Ionic Strength-Mediated Phase Transitions of Surface-Adsorbed DNA

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Ionic strength mediated phase transitions of surface adsorbed DNA on single-walled carbon nanotubes Daniel P. Salem, Xun Gong, Albert Tianxiang Liu, Volodymyr B. Koman, Juyao Dong, and Michael S. Strano J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09258 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 20, 2017

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Journal of the American Chemical Society

Ionic strength mediated phase transitions of surface adsorbed DNA on single-walled carbon nanotubes Daniel P. Salem, Xun Gong, Albert Tianxiang Liu, Volodymyr B. Koman, Juyao Dong and Michael S. Strano* Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139 KEYWORDS: Single walled carbon nanotubes, nanosensors, fluorescence, ionic strength, molecular recognition

ABSTRACT Single-stranded DNA (ssDNA) oligonucleotides have unique, and in some cases, sequencespecific molecular interactions with the surface of carbon nanotubes that remain the subject of fundamental study. In this work, we observe and analyze a generic, ionic strength mediated phase transition exhibited by over 25 distinct oligonucleotides adsorbed to single-walled carbon nanotubes (SWCNTs) in colloidal suspension. The phase transition occurs as monovalent salts are used to modify the ionic strength from 500 mM to 1 mM, causing a reversible reduction in the fluorescence quantum yield by as much as 90 percent. The phase transition is only observable by fluorescence quenching within a window of pH and in the presence of dissolved O2, but occurs independently of this optical quenching. The negatively charged phosphate backbone increases (decreases) the DNA surface coverage on an areal basis at high (low) ionic strength, and is well described by a two state equilibrium model. The resulting quantitative model is able

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to describe and link, for the first time, the observed changes in optical properties of DNAwrapped SWCNTs with ionic strength, pH, adsorbed O2, and ascorbic acid. Cytosine nucleobases are shown to alter the adhesion of the DNA to SWCNTs through direct protonation from solution, decreasing the driving force for this phase transition. We show that the phase transition also changes the observed SWCNT corona phase, modulating the recognition of riboflavin. These results provide insight into the unique molecular interactions between DNA and the SWCNT surface, and have implications for molecular sensing, assembly and nanoparticle separations.


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Introduction The unique electronic and fluorescence properties of semiconducting single-walled carbon nanotubes (SWCNTs) have made them attractive transducers and scaffolds for biosensor applications1-3. Individualized, polymer-wrapped SWCNT optical sensors capitalize on the sensitivity of 1D excitons to the chemical environment surrounding the nanotube. Perturbations in the polymer wrapping induced by a molecular recognition event result in changes to the local dielectric environment4-6 or the density of exciton quenching sites7-10. Therefore, not surprisingly, baseline SWCNT fluorescence is heavily influenced by both the specific polymer wrapping as well as the solution conditions of the nanoparticle dispersion. Better understanding how the polymer wrapping and solution conditions interact with each other to influence the wrapping structure and SWCNT optical properties is needed to improve SWCNT-based sensor design11,12 as well as for other applications including chirality-based SWCNT separation13,14. We note that mechanisms with molecular level description have been lacking for relating corona phase properties, solution conditions and the SWCNT exciton for predictive models of optical properties. Such mechanisms would not only help improve the synthesis and design of nanotubebased sensors, but also inform structure-property relations for polymers adsorbed onto all nanoparticles, an endeavor that has also been lacking. In this work, we observe and quantitatively describe a generic phase transition undergone by surface adsorbed single-stranded DNA (ssDNA) oligonucleotides on the SWCNT which optically modulates both the photoabsorption and photoluminescence of the SWCNT. This phase transition is in turn linked to the solution pH, the corona phase, molecularly adsorbed O2 and ionic strength in a ssDNA sequence dependent manner, hence forming a comprehensive mechanistic description of these connected phenomena.


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Single-stranded (ssDNA) and double-stranded DNA (dsDNA) has been shown to be a very effective dispersing agent for carbon nanotubes15,16, and provides an ideal polymer system for studying nanoparticle dispersions due to the precisely controlled composition of DNA. As a result, the interaction of ssDNA and dsDNA oligonucleotides with the carbon nanotube surface is of fundamental importance in nanotechnology and has been heavily investigated15,17-19. Despite being composed of four structurally similar monomers as nucleobases, it is known that the ssDNA-SWCNT interaction is sequence dependent13,20,21, with specific lattice registration of sequences with the cylindrical graphene surface which depends on both diameter and chiral vector. This interaction has been utilized for precision sorting of ssDNA-wrapped carbon nanotubes using ion-exchange chromatography by Zheng and co-workers13,15 because of the observed sequence dependence of the corona phase on specific (n,m) carbon nanotubes. To date, the link between this sequence dependence and lattice registration is not fully understood. Many previous studies have used molecular dynamics simulations19,22-25 and coursegrained models26,27 to predict the corona structure and binding energies of ssDNA-wrapped SWCNTs. Johnson and coworkers reported that (GT)20 and (GT)30 ssDNA strands spontaneously wrap SWCNTs in a helical manner19, while the free energy landscape of (GT)7 ssDNA indicates six potential wrapping conformations28. Roxbury et al. studied the wrapping conformations of several short ssDNA oligonucleotides and found the corona structures to vary significantly across different SWCNT chiralities, influenced by inter- and intra-strand hydrogen bonding20,24,29,30. In addition, a course-grained model developed by Shankar and coworkers indicates that binding energies and corona structures can vary across SWCNT enantiomers for ssDNA wrappings that exhibit a nonzero spontaneous torsion27.


