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Nanomanipulation of Individual DNA Molecules Covered by SingleLayered Reduced Graphene Oxide Sheets on a Solid Substrate Ying Wang, Yue Shen, Bin Li, Shuo Wang, Jinjin Zhang, Yi Zhang, and jun hu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05175 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 26, 2017

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The Journal of Physical Chemistry

Nanomanipulation

of

Individual

DNA

Molecules

Covered by Single-Layered Reduced Graphene Oxide Sheets on a Solid Substrate Ying Wang , Yue Shen , Bin Li , Shuo Wang , Jinjin Zhang , Yi Zhang *, Jun Hu * †

†

§

†

†

†

†

†

Key Laboratory of Interfacial Physics and Technology and Laboratory of Physical Biology,

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China. §

Key Laboratory of Salt Lake Resources Chemistry of Qinghai Province, Qinghai Institute of Salt

Lakes, Chinese Academy of Sciences, Xining, Qinghai 810008, China.

ABSTRACT Nanomanipulation of single DNA molecules has great potential in fundamental genetic research and clinical analysis, and is a good model system for studying the interfacial effects on physiochemical processes, which occur when manipulating the linear DNA molecules with an atomic force microscope (AFM) tip. Here, we demonstrate that AFM nanomanipulation can be carried out on DNA molecules covered by a single-layered reduced graphene oxide sheet. Nanomanipulation, which includes cutting, pushing, and sweeping operations, specific to the covered DNA molecules can be achieved in a well-controlled manner using AFM in the PeakForce Quantitative Nano-Mechanics mode. It was found that the normal force required to cut covered DNA strands is over five times greater than that required for naked strands. This technique provides a distinctive method for the construction of graphene architecture by tailoring the underlying artificial DNA nanostructures.

INTRODUCTION Atomic force microscopy (AFM)-based nanomanipulation of single DNA molecules has great potential in fundamental genetic research and clinical analysis, and is a good 1

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model system for studying the interfacial effects on the physiochemical processes during the manipulation of linear DNA molecules with an AFM tip. The research on the dissection of single DNA strands by an AFM tip on a solid surface can be dated back to 1996,1 which was carried out in Salmeron’s group. Later, we showed how to cut, push, and fold single DNA molecules precisely using AFM,2 with which complex artificial patterns of individual DNA molecules could be fabricated. Several research groups have demonstrated that AFM can be further developed into operative tools to isolate biological objects. For example, it was reported that AFM tips could occasionally pick up plasmid DNA from a solid surface.3 We have shown that single DNA fragments dissected from long DNA molecules at the designated positions can be further isolated by precise AFM nanomanipulation, and the isolated DNA fragments can be amplified by single-molecule polymerase chain reaction (PCR).4, 5 Recently, it has been demonstrated that graphene sheets can precisely replicate the morphology of the underlying DNA nanostructures residing on a solid substrate,6 which can not only enhance the stability of DNA nanostructures,7 but also provide a promising method for designing new graphene nanostructures through deformation induced by nano-objects.8-11 This finding inspires the imagination with respect to the manipulation of DNA molecules covered by graphene sheets. Although nanoscale pushing of spherical water nanodroplets covered by graphene has been achieved using AFM tips,12 it is still an open question to manipulate the soft and viscous DNA molecules with high precision in a designed manner without causing damage to the graphene sheet that acts as a single-atom-thick partition separating the manipulating tool and the object. Since AFM manipulation of DNA molecules may involve the breaking of covalent bonds,2 such an operation is expected to differ from the pushing of water nanodroplets around on the substrate with AFM tips. In addition, AFM nanomanipulation may result in complicated graphene-DNA hybrid nanostructures. In this paper, we demonstrate that it is feasible to nanomanipulate DNA molecules covered by single-atom-thick graphene sheets in a highly controllable manner. For this purpose, the DNA molecules were firstly stretched on a mica substrate. For better control, chemically reduced graphene oxide (CRGO) rather than mechanically 2


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exfoliated graphene was adopted to act as a protective layer for the stretched DNA molecules. It was found that the morphology of the underlying DNA patterns could be perfectly transferred to the covering monolayer CRGO sheets, and even the very small gaps caused by overstretching could be clearly resolved by AFM imaging. The threshold forces required to mechanically break the bare and monolayer CRGO sheet-covered DNA molecules were measured. On this basis, the cutting, pushing, and sweeping of the covered DNA molecules were performed successfully (Figure 1).

