Klint A. Rose

High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.

Advanced carbon aerogels for energy applications

Carbon aerogels are a unique class of high-surface-area materials derived by sol–gel chemistry. Their high mass-specific surface area and electrical conductivity, environmental compatibility and chemical inertness make them very promising materials for many energy related applications, specifically in view of recent developments in controlling their morphology. In this perspective we will review the synthesis of monolithic resorcinol–formaldehyde based carbon aerogels with hierarchical porosities for energy applications, including carbon nanotube and graphene composite carbon aerogels, as well as their functionalization by surface engineering. Applications that we will discuss include hydrogen and electrical energy storage, desalination and catalysis.

Capacitive desalination with flow-through electrodes

Capacitive desalination (CD) is a promising desalination technique as, relative to reverse osmosis (RO), it requires no membrane components, can operate at low (sub-osmotic) pressures, and can potentially utilize less energy for brackish water desalination. In a typical CD cell, the feed water flows through the separator layer between two electrically charged, nanoporous carbon electrodes. This architecture results in significant performance limitations, including an inability to easily (in a single charge) desalinate moderate brackish water feeds and slow, diffusion-limited desalination. We here describe an alternative architecture, where the feed flows directly through electrodes along the primary electric field direction, which we term flow-through electrode (FTE) capacitive desalination. Using macroscopic porous electrode theory, we show that FTE CD enables significant reductions in desalination time and can desalinate higher salinity feeds per charge. We then demonstrate these benefits using a custom-built FTE CD cell containing novel hierarchical carbon aerogel monoliths as an electrode material. The pore structure of our electrodes includes both micron-scale and sub-10 nm pores, allowing our electrodes to exhibit both low flow resistance and very high specific capacitance (>100 F g−1). Our cell demonstrates feed concentration reductions of up to 70 mM NaCl per charge and a mean sorption rate of nearly 1 mg NaCl per g aerogel per min, 4 to 10 times higher than that demonstrated by the typical CD cell architecture. We also show that, as predicted by our model, our cell desalinates the feed at the cells RC timescale rather than the significantly longer diffusive timescale characteristic of typical CD cells.

Electroosmotic pumps fabricated from porous silicon membranes

N-type porous silicon can be used to realize electroosmotic pumps with high flow rates per applied potential difference. The porosity and pore size of porous silicon membranes can be tuned, the pore geometry has near-unity tortuosity, and membranes can be made thin and with integrated support structures. The size of hexagonally packed pores is modified by low-pressure chemical vapor deposition (LPCVD) polysilicon deposition, followed by wet oxidation of the polysilicon layer, resulting in a pore radius varying from 1 to 3 /spl mu/m. Pumping performance of these devices is experimentally studied as a function of pore size and compared with theory. These 350-/spl mu/m-thick silicon membranes exhibit a maximum flow rate per applied field of 0.13 ml/min/cm/sup 2//V. This figure of merit is five times larger than previously demonstrated porous glass EO pumps.

The zeta potential of surface-functionalized metallic nanorod particles in aqueous solution

Metallic nanoparticles suspended in aqueous solutions and functionalized with chemical and biological surface coatings are important elements in basic and applied nanoscience research. Many applications require an understanding of the electrokinetic or colloidal properties of such particles. We describe the results of experiments to measure the zeta potential of metallic nanorod particles in aqueous saline solutions, including the effects of pH, ionic strength, metallic composition, and surface functionalization state. Particle substrates tested include gold, silver, and palladium monometallic particles as well as gold/silver bimetallic particles. Surface functionalization conditions included 11‐mercaptoundecanoic acid (MUA), mercaptoethanol (ME), and mercaptoethanesulfonic acid (MESA) self‐assembled monolayers (SAMs), as well as MUA layers subsequently derivatized with proteins. For comparison, we present zeta potential data for typical charge‐stabilized polystyrene particles. We compare experimental zeta potential data with theoretically predicted values for SAM‐coated and bimetallic particles. The results of these studies are useful in predicting and controlling the aggregation, adhesion, and transport of functionalized metallic nanoparticles within microfluidic devices and other systems.

