pubs.acs.org/NanoLett
An Electrochemically Actuated Reversible DNA Switch Yang Yang,† Gang Liu,‡ Huajie Liu,† Di Li,‡ Chunhai Fan,*,‡ and Dongsheng Liu*,†,§ †
National Center for NanoScience and Technology, China, Beijing 100190, China, ‡ Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China, and § Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China ABSTRACT In this Letter, we have realized the electrical actuation of a DNA molecular device in a rapid and reliable manner with a microfabricated chip. The three-electrode chip containing Ir, IrO2, and Ag electrodes deposited in designed shapes and positions on the SiO2 surface was made by photolithography and magnetron reaction sputter deposition technology. In this design, the negative feedback property enabled the system to rapidly change and maintain the solution pH at arbitrary value by water electrolysis. As a proof-of-concept, we can drive a DNA switch based on the opening and close of an i-motif structure by switching the potential between the working and reference electrodes between -304 and -149 mV. We have demonstrated that DNA can be electrically switched within seconds, without obvious decay of the fluorescence amplitudes for at least 30 cycles, suggesting that this DNA switch is rapid in response and fairly robust. We have also demonstrated that this device could manipulate the DNA switch automatically by using chronoamperometry. KEYWORDS DNA, molecular switch, pH change, electrochemistry
A
simple bielectrode system, which thus cannot satisfy the requirement of driving the DNA machine via pH change. An ideal system that can controllably and reliably change pH values with high precision requires small reaction volumes (rapid bulk electrolysis) and an elaborate feedback circuit (precise control of pH). We reason that a microfluidic device incorporating pH-sensitive electrodes, which was previously designed to realize pH-stat,7 could meet such requirements. The device design is shown in Scheme 1 (bottom): the Ir, IrO2, and Ag electrodes were deposited in designed shapes and positions on the SiO2 surface via photolithography and magnetron reaction sputter deposition technology, and a layer of AgCl was generated on the Ag film by electroplating (see the Supporting Information for detailed procedures). Among them, Ag/AgCl served as the working electrode (W.E.), Ir as the auxiliary electrode (A.E.), and the pH-sensitive IrO2 as the reference electrode (R.E.). The head of R.E. was inserted into the fork-shaped A.E. head, forming a response/reaction region, while the head of W.E. formed a control region. The chip was covered by a cuboid poly(dimethylsiloxane) (PDMS) with two 10 µL wells at corresponding electrode positions. The cells were filled with the DNA switch system and the KCl solution of 0.1 M, respectively, which were linked by a liquid junction filled by 1% agarose gel. This microfabricated electrochemical device could rapidly respond to the potential variation and lead to stable pH change in the bulk solution of the well, which was suitable to be integrated with our established proton-driven DNA switch system.3 Since IrO2 is pH-sensitive, the potential between the W.E. and the R.E. in this three-electrode system
fter 3 decades, DNA nanotechnology has been sufficiently developed to realize complicated fabrication of two- and three-dimension static nanostructures.1 However, it remains challenging to design dynamic nanostructures, namely, DNA molecular switches or motors. Early models using chain-exchange-reaction are inherently slow in kinetics;2 more recently developed pH-driven strategy3 is rapid and robust,4 nevertheless energy supply is still the bottleneck for reliable real-world applications of DNA motors. Either chemical oscillation or light-driven pH variation has been proposed as an energy supply to trigger DNA motions in previous studies.5 Here we demonstrate an electrically actuated DNA switch by using a microfabricated electrochemical device. Thanks to the fast and accurate operation of electric signals,6 this electrical driven method could improve the operation speed of the DNA switch within seconds, in a convenient, reliable, and programmable way. Water electrolysis is known to accumulate protons or hydroxide ions to the proximity of anodes/cathodes, leading to local pH variations in strong electrolyte solution. In a preliminary study, we found that water electrolysis using a bielectrode system led to a pH gradient between the electrodes, which could actuate DNA switches with fluorescent readout (see the Supporting Information). However, it was difficult to rapidly and stably obtain the required pH values via controlling electrolysis speed and time course with this
