Microfluidic Routes to the Controlled Production of Nanoparticles

Chemical Communications (Cambridge, England). May, 2002  |  Pubmed ID: 12122702

A microfluidic procedure for the controlled production of cadmium sulfide nanoparticles is described.

Solution-phase Electroluminescence

Chemical Communications (Cambridge, England). Sep, 2002  |  Pubmed ID: 12271691

We report emissive devices exhibiting electroluminescence in the solution phase. The principle operating mechanism for these devices--direct electronic carrier injection from the electrodes into the carrier bands of the dissolved polymer--resembles that of a conventional solid-state organic light-emitting diode and is distinct from the solvent-mediated electrochemical devices recently reported by Chang et al.

Single Particle Confocal Fluorescence Spectroscopy in Microchannels: Dependence of Burst Width and Burst Area Distributions on Particle Size and Flow Rate

Analytical Sciences : the International Journal of the Japan Society for Analytical Chemistry. Jul, 2003  |  Pubmed ID: 12880094

This article presents a non-invasive, optical technique for measuring particulate flow within microfluidic channels. Confocal fluorescence detection is used to probe single fluorescently labeled microspheres (200-930 nm diameter) passing through a focused laser beam at a variety of flow rates (100 - 1000 nL/min). Simple statistical methods are subsequently used to investigate the resulting fluorescence bursts and generate single-particle burst width and burst area distributions. Analysis of such distributions demonstrates that the average burst width and burst area decrease as particle size increases. In addition, both burst width and burst area (for a given particle size) are observed to decrease as volumetric flow rate is increased. The dependence of such distributions on particle size is proposed as a potential route to sizing single particles and molecules in microfluidic systems.

Thin-film Polymer Light Emitting Diodes As Integrated Excitation Sources for Microscale Capillary Electrophoresis

Lab on a Chip. Apr, 2004  |  Pubmed ID: 15052354

We report the use of a thin-film polymer light emitting diode as an integrated excitation source for microfabricated capillary electrophoresis. The polyfluorene-based diode has a peak emission wavelength of 488 nm, an active area of 40 microm x 1000 microm and a thickness of similar 2 mm. The simple layer-by-layer deposition procedures used to fabricate the polymer component allow facile integration with planar chip-based systems. To demonstrate the efficacy of the approach, the polyfluorene diode is used as an excitation source for the detection of fluorescent dyes separated on-chip by electrophoresis. Using a conventional confocal detection system the integrated pLED is successfully used to detect fluorescein and 5-carboxyfluorescein at concentrations as low as 10(-6) M with a mass detection limit of 50 femtomoles. The drive voltages required to generate sufficient emission from the polymer diode device are as low as 3.7 V.

Integrated On-chip Derivatization and Electrophoresis for the Rapid Analysis of Biogenic Amines

Electrophoresis. Jul, 2004  |  Pubmed ID: 15274019

We demonstrate the monolithic integration of a chemical reactor with a capillary electrophoresis device for the rapid and sensitive analysis of biogenic amines. Fluorescein isothiocyanate (FITC) is widely employed for the analysis of amino-group containing analytes. However, the slow reaction kinetics hinders the use of this dye for on-chip labeling applications. Other alternatives are available such as o-phthaldehyde (OPA), however, the inferior photophysical properties and the UV lambdamax present difficulties when using common excitation sources leading to a disparity in sensitivity. Consequently, we present for the first time the use of dichlorotriazine fluorescein (DTAF) as a superior in situ derivatizing agent for biogenic amines in microfluidic devices. The developed microdevice employs both hydrodynamic and electroosmotic flow, facilitating the creation of a polymeric microchip to perform both precolumn derivatization and electrophoretic analysis. The favorable photophysical properties of the DTAF and its fast reaction kinetics provide detection limits down to 1 nM and total analysis times (including on-chip mixing and reaction) of <60 s. The detection limits are two orders of magnitude lower than current limits obtained with both FITC and OPA. The optimized microdevice is also employed to probe biogenic amines in real samples.

