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Use of Atomic Force Microscopy for Making Addresses in DNA Coatings Dejian Zhou, Kumar Sinniah, Chris Abell, and Trevor Rayment* Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Received April 22, 2002. In Final Form: August 7, 2002 Nanoscale DNA surfaces are manipulated by coating ultraflat gold with short thiolated single-stranded (ss) and double-stranded (ds) DNA. Hybridization and 6-mercapto-1-hexanol (MCH) are used to modulate the thickness of the layers, all the changes being monitored using atomic force microscopy (AFM) in situ. Short thiolated ssDNA forms irregular 3.5 nm thick coatings, which are converted into more uniform 6.0 nm thick layers upon hybridization with complementary DNA. Holes formed in a monolayer of short thiolated dsDNA can be partially filled with a longer length thiolated ssDNA, which then protrudes from the surface after hybridization with a complementary strand. MCH is used to modulate the depth of a ssDNA layer and improve hybridization to it.
Two of the key foci of nanotechnology are the construction of nanoscale devices and the characterization and manipulation of surface properties. The potential of DNA as a building block in nanotechnology is recognized in its use in the creation of novel structures1 and as a molecular scaffold.2 The interaction between surface-immobilized DNA and free oligonucleotides in solution plays an important role in a wide range of array-based genetic diagnostic devices.3 The density, organization, and properties of surface immobilized DNA have been studied by a range of techniques that include surface force,4 quartz crystal microbalance (QCM),5 surface plasmon resonance (SPR),6 X-ray photoelectron spectroscopy (XPS),7 neutron reflection,8 and electrochemical response.9 Although these methods can provide averaged quantitative information such as surface coverage and hybridization efficiency, they are not suitable for detecting local surface variations on a nanometer scale. On the other hand, AFM has the advantage of being capable of detecting surface variations at the sub-nanometer scale, which has been used to measure DNA surface coverage,5 to measure layer thickness,10 and as a tool to construct and read DNA and protein micro- or nanoarrays.11,12 This study builds on these results * Corresponding author. E-mail:
[email protected]. Fax: +441223-336362. (1) (a) Seeman, N. C. Trends Biotechnol. 1999, 17, 437-443. (b) Seeman, N. C. Angew. Chem., Int. Ed. 1998, 37, 3220-3238. (2) Tomkins, J. M.; Nabbs, B. K.; Barnes, K.; Legido, M.; Blacker, A. J.; McKendry, R. A.; Abell, C. ChemBioChem 2001, 2, 375-378. (3) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. 1999 (suppl.), 21, 5-9. (4) Cho, Y. K.; Kim, S.; Lim, G.; Granick, S. Langmuir 2001, 17, 7732-7734. (5) (a) Satjapipat, M.; Sanedrin, R.; Zhou, F. Langmuir 2001, 17, 7637-7644. (b) Huang, E.; Satjapipat, M.; Han, S.; Zhou, F. Langmuir 2001, 17, 1215-1224. (6) (a) Peterlinz, K. P.; Georgiadis, R. M. J. Am. Chem. Soc. 1997, 119, 3401-3402. (b) Georgiadis, R. M.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (7) (a) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (b) Lin, Z.; Strother, T.; Cai, W.; Cao, Z.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 788-796. (8) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (9) (a) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975-981. (b) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 4670-4677. (10) (a) Sam, M.; Boon, E. M.; Barton, J. K.; Hill, M. G.; Spain, E. S. Langmuir 2001, 17, 5727-5730. (b) Kelley, S. O.; Barton, J. K.; Jackson, N. M.; McPherson, L. D.; Potter, A. B.; Spain, E. M.; Allen, M. J.; Hill, M. G. Langmuir 1998, 14, 6781-6784.
