Anita Rogacs

Purification of nucleic acids using isotachophoresis.

Reviewed are methods of nucleic acid (NA) extraction and sample preparation using an electrophoretic purification and focusing method called isotachophoresis (ITP). ITP requires no special surface chemistries or geometric structures, and can be achieved in a compact system with no moving parts. ITP is also compatible with a wide range of samples and lysing methods. Described are general principles of ITP, considerations around the application of ITP to biological samples (e.g., blood, urine and saliva), ITP electrolyte design considerations for fast and selective NA purification, and examples of ITP compatible lysing methods. Several of the challenges associated with purification of NAs are presented as well as methods to address these. Lastly, specific examples of lysing methods and ITP chemistries are described for purification of NA including host and pathogenic DNA, pathogenic rRNA, and host micro-RNA from complex sample matrices.

Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis

We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida . This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.

An injection molded microchip for nucleic acid purification from 25 microliter samples using isotachophoresis.

We present a novel microchip device for purification of nucleic acids from 25μL biological samples using isotachophoresis (ITP). The device design incorporates a custom capillary barrier structure to facilitate robust sample loading. The chip uses a 2mm channel width and 0.15mm depth to reduce processing time, mitigate Joule heating, and achieve high extraction efficiency. To reduce pH changes in the device due to electrolysis, we incorporated a buffering reservoir physically separated from the sample output reservoir. To reduce dispersion of the ITP-focused zone, we used optimized turn geometries. The chip was fabricated by injection molding PMMA and COC plastics through a commercial microfluidic foundry. The extraction efficiency of nucleic acids from the device was measured using fluorescent quantification, and an average recovery efficiency of 81% was achieved for nucleic acid masses between 250pg and 250ng. The devices were also used to purify DNA from whole blood, and the extracted DNA was amplified using qPCR to show the PCR compatibility of the purified sample.

Performance-cost optimization of a diamond heat spreader

The steady-state thermal resistance of a small heat source applied to a diamond heat spreader attached to a larger substrate with Newtonian cooling on the opposite side was evaluated using numerical simulation. The substrate with Newtonian cooling models a range of convection-cooled designs, such as a heat sink base with a finned surface or the base of a cold plate with liquid cooling. The objective of this work is to quantify the thermal performance of the modeled system as a function of the diamond heat spreader size and properties. The resulting maximum thermal resistance as a function of diamond spreader thickness, lateral dimension, and conductivity are presented, as well as some guidelines for effective thermal design. In addition, a range of convection conditions typical for these applications are examined. Synthetic diamond is still an expensive material, ranging from

Temperature-Dependent Permeability of Microporous Membranes for Vapor Venting Heat Exchangers

1 to

Computational Modeling of Vapor Chambers With Nanostructured Wicks

20 per square millimeter in the lateral plane. The price increases sharply with conductivity, thickness, and lateral dimension, all of which increase thermal performance of the diamond spreader. For this reason, the cost per thermal performance is a distinctly different optimization problem from the thermal performance alone. The results of this optimization are presented for a Biot number typical for forced convection through a finned surface using direct air cooling.

Particle Tracking and Multispectral Collocation Method for Particle-to-Particle Binding Assays

Improved flow regime stability and lower pressure drop may be possible in two-phase microfluidic heat exchangers through the use of a hydrophobic membrane for phase separation. Past research on vapor-venting heat exchangers showed that membrane mechanical and hydrodynamic properties are crucial for heat exchanger design. However, previous characterizations of hydrophobic membranes were primarily carried out at room temperatures with air or nitrogen, as opposed to liquid water and steam at the elevated operating temperature of the heat exchangers. This work investigates laminated PTFE, unlaminated PTFE, and nylon membranes and quantifies the permeability of the membranes to air and steam. The pressure drop across the membrane as a function of fluid flow rate and temperature characterizes the membrane permeability. This work will facilitate more focused experimental work and predictive modeling on optimizing membrane properties and will help with the development of more effective vapor venting heat exchangers.Copyright

VOLUME OF FLUID SIMULATION OF BOILING TWO-PHASE FLOW IN A VAPOR-VENTING MICROCHANNEL

As the power and heat output of modern CPUs climb ever higher and the interest in compact, passively cooled devices grows, there is an urgent need for thinner and more effective vapor chamber technologies. Nanostructured wick technologies based on oxide and organic nanowires have been proposed as a method of improving heat pipe performance in such applications. This work performs finite difference simulations of a 2D heat pipe accounting for variable porosity in the wick. For heat fluxes of 10 and 100 W/cm2 , we find that temperature difference between the evaporator and condenser regions decreases by 10%, which is promising for spreading thermal energy. We find that spatially varying porosity yields improvements in spreading heat throughout the entire wick region. Finally, we observe that boiling is depressed in the evaporator region. These results verify the benefits of nanostructured wicks. This simulation tool provides the groundwork for future studies of 3D flat package heat pipes.© 2009 ASME

On-chip isotachophoresis for separation of ions and purification of nucleic acids.

We present a simple-to-implement method for analyzing images of randomly distributed particles transported through a fluidic channel. We term this method particle imaging, tracking and collocation (PITC). Our method uses off-the-shelf optics including a CCD camera, epifluorescence microscope, and a dual-view color separator to image freely suspended particles in a wide variety of microchannels (with optical access for image collection). The particles can be transported via electrophoresis and/or pressure driven flow to increase throughput of analysis. We here describe the implementation of the algorithm and demonstrate and validate three of its capabilities: (1) identification of particle coordinates, (2) tracking of particle motion, and (3) monitoring of particle interaction via collocation analysis. We use Monte Carlo simulations for validation and optimization of the input parameters. We also present an experimental demonstration of the analysis on challenging image data, including a flow of two, interacting Brownian particle populations. In the latter example, we use PITC to detect the presence of target DNA by monitoring the hybridization-induced binding between the two populations of beads, each functionalized with DNA probes complementary to the target molecule.