Single Molecule Studies of Quantum Dot Conjugates in a Submicrometer Fluidic Channel

Lab on a Chip. Mar, 2005  |  Pubmed ID: 15726210

A microfluidic and optical system was created for the detection and analysis of single molecules in solution. Fluidic channels with submicrometer dimensions were used to isolate, detect and identify individual quantum dots conjugated with organic fluorophores. The channels were fabricated in fused silica with a 500 nm square cross section. The resulting focal volume of approximately 500 aL reduced fluorescent background and increased the signal to noise ratio of single molecule detection. The channels also enabled the rapid detection of 99% of quantum dots and organic fluorophores traversing the focal volume. Conjugates were driven through the channels electrokinetically at 2.3 kV cm(-1), excited with a single 476 nm wavelength laser and detected with a confocal microscope. Fluorescence emission was collected simultaneously from green (500-590 nm) and red (610-680 nm) regions of the spectrum. Signal rejection was minimized by the narrow and symmetric emission spectra of the quantum dots. To demonstrate efficient multicolor detection and characterization of single molecule binding, Qdot 655 Streptavidin Conjugates were bound to Alexa Fluor 488 molecules and individually detected. Photon counting histogram analysis was used to quantify coincident detection and degree of binding. Fluorescence correlation spectroscopy was used to measure the mobility of bound and unbound species. The union of fluidic channels with submicrometer dimensions and quantum dots as fluorescent labels resulted in efficient and rapid multiplexed single molecule detection and analysis.

High Spatial Resolution Observation of Single-molecule Dynamics in Living Cell Membranes

Biophysical Journal. Jun, 2005  |  Pubmed ID: 15821167

Self-organized lipid bilayers together with proteins are the essential building blocks of biological membranes. Membranes are associated with all living systems as they make up cell boundaries and provide basic barriers to cellular organelles. It is of interest to study the dynamics of individual molecules in cell membranes as the mechanism of how biological membranes function at the single molecule remains to be elucidated. In this letter we describe a study in which we incubate rat basophilic leukemia cells with a fluorescently labeled cell membrane component on a surface containing zero-mode waveguides (ZMWs). We used the ZMW to confine fluorescent excitation to an approximately 100-nm region of the membrane to monitor lipid diffusion along the cellular membrane. We showed that confinement with a ZMW largely reduced fluorescent contributions from the cytosolic pool that is present when using a more standard technique such as laser-induced confocal microscopy. We show that optical confinement with ZMWs is a facile way to probe dynamic processes on the membrane surface.

Micrometer-sized Supported Lipid Bilayer Arrays for Bacterial Toxin Binding Studies Through Total Internal Reflection Fluorescence Microscopy

Biophysical Journal. Jul, 2005  |  Pubmed ID: 15833994

In this article, we present the use of micron-sized lipid domains, patterned onto planar substrates and within microfluidic channels, to assay the binding of bacterial toxins via total internal reflection fluorescence microscopy. The lipid domains were patterned using a polymer lift-off technique and consisted of ganglioside-populated distearoylphosphatidylcholine:cholesterol supported lipid bilayers (SLBs). Lipid patterns were formed on the substrates by vesicle fusion followed by polymer lift-off, which revealed micron-sized SLBs containing either ganglioside G(T1b) or G(M1). The ganglioside-populated SLB arrays were then exposed to either cholera toxin B subunit or tetanus toxin C fragment. Binding was assayed on planar substrates by total internal reflection fluorescence microscopy down to 100 pM concentration for cholera toxin subunit B and 10 nM for tetanus toxin fragment C. Apparent binding constants extracted from three different models applied to the binding curves suggest that binding of a protein to a lipid-based receptor is influenced by the microenvironment of the SLB and the substrate on which the bilayer is formed. Patterning of SLBs inside microfluidic channels also allowed the preparation of lipid domains with different compositions on a single device. Arrays within microfluidic channels were used to achieve segregation and selective binding from a binary mixture of the toxin fragments in one device. The binding and segregation within the microfluidic channels was assayed with epifluorescence as proof of concept. We propose that the method used for patterning the lipid microarrays on planar substrates and within microfluidic channels can be easily adapted to proteins or nucleic acids and can be used for biosensor applications and cell stimulation assays under different flow conditions.

Detection and Identification of Nucleic Acid Engineered Fluorescent Labels in Submicrometre Fluidic Channels

