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Electron Transfer of Plurimodified DNA SAMs Alessandro Rospigliosi,*,† Rudolf Ehlich,‡ Heinrich Hoerber,‡ Anton Middelberg,§ and Geoff Moggridge† Department of Chemical Engineering, UniVersity of Cambridge, Pembroke Street, New Museum Site, Cambridge CB2 3RA, United Kingdom, H. H. Wills Physics Laboratory, Tyndall AVenue, UniVersity of Bristol, Bristol BS8 1TL, United Kingdom, and Australian Institute for Bioengineering and Nanotechnology (AIBN), LeVel 6, Queensland Bioscience Precinct (Bldg 80), The UniVersity of Queensland, Brisbane Qld 4072, Australia ReceiVed December 21, 2006. In Final Form: April 30, 2007 An STM-based current-voltage (I/V) investigation of deoxyribonucleic acid (DNA) 18 base pair (bp) oligonucleotide monolayers on gold is presented. Three bases of each of the immobilized and complementary strands were modified with either iodine or phenylethylene moieties. The oligonucleotides were immobilized on template stripped gold (tsg) surfaces and characterized by atomic force microscopy (AFM) and scanning tunneling microscopy (STM). AFM imaging showed that monolayers of the expected height were formed. A comparative study of normal, halogenated, and phenyl-modified DNA was made with the STM in tunneling spectroscopy (TS) mode. I/V spectroscopic measurements in the range (250 mV on both single- and double-stranded (ds) DNA monolayers (modified and unmodified) showed that for negative substrate bias (Usub) electron transfer is more efficient through a phenyl-modified monolayer than through normal or halogenated DNA. This effect was particularly clear below a threshold bias of -100 mV. For positive Usub, unmodified ds DNA was found to conduct slightly better than the modified strands. This is presumably caused by greater order in the unmodified versus modified DNA monolayers. Modifications on the immobilized (thiolated) strand seem to improve electron transport through the DNA monolayer more than modifications on the complementary (not surface-bound) strand.
1. Introduction The conduction properties of DNA have been studied by a variety of researchers using rather different methods of preparation and measurement.1-9 If double-stranded (ds) DNA could be modified in such a way as to conduct electrons well enough, it could become a significant component of future nanoelectronic circuits and/or biosensors. The interpretation of experiments that measure the electrical conduction of surface-bound monolayers or molecules can be very difficult. It has become an accepted fact that a very thin layer of water in humid air conditions makes even insulating glass or mica conductive enough to obtain STM images.19 * Corresponding author: Alessandro Rospigliosi. E-mail: alessandro@ rospigliosi.com. † University of Cambridge. ‡ University of Bristol. § The University of Queensland. (1) Giese, B. Electron transfer in DNA. Curr. Opin. Chem. Biol. 2002, 6 (5), 612-618. (2) Giese, B. Long-distance electron tranfer through DNA. Annu. ReV. Biochem. 2002, 71, 51. (3) Ishida, T.; Mizutani, W.; Aya, Y.; Ogiso, H.; Sasaki, S.; Tokumoto, H. Electrical conduction of conjugated molecular SAMs studied by conductive atomic force microscopy. J. Phys. Chem. B 2002, 106 (23), 5886. (4) Jortner, J.; Bixon, M.; Langenbacher, T.; Beyerle, M. E. Charge transfer and transport in DNA. Proc. Natl. Acad. Sci. U.S.A. 1998, 95 (22), 12759. (5) Kelley, A. M.; Michalet, X.; Weiss, S. Chemical physics - Single-molecule spectroscopy comes of age. Science 2001, 292 (5522), 1671. (6) Kelley, S. O.; Barton, J. K. DNA-mediated electron transfer from a modified base to ethidium: π-stacking as a modulator of reactivity. Chem. Biol. 1998, 5 (8), 413. (7) Lemieux, B.; Aharoni, A.; Schena, M. Overview of DNA chip technology. Mol. Breed. 1998, 4 (4), 277. (8) Lewis, F. D.; Wu, T.; Zhang, Y.; Letsinger, R. L.; Greenfield, S. R.; Wasielewski, M. R. Distance-dependent electron transfer in DNA hairpins. Science 1997, 277, 673. (9) Olson, E. J. C.; Hu, D. H.; Hormann, A.; Barbara, P. F. Quantitative modeling of DNA-mediated electron transfer between metallointercalators. J. Phys. Chem. B 1997, 101 (3), 299.
