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Motile bacteria are often found in complex, polymer-rich environments in which microbes can aggregate via polymer-induced depletion forces. Bacterial aggregation has many biological implications; it can promote biofilm formation, upregulate virulence factors, and lead to quorum sensing. The steady state aggregation behavior of motile bacteria in polymer solutions has been well studied and shows that stronger depletion forces are required to aggregate motile bacteria as compared with their nonmotile analogs. However, no one has studied whether these same trends hold at the initial stages of aggregation. We use experiments and numerical calculations to investigate the polymer-induced depletion aggregation of motile Escherichia coli in polyethylene glycol solutions on short experimental timescales (∼10 min). Our work reveals that in the semi-dilute polymer concentration regime and at short timescales, in contrast to what is found at steady state, bacterial motility actually enhances aggregate formation by increasing the collision rate in viscous environments. These unexpected findings have implications for developing models of active matter, and for understanding bacterial aggregation in dynamic, biological environments, where the system may never reach steady state.

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.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies., Changes in microbial taxa are commonly derived from estimations of relative abundance, which inherently limits the depth of the analysis. Here, the authors present an absolute quantification method based on digital PCR anchoring and 16S rRNA gene sequencing and show how a ketogenic diet affects individual taxon’s absolute abundance in stool and small-intestine mucosa samples in mice

We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the “fate” (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and were not sensitive to fate of the rest of the molecules. We show how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings.

Equipment-free pumping is a challenging problem and an active area of research in microfluidics, with applications for both laboratory and limited-resource settings. This paper describes the pumping lid method, a strategy to achieve equipment-free pumping by controlled generation of pressure. Pressure was generated using portable, lightweight, and disposable parts that can be integrated with existing microfluidic devices to simplify workflow and eliminate the need for pumping equipment. The development of this method was enabled by multi-material 3D printing, which allows fast prototyping, including composite parts that combine materials with different mechanical properties (e.g. both rigid and elastic materials in the same part). The first type of pumping lid we describe was used to produce predictable positive or negative pressures via controlled compression or expansion of gases. A model was developed to describe the pressures and flow rates generated with this approach and it was validated experimentally. Pressures were pre-programmed by the geometry of the parts and could be tuned further even while the experiment was in progress. Using multiple lids or a composite lid with different inlets enabled several solutions to be pumped independently in a single device. The second type of pumping lid, which relied on vapor–liquid equilibrium to generate pressure, was designed, modeled, and experimentally characterized. The pumping lid method was validated by controlling flow in different types of microfluidic applications, including the production of droplets, control of laminar flow profiles, and loading of SlipChip devices. We believe that applying the pumping lid methodology to existing microfluidic devices will enhance their use as portable diagnostic tools in limited resource settings as well as accelerate adoption of microfluidics in laboratories.

This paper evaluates the potential of 3D printing, a semi-automated additive prototyping technology, as a means to design and prototype a sample-to-device interface, amenable to diagnostics in limited-resource settings, where speed, accuracy and user-friendly design are critical components. As a test case, we built and validated an interlock meter-mix device for accurately metering and lysing human urine samples for use in downstream nucleic acid amplification. Two plungers and a multivalve generated and controlled fluid flow through the device and demonstrate the utility of 3D printing to create leak-free seals. Device operation consists of three simple steps that must be performed sequentially, eliminating manual pipetting and vortexing to provide rapid (5 to 10 s) and accurate metering and mixing. Bretherton's prediction was applied, using the bond number to guide a design that prevents potentially biohazardous samples from leaking from the device. We employed multi-material 3D printing technology, which allows composites with rigid and elastomeric properties to be printed as a single part. To validate the meter-mix device with a clinically relevant sample, we used urine spiked with inactivated Chlamydia trachomatis and Neisseria gonorrhoeae. A downstream nucleic acid amplification by quantitative PCR (qPCR) confirmed there was no statistically significant difference between samples metered and mixed using the standard protocol and those prepared with the meter-mix device, showing the 3D-printed device could accurately meter, mix and dispense a human urine sample without loss of nucleic acids. Although there are some limitations to 3D printing capabilities (e.g. dimension limitations related to support material used in the printing process), the advantages of customizability, modularity and rapid prototyping illustrate the utility of 3D printing for developing sample-to-device interfaces for diagnostics.

