Comprehensive two-dimensional manipulations of picoliter microfluidic droplets sampled from nanoliter samples

A facile method is presented for achieving comprehensive two-dimensional droplet manipulations in closed microstructures consisting of microwell arrays and a straight microchannel. In this method, picoliter/nanoliter droplets with controllable sizes and numbers are sampled from nanoliter samples/reagents with almost 100% efficiency. Droplet motions are precisely controlled in the ±X-direction and ±Y-direction by managing hydrostatic pressure and magnetic repulsion, respectively. As a demonstration, a fluorescein-labeled droplet and a deionized droplet are successively generated and trapped in adjacent microwells. Then their positions are quickly exchanged without cross-contamination and fusion is implemented on-demand. After operations, hydrophobic ferrofluid can be completely replaced by mineral oil and droplets still remain in microwells safely. A typical fluorescence intensity-based assay is demonstrated: droplet arrays containing copper ion are diluted disproportionately first and then detected by addition of droplet arrays containing Calcein. With the ability of comprehensive two-dimensional droplet manipulations, this method could be used in various miniaturized biochemical analyses including requirements of multistep procedures and in situ monitoring.     

On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets

The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 106 smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet motion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of approximately 18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.     

Analysis in ultrasmall volumes: microdispensing of picoliter droplets and analysis without protection from evaporation

A new approach is reported for analysis of ultrasmall volumes. It takes advantage of the versatile positioning of a dispenser to shoot approximately 150-pL droplets of liquid onto a specific location of a substrate where analysis is performed rapidly, in a fraction of the time that it takes for the droplet to evaporate. In this report, the site where the liquid is dispensed carries out fast-scan cyclic voltammetry (FSCV), although the detection method does not need to be restricted to electrochemistry. The FSCV is performed at a microcavity having individually addressable gold electrodes, where one serves as working electrode and another as counter/pseudoreference electrode. Five or six droplets of 10 mM [Ru(NH(3))(6)]Cl(3) in 0.1 M KCl were dispensed and allowed to dry, followed by redissolution of the redox species and electrolyte with one or five droplets of water and immediate FSCV, demonstrating the ability to easily concentrate a sample and the reproducibility of redissolution, respectively. Because this approach does not integrate detection with microfluidics on the same chip, it simplifies fabrication of devices for analysis of ultrasmall volumes. It may be useful for single-step and multistep sample preparation, analyses, and bioassays in microarray formats if dispensing and changing of solutions are automated. However, care must be taken to avoid factors that affect the aim of the dispenser, such as drafts and clogging of the nozzle.     

A study on liquid-liquid distribution based on single picoliter droplets and in situ electrochemical measurements

A microelectrode technique combined with the microcapillary injection/manipulation of a single organic droplet in water was developed. The technique was applied to the study of the distribution of a ferrocene derivative across a single-picoliter-nitrobenzene-droplet/water interface and to the simultaneous in situ electrochemical determination of the distributed solute in the picomole-femtomole range. The liquid-liquid distribution processes were discussed in terms of droplet size and solute concentration (in water) dependencies of the interfacial mass transfer rate.     

Vortex-trap-induced fusion of femtoliter-volume aqueous droplets

This paper describes the use of an optical vortex trap for the transport and fusion of single femtoliter-volume aqueous droplets. Individual droplets were generated by emulsifying water in acetophenone with SPAN 80 surfactant. We demonstrate the ability of optical vortex traps to position trapped droplets precisely while excluding surrounding aqueous droplets from entering the trap, thereby preventing unwanted cross contamination by other nearby droplets. Additionally, the limitation of optical vortex traps for inducing droplet fusion is illustrated, and a remedy is provided through modulation of the spatial intensity profile of the optical vortex beam. Spatial modulation was achieved by translating the computer-generated hologram (CGH) with respect to the input Gaussian beam, thereby shifting the location of the embedded phase singularity (dark core) within the optical vortex beam. We present both simulated and experimentally measured intensity profiles of the vortex beam caused by translation of the CGH. We further describe the use of this technique to achieve controlled and facile fusion of two aqueous droplets.     

Electrohydrodynamic generation and delivery of monodisperse picoliter droplets using a poly(dimethylsiloxane) microchip

We developed a drop-on-demand microdroplet generator for the discrete dispensing of biosamples into a bioanalytical unit. This disposable PDMS microfluidic device can generate monodisperse droplets of picoliter volume directly out of a plane sidewall of the microfluidic chip by an electrohydrodynamic mechanism. The droplet generation was accomplished without using either an inserted capillary or a monolithically built-in tip. The minimum droplet volume was approximately 4 pL, and the droplet generation was repeatable and stable for at least 30 min, with a typical variation of less than 2.0% of drop size. The Taylor cone, which is usually observed in electrospray, was suppressed by controlling the surface wetting property of the PDMS device as well as the surface tension of the sample liquids. A modification of the channel geometry right before the opening of the microchannel also enhanced the continuous droplet generation without applying any external pumping. A simple numerical simulation of the droplet generation verified the importance of controlling the surface wetting conditions for the droplet formation. Our microdroplet generator can be effectively applied to a direct interface of a microfluidic chip to a biosensing unit, such as AMS, MALDI-MS or protein microarray-type biochips.     