Figure 1. Schematic diagram indicating AFM manipulation of DNA molecules covered by single-layered CRGO sheets. (a) A drop of DNA solution was placed on a mica substrate. (b) DNA molecules were spread and adsorbed on the substrate. (c) CRGO sheets were placed on the substrate to cover the DNA molecules. (d) Manipulation of the DNA molecules under the CRGO sheet by an AFM tip.

METHODS Double-stranded (ds) Lambda (λ) DNA molecules (Sigma) were diluted with TE buffer (pH 8.0, 10 mM Tris-HCl, 1.0 mM EDTA) to a final concentration of 10 ng/µL. Before DNA deposition, a freshly cleaved mica surface was treated with 10 mM nickel nitrate for about 3 min, followed by washing with Milli-Q water (18.2 MΩ) and drying with airflow. A drop of 5-µL DNA solution was then placed onto the mica substrate. Using a modified “molecular combing” technique,13,14 linear DNA molecules were stretched and adsorbed on the surface (schematically represented in Figure 1(a) and (b)). The sample was then rinsed with Milli-Q water and blown dry with clean air. Aqueous solution of graphene oxide (GO) was prepared from graphite powder through a modified Hummer’s method.15-17 The graphite powder was first oxidized 3



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into graphite oxide by KMnO4 and H2SO4, which was then exfoliated into GO sheets by ultrasonication in water. The mixture was repeatedly washed using Milli-Q water and separated through filtration. Chemical reduction of GO was achieved by blending GO solution with hydrazine hydrate, as previously reported.18 Specifically, the diluted GO aqueous solution (0.5 mg/µL) was mixed with hydrazine hydrate (85 wt% in water, Sinopharm) with a volume ratio of 100:1 in a glass vial. After being mildly shaken for several minutes, the vial was put in a water bath (90 °C) for 1 h. Then, the as-generated CRGO sample was processed by ultrasonication in water for about 3 min. Finally, a drop of 5 µL CRGO solution was dropped onto the stretched DNA sample and blown off with clean air (Figure 1(c)). The DNA samples were characterized and manipulated by a MultiMode 8 AFM (Bruker) equipped with a J scanner. Silicon cantilevers (NSC11, MikroMasch) with a nominal force constant of ∼42 N/m and oscillating frequency of ~320 kHz were used. In order to improve the accuracy and reliability in force control, both imaging and manipulation

experiments

were

conducted

in

PeakForce

Quantitative

Nano-Mechanics (PF-QNM) mode, in which the maximum force (peak force) applied on the sample by the tip was directly regulated through the peak force setpoint and kept constant throughout the whole scan. In this mode, the peak force amplitude was set at 150 nm, the Z-piezo oscillation frequency at 1.0 kHz, and the scan rate at 1 Hz. The normal force applied by the AFM tip was calculated based on the spring constants of the AFM cantilevers provided by the manufacturer. During normal imaging, the peak force setpoint was set at a relatively small value (typically no more than 20 nN) to avoid damage to the DNA molecules by the scanning AFM tip. All experiments were conducted under ambient conditions at a room temperature of 20– 25 °C and relative humidity of 35–50%. The AFM topography images of the samples were processed using the software Nanoscope Analysis v1.7. For each image, a first-order flatten correction was applied to remove sample inclination.