Nanotechnology. Jul, 2005  |  Pubmed ID: 21727447

Nucleic acid engineers have created nanoscale fluorescent labels that are uniquely identifiable by the number of conjugated fluorophores, and with binding characteristics that permit recognition of individual specific biomolecules. The viability of this technology for use in multi-analyte homogeneous assays depends on the ability to optically detect individual labels, and distinguish the fluorescence emission of each label. We describe the use of fluidic channels with submicrometre dimensions to rapidly detect individual labels in solution. Labels with small differences in fluorophore composition were differentiated with varying degrees of accuracy. Labels were synthesized at the molecular level from dendrimer-like DNA, with the identity encoded into the number of Alexa Fluor 488 and BODIPY 630/650 fluorophores conjugated with the structure. To explore the decoding resolution limit, labels with a single fluorophore of each colour were detected, and were found to be distinguishable as a group, but not individually, from labels with one additional red fluorophore. Labels with one green and three red fluorophores were individually distinguishable with greater than 80% accuracy from labels with one red and three green fluorophores. Photon counting histograms were analysed to differentiate the various labels, and fluorescence correlation spectroscopy was used to measure their mobilities. Fluidic channels were fabricated in fused silica with a 500 nm square cross section, resulting in a focal volume of approximately 500 al. Because the entire channel width was illuminated, every fluorescent molecule in solution passing through the channel was uniformly excited and analyzed. Flow control enabled a balance of rapid data acquisition and efficient fluorescence collection with these nanoscale systems.

Electrospun Polymer Nanofibers As Subwavelength Optical Waveguides Incorporating Quantum Dots

Small (Weinheim an Der Bergstrasse, Germany). Apr, 2006  |  Pubmed ID: 17193073

Discrimination Between Single Escherichia Coli Cells Using Time-resolved Confocal Spectroscopy

The Journal of Physical Chemistry. B. Feb, 2007  |  Pubmed ID: 17266266

We describe a technique for rapidly discriminating between single-cell populations within a flowing microfluidic stream. Single-cell time-correlated single-photon counting (scTCSPC) as well as photon burst spectroscopy are used to characterize individual Escherichia coli cells expressed with either green, cyano, or yellow fluorescent protein. The approach utilizes standard confocal fluorescence microscopy incorporating femtoliter detection volumes. The measured burst width characteristics are predominately governed by the fluorescence quantum yield and absorption cross section of the proteins used. It is these characteristics which were used to distinguish between cells with high precision. By utilizing scTCSPC individual fluorescence lifetimes originating from single cells could also be determined. Average fluorescence lifetimes are determined using standard deconvolution procedures. The simplicity of the approach for obtaining well-defined burst width distributions is expected to be extremely valuable for single-cell sorting experiments.

Accurate Single Molecule FRET Efficiency Determination for Surface Immobilized DNA Using Maximum Likelihood Calculated Lifetimes

The Journal of Physical Chemistry. B. Mar, 2007  |  Pubmed ID: 17388421

Single molecule fluorescent lifetime trajectories of surface immobilized double-stranded DNA coupled with a tetramethylrhodmaine and Cy5 FRET pair were directly measured using time-tagged single-photon counting and scanning confocal microscopy. A modified maximum likelihood estimator (MLE) was developed to compensate for localized background fluorescence and instrument response. With this algorithm, we were able to robustly extract fluorescent lifetimes from their respective decays with as few as 20 photons. Fluorescent lifetimes extracted using an MLE were found to be highly dependent on background fluorescence. We show that appropriate factors are required to extract true lifetime trajectories from single fluorophores.

High-throughput DNA Droplet Assays Using Picoliter Reactor Volumes

Analytical Chemistry. Sep, 2007  |  Pubmed ID: 17676925

The online characterization and detection of individual droplets at high speeds, low analyte concentrations, and perfect detection efficiencies is a significant challenge underpinning the application of microfluidic droplet reactors to high-throughput chemistry and biology. Herein, we describe the integration of confocal fluorescence spectroscopy as a high-efficiency detection method for droplet-based microfluidics. Issues such as surface contamination, rapid mixing, and rapid detection, as well as low detections limits have been addressed with the approach described when compared to conventional laminar flow-based fluidics. Using such a system, droplet size, droplet shape, droplet formation frequencies, and droplet compositions can be measured accurately and precisely at kilohertz frequencies. Taking advantage of this approach, we demonstrate a high-throughput biological assay based on fluorescence resonance energy transfer (FRET). By attaching a FRET donor (Alexa Fluor 488) to streptavidin and labeling a FRET acceptor (Alexa Fluor 647) on one DNA strand and biotin on the complementary strand, donor and acceptor molecules are brought in proximity due to streptavidin-biotin binding, resulting in FRET. Fluorescence bursts of the donor and acceptor from each droplet can be monitored simultaneously using separate avalanche photodiode detectors operating in single photon counting mode. Binding assays were investigated and compared between fixed streptavidin and DNA concentrations. Binding curves fit perfectly to Hill-Waud models, and the binding ratio between streptavidin and biotin was evaluated and found to be in agreement with the biotin binding sites on streptavidin. FRET efficiency for this FRET pair was also investigated from the binding results. Efficiency results show that this detection system can precisely measure FRET even at low FRET efficiencies.

