Maximizing DNA Loading on a Range of Gold Nanoparticle Sizes Sarah J. Hurst, Abigail K. R. Lytton-Jean, and Chad A. Mirkin* Department of Chemistry and International Institute for Nanotechnology, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113 We have investigated the variables that influence DNA coverage on gold nanoparticles. The effects of salt con- centration, spacer composition, nanoparticle size, and degree of sonication have been evaluated. Maximum loading was obtained by salt aging the nanoparticles to ���0.7 M NaCl in the presence of DNA containing a poly- (ethylene glycol) spacer. In addition, DNA loading was substantially increased by sonicating the nanoparticles during the surface loading process. Last, nanoparticles up to 250 nm in diameter were found have ���2 orders of magnitude higher DNA loading than smaller (13-30 nm) nanoparticles, a consequence of their larger surface area. Stable large particles are attractive for a variety of biodi- agnostic assays. Gold nanoparticles exhibit several interesting physical and chemical properties that have made them an integral part of research in nanoscience.1 In addition to their striking optical properties, gold nanoparticles are important because they can be stabilized with a wide variety of molecules by taking advantage of well-known chemistry involving alkanethiol adsorption on gold.2 In particular, thiol-modified oligonucleotides (short synthetic DNA sequences) can be loaded onto the surface of Au nanoparticles. The resulting DNA-functionalized Au nanoparticles have become widely used as nanoscale building blocks in assembly strategies,3,4 as antisense agents in nanotherapeutics for gene regulation,5 and as probes in many biodiagnostic systems.6 In all of these applications, it is essential to understand the coverage of the DNA on the nanoparticle, and in many cases, it is favorable to have higher DNA loadings. The advantages provided by higher DNA loading have the potential to dramatically impact biodetection and nanotherapeutics. The first biodetection assays using DNA-functionalized gold nanoparticles involved colorimetric readout strategies.7-9 These procedures were inspired by the aggregation-induced red-to-blue color transition, which is due to a dampening and red shifting of the nanoparticle surface plasmon resonance band. Since the development of the initial colorimetric assays, DNA-functionalized Au nanoparticles have become a central component in a wide variety of schemes that use readout strategies including fluores- cence,10,11 radioactivity,12 quartz crystal microbalance,13 Raman spectroscopy,14,15 light scattering,16 and electrical signal.17 In addition, the bio-bar-code method is a strategy that has made a marked impact on the field of gold nanoparticle-based biodiag- nostics, by providing a protocol to detect proteins,18,19 DNA,20,21 and other biomolecules22,23 at remarkably low concentrations both serially18,20 and, in some cases, in a multiplexed format.19,21 The high sensitivity of this assay stems from the indirect amplification of the target sequence by the sizable number of DNA strands that can be loaded on a single gold nanoparticle. Therefore, the amount of DNA on each nanoparticle directly correlates to the amount of amplification possible and therefore the sensitivity attainable in this system. * Towhomcorrespondenceshouldbeaddressed.E-mail: chadnano@northwestern.edu. Phone: (847) 491-2907. Fax: (847) 467-5123. (1) Burda, C. Chen, X. Narayanan, R. El-Sayed, M. A. Chem. Rev. 2005, 105, 1025-1102. (2) Love, J. C. Estroff, L. A. Kriebel, J. K. Nuzzo, R. G. Whitesides, G. M. Chem. Rev. 2005, 105, 1103-1169. (3) Mirkin, C. A. Letsinger, R. L. Mucic, R. C. Storhoff, J. J. Nature 1996, 382, 607-609. (4) Alivisatos, A. P. Johnsson, K. P. Peng, X. Wilson, T. E. Loweth, C. J. Jr, M. P. B. Schultz, P. G. Nature 1996, 382, 609-611. (5) Rosi, N. L. Giljohann, D. A. Thaxton, C. S. Lytton-Jean, A. K. R. Han, M. S. Mirkin, C. A. Science 2006, 312, 1027-1030. (6) Rosi, N. L. Mirkin, C. A. Chem. Rev. 2005, 105, 1547-1562. (7) Elghanian, R. Storhoff, J. J. Mucic, R. C. Letsinger, R. L. Mirkin, C. A. Science 1997, 227, 1078-1081. (8) Storhoff, J. J. Elghanian, R. Mucic, R. C. Mirkin, C. A. Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (9) Reynolds, R. A. Mirkin, C. A. Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (10) Zhao, X. Tapec-Dytioco, R. Tan, W. J. Am. Chem. Soc. 2003, 125, 11474- 11475. (11) Xu, H. Wu, H. Huang, F. Song, S. Li, W. Cao, Y. Fan, C. Nucleic Acids Res. 2005, 33, e83. (12) Weizmann, Y. Patolsky, F. Willner, I. Analyst 2001, 126, 1502-1504. (13) Patolsky, F. Lichtenstein, A. Willner, I. J. Am. Chem. Soc. 2000, 122, 418- 419. (14) Cao, Y. C. Jin, R. Mirkin, C. A. Science 2002, 297, 1536-1539. (15) Cao, Y. C. Jin, R. Nam, J.-M. Thaxton, C. S. Mirkin, C. A. J. Am. Chem. Soc. 2003, 125, 14676-14677. (16) Taton, T. A. Mirkin, C. A. Letsinger, R. L. Science 2000, 289, 1757-1760. (17) Park, S.-J. Taton, T. A. Mirkin, C. A. Science 2002, 295, 1503. (18) Nam, J.-M. Thaxton, C. S. Mirkin, C. A. Science 2003, 301, 1884-1886. (19) Stoeva, S. I. Lee, J.-S. Smith, J. E. Rosen, S. T. Mirkin, C. A. J. Am. Chem. Soc. 2006, 126, 8378-8379. (20) Nam, J.-M. Stoeva, S. I. Mirkin, C. A. J. Am. Chem. Soc. 2004, 126, 5932- 5933. (21) Stoeva, S. I. Lee, J.-S. Thaxton, C. S. Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 3303-3306. (22) Georganopoulou, D. G. Chang, L. Nam, J.-M. Thaxton, C. S. Mufson, E. J. Klein, W. L. Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 2273- 2276. (23) Thaxton, C. S. Hill, H. D. Georganopoulou, D. G. Stoeva, S. I. Mirkin, C. A. Anal. Chem. 2005, 77, 8174-8178. Anal. Chem. 2006, 78, 8313-8318 10.1021/ac0613582 CCC: $33.50 �� 2006 American Chemical Society Analytical Chemistry, Vol. 78, No. 24, December 15, 2006 8313 Published on Web 11/10/2006

Moreover, recent work in the area of nanotherapeutics has demonstrated the use of DNA-functionalized gold nanoparticles as antisense agents for intracellular gene regulation.5 The nano- particles act as a nontoxic and highly efficient antisense agent that by virtue of cooperative binding properties can very effectively scavenge mRNA within the cell.5,24 In addition, the tight packing of the DNA on the surface of the nanoparticle likely plays a role in the inhibition of its degradation by nucleases. This opens the door for the use of functionalized gold nanoparticles in several very efficient gene regulation therapies. The diagnostic and therapeutic applications of oligonucleotide- modified nanoparticles benefit from the ability to maximize and tailor the amount of DNA on the gold nanoparticle surface. Herein, we fully quantify the loading of DNA on a range of gold nanoparticle sizes while examining the effects of several different parameters. We determine the dependence of DNA loading on the following: (1) salt concentrations from 0 to 1.0 M NaCl, (2) adenine (A), thymine (T), and non-DNA base (poly(ethylene glycol, PEG) spacers (the region of the oligonucleotide between the recognition sequence and the thiol functionality), and (3) sonication. Importantly, we determine the DNA loading obtained from these parameters on several sizes of gold nanoparticles: 15, 30, 50, 80, 150, and 250 nm in diameter. Through these studies, two new parameters that are significant in achieving particles with the highest DNA loading have been determined. They are (1) the use of PEG as a spacer and (2) the use of sonication. We have discovered that even 250-nm particles can be heavily loaded with DNA and made indefinitely stable. This is a significant advance since these large nanoparticles have the potential to be loaded with several orders of magnitude more DNA strands than the smaller particles (13-30 nm) that are often used in biodiag- nostic assays that rely on gold clusters as probes. PROCEDURES Materials. Gold nanoparticles were purchased from Ted Pella (Redding, CA). Olionucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville, IA) (5���HS-spacer-ATC CTT TAC AAT ATT 6���FAM 3���, where spacer ) A10, T10, or 3[(CH2- CH2O)6-phosphoramidite]. Dithiothreitol (DTT) was purchased from Pierce Biotechnology, Inc. (Rockford, IL). NAP-5 columns (Sephadex G-25 DNA grade) were purchased from G. E. Health- care (Piscatiway, NJ). Carbowax 20 M was purchased from Supelco, Inc. (Bellefonte, PA). All other salts and reagents, unless specified, were purchased from Sigma-Aldrich (St. Louis, MO). Clear 96-well plates (Costar 3696) and black