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Mechanism of DNA Adsorption and Desorption on Graphene Oxide Joon Soo Park, Nam-In Goo, and Dong-Eun Kim Langmuir, Just Accepted Manuscript • DOI: 10.1021/la503401d • Publication Date (Web): 06 Oct 2014 Downloaded from http://pubs.acs.org on October 8, 2014

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Mechanism of DNA Adsorption and Desorption on Graphene Oxide Joon Soo Park, Nam-In Goo, and Dong-Eun Kim* Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.

KEYWORDS: Graphene oxide, Fluorescent DNA, Desorption, Base complementarity.

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ABSTRACT Graphene oxide (GO) adsorbing a fluorophore-labeled single-stranded (ss) DNA serves as a sensor system, because subsequent desorption of the adsorbed probe DNA from GO in the presence of complementary target DNA enhances the fluorescence. In this study, we investigated the interaction of single- and double-stranded (ds) DNAs with GO by using a fluorescently labeled DNA probe. Although GO is known to preferentially interact with ssDNA, we found that dsDNA can also be adsorbed on GO, albeit with lower affinity. Furthermore, the status of ssDNA or dsDNA previously adsorbed on the GO surface was investigated by adding complementary or noncomplementary DNA (cDNA or non-cDNA) to the adsorption complex. We observed that hybridization occurred between the cDNA and the probe DNA on the GO surface. Based on the kinetics driven by the incoming additional DNA, we propose a mechanism for the desorption of the preadsorbed probe DNA from the GO surface: the desorption of the GO-adsorbed DNA was facilitated following its hybridization with cDNA on the GO surface; when the GO surface was almost saturated with the adsorbed DNA, nonspecific desorption dominated the process through a simple displacement of the GO-adsorbed DNA molecules by the incoming DNA molecules because of the law of mass action. Our results can be applied to design appropriate DNA probes and to choose proper GO concentrations for experimental setups to improve specific signaling in many biosensor systems based on the GO platform.

INTRODUCTION Carbon-based nanomaterials, including carbon nanofibers and carbon nanotubes, have recently attracted immense interest as biosensing materials.1-3 Graphene—a flat, two-dimensional, singlelayered, honeycomb-shaped carbon nanomaterial—is one of the most remarkable materials because of its peculiar physical properties such as good thermal stability, high electronic conductivity, and excellent mechanical strength.4,5 Graphene has been well studied and widely


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applied in chemical and biological sensing systems.6 It has been reported that graphene interacts with DNA nucleobases through van der Waals interaction.7 Despite these characteristics, graphene is usually insoluble in water because of the strong hydrophobicity on the large surface area of a carbon layer. Graphene oxide (GO) is a water-soluble material that is prepared by the oxidation of graphite using Hummers method.8 During oxidation, large quantities of oxygen-containing functional groups such as epoxy, hydroxyl, and carboxyl groups are incorporated on the surface of GO.9,10 GO is known to preferentially interact with single-stranded (ss) nucleic acids through hydrogen bonding and because of pi-stacking interactions between nucleobases and the GO surface,11,12 while it has a lower affinity for double-stranded (ds) nucleic acids.13-15 It has been reported that adsorbed nucleic acids are effectively protected from enzymatic digestion by nucleases.16-17 The long-range nanoscale energy-transfer in the GO pi-system allows GO to quench the fluorescence near the GO surface.18 Moreover, it has been demonstrated that nonfunctionalized GO inhibits enzyme activity by interacting with proteins via hydrophobic interactions.19 On the basis of these properties, GO has been employed in DNA or protein sensing,20,21 cell imaging,22 and enzyme assays.23,24 Although practical applications of GO in biochemical analysis have been successfully demonstrated, the interaction between nucleic acids and GO has not been fully investigated. Previous reports show the adsorption of ssDNA or single nucleotides/nucleosides on graphite, examined by atomic force microscopy (AFM)25 and theoretical calculations.26,27 The adsorption and desorption of ssDNA on GO have also been examined as functions of DNA length, pH, and salt.28 In addition, the length-dependent fluorescence signal of DNA on the GO surface was analyzed by conjugating DNA to a GO sheet.29 Recently, a remarkable mechanism on desorption