Single-molecule Spectroscopy Using Nanoporous Membranes

Nano Letters. Sep, 2007  |  Pubmed ID: 17718589

We describe a novel approach for optically detecting DNA translocation events through an array of solid-state nanopores that potentially allows for ultra high-throughput, parallel detection at the single-molecule level. The approach functions by electrokinetically driving DNA strands through sub micrometer-sized holes on an aluminum/silicon nitride membrane. During the translocation process, the molecules are confined to the walls of the nanofluidic channels, allowing 100% detection efficiency. Importantly, the opaque aluminum layer acts as an optical barrier between the illuminated region and the analyte reservoir. In these conditions, high-contrast imaging of single-molecule events can be performed. To demonstrate the efficiency of the approach, a 10 pM fluorescently labeled lambda-DNA solution was used as a model system to detect simultaneous translocation events using electron multiplying CCD imaging. Single-pore translocation events are also successfully detected using single-point confocal spectroscopy.

Development of Quantitative Cell-based Enzyme Assays in Microdroplets

Analytical Chemistry. May, 2008  |  Pubmed ID: 18399662

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

Gold Nanoparticles for One Step DNA Extraction and Real-time PCR of Pathogens in a Single Chamber

Lab on a Chip. May, 2008  |  Pubmed ID: 18432353

The optothermal properties of nanoparticles are of interest for biosensors and highly sensitive biochip applications. In this respect, the longitudinal resonance of Au nanorods was used to transform near infrared energy into thermal energy in a microfluidic chip. The resulting heat generated effectively caused pathogen lysis. Consequently the DNA was extracted out of the cell body and transferred to a PCR system. This resulted in the successful demonstration of a one step real-time PCR system for pathogen detection without removal or changing of reagents.

Microdroplets: a Sea of Applications?

Lab on a Chip. Aug, 2008  |  Pubmed ID: 18651063

The exploitation of microdroplets produced within microfluidic environments has recently emerged as a new and exciting technological platform for applications within the chemical and biological sciences. Interest in microfluidic systems has been stimulated by a range of fundamental features that accompany system miniaturization. Such features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit cost, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Moreover, microfluidic systems that generate and utilize a stream of sub-nanolitre droplets dispersed within an immiscible continuous phase have the added advantage of allowing ultra-high throughput experimentation and being able to mimic conditions similar to that of a single cell (in terms of volume, pH, and salt concentration) thereby compartmentalizing biological and chemical reactions. This review provides an overview of methods for generating, controlling and manipulating droplets. Furthermore, we discuss key fields of use in which such systems may make a significant impact, with particular emphasis on novel applications in the biological and physical sciences.

Monitoring of Real-time Streptavidin-biotin Binding Kinetics Using Droplet Microfluidics

Analytical Chemistry. Sep, 2008  |  Pubmed ID: 18712935

Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0 x 10 (6)-4.5 x 10 (7) M (-1) s (-1).

Fluorescence Lifetime Imaging of Mixing Dynamics in Continuous-flow Microdroplet Reactors

Physical Review Letters. Jul, 2008  |  Pubmed ID: 18764117

Water-in-oil microdroplets within fluidic channels have the potential to serve as isolated reaction compartments for monitoring real-time dynamics with high efficiency and repeatability. Droplets, usually generated from aqueous and oil solutions using standard microfluidic formats, can be produced at frequencies in excess of 1 kHz. Although mixing within such microdroplets is normally enhanced by chaotic advection, the mixing pattern from droplet to droplet is almost identical and reproducible in form. Herein, we demonstrate that fluorescence lifetime imaging can be used to reconstruct mixing patterns within a droplet with a time resolution of 5 micros.

Pillar-induced Droplet Merging in Microfluidic Circuits

Lab on a Chip. Nov, 2008  |  Pubmed ID: 18941682

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.

Design of a Solid-state Nanopore-based Platform for Single-molecule Spectroscopy

Nanotechnology. Apr, 2008  |  Pubmed ID: 21825639

We numerically assess the light propagation and distribution characteristics of electromagnetic fields on nanopores formed in dielectric and metal/dielectric membranes using a frequency-domain finite element method (3D full-wave electromagnetic field simulation). Results of such studies were used to identify aluminum as an ideal material for use in optically thick metal/dielectric membranes. The comparison between SiN and Al/SiN membranes (with and without a submicron sized aperture) was numerically and experimentally shown to verify the effect of optically thick metal layers on light propagation and fluorescence excitation. The cut-off behavior for Al/SiN membranes with varying pore diameters was investigated in terms of light propagation, distribution of electromagnetic fields, and light attenuation characteristics.

