Microliter Droplets Research Articles

Motion of viscous drops on superhydrophobic surfaces due to magnetic gradients

Microliter droplets that contain paramagnetic particles can be moved on superhydrophobic surfaces using magnetic fields. Paramagnetic microparticles form chains at concentrations ranging from 0.1 to 10 wt% and are used to move, coalesce, and split drops of water as well as drops of biological fluids. Video analysis of dextran solution drops to investigate the effect of viscosity on drop movement on LPDE surfaces suggests that paramagnetic particle chain orientation compensates for viscosity increases from 1.2 mPa s (water) to 125 mPa s (20%, w/v – Dextran 428) in order to maintain drop movement. Interestingly, such changes in chain orientation are not present for drops moving on silicon nanowire (Si NW) superhydrophobic surfaces even at higher viscosities 470 mPa s (30% w/v – Dextran 428). On Si NW surfaces, drops with high viscosity can be moved even with particle concentrations as low as 0.5%. Higher particle concentrations (2%) are needed to displace drops on LPDE surfaces. This new approach to so-called “discrete” microliter-scale fluidics has the advantages of faster and more flexible control over drop movement, manipulation, and detection of solution components.

Development and evaluation of realistic microbioassays in freely suspended droplets on a chip

A novel technique for biomolecular detection in microliter droplets floating on the surface of high density oil is presented. Each droplet was captured and manipulated dielectrophoretically and was used as a site for a microscopic bioassay based on agglutination of antibody-conjugated particles. The results were read out by the pattern of unagglomerated gold nanoparticles collected on the droplet surface. Two formats of bioassays, namely gold only agglutination and gold and latex agglutination, were investigated experimentally by varying analyte concentration, particle size and concentration, number of antigen binding sites per particle, time for incubation, and rate of particle collection on the droplet surface. The microbioassays performance was also evaluated with ricin antibodies and compared to the ricin assays in field use. It is estimated that the droplet based assays require 100x smaller sample volume and are ten times more sensitive, though they require longer times to complete. The experiments were interpreted by modeling the kinetics of particle agglutination and mass transfer processes inside the droplets. The incubation time and antigen concentration values calculated by the model correlate well with the experimental results. The results could allow for development of efficient immunoassays on a chip requiring even smaller sample volumes.

Dynamic spreading of a liquid finger driven by electrowetting: Theory and experimental validation

Electrowetting on dielectrics is now a major field of research for lab-on-chip applications. Droplets and liquid fingers are used to displace, mix, and analyze biological compounds. The creation and propagation of a liquid finger are investigated here for a channel with an elliptical cross section. Forces acting on the liquid are described and calculated, and the problem is solved using a steady-state equation. The theoretical length of the finger as a function of time is expressed and the threshold voltage for finger formation is deduced. Test devices fabricated in highly doped silicon are used to validate our theory. Liquid fingers propagating into the fabricated microchannels are extracted from microliter droplets using 1kHz ac voltages ranging from 80to180Vrms. The resulting experimental threshold voltages and the dynamic lengths are compared to the theoretical predictions. Additional discussions and calculations are proposed to improve our model so that the experimental conditions are more accurately described.

Two-dimensional magnetic manipulation of microdroplets on a chip as a platform for bioanalytical applications

Manipulating biological samples in microliter droplets has vast potential in molecular diagnostics. We present a system for the two-dimensional (2D) magnetic manipulation of aqueous droplets suspended in silicone oil as a platform for on-chip bioanalysis. Superparamagnetic microparticles inside the droplets provide the means of the magnetic actuation. The droplets can be displaced, merged, mixed and separated on the chip without the use of external moving magnets or parts. In our system three-dimensional surface structures are replaced by an optimised hydrophilic/hydrophobic patterning of the chip surface to facilitating droplet splitting. We compare the functionality of the droplet manipulation system for magnetic particles of different sizes and demonstrate a dependency between particle size and performance. Finally we present the potential of the proposed system in droplet-based biomedical analysis by performing the dilution and detection of the secondary antibody of an enzyme-linked immuno-sorbent assay (ELISA) on-chip.