DNA strands and plasmids deposited on mica, gold, or graphite (HOPG) can be displaced by interaction with the STM tip, and they are usually fixed by covalent bonding20 with a thiol group or the deposition of a metallic mask.21 Spontaneous changes and (10) Braun, E.; Eichen, Y.; Sivan, U.; Ben-Yoseph, G.; DNA-templated, assembly and electrode attachment of a conducting silver wire. Nature (London) 1998, 391, 775. (11) Richter, J. Metallization of DNA. Physica E (Amsterdam, Neth.) 2003, 16 (2), 157-173. (12) Kelley, S. O.; Holmin, R. E.; Barton, J. K. Photoinduced electron transfer in ethidium-modified DNA duplexes. J. Am. Chem. Soc. 1997, 119, 9861. (13) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Long-range photoinduced electron-transfer through a DNA helix. Science 1993, 262 (5136), 1025. (14) Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.; Hormann, A.; Olson, E. J. C.; Barbara, P. F. Rates of DNA-mediated, electron transfer between metallointercalators. Science 1996, 273 (5274), 475-480. (15) Harriman, A. Electron tunneling in DNA. Angew. Chem., Int. Ed. 1999, 38 (7), 945. (16) Giese, B.; Meggers, E.; Wessely, S.; Spormann, M.; Biland, A. DNA as a supramolecule for long-distance charge transport. Chimia 2000, 54 (10), 547. (17) Grinstaff, M. W. How do charges travel through DNA? An update on a current debate. Angew. Chem., Int. Ed. 1999, 38 (24), 3629. (18) Amann, N.; Pandurski, E.; Fiebig, T.; Wagenknecht, H. A. Electron injection into DNA: Synthesis, and spectroscopic properties of pyrenyl-modified oligonucleotides. Chem.sEur. J. 2002, 8 (21), 4877-4883. (19) Guckenberger, R.; Heim, M.; Cevc, G.; Knapp, H.; Wiegraebe, W.; Hillebrand, A. Scanning tunneling microscopy of insulators and biological specimens based on lateral conductivity of ultrathin water films. Science 1994, 266, 1538-1540. (20) Allison, D. P.; Bottomley, L. A.; Thundat, T.; Brown, G. M.; Woychik, R.; Schrick, J.; Jacobson, K.; Warmack, R. Immobilization of DNA for scanning probe microscopy. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10129-10133. (21) Dunlap, D. D.; Garcia, R.; Schabtach, E.; Bustamante, C. Masking generates contiguous segments of metal-coated and bare DNA for scanning tunnelling microscope imaging. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7652-7655. (22) Cui, X. D.; Primak, A.; Zarate, X.; Tomfohr, J.; Sankey, O. F.; Moore, A. L.; Moore, T. A.; Gust, D.; Harris, G.; Lindsay, S. M. Reproducible measurement of single-molecule conductivity. Science 2001, 294 (5542), 571. (23) Patole, S. N.; Pike, A. R.; Connolly, B. A.; Horrocks, B. R.; Houlton, A. STM study of DNA films synthesized on Si(111) surfaces. Langmuir 2003, 19 (13), 5457-5463. (24) Wagenknecht, H. A. From mechanism to application - Charge, transfer via DNA. Chem. Unserer Zeit 2002, 36 (5), 318-330.
10.1021/la063704g CCC: $37.00 © 2007 American Chemical Society Published on Web 06/23/2007
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reversion of the image contrast of single molecules on surfaces have been observed.20,32 Recent STM studies emphasize the importance of ambient conditions for DNA conductance measurements. Poly-GC oligomers of 8-14 base pairs showed a linear conductance in aqueous conditions below 500 mV. The molecules were thiol bound to two Au electrodes, bridging the gap by hybridization. The conductance was found to be inversely proportional to the length of the molecules.33 A thiolated 12 base pair polyGCpolyGC DNA on Au was studied by M. S. Xu and co-workers34 with current/voltage spectroscopy under UHV conditions. The measurements showed semiconductor characteristics with a wide band gap of 2.4-3.4 V. The interest in DNA as an electron conductor and in strategies to improve the conductivity grew due to a paper published in 1998 by Braun and co-workers.10 This paper describes how λ-DNA, functionalized with appropriate