Oscillatory Reaction Induced Periodic C-Quadruplex DNA Gating of

Feb 22, 2017 - Many biological ion channels controlled by biochemical reactions have autonomous and periodic gating functions, which play important ro...
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Oscillatory Reaction Induced Periodic C‑Quadruplex DNA Gating of Artificial Ion Channels Jian Wang,† Ruochen Fang,∥ Jue Hou,‡ Huacheng Zhang,*,‡ Ye Tian,*,§ Huanting Wang,‡ and Lei Jiang†,‡ †

Key Laboratory of Bio-inspired Materials and Interface Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ‡ Department of Chemical Engineering, Monash University, Clayton, Victoria 3800, Australia § Beijing National Laboratory for Molecular Sciences, Key Laboratory of Green Printing, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ∥ Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *

ABSTRACT: Many biological ion channels controlled by biochemical reactions have autonomous and periodic gating functions, which play important roles in continuous mass transport and signal transmission in living systems. Inspired by these functional biological ion channel systems, here we report an artificial self-oscillating nanochannel system that can autonomously and periodically control its gating process under constant conditions. The system is constructed by integrating a chemical oscillator, consisting of BrO3−, Fe(CN)64−, H+, and SO32−, into a synthetic protonsensitive nanochannel modified with C-quadruplex (C4) DNA motors. The chemical oscillator, containing H+-producing and H+-consuming reactions, can cyclically drive conformational changes of the C4-DNA motors on the channel wall between random coil and folded i-motif structures, thus leading to autonomous gating of the nanochannel between open and closed states. The autonomous gating processes are confirmed by periodic high−low ionic current oscillations of the channel monitored under constant reaction conditions. The utilization of a chemical oscillator integrated with DNA molecules represents a method to directly convert chemical energy of oscillating reactions to kinetic energy of conformational changes of the artificial nanochannels and even to achieve diverse autonomous gating functions in artificial nanofluidic devices. KEYWORDS: C-quadruplex DNA, gating, self-oscillating, conformational changes, artificial nanochannel pH,13−17 light,18−20 temperature,21,22 voltage,23,24 pressure,25 specific ions,26−28 or molecules,29−31 or by dual stimuli such as pH and temperature,32−34 pH and ion,35 pH and voltage,36 or pH and light.37,38 To date, the gating processes of the artificial nanochannels are discontinuous and not real-time, because the switchable stimuli have to be controlled externally by manual switching.16,39 In addition to the simple stimuli-responsive gating, many biological ion channels with much more complex gating processes are intelligently controlled by biochemical reactions.1,40 However, it is still a challenge to use chemical

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rtificial nanochannels have attracted enormous research attention in the past few years owing to their potentials in mimicking smart ion transport features of biological ion channels1,2 and wide applications in nanofluidics,3−5 biosensors,6−8 and energy conversions.9−11 In the presence of specific stimuli, an artificial ion channel can be switched from a closed state, in which no or low ionic current passes through the channels, to an open state that is evidenced by a high transmembrane ionic current. This smart gating function gives rise to an artificial molecular gate modified on the nanochannel. An artificial stimulus-gated nanochannel system is generally constructed by functionalizing the channel with responsive molecules and introducing corresponding physical or chemical stimuli. With diverse smart functionalization methods,12 the nanochannels can be gated by a single stimulus, such as © 2017 American Chemical Society

Received: December 30, 2016 Accepted: February 22, 2017 Published: February 22, 2017 3022

DOI: 10.1021/acsnano.6b08727 ACS Nano 2017, 11, 3022−3029

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Figure 1. Design strategy for autonomous and periodic gating of the C4-DNA-modified nanochannel in a constant reaction solution by the a BrO3−−Fe(CN)64−−HSO3− oscillator. The oligonucleotide sequence (5′ to 3′) of C4-DNA is 5′-(NH2)-(CH2)6-AAAAAAAAAA CCC TAA CCC TAA CCC TAA CCC (Bodipy493/503)-3′. (a) Schematic illustration of the autonomous gating cycle of the nanochannel driven by the chemical oscillator. In one-half of the reaction cycle, C4-DNA molecules on the channel wall are protonated by the H+-producing reaction and fold into four-stranded i-motif structures upon formation of C+−C base pairs, resulting in a closed state of the channel (right). In the other half of the reaction cycle, the folded DNA molecules are redeprotonated by the H+-consuming reaction and unfold to single-stranded structures, and the channel is reopened (left). As the oscillating reaction proceeds, the nanochannel can be opened and closed autonomously and periodically by the chemical oscillator. (b) Structure of protonation of cytosine. (c) Molecular structure of a C+−C base pair between protonated and nonprotonated cytosines. (d) Ion conductance of the C4-DNA-modified nanochannel against pH values. A sigmoidal fit to the conductance values is drawn as a continuous line.

