Anal. Chem. 2005, 77, 5583-5588
Development of a Microfluidic Platform with an Optical Imaging Microarray Capable of Attomolar Target DNA Detection Michaela Bowden, Linan Song, and David R. Walt*
Department of Chemistry, Tufts University, Medford, Massachusetts 02155
In this paper, DNA hybridization in a microfluidic manifold is performed using fluorescence detection on a fiberoptic microarray. The microfluidic device integrates optics, sample transport, and fluidic interconnects on a single platform. A high-density optical imaging fiber array containing oligonucleotide-labeled microspheres was developed. DNA hybridization was observed at concentrations as low as 10 aM with response times of less than 15 min at a flow rate of 1 µL/min using 50 µL of target DNA samples. The fast response times coupled with the low sample volumes and the use of a high-density, fiberoptic microarray format make this method highly advantageous. This paper describes the initial development, optimization, and integration of the microfluidic platform with imaging fiber arrays. Optical imaging fiber arrays are composed of a high density of individually addressable fibers, each with their own core and cladding, bound in a coherent bundle.1 Typical fiber bundles have an outer diameter of between 0.5 and 1.2 mm containing between 5000 and 50 000 individual fibers, each with core diameter dimensions between 3 and 7 µm.2 By selectively etching the core material at the fiber bundle’s distal end, an array of microwells of defined depth is created, which can then be loaded with microspheres. The microspheres contain various sensing materials, for example, oligonucleotide probes attached to amine-functionalized microspheres. To interrogate the microspheres, excitation light is transmitted through the proximal end of the fiber bundle, whereby the light propagates via total internal reflection through the bundle to the distal end.3 The light excites a fluorescently labeled material attached to the microspheres in the microwells, and the emitted light is carried back through the fiber, collected with a CCD camera, and processed with imaging software. Changes in fluorescence intensity correspond to analyte concentration. This array platform permits micrometer-scale multianalyte sensing using small sample volumes and enables high local concentrations of analytes to be achieved even with low absolute * To whom correspondence should be addressed. E-mail: david.walt@ tufts.edu. (1) Walt, D. R. Curr. Opin. Chem. Biol. 2002, 6, 689-695. (2) Epstein, J. R.; Biran, I.; Walt, D. R. Anal. Chim. Acta 2002 459, 62826286. (3) Walt, D. R.; Agayn, V.; Bronk, K.; Barnard, S. Appl. Biochem. Biotechnol. 1993 41, 129-138. 10.1021/ac050503t CCC: $30.25 Published on Web 08/04/2005
© 2005 American Chemical Society
numbers of analyte molecules.4 The fiber-optic microarray format has been implemented in a multitude of biological and chemical applications including the development of multianalyte and biosensor arrays, oligonucleotide detection systems, artificial olfaction, and cell-based microarrays.2,5-8 For detecting specific oligonucleotide sequences, the Southern method was originally employed to identify single, specific radiolabeled oligonucleotide fragments from a complex mixture.9 Today, microarrays are employed for simultaneous detection of hundreds to hundreds of thousands of target sequences with highthroughput and rapid response times. Microarrays for DNA analysis can be constructed by numerous methods including photolithography,10,11 spotting with piezoelectric dispensers,12,13 ink-jet printing,14,15 photodeposition,16,17 or bead assemblies.8,18-21 In the latter approach, oligonucleotide probe sequences are attached to surface-modified microspheres and fluorophores are attached to the complementary target sequences. By attaching different probes to different microspheres, a large number of (4) Epstein, J. R.; Walt, D. R. Chem. Soc. Rev. 2003, 32, 203-214. (5) Epstein, J. R.; Ferguson, J. A.; Lee, K.-H.; Walt, D. R. J. Am. Chem. Soc. 2003, 125, 13753-13759. (6) Kuang, Y.; Biran, I.; Walt, D. R. Anal. Chem. 2004, 76, 2902, 2909. (7) Albert, K. J.; Walt, D. R. Anal. Chem. 2001, 73, 2501-2508. (8) Epstein, J. R.; Leung, A. P. K.; Lee, K.-H.; Walt, D. R. Biosens. Bioelectron. 2003, 18, 541-546. (9) Southern, E. M. J. Mol. Biol. 1975, 98, 503. (10) Warrington, J. A.; Shah, N. A.; Chen, X.; Janis, M.; Liu, C.; Kondapalli, S.; Reyes, V.; Savage, M. P.; Zhang, Z.; Watts, R.; De Guzman, M.; Berno, A.; Snyder, J.; Baid, J. Hum. Mutat. 2002, 19 (4), 402-409. (11) Shin, D. S.; Lee, K.-N.; Jang, K.-H.; Kim, J.-K.; Chung, W.-J.; Kim, Y.-K.; Lee, Y.-S. Biosens. Bioelectron. 