potential for both fundamental research and industrial application.

Book

Mello, A. J. de; Manz, A. Chip technology for micro-separation. In Microsystem Technology; BioMethods; Birkhäuser, Basel, 1999; pp. 129–177 ISBN 9783034897846
deMello, A. J. Chapter 1. Total Internal Reflection Fluorescence Spectroscopy. In Surface Analytical Techniques for Probing Biomaterial Processes; Davies, J. Surface Analytical Techniques for Probing Biomaterial Processes; CRC Press, 1996; ISBN 9780849383526

Conference

Simon Berger, Evelyn Lattmann, Xavier Casadevall i Solvas, Stavros Stavrakis, Tinri Aegerter-Wilmsen, Alex Hajnal, Andrew deMello. Long-term C. elegans immobilization enables high-resolution developmental studies in vivo. 21st International C. elegans Conference
Simon Berger, Evelyn Lattmann, Xavier Casadevall i Solvas, Stavros Stavrakis, Tinri Aegerter-Wilmsen, Alex Hajnal, Andrew deMello. Long-term C. elegans immobilization enables high-resolution developmental studies in vivo. 2017 Nobel Symposium in Microfluidics
Andrew deMello. Microfluidics for Ultra High-Throughput Experimentation: Droplets, Dots & Photons. Nobel Symposium 162, Stockholm, Sweden, 2017

The past 25 years have seen considerable progress in the development of microfabricated systems for use in the chemical and biological sciences. Interest in “microfluidic” technology has driven by concomitant advances in the areas of genomics, proteomics, nanoscale science, drug discovery, high-throughput screening and diagnostics, with a clearly defined need to perform rapid measurements on small sample volumes. At a fundamental level, microfluidic activities have been stimulated by the fact that physical processes can be more easily controlled when instrumental dimensions are reduced to the micron scale. The relevance of such technology is significant and characterized by an array of features that accompany system miniaturization. These features include the ability to process small volumes of fluid, enhanced analytical performance, reduced instrumental footprints, low unit costs, facile integration of functional components and the capacity to exploit atypical fluid behaviour to control chemical and biological entities in both time and space.

Simon Berger, Evelyn Lattman, Xavier Casadevall i Solvas, Tinri Aegerter-Wilmsen, Alex Hajnal, Andrew deMello. Microfluidic long term immobilization, enabling C. elegans developmental studies at the subcellular level.. LATSIS Symposium EPFL 2016 Multicellular organisms in microfluidic systems
Nadia Vertti-Quintero, Xavier Casadevall i Solvas, Oliver Dressler, Stavros Stavrakis, Jan Gruber, Rudiyanto Gunawan, Andrew deMello. A microfluidic platform for the study and characterization of intrinsic stochastic variability in the stress response system of C. elegans. EMBL Conference Microfluidics 2016. EMBL Advanced Training Centre. Heidelberg, Germany
Simon Berger, Xavier Casadevall i Solvas, Stavros Stavrakis, Tinri Aegerter-Wilmsen, Evelyn Lattmann, Alex Hajnal, Andrew deMello. Microfluidic long term immobilization enabling developmental studies at the subcellular level. European Worm Meeting 2016
Simon Berger, Xavier Casadevall i Solvas, Stavros Stavrakis, Tinri Aegerter-Wilmsen, Ralf Eberhard, Alex Hajnal, Michael Hengartner, Andrew deMello. Microfluidic C. elegans immobilization, enabling long-term studies of germline development at a single-cell resolution. 20th International C. elegans Conference. University of California, Los Angeles.
Nadia Vertti-Quintero, Xavier Casadevall i Solvas, Oliver Dressler, Stavros Stavrakis, Jan Gruber, Rudiyanto Gunawan, Andrew deMello. Microfluidic high-throughput fluorescence-based sorter for studying stochastic expression of heat shock proteins on C. elegans. 20th International C. elegans Conference. University of California. Los Angeles, USA
Justina Rutkauskaite, Xavier Casadevall I Solvas, Anand Rane, Stavros Stavrakis, Linas Mazutis, Andrew J. deMello. Focusing and ordering of mammalian cells. EMBL Conference: Microfluidics 2014