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of fluorophore-labeled ssDNA probe has been proposed, in which the GO-preadsorbed probe DNA is nonspecifically displaced by incoming DNAs.30 Thus, DNA-induced DNA desorption from GO surface is an important issue to be verified to account for detailed mechanism on DNA adsorption and desorption on GO. In this study, we investigated the adsorption and desorption of several types of ssDNAs or dsDNAs on a GO surface by monitoring the changes in fluorescence of the probe ssDNA. Desorption of ssDNA from the GO surface was investigated using either complementary (c) or noncomplementary (non-c) ssDNA to compare the desorption efficiency of base-paired duplex DNA with that of simple nonspecific displacement of adsorbed ssDNA from the GO surface. As dsDNA is also known to bind to GO albeit with a lower affinity than ssDNA,13-15 we further investigated whether the base pairing of dsDNA is retained when it is adsorbed on the GO surface. In addition, we analyzed the dissociation kinetics of ssDNA adsorbed on GO surface by adding complementary or noncomplementary strands. Our mechanistic study regarding binding and desorption of ssDNA or dsDNA on the GO surface can help us understand the basic principles underlying the interactions between GO and nucleic acids.

RESULTS AND DISCUSSION DNA adsorption on GO with fluorescence quenching. To evaluate the uniform properties of GO, the GO used in this study was imaged by an AFM after it was loaded on a freshly cleaved mica surface. The height profile of the GO sheet obtained in three repeated experiments yielded an average thickness of 1.377 nm (Figure 1), which shows that the GO sheet was monolayered and existed as an exfoliated, single-layered structure in the solution. The size of the GO sheet varied from nanometers to micrometers. To compare the binding affinity of ssDNA to the GO


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surface in the presence of monovalent and divalent ions, the fluorescence quenching of FITClabeled ssDNA (F-ssDNA, 1) was examined by reacting it with increasing amounts of GO in each buffer (25 mM TrisHCl, pH 6.8) containing 25 mM of metal cations (NaCl, KCl, MgCl2, or CaCl2). When 0.1 µM of F-ssDNA was mixed with increasing concentrations of GO, the FITC fluorescence was gradually quenched and reached the saturation value of fluorescence quenching (Figure S1A). The values of the apparent dissociation constant (Kd) reflecting the affinity of the metal cations for GO, which were obtained by fitting the fluorescence quenching profile to a hyperbolic equation, were determined to be 13.5, 5.2, 1.3, and 0.6 µg/mL of GO for Na+, K+, Mg2+, and Ca2+, respectively. These values show that as previously reported, the divalent ions were more effective in fluorescence quenching, with higher affinity for binding to GO than the monovalent ions.28 Next, the adsorption kinetics of F-ssDNA (25 nM) on GO (2.5 µg/mL) were investigated in the presence of 25 mM of each metal ion (Na+ or Mg2+). The kinetic traces of decrease in fluorescence were monitored and the signal to background (S/B) ratios were obtained using the equation ((F - Fmin)/(Fmax - Fmin)), where F, Fmax, and Fmin are the fluorescence intensity at a certain point in time, the maximum fluorescence intensity, and the minimum fluorescence intensity, respectively. As shown in Figure S1B, fluorescence quenching occurred within 10 s in the presence of Mg2+, while it took about 10 min for complete binding of F-ssDNA to GO in the presence of Na+. These results indicate that the divalent metal ion facilitates ssDNA binding to the GO surface, leading to enhanced kinetics of ssDNA binding to the GO surface. Our result is consistent with a previous observation that higher concentration of Mg2+ enhances DNA adsorption kinetics by lowering electrostatic energy barrier between negatively charged DNA and GO surface.30