In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of compartments. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2 × 10(2) to 4.0 × 10(6) molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8 × 10(3) to 1.2 × 10(7) molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at a dynamic range of 1.7 × 10(2) to 2.0 × 10(7) molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in other work by Kreutz et al.

This paper describes microfluidic experiments with human blood plasma and numerical simulations to determine the role of fluid flow in the regulation of propagation of blood clotting. We demonstrate that propagation of clotting can be regulated by different mechanisms depending on the volume-to-surface ratio of a channel. In small channels, propagation of clotting can be prevented by surface-bound inhibitors of clotting present on vessel walls. In large channels, where surface-bound inhibitors are ineffective, propagation of clotting can be prevented by a shear rate above a threshold value, in agreement with predictions of a simple reaction-diffusion mechanism. We also demonstrate that propagation of clotting in a channel with a large volume-to-surface ratio and a shear rate below a threshold shear rate can be slowed by decreasing the production of thrombin, an activator of clotting. These in vitro results make two predictions, which should be experimentally tested in vivo. First, propagation of clotting from superficial veins to deep veins may be regulated by shear rate, which might explain the correlation between superficial thrombosis and the development of deep vein thrombosis (DVT). Second, nontoxic thrombin inhibitors with high binding affinities could be locally administered to prevent recurrent thrombosis after a clot has been removed. In addition, these results demonstrate the utility of simplified mechanisms and microfluidics for generating and testing predictions about the dynamics of complex biochemical networks.

This paper describes a method of time-controlled seeding to separate the stages of nucleation and growth in protein crystallization using a microfluidic device.

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.

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.

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.

This paper describes a microfluidic chip for performing kinetic measurements with better than millisecond resolution. Rapid kinetic measurements in microfluidic systems are complicated by two problems: mixing is slow and dispersion is large. These problems also complicate biochemical assays performed in microfluidic chips. We have recently shown (Song, H.; Tice, J. D.; Ismagilov, R. F. Angew. Chem., Int. Ed. 2003, 42, 768-772) how multiphase fluid flow in microchannels can be used to address both problems by transporting the reagents inside aqueous droplets (plugs) surrounded by an immiscible fluid. Here, this droplet-based microfluidic system was used to extract kinetic parameters of an enzymatic reaction. Rapid single-turnover kinetics of ribonuclease A (RNase A) was measured with better than millisecond resolution using sub-microliter volumes of solutions. To obtain the single-turnover rate constant (k = 1100 +/- 250 s(-1)), four new features for this microfluidics platform were demonstrated: (i) rapid on-chip dilution, (ii) multiple time range access, (iii) biocompatibility with RNase A, and (iv) explicit treatment of mixing for improving time resolution of the system. These features are discussed using kinetics of RNase A. From fluorescent images integrated for 2-4 s, each kinetic profile can be obtained using less than 150 nL of solutions of reagents because this system relies on chaotic advection inside moving droplets rather than on turbulence to achieve rapid mixing. Fabrication of these devices in PDMS is straightforward and no specialized equipment, except for a standard microscope with a CCD camera, is needed to run the experiments. This microfluidic platform could serve as an inexpensive and economical complement to stopped-flow methods for a broad range of time-resolved experiments and assays in chemistry and biochemistry.

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.

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype-based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen’s susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single-molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point-of-care AST and strengthen global antibiotic stewardship.

This communication shows that the concept of Brownian trapping with drift can be applied to improve quantitative molecular measurements. It has the potential to combine the robustness of end-point spatially resolved readouts, the ultrasensitivity of digital single-molecule measurements, and the large dynamic range of qPCR; furthermore, at low concentrations of analytes, it can provide a direct comparison of the signals arising from the analyte and from the background. It relies on the finding that molecules simultaneously diffusing, drifting (via slow flow), and binding to an array of nonsaturable surface traps have an exponentially decreasing probability of escaping the traps over time and therefore give rise to an exponentially decaying distribution of trapped molecules in space. This concept was tested with enzyme and protein measurements in a microfluidic device.