sticky ends, was used to bridge a 12 µm wide gap between two gold electrodes and was then used as a template for the deposition of silver ions and atoms which created a continuous silver wire. The not-veryconductive DNA became a conducting wire after silver deposition. Similar metallization strategies have been used, and a review is available in the literature.11 Studies involving covalently8,12,13 and noncovalently14,15 bound intercalators, photochemical reactions,16 and optical measurements to investigate the rate of electron transfer along short oligonucleotide sequences have given inconsistent results,17 but with a clear indication that the preparation and measuring technique has a major influence. Wagenknecht and his group presented two modification strategies of the uridine base using C-C coupling reactions: (i) first, a Suzuki coupling was used to attach a pyrene moiety to the 5 position of 5-iodo-2′-deoxyuridine (5-I 2′-deU),18 and then, (ii) a Sonogashira coupling was used to attach a pyrene group via an ethylene bridge to the same position of 5-I 2′-deU. These analogues where incorporated into short oligonucleotides and studied by UV/vis, fluorescence spectroscopy, and circular dichroism (CD). By photoexciting the chromophores, they found that the fluorescence through DNA was quenched by electron transfer only when the modified U was adjacent to a thymine (T) or a cytosine (C) nucleotide, thus proving the importance of the base sequence and of the redox potentials of the bases in a given strand. A series of 2-D and 3-D NMR experiments (COSY, HMQC, and NOESY) showed that the pyrene moiety was sticking out of the double helix, into the major groove, which ensured (25) Eckstein, F. Oligonucleotides and Analogues; Oxford University Press (IRL Press): Oxford, 1991. (26) Herne, T. M.; Tarlov, M. J. Characterization of DNA probes immobilized on gold surfaces. J. Am. Chem. Soc. 1997, 119 (38), 8916. (27) Zhou, D. J.; Sinniah, K.; Abell, C.; Rayment, T. Use of atomic force microscopy for making addresses in DNA coatings. Langmuir 2002, 18 (22), 8278-8281. (28) Sam, M.; Boon, E. M.; Barton, J. K.; Hill, M. G.; Spain, E. M. Morphology of 15-mer duplexes tethered to Au(111) probed using scanning probe microscopy. Langmuir 2001, 17, 5727. (29) Smith, D. P. E.; Hoerber, J. K. H.; Gerber, C.; Binnig, G. Smectic Liquid Crystal Monolayers on Graphite Observed by Scanning Tunneling Microscopy. Science 1989, 245, 43. (30) Guenebaut, V.; Hoerber, J. K. H.; Binnig, G. TEM moire patterns explain STM images of bacteriophage T5 tails. Ultramicroscopy 1997, 69, 129. (31) Porath, D.; Bezryadin, A.; de Vries, S.; Dekker, C. Direct measurement of electrical transport through DNA molecules. Nature (London) 2000, 403, 635. (32) Shapir, E.; Yi, J.; Cohen, H.; Kotyar, A. B.; Cuniberti, G. The puzzle of contrast inversion in DNA STM imaging. J. Phys. Chem. B 2005, 109, 1427014274. (33) Xu, B.; Zhang, P.; Li, X.; Tao, N. Direct conductance measurement of single DNA molecules in aqueous solution Nano Lett. 2004, 4, 1105-1008. (34) Xu, M. S.; Tsukamoto, S.; Ishida, S.; Kitamura, M.; Arakawa, Y. Conductance of single thiolated poly(GC)-poly(GC) DNA molecules Appl. Phys. Lett. 2005, 87, 083902.
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Figure 1. Schematic representation of the synthesis of the 5-iodo2′-deoxyuridine (3) and 5-phenylethylene-2′-deoxyuridine (5) monomers used in automated oligonucleotide synthesis. (a) 4,4′dimethoxytrityl chloride (DMTCl), dimethylaminopyridine (DMAP), dry pyridine, 20 °C, 17 h. (b) 2-cyanoethyl-N,N′-diisopropylchlorophosphoramidite, anhydrous diisopropylethylamine (DIPEA), anhydrous dichloromethane (DCM), N2, room temperature, 30 min. (c) Phenylacetylene, tetrakistriphenylphosphine palladium (Pd(PPh3)4), 5-iodo 5′-DMT-2′-deU, dimethylformamide (DMF), cuprous iodide (CuI), triethylamine (Et3N), 80 °C, 2 h.