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Since ssDNA is a charged polymer, it stabilizes nanoparticle dispersions primarily through electrostatic repulsion, and this anticipates a sensitivity to solution ionic strength31-33. Qiu and coworkers recently reported that forces between ssDNA-SWCNT hybrids become attractive at high monovalent salt concentrations, in contrast to the electrostatic repulsion that dominates at low salt concentrations32. Ghosh et al. recently studied the structure of an adsorbed ssDNA 12mer using molecular dynamics33 at NaCl concentrations of 18 mM and 150 mM. The authors found that the ssDNA strand forms a compact configuration at high salt concentration, influenced by self-stacking of ssDNA bases, versus a more elongated structure at low concentrations dominated by interactions between the ssDNA bases and the SWCNT surface. In this work, we observe that for 29 total ssDNA oligonucleotides tested, the solution ionic strength appears to modulate the photoabsorption and fluorescence of SWCNTs between two states in equilibrium. Through colloidal experiments, spectroscopy and separation, we develop a mechanistic picture of these two states as adsorbed ssDNA in high areal and low areal surface coverage corresponding to high and low ionic strengths respectively. Moreover, this discovery allows us to link, via the same mechanistic description, the influence of pH and dissolved oxygen concentration in solution, resolving several longstanding and seemingly distinct phenomena for ssDNA-wrapped SWCNTs. The mechanism is quantitatively described by a 2nd order, two state equilibrium surface balance. Application of this model to a collection of ssDNA wrappings reveals that the influence of ionic strength on corona structure and photoluminescence is dependent on nucleotide composition. These findings suggest that ionic strength is a tunable parameter for ssDNA-based SWCNT separation processes, SWCNT mediated drug delivery and ssDNA-SWCNT biosensor design.


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Results Effect of NaCl concentration on Photoabsorption and Fluorescence of ssDNA-SWCNTs Table 1 provides a list of all ssDNA wrappings studied. SWCNT stock solutions (prepared in 100 mM NaCl) were diluted to 2 mg/L at NaCl concentrations ranging from 1 mM to 500 mM. Colloidal stability was observed for all ssDNA-SWCNT solutions over this concentration range. All SWCNT solutions were allowed to equilibrate while covered at room temperature overnight before collecting fluorescence and absorbance spectra. Table 1. List of ssDNA sequences studied. Asterisks are used to note the ssDNA sequences used in Figure 5d. Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Sequence T12 T30 C12 C30 (TAT)4 (ATTT)3 (TATT)3T * (GTC)3 * (TCG)2TC * (GT)15 CT10C C2T8C2 T4C4T4 T5C2T5 C4T4C4 C5T2C5 (GT)6 * (TTA)4TT * (CGT)3C * (ATT)4 * (TATT)2TAT * (GTC)2GT * (CCG)4 * (GTT)3G * (TGT)4T * (GTC)2 * (TGTT)2TGT


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28 29

(TTA)3T * (CCG)2CC *

The effect of sodium chloride concentration on SWCNT fluorescence was rigorously mapped for 16 ssDNA wrappings (1-16 in Table 1). Of these 16 wrappings, we find that lowering the NaCl concentration from 500 mM to 1 mM causes the fluorescence of the ssDNASWCNTs to monotonically decrease for 12 wrappings (1-2, 5-14 in Table 1), monotonically increase for 2 wrappings (3-4 in Table 1), and remain relatively constant (< 20% change) for 2 wrappings (15-16 in Table 1). Figure 1a-d provides the fluorescence spectra of (ATTT)3-, (TAT)4-, T12- and C12-wrapped SWCNTs, respectively, at various NaCl concentrations between 500 mM and 1 mM. A reduction in salt concentration by two orders of magnitude causes fluorescence to monotonically decrease by as much as 90 percent for several SWCNT chiralities of (ATTT)3-, (TAT)4- and T12-wrapped SWCNTs, while fluorescence increases for C12SWCNTs. In addition, the absorbance spectra at high and low salt concentration for (ATTT)3SWCNTs (Figure 1e) show that the photoabsorption values at the excitation wavelength (785 nm) are nearly equivalent, indicating that aggregation is not responsible for the change in fluorescence. To evaluate whether SWCNT photoluminescence can be restored by raising the monovalent salt concentration, we added a constant volume of 4M NaCl to the (ATTT)3SWCNT solutions previously measured in Figure 1a. These solutions were allowed to incubate while covered at room temperature for one hour before re-collecting fluorescence spectra. Figure 1f provides the fluorescence peak heights of (ATTT)3-wrapped (7,5) SWCNTs obtained from deconvolution of the fluorescence spectra, before and after the addition of concentrated sodium chloride. The addition of salt causes the fluorescence intensities to increase along the same curve


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traced by the original measurements, indicating that the effect of salt concentration on ssDNASWCNT fluorescence is reversible. This procedure was repeated for sequences 1-10 in Table 1 and identical results were observed (Figure S1).