reactions to control the gating processes of the synthetic nanochannels. Oscillatory reaction systems are well established in chemistry,41 and oscillating reactions were widely implemented to develop self-oscillating polymers and hydrogels.42−44 For example, the Belousov−Zhabotinsky (BZ) reaction was used to develop cyclic hydrogels43 that exhibit spontaneous and autonomous periodic swelling−deswelling changes under constant conditions and to design autonomic functional surfaces composed of synthetic self-oscillating polymers.45 Also, pH oscillating reactions were used to autonomously switch conformations of DNA motors,39,46 to drive macroscopic pendulum-like motion of a gel actuator,47 to stimulate a synthetic muscle,48 to switch color of a photonic crystal hydrogel,49 and to control rhythmic aggregation of nanoscopic particles.50 In our previous work, the switchable conformational changes of C-quadruplex (C4) DNA motors were found to open and close the synthetic nanopores in the smart gating nanochannel−DNA system.9 Therefore, on the basis of these artificial self-oscillating systems, it is of considerable interest in using oscillating chemical reactions combined with DNA molecules to autonomously control gating processes of the artificial nanochannels.

Herein, we report an artificial self-oscillating nanochannel system that displays both autonomous and periodic gating functions under constant conditions by using a chemical oscillator integrated with C4-DNA molecules to open and close the artificial nanochannel. Different from previous nanofluidic systems simply gated by stimuli, this oscillatory nanochannel system can directly convert the chemical energy of oscillating chemical reactions to the kinetic energy for opening and closing of the nanochannel. The autonomous and periodic gating processes of the nanochannels are confirmed by the cyclic and periodic high−low ionic current oscillations of the nanochannel system measured under constant conditions. The system also shows a temperature-dependent gating period ranging from 40 to 10 min on increasing the working temperature from 30 °C to 45 °C. As an example of building autonomously gated nanofluidic systems, both the strategy and experimental results provide an avenue to construct future self-gating nanochannel devices for much more intelligent mass transport control at the nanoscale. The design strategy of the oscillatory nanochannel system is shown in Figure 1a. A well-studied chemical oscillator, consisting of BrO3−, Fe(CN)64−, H+, and SO32−,51 is integrated into a single bullet-shaped nanochannel modified with C4-DNA 3023

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nanochannel was opened and closed alternatively, resulting in an uncontinuous ionic current change (Figure S6c in the SI). The pH-dependent I−V response is correlated to channel conductance values of 1.1, 1.7, and 1.9 nS for pH 4, 7, and 9, respectively (Figure 1d). The conductance values of the nanochannel were observed by fitting the slopes of the I−V curves. As shown in Figure 1d, these results reveal that the pH required to activate the channel is close to the pKa of the cytosine groups, indicating that the DNA conformational change is responsible for the channel gating. Moreover, as the DNA strands contain both negatively charged phosphates and protonatable bases (Figure S7 in the SI), protonation of bases could reduce the net negative charges of the DNA strands. Therefore, the pH-regulated surface charge change is the other important factor for the channel gating. Figure 2 describes the experimental apparatus of the oscillatory nanochannel system. The C4-DNA-modified nano-