that the Watson-Crick base pairs between the modified U and the adenine base on the complementary strand were not disrupted. The present paper focuses on similar modifications: 5-phenylethylene 2′-deoxyuridine (Ph - ≡ - dU) and 5-iodo 2′deoxyuridine were incorporated into DNA strands. In this case, however, instead of attaching just one base analogue per strand, three modified bases were incorporated into both the immobilized strand and its complementary strand of an 18-mer. We present I/V results showing that the modified DNA exhibited altered electronic conduction properties, and therefore, a novel approach to designing conductive DNA wires is anticipated. The I/V curves also suggest that modified thiolated (i.e., immobilized) strands contribute to a greater extent to the improvement of electron transport than modifications on the complementary strands. 2. Materials and Methods A. Synthesis Scheme of 5-Ph - ≡ - 2′deU and 5-I-2′deU Modified Oligonucleotides. Two types of modifications were prepared as phosphoramidites for the subsequent incorporation into 18 base long oligonucleotides by automated synthesis techniques (Figure 1): (i) first, 5-iodo-2′-deoxyuridine, 1, was protected with a dimethyltritoxy (DMT) group to make 2. This product was then protected with standard 2-cyanoethyl-N,N′-diiospropylchlorophosphoramidite that converts it to 3 ready for oligonucleotide synthesis. (ii) A second fraction of 2 was reacted in a Sonogashira coupling to form the 5-Ph - ≡ -2′deU, 4. This product was then reacted again with 2-cyanoethyl-N,N′-diiospropylchlorophosphoramidite to make the necessary product. It was noted that in all cases it was useful to protect the free 5′-hydroxy with the DMT group in the first step, because the solubility was enhanced and the purification of the nucleosides by flash
8266 Langmuir, Vol. 23, No. 15, 2007 chromatography became easier, thus giving better yields. A Sonogashira coupling reagent mixture containing phenylacetylene (1.15 mmol), tetrakistriphenylphosphine palladium (Pd(PPh3)4) (0.046 mmol), 5-I-5′-DMT-2′deU (300 mg, 0.46 mmol), DMF (8 mL), CuI (0.09 mmol), and Et3N (0.92 mmol) under dry conditions at 80 °C for 2 h was used to synthesis 4. All the reactions shown in Figure 1 gave reasonably good yields: a ) 89%, b ) 67%, and c ) 82%. It was noticed that the coupling reactions gave better yields, if several small batches of 350-400 mg of starting material 2 were used. As later confirmed by personal communication from other researchers (at the MRC-LMB and at Sheffield University), scaling up such coupling reactions (also for Suzuki and the Heck couplingssdata not shown here) was found to give poorer yields. Sufficient fully protected 5-iodo- and 5-phenylethylene-2′deU, 3 (2.9 g) and 5 (3.3 g), respectively were purified, for the threefold insertion of the DNA analogues (generically labeled U ˆ ) into a thiolated single strand (ss) and its complementary ss oligonucleotide. The sequences shown below were synthesized by IBA (Goettingen, Germany):
Oligonucleotides containing the phenyl-modified uridine will be labeled from now on U*, and the iodo-modified ones Ui. The sequence of the DNA oligonucleotides and the positions of Ui and U* have been chosen with the following considerations in mind: (i) On the thiolated strand, the distance between a U* or Ui and T (or U*or Ui) nucleotide should not exceed two bases, because these have the lowest reduction potentials a key factor for excess electron transfer;24 (ii) U* or Ui on the complementary strand was incorporated between two U* or Ui on the thiolated strand, so as to enhance the electronic couplingsif interstrand coupling does occur; (iii) a random sequence was chosen because it reduces intramolecular steric and electronic interference; and finally, (iv) an 18-mer with approximately 50% GC vs AT bp ensured good thermal stability (i.e., no strand melting) at room temperature. We already were very confident that the Watson-Crick base pairing of the duplex DNA would not be disrupted by these modifications because, as described by Amann and co-workers,18 even the sterically bulkier and rigid pyrene-ethylene substituents were shown to be directed into the major groove of the DNA helix, therefore allowing proper hybridization to occur. Solvents were dried according to standard procedures. All reactions were carried out under argon. 2′-Deoxyuridine, anhydrous N,N′dimethylformamide (DMF) (99.9%), and anhydrous dichloromethane (DCM) were obtained from Sigma-Aldrich Chemical Co. Methanol (>99%), glacial acetic acid, and dichloromethane were purchased from Fisher Chemicals. Triethylamine (99%), pyridine, and 1,4dioxane (99.5%) were purchased from Lancaster Synthetics and were distilled from and stored over potassium hydroxide pellets and molecular sieves, respectively. Phenylacetylene (98%), dichlorobis(diphenylphosphino)ferrocenyl palladium (PdCl2(dppf) (99%)), tetrakistriphenylphosphine palladium (Pd(PPh3)4) (99.9%), cuprous iodide (CuI) (99.99%), 2-dimethyltritoxy chloride (DMT-Cl) (99%), dimethylamino pyridine (DMAP) (99%), and diisopropylethylamine (DIPEA) (98.5%) were purchased from Sigma-Aldrich Chemical Co. Each batch of 2-cyanoethyldiisopropylchlorophosphoramidite (99.9%), which was bought from the same supplier in the smallest possible quantities (2 mL), was used only for one reaction at a time. All other chemicals were used as received. Flash chromatography was performed using either Merck silica gel (particle size 40-63 nm) or Fisher silica gel (particle size 35-70 µm). Thin layer chromatography (TLC) was performed on both Merck TLC glass sheets on silica gel 60 F254 and on precoated TLC plates from Machery-Nagel SIL G-25 UV254. Visualization was achieved by UV light (254 nm) using Mineralight lamp model UVGL25 and/or dipping the TLC plate into potassium permanganate solution, followed by heating with a heat gun.
