Cross-linked Polyacrylamide Gel Electrophoresis of Single-stranded DNA for Microfabricated Genomic Analysis Systems

Electrophoresis. May, 2002  |  Pubmed ID: 12116155

Microfabricated devices are poised to offer inexpensive self-contained alternatives to conventional benchtop-scale laboratory equipment for performing a variety of important DNA analysis assays. In order to realize the dramatic cost savings possible through photolithographic fabrication techniques, these devices must occupy an extremely compact footprint on the silicon wafer. This requirement implies that electrophoretic separations must be performed over ultrashort distances. Employing cross-linked polyacrylamide gels in place of conventional uncross-linked sieving media offers a convenient strategy to achieve this goal. In this paper, we show how the increased resolving power offered by cross-linked polyacrylamide gels, along with improved sample injection techniques, can be exploited to enhance separation performance in microscale systems. We use these techniques to perform high-resolution gel electrophoresis of single-stranded DNA fragments in microfabricated devices over separation distances of 1.5 cm or less. The results presented here are in agreement with theoretical predictions and suggest that it is possible to perform DNA sequencing on compact microchips. More importantly, the separation performance demonstrated in this work is already more than adequate to perform a number of important genomic assays imposing less stringent resolution requirements than sequencing. Successfully adapting even a few of these assays to the microdevice format has the potential to provide a new generation of inexpensive and portable devices suitable for direct end-user applications.

Microdevice-based Measurements of Diffusion and Dispersion in Cross-linked and Linear Polyacrylamide DNA Sequencing Gels

Electrophoresis. Aug, 2002  |  Pubmed ID: 12210182

We use microfabricated gel electrophoresis devices incorporating integrated on-chip electrodes, heaters, and temperature sensors to measure diffusion and dispersion of single-stranded DNA fragments in cross-linked and uncross-linked polyacrylamide gels. The microdevice format allows a complete set of diffusion and dispersion data to be collected in approximately one hour. These results are compared with corresponding data obtained in a macroscale DNA sequencer, and the effects of gel composition and initiation chemistry are explored. Although the diffusion and dispersion data exhibit similar qualitative trends both on chip and on the macroscale, the magnitudes of the coefficients measured in the microdevice are somewhat higher. This discrepancy is likely due to altered polymerization kinetics arising as a consequence of using a UV-initiated polymerization chemistry to cast the on-chip gels as opposed to the standard chemical polymerization employed on the macroscale. We also find that reductions in the magnitudes of diffusion and dispersion coefficients are achieved at higher polymer concentrations and at operating temperatures in the vicinity of 50 degrees C. Finally, we find that cross-linked polyacrylamide gels yield significantly lower diffusion and dispersion coefficients than linear polyacrylamide. These findings can be used to identify rational strategies to improve separation performance in both micro- and macroscale gel electrophoresis systems.

PCR in a Rayleigh-Bénard Convection Cell

Science (New York, N.Y.). Oct, 2002  |  Pubmed ID: 12399582

A Versatile Microfabricated Platform for Electrophoresis of Double- and Single-stranded DNA

Electrophoresis. Jan, 2003  |  Pubmed ID: 12652585

We demonstrate a versatile microfabricated electrophoresis platform, incorporating arrays of integrated on-chip electrodes, heaters, and temperature sensors. This design allows a range of different sieving gels to be used within the same device to perform separations involving both single- and double-stranded DNA over distances on the order of 1 cm. We use this device to compare linear and cross-linked polyacrylamide, agarose, and thermo-reversible Pluronic-F127 gels on the basis of gel casting ease, reusability, and overall separation performance using a 100 base pair double-stranded DNA ladder as a standard sample. While cross-linked polyacrylamide matrices provide consistently high-quality separations in our system over a wide range of DNA fragment sizes, Pluronic gels also offer compelling advantages in terms of the ability to remove and reload the gel. Agarose gels offer good separation performance, however, additional care must be exercised to ensure consistent gel properties as a consequence of the need for elevated gel loading temperatures. We also demonstrate the use of denaturing cross-linked polyacrylamide gels at concentrations up to 19% to separate single-stranded DNA fragments ranging in size from 18 to 400 bases in length. Primers differing by 4 bases at a read length of 30 bases can be separated with a resolution of 0.9-1.0 in under 20 min. This level of performance is sufficient to conduct a variety of genotyping assays including the rapid detection of single nucleotide polymorphisms (SNPs) in a microfabricated platform. The ability to use a single microelectrophoresis system to satisfy a wide range of separation applications offers molecular biologists an unprecedented level of flexibility in a portable and inexpensive format.

