Hybridization Enhancement Using Microfluidic Planetary Centrifugal

Oct 22, 2004 - Magdalena A. Bynum* and Gary B. Gordon. Agilent Laboratories, Agilent Technologies, 3500 Deer Creek Road, Palo Alto, California 94304...
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Anal. Chem. 2004, 76, 7039-7044

Hybridization Enhancement Using Microfluidic Planetary Centrifugal Mixing Magdalena A. Bynum* and Gary B. Gordon

Agilent Laboratories, Agilent Technologies, 3500 Deer Creek Road, Palo Alto, California 94304

DNA microarrays produce their greatest sensitivities when hybridized using concentrated samples and effective mixing; however, these goals have proved elusive to combine. If samples are diluted enough to fill larger chambers, then mixing works well using either pumping or gravity with rotation, although sensitivities will suffer. Various techniques for mixing concentrated samples in small thin chambers have been proposed; however, they often leave streaks or scars, and their reusable components require careful cleaning. Here we introduce a versatile new microfluidics platform, a two-axis centrifuge whose fluidic chambers rotate in a planetary relationship to a radial gravitational field. This paradigm readily overcomes surface and viscous forces even in chambers only 50 µm thin. Thin chambers obviate the need for sample dilution and increase sensitivities and dynamic ranges 10-fold. In comparisons against conventional mixing using the same 10 µg of starting total RNA on 22 000-probe arrays, 10 000 more usable signals rose above the noise. In other experiments, planetary mixing was able to produce comparable results while using only one-tenth the starting sample. The benefits of planetary mixing include sample conservation, shorter hybridizations, less reliance on amplification, and the ability to quantify many gene signals otherwise obscured by noise. The microarray has evolved as a premiere information-rich assay for molecular biology. Arrays of tens of thousands of different probes on a single slide allow simultaneous detection of a large number of genetic sequences with unmatched speed, specificity, and sensitivity. Such arrays have entered the mainstream for studies of diseases and therapeutics.1-6 For example, * To whom correspondence should be addressed. Phone: 650-485-3152. Fax: 650-485-8502. E-mail [email protected]. (1) DeRisi, J.; Penland, L.; Brown, P. O.; Bittner, M. L.; Meltzer, P. S.; Ray, M.; Chen, Y.; Su, Y. A.; Trent, J. M. Nat. Genet. 1996, 14, 457-460. (2) Golub, T. R.; Slonim, D. K.; Tamayo, P.; Huard, C.; Gaasenbeek, M.; Mesirov, J. P.; Coller, H.; Loh, M. L.; Downing, J. R.; Caliguiri, M. A.; Bloomfield, C. D.; Lander, E. S. Science 1999, 286, 531-537. (3) Yeoh, E. J.; Ross, M. E.; Shurtleff, S. A.; Williams, W. K.; Patel, D.; Mahfouz, R.; Behm, F. G.; Raimondi, S. C.; Relling, M. V.; Patel, A.; Cheng, C.; Campana, D.; Wilkins, D.; Zhou, X.; Li, J.; Liu, H.; Pui, C. H.; Evans, W. E.; Naeve, C.; Wong, L.; Downing, J. R. Cancer Cell 2002, 1, 133-143. (4) Ono, K.; Tanaka, T.; Tsunoda, T.; Kitahara, O.; Kihara, C.; Okamoto, A.; Ochiai, K.; Takagi, T.; Nakamura, Y. Cancer Res. 2000, 60, 5007-5011. (5) Schwartz, D. R.; Kardia, S. L.; Shedden, K. A.; Kuick, R.; Michailidis, G.; Taylor, J. M.; Misek, D. E.; Wu, R.; Zhai, Y.; Darrah, D. M.; Reed, H.; Ellenson, L. H.; Giordano, T. J.; Fearon, E. R.; Hanash, S. M.; Cho, K. R. Cancer Res. 2002, 62, 4722-4729. 10.1021/ac048840+ CCC: $27.50 Published on Web 10/22/2004

