pH-Responsive DNA Nanolinker Conjugated Hybrid Materials for

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pH-Responsive DNA Nanolinker Conjugated Hybrid Materials for Electrochemical Microactuator and Biosensor Applications Yasamin A. Jodat, Parisa Lotfi, Parisa Pour Shahid Saeed Abadi, Ji-Young Mun, Jungmok Seo, Eun Ae Shin, Sung Mi Jung, Chang Kee Lee, and Su Ryon Shin ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01429 • Publication Date (Web): 26 Oct 2018 Downloaded from http://pubs.acs.org on November 4, 2018

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ACS Applied Nano Materials

pH-Responsive DNA Nanolinker Conjugated Hybrid Materials for Electrochemical Microactuator and Biosensor Applications

Yasamin A. Jodat†,‡, Parisa Lotfi†,¶, Parisa Pour Shahid Saeed Abadi†, Δ, Ji-Young Mun ω, Jungmok Seo†,π, Eun Ae Shinψ, Sung Mi Jungϴ, Chang Kee Leeψ*, Su Ryon Shin†*

† Division

of Engineering in Medicine, Department of Medicine, Brigham and Women’s Hospital,

Harvard Medical School, Cambridge, MA 02139, USA. ‡ Department of Mechanical Engineering, Stevens Institute of Technology, New Jersey, 07030, USA. ¶

Division of Biomedical Engineering, The Hong Kong University of Science and Technology,

Kowloon, Hong Kong. Δ

Department of Mechanical Engineering-Engineering Mechanics, Michigan Technological University,

1400 Townsend Drive, Houghton, Michigan 49931, USA. ω

Department of Structure and Function of Neural Network Ultrastructural Neuroimaging Laboratory,

Korea Brain Research Institute, Daegu, 41068, Republic of Korea. π

Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology,

Seoul, 02792, Republic of Korea. 1

ACS Paragon Plus Environment

ACS Applied Nano Materials 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 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ϴ

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Future Environmental Research Center, Korea Institute of Toxicology, Jinju, 52834, Republic

of Korea. ψ

Korea Packaging Center, Korea Institute of Industrial Technology, Bucheon, 14449, Republic of

Korea.

* Corresponding authors E-mail address: [email protected] (S.R.S.), [email protected] (C.K.L.) *These authors contributed equally as corresponding authors to this work.

Abstract Carbon nanotube (CNT)-based composite or hybrid materials have been broadly used for various biomedical applications such as microactuators, sensors, capacitors, and flexible electronic textiles owing to their appealing physical and electrical properties and energy-storage functions. However, to enable application-based specific functionalities (e.g. sensing, responding, and deformation) it is essential that smart stimulus-responsive elements be incorporated into the CNT-based materials. A pioneering approach in integrating stimulus-responsive molecules or linkers is to utilize multi-stranded DNA structures, such as i-motif DNA with a four-folded structure, which shows reversible conformational changes upon pH alteration. Herein, a pH-responsive CNT-based hybrid material is developed by conjugating i-motif DNA as a pH-responsive nano-sized crosslinker. To fabricate microfibers, the i-motif DNA nanolinker-conjugated CNT-based hybrid material is spun in a proton-rich coagulation bath. The attained hybrid microfibers are composed of partially aligned nanowires with ~ 50 nm diameters that are formed in the protonation process by self-assembly of the i-motif DNA nanolinker-conjugated CNT-based hybrid material. The hybrid microfibers showed high electrical conductivity (~ 27 S/cm), excellent capacitance in a biological medium (~59.9 F/g at pH 5 and ~47.8 2


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F/g at pH 8), and stable microactuation without creep behavior. Furthermore, the conjugated i-motif DNA in the hybrid microfibers undergoes conformational changes from a four-folded structure (pH 5) to a random coil structure (pH 8), thus enabling unique dual-pH reversibility in the microfibers, namely switchable microporosity, electrochemical redox activity, and hydrogen peroxide sensing activity. Consequently, the designed stimulus-responsive hybrid microfiber can be used for microactuation and biosensing applications.

Keywords: i-motif DNA, Carbon nanotubes, Microfibers, Biosensor, Electrochemical actuator.

1.

Introduction Micro- or nano-scale electrically conductive materials with functional capabilities such as sensing,

actuation, or capacitance as well as flexibility and stretchability, provide a platform to develop intelligent electronic devices for various biomedical applications.1 Such devices are expected to be responsive in physiological environments and be capable of converting external stimuli into a perceivable dynamic physical response, similar to human body’s regulatory mechanisms.2 To create reversible stimulus-responsive materials, the constituents must be sufficiently robust and durable while possessing a soft, porous and stimulus-responsive structure at the same time. The robust framework would maintain the integrity of the macrostructure and prevent degradation prior to the exertion of any external stimuli. Furthermore, the nano- and micro- scale porosity would contribute to fast penetration of the stimulants. In addition, presence of stimulus-responsive soft subsegments in the material empowers conformational alterations when exposed to external stimuli. As a result, physical and electrical characteristics of such hybrid materials can be tuned via controlling the internal structural and functional modifications. Carbon nanotubes (CNTs) are one of the most apposite candidates for building vigorous frameworks with high electrical conductivity. Endowed with fascinating properties such as mechanical 3



