Letter pubs.acs.org/NanoLett
Electric-Field Dependent Conformations of Single DNA Molecules on a Model Biosensor Surface Eric A. Josephs† and Tao Ye*,‡ †
School of Engineering and ‡School of Natural Sciences, University of California, Merced, California 95343, United States S Supporting Information *
ABSTRACT: Despite the variety of nucleic acid sensors developed, we still do not have definite answers to some questions that are important to the molecular binding and, ultimately, the sensitivity and reliability of the sensors. How do the DNA probes distribute on the surface at the nanoscale? As the functionalized surfaces are highly heterogeneous, how are the conformations affected when the probe molecules interact with defects? How do DNA molecules respond to electric fields on the surface, which are applied in a variety of detection methods? With in situ electrochemical atomic force microscopy and careful tailoring of nanoscale surface interactions, we are able to observe the nanoscale conformations of individual DNA molecules on a model biosensor surface: thiolated DNA on a gold surface passivated with a hydroxyl-terminated alkanethiol self-assembled monolayer. We find that under applied electric fields, the conformations are highly sensitive to the choice of the alkanethiol molecule. Depending on the monolayer and the nature of the defects, the DNA molecules may either adopt a highly linear or a highly curved conformation. These unusual structures are difficult to observe through existing “ensemble” characterizations of nucleic acid sensors. These findings provide a step toward correlating target-binding affinity, selectivity, and kinetics to the nanoscale chemical structure of and around the probe molecules in practical nucleic acid devices. KEYWORDS: Nucleic acid sensors, DNA structures, electrochemical atomic force microscopy, scanning probe microscopy, alkanethiol self-assembled monolayers
D
can provide the end-to-end distance of tethered molecules and insights into the strength of interactions between strands of the double helix,22,23 but this method is largely insensitive to the internal conformations of the molecule. In addition, these techniques provide limited information about the structure of the local chemical environment, that is, the region of the functionalized surface that is interacting with a probe molecule. On the other hand, atomic force microscopy (AFM)24 imaging would appear to be well suited to characterize single DNA molecules on biosensor surfaces. Although single molecule AFM imaging of DNA immobilized on mica has become routine,25−27 resolving individual DNA probes on biosensor surfaces in solution has not been successful28−31 for two reasons. First, unlike mica surfaces, which electrostatically bind DNA in the presence of divalent cations,32 sensor surfaces are often passivated by functional groups to reduce the nonspecific interactions with the surface,1 leaving the tethered DNA too mobile to be clearly resolved by AFM under aqueous conditions. Second, while mica presents an ultraflat surface for imaging, biosensor and microarray surfaces often possess nanometer-scale roughness and exhibit poor control over
NA immobilized on surfaces is widely used for microarrays,1,2 biosensors,3−7 and other biotechnological platforms.8,9 Often these functional surfaces are prepared by tethering chemically modified DNA to the surface using covalent attachment or strong, noncovalent interactions.1 While limited structural information about the DNA molecules under these conditions can be inferred from macroscopic measurements of DNA monolayer thickness, orientation,10−13 or density,14,15 the nanoscale structural details of DNA on these surfaces remain lacking. Single-molecule level understandings of DNA conformations, spatial arrangement on the surface, and interactions between the DNA and the sensor surface, microscopic details that will ultimately affect target binding or catalysis,16−19 will become increasingly vital for reproducible, robust and ultrasensitive detection schemes. There are few surface characterization techniques that can directly resolve complex, nanometer-scale biomolecular structures on a sensor surface. While single molecule fluorescence provides important insight into the dynamics of conformational changes and reactions,20 direct nanometer scale structural information that can be obtained from fluorescence remains limited. Although single molecule fluorescence resonant energy transfer (FRET) can measure the relative distances between a few points within a molecule,21 it remains difficult to obtain the global structure of a biomolecule. Likewise single-molecule force spectroscopy © 2012 American Chemical Society
Received: June 30, 2012 Published: September 10, 2012 5255
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Figure 1. Schematic of tethered DNA on an electrode surface. (A) The thiol tether at one end of the DNA molecule is the same length as the passivating 6-mercaptohexanol (MCH) monolayer (i), so that DNA molecules may covalently attach to the surface at defect sites without disrupting the structure of the MCH layer. After pretreatment of the monolayer and repeatedly cycling the potential, the DNA molecules disrupt the MCH monolayer preferentially along line defects in the monolayer and lie directly on the gold surface (ii), leaving the rest of the MCH structure undisturbed. (B) Topographical image of DNA taken by tapping mode AFM in 0.5× TAE at +200 mV after cycling the potential between +200 mV and −200 mV. The rodlike protusions appear elongated and preferentially oriented at discrete 60° intervals. Larger amorphous protrusions are likely DNA molecules that have not yet inserted into the line defects. (C) Maximum observed height of each rodlike protrusion on the surface and likely model of DNA adsorption consistent with the observed height. (D) Proposed model of the DNA in MCH, lying flat on the gold surface in a line defect within a mostly ordered MCH monolayer. Segments not adsorbed within the defect are too mobile to resolve.
