DNA-Based Programmed Assembly of Gold Nanoparticles on

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DNA-Based Programmed Assembly of Gold Nanoparticles on Lithographic Patterns with Extraordinary Specificity

2004 Vol. 4, No. 8 1521-1524

Balaji Kannan,† Rajan P. Kulkarni,‡ and Arun Majumdar*,†,§ Department of Mechanical Engineering, UniVersity of California, Berkeley, California 94720, Option in Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California 91125, and Materials Sciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720 Received May 19, 2004; Revised Manuscript Received June 8, 2004

ABSTRACT We demonstrate the highly specific and programmed assembly of oligonucleotide-conjugated gold nanoparticles on lithographically defined microscale gold patterns. A key feature of our fabrication technique is the use of poly(ethylene glycol) (PEG) groups to form an inert coating on regions of the chip where no nanoparticle assembly is desired. By assembling multiple layers of DNA-conjugated nanoparticles we illustrate the capability of PEG surface coatings to exquisitely direct the nanoparticles onto the lithographic patterns with almost zero nonspecific reaction per square micron. We further suggest that the use of PEG to eliminate nonspecific reaction may be extended to micro- and nanoscale fabrication systems that make use of a variety of different nanostructures.

Over the past two decades, a variety of nanoscale materials, such as nanowires1, nanotubes,2 and quantum dots3 have been synthesized that exhibit unique physical, optical, and electronic properties not observed in bulk materials. From a technological standpoint, the true potential of these novel materials will be realized only when they are interfaced with the microscale and subsequently macroscale world by assembling them into higher-level structures, devices, and systems of well-defined shapes, sizes, and functionality. Furthermore, it is becoming increasingly clear that in order to achieve this, bottom-up and top-down manufacturing processes need to be seamlessly integrated. From among the different bottom-up nanofabrication approaches, programmed assembly4-6 has emerged as a particularly powerful platform to assemble nanomaterials into a variety of superstructures. A key advantage of this assembly process is that it provides for highly parallel manufacturing as opposed to other sequential nanofabrication techniques, such as those using scanning probe microscopes. It is also possible to exquisitely control the assembly process by tuning factors such as physical and chemical properties of the nanostructures, solvent properties, and surface chemistry. On the other hand, there are well-established top-down manufacturing processes such as optical, electron beam, or dip* Corresponding author. E-mail: [email protected]. † University of California, Berkeley. ‡ California Institute of Technology. § Lawrence Berkeley National Laboratory. 10.1021/nl049247a CCC: $27.50 Published on Web 07/01/2004

© 2004 American Chemical Society

pen lithography that are capable of creating well-defined shapes and sizes at the micro- and nanoscale. By combining these two different approaches to manufacturing, it should be possible to manufacture functional devices and structures composed of nanoscale entities in a high-throughput fashion. A few research groups have already demonstrated methods by which lithographic processes can be combined with the physically or chemically programmed assembly of various nanostructures such as quantum dots and nanowires. For instance, Musick et al.7 have shown the preferential building of multilayers of gold nanoparticles onto chemically patterned gold substrates, with adjacent layers of nanoparticles connected by ligands having affinity to gold surfaces on either of their ends. Vossmeyer et al.8 have also demonstrated a combination of self-assembly and lithography using quantum dots and photoreactive silane groups. In their work, they functionalized a silicon chip with a photosensitive silane molecule and exposed specific regions of the chip to UV radiation through a photolithographic mask, thereby exposing amine groups at those locations. The versatility of both these processes is limited by the fact that there are only a few kinds of ligands and, therefore, a programmed assembly process (as described below) cannot be realized using this approach. Programmed assembly is best exemplified4,5 through the use of DNA molecules as highly specific “glue” to generate a variety of assemblies of gold nanoparticles in solution. The

Figure 1. (a) Deposition of Au film on silicon (Si) substrate; (b) Patterning of the Au film by photolithography and wet-etching; (c) Functionalization of the surrounding Si substrate by PEG-silane; (d) Selective assembly of single stranded thiolated DNA on Au patterns; (e) Assembly of first layer of DNA-conjugated Au nanoparticles on Au patterns; (f) Assembly of second layer of nanoparticles on first nanoparticle layer.

