Dynamic DNA Hybridization on a Chip Using Paramagnetic Beads

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Anal. Chem. 1999, 71, 4851-4859

Dynamic DNA Hybridization on a Chip Using Paramagnetic Beads Z. Hugh Fan,*,† Shakuntala Mangru,† Russ Granzow,‡ Paul Heaney,‡ Wen Ho,† Qianping Dong,† and Rajan Kumar†

Sarnoff Corporation, CN 5300, Princeton, New Jersey 08543, and Orchid Biocomputer Inc., 303 College Road East, Princeton, New Jersey 08540

Dynamic DNA hybridization is presented as an approach to perform gene expression analysis. The method is advantageous because of its dynamic supplies of both DNA samples and probes. The approach was demonstrated on a microfluidic platform by incorporating paramagnetic beads as a transportable solid support. A glass chip was fabricated to allow simultaneous interrogation of eight DNA target samples by DNA probes. DNA targets were immobilized on beads via streptavidin-biotin conjugation or base pairing between oligonucleotide residues. The DNA/bead complex was introduced into the device in which hybridization took place with a complementary probe. The hybridized probe was then removed by heat denaturation to allow the DNA sample to be interrogated again by another probe with a different sequence of interest. A pneumatic pumping apparatus was constructed to transport DNA probes and other reagents into the microfluidic device while hydrostatic pumping was used for the introduction of paramagnetic beads with samples. After investigating three types of paramagnetic beads, we found Dynabeads Oligo(dT)25 best suited this application. Targets on the beads could be sequentially interrogated by probes for 12 times, and the hybridization signal was maintained within experimental variation. Demonstration of specific hybridization reactions in an array format was achieved using four synthesized DNA targets in duplicate and five probes in sequence, indicating the potential application of this approach to gene expression analysis. The use of genomic information obtained from the human genome project will be aided by comparison of the gene expression patterns in normal and diseased tissues. Additionally, pharmacogenomic approaches relating genetic information to drug efficacy and toxicity will require information about gene expression patterns.1 Recently, a number of technologies to monitor gene expression patterns have been described. These include differential display polymerase chain reaction,2 serial analysis of gene * To whom correspondence should be addressed: (e-mail) [email protected]; (fax) 609-734-2595. † Sarnoff Corp. ‡ Orchid Biocomputer Inc. (1) Schena, M.; Heller, R. A.; Theriault, T. P.; Konrad, K.; Lachenmeier. E.; Davis, R. W. Trends Biotechnol. 1998, 16, 301-306. (2) Liang, P.; Pardee, A. B. Science 1992, 257, 967-971. 10.1021/ac9902190 CCC: $18.00 Published on Web 09/28/1999

© 1999 American Chemical Society

expression,3 and microarray hybridization.4 Efforts are underway to make these methods suitable for high-throughput analysis required for genomic-scale investigations. The microfluidic platform being developed for automated analysis of nanoliter volumes presents an opportunity for highthroughput gene expression studies. The advantages of the microfluidics platform have been demonstrated in the representative works, including on-chip PCR,5 DNA analysis,6-8 enzyme assay,9,10 and chemical synthesis.11 Some advantages, such as rapid reactions in microscale channels and parallel sample processing, could be leveraged with DNA hybridization to develop a new strategy for gene expression analysis. DNA hybridization has been used in a chip format for DNA sequencing and analysis.12-15 These methods involve immobilization or synthesis of an array of DNA probes on a solid support. A DNA sample, which is often fluorescently labeled, interacts with DNA probes on the support. After washing, fluorescence signals at certain locations in the array reveal sequence information. One major limitation of this approach is that such a DNA chip can be used to analyze only one sample at a time. Other disadvantages include the slow reaction rate and the inability to remake the chip with different probes. We are developing a DNA hybridization (3) Velculescu, V. E.; Zhang, L.; Vogelstein, B.; Kinzler, K. W. Science 1995, 270, 484-487. (4) Lashkari, D. A.; DeRisi, J. L.; McCusker, J. H.; Namath, A. F.; Gentile, C.; Hwang, S. Y., Brown; P. O., Davis; R. W. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 13057-13062. (5) Kopp, M. U.; de Mello, A. J.; Manz, A. Science 1998, 280, 1046-1048. (6) Woolley, T.; Hadley, D.; Landre, P.; deMello, A. J.; Mathies, R. A.; Northrup, M. A. Anal. Chem. 1996, 68, 4081-4086. (7) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (8) Waters, C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (9) Hadd, A. G.; Raymound, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (10) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378. (11) Hossein, S.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 87168717. (12) Drmanac, R.; Drmanac, S.; Strezoska, Z.; Paunesku, T.; Labat, I.; Zeremski, M.; Snoddy, J.; Funkhouser, W. K.; Koop, B.; Hood, L.; Crkvenjakov, R. Science 1993, 260, 1649-1652. (13) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (14) Sosnowski, R. G.; Tu, E.; Butler, W. F.; O’Connell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119-1123. (15) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich, D. J.; Fowler, R.; Hollis, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. E.; Varma, R. S.; Hogan, M. E. Nucleic Acids Res. 1994, 22, 2121-2125.

