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Direct mapping of heterogeneous surface coverage in DNA-functionalized gold surfaces with correlated electron and fluorescence microscopy Isaac Martens, Elizabeth A Fisher, and Dan Bizzotto Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03766 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 25, 2018

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Direct mapping of heterogeneous surface coverage in DNA-functionalized gold surfaces with correlated electron and fluorescence microscopy Isaac Martens,†,‡ Elizabeth A Fisher,†,‡ and Dan Bizzotto∗,†,‡ †AMPEL, University of British Columbia, Vancouver, Canada ‡Department of Chemistry E-mail: [email protected] Abstract The characterization of bio-functionalized surfaces such as alkanethiol self-assembled monolayers (SAMs) on gold modified with DNA or other biomolecules is a challenging analytical problem and access to a routine method is desirable. Despite substantial investigation from structural and mechanistic perspectives, robust and high-throughput metrology tools for SAMs remain elusive but essential for the continued development of these devices. We demonstrate that scanning electron microscopy (SEM) can provide image contrast of the molecular interface at during SAM functionalization. The highspeed, large magnification range, and ease-of-use make this widely available technique a powerful platform for measuring the structure and composition of SAM surfaces. This increased throughput allows for a better understanding of the non-ideal spatial heterogeneity characteristic of SAMs utilized in real-world conditions. SEM image contrast is characterized through the use of fluorescently labeled DNA which enables

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correlative SEM and fluorescence microscopy. This allows identification of the DNA modified regions at resolutions that approach the size of the biomolecule. The effect of electron beam irradiation dose are explored, which leads to straightforward lithographic patterning of DNA SAMs with nanometer resolution and with control over the surface coverage of specifically adsorbed DNA.

Keywords SEM, self-assembled monolayers, fluorescence, reductive desorption, DNA SAMs

Introduction Self-assembled monolayers (SAMs) are among the leading platforms for next-generation biomolecular recognition and detection. 1–6 Alkanethiols on gold remain the standard configuration for SAM-based sensors, despite well-known limitations and the recent emergence of alternative architectures. 7–9 Reproducibly and controllably functionalizing and characterizing the SAM surface remains a challenge in the ongoing commercialization of these devices. 10,11 While single component alkanethiolate SAMs exhibit well-defined, close-packed structures, the organization and reactivity of mixed or labelled thiolate films is considerably more complex, especially when tethered to biomolecules 12 . Several specialized imaging techniques have been developed for probing the local structure and heterogeneity of composite thiolate films. 10,13,14 All suffer from a limited dynamic range of spatial resolution, requiring a trade-off between field of view and imaging the structure at molecular scale. In contrast, scanning electron microscopy (SEM) benefits from an unusually large magnification range, capable of imaging features from several centimetres to 99.999%, 1 mm diameter, Alfa Aesar) as described previously. 23 Before each use, gold bead electrodes were cleaned of any surface contaminants by flame annealing. 23,24 The electrode was heated using a butane torch until the metal glowed red-hot for ~30 seconds and was then removed from the flame, allowed to cool slightly, and then rinsed with MilliQ water (>18 MΩ·cm). 3


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This flame annealing and rinsing process was repeated a minimum of three times.Clean gold bead electrodes were then immersed in a 1 mM ethanolic solution of 11-mercaptoundecanoic acid (MUA, 95%, Sigma-Aldrich) for 90 minutes to assemble a SAM. 25 The electrodes were removed from the MUA solution, rinsed with ethanol and stored immersed in fresh ethanol for no more than 8 hours prior to use.

Partial desorption of MUA SAM MUA SAMs were partially reductively desorbed from the gold bead working electrodes via the application of a negative potential. 26 Electrochemical measurements were performed with a gold counter electrode and a saturatedAg|AgCl reference electrode in 10 mM phosphate buffer, pH 8 (PB, NaH2 PO4 , 99.0% and Na2 HPO4 · 2H2 O, 99.5%, Sigma-Aldrich) made up in MilliQ water. First, the open circuit potential (OCP) was measured for 10 seconds. This potential was then applied using a potentiostat (Autolab PGSTAT12) for 15 seconds, followed by a step to the desired desorption potential (either -650, -700, or -750 mV vs. Ag|AgCl) which was held for 5 minutes. These potentials were chosen based on our previous work on similar systems. 26 This results in a MUA coverage on the Au{111} facets after the potential treatment of approximately 0.99, 0.94-0.98, and 0.61 respectively (determined using electrochemical impedance measurements as detailed in SI). 26 The bead was removed from the electrolyte while still under potential control, disconnected and then rinsed with MilliQ water.