© 2004 American Chemical Society

tumor samples that appear identical under the microscope yet have entirely different clinical outcomes can be differentiated using microarrays.7-9 In addition to tumor classification, researchers can use subsets of microarray probes as genetic markers to separate healthy cells from cancerous cell types.10-12 Sensitivity is very important to users reluctant to sacrifice portions of their irreplaceable tissue samples to destructive testing. For others, dissecting and isolating large numbers of cells just to gain sensitivity is time-consuming and expensive. Still others seek to avoid the labor, reagent cost, and uncertainties of amplification. Sensitivity can be enhanced in a number of ways. One way is to improve the quality of the detection probes within each feature on the array. For example, the synthesis process used by some manufacturers produces a high yield at each step, permitting longer probes, which have higher specificities.13 Thus, fewer features are needed per gene, so space on arrays may be beneficially reallocated to making each feature larger. Larger features are less noisy and therefore are more sensitive. Scanners, RNA extraction, labeling, and washing and drying procedures also influence sensitivity. Thus, the judicious choice among the popular array and scanner platforms can substantially influence detection limits.14 In addition to arrays and scanners, a third way to enhance sensitivity is to improve the hybridization conditions. The microarray hybridization regime is one of slow reaction kinetics15 and directly benefits from increased target concentration. Hybridizations also benefit from using active mixing because lateral diffusion alone only allows several millimeters of target travel, and mixing is required to replenish the target concentration in the vicinity of each probe. (6) van’t Veer, L. J.; Dai, H.; van de Vigver, M. J.; He, Y. D.; Hart, A. A. M.; Mao, M.; Peterse, H. L.; van der Kooy, K.; Marton, M. J.; Witteveen, A. T.; Schreiber, G. J.; Kerkhoven, R. M.; Roberts, C.; Linsley, P.; Bernards, R.; Friend, S. H. Nature 2002, 406, 530-536. (7) Afshari, C. A.; Nuwaysir, E. F.; Barrett, J. C. Cancer Res. 1999, 59, 47594760. (8) Khan, J.; Simon, R.; Bittner, M.; Chen, Y.; Leighton, S. B.; Pohida, T.; Smith, P. D.; Jiang, Y.; Gooden, G. C.; Trent, J. M.; Meltzer, P. S. Cancer Res. 1998, 58, 5009-5013. (9) Marx, J. Science 2000, 289, 1670-1672. (10) Hegde, P.; Qi, R.; Gaspard, R.; Abernathy, K.; Dharap, S.; Earle-Hughes, J.; Gay, C.; Nwokekeh, N. U.; Chen, T.; Saeed, A. I.; Sharov, V.; Lee, N. H.; Yeatman, T. J.; Quackenbush, J. Cancer Res. 2001, 61, 7792-7797. (11) Clark, E. A.; Golub, T. R.; Lander, E. S.; Hynes, R. O. Nature 2000, 406, 532-535. (12) Ismail, R. S.; Baldwin, R. L.; Fang, J.; Browning, D.; Karlan, B. Y.; Gasson, J. C.; Chang, D. D. Cancer Res. 2000, 60, 6744-6749. (13) Hughes, T. R.; et al. Nat. Biotechnol. 2001, 19, 342-7. (14) Stafford, P. CHI Microarray Data Analysis Conference. http:// www.healthtech.com/2003/mda/, 2003. (15) Dai, H.; Meyer, M.; Stepaniants, S.; Ziman, M.; Stoughton, R. Nucleic Acids Res. 2002, 30, e86.

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Figure 1. Unmixed hybridization with exaggerated tilt confirming that coverslip hybridization signals are directly proportional to the chamber height. Coverslip hybridizations lack uniformity and repeatability because their thickness is not accurately controlled.

Early hybridizations were done without mixing, applying undiluted samples to arrays and covering them with thin glass coverslips in order to contain and distribute small 25-µL sample volumes across the arrays. Such reactions started out rapidly because the samples were undiluted, but in the absence of mixing, reactions soon slowed due to local target depletion. Hemispherical depletion volumes form around each probe with a radius set by diffusion to ∼5 mm in a typical 17-h hybridization. In a typical thin hybridization chamber, these diffusion volumes equate to short cylinders with a height of 0.5 mm and radius of 5 mm. The cylinder heights were problematic as well because they were not constant, but rather dependent upon the coverslips being flat and parallel to the array surface. Corner spacers were only a partial solution, since coverslips warp inward under the influence of capillary attraction. The consequence of the height varying was explored in our prior work and is shown in Figure 1, where a coverslip was intentionally sloped such that it touched at one edge, while maintaining a gap of 50 µm at the other edge. It is dramatically evident that the signals from eight replicate probes on the array are not only influenced by the chamber height but are in fact, as predicted, directly proportional to it. Even the slightly weaker signals from closely paired probes are readily explained by their slightly overlapping depletion cylinders competing for the same target. In response to these limitations of coverslip hybridizations, most users turned to active mixing. The easiest way to mix is to make the chamber much thicker, add a surfactant, dilute the sample to fill the volume, and mix by either active pumping or by slowly rotating the chamber. These so-called large-volume mixing techniques improve repeatability by removing the dependency on chamber thickness. Further, since target replenishment around each feature can now take place, the initial sensitivity loss to coverslips from diluting the sample is largely recovered. Overall, large-volume mixing produces easy sample containment, improved uniformity and repeatability, and comparable sensitivity to coverslips. An ideal hybridization environment would combine the thin reaction chamber of the coverslip with some form of active mixing. It would need to somehow overcome the twin nemeses of surface tension and viscosity. One approach, used in several commercial products, is to somewhat reduce the volume and then use bladders 7040