Integrated Microsystems for Controlled Drug Delivery

Advanced Drug Delivery Reviews. Feb, 2004  |  Pubmed ID: 14741115

Efficient drug delivery and administration are needed to realize the full potential of molecular therapeutics. Integrated microsystems that incorporate extremely fast sensory and actuation capabilities can fulfill this need for efficient drug delivery tools. Photolithographic technologies borrowed from the semiconductor industry enable mass production of such microsystems. Rapid prototyping allows for the quick development of customized devices that would accommodate for diverse therapeutic requirements. This paper reviews the capabilities of existing microfabrication and their applications in controlled drug delivery microsystems. The next generation of drug delivery systems--fully integrated and self-regulating--would not only improve drug administration, but also revolutionize the health-care industry.

Printed Circuit Technology for Fabrication of Plastic-based Microfluidic Devices

Analytical Chemistry. Jun, 2004  |  Pubmed ID: 15167806

One of the primary advantages of using plastic-based substrates for microfluidic systems is the ease with which devices can be fabricated with minimal dependence on specialized laboratory equipment. These devices are often produced using soft lithography techniques to cast replicas of a rigid mold or master incorporating a negative image of the desired surface structures. Conventional photolithographic micromachining processes are typically used to construct these masters in either thick photoresist, etched silicon, or etched glass substrates. The speed at which new masters can be produced using these techniques, however, can be relatively slow and often limits the rate at which new device designs can be built and tested. In this paper, we show that inexpensive photosensitized copper clad circuit board substrates can be employed to produce master molds using conventional printed circuit technology. This process offers the benefits of parallel fabrication associated with photolithography without the need for cleanroom facilities, thereby providing a degree of speed and simplicity that allows microfluidic master molds with well-defined and reproducible structural features to be constructed in approximately 30 min in any laboratory. Precise control of channel heights ranging from 15 to 120 microm can be easily achieved through selection of the appropriate copper layer thickness, and channel widths as small as 50 microm can be reproducibly obtained. We use these masters to produce a variety of plastic-based microfluidic channel networks and demonstrate their suitability for DNA electrophoresis and microfluidic mixing studies.

Microfabricated Electrophoresis Systems for DNA Sequencing and Genotyping Applications: Current Technology and Future Directions

Philosophical Transactions. Series A, Mathematical, Physical, and Engineering Sciences. May, 2004  |  Pubmed ID: 15306487

Many routine genomic-analysis assays rely on gel electrophoresis to perform size-selective fractionation of DNA fragments in the size range below 1 kb in length. Over the past decade, impressive progress has been made towards the development of microfabricated electrophoresis systems to conduct these assays in a microfluidic lab-on-a-chip format. Since these devices are inexpensive, require only nanolitre sample volumes, and do not rely on the availability of a pre-existing laboratory infrastructure, they are readily deployable in remote field locations for use in a variety of medical and biosensing applications. The design and construction of microfabricated electrophoresis devices poses a variety of challenges, including the need to achieve high-resolution separations over distances of a few centimetres or less, and the need to easily interface with additional microfluidic components to produce self-contained integrated DNA-analysis systems. In this paper, we review recent efforts to develop devices to satisfy these requirements and live up to the promise of fulfilling the growing need for inexpensive portable genomic-analysis equipment.