Immobilization of Fluorophore-labelled DNA Mixed DNA/MUA SAMs were prepared using ssDNA (5’-CTG-TAT-TGA-GTT-GTA-TCGTGT-GGT-GTA-TTT-3’) modified with a thiol attached to a six carbon linker on the 5’ end and an AlexaFluor 488 fluorophore (shown in Fig S1) on the 3’ end (referred to as HSC6-DNA-AF488). This sequence was selected to avoid self-complementarity and secondary structure. The doubly-modified ssDNA was purchased as a protected disulfide (Integ4


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rated DNA Technologies, Iowa). Prior to use, the thiol was deprotected using a tris(2carboxyethyl)phosphine (TCEP) reduction procedure as described in the Supporting Information. Following the partial desorption of MUA, the gold bead electrodes were rinsed with MilliQ water and immersed in a 1 µM solution of HSC6-DNA-AF488 in immobilization buffer (0.806 mM Tris-HCl (Bioperformance, Sigma-Aldrich), 0.195 mM Tris-base (Bioperformance, Sigma-Aldrich), 100 mM NaCl (BioXtra, Sigma-Aldrich) and 50 mM MgCl2 , pH = 7.5) for 10 minutes. The gold bead electrodes were then stored in fresh immobilization buffer (IB) overnight.

Fluorescence Microscopy To visualize changes in the SAM, in-situ fluorescence microscopy imaging of the MUA/HSC6DNA-AF488 SAM-modified gold beads was performed in a argon-sparged solution of 10 mM Tris and 10 mM KNO3 (=99.0, Sigma-Aldrich), pH = 7.5, using an Olympus IX70 inverted fluorescence microscope equipped with a Photometrics Evolve 512 EM-CCD camera and an X-Cite eXacte light source following the methods described in ref 27 . All images were acquired using an Olympus LMPlanFl 5 × objective (NA = 0.13) through a Chroma filter set (450-490 nm excitation filter, 495 nm dichroic mirror, and 500-550 nm emission filter) in a specially designed spectroelectrochemical cell. 27 Prior to fluorescence imaging, the surfaces were conditioned using three electrochemical cycles (0 to -0.4 V vs. SCE using sequential -50 mV increments that are each 4 s long) to remove nonspecifically adsorbed DNA while preserving the thiol-bound fraction. 28,29

Electron Microscopy HSC6-DNA-AF488/MUA SAM functionalized gold beads were rinsed with water and dried in air before imaging. All images were collected on a FEI Helios 650 SEM at a range of landing voltages (50 V-10 kV). The best images, balancing image quality, surface contrast, and sample damage, were collected at 1 kV using a 50-100 pA probe. Dwell times of 305


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300 µs were used. An in-lens detector was used for high resolution imaging at working distances of 3-4 mm with immersion mode. The microscope chamber vacuum was better than 10-5 Pa during imaging. An internal plasma generator was utilized to clean the vacuum chamber of residual hydrocarbon contamination. This greatly reduces the deposition of carbonaceous impurities onto samples during imaging. Fresh samples were prepared for microscopy at each step of SAM assembly to avoid any effects of repeated imaging and transfers between the electrochemical cell and vacuum chamber. To minimize beam-damage artifacts, the microscope was focused and optimized on a small region of the sample, before acquiring high-quality images on an adjacent unexposed region. Multiple locations on each sample were imaged to ensure reproducibility. The Au{111} facet surfaces were oriented to be normal to the incident electron beam. Figure 1 has been gently 2D Fourier filtered to remove high frequency raster scanning noise in the horizontal direction. The raw data and filter mask are shown in SI (Fig. S2). All SEM images have been processed with linear histogram equalization to maximize contrast.