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To examine the effect of increasing length of the ssDNA portion in a partial duplex DNA on the binding affinity to the GO surface, we analyzed the binding affinities of four duplex DNAs, with each containing a different length of the single-stranded region (Table 1). The fluorescence intensities of the reaction mixtures were measured when 0.1 µM of each duplex DNA was mixed with increasing amounts of GO, and the relative fluorescence quenching was fitted to the hyperbolic equation. The partial duplex DNA with a longer single-stranded region was preferentially bound to GO with a higher binding affinity in the buffer containing divalent metal ions (Figure 2A), whereas the binding affinities of all duplex DNAs were observed to be similar regardless of the ssDNA portion in the buffer containing monovalent metal ions (Figure 2B). The apparent Kd value was estimated to be 1.9, 3.0, 4.9, and 6.2 µg/mL of GO for the dsDNA with an ssDNA tail of 30, 20, 10, and 0 (in the presence of Mg2+) nucleotides, respectively (inset in Figure 2A). Thus, the single-stranded region in the duplex DNA was positively correlated with the binding affinity of DNA to the GO surface. However, in the presence of monovalent cations (i.e., Na+) in the buffer, the Kd value of each DNA was not distinguishable, indicating that divalent metal ions were required to distinguish the different affinities of the single-stranded region in the duplex DNA. This suggests that the divalent metal ion (i.e., Mg2+) is more effective than the monovalent metal ion (i.e., Na+) in binding ssDNA to the GO surface, showing a correlation between the ssDNA length in dsDNA and the binding affinity. The facilitation of DNA binding in the presence of Mg2+ was likely due to the ability of a divalent ion to act as a bridging cation between the phosphodiester backbone in DNA and carbonyl oxygens on the GO surface, while the monovalent ion simply shielded the negative charge of the DNA phosphodiester backbone and reduced the charge repulsion between the GO surface and DNA.28,31 At high concentrations of GO, the fluorescence of all DNAs was observed to be


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quenched to saturation, indicating complete adsorption of the DNAs on the GO surface. This result suggests that a binding equilibrium existed between GO and the nucleic acid, as observed in a previous report.30 To further confirm that the single-stranded region facilitated adsorption of duplex DNAs on GO, we analyzed the binding affinities of dsDNAs containing identical duplex regions without a ssDNA tail (i.e., a full duplex DNA (1+6)) and with a ssDNA tail (i.e., a partial duplex DNA (1+2)). Because dsDNAs with different single-stranded portions were not distinguishable in the presence of monovalent metal ions (Figure 2B), a comparison of the binding affinities between duplex DNAs (full duplex or partial duplex) and GO was performed in the presence of divalent metal ions (Mg2+). The dsDNA with a single-stranded region showed a higher binding affinity than the dsDNA without a ssDNA tail (Figure 3). The apparent Kd values for GO toward full duplex and partial duplex DNAs (0.1 µM) were 6.4 and 2.0 µg/mL of GO, respectively, which show that the presence of a single-stranded region further increased the binding affinity between the duplex DNA and GO.

Desorption of preadsorbed single-stranded DNA from GO. To examine the status of ssDNA adsorbed on GO, F-ssDNA (1) was first mixed with GO in the buffer containing 25 mM Tris-HCl, pH 6.8 and 25 mM MgCl2 (MgCl2 buffer), followed by the addition of 20-mer ssDNA (complementary DNA 6 or noncomplementary DNA 4, Figure 4). When 0.1 µM of F-ssDNA was preadsorbed on 3 µg/mL of GO, the addition of noncomplementary ssDNA (non-c-ssDNA) and complementary ssDNA (c-ssDNA) both led to an increase in fluorescence (Figure 4A). It is likely that GO-adsorbed F-ssDNA was displaced by the incoming DNA regardless of its complementarity. At higher concentration of GO (10 µg/mL), however, fluorescence recovery


