UV Graft Polymerization of Polyacrylamide Hydrogel Plugs in

Nelson, C.; Hendy, S.; Reid, K.; Cavanagh, J. Biotechniques 2002, 32, ...... Ryan L. Wood , Israel Guerrero-Arguero , Richard A. Robison , William G. ...
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Langmuir 2003, 19, 6901-6904

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UV Graft Polymerization of Polyacrylamide Hydrogel Plugs in Microfluidic Channels Rebecca A. Zangmeister* and Michael J. Tarlov Chemical Science and Technology Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899 Received March 12, 2003. In Final Form: May 8, 2003 It has been recently demonstrated that single-stranded DNA, modified on the 5′ end with an acrylic acid functionality, can be incorporated into a polyacrylamide hydrogel matrix. These types of DNA-containing gels have recently been spatially immobilized in plastic microfluidic channels by photopolymerization, creating selective DNA three-dimensional capture elements. The DNA oligomers retain activity and are able to bind complementary target strands as they migrate through the gel plug under electrophoretic conditions. One problem that has compromised the performance of the DNA hydrogels is gel plug breakdown under continuous electrophoretic operation. When gel plugs fail, the gel delaminates from the microchannel wall and electroosmotic flow occurs between the plug and the microchannel wall. Past measurements have shown that the lifetime of the polyacrylamide gel plugs rarely exceeds 25 min under continuous use. Here we report a method to increase the stability of the polyacrylamide gel plugs by the introduction of polymerization attachment points to the polymeric microchannel surfaces prior to gel plug formation. Ultraviolet/ozone (UV/O3) treatment is used to oxidize both top poly(methyl methacrylate) (PMMA) and bottom poly(carbonate) (PC) surfaces. A methacrylate functionality, which can cross-link with the polyacrylamide gel, is introduced by reacting the oxidized surfaces with 3-methacryloxypropyltrimethoxysilane. Polyacrylamide is then UV grafted onto the chemically modified model surfaces. Contact angle measurements and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra confirm polyacrylamide grafting on PC and PMMA surfaces. The success of graft polymerization within the microchannel devices is also corroborated by scanning electron microscopy of delaminated devices and timed performance measurements under electrophoretic conditions that demonstrate an increase in the lifetime of the gel plugs by 2.5 times on average.

Introduction Polymeric hydrogel materials provide stable supports for oligonucleotide immobilization.1-17 Oligonucleotides have been immobilized through cross-linking during * Corresponding author. E-mail: [email protected]. (1) Kenney, M.; Ray, S.; Boles, T. C. Biotechniques 1998, 25, 516521. (2) Nelson, C.; Hendy, S.; Reid, K.; Cavanagh, J. Biotechniques 2002, 32, 808-815. (3) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hammond, P. W.; Kenney, M.; Boles, T. C. Nucleic Acids Res. 1999, 27, 649-655. (4) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (5) Livshits, M. A.; Mirzabekov, A. D. Biophys. J. 1996, 71, 27952801. (6) Guschin, D.; Yershov, G.; Zaslavsky, A.; Gemmell, A.; Shick, V.; Proudnikov, D.; Arenkov, P.; Mirzabekov, A. Anal. Biochem. 1997, 250, 203-211. (7) Fotin, A. V.; Drobyshev, A. L.; Proudnikov, D. Y.; Perov, A. N.; Mirzabekov, A. D. Nucleic Acids Res. 1998, 26, 1515-1521. (8) Proudnikov, D.; Timofeev, E.; Mirzabekov, A. Anal. Biochem. 1998, 259, 34-41. (9) Vasiliskov, A. V.; Timofeev, E. N.; Surzhikov, S. A.; Drobyshev, A. L.; Shick, V. V.; Mirzabekov, A. D. Biotechniques 1999, 27, 592-605. (10) Tillib, S. V.; Mirzabekov, A. Curr. Opin. Biotechnol. 2001, 12, 53-58. (11) Kolchinsky, A.; Mirzabekov, A. Hum. Mutat. 2002, 19, 343360. (12) Broude, N. E.; Woodward, K.; Cavallo, R.; Cantor, C. R.; Englert, D. Nucleic Acids Res. 2001, 29, e92. (13) Mitra, R.; Church, G. Nucleic Acids Res. 1999, 27, e34. (14) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E. G.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-4914. (15) Sosnowski, R. G.; Tu, E.; Butler, W. F.; Oconnell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119-1123. (16) Gurtner, C.; Tu, E.; Jamshidi, N.; Haigis, R. W.; Onofrey, T. J.; Edman, C. F.; Sosnowski, R.; Wallace, B.; Heller, M. J. Electrophoresis 2002, 23, 1543-1550. (17) Heller, M. J.; Forster, A. H.; Tu, E. Electrophoresis 2000, 21, 157-164.