specific sequence of DNA molecules conjugated to the nanoparticle provides the encoding or programming required for the assembly process. Oligonucleotides, which are short, synthetic DNA molecules, can be synthesized having the desired base sequence, length, and chemical modifications that enable their assembly onto different surfaces (such as gold, silicon, and so on). Most importantly, under appropriate conditions, two complementary single-stranded DNA molecules can specifically and reversibly assemble to form a double-stranded DNA molecule through complementary base pairing. While previous work4,5 has focused on programmed assembly in solution, i.e., in a three-dimensional volume, nanostructure assembly on a solid surface is perhaps more useful in making devices and systems. Zhang et al.9 have recently described the use of dip-pen nanolithography (DPN) and wet etching to define nanoscale gold lines on a substrate onto which a single layer of gold nanoparticles was subsequently assembled using the complementary base pairing of DNA. It must be noted, however, that any surfacebased programmed assembly is susceptible to nonspecific binding of nanostructures on undesirable regions. This is a serious issue since such nonspecific binding can lead to random shapes and structures as opposed to programmed ones. A robust process requires assembly only at predetermined locations on a surface. We demonstrate in this paper how programmed assembly can be performed in conjunction with standard lithography, with DNA strands and chemically inert PEG molecules guiding nanostructures exclusively to specific locations on a chip surface. The extraordinary specificity and selectivity of this fabrication process make it an ideal starting point for addressing the question of how top-down and bottom-up manufacturing paradigms may be combined to build functional devices and structures out of nanoscale materials. The fabrication process sequence is outlined in Figure 1. Detailed descriptions of the various individual steps and the exact procedures for the chemical reactions involved are provided as Supporting Information. The fabrication process 1522

starts with the evaporation of a thin film of gold onto a single-crystal silicon wafer. The film is then patterned using standard optical lithography followed by wet etching. At this stage the wafer has patterns of gold surrounded by regions of silicon. The wafer is then diced into chips, stripped of photoresist, and then cleaned with Piranha solution to make the native oxide of the silicon substrate hydrophilic by generating hydroxyl groups on the surface. A hydrophilic SiO2, presenting hydroxyl groups for surface reaction, enables the functionalization of the silicon substrate with organosilane molecules with high density. The chips are then dried and reacted with a solution of PEG silane in anhydrous toluene containing hydrochloric acid as catalyst. After removing excess PEG silane, the chips are dried and reacted with a solution of thiolated oligonucleotide of a specific sequence (see Supporting Information) to functionalize the gold patterns with a single layer of DNA molecules. The sequence is chosen so that the length and sequence of bases are sufficient for hybridization to occur at room temperature, i.e., the melting point of the duplex is much higher than room temperature. The thiolated DNA is reacted in a buffer of very high salt concentration in order to reduce electrostatic interactions between DNA strands, thereby increasing the surface density of the single-stranded DNA molecules on gold. It is important to note that since the chips are dried before reacting with the PEG silane, the reaction of the organosilane groups takes place only with the silicon substrate and not the gold. If some amount of moisture is present on the gold lines, the organosilane moieties would get hydrolyzed and then polymerize on the gold surface.10 The chips are then reacted with a solution of gold nanoparticles conjugated to single-stranded thiolated DNA molecules that have a base sequence complementary to those attached to the gold surface. The nanoparticles are suspended in a buffer with high concentration of NaCl salt to ensure hybridization of the complementary DNA strands by minimizing electrostatic interactions. Thus the first layer of gold nanoparticles is built on the gold patterns by programmed assembly. Another layer of gold nanoparticles is then built on the first layer by reacting the chip with a solution of nanoparticles modified with DNA strands complementary to that in the first nanoparticle layer. This process can be repeated multiple times to build many layers of nanoparticles one on top of the other, connected by complementary oligonucleotides. As control experiments, chips were also reacted with solutions of gold nanoparticles conjugated to noncomplementary DNA strands. Before imaging using a field-emission scanning electron microscope, all chips were washed in a solution of ammonium acetate, a volatile salt, to prevent salt crystallization.9 Figure 2 presents the results of experiments performed to build multiple layers of gold nanoparticles on patterns of gold surrounded by PEG-passivated regions of silicon. In all pictures, the first layer consists of gold nanoparticles of 15 nm diameter (nominal) while the second layer is made of 10 nm size (nominal) gold particles. We used this binary system of nanoparticles to help monitor the growth of Nano Lett., Vol. 4, No. 8, 2004

Figure 3. (a) One layer of DNA-conjugated gold nanoparticles assembled on gold patterns, with no passivating layer on the surrounding silicon substrate. (b) Inset from figure a showing nonspecific reaction on the bare silicon surface.

