Controlled droplet microfluidic systems for multistep chemical and biological assays.

Droplet microfluidics is a relatively new and rapidly evolving field of science focused on studying the hydrodynamics and properties of biphasic flows at the microscale, and on the development of systems for practical applications in chemistry, biology and materials science. Microdroplets present several unique characteristics of interest to a broader research community. The main distinguishing features include (i) large numbers of isolated compartments of tiny volumes that are ideal for single cell or single molecule assays, (ii) rapid mixing and negligible thermal inertia that all provide excellent control over reaction conditions, and (iii) the presence of two immiscible liquids and the interface between them that enables new or exotic processes (the synthesis of new functional materials and structures that are otherwise difficult to obtain, studies of the functions and properties of lipid and polymer membranes and execution of reactions at liquid-liquid interfaces). The most frequent application of droplet microfluidics relies on the generation of large numbers of compartments either for ultrahigh throughput screens or for the synthesis of functional materials composed of millions of droplets or particles. Droplet microfluidics has already evolved into a complex field. In this review we focus on 'controlled droplet microfluidics' - a portfolio of techniques that provide convenient platforms for multistep complex reaction protocols and that take advantage of automated and passive methods of fluid handling on a chip. 'Controlled droplet microfluidics' can be regarded as a group of methods capable of addressing and manipulating droplets in series. The functionality and complexity of controlled droplet microfluidic systems can be positioned between digital microfluidics (DMF) addressing each droplet individually using 2D arrays of electrodes and ultrahigh throughput droplet microfluidics focused on the generation of hundreds of thousands or even millions of picoliter droplets that cannot be individually addressed by their location on a chip.

A precise and accurate microfluidic droplet dilutor.

We demonstrate a microfluidic system for the precise (coefficient of variance between repetitions below 4%) and highly accurate (average difference from two-fold dilution below 1%) serial dilution of solutions inside droplets with a volume of ca. 1 µl. The two-fold dilution series can be prepared with the correlation coefficient as high as R2 = 0.999. The technique that we here describe uses hydrodynamic traps to precisely meter every droplet used in subsequent dilutions. We use only one metering trap to meter each and every droplet involved in the process of preparation of the dilution series. This eliminates the error of metering that would arise from the finite fidelity of fabrication of multiple metering traps. Metering every droplet at the same trap provides for high reproducibility of the volumes of the droplets, and thus high reproducibility of dilutions. We also present a device and method to precisely and accurately dilute one substance and simultaneously maintain the concentration of another substance throughout the dilution series without mixing their stock solutions. We compare the here-described precise and accurate dilution systems with a simple microdroplet dilutor that comprises several traps - each trap for a subsequent dilution. We describe the effect of producing more reproducible dilutions in a simple microdroplet dilutor thanks to the application of an alternating electric field.

Optimized droplet digital CFU assay (ddCFU) provides precise quantification of bacteria over a dynamic range of 6 logs and beyond.

Standard digital assays need a large number of compartments for precise quantification of a sample over a broad dynamic range. We address this issue with an optimized droplet digital approach that uses a drastically reduced number of compartments for quantification. We generate serial logarithmic dilutions of an initial bacterial sample as an array of microliter-sized droplet plugs. In a subsequent step, these droplets are split into libraries of nanoliter droplets and pooled together for incubation and analysis. We show that our technology is at par with traditional dilution plate count for quantification of bacteria, but has the advantage of simplifying the experimental setup and reducing the manual workload. The method also has the potential to reduce the assay time significantly.

A passive microfluidic system based on step emulsification allows the generation of libraries of nanoliter-sized droplets from microliter droplets of varying and known concentrations of a sample.

We present a novel geometry of microfluidic channels that allows us to passively generate monodisperse emulsions of hundreds of droplets smaller than 1 nL from collections of larger (ca. 0.4 µL) mother droplets. We introduce a new microfluidic module for the generation of droplets via passive break-up at a step. The module alleviates a common problem in step emulsification with efficient removal of the droplets from the vicinity of the step. In our solution, the droplets are pushed away from the step by a continuous liquid that bypasses the mother droplets via specially engineered bypasses that lead to the step around the main channel. We show that the bypasses tighten the distribution of volume of daughter droplets and eliminate subpopulations of daughter droplets. Clearing away the just produced droplets from the vicinity of the step provides for similar conditions of break-up for every subsequent droplet and, consequently, leads to superior monodispersity of the generated emulsions. Importantly, this function is realized autonomously (passively) in a protocol in which only a sequence of large mother droplets is forced through the module. Our system features the advantage of step emulsification systems in that the volumes of the generated droplets depend very weakly on the rate of flow through the module - an increase in the flow rate by 300% causes only a slight increase of the average diameter of generated droplets by less than 5%. We combined our geometry with a simple T-junction and a simple trap-based microdroplet dilutor to produce a collection of libraries of droplets of gradually changing and known concentrations of a sample. The microfluidic system can be operated with only two syringe pumps set at constant rates of flow during the experiment.

FOXO1 is a TXN- and p300-dependent sensor and effector of oxidative stress in diffuse large B-cell lymphomas characterized by increased oxidative metabolism.

Molecular profiling has led to identification of subtypes of diffuse large B-cell lymphomas (DLBCLs) differing in terms of oncogenic signaling and metabolic programs. The OxPhos-DLBCL subtype is characterized by enhanced mitochondrial oxidative phosphorylation. As increased oxidative metabolism leads to overproduction of potentially toxic reactive oxygen species (ROS), we sought to identify mechanisms responsible for adaptation of OxPhos cells to these conditions. Herein, we describe a mechanism involving the FOXO1-TXN-p300 redox-dependent circuit protecting OxPhos-DLBCL cells from ROS toxicity. We identify a BCL6-dependent transcriptional mechanism leading to relative TXN overexpression in OxPhos cells. We found that OxPhos cells lacking TXN were uniformly more sensitive to ROS and doxorubicin than control cells. Consistent with this, the overall survival of patients with high TXN mRNA expression, treated with doxorubicin-containing regimens, is significantly shorter than of those with low TXN mRNA expression. TXN overexpression curtails p300-mediated FOXO1 acetylation and its nuclear translocation in response to oxidative stress, thus attenuating FOXO1 transcriptional activity toward genes involved in apoptosis and cell cycle inhibition. We also demonstrate that FOXO1 knockdown in cells with silenced TXN expression markedly reduces ROS-induced apoptosis, indicating that FOXO1 is the major sensor and effector of oxidative stress in OxPhos-DLBCLs. These data highlight dynamic, context-dependent modulation of FOXO1 tumor-suppressor functions via acetylation and reveal potentially targetable vulnerabilities in these DLBCLs.