molecules. Each oscillatory reaction cycle of the chemical oscillator contains two primary composite reactions. One halfreaction cycle produces protons (H+) during the oxidation of sulfite by bromate, while the other consumes H+ during the oxidation of ferrocyanide by bromate. When the oscillating reactions proceed inside the C4-DNA-modified nanochannel, the reactions can be coupled to the conformational changes of the DNA motors immobilized on the channel wall. The C4DNA molecules initially form single-stranded random coil structures under neutral conditions (Figure 1a, left) because the pKa value of cytosine is approximately 4.252 (Figure 1b). Then, as the cytosines of the C4-DNAs are grdually protonated by the H+-producing reaction of the chemical oscillator, the singlestranded DNA structures are transformed to i-motif structures53 (Figure 1a, right). The four-stranded i-motif structure is formed on C+−C base pairs39,52 between protonated and unprotonated cytosine residues (Figure 1c). A C4-DNA strand containing 12 cytosines can form six intramolecular C+−C base pairs (Figure 1a, right). Accordingly, the folded i-motif structures can be retransformed to random coil structures by the H+-consuming reaction of the chemical oscillator (Figure 1a, left). As a result, the chemical energy of oscillating reactions inside the nanochannel is directly converted to the cyclic conformational change of the DNA motors on the channel wall. Due to the C4-DNA motors with both negatively charged phosphates and protonatable bases, the cyclic conformational transitions of the DNA motors between random coil structures and i-motif structures driven by the chemical oscillator can induce an autonomous switching system of the nanochannel between highly charged, large-sized, and hydrophilic state (open state) and lowly charged, small-sized, and hydrophobic state (closed state), thus achieving autonomous control of the gating process of the nanochannel under constant conditions.

Figure 2. Experimental setup for the oscillatory nanochannel system. A NaBrO3 solution (I) and a mixed solution of Na2SO3, K4Fe(CN)6, and H2SO4 (II) are pumped slowly and simultaneously from reservoirs into the continuously stirred two half-cells to form the oscillating reaction solution (III). Initial mixture in each cell: V = 40 mL; [NaBrO3]0 = 0.065 M; [Na2SO3]0 = 0.075 M; [K4Fe(CN)6]0 = 0.02 M; [H2SO4]0 = 0.01 M. The input rate of solutions I and II controlled by one peristaltic pump is 1.37 × 10−2 mL·s−1, while the output rate of solution III controlled by the other peristaltic pump is 2.74 × 10−2 mL·s−1. The stirring rate is 625 rpm. The temperature of the system is controlled by a thermostat. Realtime pH variation of the oscillating reaction solution and real-time ionic current oscillation induced by the channel gating are recorded simultaneously by a pH meter and a picoammeter, respectively (see Figure S8 in the SI). The insets are photographs of solutions I, II, and III, respectively.

RESULTS AND DISCUSSION Single bullet-shaped nanochannels with a length of 12 μm were prepared by asymmetrically etching single-ion-tracked polyethylene terephthalate (PET) membranes (see Experimental Section in the Supporting Information (SI)). Scanning electron microscope (SEM) images well illustrate the small tip, the large base, and the cross section of the bullet-shaped nanochannel etched for about 3 min (Figure S1 in the SI). The tip diameter (dt) of the nanochannel is 33.2 ± 3.7 nm, while the base diameter (db) is 239.2 ± 19.5 nm. Statistic results also showed that the bases of the bullet-shaped nanochannels fabricated under different etching times were about 6 times larger than the tips (Figure S2 in the SI). After observation of the single nanochannels, the inner surfaces of the nanochannels were chemically modified with C4-DNA molecules by a two-step modification method (Figure S3 in the SI). X-ray photoelectron spectroscopy, contact angle, and ionic current measurements of the nanochannel membranes before and after modification confirmed that C4-DNA molecules were successfully grafted on the channel walls (Figures S4 and S5 in the SI). The pH-gating property of the C4-DNA-modified nanochannel was systematically investigated by measuring current− voltage (I−V) curves of the nanochannel using 0.1 M KCl with different pH values as an electrolyte solution. Figure S6a in SI depicts the I−V curves of the single nanochannel modified with C4-DNA molecules. By manually increasing the pH from 3 to 9, a significant increase in the transmembrane ionic current was observed under the same applied bias (Figure S6b in the SI). By manually switching the pH value between 4 and 8, the

channel membranes were mounted in two halves of a large electrochemical cell (Figure S8a). A NaBrO3 solution (I) and a mixed solution (II) containing Na2SO3, K4Fe(CN)6, and H2SO4 were pumped slowly into the continuously stirred cells to form the oscillating reaction solution (III). The inset photographs are solutions I, II, and III, respectively. The color of solution III changed to yellow due to the oxidation of ferrocyanide to ferricyanide. Initial concentrations of the reactants are [NaBrO3] = 0.065 M, [Na2SO3] = 0.075 M, [K4Fe(CN)6] = 0.02 M, and [H2SO4] = 0.01 M. The volume of the reaction solution in each cell is 40 mL. The input rate of solutions I and II controlled by one peristaltic pump is 1.37 × 10−2 mL·s−1. To keep the solution volume in each cell constant at 40 mL, solution III was also pumped out with an output rate of 2.74 × 10−2 mL·s−1. The stirring rate is 625 rpm. The temperature of the system is well controlled by a thermostat. 3024