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approach of coupling microfluidics with paramagnetic beads, which not only reduces hybridization time from hours to seconds but also enables the simultaneous analysis of multiple samples in a chip. Paramagnetic beads have been extensively used for the preparation, separation, and detection of biological molecules such as DNA, mostly because of their efficiency, simplicity, mild operation conditions, and low cost.16 Pe´rez et al. employed antibody-derivatized beads to selectively separate Escherichia coli from a matrix for amperometric flow analysis of bacteria.17 Paramagnetic beads have recently been extended into the field of capillary electrophoresis (CE). Karger’s group incorporated beads into a commercial CE machine to perform immunoassays.18 The electrophoretic mobility of polystyrene beads in a capillary has also been investigated.19 We have applied biomagnetic techniques in microfabricated devices to perform capture and lysis of E. coli and oligonucleotide ligation reactions.20 The transportation, localization, and manipulation of paramagnetic beads have been reproducibly performed in chips. In this report, we describe dynamic DNA hybridization (DDH) by incorporating paramagnetic beads into microfluidic devices. Pneumatic pumping was employed to deliver DNA probes and washing solutions while hydrostatic pumping was used to transport beads. We also discuss our investigation to identify a type of beads suitable for multiple hybridizations and gene expression analysis. The demonstration of DDH among four DNA targets and five probes in an array format shows the considerable promise of this technique. EXPERIMENTAL SECTION Device and Fabrication. Figure 1a shows the layout of the device used for DDH. The device is composed of two 54 × 54 × 0.5 mm glass plates laminated together using a modified anodic bonding method described previously.21 One of the plates has channels that were defined using photolithographic techniques, while the other contains holes that provided access to the channels.22 The channels are about 120 µm wide and 40 µm deep. There are two types of channels, one for target introduction and one for probe introduction. Eight parallel channels, designated as target channels, are indicated in Figure 1a and numbered from 1 to 8 for later reference. The ends of target channels are staggered with each other to accommodate off-chip connections. The reservoirs on the right end of the target channels were used for sample introduction while those on the left end were for solution waste. There is only one inlet for probe channels, as indicated in Figure 1a. The probe channel is then consecutively split four times before they are connected to the target channels. The total length of each target channel is 42 mm. The distance (16) Dynal A. S. Biomagnetic techniques in molecular biology, 3rd ed.; Dynal Corp.: Oslo, Norway, 1998. (17) Pe´rez, F. G.; Mascini, M.; Tothill, I. E.; Turner, A. P. F. Anal. Chem. 1998, 70, 2380-2386. (18) Rashkovetsky, L. G.; Lyubarskaya, Y. V.; Foret, F.; Hughes, D. E.; Karger, B. L. J. Chromatogr., A 1997, 781, 197-204. (19) Huff, B. V.; McIntire, G. L. J. Microcolumn Sep. 1994, 6, 591-594. (20) Fan, Z. H.; Kumar, R.; Deffley, G.; Dong, Q.; Stabile, P.; Fare, T. Technical Digest of 1998 Solid-State Sensor and Actuator Workshop, Transducers Research Foundation, Inc., 1998; pp 97-100. (21) Fan, Z. H.; York, P.; Cherukuri, S. Microstructures and Microfabricated Systems IV; Hesketh, P. J., Barna, G., Hughes, H. G., Eds.; The Electrochemical Society: Pennington, NJ, 1997; pp 86-93.

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Figure 1. (a) Layout of the microfabricated device used. Eight parallel channels are used for the introduction of DNA target/bead complexes. DNA probes are pumped from the probe inlet and bifurcated into all channels. The detection area is also indicated. (b) The cross-sectional view of the device and peripheries. A magnet is placed on the top of the device at the detection area where beads are localized. Two heaters are attached to the bottom on both sides of the captured beads. The temperature near the beads is monitored and controlled by a thermocouple.

between target channels is 4 mm, except for 8 mm between channels 4 and 5. The total length of each probe channel from the inlet to the intersection at a target channel is 38.5 mm. All probe channels were designed to have the same length, so that a probe was uniformly distributed into all target channels. The T-intersection crossing the probe and target channels is 16 mm from the left reservoir of a target channel. The device was assembled by sandwiching it between a Plexiglas fixture and a modified microscope stage insert. The fixture, fabricated in-house, consisted of ports that allow tightly screwed connections with Upchurch connectors (Oak Harbor, WA). The ports were constructed so that they aligned with the exit holes of the device. A hole of 0.030 in., matching the size of the exit holes in the device, was drilled in the center of each connector port to permit introduction of solution to the device. The modified stage insert was also made in-house from a stainless steel plate. A 1.75 in. × 1.75 in. square aperture was cut into the plate with a 0.22-in. ledge for seating the device. The aperture also provided a view of the device through the microscope. Intimate contact between the fixture and the device was made using O-rings placed between the top of the device and the bottom of the Plexiglas around the ports. The Plexiglas fixture and the metal stage insert were screwed together to complete the assembly. (22) Fan, Z. H.; Harrison, D. J. Anal. Chem. 1994, 66, 177-184.