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was observed only for c-ssDNA (6) and it was not observed for non-c-ssDNA (4) (Figure 4B). These results suggest that an ample amount of GO allowed non-c-ssDNA to be adsorbed on the GO surface rather than letting it displace the GO-adsorbed F-ssDNA, resulting in minimal release of F-ssDNA from GO. In contrast, the complementary ssDNA molecules encountering the preadsorbed F-ssDNA molecules were readily facilitating the desorption of F-ssDNA from GO by forming duplex DNA molecules. Next, we tested the hypothesis that F-ssDNA preadsorbed on GO was simply displaced by incoming ssDNA regardless of its sequence complementarity at low GO concentration. Depending on the affinity of incoming DNA strand to the GO surface, desorption pattern by strand displacement would be changed. To verify this hypothesis, the incoming ssDNA molecules were replaced with partial dsDNA molecules containing a ssDNA tail to reduce their binding affinity to the GO surface; the partial dsDNA with a tail (20-mer) of either complementary (2+5) or noncomplementary (2'+5) sequence to F-ssDNA was used to hybridize F-ssDNA that was preadsorbed on the GO complex. After 0.1 µM of F-ssDNA was adsorbed on GO, an increasing concentration of partial dsDNA was added to the mixture of F-ssDNA–GO complex. At a GO concentration of 10 µg/mL, an increase in fluorescence was observed only when complementary dsDNA (2+5) was added (Figure 5A), which is consistent with the result shown in Figure 4B. However, when a smaller amount of GO (3 µg/mL) was used, an increase in fluorescence resulting from the release of F-ssDNA was observed in both mixtures with added cpdsDNA (partial dsDNA with complementary ssDNA tail, 2+5) and non-c-pdsDNA (partial dsDNA with noncomplementary ssDNA tail, 2′′+5) (Figure 5B). It is important to note that different increases in fluorescence were observed for the addition of complementary and noncomplementary partial dsDNAs (c-pdsDNA and non-c-pdsDNA), which was not observed


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for the addition of complementary and noncomplementary fully single-stranded DNAs (c-ssDNA and non-c-ssDNA, see Figure 4A). Such difference was likely due to the lower binding affinity of partial duplex DNA (pdsDNA) than that of ssDNA to GO, and the displacement of F-ssDNA by the incoming partial dsDNA was less favorable than that by the full ssDNA. Based on these results, in order to minimize nonspecific adsorption of the probe DNA on the GO surface, we propose that a partial double-stranded DNA harboring a single-stranded DNA tail that is complementary to the target sequence, rather than fully single-stranded DNA, is more appropriate as the probe DNA for sensing target DNAs adsorbed on GO.

Desorption of preadsorbed double-stranded DNA from GO. The status of FITC-dsDNA (F-dsDNA, 1+6) on GO surface was investigated by adding single-stranded cDNA and noncDNA (c-ssDNA, 6 and non-c-ssDNA, 4) to F-dsDNA preadsorbed on GO. When 0.1 µM of FdsDNA was incubated with 3 µg/mL of GO, unlike the preadsorption of F-ssDNA on GO, the fluorescence of dsDNA was not significantly quenched, representing a weak interaction between dsDNA and GO (Figure 6A). As the apparent dissociation constant was estimated to be 6.2 µg/mL for 0.1 µM of dsDNA in a buffer containing 25 mM Mg2+ (inset in Figure 2A), 10 µg/mL of GO was used for effective binding of F-dsDNA (0.1 µM) to the GO mesh (Figure 6A). If FdsDNA existed in duplex form on the GO surface by maintaining base-pairing, hybridization by the incoming DNA would not occur and would result in indistinguishable change in fluorescence regardless of the incoming DNA’s complementarity. At a GO concentration of 10 µg/mL, no increase in fluorescence was detected when non-c-ssDNA was added, whereas the addition of cssDNA enhanced the fluorescence of dsDNA (Figure 6B). These results suggest that the base complementarity in F-dsDNA preadsorbed on the GO surface was disrupted to some extent as


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the incoming c-ssDNA encountered. The increase in fluorescence was due to the base recognition between the added c-ssDNA and the GO-adsorbed F-dsDNA, suggesting that the bases in the preadsorbed F-dsDNA were exposed and ready to be desorbed when the incoming cssDNA strand formed hydrogen bonds with the exposed bases in the GO-adsorbed F-dsDNA strand. On the other hand, at a lower concentration of GO (7 µg/mL), an increase in fluorescence was also detected upon the addition of non-c-ssDNA (Figure 6C), possibly because it displaced a strand of the GO-preadsorbed F-dsDNA. The simple DNA displacement at a low concentration of GO is consistent with the earlier results shown in Figures 4A and 5B. Therefore, when ample space on the GO surface was available, the preadsorbed F-dsDNA on GO was readily desorbed owing to the c-ssDNA, while the non-c-ssDNA was adsorbed on the GO surface without desorption of the preadsorbed F-dsDNAs. Thus, it is conceivable that DNA hybridization occurred on the GO surface without inhibiting the base recognition between the preadsorbed FdsDNA and the incoming ssDNA.