Rospigliosi et al. Mass spectra and MALDI-TOF spectra (in both linear and reflector modes) were recorded on a Micromass Q-TOF and an Applied Biosystems 4700 Proteomics Analyzer spectrometer, respectively. Melting points were obtained using a Gallenkamp melting point apparatus and were uncorrected. NMR spectra were recorded in DMSO-d6 (unless otherwise stated) at room temperature on a Brucker DRX 500 (500 MHz 1H NMR, 125 MHz 13C NMR) with either a 13C/ 1H cryoprobe or a 13C/ 1H dual probe. Chemical shifts are given in ppm. B. Synthesis and Purification of 5-Iodo-5′-dimethyltritoxy2′-deoxyuridine (2). 1.33 g (3.75 mmol, 1 equiv) 5-iodo-2′deoxyuridine (1) was added to a dry 250 mL reaction flask and dissolved in 2 × 20 mL dry pyridine. The solvent was evaporated and the flask containing the starting material dried overnight in an oven at 40 °C under vacuum. Dry pyridine (100 mL) was added to the flask under stirring. Then, a solution of 1.54 mg (4.34 mmol, 1.2 equiv) demethoxytritylchloride (DMT-Cl) in approximately 4 mL dry pyridine and 2 mL anhydrous DCM was added dropwise to the reaction flask. The reaction was left under argon for 30 min at room temperature before adding a few grains ( 18 MΩ cm) to give approximately 100 µM solutions of single-stranded oligonucleotide and was stored in appropriate aliquots. The thiolated
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Figure 3. Schematic representation of a self-assembled monolayer (SAM) of DNA on gold; adapted from Zhou and co-workers.27 oligonucleotides (8618-SH (normal), 8522-SH (iodo-modified), 8491-SH (phenylethylene-modified)) were first treated for 30 min in a 0.1 mM DTT solution, a reducing agent that restored the chemically reactive SH groups of the oligonucleotides’ alkanethiol (prolonged periods of storage can cause the formation of disulfide bonds between thiolated oligonucleotides). Dialysis of 200 µL samples was then performed on Pierce Slide-A-Lyser dialysis cartridges (MW cutoff 2000 Da) overnight in a 2 L deionized water bath with stirring and triple water changes. Depending on the size of the gold surface, 10-20 µL of the activated oligonucleotide in 10 mM MgCl2 and 10 mM Tris-HCl solutions were then spotted onto freshly cleaved template stripped gold slides.22 All solutions were filtered through a disposable 0.2 µm membrane before use. After incubation in a humidity chamber for 3 h at room temperature, the slides were rinsed with MilliQ water and incubated for 2 h in a 1 mM MCH solution to remove nonspecifically bound DNA from the surface and to block any uncoated patches of gold. The slides were again thoroughly rinsed in deionized water and blown dry with nitrogen. Then, a similar volume (10-20 µL) of 100 µM complementary oligonucleotides (8618, 8522, or 8491) in 100 mM MgCl2 and 10 mM Tris-HCl solution was added to the spot where the thiolated DNA was adsorbed. To facilitate the hybridization, these slides were warmed up in a humidity chamber (surrounded by, but not in contact with, water at 75 °C) for 8-10 min and allowed to cool down slowly to room temperature. Figure 3 shows a schematic representation of how such a self-assembled monolayer (SAM) of DNA on a gold surface is thought to look like. Finally, after a final rinse in deionized water and blow-drying with nitrogen, the samples were investigated by scanning probe microscopy and I/V spectroscopy. G. AFM Investigation. The monolayers prepared with the procedure in section 2.F were imaged by AFM in contact mode (CM). All AFM experiments were carried out on a Digital Instrument (DI) dimension 3100 atomic force microscope with a Nanoscope IIIa controller (CA) either in tapping mode or with a liquid cell in Tris buffer at 22 ( 1 °C in contact mode. For tapping mode, standard silicon tips (Veeco) with nominal spring constant of 20-100 N/m and nominal tip radius of curvature of 5-10 nm were used. For contact mode in liquid, standard oxide-sharpened Si3N4 tips (Veeco) with nominal spring constants of 0.06 or 0.12 N/m and nominal tip radius of curvature of 20-40 nm were used. During topographic imaging, the feedback loop was constantly adjusted to ensure that only minimal force was applied. Particular care was taken when imaging DNA monolayers (usually in the range 0.2-0.4 nN) in liquid. Tris solution (10 mM Tris-HCl, 10 mM