Figure 2. Block diagram of the pneumatic pumping apparatus. A pneumatic pressure is generated from a N2 tank and controlled by a proportional servovalve. A gas chamber is used to damp the sudden pulse from valve operations. The pressure in the system is measured by a pressure transducer. Gas is connected to a manifold that splits into 10 branches. Each branch is controlled by a shut-off valve and connected with a reservoir. Only one reservoir is shown for clarity. The solution in the reservoir is pumped through a tube and into the chip device. See text for the detail.

Figure 1b illustrates a cross-sectional view of the device with all other peripherals attached. Magnets (Magnet Applications, Horsham, PA) seated on top of the device over the channels were used to localize the beads in the detection area. Each magnet was 0.2 in. × 0.1 in. × 0.02 in. with a strength at the pole of ∼600 G. Adhesive strip heaters (Minco Products, Minneapolis, MN) were placed on the bottom of the device, on each side of the magnet, to adjust temperature. The heaters were connected to a power supply (model 6216A, Hewlett-Packard, Wilmington, DE) via an Omega temperature controller (model CN8500, Stamford, NJ). A thermocouple seated on top of the device directly above one of the heaters monitored the temperature and provided feedback to the controller. It took ∼30 s to reach 87 °C at the heated region and less than 10 s to cool. A faster temperature rise could be obtained by supplying a larger current or using a heater with a higher resistance. However, it will increase the variation at the elevated temperature and decrease hybridization stringency. Apparatus. A pneumatic pumping apparatus was assembled in-house and is illustrated in Figure 2. A compressed N2 tank set at ∼5 psi provided the pressure source. Precise control of pressure was obtained using a Proportion-Air (McCordsville, IN) QB1 servovalve. The servovalve consists of two solenoid valves to control pressure, and the output is proportional to an electrical signal input. The maximum pressure from this valve is 30 in. of water with a precision of ∼0.1 in. A 4-in. long Bimba gas reservoir with a bore size of 2 in. (Knotts Co., Berkeley Heights, NJ) was used to damp sudden pulses from valve operations. The pressure in the system was measured by an Omega PX164 transducer. Gas was fed to a PEEK manifold that consists of 1 center and 10 branch channels. The diameter of channels in the gas manifold is 0.375 in. Each branch channel was connected to a shut-off valve (Upchurch), which was then connected to a sealed sample vial. Only one sample reservoir is shown in Figure 2 for clarity. Among 10 branch channels, one was designed for probe delivery, eight for the introduction of samples on beads, and one as an extra. As discussed in the Results and Discussion, we chose hydrostatic

Figure 3. The process of DNA hybridization on beads. (a) (1) A biotinylated DNA target (diamond portion) is conjugated to an M-280 streptavidin bead (filled circle portion). (2) A DNA probe is hybridized to the target on bead. Fluorescent label (F) is for detection. (b) (1) An Oligo(dT)25 bead with a covalently bound poly(T) chain. (2) An oligonucleotide-A is attached to the bead by base pairing between poly(A) and poly(T). (3) Poly(T) tail on the bead is extended by DNA polymerization. (4) Single-stranded DNA target on the bead is obtained by heat denaturation. (5) Fluorescently labeled probe is hybridized to the target on the bead and then detected.

pumping for the introduction of samples on beads after evaluating pneumatic pumping of beads. As a result, only one shut-off valve was used for most of this work. The typical size of the vials used was 300 µL, and the volume of sample solution ranged from 50 to 100 µL. The gas from the inlet tube in the vial pumped the sample through the outlet tube into a chip when the shut-off valve was turned on. The diameter of the PEEK outlet tube (Upchurch) is 0.010 in., and its total dead volume, including the connectors, is ∼10 µL. The device was mounted on an inverted fluorescence microscope for detection (Olympus IX70, Melville, NY). The microscope consisted of a halogen lamp for top illumination and a 70-W xenon lamp for sample illumination from the bottom. A cooled chargecoupled device (CCD) camera (model TEA/CCD-512TK.BM, Princeton Instruments, Trenton, NJ) was used for monitoring transport of magnetic beads and measuring fluorescence intensity. An interference filter cube was used for detecting fluorescence signals. The bandwidth of the excitation filter was 459-498 nm and 512-559 nm for the emission filter. The images of beads and fluorescence were acquired and analyzed by an imaging software (MetaMorph, Universal Imaging, Hollis, NH). Materials and Reagents. Corning 7740 (Pyrex) glass plates were purchased from Specialty Glass Products (Willow Grove, PA). Paramagnetic beads (Dynabead M-280 streptavidin, M-270 streptavidin, M-450, and Oligo(dT)25) were procured from Dynal A.S (Oslo, Norway). Tris-acetate buffer was prepared by dissolving 100 mM tris(hydroxymethyl)aminomethane (tris) in water from Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

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Table 1. Sequences of Oligonucleotides Used in This Worka mActin probe (30-mer) UPB probe (27-mer) ECP probe (25-mer) SAP probe (25-mer) BG probe (30-mer) mActin target (30-mer) mActin-A (50-mer)

