Anal. Chem. 2008, 80, 2849-2856 Effects of Microbead Surface Chemistry on DNA Loading and Hybridization Efficiency T. L. Jennings, K. S. Rahman, S. Fournier-Bidoz, and W. C. W. Chan* Institute of Biomaterials and Biomedical Engineering, Terrence Donnelly Center for Cellular and Biomoecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3G9, Canada Polymer microbeads are witnessing renewed interest for of more efficient and cost-effective nucleic acid detection in a performing biomolecule recognition assays with distinct shorter period of time would lead to a significant improvement advantages over planar microarray technology. In this for DNA hybridization applications such as in vitro DNA cloning,11 study, DNA hybridization assays are performed on the single nucleotide polymorphism genotyping,12 and genomic detec- surfaces of 1-µm-diameter, synthetically modified poly- tion.13 Microbead-based detection assays are in a position to styrene microbeads. The microbead surfaces contain circumvent the issues of planar arrays by offering specific varying amounts of poly(acrylic acid) as a source of advantages. carboxylate groups to which a DNA capture strand may Microbead particles are colloidal polymer spheres ranging from bind. Through a series of controlled experiments in which a few hundred nanometers to tens of micrometers in diameter the microbead carboxylate density and DNA:surface area and offer many choices for synthetic customization in magne- ratios are systematically altered, we find that the density tism,14,15 fluorescence properties,16,17 and surface binding proper- of carboxylate groups on the microbead surface may be ties.18 Microbead detection schemes have recently become the most important parameter affecting not only the total particularly exciting by doping them internally with quantum dot number of DNA strands that may bind to the microbead fluorophores, which can be used for ªbar codingº and high- surface but, surprisingly, also the efficiency of DNA throughput multiplexed detection of genome and proteome hybridization with complementary strands. These studies targets,19-26 allowing their utility in many of the same applications are aimed directly at understanding the physical interac- as microarrays. Microbeads distinguish themselves from planar tions between DNA strands and an anionic microbead array techniques by their ability to automatically refresh their surface. depletion layer by diffusing through solution and, particularly, offering a polymer surface that provides controllable charge The monumental success of DNA microarray assays and their density and increased surface area through microscopic rough- importance in genetic screening has led to the in-depth optimiza- ness. tion of planar support detection schemes with regard to surface Microbeads have great potential, therefore, to significantly - probe density,1 3 nucleotide length,4,5 and even the effects of advance the rapid multiplexed analysis of biomolecules such as nucleotide conformations6 and surface diffusion rates.7 Microar- rays, however, suffer from inhomogenous surface density profiles (11) Brenner, S.; Williams, S. R.; Vermaas, E. H.; Storck, T.; Moon, K.; McCollum, on individual spots, inefficient and slow surface hybridization (∼1% C.; Mao, J. I.; Luo, S.; Kirchner, J. J.; Eletr, S.; DuBridge, R. B.; Burcham, Downloaded via UNIV OF TORONTO on August 8, 2020 at 23:29:05 (UTC). - hybridization in 2 h),8 and high expenses for operation, sample T.; Albrecht, G. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1665 1670. (12) Armstrong, B.; Stewart, M.; Mazumder, A. Cytometry 2000, 40, 102-108. 9,10 preparation, and microarray plate synthesis. The development (13) Fuentes, M.; Mateo, C.; Rodriguez, A.; Casqueiro, M.; Tercero, J. C.; Riese, H. H.; Fernandez-Lafuente, R.; Guisan, J. M. Biosens. Bioelectron. 2006, See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. * To whom correspondence should be addressed. E-mail: warren.chan@ 21, 1574-1580. utoronto.ca. (14) Nam, J. M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886. (1) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, (15) Xu, H.; Wu, H.; Huang, F.; Song, S.; Li, W.; Cao, Y.; Fan, C. Nucleic Acids 29, 5163-5168. Res. 2005, 33, e83. (2) Shchepinov, M.; Case-Green, S.; Southern, E. Nucleic Acids Res. 1997, 25, (16) Battersby, B. J.; Lawrie, G. A.; Johnston, A. P.; Trau, M. Chem. Commun. 1155-1161. (Camb) 2002, 14, 1435-1441. (3) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, (17) Battersby, B. J.; Trau, M. Trends Biotechnol. 2002, 20, 167-173. 124, 14601-14607. (18) Brenner, S.; et al. Nat. Biotechnol. 