MgCl2, pH 7.5) prepared with ultrapure MilliQ water (resistance >18 MΩ cm) was used as buffer. In order to scratch the DNA layer, a small scan area was selected at a specific region, and then the set-point voltage that controls the cantilever deflection was increased until the interatomic steps of the gold terraces were observed. The required force for the scratching of the DNA layer varied from sample to sample and was typically in the region 30-60 nN. Hooke’s law (F ) kx) was used to obtain these values for the force. However, the nominal spring constant values supplied by the manufacturers, which are known to vary between (30% were used to make this calculation. Hence, a drawback of this technique is the fact that the force cannot be controlled with great precision at the nanonormal scale. After the nanoshaving procedure, the AFM tip was fully retracted from the surface by decreasing the setpoint and then re-engaged with minimal force, just enough to obtain a stable image and to take topographic images (such as Figure 4). H. STM Investigation. STM imaging was performed on a homebuilt STM at the EMBL laboratory, Heidelberg, Germany. For
Figure 4. AFM image of a scratched hole in a ds DNA monolayer of 8618d8618-SH (i.e., 8618 hybridized to the complementary thiolated 8618-SH): (a) top view with scale bar and (b) height profile average of the area encompassed by the black box (bottom left image) with three different measurements (red ) 4.516 nm; black ) 4.296 nm; green ) 4.883 nm). Table 2. Summary of the Measured and Expected Height Ranges of the SAM of Single- (1-3) and Double-Stranded (4-8) Monolayers entry
oligonucleotide
measured height range/nm
expected height range/nm
1 2 3 4 5 6 7 8
8618-SH 8522-SH 8491-SH 8618d8618-SH 8522d8618-SH 8491d8618-SH 8522d8522-SH 8491d8491-SH
2.6-3.0 2.5-2.9 2.4-2.9 4.3-4.9 4.2-5.0 3.9-4.8 4.1-4.9 4.2-4.8
2.9-3.1 2.9-3.1 2.9-3.1 4.8-5.2 4.8-5.2 4.8-5.2 4.8-5.2 4.8-5.2
imaging of the monolayers, setpoint currents of 5-50 pA were employed in conjunction with substrate potentials of less than 500 mV. Tungsten tips of 0.5 mm diameter were cut to 5-7 mm length and etched in a concentrated KOH solution with initial currents ranging between 0.5 and 1.0 A. The tip was carefully rinsed in boiling, doubly distilled water (MilliQ) to remove salt ions. Imaging of the monolayers was done in constant-current mode. This implied that the feedback loop kept the current at a constant value following in this way the surface contour mapping the sample topography. Before taking current-voltage (I/V) measurements, small areas (typically 100 × 100 nm2) were scanned to check that they were clean and without defects. After the I/V curves were taken, the same area was reimaged to check that the monolayer was still intact. A free software package called WSxM (v 4.0), made available online by a Spanish company (Nanotec Electronica S. L.; www.nanotec.es), was used to visualize and process (i.e., flatten and remove noise signals of) the STM images. After a clear image of an approximately 100 × 100 nm2 area was obtained for a given sample, the scan area was set to zero and the tip stopped at the center of the imaged area for tunneling spectroscopy
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Figure 5. STM images of double-stranded 8491d8491-SH (i.e., 8491 hybridized to the complementary thiolated 8491-SH) monolayer on gold: (a) 100 × 100 nm2 area before I/V curves were taken and (b) 500 × 500 nm2 area after I/V curves were taken; the scale bar on the right applies to both images. measurements. The tip was positioned by the feedback loop at a particular height above the sample at substrate voltage (Usub) of +80 mV and current setpoint (Isp) of 40 pA. Current-voltage measurements were also taken by setting Usub to -80 mV in order to check that the current behavior was similar and consistent. In order to allow comparison of the results under the same conditions, Usub and Isp were kept constant for all I/V scans. The feedback was switched off during spectroscopic measurements but allowed to control the distance in between. The tip voltage was always kept at 0 V, and only the substrate potential (Usub) was varied. The scan range was set to 500 mV (i.e., (250 mV), and the scan frequency used was either 10 or 20 Hz (for ds and ss SAMs, respectively).
