High-throughput direct screening of restriction endonuclease using a microfluidic fluorescence-activated drop sorter based on the SOS response in Escherichia coli.

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route to expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of ß-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of ∼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples - especially unculturable samples - and can be tailored to high-throughput screening of a wide range of genotoxic targets.

Observation of spatial propagation of amyloid assembly from single nuclei.

The crucial early stages of amyloid growth, in which normally soluble proteins are converted into fibrillar nanostructures, are challenging to study using conventional techniques yet are critical to the protein aggregation phenomena implicated in many common pathologies. As with all nucleation and growth phenomena, it is difficult to track individual nuclei in traditional macroscopic experiments, which probe the overall temporal evolution of the sample, but do not yield detailed information on the primary nucleation step as they mix independent stochastic events into an ensemble measurement. To overcome this limitation, we have developed microdroplet assays enabling us to detect single primary nucleation events and to monitor their subsequent spatial as well as temporal evolution, both of which we find to be determined by secondary nucleation phenomena. By deforming the droplets to high aspect ratio, we visualize in real-time propagating waves of protein assembly emanating from discrete primary nucleation sites. We show that, in contrast to classical gelation phenomena, the primary nucleation step is characterized by a striking dependence on system size, and the filamentous protein self-assembly process involves a highly nonuniform spatial distribution of aggregates. These findings deviate markedly from the current picture of amyloid growth and uncover a general driving force, originating from confinement, which, together with biological quality control mechanisms, helps proteins remain soluble and therefore functional in nature.

High-throughput injection with microfluidics using picoinjectors.

Adding reagents to drops is one of the most important functions in droplet-based microfluidic systems; however, a robust technique to accomplish this does not exist. Here, we introduce the picoinjector, a robust device to add controlled volumes of reagent using electro-microfluidics at kilohertz rates. It can also perform multiple injections for serial and combinatorial additions.

Ultrahigh-throughput screening in drop-based microfluidics for directed evolution.

The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify approximately 100 variants comparable in activity to the parent from an initial population of approximately 10(7). After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen approximately 10(8) individual enzyme reactions in only 10 h, using < 150 microL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.

Tracking lineages of single cells in lines using a microfluidic device.

Cells within a genetically identical population exhibit phenotypic variation that in some cases can persist across multiple generations. However, information about the temporal variation and familial dependence of protein levels remains hidden when studying the population as an ensemble. To correlate phenotypes with the age and genealogy of single cells over time, we developed a microfluidic device that enables us to track multiple lineages in parallel by trapping single cells and constraining them to grow in lines for as many as 8 divisions. To illustrate the utility of this method, we investigate lineages of cells expressing one of 3 naturally regulated proteins, each with a different representative expression behavior. Within lineages deriving from single cells, we observe genealogically related clusters of cells with similar phenotype; cluster sizes vary markedly among the 3 proteins, suggesting that the time scale of phenotypic persistence is protein-specific. Growing lines of cells also allows us to dynamically track temporal fluctuations in protein levels at the same time as pedigree relationships among the cells as they divide in the chambers. We observe bursts in expression levels of the heat shock protein Hsp12-GFP that occur simultaneously in mother and daughter cells. In contrast, the ribosomal protein Rps8b-GFP shows relatively constant levels of expression over time. This method is an essential step toward understanding the time scales of phenotypic variation and correlations in phenotype among single cells within a population.

Beating Poisson encapsulation statistics using close-packed ordering.

Loading drops with discrete objects, such as particles and cells, is often necessary when performing chemical and biological assays in microfluidic devices. However, random loading techniques are inefficient, yielding a majority of empty and unusable drops. We use deformable particles that are close packed to insert a controllable number of particles into every drop. This provides a simple, flexible means of efficiently encapsulating a controllable number of particles per drop.

A multi-color fast-switching microfluidic droplet dye laser.

We describe a multi-color microfluidic dye laser operating in whispering gallery mode based on a train of alternating droplets containing solutions of different dyes; this laser is capable of switching the wavelength of its emission between 580 nm and 680 nm at frequencies up to 3.6 kHz-the fastest among all dye lasers reported; it has potential applications in on-chip spectroscopy and flow cytometry.

Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity.

We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s(-1). To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme beta-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of approximately 300 droplets s(-1). The false positive error rate of the sorter at this throughput was <1 in 10(4) droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( approximately 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.

Drop-based microfluidic devices for encapsulation of single cells.

We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.

DNA sequence analysis with droplet-based microfluidics.

Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.

Controlling droplet incubation using close-packed plug flow.

Controlling droplet incubation is critical for droplet-based microfluidic applications; however, current techniques are either of limited precision or place strict limits on the incubation times that can be achieved. Here, we present a simple technique to control incubation time by exploiting close-packed plug flow. In contrast to other techniques, this technique is applicable to very short and very long incubation times.

Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.

In vitro compartmentalization (IVC) has previously been used to evolve protein enzymes. Here, we demonstrate how IVC can be applied to select RNA enzymes (ribozymes) for a property that has previously been unselectable: true intermolecular catalysis. Libraries containing 10(11) ribozyme genes are compartmentalized in the aqueous droplets of a water-in-oil emulsion, such that most droplets contain no more than one gene, and transcribed in situ. By coencapsulating the gene, RNA, and the substrates/products of the catalyzed reaction, ribozymes can be selected for all enzymatic properties: substrate recognition, product formation, rate acceleration, and turnover. Here we exploit the complementarity of IVC with systematic evolution of ligands by exponential enrichment (SELEX), which allows selection of larger libraries (>/=10(15)) and for very small rate accelerations (k(cat)/k(uncat)) but only selects for intramolecular single-turnover reactions. We selected approximately 10(14) random RNAs for Diels-Alderase activity with five rounds of SELEX, then six to nine rounds with IVC. All selected ribozymes catalyzed the Diels-Alder reaction in a truly bimolecular fashion and with multiple turnover. Nearly all ribozymes selected by using eleven rounds of SELEX alone contain a common catalytic motif. Selecting with SELEX then IVC gave ribozymes with significant sequence variations in this catalytic motif and ribozymes with completely novel motifs. Interestingly, the catalytic properties of all of the selected ribozymes were quite similar. The ribozymes are strongly product inhibited, consistent with the Diels-Alder transition state closely resembling the product. More efficient Diels-Alderases may need to catalyze a second reaction that transforms the product and prevents product inhibition.