Microfluidic Systems for Integrated, High-Throughput DNA Analysis
Integrating sample preparation and parallel, high-throughput, microchip-based electrophoresis could reduce the cost of obtaining DNA sequence information.
Ryan T. Kelly Adam T. Woolley Brigham Young University
B
ecause DNA is the blueprint for life in all its forms, it is no wonder that nucleic acids are of such interest to scientists. The Human Genome Project, completed in 2003, produced a wealth of information, including the number and average size of human genes, the fraction of the genome that codes for proteins, and the degree of sequence similarity, both among humans and compared with other organisms (1, 2). More importantly, deciphering the human genome should allow researchers to unravel the complex interplay between genetic and environmental factors involved in many diseases, and this should facilitate the development of therapeutic agents (1). The abundance of genomic information has been made possible in large part by improved analytical methods for nucleic acids. If all of the sequencing for the Human Genome Project had been performed with the slab gel technology that was available when the project began, the raw data would have been obtained at a rate of ~600 bases/h (3). The electrophoretic separations for just onefold coverage of the human genome would have taken millions of instrument-hours, in addition to the time required for sample preparation and data analysis. Development of capillary array electrophoresis (CAE), in which multiple samples are electrophoretically analyzed in parallel in a bundle of ~100-µm-diam capillaries, was a major breakthrough in DNA analysis (4 –7 ). High-throughput instruments with parallel capillaries for electro-
© 2005 AMERICAN CHEMICAL SOCIETY
M A R C H 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
97 A
(a)
phoretic separations played a key role in the sequencing of the human genome (8, 9). Even as analytical researchers were reaping the benefits of high-throughput CAE, new technologies were being developed to push DNA analysis further. For example, miniaturized planar CE microdevices (10) were quickly adapted to perform extremely fast (~1-min) DNA separations on individual samples (11, 12). Recent advances have led to portable, integrated genetic analyzers for rapid clinical diagnosis (13) and on-site pathogen detection (14). Furthermore, combining integrated sample preparation with parallel, high-throughput, microchip-based electrophoresis should reduce the cost of obtaining DNA sequence information. Lower costs would enable greater use of genetic data in diagnosing and treating diseases. In this article, we discuss recent developments in the miniaturization of DNA analysis with planar microfabrication technology. Specifically, we highlight improvements to both separations and throughput in micromachined devices. On-chip sample preparation and amplification techniques are discussed, as well as efforts to combine multiple analytical functions into integrated microsystems. Although nucleic acid hybridization arrays are an important area of miniaturized DNA analysis, we do not have sufficient space to cover this area here (15, 16).
(b)
Moat rings (c)
Sample
Separations Since DNA sequencing was first performed on a chip (17 ), several optimization studies have produced high-quality microchip DNA sequencing data with read lengths comparable to those obtained with commercial CAE instruments (18–20). Moreover, the 20–30-min separation times for these experiments compared very favorably with the >1-h separation times obtained with commercial CAE instruments. Recent efforts to improve microchip DNA separations have focused on developing new sieving solutions that are more compatible with the microfluidic format. Between runs, the sieving matrix [typically linear polyacrylamide (LPA)] that separates DNA fragments according to length must be pumped from the channel or capillary and replaced with fresh solution to avoid sample cross-contamination. LPA’s viscosity impedes on-line replenishment and necessitates the application of a significant amount of pressure (>300 psi) to pump the matrix through the microchannels or capillaries (21). In some cases, the high pressure can separate the thermally bonded glass substrates that make up a microchip (22). These issues have led to the development of alternative polymer solutions with desirable sieving properties but lower viscosities. Especially promising has been work with polymers whose viscosities increase abruptly above a critical temperature (22, 23). These polymers can be easily pumped into channels at room temperature; their sieving properties are activated at temperatures above 40 °C, where higher resolution and faster separation are achieved. Such formulations have already produced impressive sequencing separations in capillaries (24) and have rheological properties that are more amenable to the microchip format. 98 A
A N A LY T I C A L C H E M I S T R Y / M A R C H 1 , 2 0 0 5
Cathode Waste (d)
FIGURE 1. Microdevice layout used for 96-channel CAE sequencing. (a) Top view. (b) Vertical cutaway of a CAE microdevice. Concentric plastic rings create separate “moats” used to provide electrical contact to the drilled cathode and waste ports. Expanded views of the (c) injector region and (d) channel turn geometry used to reduce dispersion. (Adapted with permission from Ref. 27.)
