Development of Microdroplet Generation Method for Organic Solvents Used in Chemical Synthesis

Recently, chemical operations with microfluidic devices, especially droplet-based operations, have attracted considerable attention because they can provide an isolated small-volume reaction field. However, analysis of these operations has been limited mostly to aqueous-phase reactions in water droplets due to device material restrictions. In this study, we have successfully demonstrated droplet formation of five common organic solvents frequently used in chemical synthesis by using a simple silicon/glass-based microfluidic device. When an immiscible liquid with surfactant was used as the continuous phase, the organic solvent formed droplets similar to water-in-oil droplets in the device. In contrast to conventional microfluidic devices composed of resins, which are susceptible to swelling in organic solvents, the developed microfluidic device did not undergo swelling owing to the high chemical resistance of the constituent materials. Therefore, the device has potential applications for various chemical reactions involving organic solvents. Furthermore, this droplet generation device enabled control of droplet size by adjusting the liquid flow rate. The droplet generation method proposed in this work will contribute to the study of organic reactions in microdroplets and will be useful for evaluating scaling effects in various chemical reactions.     

A homogeneous assay for protein analysis in droplets by fluorescence polarization

We present a novel homogeneous (“mix‐incubate‐read”) droplet microfluidic assay for specific protein detection in picoliter volumes by fluorescence polarization (FP), for the first time demonstrating the use of FP in a droplet microfluidic assay. Using an FP‐based assay we detect streptavidin concentrations as low as 500 nM and demonstrate that an FP assay allows us to distinguish droplets containing 5 μM rabbit IgG from droplets without IgG with an accuracy of 95%, levels relevant for hybridoma screening. This adds to the repertoire of droplet assay techniques a direct protein detection method which can be performed entirely inside droplets without the need for labeling of the analyte molecules.     

Nebulizing conditions of pneumatic electrospray ionization significantly influence electrolyte effects on compound measurement

Composition of mobile phase can greatly influence the success of electrospray ionization (ESI)‐interfaced liquid chromatography–mass spectrometry analysis. To investigate the relationship between formic‐acid‐based modification of mobile phase and ESI nebulizing conditions, an API 4000 ESI source and a TSQ Quantum one were compared under the same chromatographic conditions. Ginkgo terpene lactones and flavonols were measured in plasma, which involved using ascorbic acid to circumvent cross‐interference between the analytes. ESI responses to using formic acid included changes in signal intensity, matrix effect, and upper limit of quantification. Significant disparities in the responses were observed between the two ESI sources, suggesting that the use of electrolyte modifier in liquid chromatography mobile phase and the pneumatic nebulization for ESI should be properly balanced to accomplish optimal ESI‐based analysis. The distribution of unpaired ions toward the surface of the initial droplet was assumed to be an important step in the pneumatic ESI process. When using the electrolyte in mobile phase, a too fast droplet reduction by rapid‐heating‐assisted pneumatic nebulization could negatively decrease the time available for the unpaired ions to migrate from droplet interior to its surface. Ascorbic acid was identified as a major interfering substance for the bioanalytical assay; the interference mechanism might be associated with hindering the unpaired analyte ions from distributing toward the droplet surface rather than outcompeting the analyte ions for the limited excess charge on droplets surface. The current work extends the knowledge base of pneumatic ESI, which has implication for optimal use of the ESI‐interfaced liquid chromatography–mass spectrometry technique. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass Activated Droplet Sorting (MADS) Enables High-Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s⁻¹ is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.     

Highly productive droplet formation by anisotropic elongation of a thread flow in a microchannel

We developed a microfluidic device to form monodisperse droplets with high productivity by anisotropic elongation of a thread flow, defined as a threadlike flow of a dispersed liquid phase in a flow of an immiscible, continuous liquid phase. The thread flow was anisotropically elongated in the depth direction in a straight microchannel with a step, where the microchannel depth changed. Consequently, the elongated thread flow was given capillary instability (Rayleigh-Plateau instability) and was continuously transformed into monodisperse droplets at the downstream area of the step in the microchannel. We examined the effects of the flow rates of the dispersed phase and the continuous phase on the droplet formation behavior, including the droplet diameter and droplet formation frequency. The droplet diameter increased as the fraction of the dispersed-phase flow rate relative to the total flow rate increased and was independent of the total flow rate. The droplet formation frequency proportionally increased with the total flow rate at a constant dispersed-phase flow rate fraction. These results are explained in terms of a mechanism similar to that of droplet formation from a cylindrical liquid thread flow by Rayleigh-Plateau instability. The microfluidic device described was capable of forming monodisperse droplets with a 160-microm average diameter and 3-microm standard deviation at a droplet formation frequency of 350 droplets per second from a single thread flow. The highest total flow rate achieved was 6 mL/h using the present device composed of a straight microchannel with a step. We also demonstrated parallel droplet formation by anisotropic elongation of multiple thread flows; the process was applied to form W/O and O/W droplets. The highly productive droplet formation process presented in this study is expected to be useful for future industrial applications.     