Reactions and Fluidics in Miniaturized Natural Convection Systems

Analytical Chemistry. Nov, 2004  |  Pubmed ID: 15516116

Buoyancy-driven convection offers a novel and greatly simplified mechanism for generating continuous nonpulsatile flow fields and performing thermally activated biochemical reactions. In this paper, we build on our previous work by constructing a multiwell device incorporating an array of 35-microL cylindrical cavities to perform polymerase chain reaction (PCR) amplification of a 191-base pair fragment associated with membrane channel proteins M1 and M2 of the influenza-A virus in as little as 15 min with performance comparable to conventional thermocyclers. We also describe entirely new adaptations of convective flows by conducting a series of coordinated flow visualization and computational studies to explore the design of closed-loop systems to execute tunable thermocycling, pumping, and mixing operations in a format suitable for integration into miniaturized biochemical analysis systems. Using 15-microL convective flow loops, we are able to perform PCR amplification of the same 191-base pair fragment associated with the influenza-A virus, as well as a 295-base pair segment of the human beta-actin gene in a format offering an enhanced degree of control and tunability. These convective flow devices can be further scaled down to nanoliter volumes and are ideally suited as a platform for a new generation of low-power, portable microfluidic DNA analysis systems.

Thermoplastic Elastomer Gels: an Advanced Substrate for Microfluidic Chemical Analysis Systems

Analytical Chemistry. Aug, 2005  |  Pubmed ID: 16097755

We demonstrate the use of thermoplastic elastomer gels as advanced substrates for construction of complex microfluidic networks suitable for use in miniaturized chemical analysis systems. These gels are synthesized by combining inexpensive polystyrene-(polyethylene/polybutylene)-polystyrene triblock copolymers with a hydrocarbon extender oil for which the ethylene/butylene midblocks are selectively miscible. The insoluble styrene end blocks phase separate into localized nanodomains, resulting in the formation of an optically transparent, viscoelastic, and biocompatible gel network that is melt-processable at temperatures in the vicinity of 100 degrees C. This unique combination of properties allows microfluidic channels to be fabricated in a matter of minutes by simply making impressions of the negative relief structures on heated master molds. Melt processability allows multiple impressions to be made against different masters to construct complex geometries incorporating multi-height features within the same microchannel. Intricate interconnected multilayered structures are also easily fabricated owing to the ability to bond and seal multiple layers by briefly heating the material at the bond interface. Thermal and mechanical properties are tunable over a wide range through proper selection of gel composition.

Separation Performance of Single-stranded DNA Electrophoresis in Photopolymerized Cross-linked Polyacrylamide Gels

Electrophoresis. Feb, 2006  |  Pubmed ID: 16331587

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models.

An Inexpensive Microslab Gel DNA Electrophoresis System with Real-time Fluorescence Detection

Electrophoresis. Feb, 2006  |  Pubmed ID: 16342324

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.

DNA Mutation Detection and Analysis Using Miniaturized Microfluidic Systems

Expert Review of Molecular Diagnostics. Jan, 2006  |  Pubmed ID: 16359265

Identification of genetic sequence variations occurring on a population-wide scale is key to unraveling the complex interactions that are the underlying cause of many medical disorders and diseases. A critical need exists, however, for advanced technology to enable DNA mutation analysis to be performed with significantly higher throughput and at significantly lower cost than is currently attainable. Microfluidic systems offer an attractive platform to address these needs by combining the ability to perform rapid analysis with a simplified device format that can be inexpensively mass-produced. This paper will review recent progress toward developing these next-generation systems and discuss challenges associated with adapting these technologies for routine laboratory use.

