SEPARATION MEDIA
for MICROCHIPS Mary J. Wirth University of Arizona
DNA analysis and proteomics case studies
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illustrate the research activity in microchip analyses with chemical separations.
hemical separations are essential for analyses of complex samples. Biological samples, in particular, are extremely complex and require multiple preparatory steps before the separation, yet the sample size is often limited. In view of these challenges, Manz et al. pointed out the need for miniaturized total chemical analysis systems, or µTAS (1). The idea is that tiny samples can best be injected, prepared, separated, and analyzed in one integrated system to minimize sample loss or contamination. Microchip, or lab-on-a-chip, technology has emerged from this vision, in which all of the steps are performed on a miniaturized platform. Each of these steps has been done individually on a microchip, and multiple steps have been combined on microchips. Specimens such as saliva, urine, and plasma obviously do not require cell lysis. Often, the chemical separation can be avoided by use of a microarray, as in hybridization assays or immunoassays. Today, the interest in microchips is growing because of the need for ever-increasing speed and throughput in biological analyses. Target applications for µTAS devices include point-of-care clinical diagnostics (2), forensic DNA fingerprinting (3), and real-time monitoring for biodefense (4). Here, two case studies illustrate the research activity in microchip analyses with chemical separations—one involves DNA analysis and the other proteomics. The goal is to see why a particular type of analysis might work better when integrated on a chip. We can then assess which separation media exist and what needs to be developed to make µTAS on microchips possible.
DNA analysis DNA analysis is one area that is highly suited to the µTAS approach—limited sample size, multiple processing steps, a challenging analytical separation, and a demand for high speed and throughput. The main driving forces are clinical diagnostics, forensics, and the threat of bioterrorism. Capillaries have largely replaced gels for DNA sequencing, and the separation medium currently used for CE of DNA fragments is a solution of a neutral hydrophilic polymer of high molecular weight, such as polyacrylamide, hydroxyethylcellulose, or poly(ethylene glycol) (5). The principle is that the polymer solution mimics a cross-linked gel, in which larger DNA fragments have progressively lower velocities. These replaceable sieving media are applicable to both capillaries and microchips. © 2007 AMERICAN CHEMICAL SOCIETY
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increases with the square root of the separation length, many separations are not feasible on a miniaturized platform. This particular type of separation is feasible because the short separation length still has adequate resolution to distinguish the sharp band for malignant cells from the broad distribution for normal cells, thereby speeding up the analysis. The microchip also provides the advantage of integration—PCR and separation performed with one device—which would likely reduce the cost of the tests. Thus far, though, only the separation step has been considered and not the total analysis. In the aforementioned example, both the slab gel and the microchip separations used off-line PCR amplification, which took longer than either of the separations. PCR and electrophoresis were first integrated on a microchip in 1996, with a net analysis time of 45 min for detection of a DNA fragment of Salmonella (7 ). In 2006, the same two steps were integrated on a microchip with a faster net analysis time of 12 min, also for a single fragment of Salmonella DNA (8). In both cases, the separation time was 2 min, as it was PAGE Microchip for the diagnosis of T-cell lymphoma in our first example. Note that what has changed in Standard 10 years is that the time for PCR has been reduced 3-fold, 396 – ve/eq because of innovations in the 350 speed of heating. The separaSample 1 tion step remained the same 222 because comparable separation – ve 179 media were used, thereby keeping the separation time a small Sample 6 fraction of the total analysis 75 time. + ve Given that PCR is the slow Lane M 1 2 3 4 5 6 7 8 9 step, would better media for –/e + + + –/e – + + – Diagnosis Sample 7 microchip separations of DNA Controls 20 40 60 80 100 120 140 160 have any significant impact? T ime (s) Better media, giving greater resolution for a particular separation length, would extend FIGURE 1. Comparison of data from polyacrylamide gel electrophoresis (PAGE) and microchip electrophothe use of microchips to more resis. Lanes M, 1, 6, and 7 for the gel correspond to the standard and samples 1, 6, and 7, respectively, for complex analyses that require the microchip. (Images courtesy of James Landers.) high speed. One important exunequivocal diagnosis of no malignancy. By contrast, lane 7 ample is DNA fingerprinting in forensics, where a much faster (sample 7) is a control. Both the gel and the microchip analyses analysis would reduce the backlog of samples. Integration of cell have a dominant spike, which shows how the electropherogram lysis and DNA extraction for forensics on a chip has been demwould appear for an unequivocal diagnosis of malignancy. For onstrated (3), and PCR and electrophoresis are now routinely insample 1, both the gel and the microchip have broad distribu- tegrated on microchips. In DNA