Travis D. Boone

Plastic microfluidic devices: Electrokinetic manipulations, life science applications, and production technologies

Publisher Summary This chapter describes the development and current technological status of a particular subset of microfluidic systems, namely those chips made from organic polymers and utilizing electrokinetic techniques for fluidic motivation and analyte separation. Microfluidic systems are interconnected networks of channels and reservoirs containing nano and microliter volumes. They are particularly well suited to biological assay applications that demand highly parallel, rapid, accurate, low-volume experiments requiring minimal crosscontamination. The DC electrokinetic technique of capillary electrophoresis (CE) is the application of a homogeneous electric field between the ends of a small diameter column, providing a means both to transport and to separate dissolved species in ionically conductive solutions. The surface properties of the plastic are critical to device performance, with changes in morphology or chemistry contributing to variations in adsorption. Typical plastics used for microfluidic devices are thermoplastics. Microfluidic devices demand extreme uniformity of channels that are long compared to their lateral dimensions, as well as accurate replication of micrometer-scale features, challenging production methods. A variety of different plastic devices can be successfully realized using a combination of (MEMS) microfabrication technologies and traditional plastics processing. Thermoplastic microfluidic devices made by any of the molding or embossing methods sealed with a cover to create closed capillaries, minimizing evaporation during analytical applications of the device. Plastic microfluidic devices offer several advantages over glass or silicon structures, including lower processing temperatures, high-volume manufacture at low cost, and simpler extension to multilayer device fabrication.

Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station

The International Space Station (ISS) National Laboratory is dedicated to studying the effects of space on life and physical systems, and to developing new science and technologies for space exploration. A key aspect of achieving these goals is to operate the ISS National Lab more like an Earth-based laboratory, conducting complex end-to-end experimentation, not limited to simple microgravity exposure. Towards that end NASA developed a novel suite of molecular biology laboratory tools, reagents, and methods, named WetLab-2, uniquely designed to operate in microgravity, and to process biological samples for real-time gene expression analysis on-orbit. This includes a novel fluidic RNA Sample Preparation Module and fluid transfer devices, all-in-one lyophilized PCR assays, centrifuge, and a real-time PCR thermal cycler. Here we describe the results from the WetLab-2 validation experiments conducted in microgravity during ISS increment 47/SPX-8. Specifically, quantitative PCR was performed on a concentration series of DNA calibration standards, and Reverse Transcriptase-quantitative PCR was conducted on RNA extracted and purified on-orbit from frozen Escherichia coli and mouse liver tissue. Cycle threshold (Ct) values and PCR efficiencies obtained on-orbit from DNA standards were similar to Earth (1 g) controls. Also, on-orbit multiplex analysis of gene expression from bacterial cells and mammalian tissue RNA samples was successfully conducted in about 3 h, with data transmitted within 2 h of experiment completion. Thermal cycling in microgravity resulted in the trapping of gas bubbles inside septa cap assay tubes, causing small but measurable increases in Ct curve noise and variability. Bubble formation was successfully suppressed in a rapid follow-up on-orbit experiment using standard caps to pressurize PCR tubes and reduce gas release during heating cycles. The WetLab-2 facility now provides a novel operational on-orbit research capability for molecular biology and demonstrates the feasibility of more complex wet bench experiments in the ISS National Lab environment.

Multiplexed, Disposable, Plastic Microfluidic Systems for High-Throughput Applications

Low-cost, mass-produced, plastic microfluidic devices are being developed for a variety of microfluidic applications including DNA analysis, drug discovery, and clinical diagnostics. Plastic substrates with complex patterns of 10–100μm-sized channels are reproducibly formed against mold tools fabricated by micromachining techniques. These substrates are used to produce plastic devices on which reactions and high-efficiency electrophoretic separations of biomolecules have been achieved in timescales of seconds to minutes.

Disposable Plastic Microfluidic Arrays for Applications in Biotechnology

Plastic microfluidic bioanalytical device arrays offer biochemical compatibility, low-cost mass production, and single-use disposability. We have designed, modeled, prototyped, and manufactured a range of plastic microfluidic devices for bioanalytical applications including DNA sequencing, nucleic acid fragment analysis, and high-throughput screening of pharmaceutical candidate compounds.