10.1021/la034424t

polymerization,1-4 covalently bound to prepolymerized gels using chemical treatments,5-13 and noncovalently bound using electrophoretically driven biotin-avidin binding.14-17 Single-stranded DNA (ssDNA) modified on the 5′ end with an acrylic acid functionality can copolymerize with polyacrylamide, creating a stable gel matrix with high probe density.1-4 Electrophoresis of single-stranded sequences through such probe-containing gel matrixes results in hybridization-mediated capture of complementary targets, while noncomplementary targets migrate through.1,3-4 Mutation assays,1 isolation of DNA-binding proteins,2 solid-phase PCR,3 and multitarget identification4 have been demonstrated on such supports. Plugs of ssDNA probe-containing gels have recently been immobilized in plastic microfluidic channels using UVinitiated photopatterning.4 The high probe density of the gel plugs and enhanced mass transfer characteristics of the microfluidic channels result in efficient capture of electrophoretically driven targets. Although the effectiveness of these probe-containing plugs has been demonstrated, a problem in their use has been limited long-term stability. The useful lifetime of the polyacrylamide gel plugs under continuous electrophoretic conditions was 25 min or less at voltages of 10-25 V.4 Failure events were observed by the onset of electroosmotic flow and concurrent drop in microchannel resistance. Such failure events were hypothesized to occur when electroosmotic flow initiates through microscopic pathways that develop at the gel/ microchannel wall interface, or within the gel network itself.4 In this paper we report a method to increase the stability of the gel plugs by improving the polyacrylamide adhesion to the microchannel wall surfaces using ultraviolet/ozone

This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society Published on Web 07/03/2003

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Langmuir, Vol. 19, No. 17, 2003

Zangmeister and Tarlov

Scheme 1. Schematic Diagram of the Chemical Modification Procedure Used to Modify PMMA and PC Polymer Surfacesa

a First, the surface is oxidized by exposure to UV/O3; second, the activated surface is reacted with 3-methacryloxypropyltrimethoxysilane, forming pendant methacrylate groups that can act as polymerization anchor points for acrylamide monomers during the UV graft polymerization.

(UV/O3) and chemical modification prior to gel plug formation. Various techniques have been employed to modify polymer surfaces by polymer grafting.18-24 Here we describe a three-step process involving chemical pretreatment and surface grafting that increases the adhesion of the polyacrylamide gel plugs to the microchannel walls. First, UV/O3 treatment is used to oxidize the polymer surfaces. Second, a methacrylate functionality, which can cross-link with the polyacrylamide gel, is introduced by reacting the oxidized surfaces with 3-methacryloxypropyltrimethoxysilane. Last, polyacrylamide layers are UV grafted onto the chemically modified poly(carbonate) (PC) and poly(methyl methacrylate) (PMMA) surfaces. Contact angle measurements and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectra were used to confirm the presence of polyacrylamide graft layers on model PC and PMMA surfaces. In addition, scanning electron microscopy (SEM) and fluorescence imaging were used to examine the adhesion of the gel plugs to the microchannel walls and to determine changes in stability. The pretreatment method was found to increase the lifetime of the gel plugs by 2.5 times on average. Experimental Section Materials and Chemicals.25 The two-component microchannel devices were made from poly(carbonate) (McMasterCarr, Atlanta, GA) and UV-transparent Acrylite OP-4 (PMMA, Cyro Industries, Mt. Arlington, NJ). Acrylamide (Sigma, St. Louis, MO), 3-methacryloxypropyltrimethoxysilane (PlusOne BindSilane, Pharmacia Biotech, Piscataway, NJ), sodium chloride (Mallinckrodt, Inc., Paris, KY), and 10× TE buffer (pH 7.4, 100 mmol/L Tris‚HCl, 10 mmol/L EDTA, Research Genetics, Huntsville, AL) were used as received. Acrydite-modified single-strand (18) Henry, A. C.; Waddell, E. A.; Shreiner, R.; Locascio, L. E. Electrophoresis 2002, 23, 791-798. (19) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Anal. Chem. 2002, 74, 4117-4123. (20) Uyama, Y.; Kato, K.; Ikada, Y. Adv. Polym. Sci. 1998, 137, 1-39. (21) Loh, F. C.; Tan, K. L.; Kang, E. T.; Neoh, K. G.; Pun, M. Y. J. Vac. Sci. Technol., A: Vac. Surf. Films 1994, 12, 2705-2710. (22) Loh, F. C.; Tan, K. L.; Kang, E. T.; Neoh, K. G.; Pun, M. Y. Eur. Polym. J. 1995, 31, 481-488. (23) Ichijima, H.; Okada, T.; Uyama, Y.; Ikada, Y. Makromol. Chem. 1991, 192, 1213-1221. (24) Chen, W.; Neoh, K. G.; Kang, E. T.; Tan, K. L.; Liaw, D. J.; Huang, C. C. J. Polym. Sci., Polym. Chem. 1998, 36, 357-366. (25) Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose.

DNA probes (Acrydite-5′-AGGCCCGGGAACGTATTCAC-3′) were provided by Mosaic Technologies (Waltham, MA). TAMRAlabeled targets (TAMRA-5′-GTGAATACGTTCCCGGGCCT-3′) were purchased from Operon Technologies (Huntsville, AL). All solutions were prepared using 18.2 MΩ water from a NANOpure UV system (Barnstead, Dubuque, IA). Polymer Surface Modification.25 Native PMMA and PC substrate surfaces were activated (as illustrated in the first step of Scheme 1) by placing in a UV/O3 cleaner (UVOCS, Montgomeryville, PA)