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robustness and outstanding electrical conductivity, effortlessly modifiable with various chemical reagents, as well as high surface area 3, CNTs have shown to be extensively serviceable in developing microactuators, biosensors, capacitors, energy-storage devices, and flexible electronic textiles in the biomedical research fields.4-7 Additionally, deoxyribonucleic acid (DNA) proves to be an excellent candidate for stimulus-responsive soft segments. With its unique biophysical and biochemical properties as well as distinctive electrochemical activity, DNA acts as a recognition element under various external stimuli, and as such, distinguishes itself from other smart materials such as stimuli-responsive synthetic or conducting polymers and biologically-responsive proteins.8 Specifically, multi-stranded DNA structures possess electrostatic properties and Hoogsteen pairing interactions dictated by hydrogen bonding or π-π stacking interactions, thus enabling fabrication of machine-like devices capable of pulling and stretching, as well as rotational, and even unidirectional motion under certain circumstances.9-10 Furthermore, DNA represents a powerful material in creating versatile building blocks to fabricate microstructures and devices. Among various multi-stranded DNA structures, the imotif quadruplex (i.e. cytosine-rich DNA strands) has shown to undergo reversible conformational changes when exposed to an external stimulus (e.g. pH alteration).11 The noncanonical interactions between the C+ and C base pairs (i.e., unprotonated and protonated cytosine) in the in-motif conformation form a four-folded structure with high stability at low pH levels (e.g., pH=5).11-13 Reversible interchange between a slack conformation at higher pH levels (e.g., pH=8) and a confined quadruplex at lower pH empowers the i-motif DNA polymers to be useful for synthesizing pH-fueled nanodevices. Some advantages of using CNTs and i-motif DNA nanolinker to develop pH-responsive electrochemical microactuators and biosensors are as follows: (i) CNT networks possess a significantly higher load-bearing capacity after being conjugated with i-motif DNA. (ii) The strong interactions between CNTs and i-motif DNA strands produce relatively low junction resistance to enhance electrical conductivity. (iii) CNTs further contribute to the i-motif stability as well as pH-triggered or 4


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temperature-driven conformational changes.14 Moreover, our previous research on i-motif DNAconjugated CNT materials demonstrated that the combination of favorable properties of i-motif DNA and CNTs results in controllable and reversible electrochemical activities of the hybrid by adjusting the pH level

14.

However, the absence of sturdy components such as polymeric binders and CNTs in the

developed material made it difficult to create free-standing micro-scale structures such as films and fibers. To address this issue, B-form double-stranded DNA (dsDNA) was used as a biosurfactant and polymeric binder to fabricate CNT fibers for electrochemical capacitors and actuators.15 The dsDNAs wrap around the surface of CNTs via π-π stacking and hydrophobic interactions through the numerous bases existing in the structure of the DNA strands. This strong physical interaction renders the CNTs water dispersible, thus preventing aggregation and increasing the concentration of CNTs in the hybrid materials. In addition, the dsDNA-wrapped CNT materials exhibit improved electrical and electrochemical properties due to the unique electrochemical properties of dsDNA via π-stacking of hydrogen bonding in DNA base pairs.16 The π-stacking array of hydrogen bonds forms a useful charge transfer state.17 Therefore, dsDNA-wrapped CNTs have the potential to make a robust framework to retain excellent electrical and mechanical properties. In this study, we developed electrically conductive free-standing CNT hybrid fibers using two different types of DNAs that possess reversible and controllable physical and electrochemical sensing and actuation properties upon pH alteration: (i) the dsDNA as a polymeric binder and biosurfactant to generate stable microfiber formation, (ii) the i-motif DNA nanolinker as a pH-responsive component which was conjugated on the dsDNA-coated CNTs via covalent bonding. Furthermore, during the wetspinning process of the hybrid material, the shear stress imposed by the inner wall of micro-sized nozzle allows for the alignment of CNTs in the microfibers, thus leading to high electrical conductivity.18-19 Lastly, CNTs have demonstrated electrocatalytic capabilites towards both oxidation and reduction of hydrogen peroxide (H2O2).20-39 Consequently, in addition to their great electrochemical actuation

5



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capabilities, i-motif DNA-conjugated CNT hybrid fibers are also apt to function as biosensing elements, due to their distinctive chemical and physical properties under certain pH conditions.40

2. 2.1

Results and Discussion i-motif DNA-conjugated dsDNA-wrapped SWCNT hybrid material To possess switchable and controllable redox properties in the hybrid materials, carboxyl