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to actively pump and effect mixing.16 Several other experimental techniques use ultrasound to move fluids within even smaller volumes. One, a concept called cavitation microstreaming, adds tiny air-filled chambers to a backing slide and reports a 5-fold kinetics acceleration.17 Others have explored sliding coverslips back and forth to encourage mixing. One company advocates using electrophoresis and porous arrays to concentrate the target, while adding membranes to avoid the acid production caused by galvanic action.18 The need for mixing small volumes is underscored by the variety of approaches, which attest to the difficulty of the problem. Mixing very small volumes to produce repeatable and uniform hybridizations without leaving mixing or bubble scars has proved an elusive goal. Here we introduce a new and robust paradigm for microarray hybridization we call planetary centrifugal mixing, which conveniently and economically achieves high sensitivity, high uniformity, and high throughput. We distinguish our use of the term from that sometimes associated with large-volume mixers for viscous materials and also from the term’s common association with orbital beaters. Microfluidic planetary centrifugal mixing combines the small chamber sizes of coverslips with the uniform mixing of largevolume hybridizers. Its robustness provides more freedom in the design of buffers, since surfactants or other additives are no longer required as mixing aids. Fluids ranging in properties from water to 50% glycerol move easily and mix in 50-µm thin chambers with this platform. The platform is amenable to hybridizing many arrays at one time and uses inexpensive consumables. In addition, with a simple change to one consumable component, a variety of array sizes and layouts may be accommodated. EXPERIMENTAL SECTION Apparatus. A new and experimental microfluidic planetary analytical platform that we will call a microfluidic planetary centrifuge was constructed, which is capable of performing a number of fluidic manipulations including mixing, aliquoting, filtering, and separation (Figure 2). For the present work, it was only used to accomplish mixing. The apparatus includes a method of sample containment (Figure 3). Sample containment consists of clamping a “gasket slide” against a 1 × 3 in. glass microarray slide. The gasket slides are microscope slides fitted with formedin-place inert elastomeric gaskets, developed in-house and precisely placed using a computer-controlled dispenser. The gasket cross sections are approximately 50 µm thick and 200 µm wide. Figure 2 shows the planetary centrifuge in operation using strobe light illumination. The menisci of the green fluids in the four mixing chambers are visible as arcs, centered about the axis of rotation of the centrifuge. Each of the four chambers rotates relative to the turntable of the centrifuge, as can be seen in this double-exposure photograph. Chamber rotation causes the fluids to redistribute and maintain their menisci relative to the central rotor axis and not the chambers themselves. Thus, the constantly (16) Adey, N. B.; Lei, M.; Howard, M. T.; Jensen, J. D.; Mayo, D. A.; Butel, D. L.; Coffin, S. C.; Moyer, T. C.; Slade, D. E.; Spute, M. K.; Hancock, A. M.; Eisenhoffer, G. T.; Dalley, B. K.; McNeely, M. R. Anal. Chem. 2002, 74, 6413-6417. (17) Liu, R. H.; Lenigk, R.; Druyor-Sanchez, R. L.; Yang, J.; Grodzinski, P. Anal. Chem. 2003, 75, 1911-1917. (18) Cheng, J.; Sheldon, E. L.; Wu, L.; Uribe, A.; Gerrue, L. O.; Carrino, J.; Heller, M.; O’Connell, J. Nat. Biotechnol. 1998, 16, 541-546.

Figure 2. Stroboscopic image of the experimental planetary centrifuge in operation. Fluids mix in each of its four 50-µm-thick planetary chambers, which slowly rotate relative to the spinning rotor of the centrifuge. Their continually changing menisci are visible as green arcs, which despite the rotation of the chambers remain concentric with the axis of the main rotor of the centrifuge.