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systematically measured conductivity, temperature, redox potential, and pH changes of the reaction solution simultaneously with ionic current oscillation. As illustrated in Figure S12a in the SI, the ionic current of the reproduced nanochannel system also oscillated simultaneously with pH oscillation. The redox potential of the solution autonomously oscillated between 260 and 285 mV, and its period was similar to the pH oscillation, while the solution temperature oscillated slightly between 35.0 and 35.4 °C (Figure S12b in the SI). The ion conductivity of the reacting solution fluctuated slightly between 1.03 and 1.06 S·m−1, which did not oscillate periodically because it was a combination of the ion-concentration oscillation and temperature oscillation of the reaction solution. Among the above four parameters, the ion conductivity fluctuations and the pH oscillations are contributing to ionic current oscillations of the nanochannel system. On the basis of the experimental conductivity variation, theoretical conductance change induced by the conductivity variation could be calculated from eqs S1−3 (in the SI). However, the calculated values of the ionic currents are very stable (Figure S12c in the SI), which proves that the ionic current oscillations are mainly induced by the pH-oscillation-driven channel gating. Besides, although thermal fluctuations of the ion conductivity of electrolyte solution,54 transient fluctuations of the nanochannel surface charge,48,49 and dynamic electrowetting of the channel wall17,50 have been proved to induce ionic current noise of the nanochannel system, the ion current noise of the oscillatory nanochannel systems (Figure S10 and Figure S12c in the SI) is much higher than the noise of the ionic currents measured in electrolyte solutions (Figure S6 in the SI). Additional experimental ionic current and pH curves measured with and without peristaltic pumps demonstrated that the high noise of the ionic current in the oscillatory system is mostly attributed to the peristaltic pumps (Figure S12d in the SI). To further confirm that C4-DNA motors can undergo reversible conformational transitions in the self-oscillating system, we systematically characterized circular dichroism (CD) spectra of C4-DNA molecules under different pH conditions from 3.5 to 6.5 and different temperatures from 20 to 45 °C. CD spectra of C4-DNA in response to different temperatures at various pH values are summarized in Figure S13 in the SI. At room temperature, the transition of C4-DNA from random coil structure to i-motif structure takes place at about pH 7.55−57 At 30 °C, a significant change of CD spectra observed at pH 6.5 indicated that the formation of the i-motif structure occurred at about pH 6.5 (Figure 4a). As the temperature increased to 35 °C, however, the i-motif conformation change started at pH 6.0 (Figure 4b), and it could be maintained at steady-state temperatures of up to 40 °C (Figure 4c). Furthermore, on increasing the temperature to 45 °C, an obvious change in the CD spectrum, corresponding to the deformation from the four-stranded i-motif to the singlestranded structures, could also be observed at pH 5.5 (Figure 4d). CD results suggest that pH and temperature are two key factors that control the conformational transition of C4-DNA between the i-motif and single-stranded structures. The reversible structural transition of C4-DNA at high temperatures (from 30 to 45 °C) provides a basis for the further study of the effect of temperature on the gating functions of the oscillatory nanochannel system. As the temperature was confirmed as one important factor for the conformational transition of the DNA molecules, the dependence of the autonomous gating features of the

Real-time pH variation of the reaction solution induced by the oscillating chemical reactions is measured with a benchtop pH meter; simultaneously, the real-time ionic current oscillation induced by the autonomous channel gating is recorded by a picoammeter. With the above experimental conditions, the pH value of the oscillating reaction solution at 35 °C initially decreased from above 7.2 to below 3.4 and then oscillated stably between 6.8 and 3.5 with a period of about 1600 s (Figure S9 in the SI). Consequently, the nanochannels are enforced to open and close periodically by the oscillator (Figure 3a). In Figure 3b and