Plastic Microfluidic Systems for High-Throughput Genomic Analysis and Drug Screening

Genomic analysis and drug discovery depend increasingly on rapid, accurate analysis of large sets of sample and extensive compound collections at relatively low cost. By capitalizing on advances in microfabrication, genomics, combinatorial chemistry, and assay technologies, new analytical systems are expected to provide order-of-magnitude increases in analysis throughput along with comparable decreases in per-sample analysis costs. ACLARA’s single-use, plastic LabCard systems, which transport fluids between reservoirs and through interconnected microchannels using electrokinetic mechanisms, are intended to address these analytical needs. These devices take advantage of recent developments in microfluidic and microfabrication technologies to permit their application to DNA sequencing; genotyping and DNA fragment analysis, as well as pharmaceutical candidate screening, and preparing biological samples for analysis. In a parallel effort, ACLARA has developed a new class of reporter molecules that are particularly well suited to capillary electrophoretic analysis. These electrophoretic mobility tags, called eTag reporters, can be used to uniquely label multiplexed sets of oligonucleotide recognition probes or proteins, thereby permitting traditionally homogeneous biochemical reporter assays to be multiplexed for CE analysis. Biochemical multiplexing is key to achieving new thresholds in analytical throughput while maintaining economically viable formats in many application areas. ACLARA’s microfluidic, lab-on-a-chip concept promises to revolutionize chemical analysis, similar to the way miniaturization revolutionized computing, making tools continually smaller, more integrated, less expensive, and higher performing. Microfluidic devices are made up of interconnected networks of microchannels and tiny volume reservoirs in which all the processes required for single analyses may be miniaturized, integrated, and automated within a single substrate the size of a human hand or smaller. However, LabCard devices are more than tiny replicates of existing equipment. They are characterized by high speed, parallel analysis using designs devised to eliminate sample cross contamination while automating processes that are otherwise undesirably cumbersome at the macro laboratory scale. Electrophoresis is one of the most widely used analytical separation methodologies for life science research. Electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules that have different charge-to-mass ratios such as proteins; or, molecules that have similar charge-to-mass ratios but different masses such as DNA fragments. In recent years, the development of capillary electrophoresis (CE), which is performed in a fused silica capillary filled with a buffer or polymer solution, has increased the speed of analytical separations. Planar, microchannel devices offer further improvements over capillary electrophoresis. In general, both electrophoresis and electroosmosis occur when a high electric field is applied along a microchannel (Figure 1). In practice, these effects can be comparable in magnitude, in which case ions of one charge move rapidlyat velocities of millimeters per second-while those of opposite charge move backward or slowly forward, depending upon which of the two effects dominates. The amount of fixed charge on the inner surface of a channel can be controlled by pH, specific adsorption of charged species onto the surface, or surface chemical modification. Therefore, the contribution of electroosmosis (the pumping mechanism) can be tuned relative to electrophoresis (the separating mechanism). The capability to transport and separate exceptionally small liquid volumes with precise electrical control is a powerful tool, which is complemented by an additional benefit of using microchannels with micrometer cross-sections. While electrophoresis channels are of similar cross section in both capillaries and microchannel devices, the planar format of the microchannel devices enables structures more complex than a single, non-intersecting channel to be used. Two or more channels can intersect to

Plastic Microfluidic Devices for DNA Sequencing and Protein Separations

Plastic microfluidic devices with 18-cm separation length and an offset “double-T” injection system were used to achieve DNA sequencing in 30 minutes with 98%-correct identification to a read length of 650 bases. We recently demonstrated two proteomics-enabling technologies on plastic fluidic devices: isoelectric focusing (IEF) and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).

Sub-Microliter Assays and DNA Analysis on Plastic Microfluidics

We report the design, fabrication, and performance characterization of plastic microfluidic systems for conducting (1) highly parallel enzyme-based assays using sub-microliter reagent volumes and (2) high-speed, high-resolution separation of double- and single-stranded DNA fragments for applications including genotyping and sequencing. To reduce reagent consumption, we have developed a zero-dead-volume dispensing technology for the simultaneous parallel pickup of nL volumes of 96 different reagents from a “library plate” and rapid delivery to a microfluidic assay card. We also demonstrate the capability to perform unlidded 200nL assays with one hour incubations. Recent DNA sequencing results are reported: up to 700 single-stranded DNA fragments are detected with single-base resolution in 40 minutes using a plastic microfluidic device with a 4-color detection system.