Results and Discussion MUA SAM Electrodesorption and DNA Incorporation SAMs of MUA were deposited on single crystal faceted gold beads. Thiols are reductively desorbed from gold surfaces at different electrochemical potentials according to the crystallographic orientation of the substrate. 30,31 By applying a negative potential to the surface, close to that required for SAM desorption, it is possible to partially disrupt the film, creating pores or defects. 32–34 In this experiment, a mild reductive potential selectively disturbs the SAM present on the Au{111} facets, while the other surface crystallographies remain pristine. While thiols are laterally mobile on Au surfaces at the molecular length scale, this process is kinetically slow. 35 In the absence of excess thiol in solution, defects are trapped in local energy minima, persisting for hours to days without healing. These vacant surface 6


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sites can be backfilled by subsequent exposure to a second thiol. A thiolated single stranded DNA functionalized with a fluorophore on the opposite end (HSC6-DNA-AF488) provides a convenient tag for following its incorporation into the SAM, allowing for characterization by both SEM and fluorescence microscopy techniques.

Characterizing defects in the SAM before and after DNA backfilling with SEM Bare gold electrodes containing Au{111} facets were imaged under the SEM. No features, except for adventitious dust particles were visible (Fig. S3). After annealing, the facets are flat enough that topographical contrast is not observed. 23 Following the deposition of a contiguous MUA SAM, no features are visible with the SEM (Fig. S3). Defects were then induced within the MUA SAM by electrochemically applying -650 to -750 mV vs. Ag|AgCl to the gold bead electrode. Images of these defected MUA-only layers reveal circular dark features tens of nm wide (Fig. 1) with an ill-defined image contrast. Examples of the types of defects that may be present after electrochemical treatment are shown in Figure 1c. Electron beams are well-known to lithographically desorb alkanethiolate SAMs, so fast, low-dose frames were required to minimize the beam influence. 36 Repeatedly scanning the same area of the MUA-only SAM results in local desorption and complete destruction of image contrast within two scans. An SEM image of the {111} facet after backfilling the partially exposed gold surface with thiolated DNA is shown in Figure 2. Similar features are observed as for the partially electrodesorbed MUA, but with higher contrast. Thiolated DNA probes do not densely pack onto bare gold surfaces in the relatively short backfilling time used(10 min). This limits the DNA backfilling to the most accessible locations on the surface. Nucleotide strands can adopt a variety of physisorbed conformations on the gold due to the non-specific interaction via nitrogenous bases. 37,38 Electrochemical potential cycling (0 to -0.4V) used here before analysis ensures the DNA is covalently bound to the gold surface. 29 Once DNA-labelled, 7


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Figure 1: a) Secondary electron SEM image of MUA modified Au{111} facet after partial electrochemical disruption of the film at potential of -700 mV vs Ag|AgCl. b) Higher magnification scan of the same film. c) Idealized schematics of three possible defective SAM morphologies resulting from partial electrochemical reductive desorption. These defects could be (top) holes in the ideal monolayer, (middle) a relaxation of the edge of the defect in the SAM so as to cover some of the bare gold defect, (bottom) or desorption creating regions that are of lower density of SAM, but not with distinquishable voids. The type of the defect are not easily identified in the SEM images. these bright structures were found to be more resistant to electron doses many orders of magnitude higher than required to ablate the pure alkanethiol SAM (Fig. S4). DNA is certainly damaged by electron irradiation during SEM imaging. However the products of those reactions are less volatile 39 than the alkylthiol which explains their persistence. Unambiguously interpreting these SEM images is challenging because secondary electron yield is notoriously unspecific. Topography, sample electron density, detector geometry, and surface electrostatics including work function and charging effects may simultaneously contribute to the image contrast mechanism. 40 The relatively weak signal to noise ratio from low dose imaging prevents rigorous analysis based on secondary electron yield alone. Examining the histogram of these images reveals a single broad curve, and not a bimodal distribution of bright and dark regions that would indicate simple phase segregation between the SAM and bare gold regions. (Fig. S4). Widefield fluorescence imaging was used to independently assess the DNA incorporation into the surface after partial desorption of the alkylthiol (Fig. 3). Fluorescence imaging confirms that DNA adsorption is localized almost exclusively to the Au{111} facets and surrounding terraces. 26 The absence of DNA on other crystallographic 8


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Figure 2: SEM image of the MUA SAM after backfilling electrochemically created defects with DNA. The desorption treatment potential was -700 mV vs Ag|AgCl.

Figure 3: a) Optical image of gold bead in SEM chamber, with facet oriented towards electron lens. b) SEM image showing additional signal from DNA incorporation near Au{111} facets. c) Fluorescence microscopy image of the gold bead, with DNA adsorbed selectively on the {111} facets. The desorption treatment potential was -750 mV vs Ag|AgCl.