Figure 3. Autonomous and periodic gating of the nanochannel by the chemical oscillator at 35 °C. (a) Schematic illustration of the cyclic opening and closure of the nanochannel driven by the chemical oscillator. (b) Observed continuous, autonomous, and periodic pH oscillation of the reaction solution. (c) Ionic current oscillation of the nanochannel induced by the cyclic opening and closing of the nanochannel driven by the pH oscillation. The applied voltage for current measurement is 400 mV.

c the pH oscillations of the chemical oscillator are displayed together with the simultaneously measured transmembrane ionic current of the nanochannel at 400 mV (see Figure S10 in the SI for original data of ionic current oscillation). The ionic current oscillations indeed follow the pH value, indicating that the nanochannel present in the oscillating reaction solution undergoes autonomous gating as designed. The full amplitude of the ionic current oscillation is approximately 0.4 nA at 400 mV. Due to the dependence of the ionic current on the applied voltage, the amplitude of the ionic current would increase with increasing applied voltage; for example, the full amplitude of the current oscillation at 2.0 V is about 1.5 nA (Figure S11 in the SI). In order to further confirm that the ionic current oscillation is actually induced by the channel gating, we further 3025

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Figure 5. Temperature-dependent gating functions of the oscillatory nanochannel system. pH oscillations and corresponding current oscillations measured under different temperatures: (a) 30 °C, (b) 35 °C, (c) 40 °C, and (d) 45 °C. The applied voltage for current measurement is 400 mV. All ionic currents oscillate simultaneously with pH oscillations, and the oscillation periods decrease with increasing temperature.

Figure 4. CD spectra of C4-DNA in response to various pH values at different temperatures: (a) 30 °C, (b) 35 °C, (c) 40 °C, and (d) 45 °C. These results indicate that the critical pH value for formation of the i-motif structure of C4-DNA decreases with increasing temperature.

of the amplitude of ionic current with increasing temperature could be attributed to the temperature-induced decrease of the pH oscillation amplitudes and temperature-dependent conformation transition of DNA molecules (Figure 4). Therefore, the oscillatory nanochannel system possesses temperaturedependent autonomous gating behaviors. As the channel size has been reported as one of the key factors that affect the gating effects of the nanochannel systems, we also investigated the influence of the channel sizes on the gating behaviors of the oscillatory nanochannel system. Four typically sized nanochannels were fabricated. The base diameters (db) of the nanochannels before modification were 161.9 ± 15.1 nm, 239.2 ± 19.5 nm, 299.7 ± 26.6 nm, and 557.9 ± 31.0 nm, respectively, and the corresponding tip diameters (dt) were 25.9 ± 3.7 nm, 33.2 ± 3.7 nm, 46.2 ± 6.6 nm, and 98.5 ± 9.6 nm, respectively (Figure S16 in the SI). SEM images of the tip sides of the nanochannels are shown in Figure 6a.

oscillatory nanochannel system on the temperature was systematically studied under different temperatures from 30 to 45 °C. pH−time curves shown in Figure 5a demonstrate that the period of pH oscillations is significantly influenced by temperature, while the amplitude decreases slightly with increasing temperature. The periods of pH oscillations of the reaction solution were 2300 ± 137 s at 30 °C, 1650 ± 140 s at 35 °C, 1330 ± 50 s at 40 °C, and 765 ± 20 s at 45 °C, respectively. pH oscillation amplitudes were 3.29 ± 0.02 at 30 °C, 3.08 ± 0.06 at 35 °C, 3.06 ± 0.01 at 40 °C, and 2.79 ± 0.09 at 45 °C, respectively. The current−time curves (Figure 5b) indicate that the ionic currents oscillated in the entire temperature range and gating periods of the system are similar to the pH oscillations (see Figures S14 and S15 in the SI for detailed information). Full amplitudes were ca. 0.89 ± 0.02 nA at 30 °C, ca. 0.53 ± 0.02 nA at 35 °C, ca. 0.52 ± 0.01 nA at 40 °C, and ca. 0.4 ± 0.02 nA at 45 °C, respectively. This decrease 3026

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functions of the artificial nanochannels. Our work provides a strategy to design artificial smart nanofluidic systems that can directly convert the chemical energy of oscillating reactions to the kinetic energy of channel gating under constant conditions. The utilization of oscillating reactions integrated with C4-DNA molecules to autonomous control the gating process of artificial nanochannels represents a method to construct future spontaneous mass-delivery devices and nanofluidic ionic current oscillators, which can boost the development of bioinspired intelligent nanochannels for real-world applications.