Micro- and Nanofluidic Systems for High-throughput Biological Screening

Drug Discovery Today. Feb, 2009  |  Pubmed ID: 18983933

High-throughput screening (HTS) is a method of scientific experimentation widely used in drug discovery and relevant to the fields of biology. The development of micro- and nanofluidic systems for use in the biological sciences has been driven by a range of fundamental attributes that accompany miniaturization and massively parallel experimentation. We review recent advances in both arraying strategies based on nano/microfluidics and novel nano/microfluidic devices with high analytical throughput rates.

Increasing the Trapping Efficiency of Particles in Microfluidic Planar Platforms by Means of Negative Dielectrophoresis

The Journal of Physical Chemistry. B. Feb, 2009  |  Pubmed ID: 19175343

We present a novel planar electrode geometry in which particles (typically 10 microm in diameter) are focused near a defined surface before being trapped using negative dielectrophoresis. The focusing element can deflect particles having speeds up to hundreds of micrometers per second. This trapping configuration results in improved trapping yields and a decrease in overall reagent consumption. Particles are trapped dynamically while flowing in a microfluidic channel.

Opportunities for Microfluidic Technologies in Synthetic Biology

Journal of the Royal Society, Interface / the Royal Society. Aug, 2009  |  Pubmed ID: 19474079

We introduce microfluidics technologies as a key foundational technology for synthetic biology experimentation. Recent advances in the field of microfluidics are reviewed and the potential of such a technological platform to support the rapid development of synthetic biology solutions is discussed.

Analysis of Protein-protein Interactions by Using Droplet-based Microfluidics

Chembiochem : a European Journal of Chemical Biology. Jul, 2009  |  Pubmed ID: 19496107

Every little drop: The K(D) values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL-sized droplets agree with data from bulk-fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti-ANG antibodies to ANG. Such an experimental platform could be applied to the high-throughput analysis of protein-protein interactions.

Electro-coalescence of Digitally Controlled Droplets

Analytical Chemistry. Sep, 2009  |  Pubmed ID: 19715363

In this paper we describe a universal mechanism for merging multiple aqueous microdroplets within a flowing stream consisting of an oil carrier phase. Our approach involves the use of both a pillar array acting as a passive merging element, as well as built-in electrodes acting as an active merging element. The pillar array enables slowing down and trapping of the droplets via the drainage of the oil phase. This brings adjacent droplets into close proximity. At this point, an electric field applied to the electrodes breaks up the thin oil film surrounding the droplets resulting in merging.

Chemical Imaging of Microfluidic Flows Using ATR-FTIR Spectroscopy

Lab on a Chip. Oct, 2009  |  Pubmed ID: 19789743

Elucidating the chemical composition of microfluidic flows is crucial in both understanding and optimising reactive processes within small-volume environments. Herein we report the implementation of a novel detection methodology based on Attenuated Total Reflection (ATR)-Fourier Transform Infra-Red (FTIR) spectroscopic imaging using an infrared focal plane array detector for microfluidic applications. The method is based on the combination of an inverted prism-shape ATR crystal with a poly(dimethylsiloxane)-based microfluidic mixing device. To demonstrate the efficacy of this approach, we report the direct measurement and imaging of the mixing of two liquids of different viscosities and the imaging and mixing of H2O and D2O with consecutive H/D isotope exchange. This chemically specific imaging approach allows direct analysis of fluid composition as a function of spatial position without the use of added labels or dyes, and can be used to study many processes in microfluidics ranging from reactions to separations.

Identification of Rare Progenitor Cells from Human Periosteal Tissue Using Droplet Microfluidics

The Analyst. Nov, 2009  |  Pubmed ID: 19838410

The isolation and characterisation of single cells from a heterogeneous population are important processes in cell biology, immunology, stem cell research, and cancer research. In the development of novel cell-based therapies, there is a considerable need to target specific cell types to allow for further analysis and amplification ex vivo. We introduce, herein, the use of droplet-based microfluidics as a platform technology for the identification and quantification of distinct cell phenotypes. Using molecular labelling of specific cell populations by antibodies and fluorescent dyes, detection of single cells encapsulated within picolitre-sized aqueous droplets can be performed using high-sensitivity confocal fluorescence detection. Specifically, rare progenitor cells were immunodetected within a heterogeneous population of cells isolated from human periosteal tissue. Using this model human cell population, the accuracy and reproducibility of the droplet system were tested and the results were verified using conventional flow cytometry. It was found that the quantitation of phenotypic subpopulations measured using both techniques is directly comparable. Accordingly, this study demonstrates the biological capacity of droplet-based microfluidics for cellular analysis and provides a necessary first step towards the development of a novel cell sorting technology.

