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We have developed a multistep microfluidic device that expands the current SlipChip capabilities by enabling multiple steps of droplet merging and multiplexing. Harnessing the interfacial energy between carrier and sample phases, this manually operated device accurately meters nanoliter volumes of reagents and transfers them into on-device reaction wells. Judiciously shaped microfeatures and surface-energy traps merge droplets in a parallel fashion. Wells can be tuned for different volumetric capacities and reagent types, including for pre-spotted reagents that allow for unique identification of original well contents even after their contents are pooled. We demonstrate the functionality of the multistep SlipChip by performing RNA transcript barcoding on-device for synthetic spiked-in standards and for biologically derived samples. This technology is a good candidate for a wide range of biological applications that require multiplexing of multistep reactions in nanoliter volumes, including single-cell analyses.

Many biochemical systems are spatially heterogeneous and exhibit nonlinear behaviors, such as state switching in response to small changes in the local concentration of diffusible molecules. Systems as varied as blood clotting, intracellular calcium signaling, and tissue inflammation are all heavily influenced by the balance of rates of reaction and mass transport phenomena including flow and diffusion. Transport of signaling molecules is also affected by geometry and chemoselective confinement via matrix binding. In this review, we use a phenomenon referred to as patchy switching to illustrate the interplay of nonlinearities, transport phenomena, and spatial effects. Patchy switching describes a change in the state of a network when the local concentration of a diffusible molecule surpasses a critical threshold. Using patchy switching as an example, we describe conceptual tools from nonlinear dynamics and chemical engineering that make testable predictions and provide a unifying description of the myriad possible experimental observations. We describe experimental microfluidic and biochemical tools emerging to test conceptual predictions by controlling transport phenomena and spatial distribution of diffusible signals, and we highlight the unmet need for in vivo tools.

This paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation of Escherichia coli and antimicrobial susceptibility testing of Pseudomonas aeruginosa PAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including four Mycobacterium isolates and a previously unknown fluoranthene-degrading Blastococcus species.

Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

Spatially defined arrays of droplets differ from bulk emulsions in that droplets in arrays can be indexed on the basis of one or more spatial variables to enable identification, monitoring, and addressability of individual droplets. Spatial indexing is critical in experiments with hundreds to millions of unique compartmentalized microscale processes—for example, in applications such as digital measurements of rare events in a large sample, high-throughput time-lapse studies of the contents of individual droplets, and controlled droplet-droplet interactions. This review describes approaches for spatially organizing and manipulating droplets in one-, two-, and three-dimensional structured arrays, including aspiration, laminar flow, droplet traps, the SlipChip, self-assembly, and optical or electrical fields. This review also presents techniques to analyze droplets in arrays and applications of spatially defined arrays, including time-lapse studies of chemical, enzymatic, and cellular processes, as well as further opportunities in chemical, biological, and engineering sciences, including perturbation/response experiments and personal and point-of-care diagnostics.

This study shows that environmental confinement strongly affects the activation of nonlinear reaction networks, such as blood coagulation (clotting), by small quantities of activators. Blood coagulation is sensitive to the local concentration of soluble activators, initiating only when the activators surpass a threshold concentration, and therefore is regulated by mass transport phenomena such as flow and diffusion. Here, diffusion was limited by decreasing the size of microfluidic chambers, and it was found that microparticles carrying either the classical stimulus, tissue factor, or a bacterial stimulus, Bacillus cereus, initiated coagulation of human platelet-poor plasma only when confined. A simple analytical argument and numerical model were used to describe the mechanism for this phenomenon: confinement causes diffusible activators to accumulate locally and surpass the threshold concentration. To interpret the results, a dimensionless confinement number, Cn, was used to describe whether a stimulus was confined, and a Damköhler number, Da2, was used to describe whether a subthreshold stimulus could initiate coagulation. In the context of initiation of coagulation by bacteria, this mechanism can be thought of as “diffusion acting”, which is distinct from “diffusion sensing”. The ability of confinement and diffusion acting to change the outcome of coagulation suggests that confinement should also regulate other biological “on” and “off” processes that are controlled by thresholds.