* To whom correspondence should be mail.tsinghua.edu.cn and
[email protected]. Received for review: 01/18/2010 Published on Web: 03/10/2010 © 2010 American Chemical Society
addressed,
liudongsheng@
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DOI: 10.1021/nl100169p | Nano Lett. 2010, 10, 1393–1397
SCHEME 1. The Principle for an Electrically Actuated i-Motif-Based DNA Switch Integrated in a Microfabricated pH-Stat Electrochemical Chip Incorporating Three Electrodes
is related to the environmental pH. Therefore, this device can work as a pH stabilizer at constant potentials, that is, slight pH change at the response/reaction area causes a potential shift to the nonpolarizable Ag/AgCl electrode, which stimulates feedback currents that electrolyze H2O in the response/reaction region and turns solution pH back to the original value.7 Significantly, the solution pH in the response/ reaction cell can be conveniently changed by altering the potential applied to the working electrode. We tested thusprepared device via titration with a series of BrittonRobinson buffer solutions with pH values ranging from 4 to 9 using the open circuit potential (OCP) method. As shown in Figure 1a, potential between the W.E. and R.E. changed along with the solution pH as anticipated, which led to a steady linear relationship (52 mV/ pH). As a result, it is possible to adjust the pH around auxiliary/reference electrodes (the response/reaction region) by simply applying corresponding potential to the device. For example, the potential was first set to -149 mV for 5 min referring to pH 8, then the potential was switched to -304 mV (referring to pH 5), a negative current of ∼6 µA was observed, which rapidly decayed to ∼(10-9 A in 30 s and stayed in this steady state for at least 5 min (Figure 1b). When the potential was switched back to -149 mV (pH 8), a positive current appeared instantly and rapidly decayed again. Of note, a small overadjusted negative current was found around 25 s during the process, which might be related to the asymmetry of the electrochemical reaction (Ag - e + Cl- T AgCl reaction in the control region or electrolysis reaction of H2O). These data clearly suggested that the pH change in the response/reaction region was rapid and could be stabilized at desired values. © 2010 American Chemical Society
FIGURE 1. Calibration of the electrochemical chip: (a) Titration of the chip with Britton-Robinson buffer solutions with pH from 4.08 to 8.90, potentials between W.E. and R.E. were recorded for 20 s each with the open circuit potential (OPC) method. Inset picture is the scatter of potential values vs pH values and its fitting curve. (b) Chronoamperometry at potential -304 mV (pH 5) and -149 mV (pH 8) one by another for 5 min each. (-149 mV had been preperformed for 5 min before 0 s). 1394
DOI: 10.1021/nl100169p | Nano Lett. 2010, 10, 1393-–1397
FIGURE 2. CD and fluorescent spectroscopy of switch DNA: (a) CD spectra of DNA X at pH 5.0 and pH 8.0, respectively; (b) fluorescent emission curves for DNA X and control DNA at both pH 5.0 and pH 8.0. All the solutions contain 20 mM MES and 20 mM Na2SO4, while DNA concentrations for CD and fluorescent spectroscopy are 2 and 0.2 µM, respectively.
FIGURE 3. Electrically driven DNA switch visualized by fluorescent intensity: (a) fluorescent images of the reaction region after applying different potentials between R.E. and W.E. for 5 min, each; (b) potential alteration operated on samples with complementary, mismatched, and control sequences.
