Protein quantification at the single vesicle level reveals that a subset of synaptic vesicle proteins are trafficked with high precision.

Protein sorting represents a potential point of regulation in neurotransmission because it dictates the protein composition of synaptic vesicles, the organelle that mediates transmitter release. Although the average number of most vesicle proteins has been estimated using bulk biochemical approaches (Takamori et al., 2006), no information exists on the intervesicle variability of protein number, and thus on the precision with which proteins are sorted to vesicles. To address this, we adapted a single molecule quantification approach (Mutch et al., 2007) and used it to quantify both the average number and variance of seven integral membrane proteins in brain synaptic vesicles. We report that four vesicle proteins, SV2, the proton ATPase, Vglut1, and synaptotagmin 1, showed little intervesicle variation in number, indicating they are sorted to vesicles with high precision. In contrast, the apparent number of VAMP2/synaptobrevin 2, synaptophysin, and synaptogyrin demonstrated significant intervesicle variability. These findings place constraints on models of protein function at the synapse and raise the possibility that changes in vesicle protein expression affect vesicle composition and functioning.

Ultrasensitive and high-throughput fluorescence analysis of droplet contents with orthogonal line confocal excitation.

This paper describes a simple modification to traditional confocal fluorescence detection that greatly improves signal-to-noise (s/n) for the high-speed analysis of droplet streams. Rather than using the conventional epi geometry, illumination of the droplet was in the form of a line that is orthogonal to both the direction of flow and the light-collection objective. In contrast to the epi geometry where we observed high levels of scattering background from the droplets, we detected more than 10-fold less background (depending on the laser power used) when orthogonal-line-confocal illumination was used. We characterized this improvement using a standard microfluidic platform over a range of analyte concentrations and observed an improvement in limits of detection of greater than 10. Using this method, we were able to analyze picomolar concentrations of analytes contained within picoliter-volume droplets at a rate of greater than 350 droplets per second.

Droplets for ultrasmall-volume analysis.

By using methods that permit the generation and manipulation of ultrasmall-volume droplets, researchers are pushing the boundaries of ultrasensitive chemical analyses. (To listen to a podcast about this feature, please go to the Analytical Chemistry Web site at pubs.acs.org/ancham.).

Microfabricating high-aspect-ratio structures in polyurethane-methacrylate (PUMA) disposable microfluidic devices.

We recently reported a new UV-curable polyurethane-methacrylate (PUMA) resin that has excellent qualities as a disposable microfluidic substrate for clinical diagnostic applications. This article discusses strategies to improve the production yield of PUMA chips that contain dense and high-aspect-ratio features, which presents unique challenges in demolding and bonding steps. These fabrication improvements were deployed to produce a microfiltration device that contained closely spaced and high-aspect-ratio columns, suitable for retaining and concentrating cells or beads from a highly diluted suspension.

Chemistry and biology in femtoliter and picoliter volume droplets.

The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases; in cancer metastasis, for example, a single cell seeds the formation of a distant tumor. Although tiny, a cell is a highly heterogeneous and compartmentalized structure: proteins, lipids, RNA, and small-molecule metabolites constantly traffic among intracellular organelles. Gaining detailed information about the spatiotemporal distribution of these biomolecules is crucial to our understanding of cellular function and dysfunction. To access this information, we need sensitive tools that are capable of extracting comprehensive biochemical information from single cells and subcellular organelles. In this Account, we outline our approach and highlight our progress toward mapping the spatiotemporal organization of information flow in single cells. Our technique is centered on the use of femtoliter- and picoliter-sized droplets as nanolabs for manipulating single cells and subcellular compartments. We have developed a single-cell nanosurgical technique for isolating select subcellular structures from live cells, a capability that is needed for the high-resolution manipulation and chemical analysis of single cells. Our microfluidic approaches for generating single femtoliter-sized droplets on demand include both pressure and electric field methods; we have also explored a design for the on-demand generation of multiple aqueous droplets to increase throughput. Droplet formation is only the first step in a sequence that requires manipulation, fusion, transport, and analysis. Optical approaches provide the most convenient and precise control over the formed droplets with our technology platform; we describe aqueous droplet manipulation with optical vortex traps, which enable the remarkable ability to dynamically "tune" the concentration of the contents. Integration of thermoelectric manipulations with these techniques affords further control. The amount of chemical information that can be gleaned from single cells and organelles is critically dependent on the methods available for analyzing droplet contents. We describe three techniques we have developed: (i) droplet encapsulation, rapid cell lysis, and fluorescence-based single-cell assays, (ii) physical sizing of the subcellular organelles and nanoparticles in droplets, and (iii) capillary electrophoresis (CE) analysis of droplet contents. For biological studies, we are working to integrate the different components of our technology into a robust, automated device; we are also addressing an anticipated need for higher throughput. With progress in these areas, we hope to cement our technique as a new tool for studying single cells and organelles with unprecedented molecular detail.