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s^?1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.     

Preliminary investigation of electrodynamic charged droplet processing to couple capillary liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

Charged droplet processing methodology, that utilizes electrodynamic levitation technology to control the trajectories of picoliter volume charged droplets and deliver them to a target plate at atmospheric pressure, has been developed. Termed wall-less sample preparation (WaSP), this methodology offers several features that could prove beneficial to the preparation of sample spots from separation column effluents for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. These features include solute pre-concentration factors of 10(1) to 10(3) due to volatile solvent evaporation prior to droplet deposition onto the target plate, high spatial accuracy of the deposition position of each processed droplet (+/-5 microm), and the ability to prepare sample spots as small as 20 microm in diameter from a single droplet. Here a new mode of operation of this methodology is described and used as an offline post-column pre-concentrating interface between capillary liquid chromatography (capLC) and a target plate for offline MALDI-MS. Using a fraction from the capLC separation of peptides produced by the proteolytic digestion of the protein cytidine 5'-triphosphate:phosphocholine cytidylyltransferase, MALDI sample spots were prepared using the dried-droplet method, direct piezoelectric droplet dispensing, and the processing of piezo-dispensed droplets by WaSP. The sample spot morphology was investigated using light microscopy, and peptide ion abundances produced by MALDI were measured using time-of-flight (TOF) MS. The advantages of developing an online capLC/WaSP interface with MALDI-MS in the future are discussed along with some of the challenges that may be encountered in such an endeavor.     

Alternating droplet generation and controlled dynamic droplet fusion in microfluidic device for CdS nanoparticle synthesis

A multifunctional and high-efficiency microfluidic device for droplet generation and fusion is presented. Through unique design of the micro-channels, the device is able to alternately generate droplets, generating droplet ratios ranging from 1 ratio 5 to 5 ratio 1, and fuse droplets, enabling precise chemical reactions in several picoliters on a single chip. The controlled fusion is managed by passive control based on the channel geometry and liquid phase flow. The synthesis of CdS nanoparticles utilizing each fused droplet as a microreactor for rapid and efficient mixing of reagents is demonstrated in this paper. Following alternating droplet generation, the channel geometry allows the exclusive fusion of alternate droplets with concomitant rapid mixing and produces supersaturated solution of Cd2+ and S2- ions to form CdS nanoparticles in each fused droplet. The spectroscopic properties of the CdS nanoparticles produced by this method are compared with CdS prepared by bulk mixing.     

Stochastic diffusion interactions and coarsening in a system of droplets growing from a supersaturated gas mixture

In this work we study diffusion interactions among liquid droplets growing in stochastic population by condensation from supersaturated binary gas mixture. During the postnucleation transient regime collective growth of liquid droplets competing for the available water vapor decreases local supersaturation leading to the increase of critical radius and the onset of coarsening process. In coarsening regime the growth of larger droplets is prevailing noticeably broadening the droplet size-distribution function when the condensation process becomes more intensive than the supersaturation yield. Modifications in the kinetic equation are discussed and formulated for a stochastic population of liquid droplets when diffusional interactions among droplets become noteworthy. The kinetic equation for the droplet size-distribution function is solved together with field equations for the mass fraction of disperse liquid phase, mass fraction of water vapor component of moist air, and temperature during diffusion-dominated regime of droplet coarsening. The droplet size and mass distributions are found as functions of the liquid volume fraction, showing considerable broadening of droplet spectra. It is demonstrated that the effect of latent heat of condensation considerably changes coarsening process. The coarsening rate constant, the droplet density (number of droplets per unit volume), the screening length, the mean droplet size, and mass are determined as functions of the temperature, pressure, and liquid volume fraction.     

Monodisperse w/w/w Double Emulsion Induced by Phase Separation

We develop an approach to fabricate monodisperse water-in-water-in-water (w/w/w) double emulsion in microfluidic devices. A jet of aqueous solution containing two incompatible solutes, dextran and polyethylene glycol (PEG), is periodically perturbed into water-in-water (w/w) droplets. By extracting water out of the w/w droplet, the solute concentrations in the droplet phase increase; when the concentrations exceed the miscibility limit, the droplet phase separates into two immiscible phases. Consequently, PEG-rich droplets are formed within the single emulsion templates. These PEG-rich droplets subsequently coalesce with each other, resulting in transiently stable w/w/w double emulsions with a high degree of size uniformity. These double emulsions are free of organic solvents and thus are ideal for use as droplet-vessels in protein purification, as microreactors for biochemical reactions, and as templates for fabrication of biomaterials.     

