NANO LETTERS
Production of Nanostructures of DNA on Surfaces
2002 Vol. 2, No. 8 863-867
Maozi Liu,† Nabil A. Amro,† Christine S. Chow,*,‡ and Gang-yu Liu*,† Department of Chemistry, UniVersity of California, DaVis, California 95616, and Department of Chemistry, Wayne State UniVersity, Detroit, Michigan 48202 Received May 27, 2002
ABSTRACT Nanopatterns of thiolated single-stranded DNA (ssDNA) are produced by using an atomic force microscopy (AFM)-based lithography technique known as nanografting. Under high shear force, AFM tips displace resist molecules within a self-assembled monolayer, while ssDNA molecules adsorb chemically onto the exposed gold area through the sulfur headgroup. Nanostructures of ssDNA are characterized directly and in situ by using the same tip. Lines as narrow as 10 nm have been produced. The ssDNA molecules stand up on the gold surfaces and adapt a stretched conformation. In situ and real-time imaging studies have revealed that DNA molecules within the nanostructures are accessible by enzyme molecules.
Arrays of DNA patterns are important in gene mapping, drug discovery, DNA sequencing and disease diagnosis.1 Various approaches have been taken to create DNA arrays on surfaces. One approach is to use light-directed oligonucleotide synthesis to attach DNA nucleotides at mask-defined areas and build subsequent DNA strands by coupling.2 Another method is to attach presynthesized DNA strands onto designated sites of a solid support.3,4 A broad range of solid supports have been used, such as gold, conductive polymer, SAMs, and carbon paste.5-15 Typically, the size of the DNA patterns is tens to hundreds of micrometers.16,17 Further miniaturization is essential for the development of ultrasmall biosensors and biochips. The performance of chips or sensors can be enhanced after miniaturization because of the higher density of receptor elements, higher detection sensitivity, and smaller amounts of reaction reagents. New generations of nanochips also offer the hope of faster analysis time, less waste of costly reagents, and massive parallelization.18 Scanning probe microscopy (SPM) techniques are well known for their ability to visualize surfaces of materials with the highest spatial resolution.19-21 Taking advantage of the sharpness of the tips and strong and local interactions between the tip and surface molecules, SPM has also been used to produce nanostructures on surfaces.22-30 “Dip-pen” nanolithography (DPN) has been used to pattern nanostructures of DNA on a gold surface. The size of the DNA pattern depends on the substrate, humidity of the environment, and fabrication speed, thus making it difficult to reach high spatial * Corresponding authors. Liu: Telephone 530-754-9678; Fax 530-7528995; E-mail:
[email protected]. Chow: Telephone 313-577-2594; Fax 313-577-8822; E-mail:
[email protected] † University of California. ‡ Wayne State University. 10.1021/nl025626x CCC: $22.00 Published on Web 06/12/2002
© 2002 American Chemical Society
precision. The tip coating process is relatively difficult, and a different tip needs to be used to characterize the produced pattern.31 Recently, a meniscus force nanografting method was used to pattern DNA on surfaces, and the patterns have been coupled with complementary oligonucleotides tagged by gold particles.32 The spatial precision and selectivity is not sufficiently high and the method used to characterize the DNA patterns involves tagging. We have developed three AFM-based lithography techniques for creating nanopatterns of self-assembled monolayers (SAMs) and biosensors: nanoshaving, nanografting, and nanopen reader and writer (NPRW).25-29 Using these methods, nanostructures of thiols as small as 2 × 4 nm2 have been successfully produced with various chain lengths and terminal groups such as -OH, -CO2H, -NH2, and -CHO.25 High-resolution images show that the thiol molecules within the nanopatterns are closely packed. In addition to SAM nanostructures, biomolecules such as proteins can be positioned on a surface via selective immobilization.33 To take full advantage of AFM lithography, we have tested the application of nanografting in patterning single-stranded DNA (ssDNA). The orientation and packing of the oligonucleotides within the patterns can be directly determined in situ using AFM. The accessibility of the oligonucleotides within the patterns can also be directly determined by using DNase I enzyme digestion. Both spatial precision and resolution are demonstrated with this method. This work provides a new, high-resolution, and simple approach for DNA nanofabrication and pattern characterization. Gold films on mica substrate were prepared according to a previously reported procedure.34 The thickness of the gold films was 180 nm. SAMs of hexanethiol and decanethiol were prepared by immersing the freshly prepared gold films
Figure 1. Molecular model of thiolated single-stranded DNA built using the AMBER program. These models aim at providing a clear picture of a stretched molecular conformation and dimension. Thus, no energy minimization was performed. The sequences of oligo 1 and oligo 2 are 5′-HS-(CH2)6-CTAGCTCTAATCTGCTAG and 5′-HS-(CH2)6-AGAAGGCCTAGA, respectively.