Mechanism of DNA-induced DNA desorption. To further elucidate the desorption mechanism of fluorescent ssDNA that was preadsorbed on the GO surface, we monitored the kinetics, driven by incoming ssDNA (both c-ssDNA and non-c-ssDNA), of the desorption of preadsorbed F-ssDNA from GO. After 50 nM of F-ssDNA, 1 was first adsorbed on 2.5 µg/mL of GO in 1 mL reaction volume, increasing amounts of ssDNA (10–500 nM of c-ssDNA, 6 or nonc-ssDNA, 4) were added to the solution of the probe DNA adsorbed on GO. From the moment of initial ssDNA addition, the fluorescence intensity was measured every 500 ms, and increases in the time-dependent fluorescence resulting from the desorption of the probe DNA were observed,


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as shown by kinetic traces in Figure 7A and B. These increases resulting from the addition of both c-ssDNA and non-c-ssDNA did not fit well with a single exponential function, (y = a[1 exp(-bt)]), with both curves deviating from the calculated plot of y (dashed lines in Figure 7A and B). However, all graphs were perfectly fitted by a sum of two exponential functions, y = a[1 - exp(-bt)] + c[1 - exp(-dt)], where a and c represent the amplitudes of kinetic phases with different rates b and d, respectively (solid lines in Figure 7A and B). The fit of the kinetic traces to the sum of two exponential functions suggests that two kinetically discernible phases were present in the desorption of F-ssDNA from GO, although the molecular details of these two different kinetic phases of the fluorescence increase are yet to be determined. When c-ssDNA was added to the F-ssDNA preadsorbed on the GO surface, the increase in fluorescence followed two different sets of exponential kinetics: one with a fast rate and another with a rate that was about 10-times slower (b and d in Figure 7C, respectively). The rates of the two kinetic phases increased gradually as the amount of c-ssDNA increased, while the rates did not increase with the addition of non-c-ssDNA. Thus, the desorption of the probe DNA was dependent on the concentration of the incoming c-ssDNA, which suggests that the molecular encounter between the probe DNA (F-ssDNA) and incoming c-ssDNA on the GO surface resulted in hybridization of these two DNAs. The amplitudes of the fast and slow kinetic phases were obtained using the two-term exponential function (a and c in Figure 7D, respectively) for the addition of both c-ssDNA and non-c-ssDNA. Hyperbolic, concentration-dependent increase in the amplitudes was observed when c-ssDNA was added to the F-ssDNA–GO complex, whereas sigmoid curves with an initial lag phase (similar to the lag phase seen in sigmoid growth curves of bacteria and plants) were observed for the addition of non-c-ssDNA (Figure 7D). This lag phase was likely caused by the displacement of the probe DNA at high concentration of non-


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c-ssDNA, accompanied by an increase in fluorescence. This nonspecific displacement of the preadsorbed probe DNA is consistent with the result of the desorption of probe DNA at low GO concentration (Figure 4A). At low concentration of the incoming non-c-ssDNA (0–100 nM in Figure 7D), the molecules had higher chance to be adsorbed on GO than to displace the FssDNA molecules on the GO surface. The lag phase was not observed, however, when 0 to 100 nM of c-ssDNA was added to the F-ssDNA–GO complex, suggesting that most of incoming non-c-ssDNA molecules were adsorbed on the GO surface, with little desorption of the probe DNA. Although the incoming non-c-ssDNA encountered the preadsorbed F-ssDNA, hybridization would not have occurred because of the unmatched bases between these two DNAs. These findings of kinetic behavior, together with the dsDNA desorption profiles obtained at different GO concentrations (Figure 6), can be interpreted in terms of desorption facilitated by hybridization and nonspecific desorption by simple displacement, as shown in Scheme 1. When the incoming ssDNA was complementary to the probe DNA adsorbed on the GO surface, despite most of the molecules being adsorbed by GO, a small portion encountered the preadsorbed probe DNA molecules and formed duplex DNA on the GO surface, which drove the desorption of GOadsorbed F-ssDNA (Scheme 1A). Thus, hybridization between the complementary DNAs on the GO surface was followed by the desorption of duplex DNA containing the probe DNA. However, when non-c-ssDNA was added to the probe ssDNA preadsorbed on GO surface, most of the incoming non-c-ssDNA molecules were adsorbed on GO, including those that encountered the probe DNA molecules without hybridization (Scheme 1B). When more non-c-ssDNA was added to the GO-adsorbed F-ssDNA such that the molecules exceeded the adsorption capacity of the GO surface, nonspecific and DNA-sequence-independent displacement of the GO-adsorbed FssDNA occurred with fluorescence enhancement (Scheme 1B).