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.

Second-order rate constants kij(obsd) measured at 25 °C in acetonitrile by stopped-flow spectrophotometry for forty-four electron transfer (ET) reactions among fourteen 0/+1 couples [three aromatic compounds (tetrathiafulvalene, tetramethyltetraselenafulvalene, and 9,10-dimethyl-9,10-dihydrophenazine), four 2,3-disubstituted 2,3-diazabicyclo[2.2.2]octane derivatives, six acyclic hydrazines, and the bridgehead diamine 1,5-diazabicyclo[3.3.3]undecane] and seventeen compounds and forty-seven reactions from a previous study (J. Am. Chem. Soc. 1997, 119, 5900) [three p-phenylenediamine derivatives, four ferrocene derivatives, and ten tetraalkylhydrazines] are discussed. When all 91 kij(obsd) values are simultaneously fitted to Marcus's adiabatic cross rate formula kij(calcd) = (kiikjjKijfij)1/2, ln fij = (ln Kij)2/4 ln(kiikjj/Z2), best-fit self-exchange rate constants, kii(fit), are obtained that allow remarkably accurate calculation of kij(obsd); kij(obsd)/kij(calcd) is in the range 0.5−2.0 for all 91 reactions. The average difference without regard to sign, |ΔΔGij|, between observed cross reaction activation free energy and that calculated using the kii(fit) values and equilibrium constants is 0.13 kcal/mol. The ΔGii(fit) values obtained range from 2.3 kcal/mol for tetramethyltetraselenafulvalene0/+ to 21.8 kcal/mol for tetra-n-propylhydrazine0/+, corresponding to a factor of 2 × 1014 in kii(fit). The principal factor affecting kii(fit) for our data appears to be the internal vertical reorganization energy (λv), but kii(fit) values also incorportate the effects of changes in the electronic matrix coupling element (V). Significantly smaller V values for ferrocenes and for hydrazines with alkyl groups larger than methyl than for aromatics and tetramethylhydrazine are implied by the observed ΔGii(fit) values.

1,4-Bis(2-tert-butyl-2,3-diazabicyclo[2.2.2]oct-3-yl)benzene-1,4-diyl (2) its 2,5-dimethyl and 2,3,5,6-tetramethyl derivatives (3 and 4), their radical cations, and bis-radical dications are studied. Crystal structures including those of 2^+BPh_4^-, 3^(2+)(BPh_4^-)_2, 4^+BPh_4^-, and 4^(2+)(BPh_4^-)_2 establish that ring methylation causes more N-lone pair, aryl π twist without changing the NAr,NAr distance significantly and that both 2^+ and 4^+ have the charge localized in one hydrazine unit. NMR measurements show that 3^+ has about 6% of its spin at the four aryl CH and CMe carbons, while 4^+ has about 1.5% of its spin at the four CMe carbons. The average distance between the unpaired electrons of 3^(2+) and 4^(2+) was obtained from the dipolar splittings of their thermally excited triplet states and, as expected, is significantly smaller for 3^(2+) (5.25 Å) than for 4^(2+) (5.63 Å). Rate constants for electron transfer between the hydrazine units of 3^+ and 4^+ in CH_2Cl_2 and CH_3CN were determined by dynamic ESR. The intervalence radical cations show charge transfer bands corresponding to vertical electron transfer between the ground state and the highly vibrationally excited electron-shifted state, allowing calculation of the parameters controlling electron transfer. Electron transfer parameters obtained from the CT bands using adiabatic energy surfaces which approximate the CT band shapes observed produce rate constants within experimental error of those extrapolated to room temperature from the ESR data for both 3^+ and 4^+ in both solvents, without using tunneling corrections. The effects of mixing of the electronic wave functions of the reduced and oxidized hydrazine units of 2^+ on d_(NN), the C(t-Bu)N,NA(Ar) twist angle, and the aryl nitrogen lone pair, aryl π twist angle which are observed by X-ray are close to those predicted from the position of the minima on the ET coordinate X of the adiabatic energy surface calculated from the CT band.