Despite the shorter separation times typically afforded by microchips, a single-channel microdevice cannot match the throughput of multiple-lane CAE instruments that are now capable of simultaneously separating hundreds of samples. Fortunately, the micromachined format is well suited to forming arrays of separation channels, because reservoir spacing can be designed for automated, parallel sample loading and multiple separation lanes can be bundled together to facilitate detection. The first reported microchip parallel DNA separation was performed in a 12-channel CAE microdevice to determine HFE
gene variants from multiple individuals and took only 160 s (25). platform. One of the most important The CAE device was placed on a translation stage and the chan- procedures for integration is PCR, in nels repeatedly probed by a stationary confocal fluorescence de- which a small number of copies of a tection system. To increase the number of lanes that could be DNA template are amplified exponenprobed simultaneously, CAE microchips were designed with a ra- tially to obtain a sufficient quantity for dial layout (26 ). Samples were introduced near the perimeter of analysis. PCR requires a mixture of tema circular substrate, and confocal fluorescence detection occurred plate DNA that contains the sequence of inwhere the lanes converged near the center of the device. terest, oligonucleotide primers that flank the sequence, individFigure 1 shows the design and layout for a 15-cm-diam, 96- ual nucleotide triphosphates, and DNA polymerase molecules to channel radial CAE microplate (27 ). For increased resolution, incorporate the nucleobases into a growing DNA strand that is each channel was folded several times in a serpentine manner complementary to the template. When heat-stable polyto increase the separation distance. Using this CAE merase enzymes are used, the DNA sequence can be microplate to sequence DNA from the phage amplified by cycling the temperature of the mixM13mp18, researchers found that the average ture between three different settings: ~55 ºC, read length in 95 successful channels was to anneal primers; ~72 ºC, for polymerase Alternative poly430 bases with 99% accuracy, which corresactivity; and ~94 ºC, to separate the newly ponded to 1.7 kb/min. These results repformed double-stranded DNA into indimer sieving solutions resented a 5-fold increase in separation vidual strands, thereby preparing the systhroughput compared with commercial tem for another amplification cycle. with lower viscosities can CAE instrumentation available at that Microfabricated reaction chambers be easily pumped into time and a 100-fold throughput improvecan handle smaller volumes and can cycle ment over initial microchip DNA sequencbetween temperatures more quickly than channels at room ing work (17). conventional thermal cycling systems. Recent efforts to increase DNA sample However, the biocompatibility of the surtemperature. capacity have led to the development of a face becomes crucial as the size of the reaction 384-lane CAE microplate with a radial design vessel shrinks, because of the greater surface-tosimilar to that of the 96-channel device described volume ratio. Thermal cycling in micromachined above (28). This system was used to simultaneously test systems can be accomplished by cycling the entire device 384 samples for a mutation in the HFE gene in just 7 min with with an external source, physically transporting the sample 98.7% accuracy. Because the resolution requirements were re- through a microchannel with fixed-temperature zones generated laxed for genotyping relative to sequencing, the sample injectors by external heating, or thermally cycling only the miniaturized could be simplified to occupy less space and each channel could reaction chamber where the sample is amplified. proceed directly from the perimeter to the center of the device. Whole-device thermal cycling with PCR chambers composed Is there even more room to increase the number of separation of micromachined silicon and glass has been used to test various lanes and thus throughput in CAE microchips? Technical hurdles surface passivation methods in an effort to identify PCR-comneed to be surmounted to increase the sample capacity by anoth- patible surfaces (29). In these experiments, the amplified DNA er order of magnitude. Two design constraints—the space need- was removed from the microdevice and analyzed using slab gel ed for each injection system and how tightly crowded the lanes electrophoresis. Ramsey et al. demonstrated microchip PCR are in the detection region—will ultimately limit the number of coupled to CE, in which amplification was performed by placing parallel channels that can be micromachined for DNA analysis. an entire device in a commercial thermal cycler. Electrophoretic For example, if each injector occupies 12.5 mm2, 1000 injectors separation took place after the device was transferred manually to take up 125 cm2, or 40% of the surface of a 20-cm-diam substrate the detection platform (30). Figure 2a shows the layouts of two (28). Similarly, with a 150-µm pitch between channels, 1000 ra- PCR /CE microchips used in whole-device thermal cycling. Manz and co-workers first demonstrated continuous-flow dial lanes would be at their closest proximity to each other ~2.5 cm from the center of a device, reducing the separation distance PCR on a micromachined platform, which eliminated the need and requiring design modifications for current high-throughput for rapid cycling of the substrate temperature (31). Instead, amdetection systems (26). To satisfy these constraints and push ca- plification was performed by using pressure to drive the reaction pacity to 1000 samples and beyond, various avenues of research mixture through a serpentine microchannel with three distinct must be pursued: reducing lane spacing, exploring new detection temperature zones heated by external sources (Figure 2b). With formats, decreasing injector size, evaluating devices with multiple this approach, the total number of cycles that the device can produce is fixed, but the speed at which the sample travels through planar layers, and using larger substrates. the channel can be varied to provide longer or shorter cycle times. Sample preparation Northrup et al. took advantage of microfabrication technoloNot only has the micromachined format proven advantageous for rapid, high-throughput analyses, but elegant microfluidic gy by using a heated microchamber to couple PCR with misample preparation steps have also been incorporated into this crochip CE to form an integrated bioanalyzer (32). The setup M A R C H 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
99 A
(b)
(a) PCR-2 PCR-3 PCR-1
PCR Buffer product
PCR-4
plification (>1 µL) and the much smaller amounts actually injected in microchip or conventional CE analysis (