Microelectrodes enable localized electrical stimulation and recording, and they have revolutionized our understanding of the spatiotemporal dynamics of systems that generate or respond to electrical signals. However, such comprehensive understanding of systems that rely on molecular signals—e.g., chemical communication in multicellular neural, developmental, or immune systems—remains elusive because of the inability to deliver, capture, and interpret complex chemical information. To overcome this challenge, we developed the “chemistrode,” a plug-based microfluidic device that enables stimulation, recording, and analysis of molecular signals with high spatial and temporal resolution. Stimulation with and recording of pulses as short as 50 ms was demonstrated. A pair of chemistrodes fabricated by multilayer soft lithography recorded independent signals from 2 locations separated by 15 μm. Like an electrode, the chemistrode does not need to be built into an experimental system—it is simply brought into contact with a chemical or biological substrate, and, instead of electrical signals, molecular signals are exchanged. Recorded molecular signals can be injected with additional reagents and analyzed off-line by multiple, independent techniques in parallel (e.g., fluorescence correlation spectroscopy, MALDI-MS, and fluorescence microscopy). When recombined, these analyses provide a time-resolved chemical record of a system's response to stimulation. Insulin secretion from a single murine islet of Langerhans was measured at a frequency of 0.67 Hz by using the chemistrode. This article characterizes and tests the physical principles that govern the operation of the chemistrode to enable its application to probing local dynamics of chemically responsive matter in chemistry and biology.

Using biological machinery to make new, functional molecules is an exciting area in chemical biology. Complex molecules containing both ‘natural’ and ‘unnatural’ components are made by processes ranging from enzymatic catalysis to the combination of molecular biology with chemical tools. Here, we discuss applying this approach to the next level of biological complexity — building synthetic, functional biotic systems by manipulating biological machinery responsible for development of multicellular organisms. We describe recent advances enabling this approach, including first, recent developmental biology progress unraveling the pathways and molecules involved in development and pattern formation; second, emergence of microfluidic tools for delivering stimuli to a developing organism with exceptional control in space and time; third, the development of molecular and synthetic biology toolsets for redesigning or de novo engineering of signaling networks; and fourth, biological systems that are especially amendable to this approach.

This review focuses on how the mechanistic approach of physical organic chemistry can be used to elucidate the mechanisms behind complex biochemical networks. The dynamics of biochemical reaction networks is difficult to describe by considering their individual reactions, just as the dynamics of organic reactions is difficult to describe by considering individual electrons and atomic nuclei. Physical organic chemists have developed a useful set of tools to predict the outcome of organic reactions by separating the interacting molecules into modules (functional groups), and defining general rules for how these modules interact (mechanisms). This review shows how these tools of physical organic chemistry may be used to describe reaction networks. In addition, it describes the application of these tools to develop a mechanistic understanding of the dynamics of the complex network of hemostasis, which regulates blood clotting.

Fundamental and applied research in chemistry and biology benefits from opportunities provided by droplet-based microfluidic systems. These systems enable the miniaturization of reactions by compartmentalizing reactions in droplets of femoliter to microliter volumes. Compartmentalization in droplets provides rapid mixing of reagents, control of the timing of reactions on timescales from milliseconds to months, control of interfacial properties, and the ability to synthesize and transport solid reagents and products. Droplet-based microfluidics can help to enhance and accelerate chemical and biochemical screening, protein crystallization, enzymatic kinetics, and assays. Moreover, the control provided by droplets in microfluidic devices can lead to new scientific methods and insights.

This article demonstrates that a simple chemical model system, built by using a modular approach, may be used to predict the spatiotemporal dynamics of initiation of blood clotting in the complex network of hemostasis. Microfluidics was used to create in vitro environments that expose both the complex network and the model system to surfaces patterned with patches presenting clotting stimuli. Both systems displayed a threshold response, with clotting initiating only on isolated patches larger than a threshold size. The magnitude of the threshold patch size for both systems was described by the Damköhler number, measuring competition of reaction and diffusion. Reaction produces activators at the patch, and diffusion removes activators from the patch. The chemical model made additional predictions that were validated experimentally with human blood plasma. These experiments show that blood can be exposed to significant amounts of clot-inducing stimuli, such as tissue factor, without initiating clotting. Overall, these results demonstrate that such chemical model systems, implemented with microfluidics, may be used to predict spatiotemporal dynamics of complex biochemical networks.

Rapid progress in identifying disease biomarkers has increased the importance of creating high-performance detection technologies. Over the last decade, the design of many detection platforms has focused on either the nano or micro length scale. Here, we review recent strategies that combine nano- and microscale materials and devices to produce large improvements in detection sensitivity, speed and accuracy, allowing previously undetectable biomarkers to be identified in clinical samples. Microsensors that incorporate nanoscale features can now rapidly detect disease-related nucleic acids expressed in patient samples. New microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes, and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells in the bloodstream more accurately than before. These advances will lead to faster and more reliable clinical diagnostic devices.