Figure 3. Hybridizer loaded by pipetting 60-µL samples onto gasket slides (normally already placed in their holder), covering them with arrays to form a stack, and clamping them in place with a cam.

changing fluid distribution with respect to the reaction chambers and microarrays produces thorough mixing. In normal use the chambers would be filled to a greater extent than shown here, since only the tiniest bubbles are necessary to effect mixing, and the tolerance on fill percentages is very wide. A side benefit of the high-g centrifugal force is preventing the formation of secondary bubbles and froth within the sample, which could otherwise lodge and cause local hybridization defects. Each stage of the experimental planetary hybridizer holds up to 3 arrays, allowing the hybridization of as many as 12 arrays at

the same time, a number that could easily be increased. The stages are mounted to a rotor, which spins at 1200 rpm generating ∼100g of acceleration. As the stages travel with the rotor, they are simultaneously changed in angular orientation with respect to that rotor, at a differential rate of 10 rpm, by a gear train interacting with concentric shafts driven from a common motor. When the apparent motion of the rotor is interrupted using a stroboscope, the stages appear to each rotate one full revolution every 6 s. The effect is as if the hybridization chambers were simply rotating within an extreme gravitational field sufficient to overwhelm all surface and viscous forces. It is the equivalent to mixing using the earth’s gravitational field, except with fluids having specific gravities of 100. To facilitate loading, the stages are lifted out and placed on the worktable for sample loading (Figure 3). Aliquots of 60 µL of sample are pipetted onto each gasket slide, whereupon they are covered by microarrays and added to the stack. (The total volume of the chamber is ∼80 uL.) Once a particular stage is filled with up to three array-gasket-slide sandwiches, it is clamped by a cam and placed in the hybridizer. For simplicity of temperature control, the entire hybridizer is placed in an incubator. Reagents and Materials. Heart and liver total RNA from rat were purchased from Ambion (Austin, TX). Rat microarrays, the Fluorescent Direct Label Kit, the Hyb Plus Kit, the 500-µm-thick large-volume gasket slides, the stainless steel microarray hybridization chamber assembly, the fluorescent scanner (type B), and Feature Extraction 7.1.1 software were provided by Agilent Technologies (Palo Alto, CA). DecisionSite 7.2 software, used to Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

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make the plots, visualize and analyze the data, was available from Spotfire (Somerville, MA). Cyanine-3-dCTP (1.0 mM) and cyanine5-dCTP (1.0 mM) dyes were purchased from Perkin-Elmer (Wellesley, MA). DNase/RNase-free distilled water and 20× SSC were purchased from Invitrogen (Carlsbad, CA). The QIAquick PCR Purification Kit, used to purify the labeled cDNA, was purchased from Qiagen. A microarray oven, model 400 (Robbins Scientific, Sunnyvale CA), was used to control the temperatures of the large-volume hybridizations. Preparation of Total RNA Targets. For each labeling reaction, mRNA from 10 µg of rat total RNA, from each of two organs, was converted to labeled cDNA according to the manufacturer’s direct labeling protocol. Rat heart mRNA was labeled with cyanine-3 and rat liver mRNA was labeled with cyanine-5. Afterward, all labeled cDNA was pooled and then aliquoted into individual centrifuge tubes. Each tube contained labeled cDNA from 20 µg of total RNA, or ∼1 µg, assuming mRNA is 5% of the total RNA. The samples were dried in a vacuum evaporator at 50 °C and stored dry at -80 °C until just prior to microarray hybridization. Experiments. Two types of experiments were performed in the course of this research. In both, we compared the performance of the small-volume planetary-mixed hybridization to that of conventional large-volume hybridization. In the first experiment, we explored whether planetary mixing could match the performance of large-volume hybridization using only one-tenth the amount of target (moles of target molecules). In the second experiment, we used the same amount of target to explore how many additional genes we could measure above the noise threshold of our measurement system. The experiments were performed with commercially available target mRNA from rat heart and rat liver to simulate up- and downregulation of gene expression done with real patient samples. In all cases, amplification was not used in the target labeling procedure to better understand the effects of the planetary centrifuge with small samples and concentrated target solutions. Sensitivity Comparison Experiment Using One-Tenth the Target. In this first experiment, we sought to demonstrate that planetary mixing could match the performance of the large-volume hybridization while using one-tenth of the sample. The planetary centrifuge hybridization chamber is 10 times thinner than the large-volume hybridization system. Both hybridization chambers were filled with the same concentration of labeled target, resulting in one-tenth the moles of target molecules in the planetary mixer. For this first experiment, just prior to hybridization, two tubes of ∼1 µg each of labeled cDNA were diluted with equal parts of 2× hybridization buffer and DNase free water and pooled, providing enough sample for two arrays. The standard volume of 600 µL was applied to the gasket slide used with the large-volume gravity-mixed system (containing ∼1 µg of labeled cDNA target), while only 60 µL (containing only ∼100 ng of labeled cDNA target) was applied to the small-volume gasket slide used with the planetary mixer. The microarrays were placed on top to form the mixing chamber and clamped in place, creating a leak-proof seal. The assemblies were immediately inserted into the respective hybridizers, both thermostated to 65 °C, and hybridized for 17 h. After the hybridizations, each array was disassembled in 200 mL of 6× SSC wash buffer and placed in a slide rack in a glass 7042 Analytical Chemistry, Vol. 76, No. 23, December 1, 2004