functionalized single-walled CNTs (SWCNTs) were dispersed in de-ionized (DI) water and were coated by dsDNA (up to 10 mg/ml of SWCNTs) through an hour of ultra-sonication (Figure 1 a). During the sonication process, the dsDNA (10,000 base pairs) was cut short, allowing for improved wrapping on the surface of SWCNTs.41 To conjugate i-motif DNAs onto the dsDNA-wrapped SWCNTs, a treatment with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/ N-hydroxysuccinimide (EDC/NHS) was used as a coupling reagent to generate covalent bonding between the amine groups on both ends of i-motif DNA strands and the carboxyl groups on the SWCNTs.42 Additionally, the abundance of phosphate groups within the dsDNA backbone induced negative charges, thus generating an electrostatic repulsion between the charged SWCNTs in the solution. As such, a well-dispersed dsDNA-wrapped SWCNT hybrid aqueous solution was eventually achieved. Next, the modified amine groups on 5’ and 3’ tails of the i-motif nanolinkers covalently bonded to the carboxyl groups in the SWCNTs. As reflected in highresolution transmission electron microscope (HRTEM) results (Figure 1 b), single or bundled SWCNTs were observed in a i-motif DNA-conjugated dsDNA-wrapped SWCNT (i-motif DNA/dsDNA-wrapped SWCNTs) dispersion solution showing a homogeneous solution achieved by coating with dsDNA and conjugation with i-motif DNA. Circular dichroism (CD) confirmed the working performance of i-motif DNA nanolinkers on the dsDNA-wrapped SWCNTs at different pH levels. Following assimilation of imotif DNA into the dsDNA-wrapped SWCNT structure, the i-motif DNA still exhibited its inherent characteristics at low and high pH levels (Figure 1c). The standard conformation of the i-motif DNA at pH 5 contains a positive band at 287 nm and a negative band at 256 nm. As the pH is gradually 6


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increased to 8, the i-motif DNA strands transform into a random coil conformation due to deprotonation of cytosine bases. Therefore, we found that the positive peak (287 nm) was shifted to 270nm and the intensity was significantly decreased. At the same time, the intensity of the negative peak (256 nm) was decreased and the peak was shifted to 250 nm. It is therefore inferred that the inherent properties of the DNA spacer are retained regardless of the presence of SWCNTs or dsDNA, i.e. a different type of DNA. Indeed, the i-motif DNA at pH 8 presented highly similar peak positions compared to dsDNA strands at pH 7.4 due to the fact that both DNAs reach random coil structures in slightly basic or neutral conditions. Nevertheless, a reduction was observed in both positive and negative peaks of the dsDNA due to the strong interstation between dsDNA strands and the surface of SWCNTs, resulting in the deformation of the random coil of dsDNA strands. To create nano- and micro-scale porosity while maintaining the reversible pH-responsiveness of the i-motif DNA, we made modifications to our previously developed self-assembly method which used ionic liquid/ethanol mixture as the coagulating agent.43 In that study, a highly porous, sponge-like microfiber composed of entangled nanofiber networks was achieved by self-assembly of dsDNAwrapped SWCNTs in an ionic liquid, namely molten salt. Removing the bound water from the dsDNA strands, the ionic liquid induced the formation of intertwined toroid structures of the dsDNA-wrapped SWCNT strands by increasing the hydrophobicity and van der Waals attractions among SWCNTs. Finally, the dsDNA-wrapped SWCNT fibers formed water-insoluble porous sponge structures while bypassing chemical crosslinking. However, utilization of ionic liquid as the coagulating agent is not preferred in the present study as it causes irreversible DNA deformation, which adversely affects the pH-responsiveness and functionality of the i-motif DNA.44 To induce the nanoparticle self-assembly while still maintaining the functionality of i-motif DNA, we chose nitric acid (HNO3) as the major coagulating agent. The self-assembly of the nanoparticles can also be controlled by adjusting the electrostatic interaction through changing pH, ionic strength or the electrolyte type.45 The fully negatively-charged i-motif DNA/dsDNA-wrapped SWCNTs were neutralized by addition and adsorption of proton ions (H+) in the HNO3 solution. Next, the electrostatic 7



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repulsion among the i-motif DNA/dsDNA-wrapped SWCNTs was decreased. At the same time, the van der Waals forces existing between SWCNTs was increased. Consequently, the i-motif DNA/dsDNAwrapped SWCNTs were aggregated and self-assembled into nanofibers (Figure 2a). To confirm the self-assembly of the i-motif DNA/dsDNA-wrapped SWCNTs, the dispersion solution was treated with different concentrations of HNO3 solutions. As such, the dispersed solution of i-motif DNA/dsDNAwrapped SWCNTs was diluted up to 0.1 mg/ml, the least amount required for Cryo-TEM. As shown in Figure 2b, no aggregation was observed in the i-motif DNA/dsDNA-wrapped SWCNTs dispersed solution, indicating full dispersion of the SWCNTs in DI water at 0 μM of HNO3. By increasing the HNO3 concentration to increase the proton (H+) molarity, we observed that the i-motif DNA/dsDNAwrapped SWCNTs aggregated into clusters at 1 μM, formed slack fibrous networks at 10 - 100 μM (Figure 2b), and assembled into much thicker and sturdier fibrous networks at 1 mM. We also observed similar assembly behavior for the dsDNA-wrapped SWCNTs with no i-motif DNA conjugation (Figure S1). Moreover, amplifying the degree of aggregation by increasing the HNO3 concentration resulted in a noticeable increase in zeta potential, confirming the electrical stabilization of the nanofiber network solution and neutralization (Figure 2c). UV-vis spectroscopy confirmed a high resemblance in the absorbance peaks of the i-motif DNA/dsDNA-wrapped SWCNTs with comparable van Hove singularity related transition peaks from the semiconducting and metallic SWCNTs before adding HNO3 (black line at 0 µM of HNO3) (Figure 2d). Usually, the van Hove singularity related transition peaks are observed in the well dispersed SWCNTs solution. As shown in Figure 2d, after adding and increasing HNO3 concentration up to 100 mM, the van Hove singularity related transition peaks were still clearly observed in the dispersed solution. Accordingly, it was inferred that, by increasing HNO3 concentration, the i-motif and double-stranded DNAs were still conserved among the SWCNTs after the i-motif DNAs were transformed into a four-folded structure, and dsDNA strands were transformed into an intertwined toroid structure in low pH conditions that were generated by adding HNO3. Therefore, this aggregation behavior stems from both the i-motif DNA and dsDNA on the surface of SWCNTs and not from the bundling of SWCNTs. 8