Microfluidic detection and analysis by integration of thermocapillary actuation with a thin-film optical waveguide

We demonstrate a nonintrusive optical method for microfluidic detection and analysis based on evanescent wave sensing. The device consists of a planar thin-film waveguide integrated with a microfluidic chip for directed surface flow. Microliter droplets are electronically transported and positioned over the waveguide surface by thermocapillary actuation. The attenuated intensity of propagating modes is used to detect droplet location, to monitor dye concentration in aqueous solutions, and to measure reaction rates with increasing surface temperature for a chromogenic biochemical assay. This study illustrates a few of the capabilities possible by direct integration of optical sensing with surface-directed fluidic devices.

Nanodroplets: a new method for dried spot preparation and analysis

AbstractThe dried spot technique is an effective method for elemental analysis. However, the current technology, using microliter volume spots, suffers from lack of repeatability and long sample preparation times. In this study, a new nanoliter dried spot sample preparation method was developed and tested. Drying time, drying pattern and detection limits were compared between spots formed from microliter‐ and nanoliter‐sized droplets. This new nanoliter deposition method reproducibly produced uniform spots. The sample throughput was increased significantly with nanoliter droplet drying times ranging from only seconds to minutes. The minimum detectable concentrations for the nanoliter spots were in the ppb range with the ability to detect picogram‐level masses. This new sample preparation method has the potential for use in situations where sample availability is limited. Furthermore, nanoliter dried spots are smaller in diameter than the exciting x‐ray beam in micro‐XRF and conventional XRF instrumentation. This allows faster drying nanoliter droplet microarrays to be used in situations where microliter droplets are primarily used. Published in 2004 John Wiley & Sons, Ltd.

Fundamental studies on micro-droplet movement by Marangoni and capillary effects

This work is a fundamental study on the movement of various sized micro-liter droplets on a surface subjected to temperature gradients. The histories of droplet movement are recorded by high-speed CCD camera and are simulated by numerical methods based on first principle equations. The agreement between computations and experimental observations are obtained. The study indicates that temperature gradients, the change of dynamic receding/advancing contact angles across the droplets, and the flowfields inside the droplet are the key parameters determining the moving behavior of the micro-droplet driven by Marangoni and capillary effects.

Extended embryo culture: use of two media simultaneously is an optimal approach to maximize blastocyst formation and utilization

Objective: The evaluation of a commercially prepared embryo culture medium (CCM-20, Vitrolife) and an in house prepared culture medium (SGB) for the development of human embryos to the blastocyst stage, up to day 7 in vitro. Design: This was a retrospective study at a private IVF clinic. Patients for this study were treated from August, 2000 to December, 2001. Patients were chosen for extended culture to day 5, 6 or 7 based on number of embryos, quality of embryos on day 3 of culture, age of patient, diagnosis and embryo development and quality in previous cycles. Materials/Methods: Once patient selection was made, embryos were equally divided and cultured in each medium preparation. In the case of an odd number of embryos, CCM-20 was the default medium. Culture dishes (NUNC, 35mm) were prepared by scratching the patients name and media type on the bottom of the dish. Five mls of oil (Sage, BioPharma) were pipetted into each dish. Ten microliter droplets were pipetted into the center of the dish. Droplets for rinsing the embryos were placed around the dish perimeter. Dishes were pre-equilibrated in the incubator for four hours before the embryos were moved into the extended culture media. This change over occurred on day 3 and day 5 of embryo culture. Each embryo was rinsed several times before being placed into one of the center droplets. All embryos were numbered and a daily record of the development of each embryo was kept as part of the patient cycle data. Embryos were chosen for ET and cryopreservation on day 5, 6 or 7 based on inner cell mass, trophectodermal development and degree of expansion. All embryos for ET on day 6 or 7 underwent assisted hatching. Results: Table I data were broken into the three separate series of the study period. This was done to monitor any change in the performance of the SGB medium. For all data points, where the series were separated and where they were combined, it appears that the CCM-20 media had a slight advantage over the SGB media. This might be explained in part, by the fact that the total number of embryos differed in the groups. (2935 CCM-20, 2822 SGB) Pregnancy rate and implantation rate for CCM-20 and SGB were 54.2, 41.3 and 58.8, 52.9, respectively. It appeared that the SGB performed marginally more successfully than the CCM-20 for PR and IR.Table ISeries% Embryos of Blast% Embryos to ET% Embryos to Cryo% Total UtilizationCCM-20SGBCCM-20SGBCCM-20SGBCCM-20SGB2453.343.417.212.520.017.137.229.62555.442.216.912.320.715.937.628.22654.448.614.814.121.117.735.931.824, 25, 2654.445.016.113.020.716.936.830.0 Open table in a new tab Conclusions: Both media performed well for the development of blastocysts for both ET and cryopreservation. Pregnancy rates and implantation rates were also comparable. A significant advantage of an in house prepared medium is less time is lost in its use due to a minimizing of testing and shipping time when compared to a commercial medium. Additionally, routine splitting of embryos on day 3 into two extended culture media allows the potential to default to one or other of the media if a pronounced drop in embryo quality is perceived due to suboptimal quality of either medium. Supported by: NA.

