Picoliter Droplets Research Articles

A hybrid axisymmetric flow-focusing device for monodisperse picoliter droplets

A combination of photolithography and stereolithography was successfully used to fabricate a hybrid axisymmetric flow-focusing device (h-AFFD) that produces monodisperse picoliter droplets. The h-AFFD achieved the same level of hydrodynamic performance as a monolithic AFFD produced by only stereolithography from acrylic resin. Since the h-AFFD had a narrower orifice (50 or 100 µm in diameter), created in an SU-8 sheet by photolithography, than the monolithic AFFD, we were able to produce picoliter droplets. We also succeeded in producing monodisperse droplets encapsulating a single cell without any surface modification.

Tuning Physical Properties of Nanocomplexes through Microfluidics-Assisted Confinement

The future of genetic medicine hinges on successful intracellular delivery of nucleic acid-based therapeutics. While significant effort has concentrated on developing nanocarriers to improve the delivery aspects, scant attention has been paid to the synthetic process of poorly controlled nanocomplex formation. Proposed here is a reliable system to better control the complexation process, and thus the physical properties of the nanocomplexes, through microfluidics-assisted confinement (MAC) in picoliter droplets. We show that these homogeneous MAC-synthesized nanocomplexes exhibit narrower size distribution, lower cytotoxicity, and higher transfection efficiency compared to their bulk-synthesized counterparts. MAC represents a physical approach to control the energetic self-assembly of polyelectrolytes, thereby complementing the chemical innovations in nanocarrier design to optimize nucleic acid and peptide delivery.

Creation of stepwise concentration gradient in picoliter droplets for parallel reactions of matrix metalloproteinase II and IX.

We present a new methodology for generating a stepwise concentration gradient in a series of microdroplets by using monolithic micro valves that act as "faucets" in micrometer-scale. A distinct concentration gradient of a substrate was generated for the determination of the kinetic parameters of two different enzymes using only 10 picoliter-scale droplets. With a single experiment on a chip, we obtained K(M) and k(cat) values of matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9), and compared the catalytic competence of the two enzymes. The present system and method are highly suitable for applications where the reagents or samples are limited and precious.

Aqueous droplet manipulation by optically induced Marangoni circulation

The manipulation of picoliter droplets is demonstrated using optically induced microscale circulatory flows. The circulation results from Marangoni effects induced by optical heating from light patterns created by a computer projector. Manipulation of single droplets and parallel manipulation of multiple droplets are achieved with induced forces of up to 1 nN and an average resolution of 146.5 μm.

The (S)lowdown on Crystallization

Materials Science One way to resolve the many steps that occur during crystallization is to use small droplets of solution, in part to avoid heterogeneous nucleation by impurities. Stephens et al. created picoliter droplets by using self-assembled monolayers (SAMs) that contained hydrophilic (carboxylic acid terminated) islands inside hydrophobic (fluorous terminated) layers. These islands supported hemispherical droplets, with radii varying from 4 to 10 µm (0.04 to 2 pl in volume), that were supersaturated with calcium carbonate. Bulk precipitation created many rhombohedral crystals, but in 90% of the droplets, a single smaller tetrahedral crystal formed. Redissolution of these tetrahedral crystals in undersaturated solution, or further growth from bulk solution, initiated recrystallization into rhombohedral crystals. The SAMs appear to slow down crystallization and capture the tetrahedron as an early intermediate crystal form. J. Am. Chem. Soc. 133 , 10.1021/ja200309m (2011).

Early Stages of Crystallization of Calcium Carbonate Revealed in Picoliter Droplets

In this work, we studied the heterogeneous nucleation and growth of CaCO(3) within regular arrays of picoliter droplets created on patterned self-assembled monolayers (SAMs). The SAMs provide well-defined substrates that offer control over CaCO(3) nucleation, and we used these impurity-free droplet arrays to study crystal growth in spatially and chemically controlled, finite-reservoir environments. The results demonstrate a number of remarkable features of precipitation within these confined volumes. CaCO(3) crystallization proceeds significantly more slowly in the droplets than in the bulk, allowing the mechanism of crystallization, which progresses via amorphous calcium carbonate, to be easily observed. In addition, the precipitation reaction terminates at an earlier stage than in the bulk solution, revealing intermediate growth forms. Confinement can therefore be used as a straightforward method for studying the mechanisms of crystallization on a substrate without the requirement for specialized analytical techniques. The results are also of significance to biomineralization processes, where crystallization typically occurs in confinement and in association with organic matrices, and it is envisaged that the method is applicable to many crystallizing systems.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.

