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Simultaneous Optimization of Monolayer Formation Factors, Including Temperature, To Significantly Improve Nucleic Acid Hybridization Efficiency on Gold Substrates Andrew D. Pris,* Sara G. Ostrowski, and Sarah D. Garaas General Electric-Global Research Center, One Research Circle, Niskayuna, New York 12309 Received September 30, 2009. Revised Manuscript Received February 1, 2010 Past literature investigations have optimized various single factors used in the formation of thiolated, single stranded DNA (ss-DNA) monolayers on gold. In this study a more comprehensive approach is taken, where a design of experiment (DOE) is employed to simultaneously optimize all of the factors involved in construction of the capture monolayer used in a fluorescence-based hybridization assay. Statistical analysis of the fluorescent intensities resulting from the DOE provides empirical evidence for the importance and the optimal levels of traditional and novel factors included in this investigation. We report on the statistical importance of a novel factor, temperature of the system during monolayer formation of the capture molecule and lateral spacer molecule, and how proper usage of this temperature factor increased the hybridization signal 50%. An initial theory of how the physical factor of heat is mechanistically supplementing the function of the lateral spacer molecule is provided.
Introduction Detection of diseases, pathogens, and metabolic pathways with nucleic acid (NA) markers has increased the analytical expectations of an analysis platform in terms of its achieved detection limit, selectivity, sensitivity, and dynamic range. It is generally considered that miniaturizing these analytical platforms will improve upon of these key parameters and also allow for onscene analysis, increased sample through-put, and reduce consumables. Toward these ends, a significant degree of effort has been displayed within the literature to reproduce NA methods on a miniaturized platform constructed through methods used in the microprocessor industry (i.e., deposition of metals, polymers, and oxides on silicon substrates).1-5 A segue between the arenas of microfabrication and biological recognition exists in the modification of gold substrates with single stranded NA probes. Gold is both readily handled in the microfabrication processes and multiple methods to modify gold with NA probes can be found in the open literature. There have been several approaches reported to modify gold with deoxyribonucleic acid (DNA), but the most common theme involves thiol-gold chemistry.6-9 Within this realm, there are several methods of attaching the DNA capture sequences to *Corresponding author. E-mail:
[email protected]. Telephone: 518387-4779.. (1) Christensen, T. B.; Pedersen, C. M.; Gr€ondahl, K. G.; Jensen, T. G.; Sekulovic, A.; Bang, D. D.; Wolff, A. J. Micromech. Microeng. 2007, 17, 1527. (2) Cheng, J.; Shoffner, M. A.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 380. (3) Roper, M. G.; Easley, C. J.; Landers, J. P. Anal. Chem. 2005, 77, 3387. (4) Shoffner, M. A.; Cheng, J.; Hvichia, G. E.; Kricka, L. J.; Wilding, P. Nucleic Acids Res. 1996, 24, 375. (5) Schneega, I.; Br€autigam, R.; K€ohler, J. M. Lab Chip 2001, 1, 42. (6) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481. (7) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (8) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4, 365. (9) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321. (10) Rabke-Clemmer, C. E.; Leavitt, A. J.; Beebe, T. P., Jr. Langmuir 1994, 10, 1796.
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the gold surface including: thiolating the nucleotide bases;10 reacting a functional group on the DNA with a pre-existing monolayer on gold;11-13 synthesizing the DNA to be connected via a short aliphatic chain to a thiol moiety.14,15 The latter option, although attractive due to its simplicity, was stymied by low hybridization efficiency until the enabling work of Tarlov and Herne employed a lateral spacer molecule which reduced the surface coverage of capture DNA but also increased the hybridization efficiency.16,17 Optimization of the various single factors within this protocol has occurred within the literature in a limited fashion. Postdeposition temperature conditioning,17 buffer compositions,18 other lateral spacing schemes,19-26 and vertical distance between substrate attachment group and capture sequence19,26-28 have been studied, but the simultaneous optimization of all parameters to (11) Peelen, D.; Smith, L. M. Langmuir 2005, 21, 266. (12) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044. (13) Devaraj, N. K.; Miller, G. P.; Ebina, W.; Kakaradov, B.; Collman, J. P.; Kool, E. T.; Chidsey, C. E. D. J. Am. Chem. Soc. 2005, 127. (14) Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. J. Am. Chem. Soc. 1992, 114, 8299. (15) Hashimoto, K.; Ito, K.; Ishimori, Y. Anal. Chem. 1994, 66, 3830. (16) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916. (17) Peterlinz, K. A.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401. (18) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219. (19) Wong, E. L. S.; Chow, E.; Gooding, J. J. Langmuir 2005, 21, 6957. (20) Hong, B. J.; Oh, S. J.; Youn, T. O.; Kwon, S. H.; Park, J. W. Langmuir 2005, 21, 4257. (21) Rant, U.; Arinaga, K.; Fujita, S.; Yokoyama, N.; Abstreiter, G.; Tornow, M. Langmuir 2004, 20, 10086. (22) Satjapipat, M.; Sanedrin, R.; Zhou, F. Langmuir 2001, 17, 7637. (23) Lee, C.-Y.; Nguyen, P.-C. T.; Grainger, D. W.; Gamble, L. J.; Castner, D. G. Anal. Chem. 2007, 79, 4390. (24) Sakao, Y.; Nakamura, F.; Ueno, N.; Hara, M. Colloids Surf., B 2005, 40, 149. (25) Dharuman, V.; Hahn, J. H. Sens. Actuators, B 2007, 127, 536. (26) Balamurugan, S.; Obubuafo, A.; Soper, S. A.; McCarley, R. L.; Spivak, D. A. Langmuir 2006, 22, 6446. (27) Shchepinov, M. S.; Case-Green, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155. (28) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. 1999, 21, 1.
Published on Web 03/26/2010
DOI: 10.1021/la903699f
5655
Article
Pris et al.
provide the best system performance has not been completed. It is commonly assumed that sequential optimization of the individual processes factors will provide the global maximum for a protocol. Although this is the case for simple systems, in most processes the factors do effect and interact with one another. Stated otherwise, it is possible for the factors to undergo higher order interactions that could have a positive or negative impact upon the end process. Only by simultaneously varying all of the factors in a controlled manner can these interactions, and their impact level, be determined to fully understand the complexity of the process, the importance of the individual factors, and the levels that optimize the entire process. A means of accomplishing this is through using a design-of-experiment (DOE) which provide a series of trials to screen the entire design space of the process. The meaningfulness of this DOE is couched upon the accuracy of defining the experimental space with the input factors and corresponding levels. Included within is a comprehensive DOE driven optimization for the formation of a single-stranded DNA (ss-DNA) capture monolayer on gold to increase the hybridization efficiency of the substrate.
Material and Methods Substrates. The gold substrates were created through taking a silicon [110] wafer (4-in., 3-7Ω-cm, 400(25 μm, warp