Plugging a gap in screening: Arrays of nanoliter-sized plugs of different compositions can be preformed in a three-phase liquid/liquid/gas flow. The arrays can be transported into a microfluidic channel to test against a target (see schematic representation), as demonstrated in protein crystallization and an enzymatic assay.

This paper presents an overview of our recent work on the use of soft lithography and two-phase fluid flow to form arrays of droplets. The crucial issues in the formation of stable arrays of droplets and alternating droplets of two sets of aqueous solutions include the geometry of the microchannels, the capillary number, and the water fraction of the system. Glass capillaries could be coupled to the PDMS microchannels and droplets could be transferred into glass capillaries for long-term storage. The arrays of droplets have been applied to screen the conditions for protein crystallization with microbatch and vapor diffusion techniques.

The proof of the model is in the function: A minimal model of hemostasis (a complex biochemical network responsible for blood coagulation) may be implemented with only three chemical reactions, which creates a biomimetic functional microfluidic system that is capable of repairing itself (as modeled in the figure). This simple system shows threshold response and sensitivity to flow similar to that observed in hemostasis.

Millisecond mixing and transport with no dispersion are achieved by unsteady flows induced in droplets of about 60 pL that travel through winding microfluidic channels (top). Fluorescence can be used to monitor mixing (bottom) or measure reaction rates. In principle, arbitrarily complex reaction networks can be created by combining and splitting streams of such droplets.

A convenient experimental system is described, with which electron transport through structurally well-defined, 2–5 nm-thick, organic films can be examined. Two types of junction J have been studied in which self-assembled monolayers (SAMs, for example, SAM(1) formed on Ag from aliphatic and aromatic thiols, and SAM(2), formed on Hg from hexadecanethiol) are in contact through either van der Waals interactions (see picture) or through covalent, hydrogen, or ionic bonds.

This Account summarizes techniques for carrying out microfabrication of structures with dimensions down to 10 microm in microchannels that are 0.02-2 mm wide. These methods are largely based on the exploitation of laminar flow at low Reynolds number (Re) to control the spatial delivery of reagents. These methods are illustrated by fabrication of fibers, microelectrode arrays, arrays of crystals, and patterns of proteins and cells.

Small with control: For miniaturization of protein aggregation experiments the interfacial chemistry must be controlled to avoid protein aggregation caused by interfacial adsorption. Plug-based microfluidics with defined surface chemistry (see schematic picture) can then be used to perform hundreds of aggregation experiments with volume-limited samples, such as cerebrospinal fluid from mice.

This paper describes a microfluidic device for dry preservation of biological specimens at room temperature that incorporates chemical stabilization matrices. Long-term stabilization of samples is crucial for remote medical analysis, biosurveillance, and archiving, but the current paradigm for transporting remotely obtained samples relies on the costly “cold chain” to preserve analytes within biospecimens. We propose an alternative approach that involves the use of microfluidics to preserve samples in the dry state with stabilization matrices, developed by others, that are based on self-preservation chemistries found in nature. We describe a SlipChip-based device that allows minimally trained users to preserve samples with the three simple steps of placing a sample at an inlet, closing a lid, and slipping one layer of the device. The device fills automatically, and a pre-loaded desiccant dries the samples. Later, specimens can be rehydrated and recovered for analysis in a laboratory. This device is portable, compact, and self-contained, so it can be transported and operated by untrained users even in limited-resource settings. Features such as dead-end and sequential filling, combined with a “pumping lid” mechanism, enable precise quantification of the original sample’s volume while avoiding overfilling. In addition, we demonstrated that the device can be integrated with a plasma filtration module, and we validated device operations and capabilities by testing the stability of purified RNA solutions. These features and the modularity of this platform (which facilitates integration and simplifies operation) would be applicable to other microfluidic devices beyond this application. We envision that as the field of stabilization matrices develops, microfluidic devices will be useful for cost-effectively facilitating remote analysis and biosurveillance while also opening new opportunities for diagnostics, drug development, and other medical fields.

