Sorting droplets into many outlets.

Droplet microfluidics is a commercially successful technology, widely used in single cell sequencing and droplet PCR. Combining droplet making with droplet sorting has also been demonstrated, but so far found limited use, partly due to difficulties in scaling manufacture with injection molded plastics. We introduce a droplet sorting system with several new elements, including: 1) an electrode design combining metallic and ionic liquid parts, 2) a modular, multi-sorting fluidic design with features for keeping inter-droplet distances constant, 3) using timing parameters calculated from fluorescence or scatter signal triggers to precisely actuate dozens of sorting electrodes, 4) droplet collection techniques, including ability to collect a single droplet, and 5) a new emulsion breaking method to collect aqueous samples for downstream analysis. We use these technologies to build a fluorescence based cell sorter that can sort with high (>90%) purity. We also show that these microfluidic designs can be translated into injection molded thermoplastic, suitable for industrial production. Finally, we tally the advantages and limitations of these devices.

Emergence of antibiotic resistance from multinucleated bacterial filaments.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

Bacteria and game theory: the rise and fall of cooperation in spatially heterogeneous environments.

One of the predictions of game theory is that cooperative behaviours are vulnerable to exploitation by selfish individuals, but this result seemingly contradicts the survival of cooperation observed in nature. In this review, we will introduce game theoretical concepts that lead to this conclusion and show how the spatial competition dynamics between microorganisms can be used to model the survival and maintenance of cooperation. In particular, we focus on how Escherichia coli bacteria with a growth advantage in stationary phase (GASP) phenotype maintain a proliferative phenotype when faced with overcrowding to gain a fitness advantage over wild-type populations. We review recent experimental approaches studying the growth dynamics of competing GASP and wild-type strains of E. coli inside interconnected microfabricated habitats and use a game theoretical approach to analyse the observed inter-species interactions. We describe how the use of evolutionary game theory and the ideal free distribution accurately models the spatial distribution of cooperative and selfish individuals in spatially heterogeneous environments. Using bacteria as a model system of cooperative and selfish behaviours may lead to a better understanding of the competition dynamics of other organisms-including tumour-host interactions during cancer development and metastasis.

In vitro microbial culture models and their application in drug development.

Drug development faces its nemesis in the form of drug resistance. The rate of bacterial resistance to antibiotics, or tumor resistance to chemotherapy decisively depends on the surrounding heterogeneous tissue. However, in vitro drug testing is almost exclusively done in well stirred, homogeneous environments. Recent advancements in microfluidics and microfabrication introduce opportunities to develop in vitro culture models that mimic the complex in vivo tissue environment. In this review, we will first discuss the design principles underlying such models. Then we will demonstrate two types of microfluidic devices that combine stressor gradients, cell motility, large population of competing/cooperative cells and time varying dosage of drugs. By incorporating ideas from how natural selection and evolution move drug resistance forward, we show that drug resistance can occur at much greater rates than in well-stirred environments. Finally, we will discuss the future direction of in vitro microbial culture models and how to extend the lessons learned from microbial systems to eukaryotic cells.

Anomalous spatial redistribution of competing bacteria under starvation conditions.

Bacterial cells evolved under prolonged stress often have a growth advantage in stationary phase (GASP); we expect GASP cells to maintain a proliferative state and dominate wild-type cells during starvation, especially when nutrients are limited and the medium has been conditioned. However, when we compete GASP mutants against wild-type cells in a chain of microfluidic microhabitat patches (MHPs) with alternating nutrient-rich and nutrient-limited regions, we observe the reverse effect: wild-type cells achieve maximum relative density under nutrient-limited conditions, while GASP cells dominate nutrient-rich regions. We explain this surprising observation in terms of ideal free distributions, where we show that wild-type cells maximize their fitness at high cell density by redistributing themselves to sparsely populated MHPs. At the microscopic level, we describe how biofilm formation also contributes to the population redistribution. We conclude by discussing the implications of these results for social interactions of more complex organisms.

Miniaturization and parallelization of biological and chemical assays in microfluidic devices.