to describe an approach to the design of novel and manipulatable DNA surface landscapes. This involves the unprecedented use of DNA as the resist layer, into which surface features are introduced by scraping DNA away in specific areas. Contact mode AFM is used to measure the thickness of the DNA layers as they are manipulated in a predictable and reproducible way by hybridization or treatment with 6-mercapto-1-hexanol (MCH). Experiemental Section Materials. The two 5′-thiol modified DNAs and their complementary ssDNAs utilized in this study were all purchased from MWG Biotech UK Ltd (Milton Keynes, U.K.) and used without further purification. The sequences were HSC6H12-5′-CTCTAGACATATGGGTACCG-3′ (HS-ssDNA-20), HS-C6H12-5′-GATCCTCATCGA-3′ (HS-ssDNA-12), 5′-AATTCGGTACCCATATGTCTAGAG-3′ (ssDNA-24), and 5′-TCGACGAGGATC-3′ (ssDNA12). 6-Mercapo-1-hexanol (MCH, 97%) was purchased from Aldrich. Two buffer solutions, PBS (100 mM phosphate, 200 mM NaCl, 5 mM EDTA, pH 7.2) and Tris (10 mM tris-HCl, 100 mM NaCl, 10 mM MgCl2, pH 7.5),7,9 and all other aqueous solutions were made with ultrapure MiliQ water (resistance > 18 MΩ cm). The prehybridized dsDNA was produced by heating a 1:1 mole mixed solution (∼100 µM) of the HS-ssDNA-20 with its complement ssDNA-24, or HS-ssDNA-12 with ssDNA-12, in Tris buffer to 90 °C and cooling slowly to room temperature over 2 h.10 The hybridized bulk solution was diluted to the desired concentration, 1.5-2.0 µM, with Tris buffer. All solutions were filtered through a Whatman syringe filter (0.2 µm pore size) before use. All gold surfaces used in this study were freshly prepared template stripped gold (TSG) Au(111) surfaces using the chemical stripping method with tetrahydrofuran (THF).13 Once stripped, the TSG surfaces were rinsed with THF, ethanol, and MilliQ water and dried over a stream of nitrogen and then immediately incubated with a desired solution to minimize contamination. These TSG surfaces are extremely flat (roughness < 3 Å over a scan area of 25 µm2 is routinely obtained) and have large atomically flat terraces hundreds of nanometers across (Figure 1), which provide an ideal base for the detection of small surface height variations by AFM. (11) (a) Demers, L. M.; Ginger, D. S.; Park, S. J.; Li, Z.; Chung, S. W.; Mirkin, C. A. Science 2002, 296, 1836. (b) Lee, K. B.; Park, S. J.; Mirkin, C. A.; Smith, J. S.; Mrksich, M. Science 2002, 295, 1702. (12) (a) Kenseth, J. R.; Harnisch, J. A.; Jones, V. W.; Porter, M. D. Langmuir 2001, 17, 4105-4112. (b) O’Brien, J. C.; Stickney, J. T.; Porter, M. D. J. Am. Chem. Soc 2000, 122, 5004-5005. (c) O’Brien, J. C.; Stickney, J. T.; Porter, M. D. Langmuir 2000, 16, 9559-9567. (13) (a) Hegner, M.; Wagner, P.; Semenza, G. Surf. Sci. 1993, 291, 39-46. (b) Wagner, P.; Hegner, M.; Gu¨ntherodt, H. J.; Semenza, G. Langmuir 1995, 11, 3867-3875.
10.1021/la0258547 CCC: $22.00 © 2002 American Chemical Society Published on Web 09/26/2002
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Figure 1. Representative AFM image of a freshly prepared template-stripped gold (TSG) surface before DNA immobilization showing atomically flat gold terraces. Scan size 500 × 500 nm2, height bar 3 nm. Atomic Force Microscopy. All AFM experiments were carried out on a Digital Instrument (DI) dimension 3100 atomic force microscope with a NanoScope IV controller (CA) with a liquid cell in Tris buffer at 24 ( 1 °C in contact mode. Standard oxide-sharpened Si3N4 tips (DI) with nominal spring constants of 0.06 or 0.12 N/m were employed. During topographic image collection, the feedback loop was constantly adjusted to ensure a minimum force was applied, just enough to obtain a stable image (usually in the range 0.2-0.4 nN).10b For scratching the DNA layer, a small scan area was selected at a specific region, and then the set point voltage was increased by several units until the interatomic steps of the gold terraces were observed. The required force for scratching the DNA layer varied from sample to sample and typically in the region 30-50 nN. After scratching, the AFM tip was fully retracted from the surface by decreasing the set point voltage, zoomed out to a bigger scan area, and then re-engaged with minimum force, just enough to obtain a stable image to take the topographic images.
Results and Discussion A DNA self-assembled monolayer (SAM) was coated by incubation of the fresh TSG surface for 72 h with a buffered solution (2 µM in PBS) of a 20 base single-stranded DNA modified at the 5′ terminus with a C6 alkanethiol (HSssDNA-20). Short oligonucleotides were used because they have been shown to form more ordered coatings.9 The topographic image obtained by AFM initially showed many bright spots randomly distributed over a flat surface; however, many of these disappeared as the surface was repeatedly scanned (Some residual spots can be seen in Figure 2). The spots protrude from the surface by up to 4.5 nm. Similar features have been reported previously and interpreted as the only DNA molecules immobilized on the gold surface.5 The data shown below indicate that a more probable interpretation is that the white spots were due to strands of DNA extending above a uniform layer of the DNA, on the surface. The depth of the main DNA coating was determined by creating holes in the film extending to the underlying TSG surface.10,14,15 This was achieved by applying higher loading forces to the AFM tip than those used for imaging, to scrape away part of the (14) Liu, G. Y.; Xu, S.; Qian, Y. Acc. Chem. Res. 2000, 33, 457-466. (15) We found that indentations on the DNA resist can be achieved by the AFM tip with a loading force of 30-50 nN, and SAMs of alkanethiols needed a loading force of well over 100 nN. A similar force for scratching alkanethiol SAMs has also been reported recently. See ref 12a.