This study is devoted to controlling the initiation and flow rate of spontaneous liquid-liquid flow passing through microfluidic channels in response to capillary action on a non-wetting droplet. Aqueous droplets were generated on a hydrophobic SlipChip in a shallow channel, and then a stepwise change in capillary force was induced by connecting the shallow channel to a deeper channel filled with immiscible oil. A model to predict the rate of spontaneous flow was developed based on the balance of net capillary pressure with viscous flow resistance; the inputs to the model were the liquid-liquid surface tension, advancing and receding contact angles at the three-phase aqueous-oil-surface interface, and the geometry of the device. The effects of contact angle hysteresis, presence or absence of a lubricating oil layer, and adsorption of surfactants at liquid-liquid or liquid-solid interfaces were quantified. Two different regimes of flow were studied. Faster (mm/s) flow rates were obtained when oil being displaced by the aqueous could escape through connected channels, and slower (μm/s) flow rates were obtained when displaced oil could escaped only through a μm-scale gap between the plates of the SlipChip (“dead-end flow”). Both diluted salt solutions and complex biological media such as human blood plasma were found to flow using this approach, and we anticipate it being useful in the future for control of flow in microfluidic designs that do not require external power, valves, or pumps. Approaches based on initiation of spontaneous flow would be useful for design and operation of the SlipChip platform as well as for other droplet-based and plug-based microfluidic devices.

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for “digital” (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12 000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, 1 000 000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA.

Rapid detection of small particles such as pathogens, environmental pollutants, and microorganisms is important for both the security and health of society at large. The main goal of this proposal is to establish the scientific foundations for rapidly detecting small particles at low concentrations by combining controlled chemical autocatalytic amplification and stochastic confinement of small particles with the microfluidic expertise that has been developed in Pi's laboratories. We focus on developing computational models to predict the optimal single step amplification and multi-step amplification cascades, and we will compare these models to existing biological amplification networks, including blood clotting and apoptosis.

We studied the self-assembly of inorganic nanocrystals (NCs) confined inside nanoliter droplets (plugs) into long-range ordered superlattices. We showed that a capillary microfluidic platform can be used for the optimization of growth conditions for NC superlattices and can provide insights into the kinetics of the NC assembly process. The utility of our approach was demonstrated by growing large (up to 200 μm) three-dimensional (3D) superlattices of various NCs, including Au, PbS, CdSe, and CoFe(2)O(4). We also showed that it is possible to grow 3D binary nanoparticle superlattices in the microfluidic plugs.

This article describes a SlipChip-based approach to perform bead-based heterogeneous immunoassays with multiple nanoliter-volume samples. As a potential device to analyze the output of the chemistrode, the performance of this platform was tested using low concentrations of biomolecules. Two strategies to perform the immunoassay in the SlipChip were tested: (1) a unidirectional slipping method to combine the well containing a sample with a series of wells preloaded with reagents and (2) a back-and-forth slipping method to introduce a series of reagents to a well containing the sample by reloading and slipping the well containing the reagent. The SlipChips were fabricated with hydrophilic surfaces on the interior of the wells and with hydrophobic surfaces on the face of the SlipChip to enhance filling, transferring, and maintaining aqueous solutions in shallow wells. Nanopatterning was used to increase the hydrophobic nature of the SlipChip surface. Magnetic beads containing the capture antibody were efficiently transferred between wells and washed by serial dilution. An insulin immunoenzymatic assay showed a detection of limit of approximately 13 pM. A total of 48 droplets of nanoliter volume were analyzed in parallel, including an on-chip calibration. The design of the SlipChip is flexible to accommodate other types of immunoassays, both heterogeneous and homogeneous. This work establishes the possibility of using SlipChip-based immunoassays in small volumes for a range of possible applications, including analysis of plugs from a chemistrode, detection of molecules from single cells, and diagnostic monitoring.

To obtain protein crystals, researchers must search for conditions in multidimensional chemical space. Empirically, thousands of crystallization experiments are carried out to screen various precipitants at multiple concentrations. Microfluidics can manipulate fluids on a nanoliter scale, and it affects crystallization twofold. First, it miniaturizes the experiments that can currently be done on a larger scale and enables crystallization of proteins that are available only in small amounts. Second, it offers unique experimental approaches that are difficult or impossible to implement on a larger scale. Ongoing development of microfluidic techniques and their integration with protein production, characterization, and in situ diffraction promises to accelerate the progress of structural biology.