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surfaces on the bead surface creates regions that can act as an internal standard for each SEM measurement. Any features detected away from the facets are likely due to nonspecific binding of DNA or to other contamination. As the surfaces away from Au{111} facets are mostly featureless in the SEM and fluorescently dark, we can more confidently assess that structures detected by SEM on the Au{111} facet are derived from electrochemical disruption of the SAM and subsequent DNA backfilling. DNA functionalization on the Au{111} facets increases the secondary electron yield as compared to the surrounding MUA SAM regions (Fig 3b). This internal standard approach using single crystal beads is critical for interpreting secondary electron images of SAMs, especially with highly adhesive carboxylated surfaces, such as MUA, that are likely to collect debris. Further evidence of this contrast difference due to the presence of DNA is given in Figure S5 through a comparison of low magnification SEM images of MUA modified and DNA modified gold bead electrodes. Resolving individual DNA strands on a surface under an SEM is difficult but has been reported for long genomic sequences. 41 In our system, the short length and flexibility of the DNA (~10 nm fully extended 42 ) combined with background contrast of the SAM render the individual strands incompletely resolved. Alternate SEM imaging approaches such as backscatter mode did not produce useful signal at the low accelerating voltages necessary for high surface sensitivity. DNA/MUA SAMs were incubated with several different cations (Cu2+ , Ru(NH3 )63+ , Tl+ ), but it was found that this did not noticeably alter the contrast of the film (data not shown). By driving the electrodesorption reaction with increasingly negative potentials, larger fractions of the alkanethiolate SAM is removed from the surface, increasing the number and size of defective sites. The reductive desorption of alkanethiolate SAMs follows a nucleationand-growth model. Desorption initiates at preexisting defect sites, step edges and packing vacancies within the monolayer. 43,44 Next, pin-hole defects are introduced within the wellordered regions of the SAM that grow as more of the thiol is desorbed. 31,34 The DNA used to backfill the electrodesorbed regions of the Au{111} facet are labelled

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with a fluorophore which provides a useful means of determining the amount of DNA present on the gold bead surface. Figure 4 a-c demonstrates higher loadings of the backfilled HSC6DNA-AF488 incorporated into the SAM on the Au{111} facets after application of more negative desorption potentials (NB: the intensity is logarithmic scaled). SEM images of the Au{111} facets (Fig. 4 d-f) show SAM morphology consistent with progressive stages of defect nucleation and growth. Due to changes in SEM collection efficiency, it is not possible to precisely compare absolute grayscale intensities across different samples. The fluorescence intensities are more easily compared and show a significant increase in DNA coverage with small changes in potential.

Figure 4: a-c) Fluorescence images of SAM functionalized gold bead electrodes following partial desorption via the application of -650, -700, and -750 mV vs. Ag|AgCl, respectively, and the subsequent immobilization of HSC6-DNA-AF488. The colour represents the intensity and has been represented on a log scale, as indicated by the calibration bar. d-f) Representative SEM images of the Au{111} facets of the beads shown in a-c. These SEM images are 2 µm x 2 µm. Uncropped images are provided in Fig S8.

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As before, repeated SEM imaging of the same location on the mixed MUA/DNA SAMs resulted in reduction in the observed contrast (shown in Fig. S6). The effect was most dramatic for SAMs containing the smallest amount of DNA. We hypothesized that the change in contrast was due to beam-induced removal of the alkanethiol component, which would be desorbed into the vacuum chamber much more easily than the thiolated DNA. We also noted that the electron beam was effective at bleaching the fluorophore 45 , and fluorescence imaging performed after electron microscopy shows dark rectangular scars at the imaged locations (Fig. S7). Collecting a sequential series of SEM images of the DNA-MUA SAM is useful in delineating the nature of the contrast observed (Fig S9 and S10). A large intensity is measured from a region of the DNA-MUA SAM that was not previously imaged. With repeated imaging (and therefore increased dose/exposure), the contrast decreases reaching a constant value but still revealing structure on the surface. During SEM imaging, the MUA component of the SAM was found to be removed after only a few SEM images thereby leaving the DNA SAM behind. The SEM induced changes to the surface composition conveniently enables locating regions where the DNA SAM is located. Moreover, these measurements also indicate that the intense regions in the SEM images correlate to an increased amount of DNA in that region of the SAM. Based on these control experiments, the dark regions in the SEM image correspond to locations where little DNA was incorporated due to the small amount of MUA that was reductively removed from these areas. This provides a convenient high resolution method for characterizing the mixed DNA - alkylthiolate SAMs which are typically prepared for biosensing applications. Furthermore, the SEM beam induced removal of the MUA alkylthiolate monolayer can be used for creating DNA SAMs at specific regions on the gold surface.