EXPERIMENTAL SECTION Materials. Potassium hexacyanoferrate(II) trihydrate (K4Fe(CN)6, 98.5−102.0%) and phosphate -buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (China). N-Hydroxysulfosuccinimide sodium salt (NHSS, 97%), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC, 98+%), sodium bromate (NaBrO3, 99.5%), and sodium sulfite (Na2SO3, 98%) were purchased from Alfa Aesar (China). Potassium chloride (KCl, 99.8%), potassium hydroxide (KOH, 98.0%), sulfuric acid (H2SO4, 95.0−98.0%), and sodium hydroxide (NaOH, 96.0%) were purchased from Sinopharm Chemical Reagent Beijing (China). Formic acid (HCOOH, ≥88%) and hydrochloric acid (HCl, 36.0−38.0%) were purchased from Beijing Chemical Works (China). DNA oligonucleotides were synthesized by Takara Biotechnology (Dalian). Nanochannel Fabrication. The single bullet-shaped nanochannels were fabricated in polyethylene terephthalate (Hostaphan RN12 Hoechst, 12 μm thick, with a single heavy ion track in the center) membranes by using a surfactant-assistant track-etching technique.16,58 During the etching process, the PET film was immobilized between the two chambers of a custom-designed etching cell. Etchant on one side of the film was 6 M NaOH, while the other side was etched by 6 M NaOH + 0.025% sodium dodecyl diphenyloxide disulfonate at 60 °C. A constant voltage of 1.0 V was applied across the film to monitor the etching process. After the current reached the desired value, both sides of the membrane were added to a 1 M KCl + 1 M HCOOH solution that was able to neutralize the etchant, thus slowing down and finally stopping the etching process, and single bullet-shaped nanochannels were produced. The etched membranes were then soaked in Milli-Q water to remove residual salts. SEM Measurement. Scanning electron microscopy measurements were taken in the field-emission mode using a FEI Magellan 400 extreme high-resolution microscope at an acceleration voltage of 5 kV. DNA Immobilization. C4-DNA molecules were successively immobilized onto the nanochannel surface by a two-step chemical reaction. Prior to the reaction, carboxyl groups on the PET film were activated in 4 mL of PBS (pH 7.4) solution containing 60 mg of EDC and 12 mg of NHSS for 1 h. Then, the film was reacted for 2 h with a solution of 1 μM DNA in PBS buffer (pH 7.4). Finally, the modified film was washed with Milli-Q water before further experiments. Current−Voltage Measurement. Ionic transport properties of the unmodified or DNA-modified nanochannels were evaluated by the current−voltage (I−V) curves with a Keithley 6487 picoammeter (Keithley Instruments, Cleveland, OH, USA) in 0.1 M KCl. Ag/AgCl electrodes were used to apply a transmembrane potential across the membranes. The scanning voltage had a 21 s period. The cathode faced the large opening (base) of the nanochannel, and the anode faced the small opening (tip) of the nanochannel. All measurements were carried out at room temperature, and each test was repeated five times to obtain the average value. CD Spectra. Circular dichroism spectra were measured from 20 to 45 °C on a JASCO J-815 CD spectropolarimeter with a Peltier temperature control accessory. Each spectrum represented an average of three scans accumulated with a scan speed of 200 nm/min, and wavelength scans were performed between 220 and 320 nm. C4-DNA (3.2 nmols) was dissolved in an alkaline PBS buffer solution (pH 7.4, 1.6 mL) containing NaCl (0.138 M) and KCl (0.0027 M), to give a

Figure 6. Influence of the channel size on the autonomous gating functions of the oscillatory nanochannel systems. (a) SEM images of the tip sides of the four nanochannels. The scale bar is 100 nm. (b) Ionic current−time curves of four oscillatory nanofluidic systems built upon four differently sized nanochannels: dt = 98.5 ± 9.6 nm, dt = 46.2 ± 6.6 nm, dt = 33.2 ± 3.7 nm, and dt = 25.9 ± 3.7 nm. The working temperature is 35 ± 2 °C, and the applied voltage is 400 mV.