5′-FTGTGGATCAGCAAGCAGGAGTACGATGAGT-3′ 5′-FGTACAAGGCCCGGGAACGTATTCACCG-3′ 5′-FCATGAATCACAAAGTGGTAAGCGCC-3′ 5′-FGCTCCTAAAAGGTTACTCCACCGGC-3′ 5′-FGGCTCGCTATACAGGTCCATCTTGGAAACT-3′ 5′-XACTCATCGTACTCCTGCTTGCTGATCCACA-3′ 5′-TGTGGATCAGCAAGCAGGAGTACGATGAGTAAAAAAAAAAAAAAAAAAAA-3′

a X and F stand for biotin and fluorescein groups. The sequences of DNA targets are complementary to the probes. Only mActin target is shown as an example. The sequences of oligonucleotide-A are the sequence of corresponding probe with 20 A at the 3′ end as shown in mActin-A. See the text for the code names of DNA probes.

a Milli-Q Plus water system (Millipore, Bedford, MA) and adjusting to pH 8.0 by adding acetic acid (J. T. Baker, Phillipsburg, NJ). TEN2M buffer at pH 7.5 contains 10 mM tris, 1 mM EDTA, and 2 M NaCl, while phosphate-buffered saline (PBS) buffer at pH 7.2 contains 10 mM sodium phosphate and 150 mM NaCl. T4 DNA ligase buffer at pH 7.8 was purchased from New England BioLabs (Beverly, MA), consisting of 50 mM tris-HCl, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM adenosine triphosphate (ATP). All buffers were filtered before use with a 0.22-µm Millipore membrane filter. Each DNA probe was prepared by diluting its stock solution to 1 µM in tris-acetate buffer. All other chemicals were of reagent grade. All oligonucleotides were obtained from Operon Technologies (Alameda, CA), and their sequences are listed in Table 1. The probe names, mActin, BG, UBP, ECP, and SAP, are abbreviated from mouse actin, Bacillus subtilis, universal bacterial probe, E. coli probe, and Staphylococcus aureus probe, respectively. Both mActin and BG probes were designed after searching the mouse actin gene sequence and BG sequence from GenBank (www. ncbi.nlm.nih.gov). UBP, ECP, and SAP probes were chosen according to Greisen et al.23 Preparation of Beads with Targets. Conjugation of biotinylated single-stranded DNA target to M-280 or M-270 streptavidin beads (schematically shown in Figure 3a) was performed using the following protocol. Ten microliters of stock beads (6.7 × 108 bead/mL) were first washed three times in 20 µL of TEN2M and then diluted in 10 µL of TEN2M buffer; 1 µL of 10 µM DNA target solution was added to the beads and the resultant mixture placed on a rotator for 15 min to allow conjugation. After conjugation, the beads were washed and diluted in 40 µL of tris-acetate buffer. Oligo(dT)25 beads with a DNA target were prepared by three steps, i.e., hybridization, polymerization, and denaturation, as shown in Figure 3b. Hybridization is used to attach an oligonucleotide to beads relying on base pairing between the polyadenylate (poly(A)) tail in the oligonucleotide and the poly(T) chain on the bead surface. An aliquot of 20 µL of Oligo(dT)25 stock beads (5.3 × 108 bead/mL) was first washed three times with 200 µL of PBS buffer; 1.2 µL of 10 µM oligonucleotide with poly(A) tail (oligonucleotide-A in Table 1), 10 µL of T4 DNA ligase buffer, and 69 µL of water were added to the beads and incubated at 37 °C for 15 min. Polymerization was then performed to obtain a specific sequence by adding into the bead solution 16 µL of mononucleotide triphosphate mix (A, T, G, and C each at 1.25 mM, Applied Biosystem, Foster City, CA) and 5 µL of Klenow DNA polymerase (23) Greisen, K.; Loeffelholz, M.; Purohit, A.; Leong, D. J. Clin. Microbiol. 1994, 32, 335-351.

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(New England BioLabs) and then incubating at 37 °C for 30 min. This polymerization is essentially the same as in PCR, but only the first half-cycle is carried out. Denaturation was accomplished by heating the bead solution at 95 °C for 2 min, immediately removing the supernatant to separate the denatured DNA template, and then washing the beads with 50 µL of TEN2M buffer. The denaturation step was repeated twice to ensure that no DNA template was retained on beads. The beads were then resuspended in 50 µL of tris-acetate buffer. General Procedures. The device was flushed with 0.1 N NaOH followed by Millipore water and tris-acetate buffer using a syringe. The probe port of the device was then connected to the pneumatic pumping apparatus, and tris-acetate buffer was pneumatically pumped through the PEEK line and the device to flush out air bubbles. After the completion of flushing, solutions in other ports were removed and each was refilled with 30 µL of buffer. Beads were introduced into the channels of the device using hydrostatic pumping. A bead sample was first added to the right port of a target channel. The levels of solutions in both ports were then adjusted so that the higher level in the bead reservoir pumped beads into the device. A magnet seated on top of the device localized the beads in place. After a sufficient number of beads had been accumulated to form a plug with a length of ∼0.5 mm, pumping was stopped by rebalancing the solution levels in both ports. The pneumatic pumping of tris-acetate buffer at the probe inlet port was then turned on to flush the device, after which it was ready for hybridization. Before the introduction of a probe for hybridization, the temperature in the bead region was raised to 37 °C by turning on heaters. After the buffer vial was replaced with a probe vial, the probe was pneumatically pumped through the same PEEK line into the device. Approximately 10 µL of probe was used due to the dead volume in the tubing and connectors. Tris-acetate buffer was then pumped into the device to drive the probe in the dead volume through beads and remove the excess probe. The completion of hybridization was indicated by observance of green fluorescence on beads. Pumping of each solution took 8-12 min, most of which was used to remove previous solution in the dead volume of the PEEK tube and connectors. After acquisition of fluorescence images of the beads, the temperature in the bead region was raised to 87 °C for denaturation while the buffer was continuously pumping. The completion of denaturation was observed by the disappearance of green fluorescence on the beads. The second probe was then pumped through the same set of beads to continue the cycle of hybridization/denaturation.