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2.2


pH-responsive hybrid fibers using wet-spinning method To fabricate the i-motif DNA/dsDNA-wrapped SWCNTs microfiber, the wet-spinning method was

used with a HNO3/ethanol coagulation bath. The i-motif DNA/dsDNA-wrapped SWCNTs dispersion solution was also extruded into a water bath as the control (neutral ambient). In the control bath, although the extruded fibers immediately coagulated, they gradually unfurled, forming loose networks (Figure 3a). In contrast, extraction in HNO3/ethanol (positively-charged bath) resulted in the formation of densely packed microfibers. We then obtained well-defined fibers with ~200 μm in diameter in dry state (Figure 3b). The density of the i-motif DNA/dsDNA-wrapped SWCNTs hybrid fiber was 0.65 g.cm-3, a significantly lower amount compared to our previously reported DNA/SWCNT hybrid fiber created with static DNA (d = 1.5 g.cm-3). Therefore, we deduced that the i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers have a more porous structure. Cross-sectional scanning electron microscope (SEM) revealed high fiber porosity as well as networks of congruent nanofibers with round crosssection (Figure 3c-e). The morphology was further affirmed by Transmission Electron Microscopy (TEM) on a cross-section of the hydrated fiber (Figure 3f). Specifically, it was observed that the i-motif DNA/dsDNA-wrapped SWCNTs aligned in parallel in each individual nanowire fiber, while the larger network of the nanowires was composed of partially-aligned and randomly directed bundles of nanofibers with a diameter of ~ 50 nm, formed in the protonation process by self-assembly of the imotif DNA nanolinker-conjugated CNT-based hybrid material. Furthermore, FT-IR spectroscopy was employed to confirm the cross-linkage bonding in the imotif DNA/dsDNA-wrapped SWCNT fibers (Figure 3g). As such, the absorbance bands exhibited by the dsDNA were recorded at 1603, 1654, 1692 and 1481 cm–1 from adenine, thymine, guanine, cytosine base pairs, respectively. Moreover, the absorbance band for the phosphate ester was at 1238 cm–1 while that of the 2-endodeoxyribose conformation was observed at 964 cm–1. The aromatic C=C stretch occurring at 1633 cm–1 is commonly seen in the IR spectra of SWCNTs. Thus, the formation of dsDNAwrapped SWCNT hybrid fibers results in intensity reduction from the base pairs on the DNA, and the 9



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cross-linked i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers exhibit a new absorbance phosphoramidate band at 1385 cm–1. Therefore, cross-linked i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers were prepared by modifying the sugar-phosphate backbone and the amine groups in the DNA base using a coupling agent, such as EDC. In the presence of EDC, the amino groups existing in the DNA base pairs partially form phosphoramidate bonds with the 5-terminal phosphate groups of DNA. In this way, DNA is able to form a crosslinking network with other compounds. To assess the interaction between the SWCNTs and DNA strands, the radial breathing modes (RBMs) were measured by Raman spectroscopy on raw SWCNTs, dsDNA-wrapped dispersion, crosslinked dsDNA-wrapped SWCNT fiber, and crosslinked dsDNA-wrapped SWCNTs using a range of i-motif DNA nanolinker concentrations. As shown in Figure 3h, we observed an upshift in the peaks after dispersing SWCNTs in dsDNA and further crosslinking by i-motif DNA. These results indicate a significant interaction between the SWCNTs and both the dispersant and crosslinker DNA strands. The pH responsive i-motif DNA nanolinkers produce microfibers with switchable porosity and surface topography, as confirmed by SEM analysis (Figure 4 and Figure S2). At pH 5, the hybrid fiber showed smooth and densely packed nanofiber networks due to i-motif DNA being at four-folded state (Figure 4 a and b). The four-folded structure is ~5 nm in length, as confirmed in our previous study,46 and is thus capable of fastening the dsDNA-wrapped SWCNT networks. On the contrary at pH 8, we observed higher roughness and numerous small pores on the surface of the hybrid fiber (Figure 4 c and d) as well as a micro-porous nanofiber network in the cross-section view of a SEM image (Figure 4 e). This can be pertained to the i-motif random coil structure at pH 8 with approximately 8 nm in length, also confirmed in our previous study. The dried fibers rapidly absorbed water resulting in a volume change from ~25 % to ~41 % at pH 5 to 8, respectively. Furthermore, we observed that pH-sensitive swellability of the fibers can be engineered such that the fibers expand and contract in a highly reversible fashion. As shown in Figure 4f, we observed reversible volume changes with ~ 12% diameter

10


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change per variation of pH from 5 to 8 due to the conformational changes of reversible pH-responsive imotif DNA nanolinkers.