molecular aggregation,33,34 further impeding single-molecule characterization of these systems by AFM. To investigate the nanometer scale conformations of tethered DNAs, we chose to study a model system: a thiolated DNA tethered to a gold electrode passivated by a selfassembled monolayer (SAM) of hydroxyl terminated alkanethiols. We hypothesized that this design, widely adopted in DNA sensors,2 offers well-defined structural elements that facilitate single molecule AFM imaging of DNA at electrode interfaces. DNA assembled on electrodes has been used for a variety of nucleic acid detection schemes based on electrochemical,5,6,19,35−37 fluorescence,3,33,38 or SPR readouts.39−43 An advantage of DNA immobilization on conductive surfaces is that the charged molecules can be manipulated by applying an electrode potential. Applied electric fields have been used to aid in hybridization or melting of probe DNA44−46 and in mismatch discrimination.42,43,46,47 However, due to the complex nanoscale structures that may affect the local electric fields, the mechanisms in which DNA molecules respond to electric fields are not well understood. By using an atomically flat gold substrate and controlling the assembly conditions to prevent aggregation of DNA, we recently have been able to resolve some of the conformations of short (100 bp or 500 bp), tethered DNA molecules in aqueous solutions using AFM.48 The DNA molecules could be sufficiently immobilized by application of a positive electrode potential, enabling high resolution AFM imaging in situ. We found that the dynamics of
switching by electrostatic interactions may be significantly more complex due to the presence of defects in the background SAM.48 While we previously focused on the dynamic response of DNA to applied electric fields, here we direct our attention to the nanoscale structure of the DNA on these surfaces. We show that by fine-tuning both the charge density of the electrode surface as well as the intermolecular interactions of the passivating alkanethiol SAM, surface tethered doublestranded DNA (dsDNA) molecules can be driven into conformations that depart significantly from those commonly observed on mica: an extended conformation exhibiting anomalously large correlation lengths, as well as a tightly coiled conformation with radii of curvature as small as 10 nm. We find that while the defects in the passivating SAMs have generally been considered of little consequence, in fact, complex electric field distributions at the defect sites may dramatically affect the conformations of the DNA. The results here reveal the extent to which the chemical environment may perturb the structure of DNA molecules and provide structural insight into biosensor surfaces not easily obtained from ensemble measurements, findings that represent a step toward elucidating how the nanoscale conformations of DNA on surfaces can be correlated with device function. The DNA monolayer was prepared by first forming a mercaptohexanol (MCH) SAM on a gold electrode then exposing the surface to a solution of double-stranded DNA possessing a mercaptohexyl tether at the 5′ end of one of the 5256
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molecule insight that is relevant to the commonly used DNA sensor surfaces. After the treatment, large protrusions became resolvable by AFM, and by cycling the potential between +200 and −200 mV these features were gradually transformed into rodlike structures (Figure 1B). We have previously attributed the protrusions to loosely adsorbed DNA making intermittent contacts along its length with multiple defect sites. While before we have focused on the evolution of the DNA molecules from amorphous to rodlike shapes, here we are interested in a structural characterization of the DNA within the nanoscale chemical environment of the MCH monolayer. Using the method of ref 60, we found the lengths of the DNA on the surface (Figure 2) to range from ∼10 to 50 nm, significantly