RESULTS AND DISCUSSION Device. Figure 1 shows the layout of the microfabricated device used to perform dynamic DNA hybridization. The device consists of parallel channels that are indicated as target channels and numbered from 1 to 8. The ends of target channels are staggered with each other to accommodate off-chip connections. The turn at one end of each channel is for pressure balance (vide infra). Each target channel can be filled with one DNA sample (target) that is attached to paramagnetic beads. Therefore, up to eight samples can be simultaneously interrogated with a single DNA probe. The probe is introduced from the probe inlet into the device. Four levels of bifurcation (two-way splitting) are employed to deliver an equal amount of probes into all target channels. Bifurcation is believed to be more uniform than the use of three-way or four-way splitting. A fluid with a parabolic flow profile under pneumatic pressure can be evenly split into two portions. When a three-way splitting takes place, however, the middle portion has a larger volume than the two portions at the walls. Experimental results (data not shown) indicate that the standard deviation among samples in channels was less than 5% when the bifurcation scheme was employed. One point to note is that all channels must have equal channel resistance to balance pressure. In our case, with the same channel width and depth, the lengths of channels were designed to be equal so that uniform splitting could be achieved. Pneumatic Pumping. Electroosmotic pumping (EO) is widely used in capillary electrophoresis-based chips.6,8-11,20,22 Pumping of paramagnetic beads using EO has been demonstrated both in a capillary19 and in a microfabricated device.20 However, DNA has relatively high negative electrophoretic mobility due to its large negative charges. As a result, EO pumping of DNA requires a buffer with a large electroosmotic mobility so that EO can overcome DNA’s negative electrophoretic mobility. Unfortunately, buffers used in DNA hybridization often contain a high concentration of salts, which reduce electroosmosis and make EO pumping a less effective approach. We have evaluated pneumatic pressure as the pumping method and used it to transport DNA probes in the experiments reported here. The major advantage of pneumatic pumping is its independence of pumping speed on the characteristics of solutions. The pneumatic pumping setup is shown in Figure 2 and has been described in the Experimental Section. The apparatus was able to pump paramagnetic beads into a device; however, beads must be homogeneously dispersed immediately before the bead vial is connected to the apparatus. Settlement and aggregation of beads in the vial could plug the end of the PEEK tube. Adsorption of beads onto the tube and device was not observed at the pumping speed used. Nevertheless, we used hydrostatic pumping for bead introduction in the experiments reported here due to its ease and simplicity, as discussed in the Experimental Section. Use of Paramagnetic Beads. Dynabeads M-280 were first evaluated for performing DDH. M-280 beads are uniform, superparamagnetic, polystyrene beads that are covalently coated with streptavidin.16 The diameter of M-280 beads is 2.8 µm and its biotin-binding capacity is ∼700 pmol of biotin/mg of beads according to the manufacturer’s specifications.24 We found that (24) Dynal A.S. Product description sheet accompanying products, Dynal Corp., Oslo, Norway.

Figure 4. Fluorescent images of beads in a microfabricated device. Beads are localized in a channel by a magnet: (a) M-280 beads conjugated with mActin target. (b) The same beads after hybridization with mActin probe.

M-280 beads are easy to pump due to their uniform size. The variation in bead size results in not only the nonuniform bead flow rate but also the problems caused by large bead settlement at low pumping speeds. The transport of beads in a device can be performed by either electroosmotic pumping20 or hydrostatic pumping (this work). Figure 3a schematically shows the process of using M-280 beads for DNA hybridization. Conjugation between a biotinylated DNA target and streptavidin-coated beads was carried out off-chip for this work, although we have demonstrated that this reaction can be executed on-chip as well.20 Hybridization reactions were performed in a microfabricated device. The number of oligonucleotides on each bead for all experiments (except where specified) was 1 × 106 molecules according to stoichiometrical calculations. The amount of probe was always in excess to drive hybridization to completion. The typical amount of probe was ∼10 times more than the amount of targets on the beads. The excess probe was removed after hybridization by pumping wash buffer through the beads. Figure 4 shows the typical images of M-280 beads conjugated with the mActin target and the same beads after hybridization with the mActin probe. The length of the bead plug in the figure is ∼750 µm. The volume of the reaction area was calculated to be 2.2 nL, based on an approximate trapezoid shape of the channel. The background fluorescence on bare beads is believed due to the impurities, such as plasticizers, in polystyrene. We observed on M-450 beads a degree of fluorescence similar to that found on M-280 beads with a target. Since M-450s are bare polystyrene beads, this result indicates both streptavidin and DNA do not significantly contribute to the background signal. DNA Hybridization. DNA hybridization discussed below was performed by pumping DNA probes through target-bearing beads in a microfluidic device. Hybridization reactions took place in trisacetate buffer, which was chosen to enhance discrimination, as well as electroosmotic pumping used in the work reported previously.20 Discrimination against 3-base-pair (bp) mismatched probes was observed much better in tris-acetate buffer than in either TEN2M or 300 mM sodium chloride and 30 mM sodium citrate (2×SSC) buffer, pH 7.0. The yield of hybridization between Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