Second-order rate constants k12(obsd) measured at 25 °C in acetonitrile by stopped-flow for 47 electron transfer (ET) reactions among ten tetraalkylhydrazines, four ferrocene derivatives, and three p-phenylenediamine derivatives are discussed. Marcus's adiabatic cross rate formula k12(calcd) = (k11 k22 k12 f12)1/2, ln f12 = (ln K12)2/4 ln(k11k22/Z2) works well to correlate these data. When all k12(obsd) values are simultaneously fitted to this relationship, best-fit self-exchange rate constants, kii(fit), are obtained that allow remarkably accurate calculation of k12(obsd); k12(obsd)/k12‘(calcd) is in the range of 0.55−1.94 for all 47 reactions. The average ΔΔGij between observed activation free energy and that calculated using kii(fit) is 0.13 kcal/mol. Simulations using Jortner vibronic coupling theory to calculate k12 using parameters which produce the wide range of kii values observed predict that Marcus's formula should be followed even when V is as low as 0.1 kcal/mol, in the weakly nonadiabatic region. Tetracyclohexylhydrazine has a higher kii than tetraisopropylhydrazine by a factor of ca. 10. Replacing the dimethylamino groups of tetramethyl-p-phenylenediamine by 9-azabicyclo[3.3.1]nonyl groups has little effect on kii, demonstrating that conformations which have high intermolecular aromatic ring overlap are not necessary for large ET rate constants. Replacing a γ CH2 group of a 9-azabicyclo[3.3.1]nonyl group by a carbonyl group lowers kii by a factor of 17 for the doubly substituted hydrazine and by considerably less for the doubly substituted p-phenylenediamine.

In this paper, we describe a nonlinear threshold chemistry based on enzymatic inhibition and demonstrate how it can be coupled with microfluidics to convert a chemical concentration (analog input) into patterns of ON or OFF reaction outcomes (chemical digital readout). Quantification of small changes in concentration is needed in a number of assays, such as that for cystatin C, where a 1.5-fold increase in concentration may indicate the presence of acute kidney injury or the progression of chronic kidney disease. We developed an analog-to-digital chemical signal conversion that gives visual readout and applied it to an assay for cystatin C as a model target. The threshold chemistry is based on enzymatic inhibition and gives sharper responses with tighter inhibition. The chemistry described here uses acetylcholinesterase (AChE) and produces an unambiguous color change when the input is above a pre-determined threshold concentration. An input gives a pattern of ON/OFF responses when subjected to a monotonic sequence of threshold concentrations, revealing the input concentration at the point of transition from OFF to ON outcomes. We demonstrated that this threshold chemistry can detect a 1.30–fold increase in concentration at 22 °C, and that it is robust to experimental fluctuations: it provided the same output despite changes in temperature (22–34 °C) and readout time (10-fold range). We applied this threshold chemistry to diagnostics by coupling it with a traditional sandwich immunoassay for serum cystatin C. Because one quantitative measurement comprises several assays, each with its own threshold concentration, we used a microfluidic SlipChip device to process 12 assays in parallel, detecting a 1.5-fold increase (0.64 mg/L (49 nM) to 0.96 mg/L (74 nM)) of cystatin C in serum. We also demonstrated applicability to analysis of patient serum samples and the ability to image results using a cell phone camera. This work indicates that combining developments in nonlinear chemistries with microfluidics may lead to the development of user-friendly diagnostic assays with simple readouts.

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.

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide-alkyne cycloaddition. Protein labeling is apparent within five minutes after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

The artificial millimeter-scale “autonomous movers” glide across the surface of a liquid without an external power source. This system is based on a combination of two processes: Motion of individual objects powered by the catalytic decomposition of hydrogen peroxide, and relative motion (self-assembly) caused by capillary interactions at the fluid/air interface. The picture shows the rotational/translational motion of a single object; the motion of a pair of these object depends on their chirality.