Fluid Mixing in Planar Spiral Microchannels

Lab on a Chip. Jan, 2006  |  Pubmed ID: 16372072

Mixing of fluids at the microscale poses a variety of challenges, many of which arise from the fact that molecular diffusion is the dominant transport mechanism in the laminar flow regime. While considerable progress has been made toward developing strategies to achieve improved mixing in microfluidic systems, many of these techniques introduce additional complexity to device fabrication and/or operation processes. In this work, we explore the use of compact spiral-shaped flow geometries designed to achieve efficient mixing in a format that can be constructed using a single planar soft lithography step without the need for multilayer alignment. A series of 150 microm-wide by 29 microm-tall channels were constructed, each of which incorporated a series of spiral shaped sections arrayed along the flow path. Five spiral designs with varying channel lengths were investigated, and mixing studies were carried out at flow rates corresponding to Reynolds numbers ranging from 0.02 to 18.6. Under appropriate conditions, transverse Dean flows are induced that augment diffusive transport and promote enhanced mixing in considerably shorter downstream distances as compared with conventional planar straight channel designs. Mixing efficiency can be further enhanced by incorporating expansion vortex effects via abrupt changes in cross-sectional area along the flow path.

Polymerase Chain Reaction in Miniaturized Systems: Big Progress in Little Devices

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 16508068

A critical need exists for advanced technologies that enable genomic-based DNA analysis to be performed with significantly higher throughput and at a significantly lower cost than is attainable with current hardware. Miniaturized polymerase chain reaction systems offer an attractive platform to address these needs, combining the ability to perform rapid thermocycling with a portable device format that can be inexpensively mass produced. We review recent efforts aimed at developing these next-generation systems and discuss some of the practical considerations involved in adapting this technology for routine laboratory use.

Collection, Focusing, and Metering of DNA in Microchannels Using Addressable Electrode Arrays for Portable Low-power Bioanalysis

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2006  |  Pubmed ID: 16551750

Although advances in microfluidic technology have enabled increasingly sophisticated biosensing and bioassay operations to be performed at the microscale, many of these applications employ such small amounts of charged biomolecules (DNA, proteins, and peptides) that they must first be preconcentrated to a detectable level. Efficient strategies for precisely handling minute quantities of biomolecules in microchannel geometries are critically needed; however, it has proven challenging to achieve simultaneous concentration, focusing, and metering capabilities with current-generation sample-injection technology. By using microfluidic chips incorporating arrays of individually addressable microfabricated electrodes, we demonstrate that DNA can be sequentially concentrated, focused into a narrow zone, metered, and injected into an analysis channel. This technique transports charged biomolecules between active electrodes upon application of a small potential difference (1 V) and is capable of achieving orders of magnitude concentration increases within a small device footprint. The collected samples are highly focused, with sample zone size and shape defined solely by electrode geometry.

Multivortex Micromixing

Proceedings of the National Academy of Sciences of the United States of America. May, 2006  |  Pubmed ID: 16645036

The ability to mix liquids in microchannel networks is fundamentally important in the design of nearly every miniaturized chemical and biochemical analysis system. Here, we show that enhanced micromixing can be achieved in topologically simple and easily fabricated planar 2D microchannels by simply introducing curvature and changes in width in a prescribed manner. This goal is accomplished by harnessing a synergistic combination of (i) Dean vortices that arise in the vertical plane of curved channels as a consequence of an interplay between inertial, centrifugal, and viscous effects, and (ii) expansion vortices that arise in the horizontal plane due to an abrupt increase in a conduit's cross-sectional area. We characterize these effects by using confocal microscopy of aqueous fluorescent dye streams and by observing binding interactions between an intercalating dye and double-stranded DNA. These mixing approaches are versatile and scalable and can be straightforwardly integrated as generic components in a variety of lab-on-a-chip systems.

Using in Situ Rheology to Characterize the Microstructure in Photopolymerized Polyacrylamide Gels for DNA Electrophoresis

Electrophoresis. Sep, 2006  |  Pubmed ID: 16892481

Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions.