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mActin target on beads and mActin probe in tris-acetate buffer is (59 ( 4)% (n ) 7) of that in TEN2M buffer. The hybridization temperature was experimentally determined to be 37 °C to achieve better discrimination against other probes. This temperature is slightly higher than the calculated melting temperature of the mActin probe (35.5 °C) using an empirical equation.25 At 37 °C, the hybridization signal between the mActin target and a probe with 3-bp mismatch in a microfabricated device was less than 0.5% of that between the target and a complementary probe. The typical hybridization time observed in dynamic DNA hybridization is a few seconds. Concentrated green fluorescence on beads was detected immediately after the flow front of a probe solution entered the bead region when the beads were attached with complementary targets. The rapid hybridization reaction using this scheme in a microfabricated device was also observed when the transport of both target-bearing beads and DNA probes was executed by electroosmotic pumping.20 The hybridization speed in DDH is much faster than that in other DNA hybridization arrays, in which hybridization time was from 3 to 18 h.26,27 Furthermore, it is comparable to the reaction rate in the electric field-assisted DNA hybridization.14 There are two primary reasons for rapid hybridization in DDH. First, a probe is pumped through a column of beads in DDH, so that it takes little time to reach targets on beads for interactions. In contrast, DNA needs to diffuse from the bulk to the surface in order to interact with complementary DNA on a solid support in other hybridization arrays.4,12,15,26,27 This is also evidenced by the fact that a mixing procedure by rotating the device reduced the hybridization time from overnight to 3 h.26 In other words, hybridization is probably reaction-limited in DDH whereas it is diffusion-limited in other hybridization arrays.4,12,15,26,27 Second, an excess amount (∼10×) of probe is supplied in DDH and this saturation effect drives hybridization to completion. In the other hybridization arrays,4,12,15,25,26 however, only a limited amount of DNA probe is fixed on a solid support (e.g., a Si chip). Although the DNA targets (samples) in free solution could be in great excess to enhance reaction rate, they are often either unavailable or it is economically unacceptable in real applications. Reuse of Beads for Multiple Hybridizations. For genomic applications, one DNA target sometimes needs to be interrogated by several probes with different DNA sequences of interest. For instance, a few probes may be utilized in a sequence to hybridize with different portions of a gene to verify its presence. Another example is high-throughput DNA hybridization screening, in which DNA targets also need to be interrogated with several probes. For multiple hybridizations between one target and several probes, reusing the same set of beads is advantageous because of less sample requirement, more accurate quantitation, and faster speed for high-throughput hybridization. However, denaturation must be performed between hybridizations in order to interrogate (25) Sambrook, J.; Fritsch, F. F.; Maniatis, T. Molecular CloningsA Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY,1989; Chapter 9. (26) Chee, M.; Yang, R., Hubbell, E.; Berno, A.; Huang, X. C., Stern, D.; Winker, J.; Lockhart, D. J., Morris, M. S., Fodor, S. P. Science 1996, 274, 610-614. (27) Schema, M.; Shalon, D.; Davis; R. W.; Brown; P. O. Science 1995, 270, 467-470.

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Figure 5. Effects of heat denaturation on the properties of functional groups on beads in a microfabricated device. (a) The fluorescence intensity decreases with the cycle of hybridization/denaturation when M-280 beads are used. (b) The fluorescence intensity is maintained when Oligo(dT)25 beads are used for 12 cycles of hybridization/ denaturation.

a single target with several probes. The experimental result shows that M-280 beads can be reused for DNA hybridization, although the fluorescence signal decreases with the number of hybridization/denaturation cycles. Figure 5a illustrates this with M-280 beads used for seven cycles of hybridization/denaturation in a microfabricated device. The beads were conjugated with mActin target and then hybridized with mActin probe. Hybridization temperature was set at 37 °C while denaturation was at 87 °C. Although the signal-to-noise ratio is larger than 3 after seven cycles, the decrease in signal renders quantitation difficult. The signal decrease is believed to be a result of the deterioration of binding between streptavidin and the M-280 beads. Each streptavidin molecule contains four binding sites. One binding site is attached to the bead. Among the remaining three, the one geometrically in the middle is primarily responsible for the binding of biotin. The two on the sides tend to spatially hinder biotinylated DNA from approaching the biotin-binding sites. This hindrance is believed to increase after several denaturations at high temperature due to the conformation change (denaturation) of streptavidin with high-temperature cycles.28-30 The partial denaturation of streptavidin at high temperature could also result in (28) Larson, F. Cytomics A.S, personal communication, 1998. (29) Reznik, G. O.; Vajda, S.; Smith, C. L.; Cantor, C. R.; Sano, T. Nat. Biotechnol. 1996, 14, 1007-1011.