Figure 2. (A) AFM image of the thiolated ssDNA (20-mer) SAM on a TSG surface prior to hybridization showing two scratched holes created by applying a higher loading force. (B) AFM image after hybridization with the complementary DNA (24-mer). Both scan sizes are 2.5 × 2.5 µm2, and line scan profile of the hole is shown below each image. The schematic shows the hybridization process of the thiolated DNA SAM.
DNA matrix. We have found that monolayers formed from DNA are displaced much more easily than self-assembled monolayers (SAMs) formed from alkanethiols and can be removed selectively using a soft cantilever.15 As shown in Figure 2A the holes created in this way were 3.5 ( 0.5 nm deep, which corresponds to about three times the cross section of the ssDNA.4,16 After incubation for 1 h with a complementary (nonthiolated) single-strand of DNA 24 bases long (ssDNA24) (4 µM in Tris), this surface was radically restructured. Figure 2B shows that the resulting surface was formed with few protruding features. The depth of the holes created in the layer increased to 6.0 ( 0.5 nm (Figure 2B). The simplest explanation for these observations is (portrayed in the schematic for Figure 2) the formation of a more densely packed monolayer composed of a mixture of the thiolated ssDNA and the hybridized dsDNA, formed by hybridization of the ssDNA on the surface with the complementary DNA in solution. There was no evidence of nonspecific adsorption of the ssDNA-24 onto the freshly exposed gold surface within the holes. Holes created at different locations of the posthybridized layer show the same thickness of 6.0 ( 0.5 nm, indicating that the hybridized layer is uniform. This is the first report of the use of AFM to follow DNA hybridization directly by monitoring thickness changes in the DNA monolayer. The closest precedent appears to be a report showing m13 phage DNA apparently hybridized to a short oligonucleotide tethered to a surface.5a DNA hybridization at surfaces has previously been studied by detecting changes in refractive index (SPR),6 mass (QCM),5 XPS,7 electrochemical response,9 and neutron reflection.8 However, (16) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Marcromolecules 1997, 30, 5763-5765.
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Figure 3. (a) AFM image of the thiolated dsDNA (12-mer) SAM on a TSG surface. (b) Exposure to thiolated ssDNA (20-mer) for 2 h. (c) On further exposure to complementary ssDNA (24-mer) for 1 h. All the scan sizes are 1.5 × 3.0 µm2, and line profiles of each image show changes in the depth of the holes. The schematic shows the filling and hybridization process of the DNA SAM within the holes.
these methods are not suitable for detection of hybridization on nanoscale regions of a surface. The use of AFM to monitor changes in the thickness of DNA layers upon applying an electric field or after enzymatic modification has also been reported.10,12 Surface features can be further manipulated by filling in the indentations created on the DNA resist with longerlength DNA to generate protrusions of various heights. To explore this idea, a double-strand DNA surface was prepared by incubating HS-dsDNA-12 (1.5 µM in Tris) with a freshly prepared TSG surface for 72 h. An AFM topographic image showed the formation of a complete SAM. Extended holes were then created in the monolayer, which was shown to be 3.1 ( 0.5 nm thick (Figure 3a). The surface was then incubated for 2 h with HS-ssDNA-20 (2 µM in Tris). The depth of the holes decreased to 1.3 ( 0.4 nm (Figure 3b), suggesting that the HS-ssDNA-20 is filling in the holes created to a depth of about 1.8 nm. This is consistent with the formation of a DNA layer tilted much closer to the underlying substrate, since the incubation time here (2 h) was much less than that used in the first experiment (72 h). When a solution of the complementary ssDNA-24 (2 µM in Tris) was introduced for 1 h, the holes were replaced by protruding plateaus extending 1.2 ( 0.5 nm above the surface (Figure 3c). This is consistent with the HS-ssDNA-20 hybridizing to the ssDNA-24 to form more rigid, closely packed, and ordered arrays. The interpretation is portrayed in the schematic for Figure 3. Holes created, posthybridization, in different areas on the surface show that the main film thickness is still 3.1 nm, indicating the background DNA matrix had not been changed and that the hybridization occurred only in the holes where HS-ssDNA-20 was adsorbed. The height of the hybridized DNA patches is 4.3 ( 0.5 nm relative to the TSG surface, which is less than the value of 6.0 ( 0.5 nm obtained from the experiments described in Figure 2, most likely due to the formation of a less compact DNA layer upon shorter incubation times. Nontheless, this is