Experimental investigation of the impact, spreading and drying of picolitre droplets onto substrates with a broad range of wettabilities

Experimental investigation of the impact, spreading and drying of picolitre droplets onto substrates with a broad range of wettabilities

Pneumatic handling of droplets on‐demand on a microfluidic device for seamless processing of reaction and electrophoretic separation

Sequential operations of pre-separation reaction process by picoliter droplets and following electrophoretic separation process were realized in a single microfluidic device with pneumatic handling of liquid. The developed device consists of a fluidic chip made of PDMS, an electrode substrate, and a temperature control substrate on which thin film heater/sensor structures are fabricated. Liquid handling, including introduction of liquid samples, droplet generation, and merging of droplets, was implemented by pneumatic manipulation through microcapillary vent structures, allowing air to pass and stop liquid flow. Since the pneumatic manipulations are conducted in a fully automated manner by using a programmable air pressure control system, the user simply has to load liquid samples on each liquid port of the device. Droplets of 420 pL were generated with an accuracy of ± 2 pL by applying droplet generation pressure in the range of 40-100 kPa. As a demonstration, a binding reaction of a 15 mer ssDNA with a peptide nucleic acid oligomer used as an oligoprobe followed by denaturing electrophoresis to discriminate a single-base substitution was performed within 1.5 min. By exploiting the droplet-on-demand capability of the device, the influence of various factors, such as reaction time, mixing ratio and droplet configurations on the ssDNA-peptide nucleic acid binding reaction in the droplet-based process, was studied toward realization of a rapid detection method to discriminate rapid single-base substitution.

Interplay of Processing, Morphological Order, and Charge-Carrier Mobility in Polythiophene Thin Films Deposited by Different Methods: Comparison of Spin-Cast, Drop-Cast, and Inkjet-Printed Films

The dependence of morphology and polymer-chain orientation of regioregular poly(3-hexylthiophene) (rrP3HT) thin films on processing conditions have been widely studied. However, their possible variation across the film thickness direction remains largely unknown. We report here a marked difference in the optical dielectric (n,k) spectra between the top and bottom interfaces of spin-cast (sc) rrP3HT films deposited from chlorobenzene solutions. These spectra were obtained from reflection variable-angle spectroscopic ellipsometry using a self-consistent graded optical model with self-imposed Kramers-Krönig consistency. The top interface shows a red-shifted absorption that is characteristic of better order than at the bottom, across a wide range of film thicknesses. This disparity diminishes in drop-cast (dc) and multipass inkjet-printed (ijp) films, and disappears in amorphous films such as those of polystyrene and of a green-emitting phenyl-substituted poly(p-phenylenevinylene). The (n,k) spectra also reveal that crystallinity increases across sc < dc < ijp films. This is supported by cross section scanning electron microscopy of the cleaved edges and measurement of the microroughness of both the film interfaces. Furthermore, optical anisotropy decreases across sc > dc > ijp films. Finally, near-edge X-ray absorption fine structure spectroscopy also shows the frontier chains in ijp and dc films are more isotropically oriented than those in sc films. These results suggest that semicrystalline conjugated polymer films can be produced far from equilibrium. This explains the marked variation in their (opto)electronic properties between the top and bottom surfaces that has sometimes been found depending on the film deposition method. In particular, an unusually pronounced crystallization is induced by ijp. We label this marked ijp-induced crystallization the "ijp morphology", which appears to be general, as it is found also in single-inkjet-droplet films. It appears also to be responsible for the lower field-effect mobility measured for ijp films deposited on a variety of linear and circular electrode arrays. This however can fortuitously be reversed by annealing in solvent vapor. As all films were deposited in the low Peclet-number regime, we can rule out surface skin formation. We attribute the extensive crystallization to the non-uniform drying of picoliter droplets, further promoted by repeated film swelling-deswelling cycles in multipass-ijp films.

Generation of Picoliter Droplets with Defined Contents and Concentration Gradients from the Separation of Chemical Mixtures

There has been an increasing drive toward miniaturizing and accelerating experiments with droplet-based microfluidics across the chemical disciplines. Current applications take advantage of the numerous techniques for manipulating nano- to femtoliter droplets within microfluidic devices. To expand the range of possible applications, we have developed a method for compartmentalizing pure compounds within droplets, at a gradient of concentrations, starting from chemical mixtures. In this technique, a mixture is injected into an ultra performance liquid chromatography (UPLC) system, and droplets are generated from the LC output at a frequency high enough to fraction each compound into approximately 10(5) droplets, compartmentalizing pure compounds into a sequence of droplets with a range of concentrations spanning 2-3 orders of magnitude. Here we used fluorescent dyes to quantify the concentration profile of the droplet collections, and to demonstrate the correspondence between the concentration profile of the droplets and the compound elution profile monitored with a UV absorbance detector, allowing the use of compounds that are not fluorescently labeled but show UV absorbance. Hence this technique is applicable to a wide variety of applications that require both compound purity and the ability to probe a variety of concentrations, such as drug screening and titrations.