the deterioration of binding between streptavidin and the biotin moiety, thus the loss of captured biotinylated DNA targets. The results using Dynabeads M-270 indirectly confirm this observation. M-270 beads are also streptavidin-coated, superparamagnetic, polyurethane (not polystyrene) beads.16 The diameter of the beads is 2.9 µm and their biotin-binding capacity is ∼900 pmol of biotin/mg of beads according to the manufacturer’s specification.24 Besides the different polymer coating, the major difference between M-270 and M-280 beads is that the former is hydrophilic whereas the latter is hydrophobic. Our experiments indicate that the fluorescence signal decreases with the number of hybridization/denaturation cycles for both M-270 and M-280 streptavidin beads, although the percentage decrease is slightly smaller for M-270 than it is for M280. The significant difference in surface properties between these two types of beads failed to change the signal decrease trend. Therefore, degradation of the streptavidin coating is the probable cause of signal decrease. One possible solution to this problem is to use beads that do not employ streptavidin-biotin conjugation. We found Dynabeads Oligo(dT)25 an ideal candidate. Oligo(dT)25 beads have properties similar to M-270 except that they contain 25-nucleotide-long chains of deoxythymidylate (poly(T)). The poly(T) chain is covalently attached to the bead surface via a 5′ linker group (Figure 3b(1)). Oligo(dT)25 beads are designed for rapid isolation of mRNA from the cell extract, as most mRNA have a poly(A) tail at the 3′ end. Figure 3b shows the protocol of using Oligo(dT)25 beads for DNA hybridization. An oligonucleotide was designed to contain a sequence complementary to the DNA target and a poly(A) tail at the 3′ end. The oligonucleotide was first attached to beads relying on base pairing between the poly(A) tail in the oligonucleotide and the poly(T) residue attached to the bead surface (Figure 3b(2)). DNA polymerization was then performed, in which the oligonucleotide functioned as a template and poly(T) as a primer, and Klenow was used as DNA polymerase (Figure 3b(3)). The template oligonucleotide was separated from the extended poly(T) residue, leaving single-stranded DNA (as a target for next step) on the beads that was then pumped into the microfabricated device (Figure 3b(4)). A fluorescently labeled probe was hybridized to the target on the beads (Figure 3b(5)). As for M-280 beads, preparation of the DNA/bead complex was carried out off-chip (steps 2-4 in Figure 3b) whereas the hybridization step was performed on-chip (step 5). Figure 5b shows the fluorescence intensities in 12 consecutive hybridizations in a microfabricated device. A denaturation step was performed at 87 °C between hybridizations. This result indicates that the binding capacity of Oligo(dT)25 beads was maintained within experimental variations in hybridization/ denaturation cycles. We conclude that Oligo(dT)25 beads are better than M-280 and M-270 beads for DDH using multiple probes. The probable reason is that the poly(T) chain in the Oligo(dT)25 beads is not a protein. Unlike streptavidin in M-280 beads, the poly(T) chain did not denature at high temperature. As a result, covalent binding between poly(T) and the beads as well as the binding between poly(T) and the extended nucleotides was kept intact without loss. (30) Sano, T.; Pandori, M. W.; Chen, X.; Smith, C. L.; Cantor, C. R. J. Biol. Chem. 1995, 270, 28204-28209.

Dynamic DNA Hybridization. DDH is a hybridization process performed by pumping DNA probes through target-bearing beads in a microfluidic device. Dynamic refers to the fact that paramagnetic beads, DNA target, and probe can be changed as needed, through pumping them into a microfabricated device. Probes are not fixed on the chip as in the DNA hybridization arrays.12-15 In addition, the amount of beads and DNA can also be adjusted as required. For example, the probe can be continuously pumped through target-bearing beads to drive hybridization to completion. In the DDH approach, DNA target samples are first attached to paramagnetic beads using streptavidin-biotin conjugation or base-pairing between oligonucleotides. These DNA/bead complexes are simultaneously introduced into parallel target channels in a microfabricated device shown in Figure 1. A magnet is used to localize these beads in the detection region. The introduction of a DNA probe through a bifurcation scheme allows all DNA targets to be interrogated by the probe simultaneously. The probe is fluorescently labeled, so that the detection of fluorescence in a channel after washing indicates that the target in that channel has a complementary sequence to the probe. After removing the first probe by denaturation, the same set of targets can be interrogated with a different probe. It is also feasible to immobilize DNA probes onto beads, followed by pumping target sample through probe-bearing beads in a microfabricated device. However, this scheme requires probe/ bead complexes to split into parallel channels if multiple samples are simultaneously interrogated by one probe as discussed in this work. Our experimental results indicate that bifurcation of beads in a microfabricated device is less uniform than bifurcation of solutions probably due to inhomogeneity of the bead suspension. In addition, rapid hybridization will be difficult to achieve because of the lack of an excess amount of DNA target in solution. As discussed previously, DNA target sample in great excess is economically unacceptable or simply unavailable in real applications. We used nine synthesized oligonucleotides as DNA targets and probes to demonstrate DDH. Four oligonucleotides were designated as targets and five as probes. The names and sequences of oligonucleotides are listed in Table 1. The device used to perform experiments is shown in Figure 1. Each DNA target was produced using Oligo(dT)25 beads as discussed previously. DNA target/bead complexes were hydrostatically pumped into the device. Channels 1 and 5, 2 and 6, 3 and 7, and 4 and 8 were respectively filled with BG, mActin, ECP, and UBP targets. To ensure all beads were captured by the magnet without straying into the side probe channels, the pumping of bead/targets was from the right to the left reservoirs. All DNA targets had a chance to react with the first probe as discussed in the Experimental Section. Hybridization reactions took place at 37 °C in trisacetate buffer. The results are shown in Figure 6, in which eight signals in green were obtained from hybridization with the BG probe. Each signal represents what was measured in the order from channel 1 to channel 8. The presence of positive signals in channels 1 and 5 indicates the targets in these two channels contain a DNA sequence complementary to the BG probe. A denaturation step was carried out before the introduction of the second probe. Denaturation can be executed either by heating Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