Then, the four nanochannels were modified by C4-DNA under the same condition. After modification, the four nanochannels were utilized to construct an oscillatory nanofluidic system as indicated in Figure 2. The applied voltage was 400 mV and the working temperature was 35 ± 2 °C. pH oscillations of the four oscillatory nanochannel systems are shown in Figure S17 in the SI, which demonstrated the oscillatory system had a good reproducibility. As shown in Figure 6b, all four nanochannel systems exhibit periodic ionic current oscillations (see Figure S18 in the SI for detailed information), which proves that the four nanochannels are autonomously gated. However, the amplitudes of the ionic current oscillations decreased from ∼0.55 nA to ∼0.30 nA as the channel size increased from 26 nm to 99 nm. The on−off ratios of ionic currents at open and closed states also illustrated that the gating ability of the oscillatory system decreased with increasing the channel size (Figure S19 in the SI). Therefore, reducing the size of the nanochannel is an effective way to observe a high gating effect in the oscillatory nanochannel system.

CONCLUSIONS In summary, we have constructed an oscillatory nanochannel system, in which BrO3−−Fe(CN)64−−H+−SO32− oscillating chemical reactions were used to autonomously open and close the C4-DNA-modified nanochannels under constant conditions. Cyclic conformational transitions of the DNA motors, attached on the channel walls, could induce a periodic and continuous gating of the nanochannel between open and closed states, resulting in continuous and periodic high−low ionic current oscillations of the channels. Our experimental results also systematically demonstrated the gating period of the nanochannel system could be gradually decreased by increasing the working temperature, and the large channel size is not favorable for the implementation of the autonomous gating 3027

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ACS Nano final concentration of 2 μM DNA−PBS buffer solution in a quartz cell. The pH value of the buffer was cycled between 3.5 and 6.5 by alternatively adding 1 M HCl and/or 1 M KOH to obtain the proper solution, and vice versa. Measurements of the Oscillatory Nanofluidic System. The pH value, temperature, redox potential, and ion conductivity of the oscillating reaction solution were measured by a Mettler SevenExcellence pH/mV/conductivity meters with time intervals of 5 s. Ionic current measurements were carried out by the Keithley 6487 picoammeter with time intervals of 5 s. Pt electrodes were used to apply a constant potential (400 mV) across the nanochannel membranes. The cathode faced the base side of the nanochannel, while the anode faced the tip side of the nanochannel. The ionic current measurement was started when the pH oscillation of the reaction solution was stabilized.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b08727. SEM images of the cross section and the surface of the bullet-shaped nanochannel membrane, contact angles, XPS data and current−voltage measurements of the nanochannel before and after modification, C4-DNA molecular structure, experimental apparatus, original data of ionic currents, pH, ion conductivity, temperature, and redox potential oscillations of the reaction solution at 35 °C, pH and ionic current oscillation under conditions with and without peristaltic pumps, and dependence of on−off ratios on the channel size (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (H. Zhang): [email protected]. *E-mail Y. Tian): [email protected]. ORCID

Jue Hou: 0000-0003-0325-6979 Huanting Wang: 0000-0002-9887-5555 Lei Jiang: 0000-0003-4579-728X Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research is financially supported by the National Research Fund for Fundamental Key Projects (2013CB932802), National Natural Science Foundation (21501185, 21671194, and 21421061), the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-M03), the 111 Project (B14009), and the Australian Research Council (DE170100006, DP150100765). REFERENCES (1) Hille, B. Ion Channels of Excitable Membranes, 3rd ed.; Sinauer Associates: Sunderland, MA, 2001. (2) Cao, C.; Ying, Y.-L.; Hu, Z.-L.; Liao, D.-F.; Tian, H.; Long, Y.-T. Discrimination of Oligonucleotides of Different Lengths with a WildType Aerolysin Nanopore. Nat. Nanotechnol. 2016, 11, 713−718. (3) Lan, W.-J.; Edwards, M. A.; Luo, L.; Perera, R. T.; Wu, X.; Martin, C. R.; White, H. S. Voltage-Rectified Current and Fluid Flow in Conical Nanopores. Acc. Chem. Res. 2016, 49, 2605−2613. (4) Haywood, D. G.; Saha-Shah, A.; Baker, L. A.; Jacobson, S. C. Fundamental Studies of Nanofluidics: Nanopores, Nanochannels, and Nanopipets. Anal. Chem. 2015, 87, 172−187. 3028

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DOI: 10.1021/acsnano.6b08727 ACS Nano 2017, 11, 3022−3029