The closing of the nuclear fuel cycle is an unsolved problem of great importance. Separating radionuclides produced in a nuclear reactor is useful both for the storage of nuclear waste and for recycling of nuclear fuel. These separations can be performed by designing appropriate chelation chemistries and liquid-liquid extraction schemes, such as in the TALSPEAK process (Trivalent Actinide-Lanthanide Separation by Phosphorus reagent Extraction from Aqueous Komplexes). However, there are no approved methods for the industrial scale reprocessing of civilian nuclear fuel in the United States. One bottleneck in the design of next-generation solvent extraction-based nuclear fuel reprocessing schemes is a lack of interfacial mass transfer rate constants obtained under well-controlled conditions for lanthanide and actinide ligand complexes; such rate constants are a prerequisite for mechanistic understanding of the extraction chemistries involved and are of great assistance in the design of new chemistries. In addition, rate constants obtained under conditions of known interfacial area have immediate, practical utility in models required for the scaling-up of laboratory-scale demonstrations to industrial-scale solutions. Existing experimental techniques for determining these rate constants suffer from two key drawbacks: either slow mixing or unknown interfacial area. The volume of waste produced by traditional methods is an additional, practical concern in experiments involving radioactive elements, both from disposal cost and experimenter safety standpoints. In this paper, we test a plug-based microfluidic system that uses flowing plugs (droplets) in microfluidic channels to determine absolute interfacial mass transfer rate constants under conditions of both rapid mixing and controlled interfacial area. We utilize this system to determine, for the first time, the rate constants for interfacial transfer of all lanthanides, minus promethium, plus yttrium, under TALSPEAK process conditions, as a first step toward testing the molecular mechanism of this separation process.

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 paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1,280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.

In this article, we developed a "plant on a chip" microfluidic platform that can control the local chemical environment around live roots of Arabidopsis thaliana with high spatial resolution using multi-laminar flow. We characterized the flow profile around the Arabidopsis root, and verified that the shear forces within the device ( approximately 10 dyne cm(-2)) did not impede growth of the roots. Our platform was able to deliver stimuli to the root at a spatial resolution of 10-800 microm. Further, the platform was validated by exposing desired regions of the root with a synthetic auxin derivative, 2,4-dichlorophenoxyacetic acid (2,4-D), and its inhibitor N-1-naphthylphthalamic acid (NPA). The response to the stimuli was observed using a DR5::GFP Arabidopsis line, where GFP expression is coupled to the auxin response regulator DR5. GFP expression in the root matched the position of the flow-focused stream containing 2,4-D. When the regions around the 2,4-D stimulus were exposed to the auxin transport inhibitor NPA, the active and passive transport mechanisms of auxin could be differentiated, as NPA blocks active cell-to-cell transport of auxin. Finally, we demonstrated that local 2,4-D stimulation in a approximately 10 microm root segment enhanced morphological changes such as epidermal hair growth. These experiments were proof-of-concept and agreed with the results expected based on known root biology, demonstrating that this "root on a chip" platform can be used to test how root development is affected by any chemical component of interest, including nitrogen, phosphate, salts, and other plant hormones.

This paper uses microfluidics to implement genetic algorithms (GA) to discover new homogeneous catalysts using the oxidation of methane by molecular oxygen as a model system. The parameters of the GA were the catalyst, a cocatalyst capable of using molecular oxygen as the terminal oxidant, and ligands that could tune the catalytic system. The GA required running hundreds of reactions to discover and optimize catalyst systems of high fitness, and microfluidics enabled these numerous reactions to be run in parallel. The small scale and volumes of microfluidics offer significant safety benefits. The microfluidic system included methods to form diverse arrays of plugs containing catalysts, introduce gaseous reagents at high pressure, run reactions in parallel, and detect catalyst activity using an in situ indicator system. Platinum(II) was identified as an active catalyst, and iron(II) and the polyoxometalate H(5)PMo(10)V(2)O(40) (POM-V2) were identified as active cocatalysts. The Pt/Fe system was further optimized and characterized using NMR experiments. After optimization, turnover numbers of approximately 50 were achieved with approximately equal production of methanol and formic acid. The Pt/Fe system demonstrated the compatibility of iron with the entire catalytic cycle. This approach of GA-guided evolution has the potential to accelerate discovery in catalysis and other areas where exploration of chemical space is essential, including optimization of materials for hydrogen storage and CO(2) capture and modifications.

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.