shell, clear bottom, 96-well plates (Costar 3603) were purchased from Corning, Inc. (Corning, NY). NANOpure H2O ( 18.0 M���), purified using a Barnstead NANOpure Ultrapure water system, was used for all experiments. Instrumentation. Absorbance measurements of oligonucle- otides and gold nanoparticles were collected using a Bio-Tek Synergy HT Microplate spectrophotometer. Fluorescence mea- surements were performed on a Molecular Devices Gemini EM Microplate spectrofluorometer. All sonication was performed using a Branson 2510 sonicator. Preparation of Alkanethiol Oligonucleotide-Modified Gold Nanoparticles. Gold nanoparticles were functionalized with fluorophore (fluorescein, 6���FAM)-modified alkanethiol oligonucle- otides. Prior to use, the disulfide functionality on the oligonucle- otides was cleaved by addition of DTT to lyophilized DNA and the resultant mixture incubated at room temperature for 1 h (0.1 M DTT, 0.18 M phosphate buffer (PB), pH 8.0). The cleaved oligonucleotides were purified using a NAP-5 column. Freshly cleaved oligonucleotides were added to gold nanoparticles (1 OD/1 mL), and the concentrations of PB and sodium dodecyl sulfate (SDS) were brought to 0.01 M and 0.01%, respectively. The oligonucleotide/gold nanoparticle solution was allowed to incubate at room temperature for 20 min. The concentration of NaCl was increased to 0.05 M using 2 M NaCl, 0.01 M PBS while maintaining an SDS concentration of 0.01%. The oligonucleotide/ gold nanoparticle solution was then sonicated for ���10 s followed by a 20-min incubation period at room temperature. This process was repeated at one more increment of 0.05 M NaCl and for every 0.1 M NaCl increment thereafter until a concentration of 1.0 M NaCl was reached. The salting process was followed by incubation overnight at room temperature. To remove excess oligonucle- otides, the gold nanoparticles were centrifuged and the superna- tant was removed, leaving a pellet of gold nanoparticles at the bottom. The particles then were resuspended in 0.01% SDS. This washing process was repeated for a total of five supernatant removals. Quantification of Alkanethiol Oligonucleotides Loaded on Gold Nanoparticles. To determine the number of oligonucle- otides loaded on each particle, the concentration of nanoparticles and the concentration of fluorescent DNA in each sample were measured. The concentration of gold nanoparticles in each aliquot was determined by performing UV-visible spectroscopy measure- ments. These absorbance values were then related to the nano- particle concentration via Beer���s law (A ) bc). The wavelength of the absorbance maximums (��) and extinction coefficients ( ) used for each particle size are as follows: 15 nm, �� ) 524 nm, ) 2.4 �� 108 L/(mol���cm) 30 nm, �� ) 526 nm, ) 3.0 �� 109 L/(mol��� cm) 50 nm, ) 531 nm, ) 1.5 �� 1010 L/(mol���cm) 80 nm, �� ) 545 nm, ) 6.85 �� 1010 L/(mol���cm) 150 nm, �� ) 622 nm, ) 2.19 �� 1011 L/(mol���cm) 250 nm, �� ) 600 nm, ) 5.07 �� 1011 L/(mol���cm). In order to determine the concentration of fluorescent oligo- nucleotides in each aliquot, the DNA was chemically displaced from the nanoparticle surface using DTT. The displacement was achieved by adding equal volumes of oligonucleotide-functional- ized gold nanoparticles and 1.0 M DTT in 0.18 M PB, pH 8.0. The oligonucleotides were released into solution during an overnight incubation, and the gold precipitate was removed by centrifugation. To determine oligonucleotide concentration, 100 ��L of supernatant was placed in a 96-well plate and the fluores- cence was compared to a standard curve. Because the 6���FAM fluorophore is sensitive to pH,25 the oligonucleotide samples for the standard curve were prepared with the same 1.0 M DTT buffer solution. During the fluorescence measurement, the fluorophore was excited at 495 nm and the emission was collected from 530 to 560 nm. The number of oligonucleotides per particle for each aliquot was calculated by dividing the concentration of fluorescent (24) Lytton-Jean, A. K. R. Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 12754- 12755. (25) Wang, L. Roitberg, A. Meuse, C. Gaigalas, A. K. Spectrochim. Acta, Part A 2001, 57, 1781-1791. 8314 Analytical Chemistry, Vol. 78, No. 24, December 15, 2006