High-throughput Confinement and Detection of Single DNA Molecules in Aqueous Microdroplets

Chemical Communications (Cambridge, England). Nov, 2009  |  Pubmed ID: 19865645

A droplet-based microfluidic system combined with high-sensitivity optical detection is used as a tool for high-throughput confinement and detection of single DNA molecules.

Mapping of Fluidic Mixing in Microdroplets with 1 Micros Time Resolution Using Fluorescence Lifetime Imaging

Analytical Chemistry. May, 2010  |  Pubmed ID: 20356052

Microdroplets generated in microfluidic channels hold great promise for use as substrates in high-throughput chemical and biological analysis. These water-in-oil compartments can serve as isolated reaction vessels, and since they can be generated at rates in excess of 1 kHz, thousands of assays can be carried out quickly and reproducibly. Nevertheless, sampling the large amount of information generated from these platforms still remains a significant challenge. For example, considering the high droplet generation rates and velocities, reproducibility and micrometer resolution are challenging requirements that must be fulfilled. Herein we combine confocal fluorescence lifetime imaging microscopy with a statistical implementation that permits the analysis of mixing phenomena within microdroplets with a temporal resolution of 1 mus. Importantly, such exquisite resolution is only possible as a result of the large number of droplets sampled and their high structural reproducibility.

High-resolution Local Imaging of Temperature in Dielectrophoretic Platforms

Analytical Chemistry. Sep, 2010  |  Pubmed ID: 20684541

The use of dielectrophoretic forces is crucially tied to the knowledge of Joule heating within a fluid, since the use of planar microelectrodes creates a temperature gradient within which the particle of interest is manipulated. Mapping temperature with sufficient spatial resolution within a dielectrophoretic trap is recognized to be of high importance. Herein, we demonstrate local temperature measurements in the vicinity of a trapped micrometer-size particle using confocal fluorescence spectroscopy. Such measurements are shown to provide a novel calibration tool for screening temperature-mediated processes with high resolution.

Passive Self-synchronized Two-droplet Generation

Lab on a Chip. Oct, 2010  |  Pubmed ID: 20717573

We describe the use of two passive components to achieve controllable and alternating droplet generation in a microfluidic device. The approach overcomes the problems associated with irregularities in channel dimensions and fluid flow rates, and allows precise pairing of alternating droplets in a high-throughput manner. We study droplet generation and self-synchronization in a quantitative fashion by using high-speed image analysis.

Rapid Prototyping of Nanofluidic Systems Using Size-reduced Electrospun Nanofibers for Biomolecular Analysis

Small (Weinheim an Der Bergstrasse, Germany). Nov, 2010  |  Pubmed ID: 20878634

Biomolecular transport in nanofluidic confinement offers various means to investigate the behavior of biomolecules in their native aqueous environments, and to develop tools for diverse single-molecule manipulations. Recently, a number of simple nanofluidic fabrication techniques has been demonstrated that utilize electrospun nanofibers as a backbone structure. These techniques are limited by the arbitrary dimension of the resulting nanochannels due to the random nature of electrospinning. Here, a new method for fabricating nanofluidic systems from size-reduced electrospun nanofibers is reported and demonstrated. As it is demonstrated, this method uses the scanned electrospinning technique for generation of oriented sacrificial nanofibers and exposes these nanofibers to harsh, but isotropic etching/heating environments to reduce their cross-sectional dimension. The creation of various nanofluidic systems as small as 20 nm is demonstrated, and practical examples of single biomolecular handling, such as DNA elongation in nanochannels and fluorescence correlation spectroscopic analysis of biomolecules passing through nanochannels, are provided.