fingerprinting, single base-pair tions, but a weak spike is seen both in the gel at 75–179 bp resolution is needed for fragments in the size range 100–1000 bp (which may not be discernable in the printed figure), and in the (9). This is routinely achieved with capillaries with separation lengths of ≥30 cm for the same sieving media used in the T-cell microchip at 140 s, which makes the diagnosis equivocal. This example illustrates how microchips, like gels, readily lymphoma example (10). The difference is that the T-cell examallow multiple lanes for running size standards and multiple con- ple used a separation length of only 4.2 cm, which is insufficient trols and samples. However, the slab gel took 2.5 h, whereas the to achieve single base-pair resolution. Long separation lengths have been achieved on microchips by microchip took 2.0 min. This 75-fold reduction in time is attributable to the higher electric field allowed for the microchip using serpentine channels, where tapering the turns preserves combined with the shorter separation length. Because resolution high efficiency, and a spiral channel that was 25 cm long has also Relative fluorescence
The first case study focuses on gene probing, in which one particular locus of the genome is analyzed for an abnormality. The suspected abnormal sequence is used as a DNA template and selectively amplified by PCR. The amplified sequence that is generated is a double strand, which is readily detectable with fluorescence if an intercalation dye is used. Such an analysis was demonstrated for T-cell lymphoma (6). The basis of the analysis is that normal cells have a distribution of mutations, whereas malignant cells have a single, abundant mutation. Figure 1 shows a comparison of results from multilane gel separations and multichannel microchip separations. The top electropherogram for the microchip is the separation of a DNA size standard, and the same separation for the gel is in the lane marked M. Lanes 1, 6, and 7 for the gel correspond to samples 1, 6, and 7 of the microchip, respectively. Lane 6 gave a broad smear in the gel, at 179–222 base pairs (bp), and also in the microchip, at 140–160 s. This distribution of mutations allows an
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Conventional
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been used (11–13). A microfabricated device for DNA sequencing uses arrays of serpentine channels 15.9 cm in length (14). Its unique ability to integrate key steps and the lower expense of providing more separation channels could make it preferable to capillary arrays (15). These advances would be accrued for clinical gene mapping and in forensics. Shorter path lengths for higher speed would require sieving media with higher selectivity. Can the selectivity in sieving electrophoresis be improved? Different matrices provide varying amounts of selectivity (16), and a considerable amount of research is directed toward increasing selectivity. The resolution R s is defined in terms of the selectivity µ/, which is the relative difference in electrophoretic mobility between two DNA fragments whose lengths differ by 1 bp. R s also depends on the separation length L and plate height H.
µ √L/H Rs = • 〈µ〉 4
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Elongated
Folded
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The electrophoretic mobility µ is the proportionality constant between velocity v and electric field strength E, as in µ = v/E. H in any separation is a measure of peak broadening, as in H = 2/L, in which 2 is the peak variance for a given L. H is a figure of merit for the engineering of the separation, including the device and the separation medium—a small H is desired. In an open channel, the contributions to H are injected width, molecular diffusion, detected width, and convection from thermal gradients (17 ). Minimizing H in microchips is important because, as Equation 1 above shows, a decrease in H can be accompanied by a decrease in L by the same factor without sacrificing Rs. Once H has been minimized, Rs can only be improved by increasing µ/. The term µ/ contributes linearly to Rs and characterizes how well the sieving medium distinguishes fragments of differing length. Two recent papers compared µ/ for a slab gel and a capillary with a soluble polymer; both used DNA fragment sizes in the range of 100–1000 bp (10, 18). The researchers found that µ/ is comparable in the two media: µ/ = 0.002 for a 100-base fragment, with the same roll-off in selectivity for longer DNA fragments, giving µ/ = 0.0005 for 1000 bp. This similarity raises the question of whether µ/ represents a fundamental limit. A physical understanding of the electrophoretic separation of DNA fragments can indicate whether improving µ/ is possible. The Ogston model is the simplest one for sieving a polymer in a random network of fibers. It is described by ln µ = ln µ0 – c (R + r)2, in which µ and µ0 are the mobilities in the presence and absence of the sieving medium, respectively; c is the polymer concentration; R is the radius of the DNA fragment, approximated as a sphere; and r is the fiber radius (19). In practice, the Ogston sieving model does not work well for DNA fragments because the applied field significantly distorts their shape (20). The DNA fragments align with the field to streamline their way through the medium, approaching linear shapes at high fields. Their hydrodynamic radii thus become similar, and the sieving medium no longer imparts significant µ/. The effect is minimal for fragments