Stretchable micro-electrode array [for retinal prosthesis]

This paper focuses on the design considerations, fabrication processes, and preliminary testing of a retinal prosthesis that has the potential to aid in vision restoration to millions of blind patients. We are developing an implantable, stretchable micro-electrode array using polymer-based microfabrication techniques. The device will serve as the interface between an electronic imaging system and the human eye, directly stimulating retinal neurons via thin film conducting traces and electroplated electrodes. The metal features are embedded within a thin (/spl sim/50 /spl mu/m) substrate fabricated using poly (dimethylsiloxane) (PDMS), a biocompatible elastomeric material that has high oxygen permeability and low water permeability. The conformable nature of PDMS is critical for ensuring uniform contact with the curved surface of the retina. To fabricate the device, we developed unique processes for metalizing PDMS to produce robust traces capable of maintaining conductivity when stretched (strain = 7%, SD 1), and for selectively passivating the conductive elements. An in situ substrate curvature measurement taken while curing the PDMS revealed a tensile residual strain of 10%, explaining the stretchable nature of the thin metalized devices.

Photocurable Liquid Core–Fugitive Shell Printing of Optical Waveguides

N Integrated optical systems require waveguides that can route light along defi ned pathways with minimal losses and negligible crosstalk. [ 1–3 ] In addition to signal transmission, optical waveguides play an important role in the area of sensing. For example, evanescent fi eld sensors are applied to the detection of analytes in the body, [ 4 ] atmosphere, [ 5 ] and liquid solutions. [ 6 ] Polymeric and hybrid materials are of increasing interest for these applications due to their low temperature processing. [ 7 ] To date, channel waveguides have been fabricated by direct lithographic patterning, [ 8 , 9 ] photoresist-templated etching, [ 10 ] or soft-lithographic approaches. [ 11 , 12 ] However, these techniques are limited either to in-plane confi gurations or require repeated developing or etching steps to produce multiple layers of waveguides. Those processing steps often have a deleterious effect on waveguide performance, leading to rough edges and, hence, higher optical loss. [ 8 , 11 , 13 ]

Polymer-Based Packaging Platform for Hybrid Microfluidic Systems

A polymer-based packaging platform for creating hybrid microfluidic systems is presented. Polydimethylsiloxane (PDMS) is cast into an acrylic mold frame with suspended elements that are removed after curing to form chip cavities, inlet and outlet ports, microchannels, and reservoirs. The packaging approach enables the integration of off-the-shelf components such as pumps and valves with glass microfluidic devices, electronic chips, sample reservoirs, and flow channels. A particle pre-concentration module with a glass capture chip and integrated micropump is shown as an example. A pneumatically driven microfluidic pumping module is also shown. Custom microfluidic interconnects for interfacing to micro-scale fluidic systems are presented. The connectors are capable of withstanding more than 1000 psi and allow microdevices to be rapidly connected to macroscopic devices and systems, without the use of tools.

Acoustic particle filter with adjustable effective pore size for automated sample preparation.

This article presents analysis and optimization of a microfluidic particle filter that uses acoustic radiation forces to remove particles larger than a selected size by adjusting the driving conditions of the piezoelectric transducer (PZT). Operationally, the acoustic filter concentrates microparticles to the center of the microchannel, minimizing undesirable particle adsorption to the microchannel walls. Finite element models predict the complex two-dimensional acoustic radiation force field perpendicular to the flow direction in microfluidic devices. We compare these results with experimental parametric studies including variations of the PZT driving frequencies and voltages as well as various particle sizes (0.5-5.0 microm in diameter). These results provide insight into the optimal operating conditions and show the efficacy of our device as a filter with an adjustable effective pore size. We demonstrate the separation of Saccharomyces cerevisiae from MS2 bacteriophage using our acoustic device. With optimized design of our microfluidic flow system, we achieved yields of greater than 90% for the MS2 with greater than 80% removal of the S. cerevisiae in this continuous-flow sample preparation device.

Shape control synthesis of fluorapatite structures based on supersaturation: prismatic nanowires, ellipsoids, star, and aggregate formation

Fluorapatite nanostructures of various shapes (prismatic, ellipsoidal, star, and aggregate) were synthesized and their structures correlated with the supersaturation of the system. Reagent concentration and pH were adjusted and the change in supersaturation was simulated by the Geochemists Workbench® software and the MINTEQ database. A higher pH caused changes to the FAP surface charge and was shown to be the dominant force behind aggregate formation. This led to nanorod aggregates and when combined with an increase in reagent concentration, FAP stars were generated. Increasing reaction temperature (room temperature to 100 °C) allowed release of calcium by the chelating agent, EDTA, which steadily increased the supersaturation as demonstrated by simulation. This condition led to ellipsoidal nanorods. As the crystal growth continued with an increasing reaction temperature of up to 150 °C, ellipsoidal nanorods transformed to prismatic nanowires. This transformation was explained by the decreasing supersaturation of the system as the growth nutrients were consumed. Microwave irradiation, the role of fluorite, and control of monodispersity for the FAP synthesis are also discussed.