This paper describes two SlipChip-based approaches to protein crystallization: a SlipChip-based free interface diffusion (FID) method and a SlipChip-based composite method that simultaneously performs microbatch and FID crystallization methods in a single device. The FID SlipChip was designed to screen multiple reagents, each at multiple diffusion equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis, against 48 different reagents at five different equilibration times each, consuming 12 microL of each protein for a total of 480 experiments using three SlipChips. The composite SlipChip was designed to screen multiple reagents, each at multiple mixing ratios and multiple equilibration times, and was validated by screening conditions for crystallization of two proteins, enoyl-CoA hydratase from Mycobacterium tuberculosis and dihydrofolate reductase/thymidylate synthase from Babesia bovis. To prevent cross-contamination while keeping the solution in the neck channels for FID stable, the plates of the SlipChip were etched with a pattern of nanowells. This nanopattern was used to increase the contact angle of aqueous solutions on the surface of the silanized glass. The composite SlipChip increased the number of successful crystallization conditions and identified more conditions for crystallization than separate FID and microbatch screenings. Crystallization experiments were scaled up in well plates using conditions identified during the SlipChip screenings, and X-ray diffraction data were obtained to yield the protein structure of dihydrofolate reductase/thymidylate synthase at 1.95 A resolution. This free-interface diffusion approach provides a convenient and high-throughput method of setting up gradients in microfluidic devices and may find additional applications in cell-based assays.

This paper describes a microfluidic approach to perform multiplexed nanoliter-scale experiments by combining a sample with multiple different reagents, each at multiple mixing ratios. This approach employs a user-loaded, equipment-free SlipChip. The mixing ratios, characterized by diluting a fluorescent dye, could be controlled by the volume of each of the combined wells. The SlipChip design was validated on an approximately 12 nL scale by screening the conditions for crystallization of glutaryl-CoA dehydrogenase from Burkholderia pseudomallei against 48 different reagents; each reagent was tested at 11 different mixing ratios, for a total of 528 crystallization trials. The total consumption of the protein sample was approximately 10 microL. Conditions for crystallization were successfully identified. The crystallization experiments were successfully scaled up in well plates using the conditions identified in the SlipChip. Crystals were characterized by X-ray diffraction and provided a protein structure in a different space group and at a higher resolution than the structure obtained by conventional methods. In this work, this user-loaded SlipChip has been shown to reliably handle fluids of diverse physicochemical properties, such as viscosities and surface tensions. Quantitative measurements of fluorescent intensities and high-resolution imaging were straighforward to perform in these glass SlipChips. Surface chemistry was controlled using fluorinated lubricating fluid, analogous to the fluorinated carrier fluid used in plug-based crystallization. Thus, we expect this approach to be valuable in a number of areas beyond protein crystallization, especially those areas where droplet-based microfluidic systems have demonstrated successes, including measurements of enzyme kinetics and blood coagulation, cell-based assays, and chemical reactions.

This paper describes a method to generate functionalizable, mobile self-assembled monolayers (SAMs) in plug-based microfluidics. Control of interfaces is advancing studies of biological interfaces, heterogeneous reactions, and nanotechnology. SAMs have been useful for such studies, but they are not laterally mobile. Lipid-based methods, though mobile, are not easily amenable to setting up the hundreds of experiments necessary for crystallization screening. Here we demonstrate a method, complementary to current SAM and lipid methods, for rapidly generating mobile, functionalized SAMs. This method relies on plugs, droplets surrounded by a fluorous carrier fluid, to rapidly explore chemical space. Specifically, we implemented his-tag binding chemistry to design a new fluorinated amphiphile, RfNTA, using an improved one-step synthesis of RfOEG under Mitsunobu conditions. RfNTA introduces specific binding of protein at the fluorous−aqueous interface, which concentrates and orients proteins at the interface, even in the presence of other surfactants. We then applied this approach to the crystallization of a his-tagged membrane protein, Reaction Center from Rhodobacter sphaeroides, performed 2400 crystallization trials, and showed that this approach can increase the range of crystal-producing conditions, the success rate at a given condition, the rate of nucleation, and the quality of the crystal formed.

This paper describes experiments for characterizing mass transfer at the hydrophilic surface of the substrate in a chemistrode. The chemistrode uses microfluidic plugs to deliver pulses of chemicals to a substrate with high temporal resolution, which requires efficient mass transfer between the wetting layer and the hydrophilic surface of the substrate. Here, total internal reflection fluorescence microscopy (TIRFM) was used to image the hydrophilic surface of the substrate as plugs were made to flow over it. The surface of the substrate was rapidly saturated with a fluorescent dye as the fluroesecent plugs passed over the substrate, confirming effective mass transfer between the wetting layer and the surface of the substrate. The dynamics of saturation are consistent from cycle to cycle, indicating that the chemistrode can stimulate surfaces with high reproducibility. The number of plugs required to reach 90% saturation of the hydrophilic surface of the substrate, ϕ(90%), only weakly depended on experimental conditions (the Peclet number or the capillary number). Furthermore, over a wide range of operating conditions, ϕ(90%) was less than 4. These results are useful for improving the chemistrode and for understanding other phenomena that involve diffusional transfer in multiphase or recirculating flows near surfaces.