into the ethanol solutions containing 1 mM thiol for at least 24 h. The atomic force microscope employed a home-constructed deflection-type scanner35,36 controlled by commercial electronics and software (RHK Technology, Inc., Troy, MI). Sharpened Si3N4 microlevers (Thermomicroscopes, Sunnyvale, CA) with a force constant of 0.1 N/m were used for AFM imaging and nanografting. The thiolated ssDNA used for fabrication were 5′-HS(CH2)6CTAGCTCTAATCTGCTAG-3′ (oligo 1), and 5′HS-(CH2)6AGAAGGCCTAGA-3′ (oligo 2) (Synthegen LLC, Houston, TX). Molecular models of ssDNA built using the AMBER program are shown in Figure 1. The predicted length of fully stretched oligo 1 and oligo 2 (including the six-carbon linker) are 84 and 59 Å, respectively. The RQ1 Rnase-free DNase was purchased from Promega (Madison, WI). The procedure for fabricating a DNA nanopattern is illustrated in Figure 2. In the first step, an alkanethiol SAM is imaged in a liquid medium containing thiolated ssDNA under a low load (Figure 2A). After a fabrication site is selected, the thiol molecules are removed by the tip with a higher force during the scan (Figure 2B). Thiolated ssDNA molecules in solution adsorb onto the freshly exposed gold area following the scanning track of the AFM tip. The DNA pattern can then be characterized by using the same AFM tip at a reduced imaging force (Figure 2C). 864
Figure 2. Schematic diagram illustrating the basic steps to produce ssDNA nanostructures using nanografting.
Figure 3A shows a 115 × 135 nm2 nanopattern of oligo 1 (18-nucleotide ssDNA) grafted within a CH3(CH2)5S/Au(111) matrix. Nanografting and imaging of the patterns were conducted in a mixed solvent of 2-butanol/water/ethanol with a ratio of 6:1:1(v/v/v) containing 40 µM ssDNA. The fabrication was accomplished under a 20 nN force and at a speed of 800 nm/s. The cursor profile in Figure 3B indicates that the oligo 1 pattern is 54-74 Å higher than the matrix SAM (or 63-83 Å of the total length). Figure 3C shows another example, in which a 190 × 255 nm2 pattern of oligo 2 (12-nucleotide ssDNA) was grafted into a different alkanethiol CH3(CH2)9S/Au(111) matrix. The height difference of 36-46 Å between the pattern and matrix was measured as shown in the corresponding cursor profile in Figure 3D. Thus, the oligo 2 molecules have the measured height ranging from 50 to 60 Å. Nano Lett., Vol. 2, No. 8, 2002
Figure 3. Positioning ssDNA on gold surface. [A] A 115 × 135 nm2 square of oligo 1 nanostructure grafted within a hexanethiol SAM. [B] Corresponding cursor profile as indicated in [A]. [C] A 190 × 255 nm2 rectangle of oligo 2 fabricated within a decanethiol SAM. [D] Corresponding cursor profile as indicated in [C]. [E] Three nanolines of oligo 2 with sizes 20 × 170 nm2, 15 × 150 nm2, and 25 × 160 nm2 were produced within a decanethiol SAM: a1 and a3 are solid lines, while a2 is a broken line. [F] Corresponding cursor profile crossing the three lines.
As shown in Figure 1, the fully stretched oligos exhibit a height of 84 and 59 Å, respectively. The measured DNA heights within the nanopattern correspond well with a standing up and nearly stretched configuration. The variation in DNA height within the pattern is likely due to variations in conformation and imperfection in packing. Achieving a standing up orientation is very important for the performance of the DNA patterns, particularly when used as sensing elements. The accessibility of the DNA pattern whose molecules are lying down will be hindered by the substrate, thus the performance of such DNA arrays is likely to be compromised. The fabrication method described here provides an efficient means to make DNA arrays in which the orientation and position of DNA molecules can be well controlled. In Figure 3E, three ssDNA nanolines of oligo 2, a1-a3, were grafted by a single scan with dimensions of 20 × 170 nm2, 15 × 150 nm2, and 25 × 160 nm2, respectively. The matrix is decanethiol SAM. The line width of the DNA pattern we have reached is as narrow as 10 nm. The apparent heights are shown in the cursor profile in Figure 3F. The overall height seems lower than the rectangular pattern because the DNA molecules within lines are more subjective to tip pressure. The middle pattern, a2, is a broken line, in which the smallest ssDNA dot is 7 × 11.5 nm2 (indicated by an arrow). If we assume that the DNA molecules exhibit a close-packed structure and the cross section diameter of ssDNA is 20 Å, then this dot consists of only 26 molecules. The actual number is likely less. The periodicity of SAMs is routinely visible under AFM; however, it is difficult to obtain molecular resolution images within the patterned ssDNA areas. This observation is likely Nano Lett., Vol. 2, No. 8, 2002