Atomic force microscopy imaging of T4 bacteriophages on silicon substrates

A new atomic force microscope incorporating microfabricated cantilevers and employing laser beam deflection for force detection has been constructed and is being applied to studies of biological material. In this study, T4 bacteriophage virus particles were deposited from solution onto electronic-grade flat silicon wafers and imaged in air with the microscope. Microliter droplets of the solution were deposited and either allowed to dry or removed with blotting paper. The images show both isolated viruses and aggregates of various sizes. The external structure as well as strands believed to be DNA streaming out of the virus could be observed. The construction of the microscope and its performance are also described.

Seed set after pollination with in-vitro-matured, isolated pollen of Triticum aestivum

Immature pollen of two varieties of Triticum aestivum, at the stage right after the first pollen mitosis, was isolated from individual anthers and cultured in microcultures of microliter droplets. In a specifically designed medium, some of the pollen grains developed to maturity. These were applied to excised stigmas on agar, where they produced pollen tubes. Application to flowers in vivo led to seed set. Pollen was matured in vitro from a variety that produced a different protein banding pattern on SDS-PAGE as compared to the variety that was pollinated. The protein banding in the produced seeds showed the hybrid pattern, demonstrating that the seeds were not produced by self-pollination in this in-breeding species but by pollination with the in-vitro-matured pollen.

Histochemical Technique: Densitometry of Nanogram Quantities of Proteins Separated in One-Dimensional Microslab Gels

We describe methods for densitometry of electrophoretically separated proteins in 25-millimeter microslab gels. The methods are sufficiently sensitive to use with several individually excised plant cells, for which we describe an extraction procedure. In brief, submicrogram samples are excised from freeze-dried plant tissue. Extraction takes place under oil in microliter droplets of detergent. Proteins are separated by one-dimensional microelectrophoresis (HM Poehling, V Neuhoff 1980 Electrophoresis 1: 90-102) and then stained by a sensitive Coomassie procedure (V Neuhoff, R Stamm, E Hansjorg 1985 Electrophoresis 6: 427-448). The resulting profile is scanned by a computerized densitometer based on the Leitz Diavert MPV Microphotometer. Evaluations and typical data demonstrate the high performance of this system.

Successful in vitro fertilization and embryo transfer in cynomolgus monkeys

We have started an in vitro fertilization program in cynomolgus monkeys in an effort to develop an appropriate animal model to improve our knowledge of early embryonic development. In 16 of 25 animals treated with menopausal gonadotropins, preovulatory follicles developed. Follicular aspiration was performed at laparotomy after human chorionic gonadotropin injection. A total of 299 follicles were aspirated, and 251 oocytes were recovered. Oocytes were cultured in 1 ml of growth medium or 100 microliter droplets of medium under mineral oil. Semen samples were obtained by electroejaculation, and the oocytes were inseminated 4 to 24 hours after aspiration. Culture under mineral oil significantly increased the fertilization and cleavage rates. Of 68 embryos produced, 24 have been used in 10 embryo transfers, resulting in two pregnancies.

A quantitative method of sample loading for field desorption mass spectrometry.

A quantitative method for loading nanogram samples onto field desorption emitter wires is described. Sample consumption is reduced and problems with emitter wettability are overcome by freezing microliter droplets onto the emitters. Using this technique, good spectral data are obtained from 10 to 50 ng of material. Field desorption sensitivity measurements are reported for neomycin B, adenosine and cholesterol. Evaluation of the nonquantitative dipping technique previously employed reveals poor and nonreproducible sample loading efficiency.