This paper describes composite patterning elements that use a commercially available acryloxy perfluoropolyether (a-PFPE) in various soft lithographic techniques, including microcontact printing, nanotransfer printing, phase-shift optical lithography, proximity field nanopatterning, molecular scale soft nanoimprinting, and solvent assisted micromolding. The a-PFPE material, which is similar to a methacryloxy PFPE (PFPE-DMA) reported recently, offers a combination of high modulus (10.5 MPa), low surface energy (18.5 mNm(-1)), chemical inertness, and resistance to solvent induced swelling that make it useful for producing high fidelity patterns with these soft lithographic methods. The results are comparable to, and in some cases even better than, those obtained with the more widely explored material, high modulus poly(dimethylsiloxane) (h-PDMS).

We describe molecular-scale soft nanoimprint lithographic replication of rubbed polyimide substrates to form alignment layers for liquid crystal devices. Systematic studies of the surface relief morphology of the polyimide and molded structures in three different polymers illustrate good lithographic fidelity down to relief heights of several nanometers, and with some capabilities at the level of approximately 1 nm. Collective results of experiments with several polymer formulations for molds and molded materials and process conditions indicate that this molecular-scale fidelity in replication can be used to produce surfaces that will effectively align liquid crystal molecules. Good electro-optical responses from liquid crystal light modulators that are formed in this manner suggest utility for fundamental studies and potential practical application.

Fraction collection following electrophoresis is of major importance for a variety of biological analyses. These assays typically need to identify specific fractions in the separated sample for further processing and require extraction of one or a group of fragments. In this paper, we have developed and characterized a technique to generate addressable electric fields for improved extraction during electrophoresis in microfluidic devices. The addressable electric field is achieved by applying a low bias voltage (1-2 V) to microelectrode pairs within the electrophoresis microchannel. Theoretical analysis shows the purity of the extracted sample can be improved as much as 30% over extraction without the shaped electric fields, and nearly 100% predicted yield can be achieved. We also describe the theoretical design of shaped electric fields by characterizing the optimal electrode geometry, field strength, channel configuration, and electrophoretic migration behavior needed for efficient band extraction.

Polymer-based microfluidic devices offer an attractive platform for single-use disposable applications due to their low cost, ease of fabrication and good biocompatibility. In this work, we investigated liquid handling in surface modified polyolefin microfluidic devices. The modification of the surface was accomplished using ultraviolet light, and the contact angle was reduced from 88° to 45°. This type of treatment is easy to implement and could be beneficial for liquid handling in microchannel networks. Capillary-driven flow, contact angle hysteresis and pulsed pumping were demonstrated in these plastic devices. This surface treatment also facilitates rapid gel loading for separation since viscous sieving media can be injected solely by capillary force. Nucleic acid separation was demonstrated in the gel-loaded devices.

An integrated microfluidic device capable of performing a variety of genetic assays has been developed as a step towards building systems for widespread dissemination. The device integrates fluidic and thermal components such as heaters, temperature sensors, and addressable valves to control two nanoliter reactors in series followed by an electrophoretic separation. This combination of components is suitable for a variety of genetic analyses. As an example, we have successfully identified sequence-specific hemagglutinin A subtype for the A/LA/1/87 strain of influenza virus. The device uses a compact design and mass production technologies, making it an attractive platform for a variety of widely disseminated applications.

We report initial steps towards development of microfluidic devices for detection and genotyping of influenza A viruses, by extending a design by Burns et al. [Science 282 (1998) 484] for human genotyping. Our first new device for influenza A genotyping carries out two reactions in series as well as an electrophoretic gel separation in a glass-silicon device around 3 cm wide and 10 cm long. Construction of the device utilizes solid-state electronics microfabricated in silicon and glass, deposition of resistive heaters for thermal cycling, a reaction chamber for restriction digestion, and a miniature gel separation column, with DNA driven by an electric field produced by miniature microfabricated electrodes. Detection of bands is by fluorescence, with an external light source; photodiode detectors can be either external or microfabricated into the device. The device is tested using restriction digestion on-chip and separating the products on an electrophoresis gel. The possible uses for devices of this type in tracking influenza variants, including H1, H3, H5, H9, and other subtypes, and in early recognition of emergence of variants with worldwide pandemic potential, are explored.

Microfabrication techniques have become increasingly popular in the development of next generation DNA analysis devices. Improved on-chip fluorescence detection systems may have applications in developing portable hand-held instruments for point-of-care diagnostics. Miniaturization of fluorescence detection involves construction of ultra-sensitive photodetectors that can be integrated onto a fluidic platform combined with the appropriate optical emission filters. We have previously demonstrated integration PIN photodiodes onto a microfabricated electrophoresis channel for separation and detection of DNA fragments. In this work, we present an improved detector structure that uses a PINN+ photodiode with an on-chip interference filter and a robust liquid barrier layer. This new design yields high sensitivity (detection limit of 0.9 ng µl−1 of DNA), low-noise (S/N ∼ 100/1) and enhanced quantum efficiencies (>80%) over the entire visible spectrum. Applications of these photodiodes in various areas of DNA analysis such as microreactions (PCR), separations (electrophoresis) and microfluidics (drop sensing) are presented.

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.

We have designed and constructed a microfabricated device for separation of double-stranded DNA fragments using a crosslinked sieving medium and spatially selective extraction of the desired fraction. Based on measuring the width and spacing of migrating bands, a narrow side channel is constructed perpendicular to the separation channel to collect the DNA fragments of interest. This selective collection technique was tested using a 100 base pair double-stranded DNA ladder. We successfully demonstrate selective extraction of the desired fragment with minimal interference from the adjacent bands in an electric field of 31 V/cm. We also achieve extraction of multiple DNA fragments using an array of microelectrodes in this side channel. The device uses cross-linked polyacrylamide gel matrix, allowing the separation to be performed in a distance of 1 cm or less and at a low electric field strength. Together with on-chip electrode, this design is amenable to integration with reaction chambers into a single device for portable genetic-based analysis.

Developing an on-chip fluorescence detection involves construction of ultrasensitive photodetectors that can be integrated onto a fluidic platform. In this work, we report a novel doping and construction of a P-I-N-N + photodiode with an on-chip interference filter and a robust liquid barrier layer. This new design yields a highly sensitive, low-noise photodiode and its applications in several areas of DNA analysis are presented.

Over the past year there have been a number of recent advances in the fields of miniaturized reaction and separation systems, including the construction of fully integrated ‘lab-on-a-chip’ systems. Microreactors, which initially targeted DNA-based reactions such as the polymerase chain reaction, are now used in several other chemical and biochemical assays. Miniaturized separation columns are currently employed for analyzing a wide variety of samples including DNA, RNA, proteins and cells. Although significant advances have been made at the component level, the realization of an integrated analysis system still remains at the early stages of development.