Development of a Miniature Calorimeter for Identification and Detection of Explosives and Other Energetic Compounds

Journal of Hazardous Materials. Apr, 2007  |  Pubmed ID: 17034941

The development of versatile systems capable of providing rapid, portable, and inexpensive detection of explosives and energetic compounds are critically needed to offer enhanced levels of protection against current and future threats to homeland security, as well as satisfying a wide range of applications in the fields of forensic analysis, emergency response, and industrial hazards analysis. Calorimetric techniques have been largely overlooked in efforts to develop advanced chemical analysis technology, largely because of limitations associated with the physical size of the instruments and the relatively long timescales (>30 min) required to obtain a result. This miniaturized calorimeter circumvents these limitations, thereby creating a first-of-its-kind system allowing thermal analysis to be performed in a portable format that can be configured for use in a variety of field operations with a significantly reduced response time (approximately 2 min). Unlike current explosives detectors, this system is based on calorimetric techniques that are inherently capable of providing direct measurements of energy release potential and therefore do not depend on prior knowledge of familiar compounds.

A Buoyancy-driven Compact Thermocycler for Rapid PCR

Clinics in Laboratory Medicine. Mar, 2007  |  Pubmed ID: 17416315

The authors have designed a novel convective flow-based thermocycling system capable of performing high-speed DNA amplification via the polymerase chain reaction in a simplified and inexpensive format. Successful amplification of a 191 bp influenza-A target is demonstrated within 25 minutes using a 10 muL reaction volume with no modification to standard laboratory protocols. The system is simple to assemble and can be readily integrated with existing laboratory instrumentation for automated operation.

A Pocket-sized Convective PCR Thermocycler

Angewandte Chemie (International Ed. in English). 2007  |  Pubmed ID: 17465434

Preface to Special Topic: Papers from the 2006 Annual Meeting of the American Electrophoresis Society, San Francisco, CA

Biomicrofluidics. 2007  |  Pubmed ID: 19693374

This Special Topic section of Biomicrofluidics is dedicated to original papers from the 2006 Annual Meeting of the American Electrophoresis Society (AES: http:/www.aesociety.org). This five-day meeting held in San Francisco, California, included five sessions on BioMEMS and Microfluidics and four sessions on Advances in Electrokinetics and Electrophoresis. AES and its corresponding symposia provide the most focused and well-organized meeting forum for diverse biological and engineering researchers working on electrokinetics. The work featured in this Special Topic section is no exception; it ranges from nanochannel electrophoresis to bioparticle sorting.

BioMEMS and Electrophoresis in 2006: Review of the 23rd Annual Meeting of the American Electrophoresis Society

Biomicrofluidics. 2007  |  Pubmed ID: 19693377

The 23rd Annual Meeting of the American Electrophoresis Society (AES) was held at the San Francisco Hilton in San Francisco, California on 12-17 November 2006. This year's meeting featured a look toward the future, with an emphasis on theoretical and experimental advances in miniaturization of BioMEMS, electrokinetics, and proteomics technologies. A total of 13 sessions accommodating 71 presentations and 18 posters were held in conjunction with the Annual Meeting of the American Institute of Chemical Engineers (AIChE). This review and corresponding special issue of Biomicrofluidics provide a sampling of some of the exciting research presented at the conference.

Review of Electrophoresis and BioMEMS in 2007: American Electrophoresis Society 24th Annual Meeting

Journal of Capillary Electrophoresis and Microchip Technology. 2008  |  Pubmed ID: 18982910

Researchers came together for the 24th Annual Meeting of the American Electrophoresis Society (AES), which was held November 4-9, 2007, at the Salt Palace Convention Center in Salt Lake City, UT, U.S.A. The Annual AES meeting is held in conjunction with the annual meeting of the American Institute of Chemical Engineers (AIChE). This year's meeting had a significant emphasis on theoretical and experimental advances in Biological Micro Electro Mechanical Systems (BioMEMS), electrokinetics, and proteomics technologies. A total of 15 sessions were held, within which 71 presentations and 18 posters were discussed. This review provides a brief sampling of the exciting research presented at the conference.