2004, 19 (6), 595-606. (12) Allain, L. R.; Stratis-Cullum, D. N.; Vo-Dinh, T. Anal. Chim. Acta 2004, 518 (1-2), 77-85. (13) Kuoni, A.; Boillat, M.; de Rooij, N. F. J. Assoc. Lab. Auto. 2003, 8 (5), 2428. (14) Goldmann, T.; Gonzalez, J. S. J. Biochem. Biophys. Methods 2000, 42 (3), 105-110. (15) Roth, E. A.; Xu, T.; Das, M.; Gregory, C.; Hickman, J. J.; Boland, T. Biomaterials 2004, 25 (17), 3708-3715. (16) Dickinson, T. A.; White, J.; Kauer, J. S.; Walt, D. R. Nature 1996, 382, 697-700. (17) Michael, K. L.; Ferguson, J. A.; Healy, B. G.; Bronk, K. S.; Walt, D. R. Anal. Chim. Acta 1997, 340, 123-131. (18) Ferguson, J.; Steemers, F. J.; Walt, D. R. Anal. Chem. 2000, 72, 56185624. (19) Michael, K.; Taylor, L. C.; Schultz, S. L.; Walt, D. R. Anal. Chem. 1998, 70, 1242-1248. (20) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122 (15), 3795-3796. (21) Nam, J. M.; Stoeva, S. I.; Mirkin, C. A. J. Am. Chem. Soc. 2004, 126 (19), 5931-5933
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sequences can be interrogated simultaneously using the fiberoptic microarray format. Microfluidic platforms are traditionally fabricated from silicon, glass, or both. This choice of materials is primarily due to their excellent physical, electrical, and optical properties.22 The recent trend toward polymer fabrication techniques is fueled by the increasing development and commercial availability of a diverse range of polymer materials, by the introduction of microfabrication techniques targeted at polymers, and also by the high cost and tedious, slow procedures involved in microsystem fabrication in silicon.23-25 In particular, the development of novel microfabrication techniques such as injection molding,26 laser ablation,27 hot embossing,28,29 and soft lithography30-33 have become powerful alternatives. Interest has grown greatly in polymer chip fabrication in recent years with many designs, techniques, and applications appearing in the literature.34-38 In particular, the development of microfluidic networks for biological applications has soared.39-42 Microfluidic platforms have traditionally been used for sample cleanup and preparation and for performing single DNA assays. In contrast, microarrays have been used for high-density hybridization experiments using samples prepared in a separate protocol. In this paper, we integrate a microfluidics module with a microarray to demonstrate the value of fluidic flow systems for enhancing the performance of microarrays. Merging microfluidics with an imaging fiber-optic microarray suitable for analysis of oligonucleotides has numerous benefits including faster response times, nano to picoliter sample volumes, and a higher density of probes compared to single-assay devices.43-46 Recently, the (22) Daridon, A.; Sequeira, M.; Pennarun-Thomas, G.; Lichtenberg, J.; Verpoorte, E.; Diamond, D.; de Rooij, N. F. Proc. Eurosensors XIV 2000, Copenhagen, Denmark, 2000; pp 815-818. (23) Becker, H.; Heim, U. Sens. Actuators, A 2000, 83, 130. (24) Boone, T. D.; Fan, Z. H.; Hooper, H. H.; Ricco, A. J.; Tan, H.; Williams, S. J. Anal. Chem. 2002, 74, 78A-86A. (25) Bowden, M.; Geschke, O.; Kutter, J. P.; Diamond, D. Lab Chip 2004, 3 (4), 221-223. (26) Mc Cormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (27) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Girault, H. H. Anal. Chem. 1997, 69, 2035-2042. (28) Jaszewski, R. W.; Schrift, H.; Gobrecht, J.; Smith, P. Microelec. Eng. 1998, 41/42, 575-578. (29) Xu, J.; Locascio, L.; Gaitan, M.; Lee, C. S. Anal. Chem. 2000, 72, 19301933. (30) Becker, H.; Dietz, W.; Danneberg, P. Proc. Micro TAS ′98; Micro Total Analysis Systems, Banff, Canada; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 253-256. (31) Xia, Y.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153-184. (32) Brettain, S.; Paul, K.; Zhao, X.-M.; Whitesides, G. M. Phys. World 1998, 11, 31-36. (33) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. Engl. 1998, 37, 550575. (34) Schwarz, A.; Rossier, J.; Bianchi, F.; Reymond, F.; Ferringo, R.; H. H. Girault, Proc. Micro TAS ‘98; Micro Total Analysis Systems, Banff, Canada; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 241-244. (35) Rossier, J. S.; Reymond, F.; Michel, P. E. Electrophoresis 2002, 23, 858. (36) Wu, Z.; Xanthopoulos, N.; Reymond, F.; Rossier, J. S.; Bercier, P.; Girault, H. Electrophoresis 2002, 23, 782-790. (37) Mc Donald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35 (7), 491499. (38) Pugmire, D. L.; Wadell, E. A.; Haasch, R.; Tarlov, M. J.; Locascio, L. E. Anal. Chem. 2002, 74, 871-878. (39) Schulte, T. H.; Bardell, R. L.; Weigl, B. H. Clin. Chim. Acta 2002, 321, 1-10. (40) Liu, Y.; Garcia, C. D.; Henry, C. S. Analyst 2003, 128, 1002-1008. (41) Verpoorte, E. Lab Chip 2003, 3 (4), 60N-76N. (42) Bilitewski, U.; Genrich, M.; Kadow, S. Anal. Bioanal. Chem. 2003, 377, 556-569.