2.3

Microactuation and Biosensing properties of the hybrid fibers The electrical conductivity of a CNT/polymer composite or hybrid material is ultimately defined

by the concentration and dispersion degree of SWCNTs within the polymeric matrix 47. In many cases, when developing CNT/polymer composites or hybrid materials, a common challenge is the tradeoff between micro-porosity and electrical conductivity. Usually, high micro-porosity of the fibers results in low electrical conductivity and high resistance due to the fact that the conduction pathway is usually occluded by the insulating polymeric materials between SWCNT fibers. However, our porous hybrid fibers showed high electrical conductivity, measured by I-V test (Figure S3). The measured conductivity of our porous hybrid fiber was ~ 27 S/cm, much higher than previous dsDNA-wrapped SWCNTs developed based on ionic liquids. This can be pertained to the fact that the hydrogen bonds between the matching base pairs fasten the dsDNA helix and the π-stacking interactions stand responsible for stabilizing the helix.17 Particularly, the i-motif DNA nanolinker has stacking i-motif formation on hydrogen bonding with protonated and unprotonated cytosine. The tunneling effect stemmed from the – interactions between the dsDNA/i-motif DNA and the walls of the SWCNTs results in a semiconductance behavior in DNA thus producing lower junction resistance.8 To further examine the hybrid fibers for electrochemical properties, we used cyclic voltammetry (CV) to measure the electroactivity of the hybrid fibers in Phosphate Buffer Saline (PBS) with pH 5 and 8 electrolytes. As shown in Figure 5a, the double layer capacitance at the fiber-electrolyte border governs the CV curve while signifying high fiber conductivity in wet conditions. In addition, the CV test of the i-motif DNA/dsDNA-wrapped SWCNTs hybrid fiber yielded strong redox peaks at pH 5 (Epa: 0.11V, Epc: -0.18V, scan rate: 100mV/sec) due to the presence of both i-motif DNA and dsDNA on the surface of SWCNTs. In our previous study, dsDNA-wrapped SWCNT fibers (no i-motif DNA added) showed weak redox peaks (Epa: 0.0V, Epc: -0.1V, scan rate: 25mV/sec, electrolyte: 2M NaCl) 11



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associated with the bonding of the phosphate groups or DNA base pairs through π-stacking charges along the nanotubes.18 Moreover, the redox peaks of i-motif DNA-conjugated SWCNTs (no dsDNA added) exhibited higher magnitudes of peak potential (Epa: 0.23V, Epc: 0.15 V, scan rate: 100mV/sec, electrolyte: pH 5 with 100 mM K+) associated with the bonding of the C+:C base-pair or TAA loops through π stacking charges along the nanotubes.48 Therefore, in the present study, the pair of strong redox peaks of the hybrid fiber at pH 5 is mainly caused by π stacking charges between the four-folded i-motif DNA and the surface of the nanotubes. While the generation of redox peaks is mainly from imotif DNAs, dsDNAs in the hybrid fiber shifted the position of the redox peak. Increasing the pH level to 8, we observed that the redox peaks were eventually diminished as a result of random coil structure formation in i-motif DNA by deprotonation of the C+:C base pair. In addition, we observed that the redox peak current of the hybrid fibers are quite stable during the twenty continuous CV test with various scan rates at pH 5 and 8, as shown Figure S4. The capacitance of the hybrid fibers was found to be ~59.9 F/g at pH 5 and ~47.8 F/g at pH 8 in PBS, which was normalized by mass of the SWCNTs. This value was significant compared to the value attained from the previous heat-treated electrospun DNA/SWCNT mats (~ 7 F/g) 49 as well as the usual SWCNT fibers (~ 30 F/g).42 Furthermore, the peak in the anodic current is proportional to the square-root of the potential scan rate which in turn is in line with the hypothesis of a diffusion-controlled electrochemical process with the Randles-Sevcik governing equation (Figure 5b and Figure S5),50 which further demonstrates facilitation of electron transfer between SWCNTs via the stacked hydrogen bonding of i-motif DNA and dsDNA. To further characterize the actuation capabilities, the fibers were imposed to an isotonic stress (~ 1.4 MPa) and square wave potential (+0.2 to -1.0 V) in a 2M NaCl electrolyte solution. As shown in Figure 5c, the actuation strain of the hybrid fiber with i-motif DNA nanolinkers showed excellent actuation stability with ~0.15% expansion/contraction. On the other hand, the fibers with no i-motif DNA (dsDNA-wrapped SWCNTs) showed high creep behavior. This observation was in agreement with the results of our previous study where we studied the crosslinking effect of the hybrid fiber to prevent creep behavior (Figure S6).8 The elasticity of the i-motif DNA networks in the hybrid fibers 12