Microchip DNA Electrophoresis with Automated Whole-gel Scanning Detection

Lab on a Chip. Dec, 2008  |  Pubmed ID: 19023477

Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment.

Investigating DNA Migration in Pulsed Fields Using a Miniaturized FIGE System

Electrophoresis. Dec, 2008  |  Pubmed ID: 19053074

PFGE is a well-established technique for fractionation of DNA fragments ranging from kilobases to megabases in length. But many of these separations require an undesirable combination of long experiment times (often approaching tens of hours) and application of high voltages (often approaching tens of kV). Here, we present a simple miniaturized FIGE apparatus capable of separating DNA fragments up to 32.5 kb in length within 3 h using a modest applied potential of 20 V. The device is small enough to be imaged under a fluorescence microscope, permitting the migrating DNA bands to be observed during the course of the separation run. We use this capability to investigate how separation performance is affected by parameters including the ratio of forward and backward voltage, pulse time, and temperature. We also characterize the dependence of DNA mobility on fragment size N, and observe a scaling in the vicinity of N(-0.5) over the size range investigated. The high speed, low power consumption, and simple design of this system may help enable future studies of DNA migration in PFGE to be performed quickly and inexpensively.

DNA Focusing Using Microfabricated Electrode Arrays

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19488694

Focusing methods are a key component in many miniaturized DNA analysis systems because they enable dilute samples to be concentrated to detectable levels while being simultaneously confined within a specified volume inside the microchannel. In this chapter, we describe a focusing method based on a device design incorporating arrays of addressable on-chip microfabricated electrodes that can locally increase the concentration of DNA in solution by electrophoretically sweeping it along the length of a microchannel. By applying a low voltage (approximately 1-2 V) between successive pairs of neighboring electrodes, the intrinsically negatively charged DNA fragments are induced to migrate toward and collect at each anode, thereby allowing the quantity of accumulated DNA to be precisely metered. We have characterized the kinetics of this process, and found the response to be robust over a range of different sample compositions and buffer environments.

Interfacial Complexation Explains Anomalous Diffusion in Nanofluids

Nano Letters. Feb, 2010  |  Pubmed ID: 20050689

A recent report describing dramatic anomalous enhancement in mass transport properties of nanofluids (>1000% increase in tracer dye diffusivity) has excited intense interest, but the findings have yet to be conclusively confirmed or explained. Here we investigate these phenomena using a microfluidic approach to directly probe tracer diffusion so that interactions between the suspension's principle components (nanoparticles, surfactant, and dye) can be clearly identified. Under conditions matching previously reported studies, we unexpectedly observe spontaneous formation of highly focused and intensely fluorescent plumes at the interface between fluid streams, suggesting strong complexation interactions between the dye and nanoparticles. These phenomena, driven by competition between the rates at which free tracer molecules are transported into the interfacial zone subsequently consumed by dye-nanoparticle complexation, have likely been incorrectly interpreted as anomalous diffusion enhancement. These interactions are important to consider when devising tracer-based studies of nanoparticle suspensions and may lay a foundation for new adsorption-based analytical methods.

Miniaturized System for Rapid Field Inversion Gel Electrophoresis of DNA with Real-time Whole-gel Detection

Analytical Chemistry. Mar, 2010  |  Pubmed ID: 20148578

Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (approximately 10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60-90 min. The compact size of the device also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields.

Vortex-assisted DNA Delivery

Lab on a Chip. Aug, 2010  |  Pubmed ID: 20563345

Electroporation is one of the most widely used methods to deliver exogenous DNA payloads into cells, but a major limitation is that only a small fraction of the total membrane surface is permeabilized. Here we show how this barrier can be easily overcome by harnessing hydrodynamic effects associated with Dean flows that occur along curved paths. Under these conditions, cells are subjected to a combination of transverse vortex motion and rotation that enables the entire membrane surface to become uniformly permeabilized. Greatly improved transfection efficiencies are achievable with only a simple modification to the design of existing continuous flow electroporation systems.