We chose the i-motif DNA sequence X: 5′-CCC TAA CCC TAA CCC TAA CCC-3′, which was integrated with the device to realize the electrically driven DNA switch. At pH 5.0, this switch DNA folds into a compact form, namely, i-motif structure8 where its 5′ and 3′ ends are held very close; when pH value is changed to 8.0, it is transformed into a random coil structure (Scheme 1, top). This conformation transition could be verified by circular dichroism (CD) spectroscopy. As shown in Figure 2a, at pH 5.0, a positive peak at 285 nm refers to the folded i-motif structure9 and the positive peak then shifts to 273 nm at pH 8, which indicates a single-strand DNA. By tagging a rhodamine green (fluorophore, RhG) and a Dabcyl (quencher) to the 5′ and 3′ ends of sequence X, respectively, this transition could also be followed by monitoring fluorescent emission of the fluorophore. As shown in Figure 2b, the fluorescent of RhG is significantly quenched by Dabcyl at pH 5.0. We also interrogated a control DNA: 5′-RhG-TCT ATG CTG TTA CTC TGA CTC-DABCYL-3′ at pH 5 and pH 8, which only led to a small variation that arose from the pH dependence of RhG itself.10 Therefore, the fluorescent change is suitable for monitoring the switching of the i-motif structure. In a typical study of DNA switch with the chip, we put a droplet of 0.5 µM RhG/Dabcyl modified DNA X in 10 mM © 2010 American Chemical Society
Na2SO4 (10 µL) to the RE/AE reaction region of the chip. Fluorescent intensities of the RhG at different applied potentials were observed via a fluorescent microscopy. As shown in Figure 3a, when a potential of -149 mV was applied, the corresponding pH around the RE/AE is 8.0, the DNA switch is in its open state, and the fluorescent intensity under this condition is bright; when the potential is set to -304 mV, where the pH around RE/AE is 5.0, the fluorescent intensity observed is much darker because the DNA switch is in the close state. We defined the fluorescent intensities under these two conditions as 100% and 0%, respectively. The transitions of fluorescent intensities under -175, -201, -227, -252, and -278 mV were then summarized in Figure 3a. From the result we can find that the fluorescent intensity reaches a plateau when the potential is higher than -220 mV, while a sharp transition occurs around -241 mV, a potential corresponding to pH 6.2, which is consistent with the reported transition pH value of i-motif structures.11 Thus we believe this transition represents the transition of the DNA switches. To further verify this, we investigated several control systems under the same conditions. As shown in Figure 3b, the non-i-motif control dsDNA only shows mini1395
DOI: 10.1021/nl100169p | Nano Lett. 2010, 10, 1393-–1397
FIGURE 4. The fluorescent changes following 24 cycling times of the DNA switch manipulated by alternating the operation potentials between -149 and -304 mV, 20 s per step.
TABLE 1. Comparison of Performance for Different Driving Approaches driving approach manual pH titration chemical oscillation light electricity
Our new method takes only seconds (in contrast to hours) for each cycle of the switch, indicating a significantly increased kinetics as compared to the previous ones. This kinetics is even comparable to the original manual protocol that occurs in homogeneous solution with little controllability. Such rapid kinetics enables the highfrequency operation of the DNA switch. More importantly, this electrically triggered DNA switch is fully automated and highly program controllable (see the Supporting Information), which shows unprecedented advantages over all these previous approaches.