Figure 1. Effect of monovalent salt concentration on photoluminescence spectra of (a) (ATTT)3SWCNTs, (b) (TAT)4-SWCNTs, (c) T12-SWCNTs, and (d) C12-SWCNTs. (e) Absorbance spectra of (ATTT)3-SWCNTs at high (200 mM) and low (4 mM) NaCl concentration. (f) Fluorescence intensity of (ATTT)3-wrapped (7,5) SWCNTs before (black) and after (red) the addition of concentrated NaCl, along with a fit to a two-state equilibrium model. Error bars represent standard deviations over three measurements.

Effect of pH and dissolved O2 on NaCl modulated ssDNA-SWCNT Photoabsorption and Fluorescence


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As shown in Figure 1e, decreasing the NaCl concentration results in a reduction in E11 absorbance intensity for several SWCNT chiralities. Such transition photo-bleaching is also observed when the pH or the concentration of oxidizing agents is changed for SWCNTs dispersed in a number of anionic surfactants34-36 and polymers including ssDNA-SWCNTs15,37. This effect has been attributed to a reversible oxidation of the nanotube, mediated by H+ and adsorbed molecular oxygen34-37. We therefore explored the influence of H+ and O2 on the ionic strength effect shown in Figure 1.

For all ssDNA wrappings tested besides C12 and C30,

increasing the pH from 6 to greater than 10 appears to reverse the fluorescence intensity decrease that occurs when ionic strength is lowered, with a corresponding change in E11 absorbance (shown in Figure 2a and Figure 2f, respectively, for T12-wrapped SWCNTs). Note that while the addition of NaOH does increase the ionic strength by a maximum of 1 mM, this is not a large enough increase to cause the fluorescence changes shown in Figure 1 and Figure 2a. We also investigated the role of adsorbed oxygen on the ionic strength fluorescence modulation by collecting fluorescence and absorbance spectra before and after removing dissolved O2 by degassing the solutions overnight with nitrogen. Figure 2b compares the photoluminescence spectra of degassed and non-degassed T12-wrapped SWCNTs in 5 mM NaCl. Removal of dissolved O2 causes a significant increase in SWCNT fluorescence despite being at a low ionic strength. A concomitant increase in E11 absorbance intensity is also observed (Fig. 2d). Upon exposing these degassed SWCNT solutions to the atmosphere, the fluorescence intensity of each SWCNT chirality decreases over the course of hours, presumably due to the readsorption of oxygen on the SWCNT (Figure 2e for (7,5) SWCNTs). It is noteworthy that the effect of O2 removal can be replicated by the addition of ascorbic acid to ssDNA-SWCNT dispersions. Previously, antioxidants including ascorbic acid have been


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reported to significantly increase SWCNT quantum yield38-41. These reducing agents are thought to passivate defect sites along the nanotube that also serve as sites for oxygen adsorption35,38. As shown in Figure 2c, addition of 100 µM ascorbic acid causes a significant ‘turn-on’ response for T12-SWCNTs in 1 mM NaCl, similar to O2 degassing. Moreover, the addition of ascorbic acid removes the effects of both pH and ionic strength on E11 photoabsorbance. Indeed, the resulting absorbance spectra all collapse on each other (Figure 2f-g), suggesting that these transformations lead to the same thermodynamic state. Therefore, an important mechanistic clue is the similarity between ascorbic acid addition and removal of dissolved oxygen via degassing. We attribute the slight differences in fluorescence spectra after degassing (Figure 2b) and after ascorbic acid addition (Figure 2c) to incomplete desorption of O2 when degassing overnight.


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Figure 2. Influence of pH and dissolved oxygen on salt dependent ssDNA-SWCNT photoabsorption and photoluminescence. Fluorescence increase of T12-SWCNTs at low NaCl concentration (< 5 mM) upon (a) adding 1 mM NaOH; (b) removing dissolved O2 by degassing overnight; (c) adding 100 µM ascorbic acid. (d) Change in absorbance spectra of T12-SWCNTs at low NaCl concentration upon overnight degassing. (e) Fluorescence decreese of degassed T12SWCNTs at low NaCl concentration upon exposure to air, (7,5) chirality. Error bars represent standard deviations over three measurements. (f) Absorbance spectra of T12-SWCNTs at low ionic strength and various pH conditions (i) before and (ii) after addition of ascorbic acid. (g) Absorbance spectra of T12-SWCNTs at pH 6 and various NaCl concentrations (i) before and (ii) after addition of ascorbic acid.

The data shown in Figure 1f suggest the effect of ionic strength on SWCNT photoluminescence involves an equilibrium-limited two state mechanism. Modulation of the solution salt concentration enables a transition between these two states and a subsequent change in optical properties. This is represented by Equation 1 below (derivation provided in the Supporting Information), where IA and IB are the fluorescence intensities of the low ionic strength and high ionic strength states, respectively, n is the number of ions involved in the state transition, and Kion is the equilibrium coefficient. I=

n I A + I B K ionCion n 1 + K ion Cion

(Eq. 1)

This equation is fit to the photoluminescence data for (ATTT)3-wrapped shown in Figure 1f.