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Nanopatterning DNA SAMs Further experiments were conducted to better understand beam damage, and the nature of the secondary electron contrast generating mechanism for pure alkanethiolate and mixed alkanethiolate/DNA SAMs. The microscope’s electron beam was used to write defects within a pristine MUA SAM instead of via electrochemical desorption. These defects were visualized following the approach outlined above using fluorophore labeled DNA. Patterning surfaces with DNA SAMs has been previously accomplished using both photolithography 46 and electron beam methods 22 . Layers produced with these methods have been characterized by AFM and UHV techniques but not evaluated as functional biomolecular surfaces. Our work relied on the use of alkanethiolate SAMs as resist films in electron beam lithography (EBL). 47–51 The dose-response that is achieved is specific to the SAM irradiated, substrate 52 , and various microscope settings. Our high resolution imaging (>500,000× magnification) results shown above required exposing samples to drastically higher incident electron doses per unit area than typically encountered in EBL applications. However, the vast majority of the electron beam’s kinetic energy is deposited deep into the gold substrate, so care should be taken when comparing the conventional dose units of µC/cm2 or electrons/cm3 between different systems. The MUA SAM on the Au{111} facet was treated with a range of electron doses (1104 µC/cm2 ) and an SEM image was collected over the different dosed regions. The average secondary electron yield was determined at each dose (Fig 5). The secondary electron yield of the MUA SAM initially increased with dose, at intermediate electron doses (>200 µC/cm2 ), the yield of the MUA-only film saturates and then decreases. The MUA SAM surface after exposure was then backfilled with HSC6-DNA-AF488 under the same conditions described above. The extent of DNA incorporation into the defects created by the electron beam was determined via fluorescence microscopy and the average fluorescence intensity is compared to the SEM signal as shown in Figure 5. Fluorescence microscopy of the DNA-functionalized surface shows the same trend as the 13


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Figure 5: a) Secondary electron yield of MUA SAM after exposure to a series of electron beam doses (black) and fluorescence intensity after backfilling the same regions with DNA. b) SEM image of the MUA SAM following exposure to electron beam of various doses. c) Fluorescence image of the same electron beam treated spot following backfilling with HSC6DNA-AF488. Uncropped versions of b &c are given in Figure S11. SEM image, directly linking the secondary electron yield to the availability of surface vacancies or MUA free defects in the SAM. At low lithographically convenient (1000

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µC/cm2 ) prevents a similar dose-response imaging strategy. After DNA backfilling, the surface was re-imaged with SEM. Little contrast was observed between the different regions due to the dominant effect DNA has on the secondary electron yield, essentially preventing observation of the MUA regions. High resolution images show mottled features on the surface similar to those created from electrochemical partial desorption (Fig S9). We estimate the lithography resolution at approximately 25 nm, although no attempt at optimization was made, and feature sizes sub-10 nm are routine for EBL. 51 It has been shown previously that tuning the electron dose to a resist can control the surface concentration of adsorbing proteins. 45,54 Here, the secondary electron contrast of the dosed SAM provides in-situ feedback regarding the defect density of the surface, before the sample leaves the clean vacuum environment. This control could be useful in the preparation of more complex SAM-based photoresists, or as a simple quality check on the lithography process. Further confirmation of the suitability of these patterned regions for biosensing was realized by studying the surfaces with in-situ electrochemical fluorescence microscopy. A common issue in assembling biomolecular films, especially with patterned formats, is undesirable nonspecific adsorption of the probe to the interface. 29,55 Fluorescence microscopy in conjunction with electrochemical potential cycling was performed to confirm the DNA backfilled into the SAM was specifically adsorbed (thiol-conjugated) and not merely physisorbed to debris at the interface. This was accomplished by immersing the surface into buffer and repeatedly cycling the potential over a range where the thiols are stably bound to Au surfaces (-0.4V to +0.3V vs Ag|AgCl), but which has been shown to dislodge non-specifically bound DNA. 29 The EBL sample exhibited only minimal change in fluorescence before and after this cycling, indicating very little nonspecific adsorption of the DNA, similar to the electrochemically prepared SAMs. The fluorescence signal was also monitored as the tethered DNA was electrostatically repelled and attracted to the metal electrode surface using the potential applied to the surface. 28,56 The metal electrode acts to quench the fluorescence when the fluorophore is near the surface. 27 Modulation of the fluorescence intensity during potential cycling in-