A new USP Class VI-compliant substrate for manufacturing disposable microfluidic devices.

As microfluidic systems transition from research tools to disposable clinical-diagnostic devices, new substrate materials are needed to meet both the regulatory requirement as well as the economics of disposable devices. This paper introduces a UV-curable polyurethane-methacrylate (PUMA) substrate that has been qualified for medical use and meets all of the challenges of manufacturing microfluidic devices. PUMA is optically transparent, biocompatible, and exhibits high electroosmotic mobility without surface modification. We report two production processes that are compatible with the existing methods of rapid prototyping and present characterizations of the resultant PUMA microfluidic devices.

Simultaneous generation of multiple aqueous droplets in a microfluidic device.

This paper describes a microfluidic platform for the on-demand generation of multiple aqueous droplets, with varying chemical contents or chemical concentrations, for use in droplet based experiments. This generation technique was developed as a complement to existing techniques of continuous-flow (streaming) and discrete-droplet generation by enabling the formation of multiple discrete droplets simultaneously. Here sets of droplets with varying chemical contents can be generated without running the risk of cross-contamination due to the isolated nature of each supply inlet. The use of pressure pulses to generate droplets in parallel is described, and the effect of droplet size is examined in the context of flow rates and surfactant concentrations. To illustrate this technique, an array of different dye-containing droplets was generated, as well as a set of droplets that displayed a concentration gradient of a fluorescent dye.

Fabrication improvements for thermoset polyester (TPE) microfluidic devices.

Thermoset polyester (TPE) microfluidic devices were previously developed as an alternative to poly(dimethylsiloxane) (PDMS) devices, fabricated similarly by replica molding, yet offering stable surface properties and good chemical compatibility with some organics that are incompatible with PDMS. This paper describes a number of improvements in the fabrication of TPE chips. Specifically, we describe methods to form TPE devices with a thin bottom layer for use with high numerical aperture (NA) objectives for sensitive fluorescence detection and optical manipulation. We also describe plasma-bonding of TPE to glass to create hybrid TPE-glass devices. We further present a simple master-pretreatment method to replace our original technique that required the use of specialized equipment.

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.

Capillary electrophoresis separation in the presence of an immiscible boundary for droplet analysis.

This paper demonstrates the ability to use capillary electrophoresis (CE) separation coupled with laser-induced fluorescence for analyzing the contents of single femtoliter-volume aqueous droplets. A single droplet was formed using a T-channel (3 microm wide by 3 microm tall) connected to microinjectors, and then the droplet was fluidically moved to an immiscible boundary that isolates the CE channel (50 microm wide by 50 microm tall) from the droplet generation region. Fusion of the aqueous droplet with the immiscible boundary effectively injects the droplet content into the separation channel. In addition to injecting the contents of droplets, we found aqueous samples can be introduced directly into the separation channel by reversibly penetrating and resealing the immiscible partition. Because droplet generation in channels requires hydrophobic surfaces, we have also investigated the advantages to using all hydrophobic channels versus channel systems with patterned hydrophobic and hydrophilic regions. To fabricate devices with patterned surface chemistry, we have developed a simple strategy based on differential wetting to deposit selectively a hydrophilic polymer (poly(styrenesulfonate)) onto desired regions of the microfluidic chip. Finally, we applied our device to the separation of a simple mixture of fluorescein-labeled amino acids contained within a approximately 10-fL droplet.

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.

Real-time sizing of nanoparticles in microfluidic channels using confocal correlation spectroscopy.

This Communication reports real-time sizing of nanoparticles in microfluidic systems using confocal correlation spectroscopy (CCS). CCS can be used to measure the size of both fluorescent and nonfluorescent particles at low concentrations (

Layered polyelectrolyte-silica coating for nanocapsules.