Evidence for an ionic strength mediated ssDNA phase transition on the CNT surface


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The negatively charged phosphate backbone of ssDNA results in a persistence length that changes with ionic strength42-44. While we expect a given ssDNA strand to stiffen as ionic strength is reduced, ssDNA-SWCNT systems contain the added complexity of neighboring and interacting ssDNA strands kinetically trapped on the SWCNT surface, which has the potential to magnify the effect of salt concentration on polymer conformation and corona structure. Ghosh and coworkers recently studied this problem using molecular dynamics to predict the structure of a single ssDNA 12-mer on the surface of a carbon nanotube at high and low sodium chloride concentrations (150 mM and 18 mM, respectively)33. The simulation results indicate that the ssDNA strand adopts a more compact conformation at high salt concentration, influenced by the tendency of nucleobases to undergo self-stacking. However, at low salt concentration, the ssDNA strand spreads out along the nanotube and participates in a greater number of π-π interactions with the SWCNT surface33. Brunecker et al. experimentally studied the structure of (GT)n-wrapped SWCNTs by measuring the effect of ssDNA length on the activation enthalpy of desorption45. The authors find the activation enthalpies to level out once ssDNA strands exceed one Kuhn length, suggesting that ssDNA incompletely adsorbs on the SWCNT surface in the limit of excess ssDNA45. Given that the Kuhn length is equal to twice the persistence length for a worm-like chain, we would expect the degree of ssDNA adsorption to be dependent on the dispersion ionic strength. Consequently, the tendency of a polymer strand to occupy more space on the nanotube surface as the salt concentration is lowered must result in the exclusion of currently adsorbed polymers from the surface. To measure whether such a conformational change takes place, we performed the experiment illustrated in Figure 3a. Briefly, we removed all free ssDNA from ssDNA-SWCNT


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stock solutions at high NaCl concentration (200 mM NaCl). These stock solutions were diluted 1:10 with 200 mM NaCl and water and allowed to incubate at room temperature overnight. The nanotubes were removed from solution via centrifugal filtration and the concentrations of free ssDNA in the high and low ionic strength solutions were quantified and compared. Figure 3a provides the resulting free ssDNA concentrations measured for 8 different ssDNA wrappings, normalized by the SWCNT concentrations in mg/L. The results of the ssDNA “kick-off” experiment confirm that a reduction in salt concentration causes some ssDNA to desorb from the nanotube surface. Nonzero concentrations of free ssDNA at high ionic strengths most likely reflect incomplete removal of free ssDNA in the original stock solutions prior to the experiment. Interestingly, the amount of free ssDNA excluded from the nanotube surface is highly dependent on the nucleotide sequence, with cytosine-containing sequences displaying significantly reduced desorption. In addition, nearly identical results were obtained when the experiment was performed at a pH of 11 using T12-SWCNTs, indicating that this structural change is not dependent on pH (Figure S2). To evaluate the reversibility of the structural change, this experiment was repeated for (TAT)4, (GTC)3 and (TATT)3T-wrapped SWCNTs by incompletely removing free ssDNA from stock solutions at low NaCl concentration. Upon diluting the ssDNA-SWCNTs at high and low salt concentration, the amount of free ssDNA in solution decreased in solutions of high salt concentration, suggesting some free ssDNA adsorbed back onto the SWCNTs (Figure S3). Most importantly, the amount of ssDNA that adsorbed back onto the SWCNTs was roughly equivalent to the amount of ssDNA kicked off the SWCNTs in Figure 3a, suggesting that the structural change is indeed reversible. Hence, we conclude that the two equilibrium states correspond to ssDNA adsorbed at high and low areal density at high and low ionic strength respectively.


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As an orthogonal, physical measure of corona structural changes, atomic force microscopy (AFM) was performed on (GT)15-SWCNTs deposited on cleaved mica at high and low salt concentration (200 mM and 2 mM, respectively). A comparison of SWCNT heights (as a proxy for corona thickness) was performed for at least 20 nanotubes at each ionic strength. As shown in Figure 3b, SWCNTs at low NaCl concentration are found to have a smaller height (0.68 ± 0.50 nm) than SWCNTs at high NaCl concentration (1.4 ± 1.1 nm), suggesting a more compact corona structure. Given that the two samples were derived from the same original dispersion of HiPCO SWCNTs, we do not expect the differences in SWCNT height to be caused by differences in chirality distributions. Moreover, while the washing step may have affected the ssDNA corona structures, it is unlikely to be responsible for the observed differences in SWCNT heights given that the washing was brief and the fluid for both samples was deionized water. This result is consistent with Figure 3a as well as the molecular dynamics work of Ghosh and coworkers33—we expect a decrease in ionic strength to reduce the thickness of the ssDNA wrapping caused by tighter binding of ssDNA strands and subsequent exclusion of incompletely adsorbed ssDNA. To further visualize the structural change of the ssDNA wrapping, we collected transmission electron microscopy (TEM) images of biotinylated ssDNA-wrapped SWCNTs in the presence of 5 nm streptavidin-functionalized gold nanoparticles. Briefly, biotin-(GT)30-biotin ssDNA was used to suspend SWCNTs according to our standard protocol. Dilutions of 5 mg/L at high (200 mM) and low (1 mM) NaCl concentration were equilibrated at room temperature overnight, after which they were incubated with streptavidin-functionalized gold nanoparticles (stock solution added at 4 vol%) for two hours. The solutions were later deposited onto TEM grids and allowed to dry without washing.


