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Chip-Based Bioassay Using Bacterial Sensor Strains Immobilized in Three-Dimensional Microfluidic Network Hirofumi Tani,* Koji Maehana, and Tamio Kamidate
Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan
A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays. Micro total analysis systems (µTAS) combined microfabrication technology and highly sensitive wet chemical analysis have attracted great attention because of their advantages including the reduction of device size, time, and sample/reagent consumption for assays.1-4 These microsystems have been successfully implemented in biochemical and clinical applications, such as enzyme assay5-7 and immunoassay,8-11 and separation of DNA12-14 * To whom correspondence should be addressed. E-mail: tani@ eng.hokudai.ac.jp. (1) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563-3576. (2) van den Berg, A.; Lammerink, T. S. J. Micro total analysis systems: Microfluidic aspects, integration concept and applications; Springer-Verla: Berlin, 1998. (3) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (4) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (5) Xue, Q. F.; Wainright, A.; Gangakhedkar, S.; Gibbons, I. Electrophoresis 2001, 22, 4000-4007. (6) Mao, H. B.; Yang, T. L.; Cremer, P. S. Anal. Chem. 2002, 74, 379-385. 10.1021/ac049401d CCC: $27.50 Published on Web 10/21/2004
© 2004 American Chemical Society
and proteins.15,16 The microfluidic devices are also utilized for the study of the manipulation and analysis of biological cells,17 such as flow cytometry,18,19 cell fusion,20 cell lysis,21,22 and intercellular enzyme reactions21,23. Recently, single-cell analysis using the microsystems has come to the front, in which single cells are entrapped in microfluidics, and the responses of the cells induced by substances introduced from microchannels are measured in real time.24,25 These approaches can throw light upon unexplained biological processes such as intra- or intercellular communications. The reporter gene technique that exploits regulation of gene expression as physiological responses of the cells has been applied for the cell analysis and cell-based bioassays.26 The whole-cell bioassays such as toxicity tests for new chemicals or environmental pollutants have been usually performed in test tubes or multiwell titer plates with long and tedious procedures, and thus, it is expected to apply the assay to the microfluidic formats due to their capability for highly efficient analysis. (7) Cohen, C. B.; Chin-Dixon, E.; Jeong, S.; Nikiforov, T. T. Anal. Biochem. 1999, 273, 89-97. (8) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 12131218. (9) Jiang, X. Y.; Ng, J. M. K.; Stroock, A. D.; Dertinger, S. K. W.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 5294-5295. (10) Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emne´us, J. Anal. Chem. 2002, 74, 2994-3004. (11) Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; Weigl, B. H.; Yager, P. Nat. Biotechnol. 2001, 19, 461-465. (12) Medintz, I. L.; Paegel, B. M.; Mathies, R. A. J. Chromatogr., A 2001, 924, 265-270. (13) Lagally, E. T.; Medintz, I.; Mathies, R. A. Anal. Chem. 2001, 73, 565-570. (14) Smith, E. M.; Xu, H. W.; Ewing, A. G. Electrophoresis 2001, 22, 363-370. (15) Bousse, L.; Mouradian, S.; Minalla, A.; Yee, H.; Williams, K.; Dubrow, R. Anal. Chem. 2001, 73, 1207-1212. (16) Liu, Y. J.; Foote, R. S.; Jacobson, S. C.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 4608-4613. (17) Andersson, H.; van den Berg, A. Sens. Actuators, B 2003, 92, 315-325. (18) Fu, A. Y.; Spence, C.; Scherer, A.; Arnold, F. H.; Quake, S. R. Nat. Biotechnol. 1999, 17, 1109-1111. (19) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2001, 73, 5334-5338. (20) Stromberg, A.; Karlsson, A.; Ryttsen, F.; Davidson, M.; Chiu, D. T.; Orwar, O. Anal. Chem. 2001, 73, 126-130. (21) Heo, J.; Thomas, K. J.; Seong, G. H.; Crooks, R. M. Anal. Chem. 2003, 75, 22-26. (22) Schilling, E. A.; Kamholz, A. E.; Yager, P. Anal. Chem. 2002, 74, 17981804. (23) Schulz, C. M.; Scampavia, L.; Ruzicka, J. Analyst 2002, 127, 1583-1588. (24) Andersson, H.; van den Berg, A. Curr. Opin. Biotechnol. 2004, 15, 44-49. (25) Huang, W. H.; Cheng, W.; Zhang, Z.; Pang, D. W.; Wang, Z. L.; Cheng, J. K.; Cui, D. F. Anal. Chem. 2004, 76, 483-488. (26) Lewis, J. C.; Feltus, A.; Ensor, C. M.; Ramanathan, S.; Daunert, S. Anal. Chem. 1998, 70, A579-A585.