DNA strands (a thiol tether) for several minutes (“the insertion method”). Thiolated DNAs on gold electrodes are often either coadsorbed with MCH49,50 or immobilized by first exposing the surface to the DNA then backfilling the surface with a solution of MCH.12,33,35 From ensemble measurements, the MCH was typically assumed to decouple the DNA molecules from the surface (with the exception of the thiol tether) and prevent them from adhering nonspecifically to the gold electrode. However, little is known about the nanoscale spatial distribution of the adsorbed DNA molecules. From a recent fluorescence study33 as well as our own using AFM (data not shown), surfaces assembled from the “backfilling” method are found to possess local domains of aggregated DNA that may not only impede hybridization15,51 and conformational switching11,34 but also make it difficult to resolve individual molecules with AFM. The phase separation of different molecules into different nanoscale domains is often energetically favored.52,53 The insertion method54−56 can anchor isolated DNA molecules to a SAM surface under kinetic control; by exposing a preformed MCH monolayer to the thiolated DNA, the DNA molecules chemisorb onto the surface preferentially at the SAM defects (which are, in general, separated by tens of nanometers). Because of the match in length of the thiol tether to the MCH molecules, it is assumed that the DNA molecules are able to anchor to the surface at the defects without significantly disrupting the background monolayer (Figure 1Ai). The MCH molecules surrounding a defect prevent the anchored DNA molecule from diffusing along the surface to form aggregated domains. Using the insertion method we can consistently generate surfaces with isolated and individually resolvable DNA molecules. Molecular resolution scanning tunneling microscopy (STM) studies of the MCH SAM have revealed that the SAM57 possesses a large number of defects in the crystalline monolayer structure. These defects are expected to dramatically alter the electric field distribution of the electrical double layer at the gold-solution interface and possess regions of exposed alkane chains which have hydrophobic interactions with the DNA. After exposing the MCH monolayer to the thiolated 503 bp DNA for 5 min, we pretreated the surface as described previously48 in order to resolve the DNA molecules on the surface. Briefly, while initially only small features, the tether points of the molecules, were observable, we applied a moderately positive potential (+600 mV) that immobilizes the DNA. The immobilization was assumed to occur at nanoscale defects in the SAM, which may be a consequence of the rearrangement of MCH molecules or the desorption of a small number of thiol molecules. Commonly used DNA SAMs prepared on polycrystalline gold surfaces using the backfill method possess higher densities of defects than on singlecrystal surfaces. A qualitative measure of the defects is the double layer capacitance,58 which is determined by the permeability of the monolayer to the electrolyte. We found that after applying +600 mV for up to 25 min to the single crystalline monolayer electrode, the capacitance increased to 3.1 μF/cm2 (Supporting Information), similar to that observed on DNA-MCH monolayer on polycrystalline surfaces.33,59 A drawback of the commonly used polycrystalline gold substrates is that it is difficult to achieve molecular resolution on these surfaces using scanning probe microscopy because the surface roughness may overwhelm molecular features. Using a single crystal substrate allows us to controllably introduce defects into the SAM while maintaining the ability to image single molecules. Therefore, our model system can offer single
Figure 2. DNA length and bending along backbone in MCH from Figure 1B. (A) Observed length d of adsorbed DNA molecules on the surface. (B) Mean cosine value of the angle between the first (discrete 2.5 nm length) segment of each DNA molecule (chosen to be one end at random) and a segment at distance d along backbone of molecule (solid blue). For comparison with double-stranded DNA randomly adsorbed on to a surface, dotted line is expected values for a wormlike chain with persistence length 53 nm. Error bars are the standard error of the mean (σ/√n, where n is the number of samples of length equal to or greater than d). (Inset, upper right). DNA adsorbed onto mica observed with tapping mode in air. The DNA match the expected contour length of ∼166 nm for the 503 bp DNA and appear significantly more flexible than in MCH. (Inset, lower left) Schematic illustration of how length d and angular difference θ were measured using an algorithm based on ref 60.