3. Results A. AFM Investigation. First, a large area was imaged; then, a greater load was applied over a selected smaller part of this area (which shaves/scratches away the DNA in that area). The initial larger area (including the part which had been scratched away) was then imaged again, using a much smaller deflection setpoint. The height profile that was obtained was thus used to demonstrate that DNA strands of the expected length were immobilized on the surface. Comparison to other literature results showed that the height measurements we took correspond well to a monolayer which protrudes at an angle of 45° from the surface.27Figure 4 shows an example of a surface area, where the DNA was removed using high loading forces on the tip: (a) the top view of a 2 µm × 2 µm hole in a ds unmodified (8618d 8618-SH) DNA monolayer; (b) the corresponding height profile. Table 2 summarizes the results obtained from at least three distinct measurements of each investigated monolayer. The “d” sign indicates that strand xxxx is hybridized with xxxx-SH. The expected heights were calculated as follows: (i) For ss monolayers, no accurate calculation was possible, because it is very difficult to model how the single strands coil up on the surface; the range 2.9-3.1 nm is given as an approximate indication and is based on literature values; Zhou and co-workers found a height of 3.5 nm for single-stranded 20-mers.26 (ii) The expected height range for the ds monolayers was calculated in two steps: first, the height value corresponding to a tilt of exactly 45° (as reported by Sam and co-workers28) was used to calculate that for a 7.1 nm long oligonucleotide (18 bases × 0.34 nm/base ) 6.1 nm + 1 nm for the alkanethiol tether) the expected height would be 5.0 nm; then, a range of 45° ( 2.5° for the oligonucleotide inclination was chosen arbitrarily, hence giving values in the
range 4.8-5.2 nm. The measured height values were always slightly lower than these calculated values. This is probably due to the nature of the imaging technique (contact-mode AFM), which inevitably touches and thereby compresses the monolayers slightly. The height profiles do show the expected increased height profile for ds over ss monolayers. A comparison with published literature shows that the heights measured are within the range of experimental error, consistent with the formation of well-ordered SAMs of DNA on gold. Having characterized these DNA monolayers on gold, we performed a comparative study of the conductivity of modified and unmodified, single- and double-stranded monolayers using the STM. B. STM and TS Investigation. It is clear from many experiments done so far that nonconductive molecular structures can be imaged using a scanning tunneling microscope (STM). The imaging of cyanobiphenyl monomolecular layers of liquid crystals, where near-atomic details were observed, confirmed the transfer of electrons through thin, nonconductive, organic materials.29 The exponential distance dependence of the current measured in these experiments decays much more slowly than under vacuum conditions, but faster through water than through, e.g., protein structures,30 leading to a positive contrast. The mechanism behind the electron transfer through nonconductive layers of organic SAMs remains unclear. Nevertheless, there is an interaction that leads to an image contrast providing information about the molecular structure. Figure 5 shows a couple of STM images of double-stranded 8491d8491-SH monolayer. The image on the left (a) was taken before the tunneling spectroscopic (TS) current voltage (I/V) investigation. The steps of the underlying gold layer seen in this image (100 × 100 nm2) are the same as those observed in the lower-left part of image b (highlighted by a black box), which was taken after the I/V scans. The greater scan area of 500 × 500 nm2 was taken after the I/V measurements, because during the experiments, the tip drifted slightly. This procedure was carried out for every DNA SAM in order to ensure that the etched tungsten tips were in good condition before STS investigation and that neither the surface nor the tip were altered by the I/V measurement. The graphs in Figures 6-8 display the results obtained for the I/V response observed with the ss and ds monolayers. In each of these graphs, the color coding is related to the monolayer
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Figure 6. I/V curves of ss DNA monolayers (errors diminish with decreasing current).
Figure 7. I/V curves of ds DNA monolayers (comparison of singly modified strands with unmodified strands).
under study (see legend). The three colored lines represent the mean and the two extremes of the standard deviation, which was calculated on the basis of at least 100 measurements per monolayer to give it statistical meaning. For negative substrate potential (Usub), electrons are transferred from the surface to the tip. The opposite is true for positive substrate voltages. The inset displays the conditions used to position the tip at approximately the same distance above the sample before the voltage sweep. During the I/V measurement, the feedback loop is deactivated, but between the spectroscopic measurements, the feedback is engaged to keep this distance as constant as possible. The key point of these results is that they provide a comparative study between modified and unmodified DNA monolayers. No quantitative conclusions about absolute conductivities can be made from the graphs/figures below, because the immobilization strategy did not allow us to exclude ionic conduction through the hydration layer surrounding the SAM. Since, however, all slides and monolayers were prepared under identical conditions (buffer type, ionic strength, and rinsing procedure), the different
I/V characteristics observed can only be due to the chemical modifications on the DNA bases. In Figure 6, it appears that, at negative Usub, electrons tunnel more efficiently through the phenyl-modified (ss 8491-SH) monolayer than the iodo-modified (8522-SH) or the unmodified (8618-SH) monolayers. This effect is less marked in the case of positive Usub. The distinction between positive and negative Usub is particularly evident in the study of ds DNA monolayers (see Figures 7 and 8). Note: the tip bias was always kept at 0 V. Figure 7 shows the trend for immobilized 8618-SH hybridized to normal (8618), iodo (8522), and phenylethylene (8491) modified DNA strands. For positive Usub, above a threshold bias of approximately 150 mV, unmodified ds 8618d8618-SH is clearly the best monolayer for the electron transport. This may be caused by (i) the modified monolayers being more susceptible to disorder by the negatively charged tip, which possibly repels and deforms the electronegative monolayer locally, and (ii) the 8522 and 8491 SAMs behaving as molecular capacitors trapping or diffusing incoming electrons within the monolayer. For negative Usub, below a threshold bias of -150 mV, the 8491d
ET of Plurimodified DNA SMAs
Langmuir, Vol. 23, No. 15, 2007 8271
Figure 8. I/V curves of ds DNA monolayers (comparison of doubly modified strands with unmodified strands).