RESULTS AND DISCUSSION 4


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The topography of CRGO-covered DNA molecules The λ DNA molecules covered by a single-layered CRGO sheet could be clearly recognized in AFM topography images (Figure 2(a)). Moreover, small gaps of a dozen nanometers wide on the stretched DNA molecules could also be distinguished clearly (see Supporting Information Figure S1). Obviously, the stretched ds-DNA molecules on bare mica and under the CRGO sheet appeared to be very similar, as shown in the section profiles of the DNA strands in Figure 2(b). This is consistent with previous works, in which circular plasmid DNA,6 triangular DNA origami7 and ribbon or double crossover DNA nanostructures8 could be replicated by mechanically exfoliated and chemical vapor deposition (CVD)-grown graphenes. Figure 2(c) showed the cross-sectional profile along the white dotted line in Figure 2(a). It reveals the heights of the naked and CRGO-covered DNA molecules as well as the roughness of the CRGO

Figure 2. (a) AFM topography image of stretched λ DNA molecules covered by a single-layered CRGO sheet on a mica surface. The image was recorded in PF-QNM mode at the peak force setpoint of 15 nN. (b) Cross sections of two different areas of a single DNA molecule, as shown in the white dashed box in (a); the red line represents the bare DNA molecule on mica while the black line represents the molecule covered by a CRGO sheet. (c) Cross-sectional profile along the white dotted line in (a). (d) The average heights of single-layered CRGO sheets, and naked and covered DNA molecules, respectively, obtained with PF-QNM mode imaging at a peak force setpoint of 15 nN. 5



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sheet. Statistical measurement (more than 50 data sets were collected for each data point) indicates the average heights of the single-layered CRGO sheets, and the naked and CRGO-covered ds-DNA molecules were 0.52 ± 0.05 nm, 0.60 ± 0.07 nm, and 0.48 ± 0.08 nm, respectively (Figure 2(d)). The apparent height of the DNA strands covered by the CRGO sheet is a little less than that of bare mica, indicating a slight deformation occurred after CRGO deposition. The above results prove that single-layered CRGO sheets have the ability to precisely replicate the underlying DNA nanostructures, although their roughness is a little greater than that of mechanically exfoliated and CVD-grown graphenes.6

The threshold forces for cutting DNA strands and CRGO In order to precisely control the DNA nanomanipulation process and cause no damage to both the underlying substrate and the covering CRGO sheet, naked DNA molecules, single layer GO, and CRGO sheets were cut and the threshold forces required to break them were recorded with AFM. The procedure of the cutting operation by AFM tips in PF-QNM mode is different to that which has been presented previously of AFM manipulation in tapping mode.2,19 Specifically, the cutting position was first selected in a routine imaging process under PF-QNM mode, in which the AFM tip scanned with a small peak force setpoint. Next, the slow scan axis was set to DISABLE so that the AFM tip scanned only a single line. Then the peak force setpoint was increased gradually until the contrast of the target sample disappeared, indicating the target was removed from its original position. In this way, a gap was generated in a linear DNA molecule. Finally, the peak force setpoint was returned to the value for routine imaging, and the slow scan axis was switched to ENABLE. Figures 3(a)-(e) displayed examples of cutting a naked DNA molecule, GO, CRGO and CRGO-covered DNA molecules according to the procedure mentioned above. GO and CRGO on a mica substrate could be identified by a previously reported method using scanning polarization force microscopy (SPFM) (see Figure S2). 20-22