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Figure 3c provides a schematic of the experiment as well as two representative TEM images of the deposited SWCNTs at high and low ionic strength in which individual nanotubes have been identified by white line segments. The images clearly show a change in distance between adjacent bound gold nanoparticles, suggesting a structural change in the ssDNA wrapping. Histograms were constructed to quantify the spacing between bound gold nanoparticles using 11 unique images at each ionic strength (38 and 22 total SWCNTs imaged at high and low ionic strength, respectively). The results in Figure 3d confirm an increase in gold nanoparticle spacing and suggest an increase in the end-to-end distance of individual ssDNA strands, consistent with the experimental results shown in Figure 3a-b.


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Figure 3. Structural changes of ssDNA-SWCNT corona upon modulation of solution ionic strength. (a) Assay schematic and experimental results for quantifying free ssDNA excluded from the SWCNT surface upon dilution at high and low ionic strength (pH = 6). Free ssDNA concentrations have been normalized by the SWCNT concentration in mg/L. Error bars represent standard deviations over three measurements. (b) AFM results measuring (GT)15-SWCNT height at high and low ionic strength. (c) Schematic and TEM images of biotinylated ssDNA-SWCNTs mixed with 5 nm streptavidin-functionalized gold nanoparticles at high and low ionic strength. SWCNTs identified by white line segments. (d) Distance between adjacent bound gold nanoparticles measured from TEM images.

Discussion A unified mechanistic description of ssDNA-SWCNT phase transitions To summarize the above observations, two pieces of physical evidence suggest that ssDNA occupies a lower (higher) areal density at low (high) ionic strength. Both the measured AFM heights and the ssDNA kick-off experiments indicate this change in density, regardless of the solution pH. When observing the change in photoluminescence of the ssDNA-SWCNTs versus salt concentration, we note that no change is observed in the absence of O2, the presence of ascorbic acid, or in the limit of highly basic pH. At an intermediate pH range and in the presence of O2, one observes that the ionic strength modulates the carbon nanotube photoluminescence between what appears to be two dominate states: high (low) intensity at high (low) ionic strength tracing a sigmodal function characteristic of an equilibrium-limited process. These observations in photoluminescence are mirrored in photoabsorption (Fig. 2e-f) and are summarized pictorially in Figure 4a. Note that the corona structures depicted in Figure 4a


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represent a simplified view for the case of helically-wrapping ssDNA, where we expect the pitch to increase as ionic strength is lowered due to an increase in the persistence length of the polymer18,42,46. This structural change may be more complex when considering short ssDNA oligonucleotides (>1 when equilibrated in air, the model simplifies to Equations 3 and 4 at low and high ionic strength, respectively. I = SWNT0

I = SWNT0

I max 1 + K AOP ( H

+ m

)

I max 1 + K BOP ( H + )

l

(Eq. 3)

(Eq. 4)

The experimental data along with model fits are provided in Figure 5c. As expected, SWCNT fluorescence decreases as pH is lowered, with a greater pH sensitivity observed at low ionic


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strength. Fitted parameters for T12-wrapped (7,5) SWCNTs are provided in the Supporting Information (Table S1).

Figure 5. (a) Reaction scheme describing ssDNA-SWCNTs in monovalent salt solution; Fluorescence intensity of T12-wrapped (7,5) SWCNTs versus (b) NaCl concentration and (c) pH at high (200 mM) and low (1 mM) ionic strength, along with model fits; (d) Change in fluorescence (I-I0) upon 200 µM ascorbic acid addition versus initial fluorescence (I0) for 15 different ssDNA wrappings at high (200 mM) and low (1 mM) ionic strength, along with a linear fit of slope -1; (e) Fluorescence of C12-wrapped (7,5)-SWCNTs versus NaCl concentration; (f) Normalized fluorescence of (7,5)-SWCNTs versus NaCl concentration for ssDNA wrappings with varying cytosine content. Error bars represent standard deviations over three measurements.

The previously described model predicts that the reversal of nanotube oxidation via degassing or antioxidant addition will maximize the quantum yield of ssDNA-SWCNTs at a


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given ionic strength. In the event that the fluorescence quantum yields of the two ssDNA states are equal (i.e., IA,max = IB,max = Imax), we predict fluorescence spectra of ssDNA-SWCNTs at various ionic strengths or pH conditions to be invariant upon oxygen removal, as shown in Equation 5.

I no O2 =

n I A,max + I B,max KionCion n 1 + KionCion

= I max

(Eq. 5)

Thus, the change in fluorescence upon excess ascorbic acid addition, ∆I AA , can be described by Equation 6 where I0 is the initial fluorescence.