Figure 4. Cy3 signals for different hybridization paradigms compared, using 1 and 10 µg of total rat heart RNA. Signals from planetary mixing were almost as strong (85%), despite using only one-tenth the sample. Each spot on the plot represents the signal for the arrays hybridized in the planetary centrifuge versus the signals for the same feature of the array hybridized with the conventional mixing system.

wash container with 6× SSC wash buffer. When all microarrays were disassembled, the wash solution was magnetically stirred gently for 10 min. Then the arrays were moved to a second wash container with 0.6× SSC wash buffer for 5 min, again with magnetic stirring. The slides were dried by slowly withdrawing them from the last wash solution. The microarrays were then scanned, feature extracted, and analyzed. Sensitivity Increase Experiment, Same Amount of Target. In this second experiment, while the moles of target were the same, the sample hybridized with the planetary mixer was more concentrated by a factor of 10 compared to the conventional hybridization, resulting in ∼1 µg of labeled cDNA target for each system. Target mixtures were prepared and hybridized to microarrays in the same manner as previously described. RESULTS Sensitivity Comparison Using One-Tenth the Target. For the first experiment, using one-tenth as much target in the planetary hybridizer, the signal levels averaged 85% as high as those when using the large-volume chamber. The data plot for the green Cy3 background-subtracted signals are shown in Figure 4. Each point on the plot corresponds to a feature on the microarray. The y-axis represents the planetary-mixed signal, while the x-axis represents the signal for the same feature in the conventional hybridizer. The straight-line fit of the data has an equation of y ) 0.85x - 38. The red Cy5 signal (data not shown) showed the same trend as the Cy3 data. The increase in scatter seen at the low end of the signals in Figure 4 results from both systems approaching their noise limits. This experiment confirmed our expectation that, over the hybridization time of 17 h, binding was very nearly proportional to concentration. In addition to measuring absolute signals to determine differences in gene expression, many researchers measure the log ratio of the green signal to the red signal within each feature. Figure 5 shows the ratios from the hybridized arrays. Despite the 10fold decrease in sample, the log ratios are well preserved between the two systems. The straight line fit through the data has the equation: y ) 1.0x - 0.04 and an R-value of 0.982. Approximately

Figure 7. Probe number versus signal intensity. When samples are limited, planetary-centrifugally mixed hybridizations produce substantially more signals above the noise floor, calculated at 3× the standard deviation of the negative controls.

Figure 5. Correlation of planetary and conventional mixing. Despite planetary mixing’s using only one-tenth the amount of sample, its ability to measure up- and down generegulation correlated well with that of conventional hybridizations. Axes show the log ratios of green/ red signals for arrays hybridized with both platforms, with 1 or 10 µg of total rat RNA per channel.

Figure 6. Comparison of sensitivities using identical 10-µg amounts of total RNA from heart. Planetary-mixed signals averaged 10 times stronger, allowing thousands more signals otherwise obscured by noise to become meaningful.