6


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Figure 3. (a) and (b) show topography images of one naked DNA molecule before and after being cut with a peak force setpoint of 260 nN. The yellow dotted line in (a) indicates the path of the tip during the cutting operation. The yellow arrow in (b) points to the gap on the naked DNA molecule induced by tip cutting. (c) A typical cutting experiment of single-layered GO and CRGO sheets at various peak force setpoints, which were 1, 1.5, 1, 1.5, 2, 2.5, 3 and 3.5 µN marked from 1 to 8, respectively. The two GO sheets could not be broken under the peak force setpoint of 1 µN; breakage of the GO sheets occurred until the peak force increased to 1.5 µN. The CRGO sheets could be cut off until the applied force reached 3 µN. GO and CRGO can be identified by scanning polarization force microscopy (SPFM), which is introduced in Supplementary Figure S2. (d) and (e) show the topography images of two CRGO-covered DNA strands before and after being cut simultaneously. The red dotted line in (d) represents the path of the tip we preset with a peak force setpoint of 1.5 µN. The red arrow in (e) points to the gaps on the covered DNA molecules induced by tip cutting. (f) The statistical threshold forces for cutting naked ds-DNA strands, single-layered GO and CRGO sheets, and CRGO-covered DNA molecules, respectively.

By this means, the threshold forces required to break naked ds-DNA strands, single-layered GO, and CRGO sheets were measured and the statistics were obtained from 30 samples (Figure 3(f)). The mean normal force required to break naked ds-DNA in PF-QNM mode is 0.26 ± 0.04 µN, which is a little larger than that in 7



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tapping mode with a negative lift height.2, 19 For monolayer GO and CRGO sheets, the threshold forces for cutting were 1.68 ± 0.45 µN and 2.90 ± 1.34 µN, respectively. We speculated that the increase of the cutting threshold for CRGO was due to its partial recovery of the sp2 carbon network, as the C:O ratio increased from 0.96:1 to 3.3:1 (see Figure S3). This result is also consistent with previous reports, which predicted that the intrinsic strength of GO would increase monotonically with the decrease of oxygen-containing groups on its surface coverage.23 In addition, we noticed that there were relatively large fluctuations in the measured threshold cutting force for both monolayer GO and CRGO sheets. These fluctuations may be attributed to the randomly selected cutting angles with respective to the lattice orientations of the GO and CRGO sheets (see Figure S4). It was reported that the cutting force for graphene along the armchair orientation was larger than that along the zigzag orientation, and the cutting forces are almost identical every 60°.24 In our experiments, the relationship between the cutting angle and the direction of the lattice was completely random, which made the fluctuation reasonable. These threshold forces also suggest that in order to break the covered ds-DNA strands without damaging the upper CRGO sheets, a normal force larger than 0.26 µN but less than 2.9 µN should be used.

Nanomanipulation of CRGO-covered ds-DNA molecules The procedure for cutting CRGO-covered ds-DNA molecules is quite similar to that of cutting naked DNA molecules, except a higher normal force exerted by the AFM tip is required. A typical image of cutting two ds-DNA strands simultaneously under a monolayer CRGO sheet is shown in Figure 3(d) and (e). The red dotted line in Figure 3(d) represents the path of the tip we preset with a peak force setpoint of 1.5 µN. The normal force should be well controlled; if the peak force setpoint is too high, the covering CRGO sheet will be scratched when the underlying DNA strands are broken which should be avoided. The gaps between the broken DNA strands were about 40 nm in the direction perpendicular to the cutting path, with small spherical aggregations at each end. These small globules at the ends of the target DNA strands are about 0.6 nm in height and 20 nm in full width at half maximum. They are 8