The SlipChip is a microfluidic device designed to perform multiplexed microfluidic reactions without pumps or valves. The device has two plates in close contact. The bottom plate contains wells preloaded with many reagents; in this paper plates with 48 reagents were used. These wells are covered by the top plate that acts as a lid for the wells with reagents. The device also has a fluidic path, composed of ducts in the bottom plate and wells in the top plate, which is connected only when the top and bottom plate are aligned in a specific configuration. Sample can be added into the fluidic path, filling both wells and ducts. Then, the top plate is “slipped”, or moved, relative to the bottom plate so the complementary patterns of wells in both plates overlap, exposing the sample-containing wells of the top plate to the reagent-containing wells of the bottom plate, and enabling diffusion and reactions. Between the two plates, a lubricating layer of fluorocarbon was used to facilitate relative motion of the plates. This paper implements this approach on a nanoliter scale using devices fabricated in glass. Stability of preloaded solutions, control of loading, and lack of cross-contamination were tested using fluorescent dyes. Functionality of the device was illustrated via crystallization of a model membrane protein. Fabrication of this device is simple and does not require a bonding step. This device requires no pumps or valves and is applicable to resource-poor settings. Overall, this device should be valuable for multiplexed applications that require exposing one sample to many reagents in small volumes. One may think of the SlipChip as an easy-to-use analogue of a preloaded multi-well plate, or a preloaded liquid-phase microarray.

This paper illustrates a plug-based microfluidic approach combining the technique of the chemistrode and the principle of stochastic confinement, which can be used to i) starting from a mixture of cells, stochastically isolate single cells into plugs, ii) incubate the plugs to grow clones of the individual cells without competition among different clones, iii) split the plugs into arrays of identical daughter plugs, where each plug contained clones of the original cell, and iv) analyze each array by an independent technique, including cellulase assays, cultivation, cryo-preservation, Gram staining, and Fluorescence In Situ Hybridization (FISH). Functionally, this approach is equivalent to simultaneously assaying the clonal daughter cells by multiple killing and non-killing methods. A new protocol for single-cell FISH, a killing method, was developed to identify isolated cells of Paenibacillus curdlanolyticus in one array of daughter plugs using a 16S rRNA probe, Pc196. At the same time, live copies of P. curdlanolyticus in another array were obtained for cultivation. Among technical advances, this paper reports a chemistrode that enables sampling of nanoliter volumes directly from environmental specimens, such as soil slurries. In addition, a method for analyzing plugs is described: an array of droplets is deposited on the surface, and individual plugs are injected into the droplets of the surface array to induce a reaction and enable microscopy without distortions associated with curvature of plugs. The overall approach is attractive for identifying rare, slow growing microorganisms and would complement current methods to cultivate unculturable microbes from environmental samples.

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.

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.

This paper utilizes cyclodextrin-based host-guest chemistry in a microfluidic device to modulate the crystallization of membrane proteins and the process of concentration of membrane protein samples. Methyl-beta-cyclodextrin (MBCD) can efficiently capture a wide variety of detergents commonly used for the stabilization of membrane proteins by sequestering detergent monomers. Reaction Center (RC) from Blastochloris viridis was used here as a model system. In the process of concentrating membrane protein samples, MBCD was shown to break up free detergent micelles and prevent them from being concentrated. The addition of an optimal amount of MBCD to the RC sample captured loosely bound detergent from the protein-detergent complex and improved sample homogeneity, as characterized by dynamic light scattering. Using plug-based microfluidics, RC crystals were grown in the presence of MBCD, giving a different morphology and space group than crystals grown without MBCD. The crystal structure of RC crystallized in the presence of MBCD was consistent with the changes in packing and crystal contacts hypothesized for removal of loosely bound detergent. The incorporation of MBCD into a plug-based microfluidic crystallization method allows efficient use of limited membrane protein sample by reducing the amount of protein required and combining sparse matrix screening and optimization in one experiment. The use of MBCD for detergent capture can be expanded to develop cyclodextrin-derived molecules for fine-tuned detergent capture and thus modulate membrane protein crystallization in an even more controllable way.

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.