A Direct Probe of the Interplay Between Bilayer Morphology and Surface Reactivity in Polymersomes

Langmuir : the ACS Journal of Surfaces and Colloids. Jul, 2010  |  Pubmed ID: 20578755

Bilayer vesicles self-assembled from amphiphilic poly(ethylene oxide)-b-polybutadiene (PEO-b-PBd) copolymers are cell-like structures whose high stability and tunable membrane properties make them ideal for use as potential drug carriers and cell mimicry templates. Understanding how the surface interactions (reaction, binding, etc.) are governed by the bilayer structure is critical to enable construction of polymersomes with tailored colloidal behavior. Here, we adapt a previously established chemical labeling method by incorporating coumarin functionalized copolymer into the vesicular structure. This allows us to probe the effect of poly(ethylene glycol) (PEG) brush and surface architecture on the bimolecular quenching reaction occurring at the polymersome surface. Using these measurements, we have tracked quenching in free solution, on bare particles, and on two types of vesicle surfaces: one where the functionalized copolymer groups are longer than the surrounding unfunctionalized copolymer, and one where both functionalized and unfunctionalized groups are the same length. We find that quenching in the presence of the PEG brush proceeds at less than half the free solution rate in both vesicle architectures. However, the quenching rate is further reduced when the functionalized and unfunctionalized groups are the same length. The surface reaction appears to be dominated by quencher diffusion, a conclusion supported by conductivity measurements and ion partition studies indicating that these effects arise as a consequence of retarded ion mobility in the presence of the PEG brush rather than ion exclusion effects. These studies reveal the interplay between the vesicle bilayer architecture (copolymer composition, chain length, local concentration surrounding the active site) and the surface reaction rate, thereby providing useful insights that can help guide the design of polymersomes with desired functional properties.

Reflection Interference Contrast Microscopy of Arbitrary Convex Surfaces

Applied Optics. Jul, 2010  |  Pubmed ID: 20648136

Current accurate applications of reflection interference contrast microscopy (RICM) are limited to known geometries; when the geometry of the object is unknown, an approximated fringe spacing analysis is usually performed. To complete an accurate RICM analysis in more general situations, we review and improve the formulation for intensity calculation based on nonplanar interface image formation theory and develop a method for its practical implementation in wedges and convex surfaces. In addition, a suitable RICM model for an arbitrary convex surface, with or without a uniform layer such as a membrane or ultrathin coating, is presented. Experimental work with polymer vesicles shows that the coupling of the improved RICM image formation theory, the calculation method, and the surface model allow an accurate reconstruction of the convex bottom shape of an object close to the substrate by fitting its experimental intensity pattern.

Tailoring the Nanoporous Architecture of Hydrogels to Exploit Entropic Trapping

Physical Review Letters. Sep, 2010  |  Pubmed ID: 20867549

Macromolecules embedded in a nanoporous matrix display anomalous transport behavior in the entropic trapping regime. But these phenomena have not been widely explored in hydrogel matrices because it has not been clear how to link them to the underlying heterogeneous nanopore morphology. Here we introduce a theoretical model that establishes this connection and describe microchip DNA electrophoresis experiments that demonstrate how entropic trapping effects can be exploited to yield a trend of increasing resolving power with DNA size (the opposite of what is conventionally observed).

Chaotically Accelerated Polymerase Chain Reaction by Microscale Rayleigh-Bénard Convection

Angewandte Chemie (International Ed. in English). Mar, 2011  |  Pubmed ID: 21404396

Tunable Synthesis of Encapsulated Microbubbles by Coupled Electrophoretic Stabilization and Electrochemical Inflation

Angewandte Chemie (International Ed. in English). Apr, 2011  |  Pubmed ID: 21442695