shorter than the expected contour length of 166 nm for a 503 bp ds DNA. The short lengths can be explained by the contrast mechanism of AFM imaging. Only segments of the DNA which are immobile over the time-scale during which the AFM tip passes over them can be observed. Therefore, the rod represents only a segment of the DNA molecule, adsorbed 5257
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Strikingly, incorporation of the DNA along the defects of the SAM results in very straight segments of DNA observed with AFM, particularly when compared to DNA adsorbed onto mica. DNA adsorbed onto mica behaves as wormlike chain (WLC) and exhibits an exponential decay of expected cosine of angular difference along its backbone with a characteristic persistence length63 (Figure 2B). In sharp contrast, the mean cosine of angular difference along the backbone of the DNA in MCH does not appear to decay with distance along the molecule. Because these molecules are embedded within a crystalline MCH monolayer, the DNA molecules are prevented from diffusing laterally on the surface, allowing for highresolution imaging. The unique surface structure, containing extended, highly linear DNA molecules embedded in a monolayer, may be useful, for example, for nucleic acid sequencing64 or protein binding studies65,66 by scanning probe microscopy. To further understand how the SAM influences DNA conformations, we used a background monolayer of 11mercaptoundecanol (C11OH), which is similar to MCH but, as a result of the five additional methylene groups, possesses significantly stronger intermolecular interactions and fewer defects in the monolayer.67 However, a mismatch between the length of the DNA’s six-carbon (C6) thiol tether and the alkane chain of C11OH is expected to affect the insertion process (Figure 4B inset). C11OH monolayers have been used as the hydroxyl-terminated background SAM in several nucleic acid sensors, even when the DNA possesses a C6 tether.68,69 A prior study using ensemble methods investigated the hybridization efficiency of single-stranded DNA (ssDNA) with a C6 tether.18 The authors found reduced hybridization efficiency for the DNA surface with a C11OH background SAM than that with the MCH background. Yet no significant disruption of the C11OH layer was observed. This was attributed to steric hindrance of the DNA by the taller background molecules; however, the mechanism has not been verified at the molecular scale. After insertion of the double-stranded DNA (dsDNA) with the C6 tether into the C11OH SAM, the morphology of the surface was revealed to be quite different from that of the MCH surface. While the C11OH SAM was mostly of high quality and crystalline, we observed large, monolayer-deep pits. Within the pits, we observed protrusions attributed to the DNA thiol tethers. While the relatively immobile tether is resolved, the rest of the molecule is lifted into the solution where it is too mobile to be observable. These defects were likely initiated during the insertion process, when a length mismatch between the DNA’s C6 tether and the surrounding C11OH disrupted the surrounding SAM structure and caused partial desorption to accommodate the large footprint of the double helix (Figure 4B). For the thiol tether to make contact with the gold, either large defects a few nanometers in diameter, which are unlikely to form during SAM growth, must be present or the DNA may need to melt close to its thiolated end to transiently form a short segment of single-stranded DNA to fit into smaller defect sites. The insertion into the defect sites may also be facilitated by the stabilization of exposed nucleobases by the exposed hydrophobic hydrocarbon chains. After applying +600 mV, the defects become occupied with donut-like structures decorating the perimeters (Figures 4A,C, and 5). The height of the features was observed to be 0.91 ± 0.31 nm above the 1.6 nm thick C11OH background SAM, consistent with DNA adsorbed directly on gold. Because the
onto the surface while the rest of the molecule is too mobile to be resolved (Figure 1D). The average topographical height of the rodlike features is 1.43 nm, which is consistent with DNA lying down directly on gold, that is, embedded in SAM defects (Figure 1Aii and 1C); however the distribution of the heights (standard deviation σ of 0.34 nm) implies that some of the DNA molecules may be adsorbed atop disordered regions of the MCH SAM. Additionally, the rodlike features preferentially align along discrete 60° orientations. We extracted an orientational order parameter61 ⟨2 cos2(3(θ1 − θ0)) − 1⟩ using the angle θ1 of the first 2.5 nm segment of each DNA molecule relative to the AFM scanning x-axis, where ⟨ ⟩ denotes the mean value of observed rodlike features, where a value of 1 indicating complete alignment at discrete 60° intervals and 0 indicating complete orientational randomness. Analysis of the rods in Figure 1B yielded an orientational order of 0.43, indicating significant angular alignment on the surface with an offset angle θ0 of 21.2°. These structural characteristics are all consistent with DNA being adsorbed at linear defects in the MCH monolayer (Figure 1D), which also occur at discrete 60° angles relative to each other.49 We used a finite element analysis to calculate the local electric fields using a modified Poisson−Boltzmann equation62 (Supporting Information) and found that they are significantly stronger above the small defect sites than above a pristine MCH monolayer far away from the defects (Figure 3A). Therefore, electrostatic interactions would
Figure 3. Calculation of potential distribution near a 2.5 nm wide defect site in (A) MCH or (B,C) C11OH using modified Poisson− Boltzmann equation.62 Each equipotential line toward the defect represents an increase (decrease) of 1 KbT/e for the different applied positive (negative) potentials; far from the surface the potential drops to 0 V. Above a defect in the SAM, the potential gradients are far greater than those above an a pristine monolayer.