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believed to be a product of the elastic retraction of the DNA strands, and were also observed previously in the breakage of stretched uncovered λ DNA molecules on a glass slide.25 No lateral dragging tail appeared at the cut ends of the covered DNA strands, which usually occurs when cutting bare DNA molecules on mica with large normal forces.19 From Figure 3 (e), it is clear that both the bottom mica substrate and the covering CRGO layer remained intact. It has been shown in Figure 3(c) that a bare mica surface would be laterally scratched when the normal force applied by the tip exceeded 1 µN. In this case, when the cutting operation was executed only on the area covered by the CRGO sheet, lateral scratching of the underlying mica substrate could be avoided under a normal force of 1.5 µN. These results suggest the flimsy sheet of carbon atoms could provide protection from mechanical damage for the underlying objects not only in the normal but also in the lateral direction. This may be attributed to low friction of the AFM tip on graphene-based materials which serve as a solid lubricant in the CRGO-DNA-mica system when suffering from a mechanical load.26 This proof-in-principle experiment confirms that manipulating DNA molecules between substrate and single-atom-thick graphene is feasible. This manipulation method is likely to provide a distinctive method for the construction of graphene architecture by tailoring the underlying artificial DNA nanostructures. The comparison study on cutting naked and covered DNA molecules was also conducted. Figure 3(a) shows the typical process of cutting a naked ds-DNA with an AFM tip whose nominal radius is 10 nm. On average, 260 nN was the threshold load to break the naked DNA strands. A normal force smaller than this value is insufficient to break a naked DNA, even over a long period of time, such as more than 100 scans, along the preset cutting path (yellow dotted line in Figure 3(a)). Once the peak force setpoint was increased to 260 nN, the naked DNA strand was broken immediately with a gap of about 10 nm (Figure 3(b)), as determined by the tip size. However, breakage of the CRGO-covered DNA strands could be observed only until the peak force setpoint was increased to about 1.5 µN (Figure 3(e)). Statistics from over 20 experiments showed that the mean threshold of the normal force to cut off CRGO-covered DNA molecules with no damage to the covering CRGO layer and 9



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underlying substrate was 1.40 ± 0.17 µN, which was much larger than that of naked DNA molecules (0.26 ± 0.04 µN) (Figure 3(f)). This value was very close to the threshold force for cutting single-layered GO sheets, which was 1.68 ± 0.45 µN on average (Figure 3(f)). In fact, when monolayer GO was used instead of CRGO, it was virtually impossible to cut off the underlying DNA molecules with no damage to the covering GO layer. It was reported that when DNA nanostructures are covered by a pristine graphene layer, the normal force required to damage them increased over an order of magnitude compared with that for unprotected nanostructures.7 Our result indicated that monolayer CRGO, which is considered to have weaker mechanical properties than pristine graphene,23,27,28 could also provide 5 times greater protection for DNA molecules from mechanical damage compared to that of unprotected molecules. Besides the threshold force required to cut DNA strands, the size of the resulting gaps on the CRGO-covered DNA strands were a little larger than that on the naked ones. Figures

4(a)

and

(b)

showed

a

representative

experiment

of

repeated

nanomanipulation of two crossed DNA strands under a monolayer CRGO sheet by one AFM tip. The four red dotted lines in Figure 4(a) indicate the path of the tip when

Figure 4. Nanomanipulation of stretched DNA molecules covered by single-layered CRGO sheets by an AFM tip in PF-QNM mode. (a) and (b) Cutting, pushing, and sweeping of two CRGO-covered DNA strands with one AFM tip. The red dotted lines, green, and blue dotted boxes in (a) represent the paths of the tip in the cutting, pushing, and sweeping operations, respectively. The red, green, and blue arrows in (b) indicate the corresponding manipulation products. All the images were recorded at a peak force setpoint of 15 nN.