High-efficiency Single-molecule Detection Within Trapped Aqueous Microdroplets

The Journal of Physical Chemistry. B. Dec, 2010  |  Pubmed ID: 21069980

Aqueous droplets were used as a tool to confine a molecular population and enable highly efficient detection at the single-molecule level. Picoliter-sized aqueous droplets were generated using classical multiphase microfluidics with an aqueous stream containing the analyte under investigation and an oil carrier phase. The droplets were then localized and isolated in specially designed trapping areas within the microfluidic channel to provide a static environment for detection of the encapsulated molecules. We show that by continuously flowing the carrier oil phase while holding the aqueous stationary, we can significantly improve on measuring repeat single-molecule events. Further, we find that the flowing oil stream induces a circulation within the trapped droplets which is proportional to the volumetric flow velocity. This circulation phenomenon allows a given molecule to be detected multiple times during an experiment and can therefore be used for performing time-dependent single-molecule analysis.

New Developments in Nanopore Research--from Fundamentals to Applications

Journal of Physics. Condensed Matter : an Institute of Physics Journal. Nov, 2010  |  Pubmed ID: 21339577

Precise Electrochemical Fabrication of Sub-20 Nm Solid-state Nanopores for Single-molecule Biosensing

Journal of Physics. Condensed Matter : an Institute of Physics Journal. Nov, 2010  |  Pubmed ID: 21339614

It has recently been shown that solid-state nanometer-scale pores ('nanopores') can be used as highly sensitive single-molecule sensors. For example, electrophoretic translocation of DNA, RNA and proteins through such nanopores has enabled both detection and structural analysis of these complex biomolecules. Control over the nanopore size is critical as the pore must be comparable in size to the analyte molecule in question. The most widely used fabrication methods are based on focused electron or ion beams and thus require (scanning) transmission electron microscopy and focused ion beam (FIB) instrumentation. Even though very small pores have been made using these approaches, several issues remain. These include the requirement of being restricted to rather thin, mechanically less stable membranes, particularly for pore diameters in the single-digit nanometer range, lack of control of the surface properties at and inside the nanopore, and finally, the fabrication cost. In the proof-of-concept study, we report on a novel and simple route for fabricating metal nanopores with apparent diameters below 20 nm using electrodeposition and real-time ionic current feedback in solution. This fabrication approach inserts considerable flexibility into the kinds of platforms that can be used and the nanopore membrane material. Starting from much larger pores, which are straightforward to make using FIB or other semiconductor fabrication methods, we electrodeposit Pt at the nanopore interface while monitoring its ionic conductance at the same time in a bi-potentiostatic setup. Due to the deposition of Pt, the nanopore decreases in size, resulting in a decrease of the pore conductance. Once a desired pore conductance has been reached, the electrodeposition process is stopped by switching the potential of the membrane electrode and the fabrication process is complete. Furthermore, we demonstrate that these pores can be used for single-biomolecule analysis, such as that of λ-DNA, which we use in a proof-of-concept study. Importantly, our approach is applicable to single nanopores as well as nanopore arrays, and can easily be extended to deposits of metal other than Pt.

Ultrafast Surface Enhanced Resonance Raman Scattering Detection in Droplet-based Microfluidic Systems

Analytical Chemistry. Apr, 2011  |  Pubmed ID: 21413700

The development of ultrafast Raman-based detection is one of the most interesting challenges underpinning the application of droplet-based microfluidics. Herein, we describe the use of surface-enhanced resonance Raman spectroscopy (SERRS) with submillisecond time resolution as a powerful detection tool in microdroplet reactors. Individual droplets containing silver nanoparticle aggregates functionalized with Raman reporters are interrogated and characterized by full spectra acquisitions with high spatial resolution in real time. Whereas previous works coupling SERRS with droplet-based microfluidics acquire a single spectrum over single or multiple droplets, we build upon these results by increasing our temporal resolution by 2 orders of magnitude. This allows us to interrogate multiple points within one individual droplet. The SERRS signals emitted from the aggregates are utilized to access the influence of flow rate on droplet size and throughput. Accordingly, our approach allows for high-throughput analysis that facilitates the study of other biological assays or molecular interactions.

Chemical, Thermal, and Electric Field Induced Unfolding of Single Protein Molecules Studied Using Nanopores

Analytical Chemistry. Jul, 2011  |  Pubmed ID: 21598904

Single-molecule experimental techniques have recently shown to be of significant interest for use in numerous applications in both the research laboratory and industrial settings. Although many single-molecule techniques exist, the nanopore platform is perhaps one of the more popular techniques due to its ability to act as a molecular sensor of biological macromolecules. For example, nanopores offer a unique, new method for probing various properties of proteins and can contribute to elucidating key biophysical information in conjunction with existing techniques. In the present study, various forms of bovine serum albumin (BSA) are detected including thermally refolded BSA, urea-denatured BSA, and multiple forms of BSA detected at elevated electric field strengths (with and without urea). We also provide excluded volume measurements for each of these states that normally are difficult to obtain due to unknown and unstable protein conformations.