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contributes to high reversibility upon deformation as well as stability with time. On the contrary, CNT fibers untreated with i-motif DNA nanolinkers undergo creep and show reduced elasticity (Figure 5c). Accordingly, further crosslinking the dsDNA-wrapped SWCNT hybrid fibers by i-motif DNA or dsDNA itself results in a significantly enhanced actuation stability which is in turn much higher than that of dsDNA-wrapped SWCNT hybrid fiber crosslinked by other crosslinking agents. In our previous paper, the dense dsDNA-wrapped SWCNT hybrid fiber showed robust electrochemical actuation at low potential ranges. However, the actuation speed was not improved by much due to the slow mass transport of the electrolyte by the compact internal network. This result was pertained to the chargedischarge performance within the internal structure. As a solution, associating the dsDNA-wrapped SWCNT nanowires with DNA spacer promotes the electrocatalytic properties of the SWCNT through electron-transfer reaction. The π-stacked hydrogen bonds among the base pairs in dsDNA along with those in the C+:C pairs in the i-motif DNA allow for improved electron transport compared to other forms of DNA such as single-stranded or the random coil structures. Therefore, we observed fast charge-discharge behavior in i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers compared with that in the hybrid fiber with no association of i-motif DNA (Figure S6 b). This charge-discharge behavior might have been impactful in improving the actuation response of the hybrid fibers. To evaluate biosensing capability, we utilized the hybrid fibers as a hydrogen peroxide (H2O2) electrochemical sensor and achieved a steady state current proportional to the H2O2 concentration at different pH conditions. Specifically, a single hybrid fiber was immersed in the electrochemical cell along with a reference and counter electrode. Then small amounts of H2O2 were added to the electrolyte gradually, and the CV test was performed on each H2O2 concentration. The redox peak (Epa: 0.1V) of the hybrid fiber at pH 5 was gradually decreased, shifted, and finally disappeared by increasing the concentration of H2O2 from nM to mM levels (Figure 6 a and d). To further understand this behavior, Circular Dichroism (CD) spectroscopy was used to characterize the i-motif DNA structure. It was then observed that the i-motif conformation maintained its four-folded structure at pH 5 when treated with up 13



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to 10mM H2O2 (Figure S7), thus confirming that the redox peak reduction is not due to the structural deformation of the i-motif DNA. It has been shown that H2O2 in the presence of sodium counterion can form stable complex with the phosphate group on the DNA strands. Also, the H2O2/DNA complex is more stable and durable than H2O/DNA.51 Therefore, we anticipated that H2O2 might be forming complex structures with the phosphate groups on the i-motif DNA and dsDNA strands. As such, the complex inhibits the charge transfer between the i-motif DNA and SWCNTs, resulting in reduction of the redox peak current at low H2O2 concentrations (< 100 µM) (Figure S8). Moreover, SWCNT by itself has shown to be an effective catalyst for H2O2 oxidation.52-53 While most i-motif DNA and dsDNA could have formed complexes with H2O2 (> 100 µM), the unbonded H2O2 could have also been oxidized on the surface of the SWCNTs, resulting in creation of a new redox peak (Epa: 0.35V). As reported by various papers, the oxidation and reduction peak potential of H2O2 is changed by electrode material and structure, type of reference electrode, electrolyte etc. For instance, it is reported that a amine functionalized multi-walled carbon nanotubes (MWCNTs) based non-enzymatic H2O2 sensor showed a similar CV behavior54. Particularly, upon increasing the H2O2 concentration, oxidation peak current increased at ~ 0.2V and reduction peak current decreased at ~ -0.3 V. The reported oxidation peak potential is therefore similarly low (~ 0.2 V in the amine functionalized MWCNTs54 versus ~ 0.32 V in our results). The difference is mainly due to different type of carbon nanotubes and coating materials. The peak current of the new redox peak was gradually increased by increasing the concentration of H2O2 within the mM range (Figure 6d). On the contrary, when the hybrid fibers were exposed to H2O2 at pH 8, no change was observed in the CV curves up to 100 µM of H2O2 (Figure 6b and c). This observation can be pertained to the random coil structure of the i-motif DNAs at pH 8 where the electron transfer cannot be inhibited by H2O2/i-motif DNA or H2O2/dsDNA complexes. Nevertheless, by adding higher concentrations of H2O2 up to 8 mM, a new redox peak (Epa: 0.35V) was observed similar to the one achieved at pH 5, with the peak current gradually increased at pH 8 (Figure 6e and f). This experiment was repeated for multiple times with different concentrations of H2O2, and the results indicated high consistency with the graphs depicted in Figure 6e and f (Figure S9). On this 14


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account, the electrochemical redox activity of the i-motif DNA/dsDNA-wrapped SWCNT hybrid fiber responded rapidly and favorably to the variation of H2O2 concentration at different pH conditions, and thus, the developed hybrid fiber can be an applicable material for simultaneous detection of pH and the target (H2O2) level by analyzing the distinctive redox peak positions and their corresponding peak currents.

3.

Conclusion

In summary, wet spinning method was utilized to prepare i-motif DNA-conjugated dsDNA-wrapped SWCNT hybrid microfibers with a hierarchical assembly of nanofiber networks. The incorporation of imotif DNA proved effectual in the debundling process of dsDNA-wrapped SWCNTs in addition to serving as a pH-responsive nanolinker after being conjugated. As such, the porosity and swellability of the hybrid fibers can be easily tuned with pH level alteration, causing the i-motif DNA to undergo conformational change from quadruplex (pH 5) to random duplex (pH 8). Moreover, coalescing the SWCNTs with both DNA types (i.e., double-stranded and i-motif) yielded in debundling and partial nanotube orientation with reduced junction resistance, thus elevating the electrical conductivity. In addition, the hybrid fiber showed excellent electrochemical behavior along with pH responsive redox activity due to the charge transfer between four-folded i-motif DNA and dsDNA on the surface of the SWCNTs. Consequently, the fabricated fibers exhibited outstanding capacitance, actuation and sensing capabilities. The crosslinked i-motif DNA networks proved specifically reliable and stable against actuation-driven creep. Lastly, we suggested a great potential in electrochemical H2O2 sensing by demonstrating the proportionality of current with concentration of H2O2. The remarkable properties of the fabricated microfibers prove them applicable for a variety of purposes. The robustness and effortlessness of the present fabrication method exploits the unique reversible transformability of the imotif DNA upon pH adjustment, a tool which can come in handy for a plethora of biomedical applications. Specifically, the remarkable electrical conductivity and strain behavior of these hybrid 15



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fibers can be effectively employed as a multifaceted sensor-actuator smart material to fabricate artificial muscles.