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Our proposed mechanism of DNA-induced DNA desorption from GO surface entails nonspecific displacement of the preadsorbed probe DNA from GO surface, which is consistent with the recently proposed mechanism of DNA sensing on GO.30 In this mechanism, nonspecific displacement of the preadsorbed DNA occurs by incoming DNA and the desorbed DNA would be hybridized with cDNA in the solution phase to prevent its readsorption.30 Consistent with this mechanism, we observed that displacement of the GO-adsorbed F-ssDNA occurred with fluorescence enhancement when the incoming DNAs regardless of its complementarity to the FssDNA exceeded the adsorption capacity of the GO surface (Figure 4A and 5B). In contrast to the previous mechanism, we propose a modified DNA desorption mechanism that desorption of the preadsorbed ssDNA from GO would be facilitated by hybridization on the GO surface if the frequency of encountering cDNA prevails over the ssDNA-GO interaction. However, we cannot rule out the possibility that different points regarding DNA hybridization were resulted from different experimental conditions, such as DNA sequences, salt concentration, and GO density, between our study and the previous one.30 From the analysis of kinetic traces of F-ssDNA desorption (Figure 7), concentration-dependent increase in the amplitudes of fluorescence enhancement showed different curves; hyperbolic curve was observed for cDNA, whereas sigmoid curve with an initial lag phase was observed for the addition of non-cDNA (Figure 7D). The presence of lag substantiates our mechanism, in which most of the incoming non-cDNA molecules were adsorbed on GO and a portion of incoming non-cDNA encountering the preadsorbed F-ssDNA would not be hybridized because of the unmatched bases between these two DNAs. In contrast, a portion of cDNA encountering the preadsorbed probe DNA molecules would form duplex DNA on the GO surface, despite


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most of cDNA being adsorbed by GO, which drove the desorption of GO-adsorbed F-ssDNA without initial lag dependency on cDNA concentration.

CONCLUSION We investigated the effect of salt in the binding of nucleic acid to GO. As previously reported, the divalent ion was more effective than the monovalent ion in the adsorption and desorption of ssDNA on the GO surface probably because of its ability to screen negative charges and to form a bridge between two molecules. By investigating the fluorescence quenching of full duplex DNA and partial duplex DNA with identical duplex regions but different overhangs, we showed that the binding affinity of nucleic acid is dependent on the presence of a single-stranded region. Next, the base recognition between the incoming ssDNA and the probe F-ssDNA preadsorbed on GO was examined by monitoring the increase in fluorescence when the probe F-ssDNA dissociated from the GO surface. We showed that the partial duplex containing a single-stranded region that was complementary to the target DNA adsorbed on GO was more applicable to signal improvement by raising the hybridization efficiency, as compared to the fully complementary ssDNA in the GO-adsorbed DNA-sensing system. Furthermore, the base complementarity of dsDNA on the GO surface was analyzed by adding ssDNA (c-ssDNA or non-c-ssDNA) to the FdsDNA–GO complex. The results showed that base pairing in dsDNA was exposed to the incoming complementary strand, indicating that hybridization with incoming c-ssDNA on the GO surface was not inhibited. Finally, the desorption kinetics of the probe F-ssDNA adsorbed on GO, driven by incoming ssDNA, were investigated. By analyzing the amplitudes and rates of two kinetically different phases of the desorption of probe DNA, we propose the following mechanism for DNA sensing on the GO surface: desorption of the GO-adsorbed DNA occurs


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either by facilitated desorption following hybridization with cDNA on the GO surface or by nonspecific desorption involving simple displacement by non-cDNA. According to the law of mass action, when the GO surface is almost saturated with adsorbed DNA, the incoming DNA molecules simply displace the preadsorbed DNA molecules on the GO surface.