Droplet merging in a straight microchannel using droplet size or viscosity difference

In the present study, a novel droplet-merging technique is proposed, which enables two nanoliter or picoliter droplets to merge in a straight microchannel with high precision. Two dispersed phase fluids are supplied respectively from two side channels in a certain range of flow rates to generate droplets of different sizes or different viscosities in a regularly alternating mode at a cross-channel intersection prior to the straight microchannel, in such a manner that the droplets are spontaneously synchronized by themselves due to the competition of the two interfaces formed at the cross-channel intersection. Then they are autogeneously merged in the downstream straight microchannel with or without a sudden expansion of the cross section, due to their velocity difference which is induced as they are transported aboard the continuous phase fluid. This droplet-merging method has no desynchronization or secondary-merging problems. Thus, it can be applied efficiently to mixing or encapsulating one target sample with another material for the purposes of nanoparticle synthesis, hydrogel-bead production, cell transplantation and so forth.

Droplet‐Based Microfluidic Synthesis of Anisotropic Metal Nanocrystals

A droplet-based microfluidic method for the preparation of anisotropic gold nanocrystal dispersions is presented. Gold nanoparticle seeds and growth reagents are dispensed into monodisperse picoliter droplets within a microchannel. Confinement within small droplets prevents contact between the growing nanocrystals and the microchannel walls. The critical factors in translating macroscale flask-based methods to a flow-based microfluidic method are highlighted and approaches are demonstrated to flexibly fine tune nanoparticle shapes into three broad classes: spheres/spheroids, rods, and extended sharp-edged structures, thus varying the optical resonances in the visible-near-infrared (NIR) spectral range.

Simultaneous Determination of Gene Expression and Enzymatic Activity in Individual Bacterial Cells in Microdroplet Compartments

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.

Dilution-free analysis from picoliter droplets by nano-electrospray ionization mass spectrometry.

Packaged, transferred, and delivered: A method has been developed that automatically transfers the contents of oil-encapsulated aqueous plugs to a co-flowing aqueous stream, which enables dilution-free electrospray ionization (ESI) mass spectrometric analysis for droplet-based microfluidics.

Integrated liquid and droplet dielectrophoresis for biochemical assays

Surface microfluidic systems have emerged as an attractive alternative to conventional closed-channel microfluidic devices. In many such systems, electric fields are leveraged for the manipulation and transport of discrete nanoliter droplets on open planar surfaces. The present research work discusses dielectrophoretic liquid and droplet actuations, which provide an attractive methodology for dispensing and manipulating nanoliter and picoliter droplets on planar surfaces. We demonstrate the integration of two independent sample actuation schemes, namely liquid dielectrophoresis (L-DEP) and droplet dielectrophoresis, and furthermore validate its applicability through model biochemical assays (DNA-PicoGreen® assay and DNA FRET assay). We also describe and present ‘tapering L-DEP’ actuation scheme, whereby we demonstrate how to simultaneously create multiple droplets of different sizes and volumes in the range of nanoliter and picoliters, from a given larger parent sample droplet.

Evaporation of inkjet printed pico-liter droplet on heated substrates with different thermal conductivity

In this work, the evaporation phenomena of 20–45 picoliter water droplet (i.e. 50–65 μm diameter) on heated substrates with different thermal conductivity are studied experimentally. The effect of thermal conductivity of substrates and inter-distance between jetted droplets on the evaporation is investigated. In addition, the model to predict evaporation rate of the picoliter droplet on different substrates at a heated condition is developed using approximations for picoliter droplet.

Cover Picture: Analysis of Protein–Protein Interactions by Using Droplet‐Based Microfluidics (ChemBioChem 10/2009)

AbstractThe cover picture shows a segmented‐flow microfluidic system that was used to analyse protein–protein interactions in picolitre droplets. The 400 pL aqueous droplets were formed, encapsulated by a carrier fluid, and then transported through a 50 μm‐wide microchannel at a constant velocity. Each droplet contained fluorescently labelled antigens and antibodies, and FRET was used to report protein–protein interactions. Angiogenin (ANG), a small polypeptide implicated in angiogenesis and tumour growth, was selected as a model protein. Specifically, an anti‐ANG antibody (anti‐ANG Ab) and an ANG antigen were labelled with fluorophores to act as donor and acceptor in the FRET measurements. KD values for ANG and anti‐ANG Ab from these experiments (KD=6.4±1.6 nM) agree closely with data from bulk fluorescence polarisation measurements KD=9.0±1.5 nM). We expect that this novel experimental platform will have significant application in high‐throughput protein expression profiling and drug discovery. For further details, see the article by S.‐I. Chang, A. J. deMello et al. on p. 1605 ff.magnified image