8618-SH monolayer conducts better than unmodified 8618, but the difference is only small. This result leads to the conclusion that the electron transfer efficiency is not greatly affected by either of the modifications on the complementary strand. The results reported in this article have been obtained by a number of repeatable experiments; the average curves shown here are qualitatively similar to all individual measurements and not the result of averaging two distinct classes of curves showing either linear conductance or a band gap. The origin of the threshold bias cannot be explained with certainty, but it is thought that the behavior of our DNA strands can be interpreted in a similar way to traditional semiconductors. Between the threshold voltages, electron conduction appears to be more difficult, and the slope resembles the conductivity of a pure water film. At the threshold bias (which may vary for different samples), the flow of electrons through the biomolecule is “favored” due to the voltage being sufficient to overcome a “barrier” analogous to that provided by the band gap in a semiconductor. By contrast, for the doubly modified DNA strands (Figure 8), a more significant difference is observed in the electron transport efficiency at negative Usub (below a threshold of -100 mV): 8491d8491-SH (red) is clearly a better conductor (outside of the standard deviation range) than unmodified DNA (yellow). In the positive Usub region (above a threshold of 150 mV), unmodified 8618d8618-SH is found to be a better conductor. This observation might be explained by the fact that the unmodified DNA monolayer, which has a smaller dipole moment than the two modified monolayers, is less susceptible to the electrostatic interaction with the STM tip. This interaction might change the molecular orientations and reduce the order of the modified monolayers and therefore impede the electron flow.35
4. Conclusion We have shown that by using a Sonogashira coupling reaction it is possible to produce a sufficient quantity of modified base analogues for multiple incorporation into single-stranded 18 base (35) Wierzbinski, E.; Arndt, J.; Hammond, W.; Slowinski, K. Langmuir 2006, 22, 2426-2429.
long oligonucleotides. Using Herne and Tarlov’s immobilization strategy, single- and double-stranded DNA monolayers were created on ultraflat template stripped gold surfaces and then studied by AFM in liquid. Tunneling spectroscopy was performed on these monolayers using a home-built STM in air at constant relative humidity, which implies that there is always a very thin layer of water covering the monolayer. Different regimes were observed for positive and negative substrate potentials. In the negative substrate potential regime, the double-stranded DNA containing phenylmodified bases was found to have higher conductivity than either unmodified DNA or iodine-modified strands. It appears that electron transport in DNA oligomers can be enhanced by a small but noticeable and defined amount when several modified bases are incorporated within DNA strands. Although in our study we do not observe a band gap of the magnitude described, e.g., by Porath,31 which might be due to remaining ions in solution, it may be possible to create synthetically a continuous electron transfer pathway if, for instance, most (if not all) DNA bases were modified with a suitable functional group. A potentially novel use for the type of SAMs proposed in this paper could be to use modified DNA monolayers as molecular capacitors rather than nanowires, because the monolayers may be modified so as to dissipate or retain electronic charge. Acknowledgment. This work was supported by the Gates Cambridge Trust, by the Department of Chemical Engineering, Selwyn College, Cambridge, and a German BMBF Nanotechnology grant. We are grateful for useful discussions on chemical matters to Dr. Matthew Gaunt and to Matthew Batchelor for his help with AFM in liquid. We also would like to acknowledge the support by EMBL visitor facilities in Heidelberg and the help given by Dr. Charles Hill and Dr. Kai Franke with the synthesis of the oligonucleotides. Our deepest gratitude for crucial help with the protecting chemistry and parts of the synthesis work goes to Dr. Steve Holmes and to Professor Mike Gait. LA063704G