Figure 2. (a-d) Scanning electron micrographs of two layers of gold nanoparticles assembled onto lithographically defined gold patterns, taken at different magnifications, with the surrounding silicon being passivated with PEG silane. The first layer consists of 15 nm diameter particles and the second layer of 10 nm size particles.

different layers of nanoparticles. While Taton et al.11 have demonstrated multilayered structures of DNA-conjugated gold nanoparticles on a glass substrate, they did not perform the experiments on lithographic patterns. On the other hand, Musick et al.7 have built multilayered structures of gold nanoparticles on lithographic patterns with adjacent layers connected by small molecules rather than oligonucleotides of a specific sequence. The use of oligonucleotides to perform nanoparticle assembly on lithographic patterns showcases the specificity and versatility of the multilayer process as discussed below. Several salient features of the assembly process are worth noting. (i) The surface density of the nanoparticles on the gold patterns is much lower than monolayer coverage because single-stranded DNA coverage on the gold lines is sub-monolayer and also because the particles are all negatively charged. (ii) Overall, the second layer of nanoparticles has a higher surface coverage than the first, as is evident from the fact that almost each 15 nm nanoparticle is surrounded by multiple 10 nm size gold particles, forming “satellite” structures. (iii) The second layer of nanoparticles builds only where nanoparticles of the first layer are present. Since no second layer builds if the DNA strands of the first and second layers are noncomplementary, this means that DNA hybridization is an extremely specific reaction and is, therefore, exclusively responsible for connecting adjacent layers of nanoparticles. (iv) As seen in Figures 2b and 2d, no gold nanoparticles are present in the region surrounding the gold patterns. As mentioned before, this region is silicon dioxide (the native oxide on single crystals silicon) functionalized with PEG silane. The PEG group thus possesses the extraordinary property of preventing nonspecific binding of all nanoparticles from its surface, irrespective of the number of layers of gold nanoparticles. Nano Lett., Vol. 4, No. 8, 2004

Figure 4. Histogram depicting the amount of nonspecific reaction (measured as the average number of nanoparticles per square micron area) occurring on the silicon portion of chips with gold patterns. The fabrication process index is 1 for nanoparticle assembly on chips whose silicon portion has no passivation layer, 2 for the fabrication process of Zhang et al.9 that uses OTS for passivation of silicon, and 3 for nanoparticle assembly in which PEG is used to passivate silicon. The error bars indicate 1 standard deviation of the measured surface density of nanoparticles.

As a comparison, the results of the assembly process when no passivating molecule is used on the native silicon dioxide are presented in Figures 3a and 3b. In this case, just one layer of gold nanoparticles is built following the functionalization of the gold film with single stranded DNA. Note that while gold nanoparticles do assemble on the gold film, there is significant nonspecific binding on the surrounding silicon surface, proving that a good passivating molecular layer is essential to achieve highly selective assembly of nanostructures at specific locations of the chip surface. We claim that the rejection capabilities of PEG are extraordinary compared to other surface coatings used in the past. Zhang et al.9 used octadecyltrimethoxysilane (OTS), while Musick et al.7 have attempted the use of carboxylterminated long-chain alkanethiols. The rationale for the latter is that the negatively charged carboxyl moieties should be capable of effectively repelling the negatively charged gold nanoparticles and hence can provide good specificity. To quantify nonspecific binding, we counted and averaged the number of nanoparticles within a 1 µm2 area of the silicon surface for the various approaches. These results, plotted as a histogram in Figure 4, clearly show the effectiveness of PEG compared to other surface coatings. We repeatedly 1523