MATERIALS AND METHODS DNA oligonucleotides and graphene oxide. The DNA oligonucleotides used in this study (the sequences are shown in Table 1) were either custom synthesized or purchased (Cosmogenetech, Seoul, Korea). Fluorescein isothiocyanate (FITC) was used for the synthesis of fluorescent oligonucleotide (20-mer probe DNA 1). Commercially available GO was used (HCGO-W-175, Graphene Laboratories, Inc., Ronkonkoma, NY, USA) in all the experiments. Atomic force microscopy. The AFM images were collected using an atomic force microscope (Nano-R2TM AFM, Pacific Nanotechnology, Blenheim, New Zealand) at room temperature in the tapping mode, with a spring constant of 40 N/m and a tip radius of ≤8 nm. After 200 ng of the graphene oxide was loaded on a freshly cleaved mica surface (Pucotech, Seoul, Korea), the sample was dried by vacuum evaporation at 85 °C for 12 h. The scanning was performed at a line frequency of 1.0 Hz, and the original images were sampled at a resolution of 256 × 256 pixels. Adsorption of fluorescent ssDNA on GO. First, 0.1 µM of fluorescent probe DNA 1 was mixed with various concentrations of GO in buffers (25 mM Tris-HCl, pH6.8), each containing 25 mM of a different metal chloride (NaCl, KCl, MgCl2, and CaCl2). The mixtures were then incubated for 10 min at room temperature and the fluorescence was measured with a multilabel plate reader (VICTOR X3; PerkinElmer, Waltham, MA, USA). The excitation wavelength was


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485 nm, and the emission wavelength was 535 nm. For the kinetic analysis of ssDNA adsorption on GO, 50 nM of the probe DNA 1 was mixed with 2.5 µg/mL of GO in a buffer (25 mM TrisHCl, pH 6.8) containing either 25 mM NaCl (NaCl buffer) or 25 mM MgCl2 (MgCl2 buffer). The fluorescence was measured every 500 ms after GO addition, using a spectrofluorophotometer (model RF-5301PC, Shimadzu Inc., Kyoto, Japan). The excitation and emission wavelengths were 485 and 535 nm, respectively. Adsorption of duplex DNAs on GO surface. To compare the binding affinity of duplex DNAs with single-stranded portions of different lengths, 1 µM of DNA 2 (51-mer) was annealed with equimolar concentration of the probe DNA 1 and the annealing DNA (3, 4, or 5; sequences shown in Table 1) in either a NaCl buffer or a MgCl2 buffer. Another experiment was set up to compare the binding affinities of the full and partial duplex DNAs, whose lengths in the duplex region were the same at 20 base pairs. First, 1 µM of the probe DNA 1 was annealed with 1 µM of DNA 2 or DNA 6 (20-mer) in a MgCl2 buffer. The oligonucleotide mixtures were heated at 95 °C for 5 min and cooled at room temperature for 1 h. Next, 5 µL of the annealed products were mixed with 45 µL of a solution containing 5 µL of 10× NaCl buffer or MgCl2 buffer and various concentrations of GO in a 96-well plate. The mixtures were further incubated at room temperature for 10 min, and FITC fluorescence was measured with a multilabel plate reader (VICTOR X3). Desorption of fluorescent ssDNA or dsDNA from GO. First, 0.1 µM of FITC-labeled ssDNA (1) or dsDNA (1+6) was mixed with different concentrations of GO (3, 7, or 10 µg/mL) in a MgCl2 buffer. After incubating at room temperature for 10 min, various amounts (0–1 µM) of 20-mer ssDNA (complementary DNA 6 or noncomplementary DNA 4) was added to the solution of DNA–GO complex, and the mixture was further incubated at room temperature for


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10 min. The FITC fluorescence was measured with a multilabel plate reader using an excitation wavelength of 485 nm and an emission wavelength of 535 nm. In addition, the desorption of the probe ssDNA (1) was also monitored when partial duplex DNAs (dsDNA with complementary ssDNA tail, 2+5; or dsDNA with noncomplementary ssDNA tail, 2′+5) were used as competing DNAs. All calculations including standard errors are performed by computer software (SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, USA)). Kinetic measurement of fluorescent ssDNA on GO. First, 50 nM of FITC-labeled ssDNA (F-ssDNA, 1) was mixed with 2.5 µg/mL of GO in a MgCl2 buffer and incubated for 10 min at room temperature. Next, increasing amounts of ssDNA (complementary DNA 6 or noncomplementary DNA 4, 10–500 nM) were added and the fluorescence intensity was measured every 500 ms with the RF-5301PC spectrofluorophotometer. The excitation wavelength was 485 nm, and the emission wavelength was 535 nm.