Wall-less sample preparation of <Emphasis Type="Italic">μ</Emphasis>m-sized sample spots for femtomole detection limits of proteins from liquid based UV-MALDI matrices

Previously, we introduced wall-less sample preparation (WaSP), technology that involves the use of an electrodynamic balance (EDB) to prepare microm-sized sample spots for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In that work we demonstrated the detection of femtomole quantities of a low molecular weight peptide and a hydrophobic ester (both <600 Da). Here we use WaSP to test the hypothesis that the use of small sample spot sizes and an instrument equipped with delayed extraction would increase the analytical utility of liquid sample spots for peptide and protein (>2500 Da) analysis by UV-MALDI-TOF-MS (Sze et al.; J. Am. Soc. Mass Spectrom. 1998, 9, 166-174). To aid the optimization of preparing microm-sized sample spots by WaSP, optical microscopy and mass spectrometry were used to investigate nonvolatile solute concentration effects on droplet fission and sample spot size, modifications of the EDB electric field to control droplet ejection, and the use of multiple droplet deposition to increase sample loading. Also described is a rapid deposition mode of operation for WaSP that allows single droplets generated at 1 Hz to be levitated briefly ( approximately 500 ms) before being ejected autonomously and deposited as a concentrated sample spot with a spatial accuracy of +/-5 microm. To test the sensitivity of the method, one hundred glycerol droplets (270 pL each, 27 nL total) each containing 32 amol lysozyme were deposited on top of each other one-at-a-time to create a single sample spot. Using a mass spectrometer equipped with delayed extraction to analyze this sample spot, we verified the hypothesis of Sze et al. by achieving detection limits three orders of magnitude below that previously observed for the detection of a protein by UV-MALDI-TOF-MS with a chemical-doped liquid matrix sample preparation.     

Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.     

Droplet fusion by alternating current (AC) field electrocoalescence in microchannels

We present a system for the electrocoalescence of microfluidic droplets immersed in an immiscible solvent, where the undeformed droplet diameters are comparable to the channel diameter. The electrodes are not in direct contact with the carrier liquid or the droplets, thereby minimizing the risk of cross-contamination between different coalescence events. Results are presented for the coalescence of buffered aqueous droplets in both quiescent and flowing fluorocarbon streams, and on-flight coalescence is demonstrated. The capillary-based system presented here is readily amenable to further miniaturization to any lab-on-a-chip application where the conductivity of the droplets is much greater than the conductivity of the stream containing them, and should aid in the further application of droplet microreactors to biological analyses.     

Pillar-induced droplet merging in microfluidic circuits

A novel method is presented for controllably merging aqueous microdroplets within segmented flow microfluidic devices. Our approach involves exploiting the difference in hydrodynamic resistance of the continuous phase and the surface tension of the discrete phase through the use of passive structures contained within a microfluidic channel. Rows of pillars separated by distances smaller than the representative droplet dimension are installed within the fluidic network and define passive merging elements or chambers. Initial experiments demonstrate that such a merging element can controllably adjust the distance between adjacent droplets. In a typical scenario, a droplet will enter the chamber, slow down and stop. It will wait and then merge with the succeeding droplets until the surface tension is overwhelmed by the hydraulic pressure. We show that such a merging process is independent of the inter-droplet separation but rather dependent on the droplet size. Moreover, the number of droplets that can be merged at any time is also dependent on the mass flow rate and volume ratio between the droplets and the merging chamber. Finally, we note that the merging of droplet interfaces occurs within both compressing and the decompressing regimes.     

Fast on-demand droplet fusion using transient cavitation bubbles

A method for on-demand droplet fusion in a microfluidic channel is presented using the flow created from a single explosively expanding cavitation bubble. We test the technique for water-in-oil droplets, which are produced using a T-junction design in a microfluidic chip. The cavitation bubble is created with a pulsed laser beam focused into one droplet. High-speed photography of the dynamics reveals that the droplet fusion can be induced within a few tens of microseconds and is caused by the rapid thinning of the continuous phase film separating the droplets. The cavitation bubble collapses and re-condenses into the droplet. Droplet fusion is demonstrated for static and moving droplets, and for droplets of equal and unequal sizes. Furthermore, we reveal the diffusion dominated mixing flow and the transport of a single encapsulated cell into a fused droplet. This laser-based droplet fusion technique may find applications in micro-droplet based chemical synthesis and bioassays.     