kinetics program (single circle) robustness automation controllability ∼10 s
low
N
low
∼1 h
low
low
N
∼1 h ∼40 s
low high
N high
N high
mal changes on fluorescent intensity. The small variation of the fluorescence could be attributed to the pH influence of fluorophore itself. This result further verifies that the fluorescent intensity changes are caused by the open and close of the DNA switch. We also found that the transition of the DNA switch could be suppressed by its fully matched complementary sequence Y, because the formed doublestranded structures are more stable than the i-motif structure even at pH 5.0. However, with proper mismatches, complementary strand Y′: 5′-GTG TTA GGT TTA GGG TTA GTG-3′, which has been reported to form double-strand structures with sequence X at pH 8.0 but not to inhibit the formation of i-motif structure at low pH,12,5b shows a fairly similar behavior as switch DNA X alone. All these results are consistent with the behavior of a DNA switch driven by manual addition of acids and bases in solution; thus we conclude that the designed device can electrically drive a DNA switch. We have also tested the robustness of this device. As shown in Figure 4 the potential between R.E. and W.E. was set to -304 and -149 mV alternatively every 20 s, which switches ON and OFF of the fluorescence almost instantly, without apparent decay of the amplitude over 24 cycles. This result demonstrates that the designed system is fast and robust. Table 1 compares the performance of our electrical-driven DNA switch with the previously reported chemical oscillation- and light-driven ones. © 2010 American Chemical Society
In summary, we have demonstrated the electrical actuation of a DNA molecular switch with a microfabricated chip with high velocity and reliability. This chip, covered with a microelectrolytic bicell unit, formed a negative feedback system, which could change and maintain the pH at arbitrary values by water electrolysis. With this system, we can conveniently drive a DNA switch based on i-motif structures open and close, and the operation is fast, robust, and programmable. As this DNA switch has been successfully used in fabricating functional materials and devices,13 we believe the electrically driven method will promote its applications in the real world. Experimental Section. Regular chemicals, including all the buffer contents, were of reagent grade or better and purchased from Sigma-Aldrich Co., Ltd. Water used in all experiments was Millipore Milli-Q deionized (15.6 MΩ). The Britton-Robinson buffer for chip calibration was a classic three-acid (H3PO4, H3BO3, CH3COOH) buffer, which had a wide pH range from 2 to 12. All the oligonucleotides were purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. The sequences include DNA X: 5′-R6G-CCC TAA CCC TAA CCC TAA CCC-DABCYL-3′; DNA Y′: 5′-GTG TTA GGT TTA GGG TTA GTG-3′; DNA Y: 5′-GGG TTA GGG TTA GGG TTA GGG-3′; Control dsDNA: 5′-R6G- TCT 1396
DOI: 10.1021/nl100169p | Nano Lett. 2010, 10, 1393-–1397
REFERENCES AND NOTES
ATG CTG TTA CTC TGA CTC-DABCYL-3′ hybridized with 3′AGA TAC GAC AAT GAG ACT GAG-5′. For spectrum detection, all the DNA samples were prepared with buffer solutions (20 mM MES, 20 mM Na2SO4, and titrated by NaOH). For the electrochemically actuating operation, DNA samples were dissolved into a 10 mM Na2SO4 solution without buffer content, because of the necessity of electrolyte and the requirement of fast pH change. CD spectra were recorded on a J-810 spectrometer (DHS Instruments Co., Ltd. Dalian). Fluorescent curves were obtained from a LS 55 fluorescence spectrometer (PerkinElmer Instruments Co., Ltd. Shanghai). All the electric performances were accomplished on a CHI 660b electrochemical workstation (CH Instruments Co., Ltd., Shanghai). The fluorescent images were observed and captured with a (ZEISS AXioSKOP 2 Plus) fluorescent microscope equipped with a CCD detection system (Axiocam MRC5). The fabrication process and all the parameters of the EC chip are demonstrated in the Supporting Information. The chronoamperometry (CA) method with parameters for automatic manipulation is also introduced in the Supporting Information, companied with a video file.
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Acknowledgment. Yang Yang and Gang Liu contributed equally to this work. The authors thank Dr. Ying Fang, Mr. Xiaojun Li, and Mr. Baogang Quan for their assistance in microfabrication and the National Science Foundation of China under Grants 20725309 (for D.L.) and 20725516 (for C.F.) and MOST under Grants 2007CB935902 (for D.L.) and 2006CB933000 and 2007CB936000 (for C.F.) for financial support.
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Supporting Information Available. Figures showing the unsatisfied bielectrode system, drawings and photos of the three-electrode chip, a summary of the calibrated chips, and automatic manipulation (with video). This material is available free of charge via the Internet at http://pubs.acs.org.
© 2010 American Chemical Society
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DOI: 10.1021/nl100169p | Nano Lett. 2010, 10, 1393-–1397