One is a quorum: As few as one to three cells of Pseudomonas aeruginosa bacteria are confined in small volumes by the use of microfluidics. These small numbers of cells are able to activate quorum sensing (QS) pathways and achieve QS-dependent growth. The results also show that at low numbers of cells, initiation of QS is highly variable within a clonal population.

Understanding the spatial dynamics of biochemical networks is both fundamentally important for understanding life at the systems level and also has practical implications for medicine, engineering, biology, and chemistry. Studies at the level of individual reactions provide essential information about the function, interactions, and localization of individual molecular species and reactions in a network. However, analyzing the spatial dynamics of complex biochemical networks at this level is difficult. Biochemical networks are non-equilibrium systems containing dozens to hundreds of reactions with nonlinear and time-dependent interactions, and these interactions are influenced by diffusion, flow, and the relative values of state-dependent kinetic parameters. To achieve an overall understanding of the spatial dynamics of a network and the global mechanisms that drive its function, networks must be analyzed as a whole, where all of the components and influential parameters of a network are simultaneously considered. Here, we describe chemical concepts and microfluidic tools developed for network-level investigations of the spatial dynamics of these networks. Modular approaches can be used to simplify these networks by separating them into modules, and simple experimental or computational models can be created by replacing each module with a single reaction. Microfluidics can be used to implement these models as well as to analyze and perturb the complex network itself with spatial control on the micrometer scale. We also describe the application of these network-level approaches to elucidate the mechanisms governing the spatial dynamics of two networks-hemostasis (blood clotting) and early patterning of the Drosophila embryo. To investigate the dynamics of the complex network of hemostasis, we simplified the network by using a modular mechanism and created a chemical model based on this mechanism by using microfluidics. Then, we used the mechanism and the model to predict the dynamics of initiation and propagation of blood clotting and tested these predictions with human blood plasma by using microfluidics. We discovered that both initiation and propagation of clotting are regulated by a threshold response to the concentration of activators of clotting, and that clotting is sensitive to the spatial localization of stimuli. To understand the dynamics of patterning of the Drosophila embryo, we used microfluidics to perturb the environment around a developing embryo and observe the effects of this perturbation on the expression of Hunchback, a protein whose localization is essential to proper development. We found that the mechanism that is responsible for Hunchback positioning is asymmetric, time-dependent, and more complex than previously proposed by studies of individual reactions. Overall, these approaches provide strategies for simplifying, modeling, and probing complex networks without sacrificing the functionality of the network. Such network-level strategies may be most useful for understanding systems with non-linear interactions where spatial dynamics is essential for function. In addition, microfluidics provides an opportunity to investigate the mechanisms responsible for robust functioning of complex networks. By creating nonideal, stressful, and perturbed environments, microfluidic experiments could reveal the function of pathways thought to be nonessential under ideal conditions.

This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as 'stochastic confinement'. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.

In this paper we describe a multijunction microfluidic device for the injection of a substrate into an array of preformed plugs carried by an immiscible fluid in a microchannel. The device uses multiple junctions to inject substrate into preformed plugs without synchronization of the flow of substrate and the array of preformed plugs of reagent, which reduces cross-contamination of the plugs, eliminates formation of small droplets of substrate, and allows a greater range of injection ratios compared to that of a single T-junction. The device was based on a previously developed physical model for transport that was here adapted to describe injection and experimentally verified. After characterization, the device was applied to two biochemical assays, including evaluation of the enzymatic activity of thrombin and determination of the coagulation time of human blood plasma, which both provided reliable results. The reduction of cross-contamination and greater range of injection ratios achieved by this device may improve the processes that involve addition and titration of reagents into plugs, such as high-throughput screening of protein crystallization conditions.

High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a "hybrid" droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as approximately 140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in approximately 10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 microl of protein solution, approximately 1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.