Selective droplet coalescence using microfluidic systems

We report a microfluidic approach, which allows selective and controlled 1 : 1, 2 : 1 or 3 : 1 droplet fusion. A surfactant-stabilized droplet with an interfacial surfactant coverage, Γ, of >98% will fuse spontaneously with a second droplet when Γ of the latter droplet is <16%. However, when Γ of the second droplet is ~66%, the two droplets will not fuse, unless they have previously been brought into contact for critical time τ. Therefore, controlling the number of droplets in contact for time τ allows precise control over the number of fused droplets. We have demonstrated efficient (proportion of droplets coalesced p(c) = 1.0, n > 1000) and selective 1 : 1, 2 : 1 or 3 : 1 droplet fusion (proportion of correctly fused droplets p(s) > 0.99, n > 1000). Coalescence in this regime is induced by hydrodynamic flow causing interface separation and is efficient at different Ca numbers and using different dispersed phases, continuous phases and surfactants. However, when Γ of the second droplet is ~96% coalescence is no longer observed. Droplet-based microfluidic systems, in which each droplet functions as an independent microreactor, are proving a promising tool for a wide range of ultrahigh-throughput applications in biology and chemistry. The addition of new reagents to pre-formed droplets is critical to many of these applications and we believe the system described here is a simple and flexible method to do so, as well as a new tool to study interfacial stability phenomena.     

Capacitance-based droplet position estimator for digital microfluidic devices

Digital microfluidic (DMF) devices manipulate minuscule droplets through basic fluidic operations including droplet transport, mixing and splitting commonly known as the building blocks for complete laboratory analyses on a single device. A DMF device can house various chemical species and confine chemical reactions within the volume of a droplet much like a micro-reactor. The automation of fluidic protocols requires a feedback controller whose sensor is capable of locating droplets independent of liquid composition (or previous knowledge of liquid composition). In this research, we present an estimator that tracks the continuous displacement of a droplet between electrodes of a DMF device. The estimator uses a dimensionless ratio of two electrode capacitances to approximate the position of a droplet, even, in the domain between two adjacent electrodes. This droplet position estimator significantly enhances the control precision of liquid handling in DMF devices compared to that of the techniques reported in the literature. It captures the continuous displacement of a droplet; valuable information for a feedback controller to execute intricate fluidic protocols including droplet positioning between electrodes, droplet velocity and acceleration control. We propose a state estimator for tracking the continuous droplet displacement between two adjacent electrodes. The dimensionless nature of this estimator means that any droplet composition can be sensed. Thus, no calibration for each chemical species within a single DMF device is required. We present theoretical and experimental results that demonstrate the efficacy of the position estimator in approximating the position of the droplet in the interval between two electrodes.     

Single-molecule emulsion PCR in microfluidic droplets

The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.     

Experimental investigation of droplet acceleration and collision in the gas phase in a microchannel

We developed a novel microfluidic system, termed a micro-droplet collider, by utilizing the spatial-temporal localized liquid energy to realize chemical processes, which achieved rapid mixing between droplets having a large volume ratio by collision. In this paper, in order to clarify the characteristics of the micro-droplet collider, dynamics of droplet acceleration, stationary motion and collision in the gas phase in a microchannel were experimentally investigated with visualized images using a microscope equipped with a high-speed camera. The maximum velocity of 450 mm s(-1) and acceleration of 1500 m s(-2) of a 1.6 nL water droplet were achieved at an air pressure of 100 kPa. Measurement results of dynamic contact angles of droplets indicated that wettability of the surface played an important role in the stability of droplet acceleration and collision. We found that the bullet droplet penetrated into the target droplet at collision, which differed from bulk scale. The deformation of the droplet was strongly suppressed by the channel structure, thus stable collision and efficient utilization of the droplet energy were possible. These results are useful for estimating the localized energy, for improving the system in order to realize extreme performance, and for extending the applications of microfluidic devices.     

Modeling of droplet traffic in interconnected microfluidic ladder devices

The problem of controlling the droplet motion in multiphase flows on the microscale has gained increasing attention because the droplet-based microfluidic devices provide great potentials for chemical and biological applications. It is critical to understand the relevant physics on droplet hydrodynamics and thus control the generation, motion, splitting, and coalescence of droplets in complex microfluidic networks. Numerical simulations using the volume of fluid algorithm are conducted to investigate the time-dependent dynamics of droplets in gas-liquid multiphase devices. An analytical model based on the electronic-hydraulic analogy is developed to describe the hydrodynamic behavior of the droplets in interconnected microfluidic ladder devices. It is found that the pressure drop caused by the droplets plays a critical role in the droplet synchronization. A fitted formula for pressure drops in the presence of surfactant is achieved by using numerical simulations. Both the numerical and the theoretical results agree well with the corresponding experimental results.