∆I AA = I max − I 0 = I max − f ( pH, salt, dissolved O 2 , ssDNA sequence )

(Eq. 6)

Accordingly, our model predicts that a simple plot of ∆I AA versus I0 should yield a line with a slope equal to -1 for a group of SWCNT dispersions at different conditions, independent of model parameters. Hence, this predicted dependence is an important test of the mechanism. To test this result, fluorescence spectra of ssDNA-SWCNTs at constant concentration and various solution conditions were collected before and after the addition of 200 µM ascorbic acid. Provided that enough antioxidant was added to completely reduce the SWCNTs, the fluorescence spectra following ascorbic acid addition corresponded to the Imax of each particular ssDNA-wrapped SWCNT. Figure 5d provides a plot of I-I0 versus I0 for 15 different ssDNA wrappings at high (200 mM) and low (1 mM) NaCl concentration, along with a fitted line with a slope of -1 (specific ssDNA sequences used are provided in Table 1). At each monovalent salt concentration, the ssDNA-SWCNTs display a variety of initial fluorescence intensities. We attribute this to the sequence dependence of the model equilibrium parameters. Moreover, the linearity of the data indicates that the maximum fluorescence intensities of the two ssDNA states


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(i.e., IA,max and IB,max) are roughly equivalent across all ssDNA wrappings tested, in support of the mechanistic model of Fig. 5a. Similar experiments were performed for single ssDNA wrappings in which initial fluorescence was modulated with ionic strength and pH. The behavior observed is equivalent to that shown in Figure 5d in which the maximum fluorescence intensity upon ascorbic acid addition is nearly constant across all solution conditions. Figure S4 provides the resulting plots of I-I0 versus I0 for (TAT)4- and T12-wrapped SWCNTs ((7,5) chirality) at 8 different pH conditions (ranging from 4 to 10) and two different salt concentrations (1 mM and 200 mM NaCl). Importantly, these experimental results show that in the absence of excess antioxidant, different ssDNA-wrapped SWCNTs represent different partially-quenched states of the same system, with a single maximum quantum yield.

Sequence dependence of the phase transition: influence of cytosine content

The results in Figure 3a suggest that cytosine-containing ssDNA sequences adsorbed on a SWCNT surface undergo a different structural reorganization upon changes in ionic strength. Specifically, the presence of cytosine groups in the ssDNA sequence appears to significantly reduce the amount of ssDNA kicked off the nanotube surface when the salt concentration is lowered. To investigate this further, we studied how NaCl concentration and solution pH affect the fluorescence properties of C12-wrapped SWCNTs. Fluorescence spectra of C12-SWCNTs were collected at various NaCl concentrations and pH conditions following the same experimental protocols previously outlined.

Figure 5e

provides the fluorescence intensity of C12-wrapped (7,5)-SWCNTs as a function of NaCl concentration at a pH of 6, indicating that the fluorescence of C12-SWCNTs increases as the salt


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concentration is lowered. In addition, the fluorescence of C12-SWCNTs increases as the solution pH is lowered at both high (200 mM) and low (1 mM) NaCl concentrations (Figure S5). As a result, the fitted β value for C12-wrapped SWCNTs (Table 2) is less than one, indicating that the high ionic strength ssDNA state is more susceptible to oxidation. While this behavior is opposite of that observed for other ssDNA wrappings tested (Table 2), removal of adsorbed oxygen via degassing or antioxidant addition does cause fluorescence to increase (Figure S6). Similar behavior was also observed for C30-wrapped SWCNTs. We believe the anomalous behavior of polyC-wrapped SWCNTs to be caused by protonation of the cytosine groups at low pH and low ionic strength. Previous studies have shown cytosine groups to become readily protonated at low pH, disrupting the structure of dsDNA and opening the potential for non-Watson-Crick nucleotide interactions56-58. Moreover, cytosine protonation appears to occur more readily at low ionic strength without large changes in pH59,60. Thus, protonation of the cytosine groups represents a competing reaction against oxygenmediated CNT reactivity with H+, resulting in the fluorescence dependence shown in Figure 5e. While the exact mechanism behind this fluorescence enhancement remains unclear, cytosine protonation results in a low ionic strength ssDNA state with a larger pristine (i.e., no sidewall oxidation) quantum yield than the high ionic strength state. Given the results in Figure 3a, we expect cytosine protonation to take place at low salt concentrations on most ssDNA sequences containing cytosine groups. While an increase in persistence length occurs as ionic strength is lowered, protonation of cytosine groups along the ssDNA strands reduces the effective charge of the overall polymer, thereby decreasing the electrostatic repulsion within and among ssDNA strands. We hypothesize that this protonation enables cytosine-containing sequences to function as “staple strands,” remaining adhered to the


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CNT surface as the salt concentration is lowered. However, it is still possible that these sequences undergo some form of structural reorganization when ionic strength is modified. To test the hypothesis of cytosine protonation at low ionic strength, we conducted the ssDNA kick-off experiment (Figure 3a) for C12-SWCNTs at pH 6 and pH 11. Briefly, C12SWCNTs at low ionic strength (1 mM NaCl) and pH 6 were diluted 1:10 with 1 mM NaCl at pH 6 and pH 11. The results shown in Figure S7 indicate that raising the pH at low ionic strength causes some C12 ssDNA to desorb from the nanotube surface, presumably caused by deprotonation of the cytosine groups. As a comparison, this experiment was repeated for C12SWCNTs in 100 mM NaCl where a raise in pH did not result in ssDNA desorption. These results further suggest that cytosine protonation takes place as ionic strength is lowered, contributing to the anomalous changes in optical properties for polyC-wrapped SWCNTs. To determine the number of cytosine groups needed to change the ssDNA-SWCNT fluorescence behavior, we tested a library of 12-nucleotide ssDNA wrappings in which we varied the thymine:cytosine ratio, including CT10C, T5C2T5, C2T8C2, T4C4T4 and C5T2C5. Figure 5f compares the normalized peak height of (7,5)-SWCNTs versus sodium chloride concentration for several of the wrappings tested. The results clearly indicate that the fluorescence dependence of ionic strength for ssDNA-SWCNT changes as more cytosine groups are added to the ssDNA sequence, becoming less sensitive to changes in salt concentration and approaching the behavior of C12-SWCNTs. Moreover, the data collected for C4T4C4-SWCNTs suggest that a proper choice of thymine and cytosine content enables the creation of ssDNA sequences that form SWCNT hybrids nearly insensitive to ionic strength. This invariance to ionic strength may be important for the design of SWCNT-based fluorescent sensors.