7000 of the 22 000 features are plotted in Figure 5. The features not represented were below the noise floor for one or the other of the arrays. The data points that are more scattered are those that fall very close to the noise floor for either the red or the green signal on either array (data not shown). Sensitivity Increase with the Same Amount of Target. Figure 6 shows the signals obtained from planetary small-volume mixing when an equal number of moles of target are hybridized in a volume 10× smaller than the conventional hybridization chamber. The horizontal and vertical axes show the green background-subtracted signal intensities for both systems. A straight line fit through the data has the equation y ) 10.7x 140 and an R-value of 0.925. In addition to measuring signals 10 times higher when using planetary mixing, we also observe that many additional probes rise out of the noise and become useful. Figure 7 shows the green background-subtracted signals, ordered by their rank intensity. A noise floor is shown at three times the standard deviation of

the negative control features. The sheer magnitude of the number of new useful signals, which roughly doubled from ∼10 000 to ∼20 000, was unexpected. Importantly, the signals for the negative controls were comparable for both platforms (data not shown), while the specific probes increased ∼10-fold, providing a clear 10-fold increase in sensitivity. DISCUSSION The typical 17-h DNA-array hybridization is characterized by reactions that are interrupted at less than 10% of the way to equilibrium. Target concentration in solution changes little from its initial value. Thus, the extent of binding after 17 h is, to first order, proportional to the initial concentration of the target. This was confirmed and leveraged in the second experiment, which used the same amount of sample in both hybridizations, only at a higher concentration in the planetary hybridizer. In this experiment, the reactions undoubtedly began at rates proportional to their target concentrations, which were 10 times greater in the planetary hybridizer. These rates were expected to change little over the course of 17 h. This is in fact what was observed; using the same amounts of sample in both hybridizations, the planetary hybridizer produced signals ∼10 times stronger. On the other hand, in the first experiment, which sought to see if comparable results could be obtained in the planetary hybridizer while using 10 times less sample, sample depletion would no longer be an insignificant second-order effect. In our regime of probe excess,19 target would be slightly consumed, resulting in a slight roll-off in the signals from the planetary hybridizer. This indeed was observed, with the signals being only 85% as strong as those from the conventional large-volume hybridization after 17 h. Losing 15% of the signal is a minor consequence of being able to use 10 times less starting material. Were identical signals the goal, as they might be for comparison with historical data, then identical signals could be obtained using the same one-tenth starting material except in one-twelfth the volume, not one-tenth the volume. We propose that this slight additional concentration increase would readily overcome the second-order effect of sample depletion. In addition to performing these types of experiments with directly labeled sample, we also generated this type of data for linearly amplified sample. We see the same results with linearly amplified samples (data not shown.) We chose to present the data (19) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168.

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for directly labeled samples because the planetary centrifuge can be used to enhance sensitivity in this sample-limited case. CONCLUSIONS We have described a new analytical apparatus we refer to as a “microfluidic planetary centrifuge”. It represents an alternative general-purpose fluidics platform that can also robustly mix, aliquot, filter, and separate solutions. It is free from contamination issues, since it can use disposable components built upon inexpensive microscope slides. Our first application was mixing small fluid volumes of full-strength target against biological microarrays. By avoiding dilution, the reaction kinetics were improved 10-fold, allowing users to conserve sample, detect weak markers, and shorten hybridizations. Experiments suggested how to obtain equivalent signals as large-volume hybridizations, while using only one-tenth the amount of often-scarce starting sample. Other experiments using the same amount of starting sample as large-volume hybridizations showed signals 10 times brighter, revealing 20 000 markers instead of the usual 10 000, on an array of 22 000 features. Small-volume mixing thus provides a new control over hybridizations, which can be set with the option to enhance sensitivity, use less starting material, provide faster results, or any combination. Planetary centrifugal mixing, together with careful selection of arrays and scanners, can make as much as a 100-fold difference in the sensitivity of DNA and other microarrays and possibly obviate the need for the expense, time, and biological noise incurred when using amplification.

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While not yet a part of our present research, planetary centrifugation is believed to represent a viable platform for performing other microfluidics tasks, standing alongside electrophoresis and active pumping. By employing the equivalent of strong gravity, and by having the direction in which gravity acts under software control, a wide variety of fluidics operations may be readily performed. Examples include mixing, separating via centrifugation, valving by “pouring”, aliquoting, filtering, and pumping through columns. While none of the three aforementioned platforms is capable of performing every microfluidic operation that might be imagined, planetary centrifugation nonetheless perhaps offers the richest subset and is amenable to working in cooperation with the other platforms. ACKNOWLEDGMENT We acknowledge the work of Arthur Schleifer in developing formed-in-place polymer gaskets. We thank Bo Curry for many engaging discussions about kinetics. We also acknowledge Jim Young for his creativity and industrial design talent on this project. We thank Willy McAllister and Ellen King for reviewing and suggesting changes to the manuscript.

Received for review August 5, 2004. Accepted August 30, 2004. AC048840+