2000, 18, 630-634. (4) Relogio, A.; Schwager, C.; Richter, A.; Ansorge, W.; Valcarcel, J. Nucleic (19) Eastman, P.; Ruan, W.; Doctolero, M.; Nuttall, R.; de Feo, G.; Park, J.; Chu, Acids Res. 2002, 30, e51. J.; Cooke, P.; Gray, J.; Li, S.; Chen, F. Nano Lett. 2006, 6, 1059-1064. (5) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, (20) Han, M.; Gao, X.; Su, J.; Nie, S. Nat. Biotechnol. 2001, 19, 631-635. 79, 975-981. (21) Li, Y.; Cu, Y.; Luo, D. Nat. Biotechnol. 2005, 23, 885-889. (6) Riccelli, P.; Merante, F.; Leung, K.; Bortolin, S.; Zastawny, R.; Janeczko, R.; (22) Rosenthal, S. Nat. Biotechnol. 2001, 19, 621-622. Benight, A. Nucleic Acids Res. 2001, 29, 996-1004. (23) Ugozzoli, L. Clin. Chem. 2004, 50, 1963-1965. (7) Chan, V.; Graves, D. J.; McKenzie, S. E. Biophys. J. 1995, 69, 2243-2255. (24) Xu, H.; Sha, M.; Wong, E.; Uphoff, J.; Xu, Y.; Treadway, J.; Truong, A.; (8) Dandy, D. S.; Wu, P.; Grainger, D. W. Proc. Natl. Acad. Sci. U.S.A. 2007, O'Brien, E.; Asquith, S.; Stubbins, M.; Spurr, N.; Lai, E.; Mahoney, W. Nucleic 104, 8223-8228. Acids Res. 2003, 31, e43. (9) Battersby, B.; Lawrie, G.; Trau, M. Drug Discovery Today 2001, 6, S19- (25) Klostranec, J.; Chan, W. Adv. Mater. 2006, 18, 1953-1964. S26. (26) Klostranec, J.; Xiang, Q.; Farcas, G.; Lee, J.; Rhee, A.; Lafferty, E.; Perrault, (10) Burbaum, J. Drug Discovery Today 1998, 3, 313-322. S.; Kain, K.; Chan, W. Nano Lett. 2007, 7, 2812-2818. 10.1021/ac7026035 CCC: $40.75 © 2008 American Chemical Society Analytical Chemistry, Vol. 80, No. 8, April 15, 2008 2849 Published on Web 02/29/2008 DNA, but surprisingly little research directed at understanding the relationship between biomolecules and the microbead surface has been performed. The complex modification of the microbead surface to maximize biomolecular binding efficiency27 could make microbead techniques impractical, whereas the biotin-streptavidin binding interaction demonstrates a sterically reduced efficiency of capture-target molecular coupling.28 An in-depth investigation of capture molecule binding to a microbead surface, using a straightforward standard approach such as carbodiimide cross- linking, is needed to promote the utility of microbead assays for multiplexed analysis. Further, the hybridization efficiency between capture and target biomolecules as a function of both surface chemistry and capture molecule density is of great importance toward maximizing the usefulness and sensitivity of microbead assays. In this report, we probe the relationships between (1) micro- bead surface chemistry and capture molecule conjugation ef- ficiency as a function of carbodiimide coupling conditions, (2) capture molecule conjugation as a function of carboxylate density on the microbead surface, and (3) hybridization efficiency of capture and target molecules as a function of capture molecule density on the microbead surface. Figure 1A illustrates the system under investigation where capture molecules of DNA are im- mobilized at a density of σ onto the surface of a microbead, and the interactions with targeted DNA molecules may be monitored optically through the incorporation of strategically chosen FoÈrster resonance energy transfer (FRET) dye pairs. Figure 1B illustrates the affect that microbead surface charge (carboxylate density) may have upon both capture molecule conjugation and target molecule hybridization. The first ∼0.7 nm distance immediately in contact with a charged surface is called the Bjerrum length and is repulsive to negative charge. The more loosely associated volume Figure 1. Microbead Surface-Based DNA Hybridization Scheme. (A) Illustration of a microbead of radius R with capture strands of further out contains screening cationic charge, which neutralizes DNA bound to the surface at density σ, and the hybridization of a the charge of the microbead and even results in charge inversion. dye-bound target strand. (B) A charged microbead surface will form The negative surface charge and the positively charged inversion different charged layers at both the solvent interface and the shell layer may both contribute to conjugation and hybridization volume just beyond, both of which could affect a biomolecular assay. molecular interactions. Finally, Figure 1C suggests different levels (C) The readout signal from any single bead is dependent upon the amount of target strand that hybridizes, which is in turn dependent of microbead preparation that are under control and will influence on the efficiency of hybridization to those strands. directly the sensitivity of a microbead biomolecular assay. Through a combination of controlled microbead surfaces and a careful selection of capture strand binding parameters, we are able to persulfate (0.2 g in 20 mL of H2O) acting as polymerization initiator demonstrate that