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.     

Thermoelectric manipulation of aqueous droplets in microfluidic devices

This article describes a method for manipulating the temperature inside aqueous droplets, utilizing a thermoelectric cooler to control the temperature of select portions of a microfluidic chip. To illustrate the adaptability of this approach, we have generated an "ice valve" to stop fluid flow in a microchannel. By taking advantage of the vastly different freezing points for aqueous solutions and immiscible oils, we froze a stream of aqueous droplets that were formed on-chip. By integrating this technique with cell encapsulation into aqueous droplets, we were also able to freeze single cells encased in flowing droplets. Using a live-dead stain, we confirmed the viability of cells was not adversely affected by the process of freezing in aqueous droplets provided cryoprotectants were utilized. When combined with current droplet methodologies, this technology has the potential to both selectively heat and cool portions of a chip for a variety of droplet-related applications, such as freezing, temperature cycling, sample archiving, and controlling reaction kinetics.     

Holographic interferometry of oil films and droplets in water with a single-beam mirror-type scheme

Application of single-beam reflective laser optical interferometry for oil films and droplets in water detection and characterization is discussed. Oil films can be detected by the appearance of characteristic interference patterns. Analytical expressions describing intensity distribution in these interference patterns allow determination of oil film thickness, size of oil droplets, and distance to the oil film from the observation plane. Results from these analyses indicate that oil spill aging and breakup can be monitored in real time by analyzing time-dependent holographic fringe patterns. Interferometric methods of oil spill detection and characterization can be automated using digital holography with three-dimensional reconstruction of the time-changing oil spill topography. In this effort, the interferometric methods were applied to samples from Chevron oil and British Petroleum MC252 oil obtained during the Deep Water Horizon oil spill in the Gulf of Mexico.     

Low-temperature pyrolysis and synthesis of nanoparticles during rapid evaporation of micron droplets of aqueous solutions

Experimental results on synthesizing nickel oxide nanoparticles during the evaporation of micron droplets of a concentrated aqueous solution of nickel chloride in a low-pressure aerosol reactor are described. It is shown that the evaporation of droplets in a stream of argon is accompanied by the formation of agglomerates of nanoparticles with a “hollow” morphology. The mechanisms of low-temperature pyrolysis as the interaction of chemical and physical processes are discussed.     

Chemical stability of teniposide in aqueous and parenteral lipid emulsions

The purpose of this study was to investigate the degradation kinetics of teniposide in lipid emulsion and aqueous solution. The chemical stability of teniposide in lipid emulsion and aqueous solution at various pH values and temperatures was monitored by high-performance liquid chromatography. In addition, the viscosities of emulsion at different temperatures were investigated. The degradation of teniposide both in emulsion and in aqueous solution was shown to follow pseudo-first-order degradation kinetics. The t (1/2) values of teniposide lipid emulsion (TLE) and the aqueous solution were 80 and 2.6 days at 10 degrees C, respectively. Under the most stable pH range of 6.0-6.5, stability of teniposide in the emulsion increased more than 30-fold compared with that in aqueous solution. Furthermore, there was a difference between the shelf life of TLE actually measured (29 days) at 10 degrees C and the one deduced (15 days) from the degradation data of high temperatures by Arrhenius equation. It could be hypothesized that the difference was due to a slower diffusion of teniposide from oil phase to aqueous phase at the lower temperatures, which would be a speed-limited process in the degradation of TLE. The results of viscosity test confirmed the presumption.     

Chemical preparation of PLZT powder from aqueous solution

A new method for preparing homogeneous lanthanum-modified lead zirconate-titanate (PLZT) powder from aqueous salt solution containing hydrogen peroxide by precipitation is described. Inorganic salts such as zirconium oxychloride and titanium tetrachloride instead of organo-metallic salts can be available as raw materials which attain well reactioned and fine-grained PLZT powder processed by decomposing the dried product at 650°C. PLZT ceramics formed by hot-pressing the powder show considerable improvements in the qualities such as optical transparency and homogeneity.     