This paper demonstrates the ability to grow silica directly on a deposited surface of polyelectrolyte. Using this strategy, we describe the deposition of layered polyelectrolyte-silica coating on negatively charged surfaces of polystyrene particles and latex nanocapsules, which could not be coated directly with silica alone. By etching away the underlying polystyrene bead, we were able to form polyelectrolyte-silica capsules that were mechanically robust. Using scanning and transmission electron microscopy, we imaged and studied the coating after the deposition of each layer of polyelectrolyte and silica. We then applied this new coating to latex nanocapsules that were loaded with fluorescein molecules. We found that the coating procedure did not cause the loaded molecules to leak out from the capsules, and we determined that the variation in the number of loaded molecules among capsules arose from differences in the volume of the nanocavities and was not caused by the loading and coating of the capsules. This layered architecture permits the thickness of the coating to be controlled in principle over a wide dynamic range, but more importantly, this coating could act as an effective seal to prevent undesired leakage from nanocapsules and thus increase the long-term storability of loaded capsules. Over a 30-day period, we determined that leakage from uncoated capsules was significant but negligible for ones that were coated with two layers of polyelectrolyte-silica. Using single-pulse UV photolysis of individual nanocapsules, we demonstrate that the molecules contained within coated capsules could be released effectively and on demand with a single laser pulse.

Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets.

This paper describes a method, which combines optical trapping and microfluidic-based droplet generation, for selectively and controllably encapsulating a single target cell or subcellular structure, such as a mitochondrion, into a picoliter- or femtoliter-volume aqueous droplet that is surrounded by an immiscible phase. Once the selected cell or organelle is encased within the droplet, it is stably confined in the droplet and cannot be removed. We demonstrate in droplet the rapid laser photolysis of the single cell, which essentially "freezes" the state that the cell was in at the moment of photolysis and confines the lysate within the small volume of the droplet. Using fluorescein di-beta-d-galactopyranoside, which is a fluorogenic substrate for the intracellular enzyme beta-galactosidase, we also assayed the activity of this enzyme from a single cell following the laser-induced lysis of the cell. This ability to entrap individual selected cells or subcellular organelles should open new possibilities for carrying out single-cell studies and single-organelle measurements.

Rapid prototyping of thermoset polyester microfluidic devices.

This paper presents a simple procedure for the fabrication of thermoset polyester (TPE) microfluidic systems and discusses the properties of the final devices. TPE chips are fabricated in less than 3 h by casting TPE resin directly on a lithographically patterned (SU-8) silicon master. Thorough curing of the devices is obtained through the combined use of ultraviolet light and heat, as both an ultraviolet and a thermal initiator are employed in the resin mixture. Features on the order of micrometers and greater are routinely reproduced using the presented procedure, including complex designs and multilayer features. The surface of TPE was characterized using contact angle measurements and X-ray photoelectron spectroscopy (XPS). Following oxygen plasma treatment, the hydrophilicity of the surface of TPE increases (determined by contact angle measurements) and the proportion of oxygen-containing functional groups also increases (determined by XPS), which indicates a correlated increase in the charge density on the surface. Native TPE microchannels support electroosmotic flow (EOF) toward the cathode, with an average electroosmotic mobility of 1.3 x 10(-4) cm(2) V(-1) s(-1) for a 50-microm square channel (20 mM borate at pH 9); following plasma treatment (5 min at 30 W and 0.3 mbar), EOF is enhanced by a factor of 2. This enhancement of the EOF from plasma treatment is stable for days, with no significant decrease noted during the 5-day period that we monitored. Using plasma-treated TPE microchannels, we demonstrate the separation of a mixture of fluorescein-tagged amino acids (glycine, glutamic acid, aspartic acid). TPE devices are up to 90% transparent (for approximately 2-mm-thick sample) to visible light (400-800 nm). The compatibility of TPE with a wide range of solvents was tested over a 24-h period, and the material performed well with acids, bases, alcohols, cyclohexane, n-heptane, and toluene but not with chlorinated solvents (dichloromethane, chloroform).

Selective electroless and electrolytic deposition of metal for applications in microfluidics: fabrication of a microthermocouple.

This paper describes a general strategy for the fabrication of a microthermocouple based on the spatially defined electroless deposition of metal, followed by annealing and electroplating. We present scanning electron microscopy and atomic force microscopy characterizations of the deposition and annealing process, as well as the performance of the microfabricated Ni-Ag thermocouple. The temperature-voltage curve for this Ni-Ag microthermocouple is linear over the range 0-50 degrees C with a slope of 61.9 degrees C mV(-1). The sensitivity of our temperature measurement, which is limited by the uncertainty of our calibration curve, is approximately 1 degrees C. The optimum figure of merit (Z(opt)) is 1.0 x 10(-5) for this type of Ag-Ni thermocouple. We have fabricated microthermocouples ranging in size from 50 to 300 microm. The microthermocouple was integrated into microchannels and used to measure the in-channel temperature rise caused by the following: (1) a simple acid-base reaction, HCl + NaOH --> H2O + NaCl, and (2) an enzyme-catalyzed biochemical reaction, H2O2 + catalase --> H2O + 1/2 O2. We have also profiled the temperature increase in the presence of electroosmotic flow for a 100-, 200-, and 300-microm channel.