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Recently, a new format for on-chip assay using a microfluidic network has been developed.27 In this assay, called a micromosaic immunoassay, a series of probes (antibody) is immobilized on a planer substrate as narrow stripes using a multichannel chip, and then a series of samples (protein A) is brought from a second multichannel chip as the probe and the sample lines make a grid pattern. The interactions between the immobilized probes and the analytes in samples take place at the intersection of the two lines, and thus, all combinations of the probes and the samples can be monitored on the single planer substrate. Additionally, threedimensional microfluidic networks have been developed for an investigation tool of multiple biochemical interactions in an array format, in which two sets of microchannels in different layers are crossed at right angles.28 In this system, enzyme- and cell-based reactions were applied and detected at each intersection of the channels. These well-conceived systems taking advantage of both microfluidics and microarray formats can be potent tools for highthroughput analysis. The microfluidic channels have been also used for patterning cells on a chip substrate.29,30 Photopolymerization of poly(ethylene glycol) was exploited for immobilization of cells inside the threedimensional microstructure of hydrogels in microchannels on a chip.31 This seems to be one of the most promising techniques for expanding the mosaic format to cell-based biosensing, but the assay is not yet realized. We have reported on the use of chemiluminescence as a detection method in µTAS,32,33 since it is widely received as highly sensitive without the necessity of light sources, which should be advantageous to realizing µTAS.34 In this paper, we have applied the whole-cell bioassay based on the expression of a bioluminescence reporter gene to µTAS in the mosaic format. In this experiment, we used Escherichia coli strains having a plasmid with firefly luciferase gene (luc) under transcriptional control of mutagen-induced bacterial SOS response as sensor bacteria for mutagenicity.35 Additionally, we attempted immobilization and induction of the sensor bacteria for on-chip expression of luc. To achieve this as an on-chip whole-cell bioassay in the mosaic format, a three-dimensional microfluidic network system composed of one perforated microwell chip bound in two microchannel chips has been employed in which the sensor cells are immobilized in agarose, one of the most benign hydrogels for cells. EXPERIMENTAL SECTION Materials. Silicon wafers (100) with a thickness of 625 µm were obtained from Osaka Tokushu-gokin. Negative photoresist SU-8 was from MicroChem Corp. Poly(dimethylsiloxane) (PDMS) prepolymer (Sylgard 184) and curing agent were from Dow (27) Bernard, A.; Michel, B.; Delamarche, E. Anal. Chem. 2001, 73, 8-12. (28) Ismagilov, R. F.; Ng, J. M.; Kenis, P. J.; Whitesides, G. M. Anal. Chem. 2001, 73, 5207-5213. (29) Tan, W.; Desai, T. A. Biomaterials 2004, 25, 1355-1364. (30) Kane, R. S.; Takayama, S.; Ostuni, E.; Ingber, D. E.; Whitesides, G. M. Biomaterials 1999, 20, 2363-2376. (31) Koh, W. G.; Pishko, M. Langmuir 2003, 19, 10310-10316. (32) Kamidate, T.; Kaide, T.; Tani, H.; Makino, E.; Shibata, T. Anal. Sci. 2001, 17, 951-955. (33) Kamidate, T.; Kaide, T.; Tani, H.; Makino, E.; Shibata, T. Luminescence 2001, 16, 337-342. (34) Roda, A.; Guarigli, M.; Michelini, E.; Mirasoli, M.; Pasini, P. Anal. Chem. 2003, 75, 463A-470A. (35) Maehana, K.; Tani, H.; Shiba, T.; Kamidate, T. Anal. Chim. Acta 2004, 522, 189-195.
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Corning. Agarose type VII with low gelling temperature (