A Microdroplet Dilutor for High-throughput Screening

Nature Chemistry. Jun, 2011  |  Pubmed ID: 21602857

Pipetting and dilution are universal processes used in chemical and biological laboratories to assay and experiment. In microfluidics such operations are equally in demand, but difficult to implement. Recently, droplet-based microfluidics has emerged as an exciting new platform for high-throughput experimentation. However, it is challenging to vary the concentration of droplets rapidly and controllably. To this end, we developed a dilution module for high-throughput screening using droplet-based microfluidics. Briefly, a nanolitre-sized sample droplet of defined concentration is trapped within a microfluidic chamber. Through a process of droplet merging, mixing and re-splitting, this droplet is combined with a series of smaller buffer droplets to generate a sequence of output droplets that define a digital concentration gradient. Importantly, the formed droplets can be merged with other reagent droplets to enable rapid chemical and biological screens. As a proof of concept, we used the dilutor to perform a high-throughput homogeneous DNA-binding assay using only nanolitres of sample.

Resizing Metal-coated Nanopores Using a Scanning Electron Microscope

Small (Weinheim an Der Bergstrasse, Germany). Oct, 2011  |  Pubmed ID: 21953785

Electron beam-induced shrinkage provides a convenient way of resizing solid-state nanopores in Si(3) N(4) membranes. Here, a scanning electron microscope (SEM) has been used to resize a range of different focussed ion beam-milled nanopores in Al-coated Si(3) N(4) membranes. Energy-dispersive X-ray spectra and SEM images acquired during resizing highlight that a time-variant carbon deposition process is the dominant mechanism of pore shrinkage, although granular structures on the membrane surface in the vicinity of the pores suggest that competing processes may occur. Shrinkage is observed on the Al side of the pore as well as on the Si(3) N(4) side, while the shrinkage rate is observed to be dependent on a variety of factors.

High-throughput Age Synchronisation of Caenorhabditis Elegans

Chemical Communications (Cambridge, England). Sep, 2011  |  Pubmed ID: 21818494

We present a passive microfluidic strategy for sorting adult C. elegans nematodes on the basis of age and size. The separation mechanism takes advantage of phenotypic differences between 'adult' and 'juvenile' organisms and their behaviour in microfluidic architectures. In brief, the microfluidic device allows worms to sort themselves in a passive manner.

DNA Tunneling Detector Embedded in a Nanopore

Nano Letters. Jan, 2011  |  Pubmed ID: 21133389

We report on the fabrication and characterization of a DNA nanopore detector with integrated tunneling electrodes. Functional tunneling devices were identified by tunneling spectroscopy in different solvents and then used in proof-of-principle experiments demonstrating, for the first time, concurrent tunneling detection and ionic current detection of DNA molecules in a nanopore platform. This is an important step toward ultrafast DNA sequencing by tunneling.

Flow-based Autocorrelation Studies for the Detection and Investigation of Single-particle Surface-enhanced Resonance Raman Spectroscopic Events

Analytical Chemistry. Feb, 2011  |  Pubmed ID: 21314203

We report on the characterization and detection of single metallic nanoparticles using a combination of correlation spectroscopy and surface-enhanced resonance Raman spectroscopy (SERRS). Minimizing the number of independent characterization steps is critical to efficiently perform such an analysis. In this article, we improve upon conventional diffusion-limited approaches by implementing a flow-based system with high temporal resolution detection. The benefits of flow over diffusion measurements allow for a higher throughput of detected events resulting in shorter analysis times. The nanoparticles are sized using their rotational diffusion time calculated with a modified autocorrelation function. Experiments are performed using Au nanoparticles labeled with the reporter molecule malachite green isothiocyanate on a custom-built Raman spectrometer.

Dielectric Cell Response in Highly Conductive Buffers

Analytical Chemistry. Jan, 2012  |  Pubmed ID: 22148418

We present a novel method for the identification of live and dead T-cells, dynamically flowing within highly conductive buffers. This technique discriminates between live and dead (heat treated) cells on the basis of dielectric properties variations. The key advantage of this technique lies in its operational simplicity, since cells do not have to be resuspended in isotonic low conductivity media. Herein, we demonstrate that at 40 MHz, we are able to statistically distinguish between live and dead cell populations.