draw the DNA onto the surface at positive potentials. By cycling the potential, the MCH molecules at the end of the defect site may be displaced, extending the linear defect and the DNA feature. We note that often nucleic acid sensors or microarrays lose the ability to hybridize effectively with their target when the probes are adsorbed directly onto gold.2 Hence, our results here suggest that care must be taken when cycling the applied potential in sensors with MCH SAMs. 5258
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Figure 4. Tethered DNA in C11OH monolayer. (A) Proposed schematic of DNA at positive potentials, where the DNA appears coiled around the edge of large defects (B inset) formed as a result of the mismatch between the tether length and C11OH. (B) At negative potentials, the DNA is lifted out of the defects, and only the thiol tether can be imaged. The defect sites maintain their structure. (C) Topographical image of DNA at positive potentials. The heights of the donut-like structures are consistent with DNA lying directly onto the gold surrounded by an ordered C11OH monolayer. Scale bar is 100 nm. (D) Imaging the surface left to right while scanning the applied potential from +600 to −400 mV the DNA appears lifted at around −200 mV (able to overcome adsorptive forces). Dotted line is the 0 mV line. (E) At 0 mV, the molecule remains lifted, and large defects maintain their shapes. Small spots in the middle are assigned to be the tether. (F) As the potential returns to +600 mV, the DNA readsorbs into the defect sites.
again (Figure 4E), indicating that these structures are stable and reversible. Close-ups of the donut-like conformations at positive potentials (Figure 5A) reveal a number of novel structural features that are distinct from the extended linear shapes commonly observed with dsDNA immobilized on mica surfaces. DNA deposited on mica surfaces often assume conformations that reflect either their structure in solution projected onto a surface or one in which they have equilibriated in two-dimensions;63,70 both these classes of conformations appear elongated with persistence length ρ approximately equal to 53 nm (e.g., Figure 2B inset). The donut-like features we observe sometimes have diameters approaching 10−20 nm. A careful comparison of the shape of the defect sites before and after lifting and of the donut-like structures’ topographical heights reveal that these donut-like structures are actually buckled, making intermittent contact with the gold surface and extending slightly beyond the perimeter of the defect (Figure 5). The significant curvature of the donut-like structures requires a significant bending energy E to overcome the natural stiffness of the double helix. The energy to coil a DNA molecule of length d into a circle in two dimensions can be approximated by E = kbTρθ2/(2d) = (2πρ/r)kbT for a radius r.63,71 For a radius of curvature of 10 nm (Figure 5Ai), the energy required to coil the DNA around the defect is 33 kbT. However, a highly charged surface has been observed to increase the flexibility of adsorbed DNA by helping to neutralize the charges along its backbone.70 At the extreme, DNA with a fully charge-compensated (neutral) backbone has a persistence length of 7 nm and the bending energy of the donut-like structure would drop to ∼4.4 kbT.57 From our calculations, we find that the electric fields are even more highly concentrated within a C11OH defect than within a defect in MCH, and the difference in the electric field strengths above
Figure 5. Close-up of six donut-like DNA structures from Figure 4. (A) The 50 nm closeups of the same defect sites occupied by DNA at (upper) +600 or (lower) 0 mV. (B) Three-dimensional rendered image to highlight the variations in height along the edge of the defects. Maximum height above the background monolayer is 1.2 nm, consistent with DNA lying directly on the gold in that instance.