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Comparison of different ssDNA wrappings containing the same thymine:cytosine ratios revealed that the location of cytosine groups and SWCNT chirality can also influence how dispersion salt concentration affects ssDNA-SWCNT fluorescence (Figure S8). This is not surprising, as many studies have shown ssDNA sequences to wrap SWCNTs in a chirality dependent manner20,21,24,30. As predicted by our model, SWCNT chiralities containing tightly bound corona structures that exclude the adsorption of oxygen or prevent sidewall oxidation will exhibit larger fluorescence and E11 absorbance peaks. Modification of the ionic strength has the potential to disrupt and/or reorganize chirality dependent corona structures, opening the opportunity for new, tightly bound structures at intermediate ionic strengths. This sequence and chirality dependent adhesion may result in optical properties that vary non-monotonically with salt concentration. Overall, these results have implications towards understanding why specific ssDNA sequences recognize specific SWCNT chiral angles.

Probing the ssDNA corona phase

Our previous results indicate that modulation of the solution ionic strength has the potential to alter the structure of the corona phase. A test of this can be provided by examining how probe molecules (i.e. analytes) adsorb onto the corona depending on the ssDNA phase. Note that this structural change can be leveraged to design new fluorescent biosensors that operate by Corona Phase Molecular Recognition (CoPhMoRe) as well. CoPhMoRe is a synthetic molecular recognition technology whereby amphiphilic polymers form synthetic molecular recognition sites upon adsorption on a nanopartcle template11,12,61. To study whether changes in ionic strength could alter molecular recognition, we measured the fluorescent responses of ssDNA-wrapped SWCNTs to the probe molecule riboflavin (vitamin B2) at high and low salt


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concentration. Riboflavin has been previously shown to induce a quenching response in polymer-wrapped SWCNTs, presumably caused by interaction of the riboflavin with the available surface of the nanotube11,39. Riboflavin calibration curves were constructed for (TAT)4-, (TCG)2TC- and T12-wrapped SWCNTs at high and low NaCl concentration (200 mM and 1 mM, respectively). Given that riboflavin is known to induce a quenching response, we increased the starting fluorescence of the ssDNA-SWCNT solutions by raising the pH to 9.5 and increasing the exposure time during the fluorescence experiments. We do not expect the pH to have changed the corona structures based on results shown in Figure S2. Riboflavin concentrations tested ranged from 1 nM to 50 µM, above which riboflavin no longer becomes soluble in water. For all ssDNA wrappings tested, lowering the solution ionic strength reduces the dissociation constant of riboflavin binding, KD, indicating stronger binding to the SWCNT surface. As shown in Table 3, this reduction is especially large for (TAT)4- and (TCG)2TCwrapped SWCNTs in which the KD drops by over an order of magnitude. Figure 6 provides the calibration curves constructed for (TCG)2TC-wrapped (7,5)-SWCNTs at high and low salt concentration. These data suggest that the ssDNA wrapping is more porous at lower salt concentration, providing riboflavin with greater accessibility to the nanotube surface. The results in Fig 6 are further validation of our two state mechanism. Moreover, these results imply that modifying the solution ionic strength has the potential to induce and/or augment molecular recognition events for fluorescent sensors designed using electrostatically-stabilized polymerSWCNT dispersions.


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Figure 6. Calibration curve of riboflavin-induced SWCNT quenching at high (200 mM) and low (1 mM) NaCl concentration showing clear indication of two distinct corona phases, consistent with the phase transition mechanism advanced in this work. Error bars represent standard deviations over three measurements.

Table 3. Fitted dissociation constants for riboflavin binding constructed from calibration curves at high (200 mM) and low (1 mM) NaCl concentration. ssDNA Wrapping (TAT)4

(TCG)2TC

T12

NaCl Concentration

Fitted KD (95% confidence intervals)

200 mM

13.3 uM (2.4, 73.6 uM)

1 mM

0.52 uM (0.23, 1.2 uM)

200 mM

5.8 uM (1.9 uM, 17.3 uM)

1 mM

73.4 nM (95.2 nM, 56.5 nM)

200 mM

0.70 uM (0.26 uM, 2.0 uM)

1 mM

0.44 uM (0.23 uM, 0.83 uM)


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Conclusion The two state, ionic strength ssDNA phase transition uncovered in this work provides a deeper understanding of how solution parameters and ssDNA sequence affect the corona phase and optical properties of SWCNTs. The mechanism also explains the influence of oxygen adsorption and pH, providing a comprehensive description of ssDNA-SWCNT dispersions. Moreover, we show that changes in the solution ionic strength induce a sequence-dependent structural change in the ssDNA corona that results in the exclusion of ssDNA strands from the nanotube surface and a reduction in corona thickness. This understanding promises to improve the utility of SWCNTs as sensors for diagnostic and therapeutic applications, drug delivery agents and chirality separation tools.