Microfluidic systems are an attractive solution for the miniaturization of biological and chemical assays. The typical sample volume can be reduced up to 1 million-fold, and a superb level of spatiotemporal control is possible, facilitating highly parallelized assays with drastically increased throughput and reduced cost. In this review, we focus on systems in which multiple reactions are spatially separated by immobilization of reagents on two-dimensional arrays, or by compartmentalization in microfabricated reaction chambers or droplets. These systems have manifold applications, and some, such as next-generation sequencing are already starting to transform biology. This is likely the first step in a biotechnological transformation comparable to that already brought about by the microprocessor in electronics. We discuss both current applications and likely future impacts in areas such as the study of single cells/single organisms and high-throughput screening.

Effect of NRG1, GDNF, EGF and NGF in the migration of a Schwann cell precursor line.

The Schwann cells are the myelinating glia of the peripheral nervous system that originated during development from the highly motile neural crest. However, we do not know what the guidance signals for the Schwann cell precursors are. Therefore, we set to test some of the known neurotrophins that are expressed early in developing embryos and have been shown to be critical for the survival and patterning of developing glia and neurons. The goal of this study was to determine more specifically if GDNF, NRG1 and NGF are chemoattractants and/or chemokinetic molecules for a Schwann cell precursor line, the Spl201. We performed live chemoattraction assays, with imaging and also presented these molecules as part of their growing substrate. Our results show for the first time that GDNF and NRG1 are potent chemoattractive and chemokinetic molecules for these cells while NGF is a chemokinetic molecule stimulating their motility.

Electronic control of elastomeric microfluidic circuits with shape memory actuators.

Recently, sophisticated fluidic circuits with hundreds of independent valves have been built by using multi-layer soft-lithography to mold elastomers. However, this shrinking of microfluidic circuits has not been matched by a corresponding miniaturization of the actuation and interfacing elements that control the circuits; while the fluidic circuits are small ( approximately 10-100 micron wide channels), the Medusa's head-like interface, consisting of external pneumatic solenoids and tubing or mechanical pins to control each independent valve, is larger by one to four orders of magnitude (approximately mm to cm). Consequently, the dream of using large scale integration in microfluidics for portable, high throughput applications has been stymied. By combining multi-layer soft-lithography with shape memory alloys (SMA), we demonstrate electronically activated microfluidic components such as valves, pumps, latches and multiplexers, that are assembled on printed circuit boards (PCBs). Thus, high density, electronically controlled microfluidic chips can be integrated alongside standard opto-electronic components on a PCB. Furthermore, we introduce the idea of microfluidic states, which are combinations of valve states, and analogous to instruction sets of integrated circuit (IC) microprocessors. Microfluidic states may be represented in hardware or software, and we propose a control architecture that results in logarithmic reduction of external control lines. These developments bring us closer to building microfluidic circuits that resemble electronic ICs both physically, as well as in their abstract model.

Design and fabrication of chemically robust three-dimensional microfluidic valves.

A current problem in microfluidics is that poly(dimethylsiloxane) (PDMS), used to fabricate many microfluidic devices, is not compatible with most organic solvents. Fluorinated compounds are more chemically robust than PDMS but, historically, it has been nearly impossible to construct valves out of them by multilayer soft lithography (MSL) due to the difficulty of bonding layers made of "non-stick" fluoropolymers necessary to create traditional microfluidic valves. With our new three-dimensional (3D) valve design we can fabricate microfluidic devices from fluorinated compounds in a single monolithic layer that is resistant to most organic solvents with minimal swelling. This paper describes the design and development of 3D microfluidic valves by molding of a perfluoropolyether, termed Sifel, onto printed wax molds. The fabrication of Sifel-based microfluidic devices using this technique has great potential in chemical synthesis and analysis.

Patterning lines by capillary flows.

We report that capillary flows in an evaporating thin film create line patterns, with widths ranging from a few micrometers to less than 100 nm. Deliberate patterning of such lines requires contact-line pinning and the presence of foaming surfactants. Large-scale photolithography can guide and control these structures by creating pinning points and steering evaporation. We provide demonstrations of this process by making self-assembling lines of colloidal quantum dots and microspheres.