AFM records images line by line, we could sweep the potential from +600 to −400 mV during image acquisition and the “slow scan” axis could be considered a “time” axis;48 thus we can observe the response of the DNA molecules to different potentials within the same AFM image (Figure 4D). Around −100 to −200 mV we found a sharp transition between defects with the donut-like structures and the ‘vacant’ defect sites originally observed before application of the positive potential. The DNA molecules remained lifted when the potential was raised back to 0 mV, while the defects retain contours similar to the donut-like structures (Figure 5A, and compare Figure 4C,E). Returning the potential to +600 mV resulted in the occupation of the defect sites with the donut-like structures 5259
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the defect and above a pristine monolayer is even greater (Figure 3B). These high-curvature conformations within or in close proximity to the opening of the defect can be rationalized by the positive surface charges that minimize electrostatic repulsion between the negatively charged segments within a DNA molecule as well as the interactions with gold and alkyl chains. Upon reversal of the potential, the electrostatic repulsion is no longer screened and stabilizing interactions with the gold or the alkane chain are no longer sufficient to hold the molecule in place. The pitlike defects in the C11OH SAM (Figure 5A) are markedly different from the linear defects found in MCH. Because C11OH is long and more hydrophobic, formation of linear defects in the C11OH SAM under aqueous conditions requires a higher energy cost, that is, line tension.72 Therefore, more circular defects, which can reduce the energy cost, may be favored. High-resolution imaging of DNA on model surfaces (gold with hydroxyl-terminated monolayers) using electrochemical AFM has provided novel real-space details of the conformations of the molecules and their local environment, which would be difficult to discover using ensemble studies. By changing the background SAM, the DNA on the surface could be driven to conformations that appear either significantly stiffer or more flexible than DNA adsorbed on mica. Our studies suggest that much of what is learned about the nanoscale conformations of adsorbed DNA by AFM studies on mica may not automatically translate to DNA adsorbed on biosensor surfaces. Additional studies will be needed to understand the detailed roles of heterogeneous surface chemical functionalities and electric fields. Our observation of the prominence of defects in the passivating background has intriguing implications in the design of biosensors that utilize shorter DNA molecules and interfacial electric fields. A significant limitation of the alkanethiol background in sensors that utilize electric fields is that the electric fields are significantly diminished on ordered SAMs (often by an order of magnitude or more) due to the large potential drop across the dielectric layer.73 The controlled introduction of defects in the SAM may enable sensors demanding greater sensitivities to the applied potential. The stability and reversibility of the DNA switching in the large defects in the C11OH SAM (Figure 4C−F) and their highly concentrated electric fields suggest that the C11OH SAM may in fact be more suitable for these devices than the commonly used MCH SAM. A possible concern for defect-mediated switching (lifting and adsorption) is the nonspecific interactions on the exposed gold. However, the application of a negative potential may repel nonspecifically adsorbed nucleic acids as shown in Figure 4E. Future studies using in situ electrochemical AFM will help to elucidate the role of background SAM, solution ionic strength, and assembly conditions in guiding the spatial organization and conformations of DNA on surfaces. Combining these measurements with single-molecule studies of hybridization74 or catalysis75 will ultimately allow us to correlate the performance of a biosensor with the nanoscale conformations of the DNA and SAM structure to gain a truly molecular-scale view of nucleic acid sensors and devices.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge helpful discussions with Professor Yanbao Ma. The authors acknowledge the support provided by UC Merced. E.A.J. was supported by the UC Merced Faculty Mentor Fellowship.
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ASSOCIATED CONTENT
S Supporting Information *
Materials and methods and double layer capacitance measured using cyclic voltammetry. This material is available free of charge via the Internet at http://pubs.acs.org. 5260
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dx.doi.org/10.1021/nl3024356 | Nano Lett. 2012, 12, 5255−5261