In traditional screening with 96-well plates, microliters of substrates are consumed for each reaction. Further miniaturization is limited by the special equipment and techniques required to dispense nanoliter volumes of fluid. Plug-based microfluidics confines reagents in nanoliter plugs (droplets surrounded by fluorinated carrier fluid), and uses simple pumps to control the flow of plugs. By using cartridges pre-loaded with nanoliter plugs of reagents, only two pumps and a merging junction are needed to set up a screen. Screening with preloaded cartridges uses only nanoliters of substrate per reaction, and requires no microfabrication. The low cost and simplicity of this method has the potential of replacing 96-well and other multi-well plates, and has been applied to enzymatic assays, protein crystallization and optimization of organic reactions.

This paper characterizes a microfluidic platform that differentially controls the temperature of each half of a living Drosophila melanogaster fruitfly embryo in space and time (E. M. Lucchetta, J. H. Lee, L. A. Fu, N. H. Patel and R. F. Ismagilov, Nature, 2005, 434, 1134-1138). This platform relies on laminar flow of two streams of liquid with different temperature, and on rapid prototyping in polydimethylsiloxane (PDMS). Here, we characterized fluid flow and heat transport in this platform both experimentally and by numerical simulation, and estimated the temperature distribution around and within the embryo by numerical simulation, to identify the conditions for creating a sharper temperature difference (temperature step) over the embryo. Embryos were removed from the device and immunostained histochemically for detection of Paired protein. Biochemical processes are sensitive to small differences in environmental temperature. The microfluidic platform characterized here could prove useful in understanding dynamics of biochemical networks as they respond to changes in temperature.

In situX-ray data collection has the potential to eliminate the challenging task of mounting and cryocooling often fragile protein crystals, reducing a major bottleneck in the structure determination process. An apparatus used to grow protein crystals in capillaries and to compare the background X-ray scattering of the components, including thin-walled glass capillaries against Teflon, and various fluorocarbon oils against each other, is described. Using thaumatin as a test case at 1.8 Å resolution, this study demonstrates that high-resolution electron density maps and refined models can be obtained fromin situdiffraction of crystals grown in microcapillaries.

Protein crystallization is important for determining protein structures by X-ray diffraction. Nanoliter-sized plugs--aqueous droplets surrounded by a fluorinated carrier fluid--have been applied to the screening of protein crystallization conditions. Preformed arrays of plugs in capillary cartridges enable sparse matrix screening. Crystals grown in plugs inside a microcapillary may be analyzed by in situ X-ray diffraction. Screening using plugs, which are easily formed in PDMS microfluidic channels, is simple and economical, and minimizes consumption of the protein. This approach also has the potential to improve our understanding of the fundamentals of protein crystallization, such as the effect of mixing on the nucleation of crystals.

This paper reports a plug-based, microfluidic method for performing multi-step chemical reactions with millisecond time-control. It builds upon a previously reported method where aqueous reagents were injected into a flow of immiscible fluid (fluorocarbons) (H. Song et al., Angew. Chem. Int. Ed., 2003, 42, 768). The aqueous reagents formed plugs – droplets surrounded and transported by the immiscible fluid. Winding channels rapidly mixed the reagents in droplets. This paper shows that further stages of the reaction could be initiated by flowing additional reagent streams directly into the droplets of initial reaction mixture. The conditions necessary for an aqueous stream to merge with aqueous droplets were characterized. The Capillary number could be used to predict the behavior of the two-phase flow at the merging junction. By transporting solid reaction products in droplets, the products were kept from aggregating on the walls of the microchannels. To demonstrate the utility of this microfluidic method it was used to synthesize colloidal CdS and CdS/CdSe core-shell nanoparticles.

Microchips: Multistep reactions may be catalyzed by enzymes immobilized on microbeads trapped inside microfluidic channels. Large integrated microfluidic circuits can be created by using multilayer soft lithography in poly(dimethylsiloxane). These circuits (see schematic representation) can be used to perform hundreds of different reactions simultaneously, and can be applied to a range of problems, from enzymatic assays to crystallization of proteins.

This account examines developments in “digital” biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide “analog-to-digital” signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in “digital” microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This “digitizing” offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more deeply, and advancing molecular and cellular analysis. Microfluidics will impact digital biology and chemistry and will also benefit from them if it becomes massively distributed.

This Account summarizes techniques for carrying out microfabrication of structures with dimensions down to 10 mum in microchannels that are 0.02-2 mm wide. These methods are largely based on the exploitation of laminar flow at low Reynolds number (Re) to control the spatial delivery of reagents. These methods are illustrated by fabrication of fibers, microelectrode arrays, arrays of crystals, and patterns of proteins and cells.