4.

Experimental Section

Materials The i-motif DNA molecules with four stretches of CCC and 5’- and 3’-terminal amino groups (H2NC6H12-5’-CCCTAACCCTAACCCTAACCCTAAA-3’-C6H12(CH2OH)NH2) were purchased from IDT. The SWCNTs were purchased from CNI Inc. The following were purchased from Sigma Aldrich: DNA (double-stranded, salmon sperm, 10,000 base pairs), N-hydroxysuccinimide (NHS), Ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC), ethanol, HNO3, NaCl, and H2O2.

Fabrication of i-motif DNA/SWCNT microfiber To perform functionalization, the SWCNTs were first sonicated for 1 hour in a solution of Sulfuric Acid (H2SO4) and Nitric Acid (HNO3), combined with 3:1 ratio. After filtration, the SWCNTs were neutralized through washing with de-ionized water. The dsDNA (0.4 wt %) was dissolved in deionized water for 6 hours. Next, the functionalized SWCNTs were sonicated with dsDNA for 1 hr. The sonication process resulted in the length of the dsDNA (10,000 base pairs) to be trimmed. The dsDNAwrapped SWCNTs solution was then allowed to be stirred for 24 hours after the pH was increased to 6.5. Then the dsDNA-wrapped SWCNT solution was treated with EDC/NHS for 30 min. Amine group functionalized i-motif DNA was added in the solution and the reaction blend kept in stir for 24 hours at room temperature. The hybrid fibers were made by injecting an aqueous solution of the obtained i-motif conjugated dsDNA-wrapped SWCNTs into a coagulation bath of 0.3M HNO3 and ethanol (v/v of 9:1) at 16 rpm. The fibers were left in the bath for 30 min to allow for completion of the coagulation process. Finally, the fibers were washed with deionized water multiple times and were air-dried in a vertically suspended condition. 16


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Characterizations The multi-scale structure of the fibers was characterized with SEM (Hitachi Model S4700, Japan) and Cryo-TEM (Tecnai-12; FEI, USA), and the images were obtained by a charge-coupled detector (CCD) camera (Multiscan 600W; Gatan) operating at −170o C. Holey carbon grids were loaded with aqueous films of SWCNT-DNA hybrids and were then frozen before drying of samples. The frozen grids were stored in liquid nitrogen until being transferred to a cryotransfer (Gatan model 630; Gatan, USA) at approximately –190o C. Positive staining of DNA was done using uranyl acetate. The zeta potential of the DNA-SWCNT hybrids was determined using a Malvern Zetasizer Nano series instrument. Cyclic voltammetry and actuation tests were carried out by a triple electrode electrochemical cell paired with CHI 600B potentiostat (USA). SWCNT-DNA hybrid fiber, Ag/AgCl, and Pt mesh were used as working, reference, and counter electrodes, respectively. The actuation of the fibers was quantified using a dual-mode force sensor/controller (Aurora Scientific) along with an AD Instruments MacLab interface. One end of the fiber was clamped while the other end was connected to the lever arm in the electrochemical cell. For the evaluation of H2O2 sensing, small amounts of H2O2 were added slowly to the electrolyte. The CV test was performed for each H2O2 concentration as formerly described. The conductivity and electrochemical capacitance of the fibers were measured as described in our previous work 8. Briefly, a custom four-probe conductivity cell was used at a constant temperature and humidity. The circular pin-shaped electrodes with 0.2 cm distance from each other were connected to the fibers by silver paint. A potentiostat/galvanostat (eDAQ) was used to apply a constant current between the outer electrodes. Next, a digital multimeter (Agilent 34401A) was used to measure the potential difference between the inner electrodes. The conductivity was then calculated using the conductivity formula: S=l/RA, where R is the electrical resistance, A is cross-sectional area, and l is the length of the specimen. The capacitance of the fiber was measured from the CV curves obtained within the potential window of -0.6 to 0.6 V with 20-100 mV/s scan rates in the PBS at pH 5 (Figure S6). The capacitance (Cmass) was

17



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then calculated using the formula: Cmass = I/(ν×m), where I is the current plateau at +0.4 V, v is a given scan rate, and m is the weight of the hybrid microfiber.

Acknowledgement The authors gratefully acknowledge funding from the Air Force Office of Sponsored Research under award (FA9550-15-1-0273). S.R.S. would like to recognize and thank Brigham and Women’s Hospital President Betsy Nabel, MD, and the Reny family, for the Stepping Strong Innovator Award through their generous funding. P.P.S.S.A. was supported by NIH grant 5T32EB016652-02, American Heart Association grant 17SDG33660925, and an American Fellowship from American Association of University Women. J.S. acknowledges the KIST project (2E27930).