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Figure 6. DDH among four DNA targets and five DNA probes in a microfabricated device. BG, mActin, ECP, and UBP targets are respectively filled in channels 1 and 5, 2 and 6, 3 and 7, and 4 and 8. The probes are introduced in the order as indicated in y axis. A denaturation procedure is performed after hybridization with each probe. Positive signals are observed in only those channels filled with complementary targets.

at a high temperature or by the introduction of 0.1 N NaOH, as suggested by the bead manufacturer. We found that denaturation was faster and more complete when heating was used. After studying various temperatures, we chose 87 °C as the temperature for all experiments. It should be noted that the actual temperature in the channels is probably slightly different from the one we measured at the spot shown in Figure 1b. The probes were introduced in the order of BG, mActin, SAP, ECP, and UBP. Probe SAP was used as a negative control since its sequence is not complementary to any targets. Figure 6 compiles all hybridization results between four targets and five probes. Hybridization signals were observed only in those channels filled with targets that have sequences complementary to the respective probes. Background signals were obtained from all other channels as expected. The whole experiment was completed in less than 3 h. The most time was used to flush the dead volume in tubes and connectors as discussed in the Experimental Section. The dead volume, accordingly the sample volume required, is in the microliter range, which is several orders of magnitude larger than the volume (2.2 nL) of the actual reaction area, i.e., the region occupied by beads. The amount of probe required for hybridization reactions in a microfabricated device is probably from tens to hundreds of nanoliters. A liquid delivery system in such a range is going to be commercially available as the current efforts in the commercialization of microfluidics technology progress. Therefore, the time used for DDH will be significantly reduced after the macro-micro interface is improved. CONCLUSIONS We have demonstrated the approach of DDH using four synthesized DNA targets and five probes in a microfabricated 4858 Analytical Chemistry, Vol. 71, No. 21, November 1, 1999

device. Simultaneous analysis of eight DNA samples in a chip suggests it should be feasible to extend DDH to analyze a large number of samples in a densely packed chip. We have investigated three types of paramagnetic beads and found Dynabeads Oligo(dT)25 are the most suitable beads for DDH. DNA targets on Oligo(dT)25 beads could be sequentially interrogated for 12 times by DNA probes and there was no significant change in hybridization signal. In addition, preliminary results using β-globin 286-basepair PCR product indicate that the method can be extended to genomic DNA. The approach proved to be very promising for both qualitative and quantitative screening of DNA samples. DDH offers several advantages. First, the hybridization process is dynamic since both the type and the amount of paramagnetic beads, DNA target, and probe can be changed as needed, through pumping them into a microfabricated device. Second, the hybridization reaction is faster and the reaction efficiency is higher, since it is conducted in a small confined area (∼2 nL) and there is a continuous pumping of fresh probe solution. Third, multiple DNA samples can be introduced in different channels in a microdevice and all samples can be analyzed at once. Fourth, the concentration of probes from solution onto beads enhances sensitivity. Finally, DDH possesses potential benefits related to miniaturization, such as less sample amount and less reagent consumption.5,7 DDH can be readily extended to gene expression analysis. For example, cDNA can be synthesized from RNA extracts from samples such as human tissues. Subsequent digestion of the cDNA with a restriction enzyme produces DNA fragments of interest. PCR amplification of the DNA fragments results in DNA samples that can be analyzed by DDH. The results provide a measure of the expressed mRNA in the original sample.

ACKNOWLEDGMENT This work was financially supported by Orchid Biocomputer Inc. Suggestions from F. Larsen and O. Dahlberg at Cytomics A.S, and from L. Korsnes and E. Finne at Dynal A.S, all at Oslo, Norway, are greatly appreciated. Thanks go to L. Cao and B. Lal at Sarnoff for their preparations of reagents. We gratefully acknowledge technical direction from S. Cherukuri, P. Stabile, and T. Fare at Sarnoff, and technical assistance from P. Coyle, K. Rudofsky, T. Davis, C. Bindra, D. Fishman, B. Hoghooghi, G. Kaganowicz, and S. Lipp at Sarnoff, and G. Tokiwa and A.

Akkapeddi at Orchid. Thanks are also extended to reviewers for their comments and suggestions. SUPPORTING INFORMATION AVAILABLE A table of the data used in Figure 6 is available. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review February 24, 1999. Accepted August 17, 1999. AC9902190

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