Materials and Methods Materials

All chemicals were purchased from Sigma-Aldrich (USA) unless state otherwise. ssDNA sequences were purchased from Integrated DNA Technologies (IDT, USA). HiPCO SWCNTs were used for all experiments and were purchased from Unidym (lot R1831). The raw SWCNTs were processed according to the manufacturer’s recommendation to remove impurities and residual solvent. Streptavidin-functionalized 5 nm gold nanoparticles were purchased from Cytodiagnostics. Preparation and characterization of SWCNT dispersions


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SWCNT dispersions were prepared by combining 1 mg of SWCNTs and 2 mg ssDNA in 1 mL of 100 mM NaCl solution. This mixture was tip sonicated (Qsonica Q125) in an ice bath with a 0.125 in. probe for 10 minutes at a power of 4W. Crude SWCNT dispersions were centrifuged two times at 16,000g for 90 min to remove SWCNT bundles and other solid impurities. The top 80% of supernatant was collected after each round of centrifugation. Absorption spectra of SWCNT dispersions were collected (Cary 5000, Agilent Technologies) to approximate the concentrations of the post-dispersion stock solutions using the absorbance at 632 nm and an extinction coefficient of ε632 = 0.036 (mg/L)-1cm-1 62. SWCNT near-infrared fluorescence measurements

SWCNT stock solutions were diluted to a concentration of 2 mg/L in solutions of varying ionic strength and pH. These solutions were incubated at room temperature overnight to allow the systems to reach equilibrium prior to collecting fluorescence and/or absorbance measurements. Fluorescence measurements were conducted in triplicate in 96-well plates (Tissue Culture Plates, Olympus Plastics) using volumes of approximately 200 µL. SWCNT solutions were excited using a 785 nm laser (450 mW, B&W Tek Inc.), inverted microscope (Zeiss AxioVision) and 20X objective. Fluorescence was collected with a PI Acton SP2500 spectrometer and a Princeton Instruments InGaAs OMA V detector. Exposure time was held constant across a given experiment and varied between two and five seconds. In all cases, fluorescence spectra were background corrected using SWCNT-free solution in an equivalent volume. During experiments in which an analyte was added (e.g., ascorbic acid), 2 uL of analyte solution was added to each well and mixed prior to collecting additional fluorescence measurements.


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Fluorescence spectra were deconvoluted to obtain the contributions of individual chiralities using a Matlab script. With the exception of the (7,6) and (9,4) chiralities, deconvoluted peak heights were used for subsequent analyses. Free ssDNA quantification

Free ssDNA was quantified using a ssDNA dye and ssDNA standard (Promega). During each quantification experiment, calibration curves were constructed using the ssDNA concentration standard provided in the kit. Individual calibration curves were constructed for each ionic strength and pH due to the potential effect these parameters have on dye fluorescence intensity. All SWCNTs were filtered from solution prior to free ssDNA quantification. Atomic Force Microscopy

SWCNT solutions at different ionic strengths were deposited onto freshly cleaved mica (Grade V-1 Muscovite, SPI Supplies, Structure Probe, Inc.) and allowed to dry for one hour. The surfaces were rapidly washed with water to remove excess salt while minimizing contact time with the water. Imaging was performed using an Asylum Research MFP-3D AFM in tapping mode. Data were processed using Gwyddion and Matlab. Transmission Electron Microscopy

TEM grids (lacey carbon support films, 300 mesh, Cu, Ted Pella, inc.) were placed on a paper based hydrophilic support film while the corresponding ssDNA-SWCNT solutions were transferred using the drop casting method. The sample was dried under ambient conditions and mounted onto the TEM microscope for further characterization. Imaging was performed at 120 kV with FEI Tecnai G2 Spirit TWIN Transmission Electron Microscope, with high resolution images captured with the monochromator activated and using a 5 µm slit to reduce the beam


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energy spread to ~0.5 eV. All samples are examined at room temperature with electron gun/column pressure lower than 8 Log.

SUPPORTING INFORMATION Detailed derivations of Equations 1 and 2, fluorescence data for other ssDNA-SWCNT hybrids and additional results from the ssDNA kickoff experiment (PDF).

AUTHOR INFORMATION Corresponding Author * Email: [email protected] ACKNOWLEDGMENTS This work was supported by the Abdul Latif Jameel World Water and Food Security Lab (JWAFS) and the US Department of Energy, Office of Science, Basic Energy Sciences under Award grant number DE-FG02-08ER46488 Mod 0008. D.P.S. is grateful for a National Science Foundation Graduate Research Fellowship under Grant No. 2388357. ABBREVIATIONS SWCNT, single walled carbon nanotubes; ssDNA, single-stranded DNA; dsDNA, doublestranded DNA; NaCl, sodium chloride.


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TOC graphic:


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Ionic Strength-Mediated Phase Transitions of Surface-Adsorbed DNA

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