In vitro studies of magnetically enhanced transfection in COS-7 cells

In the magnetically enhanced gene delivery technique, DNA complexed with polymer coated aggregated magnetic nanoparticles (AMNPs) is used for effecting transfection. The aim of this study is to examine the relationship between transfection efficiency and the physical characteristics of the polymer coated AMNPs. In vitro studies of transfection efficiency in COS-7 cells were carried out using pEGFP-N1 and pMIR-REPORT complexed polyethylenimine (PEI) coated iron oxide magnetic nanoparticles. PEI coated AMNPs (PEI-AMNPs) with average individual particle diameters in the range of 8 nm to 30 nm were studied and characterized by transmission electron microscopy, vibrating sample magnetometry, X-ray diffractometry, thermal gravimetric analysis and photon correlation spectroscopy methods. PEI-A8MNP and PEI-A30MNP yielded higher transfection efficiency compared to commercial polyMAG particles as well as PEI of equivalent molar ratio of nitrogen/phosphorous (N/P ratio). The transfection efficiency was related to the physical characteristics of the PEI-AMNPs and its complexes: transfection efficiency was strongly positively correlated with saturation magnetization (Ms) and susceptibility (χ), strongly negatively correlated with N/P ratio, moderately positively correlated to zeta potential and moderately negatively correlated to hydrodynamic diameter of the complex. PEI-A8MNP and PEI-A30MNP possessing higher Ms, χ, lower N/P ratio and smaller complex size exhibited higher transfection efficiency compared to PEI-A16MNP which have weaker magnetic properties, higher N/P ratio and larger complex size. We have demonstrated that optimization of the physical properties of PEI-AMNPs is needed to maximize transfection efficiency.     

Gene transfection of mammalian cells using membrane sandwich electroporation

To avoid safety issues such as immune response and cytotoxicity associated with viruses and liposomes, physical methods have been widely used for either in vivo or ex vivo gene delivery. They are, however, very invasive and often provide limited efficiency. Using pEGFP and pSEAP plasmids and NIH 3T3 fibroblasts as models, we demonstrate a new electroporation-based gene delivery method, called membrane sandwich electroporation (MSE). The MSE method is able to provide better gene confinement near the cell surface to facilitate gene transport into the cells and thus shows significant improvement over transgene expression of mammalian cells compared to current electroporation techniques.     

Microfluidic and optical systems for the on-demand generation and manipulation of single femtoliter-volume aqueous droplets

This paper describes a fluidic and optical platform for the generation and manipulation of single femtoliter-volume aqueous droplets. Individual droplets were generated on-demand using a microfluidic chamber that confers environmental flow stability. Optical vortex traps were implemented to manipulate and transport the generated droplets, which have a lower refractive index than the immiscible medium in which the droplets are immersed and thus cannot be trapped using conventional optical tweezers. We also demonstrated the ability to shrink and increase the refractive index of the generated droplet, thereby permitting its facile fusion with another droplet using an optical tweezer. To illustrate the versatility of this platform, we have performed both fast (<1 s) and slow (>1 h) chemical reactions in these femtoliter-volume aqueous droplets.     

Development of binding assays in microfabricated picoliter vials: an assay for biotin

A homogeneous binding assay for the detection of biotin in picoliter vials was developed using the photoprotein aequorin as the label. The binding assay was based on the competition of free biotin with biotinylated aequorin (AEQ-biotin) for avidin. A sequential protocol was used, and modification of the assay to reduce the number of steps was examined. Results showed that detection limits on the order of 10(-14) mol of biotin were possible. Reducing the number of steps provided similar detection limits but only if the amount of avidin used was decreased. These binding assays based on picoliter volumes have potential applications in a variety of fields, including microanalysis and single-cell analysis, where the amount of sample is limited. In addition, these assays are suitable for the high-throughput screening of biopharmaceuticals.     

Elaboration of perfect core-shell submicronic magnetic latexes from oil in water ferrofluid droplets for bionanotechnology applications

The aim of this paper is to present a new route leading to highly magnetic submicronic latex particles. This was performed using oil in water magnetic droplets as a seed for emulsion polymerization process with hydrophobic monomer. After a short study related to adsorption and desorption of amphiphilic polymer onto ferrofluid droplets, our work consisted in the determination of parameters which could have a rule in the core-shell morphology setting: the cross-linker ratio. Finally, these latex particles are characterized in terms of colloidal characteristics, chemical composition and intrinsic properties. The resulted particles present; submicronic diameter, high magnetic content (around 60 wt.%) and well-defined morphology. When a water-soluble initiator (potassium persulfate) was used together with a crosslinking agent (divinylbenzene) in the polymerization recipe, a more homogeneous core-shell morphology was reached.     

Chemical deposition of PbS on NASICON from aqueous solutions

Phase-pure polycrystalline lead sulfide films have been grown on sodium-ion-conducting solid-electrolyte (NASICON, Na₃Zr₂Si₂PO₁₂) ceramic substrates by chemical deposition from solution. We have studied the microstructure of the films as a function of deposition time (5–180 min) at a fixed deposition temperature of 286 K and the effect of deposition temperature on their growth rate and microstructure. Empirical relations are obtained for the average crystallite size as a function of deposition time and temperature which can be used to select conditions for the growth of PbS films with tailored microstructure.