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concept of merging microfluidics with newly developed smart, optical detection systems utilizing miniaturized platforms based on CCD or CMOS technology has been described in the literature.47-51 It is envisaged that this technology will eventually replace the bench-scale optical components currently used for the imaging fiber-optic system utilized here. In this paper, we describe our initial approach to integrating an optical fiber oligonucleotide array within a microfluidic manifold. We chose to examine DNA hybridization using two biowarfare agent probes as an initial application. EXPERIMENTAL SECTION Optical Imaging Fiber Bundles. The optical bundles utilized for this research were fabricated and supplied by Illumina, Inc. (San Diego, CA) with microwells etched at the distal end. The bundles had an outer diameter of 1 mm and individual cores of 3.1-µm diameter with center-to-center distances of 5.7 µm, resulting in a high-density microarray of 49 777 individually addressable light pathways. The core and cladding consist of germanium-doped glass with the core (1.694) having a higher refractive index than the cladding (1.56). The core is more easily etched in acid, enabling selective etching of the core to create the microwells. Illumina, Inc. carried out the well etching in-house, the specific details of which were not provided. The well depth was measured with atomic force microscopy to be 3 µm, and the well volume was calculated as 21 fL. Fiber bundles were polished using a standard polishing procedure, also carried out by Illumina, Inc.18 Instrumentation. The fluorescence-based imaging system is a custom-built, computer-controlled epifluorescence microscope (Olympus, Optical Analysis Corp., Cambridge, MA).8 The main components of the system are for illumination including the following: a light source, filter wheels for excitation and emission, and a dichroic filter; optics, including adjustable focus and a range of lenses; and detection, including a CCD camera and imaging software.52 The white light source is a xenon arc lamp, which can excite fluorescing molecules over the UV spectrum. The white light passes through the excitation filter to provide monochromatic light, which is collimated and focused at the proximal end of the fiber and transmitted to the distal end of the fiber containing the microsphere sensors. Light emitted from fluorescence occurring at the distal end is retransmitted through the fiber, where the light passes through the emission filter and is captured by a highresolution Hamamatsu digital imaging camera (ORCA-HR, Optical Analysis Corp., Cambridge, MA). The software (IPLab 6.3, Scanalytics, Inc., Fairfax, VA) controls the apparatus, automates the measurement sequences, and facilitates imaging analysis. (43) Walt, D. R. Science 2000, 287, 451-452. (44) Case-Green, S. C.; mir, K. U.; Pritchard, C. E.; Southern, E. M. Curr. Opin. Chem. Biol. 1998, 2, 404-410 (45) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467470. (46) Southern, E. M. Trends Genet. 1996, 12, 110-115. (47) Vilkner, T.; Janasek, D.; Manz, A. Anal. Chem. 2004, 76, 3373-3386. (48) Swanson, P.; Gelbart, R.; Atlas, E.; Yang, L.; Grogan, T.; Butler, W. F.; Ackley, D. E.; Sheldon, A. Sens. Actuators, B 2000, 64, 22-30. (49) Adama, M. L.; Enzelberger, M.; Quake, S.; Scherer, A. Sens. Actuators, A 2003, 104, 25-31. (50) Pittet, P.; Galvan, J. M.; Lu, G. N.; Blum, L. J.; Leca-Bouvier, B. D. Sens. Actuators, B 2004, 97, 355-361. (51) Yotter, R. A.; Warren, M. R.; Wilson, D. M. Sens. Actuators, B 2004, 103, 43-49. (52) Epstein, J. R.; Lee, M.; Walt, D. R. Anal. Chem. 2002, 74, 1836-1840.