Supporting Information: The following supporting materials are available online free of charge via http://pubs.acs.org: Cryo-TEM graphs of the coalescence of the dsDNA-wrapped SWCNT without i-motif DNA, SEM images of fiber cross-section, cyclic voltammogram with various scan rate, electrochemical actuation data and charge and discharge behavior of the i-motif DNA/dsDNA-wrapped SWCNT, CD spectra of the i-motif DNA at various H2O2 concentrations, schematic for the redox activity mechanism of the imotif DNAs/dsDNA-wrapped SWCNTs at various H2O2 concentrations (pH 5).

18


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Figures

Figure 1. (a) Schematic diagram showing synthesis procedure of i-motif DNA conjugated dsDNAwrapped SWCNTs and the working cycle between random coil (pH 8) and four-folded (pH 5) structures. (b) An HRTEM image of i-motif DNA/dsDNA-wrapped SWCNT networks. Magnified image showed single or bundled SWCNTs indicated by black arrows. (c) CD spectra of dsDNA-wrapped SWCNT conjugated with i-motif DNAs at pH 5 (red) and pH 8 (blue), as compared to dsDNA (black) and dsDNA-wrapped SWCNTs (green). Concentration of sample solution: 0.2 mg/ml.

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Figure 2. (a) Schematic illustration showed the formation process of nanofiber networks with the imotif DNA/dsDNA-wrapped SWCNTs. Left to right: morphology changes by addition of positive charges (HNO3) to the neutral liquid (water). A network of the i-motif DNA/dsDNA-wrapped SWCNT nanofibers is formed via coalescence of nanoparticle aggregates. (b) Cryo-TEM micrographs showing the coalescence of the DNA-CNT conjugates with increases in the concentration of HNO3. (c) Zeta potential and (d) UV-Vis spectroscopy of the i-motif DNA/dsDNA-wrapped SWCNTs dispersed solution (~0.1 mg/ml) as a function of the concentration of HNO3.

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Figure 3. (a) Coagulation behavior of extruded i-motif DNA/dsDNA-wrapped SWCNT dispersed solution (concentration: 10 mg/ml) in water and 0.3 M HNO3/ethanol coagulation baths. (b) SEM images of the i-motif DNA/dsDNA-wrapped SWCNT microfibers with a diameter of ~200 µm. (c) Cross-section view of the i-motif DNA/dsDNA-wrapped SWCNT microfibers consisting of nanofiber networks. (d) Cross-section image of individual nanofibers with round-shape. (e) Network junctions of nanofibers. (f) TEM of a cross-sectional slice of the i-motif DNA/dsDNA-wrapped SWCNT microfiber 28


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showed partially aligned the fibrous structure. Dark regions indicate positive staining of DNA by uranyl acetate. (g) FT-IR spectra of i) raw dsDNA, ii) dsDNA-wrapped SWCNT hybrid fiber, and iii) crosslinked i-motif DNA/dsDNA-wrapped SWCNT hybrid fiber. (h) Raman spectra in the radial breathing modes of i) raw SWNT, ii) uncrosslinked dsDNA-wrapped SWCNTs iii) crosslinked dsDNAwrapped SWCNTs by EDC/NHS, iv) crosslinked dsDNA-wrapped SWCNTs by 0.2 mg/ml i-motif DNA, v) crosslinked dsDNA-wrapped SWCNTs by 0.5 mg/ml i-motif DNA, and vi) crosslinked dsDNA-wrapped SWCNTs by 1.0 mg/ml i-motif DNA.

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Figure 4. Change of surface roughness and internal structure of the i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers by conformational change of i-motif DNA under two distinct pH conditions at pH 5 (a, b) and pH 8 (c, d, e). (f) Reversible change of diameter for the expansion and contraction of imotif DNA/SWCNT hybrid fiber during pH switching from pH 8 to pH 5.

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Figure 5. (a) Cyclic voltammogram of the i-motif DNA/dsDNA-wrapped SWCNT hybrid fibers during potential cycling (scan rate = 100 mV/s, electrolyte = PBS at pH 5 and 8). (b) Potential scan rate dependence of peak anodic current, ipa, for the CNT-DNA microfiber at pH 5. (c) Electrochemical actuation mechanism of the CNT-DNA microfiber. Strain versus time associated with application of a step function potential to microfibers with (solid line) and without (dashed line) the DNA spacer (between +0.2 V and -1.0 V vs. Ag/AgCl, Time interval = 30 s, electrolyte = 2 M NaCl aqueous solution, applied load = 1.4 MPa).

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Figure 6. Biosensing performance of the hybrid fiber; Dependence of the electrochemical behavior on the concentration of hydrogen peroxide: a, b) Change of cyclic voltammogram with the concentration of hydrogen peroxide at pH 5 (a) and 8 (b) in the H2O2 concentration in the range of 1 nM- 100 μM. c) Comparison of the dependence of current on the H2O2 concentration in the range of 1 nM- 100 μM at pH 5 and 8 associated with the potential shown with blue dotted line in (a) and (b). d, e) Change of cyclic voltammogram with the concentration of H2O2 in the range of 0.5 mM- 8 mM at pH 5 (d) and 8 (e). f) Comparison of the dependence of current on the H2O2 concentration in the range of 1 nM- 100 μM at pH 5 and 8 associated with the potential shown with blue dotted line in d and e. (Potential= V, scan rate = 100 mV/s, electrolyte = PBS)

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Table of Contents/Abstract Graphic

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pH-Responsive DNA Nanolinker Conjugated Hybrid Materials for

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