observed the complete absence of nonspecific nanoparticle binding on the PEG-passivated surface, as exemplified in Figure 2. A survey of the existing scientific literature suggests no other surface coating besides PEG that is capable of such exclusive specificity as demonstrated above. Indeed, it is well-known that substrates coated with PEG show very little nonspecific binding to a wide range of proteins.12 Research has shown that the PEG layer consists of hydrated, relatively dense-packed chains that move around rapidly and possess a high degree of conformational entropy when the substrate is immersed in solution. Applying the exclusion volume theory13 to a system consisting of PEG polymeric chains and gold nanocrystals, we maintain that the almost singlenanoparticle specificity observed in the present case is purely steric in origin, with the criss-crossing, densely packed PEG chains effectively blocking the adsorption and entry of both nanoparticles and DNA molecules. It appears likely that, since the selectivity of the PEG group has a physical rather than a chemical origin, the rejection capabilities of PEG should hold for a wide variety of nanoscale structures and molecules. Although the results presented in this article are specific to the system of gold nanoparticles and oligonucleotides, it should be possible to explore other systems such as nanowires, nanotubes, and other molecules such as proteins, small molecules (ligands), and so on for use with PEG-patterned substrates. Hence, this manufacturing process could serve as a very versatile and selective platform for fabrication of devices and structures composed of a variety of different nanoscale building blocks. Obviously, it is not necessary to limit the lithographic pattern to gold films. For instance, it should be possible to fabricate silicon chips that have one kind of functional organosilane (such as aminosilanes or mercaptosilanes) surrounded by PEG silane, which would therefore provide the capability of assembling a combination of nanostructures and molecules on the functional silanes. Such a manufacturing technique comprising chemical patterning on silicon surfaces exclusively would possibly bring the integration of nanomaterials with conventional integrated circuits (ICs) a step closer. In summary, we reach the following conclusions. (1) It is possible to program or encode the assembly of multiple layers of nanostructures on a lithographically patterned solid surface using DNA as highly specific glue. (2) In such a manufacturing process, nonspecific binding of nanostructures is highly undesirable since it produces random as opposed to programmed structures and shapes. (3) The use of poly(ethylene glycol) (PEG) to passivate a surface provides extraordinary specificity in DNA-based programmed assembly, since it almost completely prevents nonspecific binding. Quantitative analysis of nonspecific binding clearly shows the superiority of PEG compared to other surface coatings previously used.

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(4) We claim that because the extraordinary specificity of PEG arises from physical (entropic) interactions, as opposed to chemical ones, it has the potential to serve as a universal surface passivation material for a wide variety of biomolecules and nanostructures. This offers the promising prospects of: (a) programmed assembly of nanostructures with exquisite control to make devices and systems; (b) nanostructure-based high-throughput bioassays with almost singlemolecule resolution owing to extremely low background signal. Acknowledgment. The authors are grateful to Professor Paul Alivisatos of the University of California at Berkeley, who graciously allowed us to make use of all the facilities of his laboratory in the Department of Chemistry. We also thank Dr. Daniele Gerion of Lawrence Livermore National Laboratory and Professor Ram Datar of University of Southern California for useful discussions and suggestions. We acknowledge the Berkeley Microfabrication Laboratory for providing microfabrication facilities and the assistance of Dr. Gordon Vrdoljak of the Robert D. Ogg Electron Microscopy Center with SEM imaging. A.M. was initially supported by an SGER grant from the National Science Foundation and later by funding from Basic Energy Sciences, Department of Energy. He appreciates the support of SINAM, an NSF-supported NSEC. He also thanks the Miller Institute for a Professorship. B. K. was supported by a scholarship from the Department of Mechanical Engineering at UCB. R.P.K. was supported by an NSDEG fellowship. Supporting Information Available: Detailed descriptions of the various individual steps and the exact procedures for the chemical reactions involved. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Duan, X. F.; Lieber, C. M. AdV. Mater. 2000, 12(4), 298. (2) Cassell, A. M.; Raymakers, J. A.; Kong, J.; Dai, H. J. J. Phys. Chem. B 1999, 103(31), 6484. (3) Alivisatos, A. P. Science 1996, 271(5251), 933. (4) Alivisatos, A. P.; Johnsson, K. P.; Peng, X. G.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P.; Schultz, P. G. Nature 1996, 382(6592), 609. (5) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382(6592), 607. (6) Mbindyo, J. K. N.; Reiss B. D.; Martin, B. R.; Keating, C. D.; Natan, M. J.; Mallouk, T. E. AdV. Mater. 2001, 13(4), 249. (7) Musick, M. D.; Keating, C. D.; Lyon, L. A.; Botsko, S. L.; Pena, D. J.; Holliway, W. D.; McEvoy, T. M.; Richardson, J. N.; Natan, M. J. Chem. Mater. 2000, 12(10), 2869. (8) Vossmeyer, T.; Jia, S.; Delonno, E.; Diehl, M. R.; Kim, S. H.; Peng, X.; Alivisatos, A. P.; Heath, J. R. J. Appl. Phys. 1998, 84(7), 3664. (9) Zhang, H.; Li, Z.; Mirkin, C. A. AdV. Mater. 2002, 14(20), 1472. (10) Kurth, D. G.; Bein, T. Langmuir 1995, 11, 3061. (11) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122(26), 6305. (12) Zdyrko, B.; Klep, V.; Luzinov, I. Langmuir 2003, 19, 10179. (13) Hermans, J. J. Chem. Phys. 1982, 77(4), 2193.

NL049247A

Nano Lett., Vol. 4, No. 8, 2004