Table 1. Probe ssDNA Sequences Used for Both Static and Microfluidic Measurements
Figure 1. Encoding image for BTK1 and BTK2 with 0.01 and 1 M Eu(III), respectively, taken at excitation and emission wavelengths of 365 and 615 nm, respectively, in 5× PBS solution.
Microsphere Internal Encoding. The 3.1-µm amine-functionalized microspheres of poly(methylstyrene)divinylbenzene were chosen as the starting material (Bang’s Laboratories, Inc., Carmel, IN). Eu(III) thenoyltrifluoroacetonate‚3H2O was employed as the encoding dye, with excitation and emission wavelengths of 365 and 615 nm, respectively. The amine-functionalized microspheres were filtered and washed with dry tetrahydrofuran (THF). The microspheres were then added to a Eu(III) solution in THF. Two concentrations of Eu(III), 0.01 and 1 M, were used to internally encode the microspheres for BTK1 and BTK2 probe sequences, respectively. The microsphere/Eu(III) dye solution was vortexed for ∼2 h. Upon removal of THF, the microspheres contract, entrapping the Eu(III) internally. Microspheres were filtered and washed with methanol and stored in PBS buffer containing 0.01% Tween.5,18 A multitude of differently encoded microsphere stock solutions can be prepared in this way using different concentrations of Eu(III) or a combination of different encoding dyes, specific for each microsphere type. The encoding image was recorded for both BTK1 and BTK2 with internal encoding concentrations of Eu(III) of 0.01 and 1 M, respectively, with a 500-ms exposure time and at a 40× magnification. BTK1 and BTK2 sensors are differentiable from each other based upon their internal encoding concentrations as shown in Figure 1. DNA Probe Sequence Immobilization. Both 50-mer probes were specifically designed based on the target gene (Cry1A) of Bacillus thuringiensis Kurstaki (BTK), a biowarfare agent simulant. The two sequences were labeled as BTK1 and BTK2, respectively. Rapid detection of biowarfare agents is essential to provide rapid diagnosis and treatment.53 The immobilization was based upon an experimental protocol described in detail by Ferguson et al.18 To summarize, aminefunctionalized microspheres were modified with glutaraldehyde and polyethyleneimine (PEI). Cyanuric chloride-activated oligonucleotide probes were then added in excess. The PEI linker was incorporated to enhance the fluorescent signal upon hybridization with complementary target DNA in solution. To inhibit nonspecific binding of target DNA to the amine-functionalized microspheres (53) Walt, D. R.; Franz, D. R. Anal. Chem. 2000, 72, 738A-746A.
probe
sequence
BTK1
TGG-TCA-GGG-CAT-CAA-ATA-ATG-GCTTCT-CCT-GTC-GGT-TTT-TCG-GGG-CCA-GA
BTK2
GGT-AGT-TTT-CGA-GGC-TCG-GCT-CAGGGC-ATA-GAA-AGA-AGT-ATT-AGG-GGT-CC
during hybridization, succinic anhydride was used at the final stage to cap all unreactive amines. The 50-mer oligonucleotide probe sequences for BTK1 and BTK2 are shown in Table 1. Randomly Addressable Microarrays. Small volumes of the microsphere stocks for BTK1 and BTK2 were premixed and randomly loaded onto the fiber in aliquots of e1 µL with a micropipet. The fiber was loaded with a low-concentration sensor stock solution to generate a microarray containing many derelict sites, i.e., no sensors present in microwells. Preliminary studies are typically carried out with a smaller number of total microsphere sensors and sensor types on the fiber microarray. After 15-20 min, the microspheres had settled into the etched wells and excess microspheres and buffer were gently removed from the surface with a swab soaked in deionized water. Fluorescence images were recorded with the IPLab imaging software. The two different internal encoding Eu(III) concentrations of 0.01 and 1 M, used for the BTK1 and BTK2 microspheres, respectively, ensured both microsphere types were clearly distinguishable from each other. The target DNA molecules were labeled with a reporter dye, Cy3, which, upon hybridization, produces a fluorescent signal that corresponds to the concentration of target DNA in solution. The target DNA was prepared in 5× PBS solution containing 0.01% Tween. A fluorescent signal was observed for each microsphere type upon hybridization of the probe sequence with its complementary target in solution. Hybridization studies were performed at room temperature (∼26 °C). An encoding image, taken at the excitation and emission wavelengths of the Eu(III) dye, was used to locate the position and to differentiate between the BTK1 and BTK2 microsphere sensors. Microfluidic Manifold. The microfluidic chip was fabricated in a Tefzel tee consisting of a channel of 1.25-mm internal diameter and a dead volume of