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dicates that the EBL-patterned DNA strands are spaced far enough apart from each other so that they are able to change orientation. 57 This assay for DNA SAM packing density indicates the strands are separated sufficiently to be available for efficient hybridization. 58 The surface density or packing of DNA is an important aspect that must be controlled and optimized for successful recognition of targets in solution. 38,59,60 This EBL approach enables a careful control over the patterning and density of the DNA SAM enabling the construction of well defined biosensor surfaces.

Conclusion We demonstrate for the first time that defects due to partial electrodesorption in alkanethiolate SAMs can be detected directly using SEM, requiring no sample preparation or specialized hardware. The imaging of DNA-functionalized interfaces with SEM is a simple, low cost, widely available procedure, at least an order of magnitude faster than AFM/STM, and likely extendable to proteins and other biomolecules of interest. This permits the characterization of surfaces over comparatively large areas, conceivably measuring whole biosensor devices. Further work using conventional gold sputtered films instead of single crystal surfaces is required before this imaging strategy can be broadly applied to mapping the SAM interface. In addition to imaging SAM microstructure, electron microscopy is used to lithographically pattern thiolated DNA on gold in a single, maskless step with tuneable probe density. We anticipate the ease and accessibility of this technique will facilitate the characterization, fabrication, and optimization of biofunctionalized interfaces.

Acknowledgement This research was supported by funds from the NSERC Discovery and Equipment grants. IM was also supported by NSERC Doctoral scholarship (CGSD). The SEM imaging was performed in the Centre for High-Throughput Phenogenomics at the University of British 16


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Columbia, a facility supported by the Canada Foundation for Innovation, British Columbia Knowledge Development Foundation, and the UBC Faculty of Dentistry and training support from Prof. D.P. Wilkinson.

Supporting Information Available Preparation of DNA deposition solution; SEM analysis of MUA SAMs; SEM and Fluorescence images of DNA/MUA SAMs after potential treatments; fluorescence images of DNA/MUA SAMs after SEM imaging; raw uncropped SEM images used in Figure 4; analysis of the secondary electron yield during serial exposures for SEM imaging; raw SEM and fluorescence images used in Figure 5.

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(6) Odenthal, K. J.; Gooding, J. J. An introduction to electrochemical DNA biosensors. The Analyst 2007, 132, 603–610. (7) Crudden, C. M.; Horton, J. H.; Ebralidze, I. I.; Zenkina, O. V.; McLean, A. B.; Drevniok, B.; She, Z.; Kraatz, H.-B.; Mosey, N. J.; Seki, T.; Keske, E. C.; Leake, J. D.; Rousina-Webb, A.; Wu, G. Ultra stable self-assembled monolayers of N-heterocyclic carbenes on gold. 2014, 6, 409–414. (8) Liu, G.; Böcking, T.; Gooding, J. J. Diazonium salts: Stable monolayers on gold electrodes for sensing applications. Journal of Electroanalytical Chemistry 2007, 600, 335– 344. (9) Chaki, N. K.; Vijayamohanan, K. Self-assembled monolayers as a tunable platform for biosensor applications. Biosensors and Bioelectronics 2002, 17, 1–12. (10) Josephs, E. A.; Ye, T. Electric-field dependent conformations of single DNA molecules on a model biosensor surface. Nano Letters 2012, 12, 5255–5261. (11) Huang, E.; Zhou, F.; Deng, L. Studies of Surface Coverage and Orientation of DNA Molecules Immobilized onto Preformed Alkanethiol Self-Assembled Monolayers. Langmuir 2000, 16, 3272–3280. (12) Smith, R. K.; Lewis, P. A.; Weiss, P. S. Patterning self-assembled monolayers. Progress in Surface Science 2004, 75, 1–68. (13) Murphy, J. N.; Cheng, A. K. H.; Yu, H. Z.; Bizzotto, D. On the Nature of DNA SelfAssembled Monolayers on Au : Measuring Surface Heterogeneity with Electrochemical in Situ Fluorescence Microscopy. Journal of the American Chemical Society 2009, 131, 4042–4050. (14) Grubb, M.; Wackerbarth, H.; Ulstrup, J. Identification of Single-Stranded DNA by In

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