This paper describes the use of laminar flow of liquids in capillary systems to pattern the cell culture substrate, to perform patterned cell deposition, and to pattern the cell culture media. We demonstrate the patterning of the cell culture substrate with different proteins, the patterning of different types of cells adjacent to each other, the patterned delivery of chemicals to adhered cells, and performing enzymatic reactions over select cells or over a portion of a cell. This method offers a way to simultaneously control the characteristics of the surface to which cells are attached, the type of cells that are in their vicinity, and the kind of media that cells or part of a cell are exposed to. The method is experimentally simple, highly adaptable, and requires no special equipment except for an elastomeric relief that can be readily prepared by rapid prototyping.

This paper reports a strategy that uses microfluidic networks to pattern self-assembled monolayers with gradient microislands for the attachment of individual cells. A microfluidic network is first used to pattern a monolayer into square regions that present maleimide groups and then used to flow a solution having a gradient of the cell adhesion peptide Arg-Gly-Asp over the substrate. In this way, the surface is patterned with microislands approximately 33 x 33 micrometers in size and each having a defined gradient of immobilized cell adhesion ligand. B16F10 cells were allowed to attach to the patterned islands and were found to display a nonuniform distribution of cytoskeletal structures in response to the gradient of adhesion ligand. This work is significant because it permits studies of the influence of a nonuniform microenvironment on the polarization, differentiation, and signaling of adherent cells.

Protein crystallization is a major bottleneck in determining tertiary protein structures from genomic sequence data. This paper describes a microfluidic system for screening hundreds of protein crystallization conditions using less than 4 nL of protein solution for each crystallization droplet. The droplets are formed by mixing protein, precipitant, and additive stock solutions in variable ratios in a flow of water-immiscible fluids inside microchannels. Each droplet represents a discrete trial testing different conditions. The system has been validated by crystallization of several water-soluble proteins.

Rare cells can be difficult to analyze because they either occur in low numbers or coexist with a more abundant cell type, yet their detection is crucial for diagnosing disease and maintaining human health. In this tutorial review, we introduce the concept of microfluidic stochastic confinement for use in detection and analysis of rare cells. Stochastic confinement provides two advantages: (1) it separates rare single cells from the bulk mixture and (2) it allows signals to locally accumulate to a higher concentration around a single cell than in the bulk mixture. Microfluidics is an attractive method for implementing stochastic confinement because it provides simple handling of small volumes. We present technologies for microfluidic stochastic confinement that utilize both wells and droplets for the detection and analysis of single cells. We address how these microfluidic technologies have been used to observe new behavior, increase speed of detection, and enhance cultivation of rare cells. We discuss potential applications of microfluidic stochastic confinement to fields such as human diagnostics and environmental testing.

We present a novel microfluidic device for amyloid peptide aggregation research. The device relies on the control of interfacial chemistry, which allows miniaturizing of aggregation measurements to nanoliter volumes. In traditional in vitro aggregation experiments, adsorption of amyloid peptides to various interfaces has been shown to nucleate and to enhance peptide aggregation. The problem of adsorption is even more pronounced upon miniaturization of aggregation experiments. Miniaturization leads to an increase of the surface-to-volume ratio, and concomitantly to an increase of amyloid peptide aggregation if the surface is not controlled. Nevertheless, miniaturization of aggregation experiments is desirable for samples available only in small volumes, as for example cerebrospinal fluid (CSF) from mice. CSF has recently gained interest in Alzheimer research, however CSF analytics has been hampered to due the small available volume.In order to miniaturize and control the interfacial chemistry of aggregation experiments we used a plug based microfluidic approach. Plugs are nanoliter sized aqueous droplets formed in the flow of immiscible fluids inside microfluidic channels. Upon peptide encapsulation into plugs, the unfavorable interfaces are exchanged for an adjustable liquid/liquid interface. We show for one prominent amyloid peptide, the Alzheimer's peptide Aβ(1-40), that aggregation in plugs has kinetics of orders of magnitude slower than under standard conditions. Further we show the applicability of this miniaturized system to aggregation experiments by testing the inhibitory potency of CSF from wild type and ceAPPswePS1ΔE9/TTR-/- mice on Aβ aggregation. Using the plug-based approach, we were able to perform over 750 experiments with a single mouse CSF sample of 5 μl in volume. The plug system offers many new opportunities to investigate in vitro aggregation studies, as for example time controlled aging of amyloid peptides, nucleation in a confined environment, and screening of drug components.