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FIGURES

Figure 1. (A) AFM image and (B) height profile of GO. The three different colors represent three repeated experiments that yielded an average thickness of 1.377 nm for the GO sheet.


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Figure 2. Comparison of fluorescence-quenching efficiencies of partial duplex DNAs containing 20 nucleotides of duplex region and different portions of a single-stranded DNA. The partial duplex DNAs, pdsDNA1, pdsDNA2, pdsDNA3, and dsDNA, which possess the single-stranded region of 30, 20, 10, and 0 nucleotides, are represented by ●, ○,

▼,

and

▽,

respectively. Each

partial duplex DNA was mixed with an increasing amount of GO in a buffer containing (A) 25 mM MgCl2 and (B) 25 mM NaCl. Inset: Each apparent Kd (in µg/mL) value obtained from the hyperbolic curves is shown in the bar chart.


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Figure 3. Comparison of binding affinity between the full duplex DNA and GO and that between the partial duplex DNA and GO. By fitting the relative quenched-fluorescence intensities to the hyperbolic equation (continuous lines), the apparent Kd value for the interaction between GO and the full duplex DNA (0.1 µM) and that between GO and the partial duplex DNA (0.1 µM) were determined to be 6.4 and 2.0 µg/mL of GO, respectively.


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Figure 4. Fluorescence intensity changed as ssDNA was added to the F-ssDNA preadsorbed on GO. Increasing amounts of complementary or noncomplementary ssDNA were added to a solution containing 0.1 µM of F-ssDNA preadsorbed on (A) 3 µg/mL and (B) 10 µg/mL of GO. The corresponding fluorescence intensity was measured as the amount of ssDNA increased.


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Figure 5. Schematic illustration showing the addition of partial duplex DNA to the F-ssDNA– GO complex, corresponding variations in fluorescence intensity. First, 0.1 µM of F-ssDNA was preadsorbed on (A) 3 µg/mL and (B) 10 µg/mL of GO. A partial duplex DNA containing either a complementary or noncomplementary tail was then added to the F-ssDNA–GO solution, and the fluorescence intensity was measured as the partial duplex DNA concentration increased.


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Figure 6. (A) Schematic illustration showing the addition of ssDNA to the F-dsDNA–GO complex; fluorescence quenching of the F-dsDNA with increasing concentrations of GO. First, 0.1 µM of F-dsDNA was adsorbed on (B) 10 µg/mL and (C) 7 µg/mL of GO. ssDNA (cDNA or non-cDNA) was then added to a solution of the F-dsDNA–GO complex.


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Figure 7. Desorption kinetics of F-ssDNA adsorbed on GO upon addition of increasing amounts of (A) cDNA and (B) non-cDNA. Each curve was fitted to the two-term exponential equation (y = a[1 - exp(-bt)] + c[1 - exp(-dt)]). The rates (b and d) and the amplitudes (a and c) of each curve were obtained and shown in the plots in (C) and (D), respectively.


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SCHEMES

Scheme 1. Schematic of the desorption mechanism of the probe DNA adsorbed on GO, driven by the addition of (A) cDNA and (B) non-cDNA. Desorption of the GO-adsorbed DNA occurred either via facilitated desorption following hybridization with cDNA on the GO surface or via nonspecific simple displacement by non-cDNA according to the law of mass action.


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TABLES.

Table 1. Sequence and structural information of DNA oligonucleotides used in this study


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AUTHOR INFORMATION Corresponding Author *(D.-E. Kim) E-mail: [email protected]; Fax: +82-2-3436-6062; Tel: +82-2-2049-6062. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Research Foundation grants funded by the Korean Government MSIP (NRF-2010-0019306 and 2012M3A9B2028336). ABBREVIATIONS GO, graphene oxide; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; cDNA, complementary DNA.


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GRAPHICAL ABSTRACT


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Mechanism of DNA Adsorption and Desorption on ... - ACS Publications

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