This paper describes a microfluidic system to screen and optimize organic reaction conditions on a submicrogram scale. The system uses discrete droplets (plugs) as microreactors separated and transported by a continuous phase of a fluorinated carrier fluid. Previously, we demonstrated the use of a microfabricated PDMS plug-based microfluidic system to perform assays and crystallization experiments in aqueous solutions with optical detection. Here, we developed an approach that does not require microfabrication of microfluidic devices, is applicable to synthetic reactions in organic solvents, and uses detection by MALDI-MS. As a demonstration, conditions for selective deacetylation of ouabain hexaacetate were tested, and the optimum conditions for mono-, bis-, or trisdeacetylation have been identified. These conditions were validated by scale-up reactions and isolating these potentially neurotoxic products. Mono- and bisdeacetylated products are unstable intermediates in the deacetylation and were isolated for the first time. This system enables no-loss handling of submicroliter volumes containing a few micrograms of a compound of interest. It could become valuable for investigating or optimizing reactions of precious substrates (e.g., products of long synthetic sequences and natural products that can be isolated only in small quantities).

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.

Crystal structures of 2-tert-butyl-3-(2,3,5,6-tetramethylphenyl)-2,3-diazabicyclo[2.2.2]-octane radical cation nitrate (HyDU+NO_3-) [Hy = (2-tert-butyl-2,3-diazabicyclo[2.2.2]oct-3-yl]; 2-tert-butyl-3-(1-naphthyl)-2,3-diazabicyclo[2.2.2]octane radical cation hexafluoroantiminate (Hy^1NA+SbF_6-); 2-tert-butyl-3-(2-naphthyl)-2,3-diazabicyclo-[2.2.2]octane radical cation hexafluoroantiminate (Hy^2NA+SbF_6-); 1,5-bis(2-tert-butyl-2,3-diazabicyclo[2.2.2]oct-3-yl)naphthalene dication bis(tetraphenylborate) (Hy_2^(15)NA^(2+)(Ph_4B^-)_2); and 2,7-bis(2-tert-butyl-2,3-diazabicyclo[2.2.2]oct-3-yl)naphthalene dication bis(hexafluoroantiminate) (Hy_2^(27)NA^(2+)(SbF_6^-)_2·CH_3CN) are reported, and the geometries about the oxidized Hy units compared with literature data for neutral Hy-substituted analogues and the geometry changes upon electron loss for these compounds, which have a lone pair, lone pair twist angle in the neutral form (θ(0)) in the range 122−130°, are compared with those for tetraalkylhydrazines that have θ(0) values near 180, 90, and 0°.

This article reports a functional chemical reaction network synthesized in a microfluidic device. This chemical network performs chemical 5000-fold amplification and shows a threshold response. It operates in a feedforward manner in two stages: the output of the first stage becomes the input of the second stage. Each stage of amplification is performed by a reaction autocatalytic in Co^(2+). The microfluidic network is used to maintain the two chemical reactions away from equilibrium and control the interactions between them in time. Time control is achieved as described previously (Angew. Chem., Int. Ed. 2003, 42, 768) by compartmentalizing the reaction mixture inside plugs which are aqueous droplets carried through a microchannel by an immiscible fluorinated fluid. Autocatalytic reaction displayed sensitivity to mixing; more rapid mixing corresponded to slower reaction rates. Synthetic chemical reaction networks may help understand the function of biochemical reaction networks, the goal of systems biology. They may also find practical applications. For example, the system described here may be used to detect visually, in a simple format, picoliter volumes of nanomolar concentrations of Co^(2+), an environmental pollutant.

On-chip protein crystallization and diffraction: Proteins can be crystallized in nanoliter volumes by using both microbatch and vapor-diffusion techniques inside composite PDMS/glass microfluidic devices (PDMS=polydimethylsiloxane). The quality of crystals can be assessed directly by on-chip X-ray diffraction.

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.

Crown thioethers, 1,4,8,11-tetrathiacyclotetradecanes and 1,4,8,11,15,18-hexathiaheneicosanes, have been synthesized as 2:2 and 3:3 cycloadducts in a ‘one-pot’ homolytic macrocyclization of alkynes with propane-1,3-dithiol.