10


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cutting at a peak force setpoint of 1.5 µN. The corresponding red arrows in Figure 4(b) point out the cutting results. The stretched DNA strands were cut into four smaller pieces of about 20 nm in length, with four gaps left with sizes ranging from 20 nm to 50 nm along the molecules. It was found that there were two key scanning parameters that could influence the gap size during the cutting operation, namely the peak force setpoint and the scan size. Increase of the two parameters would result in wider gaps on the DNA strands. In addition, AFM tip radius was another important factor that affected the gap size. The smaller the tip radius, the narrower the gaps we got under the same conditions. Once the tip was worn out, the gaps it cut would broaden. The smallest gap size on the CRGO-covered DNA strands after AFM cutting by silicon tips with a nominal radius of about 10 nm in dozens of experiments was about 20 nm, twice larger than that of naked strands. We think this was because larger normal forces were used for cutting CRGO-covered DNA leading to more contact area between the tip and sample, as proven in previous studies on manipulation experiments by AFM tips.29 If small tips such as diamond tips with a radius of about 2 nm is used,30,31 the gap size is expected to be smaller. In addition to cutting, pushing and sweeping operations on the CRGO-covered DNA strands could also be achieved, which have been performed on naked DNA strands in a previous report.2 Representative examples of these types of manipulation are shown in Figure 4(a) and (b). The spherical particle, marked with a green arrow in the center of Figure 4(b), is a product of a controlled pushing operation. During the pushing procedure, the tip continuously scanned an area marked by the green dotted box in Figure 4(a) (with the slow scan axis set to ENABLE) at a peak force setpoint smaller than the cutting threshold (usually 100–200 nN smaller). By this means, the tiny particle, about 0.8 nm in height and 50 nm in width, was formed by tip-induced folding of a small piece of DNA of 40 nm in length. This interesting result showed that even when encapsulated tightly by a CRGO sheet and substrate, the interlayer DNA molecules still have a certain degree of freedom. The sweeping operation is similar to pushing in procedure, but at a larger peak force setpoint (usually 200 nN larger) than the cutting threshold. A stretched DNA strand about 200 nm in length, as 11



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highlighted in the blue box in Figure 4(a), was completely removed in this manner, leaving a large blank area (marked with a blue arrow). No obvious residues were found both inside and outside of the manipulation area. Therefore, we assumed that the target DNA fragment was not pushed away but crushed down by the AFM tip. Compared with the bare DNA molecules, the CRGO-covered DNA has better structural stability against mechanical damage and washing, which may extend the limits of DNA-mediated fabrication of graphene-based devices.7 In addition, it is easier to control for AFM nanomanipulation of the CRGO-covered DNA, as the bare DNA strands on mica substrate tend to move around on the substrate during manipulation operation.

CONCLUSIONS In summary, we have shown that AFM nanomanipulation of single-layered CRGO sheet-covered DNA strands could be achieved. Cutting, pushing, and sweeping operations of CRGO-covered DNA molecules can be performed in a well-controlled manner in PF-QNM mode, without damage to both the substrate and the covering CRGO layer. It has been found that a higher normal force applied by AFM tip is required for cutting the CRGO-covered DNA molecules than for naked DNA, and the size of the resulting gap can be as low as 20 nm. Through these operations, nano-sized DNA dots and line structures could be formed under a CRGO sheet. This study expands the scope of AFM-based manipulation, and provides a diversified method to build graphene nanostructures with DNA patterns as templates.

ASSOCIATED CONTENT Supporting Information Monolayer CRGO sheet could replicate the underlying DNA nanostructure with very high precision (Figure S1). Cuts or pseudo-cuts of GO and CRGO sheets could be distinguished clearly in SPFM (Figure S2). XPS spectra of GO before and after being chemically reduced (Figure S3). Cutting along different angles in the same monolayer 12


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GO and CRGO sheet (Figure S4).

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (Y.Z.) and [email protected] (J.H.) ORCID Jun Hu: 0000-0002-7282-2316

ACKNOWLEDGMENTS This work is supported by a grant from the National Natural Science Foundation of China (NSFC Nos. 11604358, 11674344 and 11405250), and Key Research Program of Frontier Sciences, Chinese Academy of Sciences ( No. QYZDJ-SSW-SLH019).

ABBREVIATIONS SPM, scanning probe microscopy; AFM, atomic force microscopy; CRGO, chemically reduced graphene oxide; GO, graphene oxide; PF-QNM, PeakForce Quantitative Nano-Mechanics; SPFM, scanning polarization force microscopy

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Nanomanipulation of Individual DNA Molecules ... - ACS Publications

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