the first report to use AFM to detect the DNA hybridization in situ on a nanometer scale at a localized surface region. As a control, a double-stranded DNA surface was formed by incubating a freshly prepared TSG surface for 72 h with the HS-ssDNA-20 (2 µM in Tris) prehybridized to the complementary ssDNA-24. Holes created within the dsDNA monolayer showed the film was 7.0 ( 0.8 nm thick. The maximum theoretical thickness of this fully hybridized dsDNA is 9.7 nm (1.2 nm for the HSC6 linker, 0.34 nm for each base pair in dsDNA, and 0.43 nm for each base in ssDNA),4,16 suggesting the dsDNA was tilted at an angle of 46 ( 6° from the surface normal, which is in excellent agreement with the literature value of ∼45°.10 The reduced film thickness observed from the in situ hybridization suggests that the surface immobilized DNA was not fully hybridized and/or not closed-packed. Incomplete hybridization of surface bound DNA has been reported in other studies of surface hybridization.5-9 6-Mercapto-1-hexanol (MCH) competes successfully with thiolated DNA on a gold surface and has been shown to extend short DNA oligonucleotides away from the surface.7-9 This effect can be used to further manipulate the thickness of the extended DNA coating on surfaces. A monolayer created by incubating TSG with HS-ssDNA20 (2 µM in Tris) for 48 h showed a thickness of 3 nm (Figure 4A). Upon treatment with a dilute buffered solution of MCH (1 mM in Tris) for 50 min, the hole depth increased to 4 nm (Figure 4B). However, the forces used in this experiment were insufficient to remove an alkanethiol such as MCH, so the depth recorded does not include the thickness of the MCH monolayer (1.2 nm).17 Upon hybridization with a complementary ssDNA-24, the apparent depth of the layer increased to 6 nm (again not including the thickness of the MCH layer) (Figure 4C). After adding the normal thickness of an MCH monolayer, the total layer thickness is 7.2 nm, which is similar to that found for the monolayer formed by incubation of dsDNA-20/24 with the TSG and greater than that formed
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Figure 4. (A) AFM image of the thiolated ssDNA (20-mer) SAM on TSG. The scratched hole shows a depth of 3 nm. (B) After treatment with MCH in 1 µM Tris for 50 min. The depth of the hole is 3.5 nm. (C) After hybridization with ssDNA-24, the depth of the hole increased to 6 nm. All scan sizes are 2.0 × 2.0 µm2. The schematic shows the hybridization process of the thiolated DNA SAM in the presence of MCH.
by in situ hybridization experiments in the absence of MCH described above (6.0 nm). These observations suggest that thicker layers incorporate more dsDNA, and they are consistent with reports that MCH may improve hybridization efficiency at surfaces.6-9 The effect of adding MCH was found to depend on the stability of the thiolated dsDNA monolayers. Holes created in a 3.1 nm thick SAM prepared from dsDNA-12 were filled with prehybridized thiolated dsDNA-20/24. It was found that the localized surface features prepared in this way were not entirely regular and protruded up to 4.2 nm above the surface. Upon incubation of this surface with MCH (1 mM in Tris buffer) for 2 h, the newly formed dsDNA-20/24 patches were completely displaced and (17) The indentation of the DNA layer was carried out in a 1 µM MCH solution in Tris buffer with a loading force of 22 nN. It was found that the presence of the competing MCH greatly facilitated the complete removal of the DNA. To scratch away the MCH SAM, a stiffer cantilever and a loading force of more than 100 nN were found to be necessary. The scratching of an alkanethiol SAM in the presence of a different competing thiol in solution has been described as “nanografting” by Liu et al., and they found the competing thiol followed the scraping track of the AFM tip to form a completely covered SAM within the scratched area. See ref 14.
replaced by a SAM of MCH. There was no evidence of erosion of the main DNA coating into which the holes had been made, suggesting that the resist monolayer is kinetically more stable than the dsDNA areas created by “scratching”. It is likely that the longer incubation period (72 h) used to prepare the main layer would produce a film with fewer defects that could act as sites for attack by MCH.9 In summary, this study utilized AFM to systematically follow specific manipulations of coatings formed with ssand dsDNA on gold. It is shown that the layer thickness can be modulated by hybridization and treatment with MCH. Surface features introduced by scratching away patches of DNA on gold can be partially or completely filled to create protrusions of various heights. These studies open up the possibility for the creation of complex surface architectures. Acknowledgment. We wish to thank the Leverhulme Trust for funding this project. K.S. thanks Calvin College and Burroughs-Wellcome Fund for sabbatical support. LA0258547