due to the fact that the DNA molecules do not form an ordered and close-packed structure on the gold surface. The cross-sectional area of single oligonucleotide is approximately 300 Å2, according to our model, which is much larger than that of the hydrocarbon thiol, and the phosphate groups are negatively charged. Therefore, the formation of ordered and closely packed structures may not be thermodynamically favorable for the thiolated ssDNA molecules. To verify the composition of the nanopatterns and the accessibility of the molecules within the pattern, an enzyme, RQ1 RNase-free DNase, was used as a probe. This enzyme degrades double-stranded and single-stranded DNA endonucleotically to produce oligonucleotide fragments at the 3′ end with a hydroxyl terminal group. The DNA nanopatterns were thoroughly washed after the fabrication, and the solvent was then replaced sequentially by ethanol, water, and finally buffer solution (40 mM Tris-HCl, 10 mM MgSO4 and 1 mM CaCl2, pH 8.0). Next, RQ1 DNase I was introduced and the surface was monitored in situ by using the same AFM probe that carried out nanofabrication and imaging. The reaction process is followed by the time evolution of AFM images and the corresponding height measurements (cursor profiles on the right). Figure 4 shows three snapshots of an oligo 2 pattern (300 × 200 nm2) within hexanethiol after the injection of the enzyme solution at critical reaction moments. Figure 4A is the topographic image of the DNA pattern before the injection of the enzyme. Figure 4B is the corresponding lateral force image. After the injection of DNase I and incubation at room temperature (21°C) for 4 h, changes in the topographic image are very evident (compare Figure 4D with 4A), accompanied by the apparent height decrease (compare 4F with 4C). The density of the ssDNA 865
Figure 4. Time-dependent AFM images of oligo 2 nanostructure during digestion by DNase I enzyme. [A] Topographic images of the nanopattern of oligo 2 inlaid in a hexanethiol matrix prior to the introduction of enzyme. [B] Corresponding lateral force image of [A]. [C] Cursor profile as indicated in [A]. [D]-[F] same as [A]-[C], after 4 h incubation with enzyme solution. [G]-[I] same as [A]-[C] after 20 h incubation with enzyme solution.
molecules within the pattern has decreased as well, because DNase I is a nonrestriction endonuclease that cleaves the ssDNA randomly. Once the oligonucleotides were cleaved, the free fragments were released into the buffer solution. Subsequently, the height of the ssDNA pattern decreased. After 20 h of digestion, the ssDNA pattern within the hexanethiol SAM is hardly recognizable as shown in Figure 4G and cursor profile Figure 4I. The enzyme cannot cleave the carbon-carbon bonds, therefore the digestion stops at the linker terminal. Since the ssDNA was modified on its 5′ end with a six-carbon thiol linker, there is no obvious height difference between the pattern and the matrix hexanethiol after the digestion. The terminal group in the patterned area is a 3′ hydroxyl in contrast to the hydrophobic matrix. The interaction between tip and hydroxyl group is stronger than that of the methyl group, thus, the pattern in the corresponding frictional force image exhibits higher contrast (Figure 4H). This enzyme digestion experiment further confirms that the molecules in the patterned area are indeed ssDNA, and the ssDNA molecules are accessible by subsequent reactants such as enzymes. A new AFM-based lithography method of producing nanopatterns of ssDNA is introduced. The nanostructures of ssDNA are produced by nanografting thiolated oligonucleotides. The thiol matrix likely guides the DNA adsorption 866
and prevents the lateral diffusion of the ssDNA, therefore, the geometry and packing of the ssDNA molecules are well controlled. The structure of the nanopattern and the relative orientation of the ssDNA molecules have been determined in situ by using AFM. DNA molecules adopt a standing up orientation. An enzyme digestion experiment confirms the composition of the nanopattern and reveals the high accessibility of the oligonucleotide molecules. The procedures reported here are easy to perform and the characterization of the products is in situ and direct. The DNA nanopatterning methodology provides a unique opportunity for engineering biostructures with nanometer precision, which shall benefit the nanofabrication of DNA biosensors and biochips. Acknowledgment. We thank Drs. Song Xu, Beatriz Llano-Sotelo, and Ms. Jayne Garno at Wayne State University for many helpful discussions, and Mr. Guanglei Cui at SUNY at Stony Brook for his assistance in constructing the ssDNA models. This work was supported by UC Davis and the National Science Foundation (Grant CHE-9733400). N.A.A. is supported by a graduate research fellowship from the NSF-IGERT program (970952 and 9972742). References (1) Bier, F. F.; Fu¨rste, J. P. In Frontiers in Biosensors; Birkha¨user Verlag: Basel, Switzerland, 1997; Vol. 1, p 97. Nano Lett., Vol. 2, No. 8, 2002
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